Polymer-Based Carriers for Controlled Delivery Of

Submitted by M.Sc. Javier Pérez Quiñones P OLYMER - BASED Submitted at Institute of Polymer Chemistry CARRIERS FOR Supervisor and First Examiner CONTROLLED DELIVERY Univ. - Prof. Dr. Oliver Brüggemann

OF DIOSGENIN, Second Supervisor and Examiner Univ. - Prof. in Dr. in Sabine Hild

AGROCHEMICALS AND October 2019 ANTICANCER DRUGS

Doctoral Thesis to obtain the academic degree of Doktor der Naturwissenschaften in the Doct oral Program Naturwissenschaften

JOHANNES KEPLER U NIVERSITY LINZ Altenberger Str. 69 4040 Linz, Austria www.jku.at DVR 0093696

STATUTORY DECLARATIO N

I hereby declare that the thesis submitted is my own unaided work, that I have not used other than the sources indicated, and that all direct and i ndirect sources are acknowledged as references.

This printed thesis is identical with the electronic versi on submitted.

Linz ,

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To my Ma m and Grandma

ACKNOWLEDGMENTS

First of all , I want to thank Univ. - Prof. Dr. Oliver Brüggemann. Thank you so much for giving me th e opportunity to work in ICP for more than 4 years! Many thanks also for being my supervisor, an example to follow and for your support to carry out the research and writing the related papers. I hope that our LIT application or other project applications will succeed.

Univ. - Prof. in Dr. in Sabine Hild is also thanked for being my second supervisor of the thesis.

I would like to thank Prof. Dr. Carlos Peniche Covas for his help and guidance during my time at University of Havana, and continued scientific coll aboration.

I am very grateful to Ms. Emma Huss at the JKU International Office for providing me with all the tips, support and great help to solve every problem during my stay in Linz.

I also want to thank the ICP “family” for your help and collaboration, specifically to :

- Aitziber Iturmendi, because you had a lot of patience with me and share your expertise about polyphosphazenes, and anything that I asked you. Thanks also for your collaboration on the writing of the manuscripts. - Helena Henke, because y ou were helpful and always sharing your lab experience. I hope to keep our collaboration on the papers, further scientific projects. - The Spanish speaking group at the ICP: Aitzi, Dra. Yolanda Salinas and Adriana Estrada, que hablamos en Español. Siempre me río mucho con Yolanda y Aitzi, que contagian a todos con su risotada! Gracias Yolanda porque siempre puedo contar contigo en mis problemas con mi mamá, por la paella! - Renate Herbrik (“Renate”) and Andreas Schnölzer (“Andy”) . Both of you helped me whenever I needed . Thank you so much for everything!

I would like to thank also Dr. Cezarina C. Mardare and Prof. Dr. Achim W. Hassel for their help doing some SEM and collaboration on our papers. Mr. G ü nter Hesser is acknowledged for his help with the TEM. Lisa M . Uiberlacker and Ines Traxler are also acknowledged for the AFM imaging of my samples. Prof. Dr. Claudia Schmidt is thanked for the elemental analysis. Dr. in Cornelia Roschger is greatly acknowledged for the biological assays of all my samples and fruitfu l collaboration.

I want to thank my family. THANKS a lot for all to my mam Nivia and grandma Nanina!

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ABSTRACT

Diosgenin ((25 R ) - spirost - 5 - en - 3β - ol) is a steroidal sapogenin prepared mostly from dioscin, the main bioactive component of Chinese medicinal herbs of the Dioscoreacea family. Diosgenin itself is known to show cardioprotective activity in animals, in vitro cytotoxicity t o cancer cells, anti tumor activity, as well as antidiabetic and antioxidant properties. However, its l ow oral bioavailability in mice and the poor aqueous solubil ity of diosgenin (10 µg/L) hold back its use. Diosgenin is the main substrate in the synthesis of steroids and analogues of brassinosteroids for medical and agricultural applications. In this sense, DI31 (trademark Biobras - 16), a synthetic analogue of brassinosteroid is synthesized from diosgenin for agrochemical use (increase of efficiency in crop s ~5 to 30%). However , the fast vegetal metabolism and low aqueous solubility of DI31 and S7 (another brassinosteroid analogue) limit achieving their full potential in agriculture, with multiple foliar spray applications to crops needed. Additionally, the anticancer potential of DI31 and S7 must be explored, as some brassinosteroid analogues are described to exhibit good anti tumor activity. On the other hand, traditional anticancer drugs like camptothecin derivatives (i.e. irinotecan, topotecan) and epirubi cin have shown limited aqueous solubility and severe to medium adverse side effects when administered. That is why the design, preparation and characterization of some polymer - based carriers for all mentioned drugs are still under active research. In this research project , cellulose ethers (methyl cellulose, hydroxyethyl cellulose, (hydroxypropyl)methyl cellulose), silk fibroin hydrolysate and poly(dichloro)phosphazene were properly functionalized and used as polymeric carriers of the studied drugs with in creased aqueous solubility or dispersibility, controlled drug delivery and maintained biological activity. This thesis focuses on the synthesis of polymer - based carriers for diosgenin, brassinosteroids DI31 and S7, camptothecin and epirubicin hydrochlorid e by two main routes: amphiphilic polymer - drug conjugates appearing in aqueous media as aggregates with a micelle - like or a c ore - shell particle structure, and loading of anticancer drugs via non - covalent interactions on synthesized amphiphilic polymer - co - d rug conjugates that self - aggregated as core - shell particles in aqueous media. The synthesis of cellulose ethers esterified with diosgenin, DI31 and S7 with good agrochemical activity, and the hydrophobic loading of camptothecin in testosterone - , tocopherol - and ergocalciferol - grafted cellulose ethers with maintained anticancer

ix activity are discussed in the first part of the thesis. Similarly, functionalization of a silk fibroin hydrolysate with steroids and vitamins allowed obtaining polymeric aggregates wi th sustained drug release and good agrochemical and anticancer activity, as shown in the second part of the thesis. Finally, functionalization of poly(dichloro)phosphazene s with the drugs and Jeffamine M - 1000 affording aqueous aggregates with excellent agr ochemical and anticancer activities is presented.

KURZFASSUNG

Diosgenin ((25R) - spirost - 5 - en - 3β - ol) ist ein steroidales Sapogenin, das hauptsächlich aus Dioscin hergestellt wird, dem wichtigsten bioaktiven Bestandteil chinesischer Heilkräuter der Dioscoreacea - Familie. Es ist bekannt, dass Diosgenin selbst eine kardiop rotektive Aktivität bei Tieren, eine in vitro - Zytotoxizität gegenüber Krebszellen, eine Antitumoraktivität sowie antidiabetische und antioxidative Eigenschaften aufweist. Die geringe orale Bioverfügbarkeit bei Mäusen und die schlechte Wasserlöslichkeit von Diosgenin (10 µg/L) schränkt jedoch dessen Verwendung ein. Diosgenin ist das Hauptsubstrat bei der Synthese von Steroiden und Analoga von Brassinosteroiden für medizinische und landwirtschaftliche Anwendungen. In diesem Sinne wird DI31 (Warenzeichen Biobr as - 16), ein synthetisches Analogon von Brassinosteroid, aus Diosgenin zur agrochemischen Verwendung synthetisiert (Steigerung der Effizienz in Kulturpflanzen ~ 5 bis 30%). Der schnelle Pflanzenstoffwechsel und die geringe Wasserlöslichkeit von DI31 und S7 (einem weiteren Brassinosteroid - Analogon) begrenzen jedoch die Ausschöpfung ihres vollen Potenzials in der Landwirtschaft. Darüber hinaus muss das Antikrebspotential von DI31 und S7 untersucht werden, da einige Brassinosteroid - Analoga beschrieben werden, d ie eine gute Anti - Tumor - Aktivität aufweisen. Andererseits haben herkömmliche Krebsmedikamente wie Camptothecinderivate (d. H. Irinotecan, Topotecan) und Epirubicin eine begrenzte Wasserlöslichkeit und schwere bis mittelschwere Nebenwirkungen bei der Verabr eichung gezeigt. Aus diesem Grund werden Design, Herstellung und Charakterisierung einiger Trägerstoffe auf Polymerbasis für alle genannten Arzneimittel noch aktiv erforscht. In dieser Forschungsarbeit wurden Celluloseether (Methylcellulose, Hydroxyethylce llulose, (Hydroxypropyl)methylcellulose), Seidenfibroinhydrolysat und Poly(dichlor)phosphazen e passend funktionalisiert und als polymere Träger der untersuchten Medikamente mit erhöhter Wasserlöslichkeit oder Dispergierbarkeit, kontrollierter Wirkstoffabga be und aufrechterhaltener biologischer Aktivität verwendet. Diese Dissertation befasst sich mit der Synthese polymerbasierter Trägerstoffe für Diosgenin, Brassinosteroide DI31 und S7, Camptothecin und Epirubicinhydrochlorid auf zwei Hauptwegen: amphiphile Polymer - Wirkstoff - Konjugate, die in wässrigen Medien als Aggregate mit einer micellartigen oder einer Kern - Schale - Partikelstruktur erscheinen, und das Laden von Krebsmedikamenten über nicht - kovalente Wechselwirkungen auf synthetisierten amphiphilen Polymer - Co - Medikamenten -

Konjugaten, die als Kern - Schale - Partikel in wässrigen Medien selbstständig aggregiert sind. Die Synthese von mit Diosgenin, DI31 und S7 veresterten Celluloseethern mit guter agrochemischer Aktivität und die hydrophobe Beladung von Testoste ron - , Tocopherol - und Ergocalciferol - gepfropften Celluloseethern mit Camptothecin bei gleichbleibender Antikrebsaktivität werden im ersten Teil der Arbeit diskutiert. Auch die Funktionalisierung eines Seidenfibroinhydrolysats mit Steroiden und Vitaminen e rmöglichte den Erhalt polymerer Aggregate mit verzögerter Wirkstofffreisetzung und guter agrochemischer und krebsbekämpfender Aktivität, wie im zweiten Teil der Arbeit gezeigt. Schließlich wird die Funktionalisierung von Poly(dichlor)phosphazen en mit den M edikamenten und Jeffamin M - 1000 vorgestellt, wobei wässrige Aggregate mit hervorragenden agrochemischen und Antikrebsaktivitäten erhalten werden.

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OVERVIEW

This thesis is divided in the following main chapters:

Chapter 1 presents the studied drugs (diosge nin, synthetic analogues of brassinosteroids DI31 and S7, anticancer drugs camptothecin and epirubicin), with emphasis on their structure, main properties and potentialities for medicine and agriculture, as well as the current drawbacks that restrain their use. The polymers (cellulose ethers, silk fibroin and polyphosphazenes) are proposed for the preparation of polymer - based carriers for controlled delivery of the drugs, with a brief outline of the thesis goals, scientific hypotheses supporting this resear ch and the general route followed for the preparation of polymer - based carriers. The synthesis, structure, main properties and applications of the studied polymers are also thoroughly described.

Chapter 2 shows the functionalization of water - soluble cell ulose ethers ( methyl cellulose, hydroxyethyl cellulose, (hydroxypropyl)methyl cellulose ) via esterification with steroid hemisuccinates (diosgenin hemisuccinate, DI31 and S7 hemisuccinate, testosterone hemisuccinate) for sustained drug release, or with vit amin hemisuccinates (α - tocopherol hemissucinate, vitamin D2 hemisuccinate) and further hydrophobic encapsulation of camptothecin for its controlled release.

Chapter 3 presents the functionalization of a water - soluble silk fibroin hydrolysate via esterific ation of silk fibroin tyrosine residues with N - hydroxysuccinimide esters of the steroid and vitamin hemisuccinates for controlled release of diosgenin, DI31 and S7, or further encapsulation of camptothecin, with observed good cell uptake and maintained cyt otoxicity to MCF - 7 human breast cancer cells of camptothecin bearing silk fibroin - based carriers.

Chapter 4 describes the synthesis of poly(organo)phosphazenes substituted with diosgenin, DI31, S7 glycinates and Jeffami ne M - 1000 for controlled drug rele ase and agrochemical applications, or with testosterone and tocoph erol glycinates and Jeffamine M - 1000 for further camptothecin and epirubicin loading for sustained release and potential anticancer applications.

Chapter 5 summarizes the results presented in this dissertation.

xiii LIST OF PUBLICATIONS

This dissertation is consisted of the following papers and manuscripts:

Chapter 2 .1 J. P. Quiñones, C. C. Mardare, A. W. Hassel, O. Brüggemann, Self - assembled cellulose particles for agrochemical applications, European Polymer Journal 93 (2017) 706 - 716. Reproduced with permission. Doi: 10.1016/j.eurpolymj.2017.02.023

Chapter 2 .2 J. P. Quiñones, C. C. Mardare, A. W. Hassel, O. Brüggemann, Testosterone - and vitamin - grafted cellulose ethers for sustained release o f camptothecin, Carbohydrate Polymers 206 (2019) 641 - 652. Reproduced with permission. Doi: 10.1016/j.carbpol.2018.11.047 J. P. Quiñones, C. C. Mardare, A. W. Hassel, O. Brüggemann, Corrigendum to “Testosterone - and vitamin - grafted cellulose ethers for su stained release of camptothecin” [Carbohydr. Polym. 2016 (2019) 641 - 652], Carbohydrate Polymers 208 (2019) 323 - 327. Reproduced with permission. Doi: 10.1016/j.carbpol.2018.12.085

Chapter 3 .1 J. P. Quiñones, C. Roschger, A. Zierer, C. Peniche, O. Brüggemann, Steroid - grafted silk fibroin conjugates for drug and agrochemical delivery, European Polymer Journal 119 (2019) 169 - 175. Reproduced with permission. Doi: 10.1016/j.eurpolymj.2019.07.025

Chapter 3 .2 J. P. Quiñones, C. Roschger, A. Zierer, C. Peniche - Covas, O. Brüggemann, Self - assembled silk fibroin - based aggregates for delivery of camptothecin, submitted.

Chapter 4 .1 J. P. Quiñones, A. Iturmendi, H. Henke, C. Roschger, A. Zierer, O . Brüggemann, Polyphosphazene - based nanocarriers for the release of agrochemicals and potential anticancer drugs, in revision.

Chapter 4 .2 J. P. Quiñones, A. Iturmendi, H. Henke, C. Roschger, A. F. Zierer, C. Peniche - Covas, O. Brüggemann, Polyphosphazene - based nanocarriers for the release of camptothecin and epirubicin, submitted.

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ABBREVIATION S

A431 A431 human epidermoid carcinoma cell line A549 A549 human non - small adenocarcinoma lung cancer cell line AFM atomic force microscopy Akt Protein Kinase B (PKB) AMIMCl 1 - allyl - 3 - methylimidazolium chloride ANOVA analysis of variance ATR - FTIR attenuated total reflectance F ourier transform infrared ATRP atom transfer radical polymerization BMIMCl 1 - butyl - 3 - methylimidazolium chloride BMP - 2 bone morphoge netic protein Boc - Gly - OH N - (tert - Butoxycarbonyl)glycine BR brassinosteroids BRI1 protein receptor - like - kinase of brassinosteroids in plants Caco - 2 Caco - 2 human epithelial colorectal adenocarcinoma cell line CD - 44 cell surface receptor CMC critical mi celle concentration CPT camptothecin DCC N,N ′ - d icyclohexylcarbodiimide DCM dichloromethane DLS dynamic light scattering DMAP 4 - (dimethylamino)pyridine DMF N,N - dimethylformamide DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DSC differential scanning calorimetry DU - 145 DU - 145 human prostate cancer cell line EDTA ethylenediaminetetraacetic acid EGF e pidermal growth factor EMIMAc 1 - ethyl - 3 - methylimidazolium acetate EPI epirubicin EPR enhanced permeability and retention

Et 3 N triethylamine EtOH ethanol

EV71 enterovirus 71 FDA U S F ederal Drug Administration FTIR Fourier transform infrared spectroscopy GPC gel permeation chromatography HCC Hepatocellular carcinoma cell line HCT - 116 HCT - 116 human colon cancer cell line HDL high - density lipoprotein HEC hydroxyethyl cellulose HEK - 293 HEK - 293 human embryonic kidney cell line HEL erythroleukemia cell line HeLa Henrietta Lacks cervical cancer cell line Hep2 Hep2 human epithelial type 2 cancer cell line HepG2 HepG2 human hepatocellular carcinoma cell line HER - 2 human epiderm al growth factor receptor 2 HIV - 1 human immunodeficiency virus 1 HL60 HL60 human leukemia cell line HPMC (hydroxypropyl)methyl cellulose

IC 50 half maximal inhibitory concentration JAK1 Janus kinase 1, human tyrosine kinase protein for signaling of t ype I cytokines JAK2 Janus kinase 2, human tyrosine kinase protein for signaling of type II cytokines JNK c - Jun N - terminal kinase pathway K562 K562 human leukemia cells

LD 50 oral median lethal dose LDL low - density lipoprotein LRR22 leucine - rich - repea t fragment of BRI1 brassinosteroid receptor MC methyl cellulose MCF - 7 MCF - 7 human breast cancer cell line M - 1000 amino - capped poly(ethylene oxide - co - propylene oxide) MDA 231 MDA 231 xenografts in mice, MDA - MB - 231 metastatic human breast carcinoma cells implanted in mice and forming tumors (xenografts) MeOH methanol MES 2 - ( N - morpholino)ethanesulfonic acid

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M n number average molecular weight

M w weight average molecular weight NMR nuclear magnetic resonance NMMO N - methylmorpholine oxide

[NPCl 2 ] n p oly(dichloro)phosphazene o/w oil - in - water PARP poly(ADP - ribose) polymerase PBS phosphate buffered saline PC - 3 PC - 3 human prostate cancer cell line PCL polycaprolactone PEG poly(ethylene glycol) pI isoelectric point PLA polylactic acid PLGA poly(l actic - co - glycolic) acid QSAR quantitative structure - activity relationship RES reticuloendothelial system RGD arginine - glycine - aspartic acid tripeptide RNA ribonucleic acid ROP ring - opening polymerization SAB synthetic analogues of brassinosteroid SEC size exclusion chromatography SEM scanning electron microscopy SF silk fibroin SK - OV - 3 SK - OV - 3 human ovarian cancer cell line SLFN11 Schlafen 11 factor inhibitor of DNA replication that promotes cell death in response to DNA damage STAT - 3 signal transducer and activator of transcription 3 SW - 480 SW - 480 colon adenocarcinoma cell line TBAF tetrabutylammonium fluoride TEM transmission electron microscopy TEMPO 2,2,6,6, - tetramethylpiperidine 1 - oxyl, catalyst for oxidation of alcohols to aldehydes and ketones TFA trifluoroacetic acid TGA t hermogravimetric analysis

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THF tetrahydrofuran TOC table of contents TPGS tocopherol polyethylene glycol succinate 3D three - dimensional U937 U937 human myeloid leukemia cell line (monoblastic leukemia) UV - Vis ultraviolet - visible WM9 WM9 human metastatic melanoma cancer cell line w/o water - in - oil WRN Werner syndrome protein found in patients with an autosomal recessive disorder w/sc - CO 2 water - in - supercritical - CO 2 XRD X - ray diffraction

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C ONTENTS

ACKNOW LEDGMENTS………………………………………………………………….. vii ABSTRACT………………………………………………………………………………… ix KURZFASSUNG…………………………………………………………………………... xi OVERVIEW………………………………………………………………………………… xiii LIST OF PUBLICATIONS………………………………………………………………… xiv ABBREVIATIONS………………………………… ………………………………………. xv CONTENTS………………………………………………………………………………... xix 1. Introduction……………………………………………………………………………. 1 1.1. Diosgenin………………………………………………………………………. 4 1.2. Brassinosteroids……………………………………………………………….. 7 1.2.1. Brassinosteroids in nature……… …………………………………... 7 1.2.2. Structure of brassinosteroids……………………………………….. 9 1.2.3. Biosynthetic pathway of BR…………………………………………. 1 1 1.2.4. Structure - activity studies…………………………………………….. 1 1 1.2.5. Synthetic analogues of brassinosteroids (SAB)………………… ... 1 2 1.2.6. Applications of BR and SAB………………………………………… 1 3 1.3. Anticancer drugs………………………………………………………………. 1 5 1.3.1. Camptothecin and its applications…………………………………. 1 5 1.3.2. Mechanism of action of camptothecin……………………………... 1 7 1.3.3. Camptot hecin derivatives…………………………………………… 1 8 1.3.4. Epirubicin……………………………………………………………… 2 0 1.3.5. Mechanism of action of epirubicin………………………………….. 2 2 1.3.6. Epirubicin - polymer carriers………………………………………….. 2 2 1.4. Controlled drug release………………………………………………… …….. 2 3 1.4.1. Agrochemicals release………………………………………………. 2 3 1.4.2. Nanomedicine and drug release……………………………………. 2 7 1.5. Cellulose………………………………………………………………………... 3 1 1.5.1. Cellulose properties and applications……………………………… 3 2 1.5.2. Modification of cellulose……………………………………………... 37 1.6. Silk fibroin………………………………………………………………………. 39 1.6.1. Silk fibroin properties and applications…………………………….. 4 2 1.6.2. Preparation of silk fibroin particles…………………………………. 47 1.7. Polyphosphazenes…………………………………… ………………………. 49 1.7.1. Polyphosphazenes properties and applications………………….. 5 2 1.8. References……………………………………………………………………... 58 2. Cellulose - based carriers for delivery of agrochemicals and drugs……………… 8 0 2.1. Self - assembled cellulose particles for a grochemical applications……….. 8 1 2.1.1. Introduction…………………………………………………………… 83

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2.1.2. Experimental………………………………………………………….. 85 2.1.3. Results and discussion……………………………………………… 88 2.1.4. Conclusions…………………………………………………………… 97 2.1.5. Reference s……………………………………………………………. 98 SUPPORTING INFORMATION……………………………………………………. 1 0 0 2.2. Testosterone - and vitamin - grafted cellulose ether for sustained release of camptothecin………………………………………………………………... 1 0 1 2.2.1. Introduction……………………………………………………………. 1 0 3 2.2.2. Experimental………………………………………………………….. 1 0 6 2.2.3. Results and discussion……………………………………………… 1 1 2 2.2.4. Conclusions…………………………………………………………… 1 2 5 2.2.5. References……………………………………………………………. 1 2 6 SUPPORTING INFORMATION……………………………………………………. . 1 29 3. Silk fibroin - based carriers for delivery of agrochemicals and drugs…………….. 1 3 0 3.1. Steroid - grafted silk fibroin conjugates for drug and agrochemical delivery…………………………………………………………………………. 1 3 1 3.1.1. Introduction……………………………………………………………. 1 3 3 3.1.2 . Experimental………………………………………………………….. 1 3 5 3.1.3. Results and discussion……………………………………………… 1 39 3.1.4. Conclusions…………………………………………………………… 1 4 6 3.1.5. References……………………………………………………………. 1 47 SUPPORTING INFORMATION…………………………………………………….. 1 49 3.2. Se lf - assembled silk fibroin - based aggregates for delivery of camptothecin…………………………………………………………………… 1 5 0 3.2.1. Introduction……………………………………………………………. 1 5 2 3.2.2. Materials and methods………………………………………………. 1 5 4 3.2.3. Results and discussion………………………………………… …… 1 57 3.2.4. Conclusions…………………………………………………………… 16 5 3.2.5. Notes and references………………………………………………... 16 6 SUPPORTING INFORMATION…………………………………………………….. 1 68 4. Polyphosphazene - based carriers for delivery of agrochemicals and drugs……. 17 4 4.1. Poly phosphazene - based nanocarriers for the release of agrochemicals and potential anticancer drugs……………………………………………….. 17 5 4.1.1. Introduction……………………………………………………………. 1 77 4.1.2. Materials and methods………………………………………………. 1 79 4.1.3. Results and discussio n……………………………………………… 18 3 4.1.4. Conclusions…………………………………………………………… 19 4 4.1.5. Notes and references………………………………………………... 19 5 SUPPORTING INFORMATION…………………………………………………….. 198 4.2. Polyphosphazene - based nanocarriers for the release of camptothecin an d epirubicin………………………………………………………………….. 22 1

4.2.1. Introduction……………………………………………………………. 22 3 4.2.2. Materials and methods………………………………………………. 22 5 4.2.3. Results………………… ……………………………………………… 2 30 4.2.4. Conclusions…………………………………………………………… 2 4 1 4.2. 5. References……………………………………………………………. 24 2 SUPPORTING INFORMATION…………………………………………………….. 24 5 5. Summary and Outlook……………………………………………………………….. 26 4 PUBLICATIONS…………………………………………………………………………… 26 7 CONFERENCE CONTRIBUTIONS……………………………………………………... 26 9

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1. Introduction

Dioscin is a widely distributed and abundant steroid saponin. It is the main bioactive ingredient of several herbs of the Dioscoreaceae family with a long history of application in traditional Chinese medicine for alleviation of asthma, rheum atoid arthritis and different pains. 1 Dioscin is reported to exert anti - inflammatory activity, protective effect against induced liver damage and cerebral infarct, cardioprotective effect, anti - platelet activity, immu ne - regulatory activity and anti tumor ac tivity. 1,2 In vitro anti - proliferative activity of dioscin on more than 22 cancer cell lines is exerted via different mechanisms. 2 Furthermore, in vivo anti tumor and anti - angiogenic effects of dioscin is observed in C26 colon cancer in mice, with low to no n - visible adverse side effects. 2 The metabolism of dioscin and other steroid saponins contained in the Dioscoreacea herbs, release the bioactive steroid diosgenin (aglycone) and saccharides. 1 Diosgenin ((25 R ) - spiro st - 5 - en - 3β - ol) itself is reported to exhibit cardioprotective activity by different mechanisms as concluded from studies in rats and dogs, as well as in vitro cytotoxicity to different cancer cells (i.e. HeLa, L929) and in vivo anti tumor activity. 1 Interes tingly, in vitro antiproliferative studies performed by Hong et al. , showed that diosgenin dose needed to inhibit normal cells growth was 2 to 11 times superior than the necessary dose to halt the growth of cancer cells. 1 Additionally, diosgenin is known f or its hypoglycemic and antidiabetic, hypocholesterolemic and antioxidant properties. However, its poor aqueous solubility and low oral bioavailability observed in rats hinder the pharmaceutical use of diosgenin. Therefore, this is the first scientific pr oblem to be solved. To this end, a main aim of this research is to synthesize and thoroughly characterize different polymer - based carriers for diosgenin. It is hypothesized that prepared polymeric carriers of diosgenin will allow obtaining stable aqueous d ispersions of diosgenin beyond parent drug solubility (10 µg/L), with controlled drug release behavio u r for potential anticancer applications. Besides, once the methodology or synthetic route is well optimized with the cheap substrate diosgenin, it will be applied to the synthesis and exhaustive characterization of polymer - based carriers of brassinosteroid analogues for agrochemical use. DI31 ((25 R ) - 3β,5α - dihydroxyspirostan - 6 - one, commercial trademark Biobras - 16) and S7 ((22 R ,23 R ) - 22,23 - epoxy - 3β,5α - dihydrox ystigmastan - 6 - one) are two synthetic analogues of brassinosteroids with excellent plant growth stimulator activity, protective effects on plants unde r biotic and/or abiotic stress, promotor activity of seed germination, pesticide activity and other benefic ial effects on plants, which significantly increase the efficiency of crops (~5 to 30%). Moreover, some synthetic analogues of

brassinosteroids have shown anticancer activity. DI31 is synthesized from diosgenin in three steps (epoxide of diosgenin, triol f ormation and oxidation of hydroxyl at C6 position) with good yield, and almost intact diosgenin backbone. DI31 and S7 present a slightly better aqueous solubility than diosgenin, but still inadequate for their direct use in agriculture. Then, DI31 and S7 a re prepared as 50% v/v hydroalcoholic suspensions at maximal concentration of 1 g/L with some additives to avoid precipitation of the brassinosteroids (surfactants, isopropanol, N,N - dimethylformamide), which are diluted with water until 10 - 5 to 10 - 7 g/L of brassinosteroid immediately before their foliar spray application to the plants. Two or three foliar spray applications of DI31 or S7 are usually needed on the crops due to the fast vegetal metabolism of exogenously applied brassinosteroids. Therefore, lo w aqueous solubility of brassinosteroid analogues DI31 and S7, together with their quick metabolism in plants hamper obtaining the full benefits of their application in agriculture. This is the second scientific problem to be solved in this research. Acco rdingly, the synthesis and characterization of polymer - based carriers for sustained release of brassinosteroids DI31 and S7 with maintained good agrochemical activity is also envisioned in this work. The agrochemical activity of obtained brassinosteroid (D I31, S7) carrying polymers will be evaluated using an in vitro radish cotyledon test for auxin type detection of plant growth enhancer effect. Besides, the study of toxicity of diosgenin and brassinosteroid (DI31, S7) bearing polymer - based carriers to non - cancer HEK - 293 human embryonic kidney cells and MCF - 7 human breast cancer cells is also envisioned in this research. Cancer is a major health public problem worldwide, being one of the most diagnosed, widespread and lethal disease. Unfortunately, there is a constant growth of new diagnosed patients, recurrence of tumors, resistant malignancies and deaths related to over hundred different cancers. The progress of medicine in the last 60 - 70 years, with the discovery of new potent anticancer drugs, combination treatments, genetically engineered medicines and other approaches, failed to eradicate and to reduce the devastating impact of this disease in the human race. Chemotherapy is still a key piece in the curative and palliative treatment of many cancers and t umors. In this sense, the design and evaluation of novel nanomedicines for anti tumor use is of vital relevance and plenty of resources and researches are devoted to this end. That is why another major aim of this research is to synthesize and characterize polymer - based carriers for controlled delivery of anticancer d rugs, with maintained good anti tumor activity and potential better therapeutic effect (enhanced anticancer activity with lower dose and reduced adverse side effects). Particularly, hydrophobic

c amptothecin (precursor of topotecan, irinotecan) and the more hydrophilic epirubicin hydrochloride are the anticancer drugs studied in this work. To evaluate the cytotoxicity of camptothecin and epirubicin bearing polymer - based carriers expressed in terms of HEK - 293 and MCF - 7 relative cell viabilities is another aim of this work. Camptothecin ( S - (+) - camptothecin or (20 S ) - camptothecin ) (CPT) is a natural product first isolated from plants, which forms the family of topoisomerase I inhibitors. It has shown s trong in vitro cytotoxicity to many cancer cell lines and in vivo anti tumor activity against different malignancies. Camptothecin and some derivatives also showed antiviral activity against HIV - 1 and hepatitis C virus. On the other hand, the low solubility of camptothecin in water (1.3 mg/L), low stability and efficacy at human body conditions (~10% of the bioactive lactone form of camptothecin occurs at pH 7.4), and severe adverse side effects paused its medical utilization since the 1970’s. Epirubicin ( 4’ - epidoxorubicin ) is an anthracycline antibiotic with anticancer activity due to its topoisomerase II inhibition effect. Epirubicin is clinically applied in combination with other anti tumor drugs against lymphomas, lung cancer, and other cancers with approx imately similar therapeutic results and reduced side effects compared to doxorubicin. The commercial form of epirubicin is the epirubicin hydrochloride, a low to moderated water - soluble drug (10 mg/mL at 25 o C), which is stable between 4.5

Scheme 1. (a) Polymer - drug conjugates that self - assembly as aggregates with a micelle - like structure or core - shell particle in aqueous media, (b) hydrophobic encapsulat ion of camptothecin in the inner core or hydrophilic loading of epirubicin in the outer shell of amphiphilic polymer - co - drug conjugates self - aggregated as core - shell particles in aq ueous media. Symbols: ( ) P=N backbone of polyphosphazenes or hydro philic backbone of celluloses and silk fibroin, ( ) hydrophobic drug (diosg enin, DI31, S7 ) or co - dr ug (tocopherol, ergocalciferol, testosterone), ( ) hydrophilic Jeffamine M - 1000 in polyphosphazenes,

( ) so lvent (DMSO or H 2 O), ( ) encapsulated CPT, ( ) epirubicin hydrochloride.

1.1. Diosgenin Diosgenin ((25 R ) - spirost - 5 - en - 3β - ol) is a steroidal sapogenin prepared by acid, basic or enzyme hydrolysis of dioscin 3 and other saponins 4 ( Figure 1 ). The yields of diosgenin preparation ranged from 0.2% - 3% to 2 - 4% (dried weight of vegetal material used as dioscin source ) when traditional acid hydrolysis is compared to modern and significantly less pollutant methods such as pressurized biphase acid hydrolysis, 5 - 7 ionic liquid hydrolysis 8 and enzyme hydrolysis. 9 Dioscin is the most common steroidal saponin. It has been iso lated and extracted from more than 20 botanic families of plants, with the roots of dioscorea composita and dioscorea floribunda considered the richest source, 4,10,11 and dioscorea zingiberensis and dioscorea nipponica considered

important sources. 3,5 - 10 D iosgenin has been thoroughly employed as starting substrate in the chemical synthesis of many steroids (i.e. progesterone, other sex hormones and several corticosteroids). 12,13 It is reported that 60% of steroidal drugs and almost all their production in I ndia was based on diosgenin. 9,10 Furthermore, several synthetic analogues of brassinosteroids (SAB) with plant growth regulator or anti - ecdysteroid effect have been synthesized from diosgenin. 14 - 16

Figure 1. Chemical structures of diosgenin (I) and d ioscin (II).

Diosgenin and its saponins are known to exert hypocholesterolemic, antioxidant and antidiabetic effects when administered in mice. 17 - 20 It is considered that diosgenin and its derivatives are capable of reducing serum cholesterol by inhibitio n of the cholesterol absorption. 21 In this sense, Marín - Medina et al. showed that diosgenin alone can diminish one half of the cholesterol activity found in liver of hypercholesterolemic rats, with a doubled fecal elimination of neutral steroids. 22 In anot her study, Manivannan et al. showed that treatment in adenine - induced chronic renal failure rats with 40 mg/kg of diosgenin significantly increased the activity of enzyme antioxidants (superoxide dismutase, catalase, glutathione peroxidase) and the level o f reduced glutathione by 1.4 times, with a reduction of lipid peroxidation products found in the heart and prevented cardiac fibrosis. 20 These results confirmed the previous findings of Patel et al. and Son et al. about the benefits of diosgenin supplement ation in rats. 17,18 It caused significant reduction of their plasma and hepatic total cholesterol and LDL - cholesterol with an increase of HDL - cholesterol via suppression of cholesterol absorption and increasing cholesterol secretion. 17,18 It was also obser ved a significant increase of antioxidative enzyme activities (superoxide dismutase, glutathione peroxidase and catalase) and hypoglycemic effects by reducing intestinal disaccharidases activities. 17,18 The anticancer effect of diosgenin, dioscin and other diosgenin saponins has been thoroughly studied to determine their potential as antitumor candidates. It is accepted that the anticancer effect of diosgenin and their saponins mainly depends on the cell

line, on the evaluated concentration and on time. 23,2 4 Besides, diosgenin has been reported to show anti - proliferative, anti - metastatic, anti - angiogenic and pro - apoptosis effects against different tumor cells such as leukemia, breast and prostate cancer, colorectal cancer, osteosarcoma and hepatocellular can cer. 23 - 30 Bairi et al. listed the anticancer activities of diosgenin in vivo and in vitro from available literature, showing that diosgenin used in doses from 5 to 100 µmol/L and 15 to 500 mg/kg resulted effective in inhibition of proliferation of 14 cance r cell lines or tumors (i.e. 1547 osteosarcoma cells, HEL erythroleukemia cells, human breast cancer MCF - 7 and MDA 231 xenografts in mice, PC - 3 human prostate cancer cells). 29 Diosgenin was also found to activate p53 gene responsible to release apoptosis - i nducing factor to modulate caspase - 3 activation in six cancer cell lines or tumors (i.e. 1547 osteosarcoma cells, MCF - 7 breast carcinoma cells) and to activate NF -  B signal pathway in five cancer cells (i.e. U937 leukemia cells, MCF - 7 breast cancer cells, PC3 human prostate cancer cells). 27 Furthermore, it can provoke PARP cleavage and inhibition of STAT - 3, JAK1 and JAK2 in human hepatocellular carcinoma HCC cells and to inhibit Akt and JNK phosphorylation in A431 and Hep2 cells, and HER2 - 27 overexpressing can cer cells. Besides, several diosgenin saponins have an increased anticancer effect, with an important influence of the saccharide fragments covalently bonded to the diosgenin on the biological activity. 30 In a recent study of Yonghua et al. , 18 synthetic glycosylated derivatives of diosgenin showed significantly better anticancer activities than diosgenin on human leukemia HEL, K562, HL60 cells and melanoma WM9 cells. 30 In addition, one of the synthetic diosgenin glycosylated derivatives and two natural s aponins exhibited excellent anticancer activity, with IC 50 (half maximal inhibitory concentration) similar or lower than of traditional anticancer drug Adriamycin. Similarly, two different diosgenin esters were synthesized using ionic liquid as a catalyst and both esters showed slightly better anticancer activity than diosgenin when evaluated in PC3 human prostate cancer cells. 31 On the other hand, the very low aqueous solubility of diosgenin (approximately 10 µg/L in distilled water at 37 o C), together wit h a scarce absolute oral bioavailability (4.5% - 7.6%) when administered to mice limit its medical applications. 32,33 Okawara et al. demonstrated that inclusion complexes of diosgenin in different β - cyclodextrin derivatives increased 100 - 20000 times the solu bility in water of diosgenin and 3 - 10 times the oral bioavailability in rats. 32 These facts and the chemical similarity of diosgenin and several SAB with agrochemical activity, motivated us to synthesize different diosgenin - and brassinosteroid - chitosan an d hyaluronic acid conjugates for drug delivery applications. 34,35 Efficient functionalization of chitosan and hyaluronic acid

with diosgenin and brassinosteroids, with a sustained release of the drugs and good agrochemical activity of polymer conjugates we re achieved. These results motivated us to synthesize new drug delivery systems based on other biomaterials (cellulose derivatives, silk fibroin and polyphosphazenes) for sustained release of diosgenin, brassinosteroids and anticancer drugs.

1.2. Brassino steroids 1.2.1. Brassinosteroids in nature Brassinosteroids (BR) are steroid plant hormones (phytohormones) with the capability to regulate the vegetal physiological processes at very low concentrations (approximately 10 - 9 mol/L). In this sense, BR togethe r with other phytohormones regulate growth, xylem differentiation, senescence and disease resistance of the plants. 36,37 The phytohormones are traditionally classified in five families: auxins, gibberellins, ethylene, cytokinins and abscicic acid ( Figure 2 ). Their biosynthesis involves the entire plant, 38 and usually the physiological processes are regulated or influenced by each one independently or in a simultaneous action of several phytohormones. 37,39 These phytohormones exert their effects in local tis sues and cells. 37,39,40 Phytohormones control the biosynthesis of proteins and carbohydrates, regulate the water content in plants, protect them from environmental stress and facilitate the transport of the photosynthetic products. In addition, they also r egulate growth and flower processes, modulate vegetal response to pathogens attack and control the fruit ripening. That is why the BR are applied as plant growth regulators in agriculture. 41 - 43

Figure 2. Chemical structures of representative phytoh ormones: (a) 3 - Indoleacetic acid (auxin), (b) gibberellic acid (gibberellin), (c) ethylene, (d) kinetin (cytokinin) and (e) abscisic acid.

Grove et al. isolated 4 mg of a steroidal compound, with potent stimulant plant cell growth activity, from 40 kg of Brassica napus var. Napus L. pollen in 1978. 37 The structure of this compound known as brassinolide ((22 R ,23 R ) - 2 α ,3 α ,22,23 - tetrahydroxy - 6,7seco - 5 α - campestan - 6,7 - lactone) was defined by NMR spectroscopy and XRD in 1979 ( Figure 3 ). 37,44 The is olation of other compounds with similar structure and biological activity in the following years end up in the rise of the brassinosteroids. 37,45

Figure 3. (I) Structure of brassinolide ((22 R ,23 R ,24 S ) - 2 α ,3 α ,22,23 - tetrahydroxy - 24 - methyl - B - homo - 7 - oxa - 5 α - cholestan - 6 - one) and (II) conformational model of BR backbone. Adapted with permission from reference 46 .

BR were classified as the sixth class of phytohormones, once discovered the protein receptor involved in their molecular recognition in 1997 (leuci ne - rich - repeat ectodomain of the receptor - like - kinase BRI1). 47,48 BR are the only phytohormones with steroid structure (cyclopentanoperhydrophenanthrenes). Currently, almost 70 BR have been isolated from plants. 37,49,50 These phytohormones control the germ ination of seeds, the flowering and the accretion of biomass in plants via modulation of cell division and enlargement, while exerting a vital role in plant photo - regulation through the synthesis 36,37,40,42,43,50,51 of the chlorophyll and CO 2 assimilation. Thus, BR protects plants from pesticides, herbicides and environmental stress (i.e. dryness or flooding, extreme temperatures, salinity, viral infections, fungal attacks, others). 50,52,53 These phytohormones are widely disseminated around the vegetal king dom (i.e. monocotyledons, dicotyledons and gymnosperms), and in algaes and cyanobacterias in contents from 10 - 1 to 10 - 7 nmol/g. 48,54 The BR content in plants depends on specie, plant component and stage of development. The higher contents of BR in plants a re usually found in the immature seeds, the pollen and the small fruits. 54,55 The regulatory effect of BR is observed at concentrations 100 smaller than the required for other phytohormones and the interaction of BR with auxins, gibberellins and other

phyt ohormones is well documented. 36,37,42,56 - 58 Therefore, BR have been employed as agrochemicals, because small quantities (from 5 to 100 mg/ha) applied can increase the yield and quality of several crops. 56,57

1.2.2. Structure of brassinosteroids The 5 α - cholestan is the basic skeleton of the BR. ( Figure 4 ). The main variations in the BR structure are observed at the oxygenated functions presented in the rings A and B, and/or in the branched chain. 54,55,57 10 structural modifications related to different oxygenated groups, their position and conformations are documented in the A ring. 54,55,57 BR often present a 2 α ,3 α - diol function. BR with 2 β ,3 α y 2 β ,3 β configurations are possible metabolites of the 2α,3α BR. 46 On the other hand, BR with only an oxygenate d function at C3 are precursors in the biosynthesis of the BR carrying a 2 α ,3 α - diol. The trend of BR biological activity follows the order: 2 α ,3 α > 2 α ,3 β > 2β,3 α > 2 β ,3 β . It seems that the orientation 2 α of a hydroxyl substituent is essential for good BR b iological activity. 46,55 The most active BR exhibits a seven - member B nucleus with a 7 - oxalactone function. The six - member B ring without oxygenated function or carrying a 6 - oxo or 6 - hydroxy groups are biosynthetic precursors of 7 - oxalactone BR. 46 However, BR with a 6 - oxo group are the majority of the isolated BR. The biological activity of the 6 - oxo BR is approximately a 50% of the observed in BR with a 7 - oxalactone moiety. 46 The branched chain of BR is classified by the number of carbon atoms as: C27, C28 y C29. 54,57 In addition, BR have a 22 R ,23 R - diol function. C24 is the most important carbon of branched chain affecting the biological activity of BR. 54 In this sense, substituents and stereochemistry at C24 influence the biological activity of BR (i.e. br assinolide presents a methyl group at C24 with stereochemistry S , 24 - epibrassinolide shows the methyl group at C24 with stereochemistry R , 28 - homobrassinolide exhibits a ethyl group at C24 with stereochemistry S , the dolicholide possess a methylene group a t C24 and the norbrassinolide have not substituent at C24). 46 Several BR show an isopropyl substituent at C25, but sometimes a tert - butyl substituent is observed at C25 (i.e. 25 - methyldolichosterone) 46,59 ( Figure 4 ).

Figure 4 . Substituents at A ring, B r ing and branched chain of BR. Reproduced with permission from reference 46 .

1.2.3. Biosynthetic pathway of BR The arabidopsis thaliana is often selected to study the vegetal physiology and genetics, and the biosynthetic pathway of BR. 38,40,42,45,47 - 49,54,5 5,58,59 The genome of this plant was fully sequenced in 2000. 60 These studies demonstrated that the biosynthesis of brassinolide starts from campesterol throughout two independent routes: early and late oxidation, with the predominant route being determine d by environmental conditions. 46,61 It is proposed that early oxidation of B ring occurs as predominant route in darkness, while early oxidation is the most important pathway in lighted conditions. 61 Chory et al. identified the protein BRI1 as the BR recep tor in this plant. 47 These authors demonstrated the link of BR and BRI1 through a 70 aminoacids section of the protein and the fragment LRR22. 62 However, the signal transduction and mechanism of action of BR is not completely known. 48 Recent crystallograph ic studies together with computational chemistry and docking simulations are getting new insights about the 3D structure of the BRI1 receptor protein and are employed to predict BR - type activity of different compounds. 50,63,64

1.2.4. Structure - activity stu dies The first quantitative structure - activity studies (QSAR), performed by Thompson and Takatsuto, 46,65 and experimental results of bioassays to evaluate the BR plant growth activity 66,67 make clear the most important structure requirements for a good agr ochemical activity of BR. These conditions are:  2 α ,3 α - diol system in the A ring.  6 - oxo - 7 - oxalactone or 6 - oxo in the B ring.  Trans fusion of the A and B rings.  22 R ,23 R - cis diol in the branched chain.  A methyl or ethyl group at C24 with configuration S. Morera - Boado 46 also showed with the use of computati onal chemistry techniques (i.e. molecular mechanics, semiempirical and DFT calculations):  The most stable conformations of the BR are the ones with the branched chain pointing out to the β side of the cyclopentanoperhydrophenanthrene skeleton.  There is int ramolecular H - bonding in the 2,3 - diol system of the A ring and in the 22,23 - diol of the branched chain of the BR.  The interaction with water seems to be more favorable for the diol system in the A ring than for 7 - oxalactone or a carbonyl group in B ring. However, Brosa et al. demonstrated that more important than these functionalities is the 3D molecular conformation of the compound because of its interactions with the BR

receptor in the plants. 66 The recent advances in the 3D structure elucidation of the BRI1 BR receptor in the plant allowed a more precise and systematic screening of different compounds as well as the design of BR mimetics with potential agrochemical activity with the use of molecular docking simulations and other modern computational chem istry techniques. 50,68,69

1.2.5. Synthetic analogues of brassinosteroids (SAB) The isolation of BR from nature is not economically suitable nowadays because their low content in plants. Therefore, the synthetic production of BR has been proposed, but low y ields and many synthetic steps make this approach unsuitable. On the other hand, the synthesis of BR analogues (compounds with structure variations when compared to natural BR), seems a promising strategy. SAB show in vitro agrochemical activities lower th an BR. However, the SAB exhibit good in vivo plant growth activity and their synthesis is efficient and economic. In this sense, more than 70 SAB have been synthesized from steroid sapogenins and cholic acids. 14,70,71 Over 100 SAB have been prepared at Cen ter of Natural Products (CNP) of University of Havana, Cuba from steroid sapogenins, cholic acids and stigmasterol. Particularly, the SAB obtained of steroid sapogenins showed good in vivo agrochemical activity. For example, the SAB DAA9, with just a 4% of in vitro biological activity of brassinolide, presented a similar in vivo activity to brassinolide. 72 Figure 5 shows the structure of the most active spirostane - based brassinosteroids prepared at CNP. Table 1 summarizes results obtained in different crops upon application of two SAB, with increased crop yields of 10 to 30%.

Figure 5. Chemical structures of bioactive spirostane - based brassinosteroids synthesized at CNP.

Table 1. Biological activity of two SAB synthesized at CNP. 73 - 75 Increase d yield SAB Crop (%) DAA6 Tomate 10 - 30 Potato 10 - 25 Onion 15 - 25 Garlic 5 - 27 Soy bean 10 - 25 Pepper 15 - 30 Rice 15 - 25 Beans 10 - 15 Tangerine 10 - 15 Watermelon (germinación).30 DI31 Potato 18 - 25 Onion 25 - 30 Garlic 5 - 25 Soy bean 15 - 20 Pepper 28 Grape 5 - 10 Rice 15 - 25 Beans 15 - 20

Another relevant SAB created at the CNP from stigmasterol is S7 ((22 R ,23 R ) - 22,23 - epoxy - 3β,5α - dihydroxystigmastan - 6 - one) ( Figure 6 ), which exhibits in vitro and in vivo agrochemical activity quite similar to the observed for DI31. 76

Figure 6. Chemical structure of bioactive stigmasterol - based SAB S7 synthesized at CNP.

1.2.6. Applications of BR and SAB The plants are affected by different biotic and abiotic stress conditions (i .e. heavy metals, dryness or flooding, salinity, extreme temperature, plagues and pathogen infections). Dryness, salinity, extreme temperatures and oxidative stress are usually interrelated and seem to cause similar cell damage. In addition, these factors are able to produce morphological, physiological, biochemical and molecular changes in plants.

Furthermore, several studies demonstrate the increased activity of antioxidant enzymes (i.e. superoxide dismutase, catalase, glutathione peroxidase and ascorbate peroxidase) in plants under oxidative stress when exogenous BR are applied. 49,77 However, there is still missing information about the molecular mechanism involved in the hormone - mediated adaptive response to environmental stress, particularly to biotic s tress conditions (plagues and pathogen infections). 36,78,79 The stress protective effect of BR is often intimately related to their interaction with the other phytohormones. 36,78,80,81 Divi et al. proposed that BR exert their stress protective effect on pl ants independent or related with other hormones sharing transcriptional targets. 81 Additionally, abscisic acid inhibits BR effects in plants under stress. 81 BR are effective in promotion of growth and development of plants under saline stress. 82,83 However , it is vital to find the optimal conditions (dosage, time, application route) for BR application to different plants under saline stress. 82,83 In another study, Houimli et al. established that external supply of 24 - epibrassinolide increases the growth and protects the cell membrane integrity in plants under saline stress. 84 On the other hand, Hayat et al. showed the alleviation of damage caused in Vigna radiata (green soy bean) crops by thermal and/or saline stress as result of exogenous supply of 28 - homob rassinolide. 85 BR (particularly brassinolide, 28 - homobrassinolide and 24 - epibrassinolide) have been exhaustively described to protect the plants exposed to extreme temperatures, dryness or flooding via modulation of the vegetal antioxidant 77 defense system, increased CO 2 assimilation and proteins biosynthesis. Kohout et al. reported that external application of 24 - epibrassinolide to rape plants is able to increase the internal levels of other phytohormones (i.e. indole - 3 - acetic acid, 6 - benzylaminopurine, tr ans - zeatin, dihydrozeatin). 86 Several studies have indicated that metabolism of heavy metals and pesticides in plants treated with BR decrease their levels in crops and reduce the soils pollution. In this sense, there are interesting reports about cucumber plantations treated with 24 - epibrassinolide and under high levels of chlorinated pesticides, 87 radish plants treated with 24 - epibrassinolide and tomato crops treated with 28 - homobrassinolide and exposed to high levels of cadmium, 88,89 pigeon pea plants tr eated with 24 - epibrassinolide and exposed to high levels of aluminium, 90 and Chlorella vulgaris algae treated with brassinolide and exposed to high levels of copper, lead and cadmium. 91 Vardhini and Anjum revised the stress protective effect of 28 - homobras sinolide and 24 - epibrassinolide on different plants (i.e. cucumber, radish, tomato, common bean, mung bean, brown mustard) under heavy metal condition (i.e. Cd, Cu, Pb, Ni, Cr, Al, Mn, As) reported until end of 2014. 77

As previously described, BR (i.e. 24 - epibrassinolide, 28 - homobrassinolide) and SAB are able to stimulate the germination of seeds, 92 the growth of plants and efficiency of crops, 93,94 to promote the photosynthesis, 95 and senescence processes. 96 Furthermore, some BR and SAB are proposed as pla guicides, due to their anti - ecdysteroid activity. 16,97 Moreover, some BR and SAB exhibit antiviral and anticancer activity. 98 - 100 A comparative study about lipid peroxidation and antioxidant activity of 28 - homobrassinolide and gibberellic acid on diabetic mice concluded that these compounds seem to improve the physiological condition through these mechanisms. 101

1.3. Anticancer drugs Cancer is one of the top five leading causes of death worldwide and estimated to be the major cause of death this century. 18 .1 million new cancer cases were diagnosed and 9.6 million cancer deaths accounted in 2018, with more than 100 different cancers affecting humans. 102 Currently, l ung cancer is the most common and deadly malignancy with 11.6% and 18.4% of the total diagnose s and deceases, followed by breast and prostate cancers, and colorectal cancer in terms of incidence and mortality. 102 The prevalence and deaths related to cancer increase annually, in spite of progress in the antitumor treatments (i.e. personalized chemot herapy, immuno - therapy and genetically designed nanomedicines). 102,103 However, traditional chemotherapeutic drugs such as camptothecin analogues, doxorubicin, cisplatin and others are still often used alone or in combination with radiotherapy, surgery rem oval of tumors and new nanomedicines for treatment of cancers. 103 - 105 Nevertheless, camptothecin derivatives, doxorubicin and cisplatin have shown negative side effects and cancer cells resistance. 106,107 To reduce the side effects of camptothecin and doxo rubicin with increased aqueous solubility, we prepared camptothecin - and doxorubicin - loaded hyaluronic acid - testosterone nanoparticles. 108 The sustained drug release and good anticancer activity of these hyaluronic acid - testosterone nanoparticles encourage d us to prepare new drug delivery systems based on water - soluble cellulose ethers, silk fibroin and polyphosphazenes for controlled release of camptothecin and/or epirubicin (4’ - epidoxorubicin).

1.3.1. Camptothecin and its applications Camptothecin (CPT) i s a natural plant alkaloid with topoisomerase I inhibitor activity first isolated from Camptotheca acuminata Decne. (Nyssaceae) by Wall et al. in 1966. 109,110 CPT has been also isolated from Nothapodytes nimmoniana (Grah.) Mabb. and other species of the fa milies of Camptotheca, Icacinaceae, Rubiaceae, Apocynaceae and Loganiaceae. 111,112 Nowadays, most of CPT is still extracted from

the fruits and stem bark of Camptotheca acuminata (from China) and N. nimmoniana (from India). 112 In spite the improvements of CPT extraction with ethanol from plants with the use of accelerated solvent extractor, 113 ultrasonic extraction 114 and microwave - assisted extraction, 115 the low yield achieved (maximal from 0.2% to 1.4%) 111 - 113 and depletion of vegetal CPT sources motivate d the complete chemical synthesis and biotechnological production of CPT as alternatives. 116,117 CPT has shown potent in vitro antiproliferative effect on several cancer cell lines (IC 50 on Caco - 2, HeLa, MCF - 7 and PC - 3 of 2.41 µmol/L, 0.62 µmol/L, 0.56 µmo l/L and 0.48 µmol/L, respectively) 118 - 120 and in vivo anticancer activity against ovarian cancer, small - cell lung cancer, breast cancer, melanoma, sarcomas and lymphomas among others. 120 - 122 CPT and 9 - nitro - (20 S ) - camptothecin exhibit good in vitro and in v ivo antiviral activity against hepatitis C virus, HIV - 1 and enterovirus 71 (EV71). 123 - 125 However, the low aqueous solubility (1.3 µg/mL in water), 126,127 severe side effects of its administration (i.e. myelosuppression, severe hemorrhagic cystitis, diarrh ea, vomiting), reduced stability and efficacy in physiological conditions, as well as cancer cell resistance mechanisms halted its medical application in chemotherapy since 1972. 126 - 129 That is why the synthesis of different CPT derivatives capable to circ umvent these problems have been extensively performed for decades. In this sense, thousands of CPT analogues are described in the literature, as well as their in vitro and/or in vivo anticancer activity evaluation. 1 09 However, only topotecan and irinotecan , two CPT analogues, have been approved by the US FDA (Food and Drug Administration, USA), European regulatory agency and Japanese regulatory agency. 127 ,1 28 Both CPT derivatives are widely used as chemotherapeutic drugs. CPT ((4 S ) - 4 - ethyl - 4 - hydroxy - 3,4,12, 14 - tetrahydro - 1H - pyrano[3’,4’:6,7]indolizino[1,2 - β]quinoline - 3,14 - dione) usually named S - (+) - camptothecin or (20 S ) - camptothecin, is a pyranoindolizinoquinoline that present a pentacyclic ring structure with a pyrrolo (3,4 - β) quinoline fragment (A, B and C rings), a pyridine fragment (D ring) and one asymmetric stereocenter at C20 with ( S ) configuration in the α - hydroxy lactone ring (E ring) ( Figure 7 ). 130 The α - hydroxy lactone ring is susceptible of hydrolysis in aqueous medium. CPT is in equilibrium betwee n the lactone (closed - ring), capable to inhibit the topoisomerase I and to exhibit anticancer activity, and the carboxylate (open - ring) ( Figure 7 ) with superior aqueous solubility, higher affinity for human serum albumin and minimal antitumor activity. 128, 130,131 It is considered that approximately 90% of the active lactone (closed form) CPT is available at pH 4.5, but only about 10% of the lactone CPT is present at pH 7.4. 127 In this sense, lactone (closed - ring) and carboxylate (open - ring) CPT equally coex ist at pH of 6.65. 131

Figure 7. Chemical structure of camptothecin and equilibrium between the lactone (closed - ring) A and the carboxylate (open - ring) B of camptothecin. Reproduced with permission from reference 131 .

1.3.2. Mechanism of action of campt othecin CPT and metabolites protects the plants against thermal stress and insect and pathogen attack. However, the biosynthetic route of CPT in plants is not completely known. 132 Camptothecin is considered an inhibitor of DNA replication. Its mode of acti on is based on formation of a ternary complex with enzyme topoisomerase I and DNA that stops the DNA replication and cell proliferation. 109 , 128,130,133 This complex avoids the DNA re - ligation of the nicked DNA and dissociation of the enzyme from the DNA st rand, with subsequent cell death. 1 33 The DNA - topoisomerase I - CPT ternary complex is proposed to be formed by several hydrogen bonds. 128 The E ring of CPT interacts from three different positions with the topoisomerase I. The formation of two hydrogen bonds between oxygen atoms in CPT lactone with the amino groups of Arg364 (arginine residue at position 364 of topoisomerase I) and between the (20 S ) – OH group of CPT with the side chain of Asp533 (aspartate residue at position 533 of topoisomerase I) stabilize the CPT - topoisomerase I adduct, whereas hydrogen bond formed between the C17 carbonyl group of CPT and the amino group of pyrimidine ring of the +1 cytosine of the non - cleaved DNA strand allows the formation of the ternary complex. 128 It is known that onl y the (20 S ) - camptothecin is active to topoisomerase I and that the planar structure of CPT is required for a proper interaction with the enzyme. 128,130,133 Therefore, CPT cytoxicity is mostly due to collisions between replication forks and the ternary comp lex of CPT - topoisomerase I - DNA with breakage of DNA double - strands during DNA replication and activation of the cell death routes by p53 (TP53) and Schlafen 11 (SLFN11). 109, 133 CPT resistance of cancer cells is related to the potential of camptothecins to downregulate the expression of topoisomerase I needed for the CPT mechanism of action and/or topoisomerase I gene mutations in cancer cells. 1 09 CPT also altered the cellular localization and induced degradation of

Werner syndrome protein (WRN) involved in DNA repair, genome stability and cells senescence. 134

1.3.3. Camptothecin derivatives Li et al. summarized the most important findings from the studies on CPT structure - activity relationship as: the lactone (closed - ring) of E ring is much more cytotoxic th an the carboxylate (open - ring) form of E ring; 109 ,128 the (20 S ) configuration of hydroxyl group at E ring is needed for CPT cytotoxicity, while (20 R ) - camptothecins are inactive; 109 ,128,130 the A and B aromatic rings and the C ring are important for antican cer activity of CPT, and their modifications are tolerated. 128 In general, substitution of CPT in C, D, E rings usually reduce the anticancer activity. 128 Surprisingly, modification of CPT lactone ring by inserting a methylene group between the asymmetric C20 and the lactone resulted in a 7 - members β - hydroxylactone CPT analogue (homocamptothecin) with good anticancer activity and better stability than CPT in serum and buffer solutions. 128,130 Other CPT analogues includes the silatecans (7 - silylcamptothecin derivatives) and the homosilatecans (7 - silylhomocamptothecin derivatives) with reduced CPT - serum albumin interaction and better stability. 130 Several CPT analogues are under different clinical phase study. In this sense, FL118 (10,11 - methylenedioxy - (20 S ) - c amptothecin) is particularly promising against colorectal and head - neck cancers in human tumor models, and it appears effective against human xenograft tumors resistant to irinotecan. 10 9 However, only irinotecan and topotecan, two water - soluble CPT analogu es, have been approved and are actively used as chemotherapeutic drugs. 109 ,127,128 Irinotecan (trademark Camptosar) is widely used for medical treatment of refractory non - Hodgkin lymphomas, metastatic breast cancer, small - cell and non - small cell lung cance rs, colon and rectum cancers, ovarian and cervix cancers, as well 128,130 as head and neck cancers. The reported values of IC 50 for irinotecan in vitro applied to HCT - 116 and SW - 480 human colorectal cancer cells are 12.1 µmol/L and

11.8 µmol/L, respectively whereas IC 50 values in MCF - 7 human breast cancer cells, A549 human non - small cell lung cancer and SK - OV - 3 human ovarian cancer cells are 1.4 µmol/L, 4.9 µmol/L and 1.0 µmol/L, respectively. 135,136 The side effects of irinotecan includes myelosuppression ( anemia and reduced leucocyte counts), pulmonary toxicity and gastrointestinal problems (vomiting, and particularly diarrhea as dose limiting when irinotecan is used on a daily basis). 128,130 On the other hand, topotecan (trademark Hycamtin) is mainly indic ated for the treatment of refractory and advanced ovarian cancers. 128,130 However, topotecan is also used in the treatment of cervical cancers, small - cell carcinoma, gastric and breast cancers, hepatocellular and pancreatic

cancers, melanoma, myeloma and p rostate cancer among others. 128 The reported values of IC 50 for topotecan applied to MCF - 7 human breast cancer cells, DU - 145 and PC - 3 human prostate cancer cells are 0.22 µmol/L, 0.19 µmol/L and 0.09 µmol/L, respectively. 137 - 139 The most frequent and main dose limiting toxicity is neutropenia, because diarrhea, vomiting and nausea can be controlled with usual supportive medication. 128,130 The structures of homocamptothecin, irinotecan, topotecan and few other relevant CPT analogues are presented in Figure 8 .

Analogue Homocamptothecin

Analogue R1 R2 R3 R4

Irinotecan CH 3 CH 2 – – H – H

Topotecan – H (CH 3 ) 2 NCH 2 – – OH – H FL118 – H – H

Rubitecan – H – NO 2 – H – H

Belotecan (CH 3 ) 2 CHNH(CH 2 ) 2 – – H – H – H Lurtotecan – H

Exatecan CH 3 – – F

Karenitecin (CH 3 ) 3 Si(CH 2 ) 2 – – H – H – H Figure 8. Chemical structures of some CPT analogues. Adapted with permission from references 128,130 .

In another approach to circumvent the problems associated with CPT use, several CPT - polymer conjugates have been prepared with different results in c linical phase I and II trials. 10 9 The most promising CPT - polymer conjugates appeared to be CRLX101 (IT - 101, a β - cyclodextrin esterified CPT at 20 – OH group), T - 0128 (a delimotecan conjugated to carboxymethyldextran with a triglycine linker), CT - 2106 (a pol y - L - glutamate conjugated CPT) with similar pharmacokinetics profile to CPT and contradictory reports on better anticancer activity. 10 9 Furthermore, CPT and some CPT analogues have been encapsulated in polymeric nanoparticles such as N - trimethyl chitosan, 10 9 2 - hydroxypropyl - β - cyclodextrin/poly(anhydride) nanoparticles, 131 biocompatible polyester dendrimer, 140 biotinylated Pluronic F127/polylactic acid nanoparticles 141 among other polymers with increased CPT bioavailability, anticancer activity, cell uptake, s olubility and stability. As an example of dual anticancer drug delivery system, Camacho et al. prepared CPT - and doxorubicin - hyaluronic acid conjugates for dual delivery of camptothecin and doxorubicin with significant tumor reduction in murine 4T1 breast cancer model. 142 However, the only CPT - polymer approved by FDA is Onivyde, a nanoliposomal formulation of irinotecan used in a combination chemotherapy regimen for the treatment of resistant metastatic pancreatic cancer. 143

1.3.4. Epirubicin Epirubicin ((7 S ,9 S ) - 7 - [(2 R ,4 S ,5 R ,6 S ) - 4 - amino - 5 - hydroxy - 6 - methyloxan - 2 - yl]oxy - 6,9,11 - trihydroxy - 9 - (2 - hydroxyacetyl) - 4 - methoxy - 8,10 - dihydro - 7 H - tetracene - 5,12 - dione) ( Figure 9 ) also known as 4’ - epidoxorubicin or just epidoxorubicin, 4’ - epi - isomer of doxorubicin, is an anth racycline antitumoral antibiotic used in combination with other drugs for treatment of several cancers. It is sold as epirubicin hydrochloride under the trade names Ellence in the USA and Pharmorubicin or Epirubicin Ebewe worldwide.

Figure 9. Chemical structure of epirubicin hydrochloride.

The epirubicin is less cardiotoxic than doxorubicin because the different direction of the hydroxyl group attached to the C4 position of the sugar ring. 144 Cardiotoxicity associated to chemotherapeutic use of anthracyclines (i.e. doxorubicin, daunorubicin, epirubicin) with dose - related cardiomyocyte damage and life - threatening left ventricular dysfunction may affect about 5% of high risk patients or long term survivors of cancers treate d with these drugs. 144,145 Epirubicin is synthesized from daunorubicin, produced by fermentation of Streptomyces peucetius , 144,146 using several steps with a final yield about 20 - 30%. 147,148 Other proposed synthetic methods to obtain epirubicin include the modification of doxorubicin, 148 mainly isolated of Streptomyces peucetius ATCC27952 149 or the purification and modification of 4’ - epi - daunorubicin 150 also extracted by fermentation of genetically modified strands of Streptomyces peucetius . 151 Epirubicin i s widely used in combination with other anticancer drugs for the treatment of breast and ovarian cancer, lymphomas, gastric cancer and lung cancer with similar to slightly better results and less side effects than doxorubicin. 144,146,152 - 154 Furthermore, e pirubicin has shown potent cytotoxicity to several cancer cell lines (IC 50 values observed in MCF - 7, HeLa, A549 and HepG2 of 0.71 µmol/L, 0.42 µmol/L, 0.63 µmol/L and 1.3 µmol/L, respectively). 155 Epirubicin hydrochloride is considered a water - soluble drug , 156,157 with low to moderate solubility in water (about 10 mg/mL in water at 25 o C), 158 5% glucose and 0.9% sodium chloride that increases at acidic pH (stable between 4.5

1.3.5. Mechanism of action of epirubicin Epirubicin h as cytotoxic and antimitotic activity. 144 Epirubicin, as most of anthracyclines, inhibits the synthesis of DNA, RNA and proteins in cells by two major mechanisms. One of them is the intercalation of Epirubicin planar fragment between DNA nucleotide base pa irs with formation of epirubicin - DNA complexes and inhibition of topoisomerase II activity, which stops the re - ligation of nicked DNA strands, the replication and transcription of DNA, and inhibits DNA helicase activity. The other one is the production of iron - mediated free radicals due to involvement of epirubicin in redox reactions at cell level, which increase lipid peroxidation, oxidative stress and levels of the mutagenic modified base 8 - oxo - 7,8 - dihydro - 2’ - deoxyguanosine as demonstrated by Mousseau et al. , 164 with subsequent DNA damage and apoptosis. 149 However, the complete mechanisms of epirubicin cytotoxicity are not completely known. The outlined mechanisms similarly explain the cardiotoxicity of epirubicin and the other anthracyclines related to c ardiomyocytes (heart cells) apoptosis after drug uptake. 144

1.3.6. Epirubicin - polymer carriers Epirubicin has been encapsulated in or covalently linked to different natural and synthetic polymers to obtain different nanoparticles, polymersomes, nanomicelle s and other drug delivery systems with enhanced bioavailability, reduced adverse side effects, and better therapeutic results. For example, liposomal anthracyclines have shown reduced cardiotoxicity and increased therapeutic potential of the drug. 144 In th is sense, Hassanzadeh et al. prepared epirubicin - loaded tocopherol succinate - pullulan micelles for folic acid targeted delivery to HeLa and MCF - 7 cancer cell lines, with higher toxicity on HeLa cell line than MCF - 7. 165 In another study, Tariq et al. prepar ed epirubicin - loaded PLGA nanoparticles for oral delivery of epirubicin, with oral bioavailability increased 3.9 fold as compared to free epirubicin and improved intestinal transport of epirubicin, which might allow replacing traditional intravenous admini stration of epirubicin with patient care at home. 156 Similarly, enhanced cytotoxicity, cell uptake and accumulation in tumor tissue was observed for epirubicin - loaded biotinylated - chitosan - PLGA nanoparticles prepared by Chen et al. 166 Another tumor - targete d delivery of epirubicin was achieved with the preparation of epirubicin - loaded CXCR4 - PLGA/TPGS nanoparticles for liver - targeted epirubicin delivery. This resulted in a 3 - fold improvement in cellular uptake compared to non - CXCR4 surface modified nanopartic les, as well as major nanoparticles accumulation in the liver tumor and enhanced epirubicin cytotoxicity on cancer cells. 167

1.4. Controlled drug release The controlled release of drugs is attracting researchers devoted to the preparation of new systems fo r applications in medicine, pharmacy and other fields. The release behavio u r and target of release are different when comparing traditional formulations with the ones aimed to sustained release when both are administered in similar conditions. 168 The main goals of controlled release are: protection of drug to be released until reaching the delivery location, and to maintain constant release rates and therapeutic concentration of delivered drug during required times, with specificity and persistence. Thus, d rug side effects are minimized, with a reduction in application frequency.

1.4.1. Agrochemicals release Hereby, the generic classification of agrochemicals must be better explained. Agrochemicals are generally understood as a group of compounds used with b eneficial purpose to increase the production of crops and their quality. 169 The main group of agrochemicals include pesticides (herbicides, insecticides, fungicides, bactericides, nematicides and others), inorganic and organic fertilizers. It also includes growth agents (i.e. phytohormones and their analogues, precursors or generators of phytohormones), manure and other beneficial agents involved in the growth and protection of the plants. 169,170 Nowadays, a lot of effort is devoted to reduce the excessive and periodic application of traditional pesticides and other agrochemicals. The intensive use of agrochemicals provoke toxicity to the plants, soil and environment contamination. Furthermore, undesired presence of pesticide residues in crop products, redu ction of biodiversity with negative impact on pollinators (i.e. bees, butterflies), induced resistance of pests and plagues, as well as human and animal health problems are also associated with indiscriminate agrochemicals application. 169,170 Particularly, the spraying of pesticides and other agrochemicals results in approximately 60 - 70% loss of active ingredients. 171 Consequently, only 0.1% of the pesticides reached the intended plagues. 170 This can be due to rainfall wash off, degradation by light, heat a nd microorganisms, leaching in the soil and volatilization. 169 - 171 Therefore, the traditional methods of agrochemical application result in loss of most active ingredients used, and fast loss of activity or reduction below necessary levels to maintain the intended biological activity in the required site. It end up in repeated and excessive application of the agrochemicals with significant economic losses and environment pollution. 169 - 171

A possible solution to these problems is the use of nanomaterials fo r controlled delivery of agrochemicals or nanoagrochemicals. 172 Nanomaterials can protect the active ingredients from degradation, increase their biocompatibility, biodegradation and availability. They can also improve the solubility and efficiency of the active ingredients, and increase the effect on the target organisms (plants or pests). Consequently, they can diminish the impact and toxicity on non - target organisms and extend release of the active ingredients with potential for single application use. A dditionally, they are capable of reducing the leaching of the agrochemicals and the nitrogen transfer to groundwater, among other benefits. 169 - 173 In this sense, imazapyr - and imazapic - loaded chitosan nanoparticles, atrazine - loaded polycaprolactone nanopar ticles have displayed lower toxicity to non - target organisms and superior activity to target - organisms than parent pesticides, with a significant reduction of applied dose. 172 Additionally, a 10 - times lower soil accumulation of pesticide imidacloprid relat ed to the use of imidacloprid - loaded chitosan nanoparticles, and significantly less pesticide residues found in leaves is reported. 170 The reduction of active ingredient dose applied to the crops is very important when using phytohormones and their analo gues with plant growth regulator activity. This is because high doses of these compounds are often toxic to the plants. 172 That is why intensive research on the preparation of chitosan, chitosan/alginate and other biopolymer - based microspheres and nanopart icles for the controlled release of brassinosteroids, gibberellins, NO - precursors and other phytohormones is ongoing. 172 Polymeric nanoparticles, hydrogels, beads, nanocomposites and other polymer - based formulations are very attractive to be used as drug delivery systems in sustainable agriculture via encapsulation and/or covalent bond of the active ingredient. It might allow the release of multiple agrochemicals and stimulus - responsive release of pesticides in presence of the plague. 170,171 For example, F ischer et al . prepared a vaccination system based on pyraclostrobin - loaded methacrylated - lignin nanocarriers for the treatment of grapevine trunk disease Esca. 171 The fungicide release occurs only after degradation of the lignin carrier by the Esca fungi. 1 71 It allowed the cure of infected plant and to observe protective effect during 4 years after administration of 10 mg of the fungicide. Pyraclostrobin was not detected in the grapes harvested 3 months after vaccination. 171 Polymer - based nanomaterials as hydrogels and beads for agriculture can also improve the water - holding capacity of the soils, with plants and soil protection effect in dryness and crop seasons. 169,173 It allows to restore degraded soils with the use of microorganisms (bio - remediation) an d plants (phyto - remediation). 170

On the other hand, nanoclays and nanocomposites of silica with different polymers (pectin - conjugates silica microcapsules, alginate - silica nanoparticles, chitosan - silica nanoparticles) have been widely employed for the del ivery of pesticides. 170 Other non - polymeric nanoparticles employed in controlled release of agrochemicals include carbon nanotubes, nanozeolites, lipid nanoparticles, metallic and metal oxide 173 - 175 nanoparticles (ZnO, TiO 2 , Au, Ag, Ag 2 O, Cu, CuO, Fe 3 O 4 ). However, ZnO and silica nanoparticles are known to cross the placenta barrier in pregnant rats, with ZnO particles being toxic to algae, bacteria and invertebrates. 175 The study of the toxicity of nanoparticles in agriculture is a complicated topic becaus e the different impact and interactions of plants, soils and microorganisms living in the soil (insects, bacteria, fungi). 174 The most common toxicity studies of nanoparticles used in agriculture are carried out in metallic nanoparticles. 174 The soil chara cteristics (pH, organic matter content, cation exchange capacity, moisture, kind of soil), kind of crop, nanoparticles concentration and size determine the overall toxicity, phyto - toxicity and effects of nanoparticles applied in agriculture. 174 For example , ZnO nanoparticles exhibited significant toxicity toward Triticum aestivum (common wheat) at 45.5 mg/kg when applied in a loamy clay soil with pH 7.4; but, non - toxicity was observed when same nanoparticles were applied towards Cucumis sativus (cucumber) a t 2 g/kg in a loamy sand soil with pH 5.5. 174 Nevertheless, biosynthetic metal nanoparticles are found to be more active and significantly less toxic than the synthetic nanoparticles, with biosynthetic Ag and ZnO nanoparticles being successfully used as fe rtilizers and antimicrobials in different crops. 174 Polymer - based nanoparticles are extensively used for sustained delivery applications in agriculture and medicine because of their easy and reproducible synthesis and their fine - tunable properties. 169,170 ,172 Particularly attractive is the existence of green synthetic approaches (i.e. biosynthetic methods using genetically - modified bacteria and organisms) for production and modification of the polymers. 169,170,172 Another advantage of using polymers for dr ug delivery is the availability of natural polymers (i.e. alginates, starch, celluloses, lignin, cyclodextrins, dextran, guar gums, pectins, chitosans, silk fibroins) and synthetic polymers (polyethylene glycol, polyglycolide, poly(citric acid), poly(vinyl alcohol), poly(lactide - co - glycolide), polyethylenimine) to choose from as needed. 169,170,172 Additionally, the polymers can be chemically modified and/or directly used for encapsulation/covalent linkage of active ingredients. 169,170,172 Lárez - Velásquez 17 6 and Fraceto et al. 177 proposed the sustained release of agrochemicals with the following intentions:

 The agrochemical effects are extended in time, while controlling the employed quantities .  A release of the agrochemical from the polymer matrix when need ed by the plant, thus smaller quantities are required than when applying directly the agrochemical alone.  A decrease in the number of applications to plants; thus diminishes the contact of workers with the agrochemicals and the vegetal stress.  A decrease of toxicity to humans and animals, due to a local application in plants and with smaller drug doses.  The employment of the required quantities of the agrochemicals, with subsequent lower costs .  Environmentally friendly formulations for delivering the requ ired quantity of agrochemicals, while the biodegradation of the biomaterial do not contaminate the soil.  Increased solubility or dispersion, stability, bioavailability and reduced degradation of the agrochemicals.  Smart stimulus - responsive drug delivery sy stems for the agrochemicals, with drug release triggered by pH, temperature, light, moisture, enzyme activity, as well as specific action of the agrochemicals to few plant species or pests. Nevertheless, deeper studies are missing to properly evaluate the toxicity of nanoagrochemicals to ensure sufficient safety of workers handing these products and of the food produced using them. 173 Thus, Fraceto et al. 172 - 174,177,178 summarized the most relevant issues and obstacles to be solved for a widespread and acce pted use of the nanoagrochemicals:  The costs of nanomaterials to be used in agriculture must be competitive with the traditionally available agrochemicals and the intended crops.  Sufficient safety demonstrated and well evaluated toxicity in field (natural habitat) of the nanoagrochemicals, to know in advance the dose limits, acute and long - term toxicity to target and non - target organisms, plants, animals and humans, accumulation and effects on the soil and plants, interaction with the soil microbiota, bioav ailability and distribution in the plants.  Modification of the toxicity of the nanoagrochemicals and accurate determination of optimal doses for each crop in relation with the soil characteristics, season, physico - chemical properties of the nanomaterials u sed.

 Transference of environmental friendly biosynthetic nanoparticles and nanoagrochemicals from the laboratory to the field, and proper upscaling from the laboratory level to the industrial process for massive use in agriculture.  Development and adaption of current analytical and toxicological techniques used in laboratory to evaluate nanoparticles and nanomaterials to be used in field conditions to assess the toxicity and properties at short - and long - term of the nanoagrochemicals.  To adapt and develop t he current regulations for use of nanomaterials for a better evaluation of toxicity, bioavailability and complex interactions of nanoagrochemicals in the field.

1.4.2. Nanomedicine and drug release Nanomedicine is considered the application of nanotechnol ogy using nanomaterials (i.e. nanoparticles, nanorobots) to medicine. 179,180 Nanoparticles are widely used in nanomedicine with diagnosis/sensory purposes and for drug delivery applications to treat several diseases such as cancers, malaria, hepatitis, Par kinson’s disease and Alzheimer’s disease. 179 - 182 Among different nanoparticles or nanocarriers for applications in nanomedicine, metallic nanoparticles (Ag, Au, Cu, Zn, ZnO, Fe, Fe 3 O 4 , 180 Fe 2 O 3 , Ti, Co, Al, Cd, Pd) with 2 nm, 4 nm and less than 10 nm sizes ar e used. They find application mostly with sensory/diagnostic purposes, for hyperthermia, controlled drug delivery, radiotherapy for cancer, antibacterial and microbiocidal applications. Silica nanoparticles (mesoporous, non - porous and porous silica) wit h different pore and particle sizes can be easily loaded and/or covalently modified with different drugs and molecules for imaging, diagnosis and drug delivery applications. 180 Carbon nanoparticles (fullerenes, nanodiamonds, graphenes) with sizes from 1 nm to 100 - 150 µm, are applied as biosensors, to immobilize enzymes and for tissue regeneration. 180 Organic nanoparticles (polymer - based nanoparticles, lipid - based nanoparticles, dendrimers, nanomicelles, microemulsions) with dimensions from 10 nm to 1 µm, ar e mostly used for drug delivery applications. 180 Nanoparticles as drug delivery systems present several advantages. They provide sustained release at the intended site, with increased solubility or dispersion and bioavailability of the drug, stability and protection of the drug from degradation, extended blood circulation times, lower side effects and decreased toxicity. This reduces the drug quantity necessary to achieve the therapeutic effects and prolonged therapeutic effects from days to weeks. Nanopar ticles can provide multi - drug delivery, combination of polymer and metals for drug delivery and

imaging/radiotherapy/hyperthermia treatments. They also allow smart stimulus - responsive drug delivery to ensure target release of the cargo and/or intracellular activation and accumulation in cancer cell lysosomes. 179,180 Polymer - based nanoparticles can be easily surface functionalized with peptides or oligosaccharides for active tumor - targeting release of single or combined anticancer drugs. 179,180 This strateg y allows to cross the blood - brain barrier and to reduce immunogenicity of some drugs and nanocarriers. 179 For example, hyaluronic acid nanoparticles or nanocarriers coated with hyaluronic acid oligomers are known to target CD - 44 receptor of cancer cells. T his allows targeted delivery of anticancer drugs to lung adenocarcinoma cells, colon and breast tumors with lowered immunogenicity. 179 Polymeric micelles are a class of polymer - based nanoparticles formed of amphiphilic block copolymers that self - assemble into a core - shell structure in aqueous medium. 179 The hydrophobic core can encapsulate lipophilic drugs such as paclitaxel and camptothecins with increased bioavailability and stability, while the hydrophilic shell allows the solubility of the system in wa ter. The micelles accumulate in the tumor tissues via the enhanced permeability and retention (EPR) effect and reduced renal excretion due to their sizes below 100 nm. 179 There are two approaches for drug incorporation into the polymeric nanoparticles: th e encapsulation or loading in the core of the nanoparticles or nanomicelles, and the covalent link of the drug and the polymer or formation of a polymer - drug conjugate. 179,181 The main advantages of polymer - drug conjugates over the free drug are similar to the ones observed for drug loaded polymeric nanomicelles. These are, passive accumulation in tumor tissues due to EPR effect, superior drug solubility in aqueous medium and reduced toxicity and side effects. 181 Furthermore, the polymer - drug conjugate appr oach is better suited for multi - drug release due to the possibility of polymer functionalization with different drugs via different linkers capable to trigger specific and separated drug release upon different stimuli (i.e. pH, ionic strength, reductive or oxidative environments). 181 It is also possible to incorporate hydrophobic drugs in the core of the nanoparticles and to bind hydrophilic and/or charged drugs (i.e. DNA, RNA, doxorubicin hydrochloride) on the outer nanoparticle shell. 181 Some combinations of drug encapsulated or loaded in polymeric nanoparticles, and polymer - drug conjugate carriers are presented in Figure 10 . 181

(a) (b) (c)

(d) (e)

Figure 10. Scheme of (a) drug encapsulated or loaded ( ) in the core of core - shell polymeric particles, (b) polym er - drug ( ) conjugate in core - she ll particles (c) two drugs ( ) bonded with different linkers (i.e. pH and reductive sensitive linkers) to the polymer in core - shell particles, (d) drug encapsulated ( ) in the core of polymer - drug ( ) con jugate core - shell particles, (e) loading in, covalently linked to, or electrostatic adso rption of a hydrophilic drug ( ) on the shell of a polymer - drug ( ) conjugate in core - shell polymeric particles.

It is reported that radiotherapy increase the therapeutic index of polymer - drug conjugates releasing doxorubicin, probably due to increased EPR effect and passive diffusion of the nanoparticles to tumor tissues. 181 Targeting of the drug delivery systems can be classified into passive and active. 179 In passive drug tar geting, the nanocarriers circulates in the blood and they accumulate in the tumor tissues due to EPR effect related to leaky tumor vasculature. 181 Active targeting is based on chemical affinity of overexpressed receptors or surface proteins in the tumor ce lls with peptides and other molecules covalently bonded to the nanocarriers surface. 181 Other aspects of critical importance when designing nanoparticles and nanomaterials for drug delivery applications in nanomedicine are the nanoparticles clearance via reticuloendothelial system (RES) due to serum protein adsorption at the nanoparticles surface (opsonization) and their uptake by normal cells and tissues mainly located in spleen, liver and bone marrow, 182,183 as well as spleen filtration of nanoparticles and renal system clearance of particles smaller than 20 nm. 182,183 It is considered that 100 - 200 nm polymeric nanoparticles might avoid the RES clearance system and exhibit less aggregation in biological medium, with a longer systemic circulation time. 184 However, mechanical properties or intrinsic deformability of nanogels seems to be more important than hydrodynamic particle sizes in the nanoparticles clearance and bloodstream circulation times. 183 In this sense, Zhang et al . demonstrated that 120 nm sof t polymeric nanogels exhibited 9 - 20 hours in vivo circulation half - lives, comparable to well - known long circulating

PEG - based particles. 183 Furthermore, Blanco et al . reported good bioavailability, constant 5 - fluorouracil concentration extended up to 11.5 hours. 185 Groups of nanogels were observed in the tissue 15 days after injection of 600 nm 5 - fluorouracil - loaded folate - N - isopropylacrylamide submicrohydrogels. 185 The blood - brain barrier must be also taken into account when designing nanoparticles intend ed to deliver drugs into the brain because of nanoparticle accumulation in the brain with related neurotoxicity. 182 All these factors are mostly determined by the size, charge, structure, composition and degradability of the nanoparticles used as nanomedic ines. 182 According to Patra et al. 179 and Roemeling et al. , 182 10 - 13 nanomedicines are FDA - approved and 36 nanomedicines are in I - III clinical phase trial by 2017 - 2018 for the treatment of different cancers and malignancies. The biopolymers (chitosan, cell ulose, dextran, xanthan gum, sodium alginate, hyaluronic acid) have been extensively employed for controlled drug release applications. 179,186 The drug is released from these polymers through different mechanisms, and sometimes a combination of mechanisms: 187  Diffusion control, the limiting step is the diffusion rate of the drug through the polymer matrix.  Chemical control, when a biodegradable matrix is employed or the drug is chemically link to the polymer matrix trough a bond amenable to chemical or enzy me hydrolysis.  Solvent control, hydrophilic polymers are employed as matrix. The particles can be morphologically classified as monolithic (the drug is homogeneously dispersed through the matrix), or core - shell particles (a core carrying the drug and a pol ymeric shell surrounding it). It is possible to prepare micro or nanocapsules (empty particles). Sometimes it is possible to find the drug adsorbed on the surface of the particles or capsules. 188 Figure 11 shows the main variations of particles and capsule s.

(a) (b) (a) (b)

Figure 11. Scheme of (a) monolithic particles, (b) core - shell particles and (c) capsules loaded with the drug or with the drug adsorbed on the surface. Adapted with permission from reference 188 .

1.5. Cellulose Cellulose is the most abundant b iomass resource in the nature, where together with hemicelluloses and lignin, it is the main structural component of plants. 189,190 Cellulose also constitutes the cell wall of algae, oomycetes and bacteria. 191 Cellulose is considered the most relevant rene wable, economical and biodegradable biopolymer. 192,193 Cellulose global production is estimated to be superior than 75000 million tons/year. 189 It is a linear homopolymer of β(1→4) linked anhydro D - glucose repeating units ( Figure 12 ), with 10000 to 15000 g lucose units depending on the source, the extraction and purification conditions. 194 - 196 The linear conformation of cellulose chains is due to intermolecular hydrogen bonds between adjacent glucose repeating units. 196 Similarly, intermolecular hydrogen bon ds between glucose units of different chains and van der Waals interactions result in a parallel array of cellulose chains to form cellulose fibrils that further arrange to form microfibrils of some microns length and 5 to 50 nm diameter. 196 Cellulose pres ents amorphous and crystalline areas in solid state, with degree of crystallinity from 40 to 60%. 190,196 Cellulose nanocrystals are obtained from the crystalline areas of cellulose. 196 Crystalline cellulose presents four main polymorphs (I, II, III, IV), w ith “native” or “natur al” cellulose I produced in nature as the polymorphs Iα (triclinic structure) and Iβ (monoclinic structure). 190,194,196 Cellulose obtained of algae and bacteria are predominantly formed by Iα cellulose, which can be partially transformed into Iβ cellulose by heating at 260 o C in NaOH aqueous solution. 190,196 Iβ cellulose is also the major component of cellulose produced in plants. 194,196 Cellulose II, with a monoclinic structure, is the most stable cellulose polymorph. 190,194,196 It can be prepared by solub ilization and recrystallization of

cellulose I, a process called “regeneration”, or by treatment of cellulose I with NaOH aqueous solution (mercerization). 190,194,196 Treatment of cellulose I or II with ammonia give cellulose III. 190,194,196 Cellulose III can be converted into cellulose IV upon heating. 190,194,196 The transformation of different cellulose polymorphs was summarized by Kamell et al. ( Figure 13 ). 190 Cellulose I presents the cellulose chains in a parallel array (all 1 →4 glycosidic bonds points in the same direction). 194,196 On the other hand, cellulose II polymorph is characterized by an antiparallel organization of the cellulose chains. 194

Figure 12. Chemical structure of cellulose. Reproduced with permission from reference 190 .

Figure 13. Transformations of cellulose polymorphs. Reproduced with permission from reference 190 .

1.5.1. Cellulose properties and applications Cellulosic materials have been widely used in fabrication of paper and cardboards, coatings (i.e. cellophane), membranes for blood purification and dialysis, composites and scaffolds for tissue engineering, construction materials, food and pill inert fillers, textiles (i.e. Ra yon and Tencel), chelating agents for wastewater treatments, nanoelectronics and sensors among others. 189 - 197 Cellulose is a semirigid polymer highly crystalline, lightweight, with a high aspect ratio, insoluble in water and most organic solvents, due to s trong and highly extended intra -

and intermolecular hydrogen bonds among the cellulose chains. 190,192,194 - 200 Yang and Li summarized the most common solvents used for dissolution of cellulose: N - methylmorpholine oxide (NMMO), LiCl/ N,N - dimethylacetamide, Li Cl/1,3 - dimethyl - 2 - imidazolidinone, N 2 O 4 / N,N - dimethylformamide, tetrabutylammonium fluoride/dimethyl sulfoxide (TBAF/DMSO), tetrabutylammonium acetate/DMSO, LiClO 4 ∙ 3H 2 O,

LiSCN ∙2H 2 O, ionic liquid systems (i.e. 1 - allyl - 3 - methylimidazolium chloride (AMIMCl), 1 - butyl - 3 - methylimidazolium chloride (BMIMCl), 1 - ethyl - 3 - methylimidazolium acetate (EMIMAc)), and alkali/urea or thiourea aqueous solvents (i.e. 7 wt% NaOH/12 wt% urea in water at - 12 o C, 9.5 wt% NaOH/4.5 wt% thiourea in water at - 5 o C, tetrabutylammonium h ydroxide/urea). 195,200 The disruption of cellulose hydrogen bonds in solution facilitates its chemical modification via homogeneous reaction to obtain water - soluble cellulose derivatives and several functionalized celluloses with new properties and applica tions. It must be noted that properties and applications of cellulose materials are highly determined by their structure, preparation conditions and kind of material. 196 Moon et al. exhaustively described the different cellulosic materials obtained of cell ulose by using mechanical, acid or enzymatic hydrolysis. These materials include: wood and plant fibers, microcrystalline cellulose (i.e. Avicel, porous particles of 10 - 50 µm diameter), microfibrillated cellulose (10 - 100 nm × 0.5 - 10 µm), nanofibrillated ce llulose (4 - 20 nm × 500 - 2000 nm), cellulose nanocrystals prepared by acid hydrolysis of aforementioned cellulose materials (3 - 5 nm × 50 - 500 nm rod - like or whisker - like particles), tunicate cellulose nanocrystals (ribbon - like shaped particles with 8 nm × 20 nm × 100 - 4000 nm), algae cellulose particles (20 nm × 20 nm square cross - section or 5 nm × 20 - 30 nm rectangular cross - section particles), and bacterial cellulose particles (6 - 10 nm × 6 - 10 nm square cross - section or 6 - 10 nm × 30 - 50 nm rectangular cross - sect ion microfibrils). 196 The relative crystallinity of these cellulosic materials spans from 27 - 100% referred to native cellulose. 196 It follows the trend cellulose II

polyvinyl alcohol to obtain cross - linked hydrogels and nanocomposites with similar mechanica l properties to cardiovascular tissues. 200 Cellulose - chitosan hydrogels were also prepared for wastewater treatment due to good chelating activity. 200 Other cellulose - based composites includes: bacterial cellulose - calcium deficient hydroxyapatite hydrogel for potential orthopedic implants, cellulose - heparin - charcoal composites for better serum compatibility, cellulose - quantum dot hydrogels. 200 Celluloses are degraded by different cellulases, a group of enzymes formed by endo - glucanases (catalyze random clea vage of celluloses), exo - glucanases or cellobiohydrolases (release cellobiose of celluloses chain ends), and β - glucosidases (release glucose of cellobiose and oligo - celluloses). 201 In this sense, Yeh et al. showed that a reduction of microcrystalline cellu lose particle size resulted in reduced crystallinity, with an increase of enzymatic hydrolysis rate and glucose production. 201 Xiao et al. studied the effect of different factors on enzymatic hydrolysis of cellulose regenerated (cellulose II) from ionic li quids. 202 The authors found a significant loss of crystallinity upon regeneration of cellulose (change of cellulose I to cellulose II), and fastest hydrolysis when cellulose was dissolved in BMIMCl at 120 o C for 20 min, regenerated using water and enzymati c hydrolysis carried out at 50 o C. 202 Cellulases are produced in actinomycetes, bacteria, fungi, protozoa, termites, cockroaches, nematodes, crayfish and other invertebrates, intestinal tract of ruminants. 203 - 205 Cellulases are added to the feed of ruminan ts, poultry, pigs, fish and other animals to improve the food digestibility for milk yield and body weight gain. 203 The absence of cellulases in humans impede the cellulose degradation, which is critical for endovenous administration of cellulose based par ticles for drug delivery applications. Therefore, design of cellulosic particles aimed to systemic administration in humans with molecular weights near to renal clearance limit (30 - 60 kDa) might ensure their excretion after their therapeutic purpose is com pleted. 206 Contrariwise, no in vivo enzymatic degradation of cellulose in humans is highly desirable for permanent intended implants (i.e. blood vessel and heart valve replacements). 191 Additionally, bacterial cellulose scaffolds appears to integrate well permanently into the host tissue. 191

Cellulose and its derivatives are considered non - toxic (oral median lethal dose (LD 50 ) of microcrystalline cellulose superior to 5 g/kg in rats), 207 biocompatible and biodegradable. 189,207,208 Bacterial cellulose was ap proved by the FDA for tissue engineering applications in 1996. 209 In this sense, Park et al. demonstrated the wound healing effect, non - toxicity and biocompatibility of bacterial cellulose films surgically implanted in the back of rats. 208 The authors foun d that bacterial cellulose implants

were non - toxic, fully degraded and resorbed without visible cellulose fragments after 4 weeks, as well as wound healing response superior to Vaseline gauze patch after 14 days. 208 Bacterial cellulose hydrogels have been proposed for meniscus and dental implants, as well as for tissue engineering scaffolds. 200 Bacterial cellulose blood vessel replacement was found highly biocompatible, functional, stable, low immunogenic and with low inflammatory response on sheep after 3 months. 191 In another study, Lima et al. showed no genotoxicity (DNA breaks) of ruby cotton nanofibrillated cellulose to vegetal Allium cepa L. (onion) root cells, 3T3 cell mouse fibroblasts and human lymphocytes. 210 Azoidis et al. showed that nanofibrilla r cellulose is biocompatible with human mesenchymal stem cells and can be used for their intensive cell culture. 211 Several in vitro cytotoxicity studies of cellulose nanocrystals on HEK - 293 (human embryonic kidney), human fibroblasts, L929, MDA - MB - 231 xen ografts in mice , MCF - 10A, PC - 3 human prostate cancer cells , HCT116 human colon carcinoma, and other cells demonstrated the non - toxicity of cellulose nanocrystals except when used in high concentrations or via oral inhalation on 3D model of human epithelial airway barrier. 197 In vivo studies of cellulose nanocrystals also showed a minimal toxicity when administered orally to rats (LD 50 ~2 g/kg), non - toxicity after intradermal injection of 1.1 mg/mL cellulose nanocrystals suspension and repeated topical appli cation of 103 mg/mL cellulose nanocrystal gel to guinea pigs. 197 Additionally, ecotoxicologic evaluation of cellulose nanocrystals on nine different aquatic species showed no genotoxicity and impact on survival below 1 g/L of cellulose nanocrystals. 197 Mic rocrystalline cellulose is intensively used as a binder in pharmaceutical industry for pill preparation by compression and as inner filler of different medicines and cosmetics. 207 It is also used as emulsifier, filler or bulking additive, thickener and oth er applications in food industry. 207 Regenerated cellulose microcapsules with highly porous structure, efficiently encapsulated (80%) hydrophilic Rodhamine B and hydrophobic Nile red, with drug delivery rate controlled by coating the cellulose surface with poly(methyl methacrylate). 192 In another study, cellulose - phosphorus hydrogels with intense fluorescence under UV irradiation, high brightness and long - lasting afterglow were prepared and detected in the stomach and under skin with and without excitation irradiation, being a good candidate for biomedical imaging. 189 In situ polymerization of conductive polyaniline in one side of cellulose hydrogel resulted in a biocompatible and good promoter for sciatic nerve regeneration of Sprague - Dawley rats. 189 Cellul ose nanocrystals are functionalized with different drugs, polymers, peptides and other compounds for smart stimulus - responsive and targeted drug delivery. 197 For

example, cellulose nanocrystals have been surface modified with tetracycline and doxorubicin, released during 24 hours. 197 Similarly, cellulose nanocrystals have been treated with cetyl trimethylammonium bromide for loading of anticancer drugs (i.e. etoposide, paclitaxel, docetaxel, luteoloside, luteolin), their sustained delivery and uptake by KU - 7 bladder cancer cells. 197 Furthermore, curcumin - cellulose nanoparticles showed superior uptake and cytotoxicity to PC - 3, C4 - 2 and LNCaP human prostate cancer cells when compared to parent curcumin. 197 Viral inhibitor 4 - sulfophenyl isothiocyanate covalentl y linked to cellulose nanocrystals inhibited 100% a fluorescent marker expressing Semliki Forest virus in Vero (B) cells with no cytotoxicity observed. 197 Folic acid - cellulose nanocrystal conjugates exhibited superior cytotoxicity due to irreversible elect roporation on folate receptor - positive cancer cells without any harm to folate receptor - negative tissue. 197 Indocyanine green - loaded poly(2 - oxazoline) - cellulose nanocrystals were prepared and displayed to be an efficient system for photothermal cancer ther apy. 197 Different gelatin - and starch - cellulose nanocrystal hydrogels have been prepared for pH and/or temperature triggered release of theophylline, cyanocobalamin (vitamin B12). 197 Complex double membrane alginate - cellulose nanocrystal hydrogels for fast release of antibiotic drug CH from the alginate shell and slow delivery of EGF growth factor from the inner alginate - cellulose nanocrystal layer were demonstrated. 197 Cellulose nanocrystals have been also used in the preparation of biocompatible scaffolds for tissue engineering application. He et al. fabricated silk fibroin - chitosan - cellulose nanowhiskers composite porous scaffolds via a layer by layer assembly process for bone tissue regeneration, with good biocompatibility, mechanical properties and biom ineralization levels. 212 Microfibrillated and nanofibrillated cellulose are both widely applied as reinforcing filler material in preparation of composites with other polymers (i.e. starch, chitosan) and inorganic compounds (Ti, CaO 2 , SrF 2 ) for preparation of drug delivery systems for antibiotics and other compounds, edible films, wound sterilization by generating O 2 , removal of heavy metals during wastewater treatments, and other applications. 189 Cellulosic materials have been also employed in agriculture for the preparation of superabsorbent hydrogels capable to slowly release water to the plants when required or as a function of the soil dryness. 213 - 215 Demitri et al. prepared and evaluated a superabsorbent cellulosic hydrogel for the cultivation of tomat oes in greenhouse conditions. 215 The authors found that the hydrogel was capable to slowly release water to the plant roots and to maintain a good hydration of the soil, with potential optimization of water use in crops and reduction of water loss due to t raditional

extensive soil irrigation. 213,215 Other uses of cellulose - based materials in agriculture include the preparation of cellulose - clay composites for slow release of herbicides (i.e. acetochlor), or of microfibrillar cellulose hydrogel composites to improve the urea leaching loss rate in sandy soils and to increase the okra plant growth. 213 Cellulose hydrogels were also employed for the controlled release of urea, inorganic salts used as NPK fertilizer and antimicrobial thymol for agrochemical applic ations. 213,216,217 For instance, Senna et al. prepared an EDTA anhydride cross - linked cellulose acetate hydrogel containing KCl and NH 4 H 2 PO 4 , which slowly released the salts to the soil and preserved the soil moisture. 217 Furthermore, cellulose - lignin hydr ogel beads were prepared for immobilizing lipase and potentially could be used for other enzymes with sensory purposes (i.e. detection of pesticides, pests, excessive salinity or dryness) in agriculture. 218 Cellulose edible coating of fruits to delay posth arvest ripening, to preserve the nutrients, to reduce the weight loss and to maintain good organoleptic properties is also under active research. 219 - 221

1.5.2. Modification of cellulose Surface functionality of cellulosic materials determines the properti es of their suspensions, the preparation of cellulose - based composites and their applications. 196 Moon et al. summarized the surface modification of cellulosics as: 196  Surface chemistry of the cellulosic materials due to their extraction process or utiliza tion of a similar process to treat the surface of the cellulose particles. The two most common methods for extraction of cellulosics from native cellulose (cellulose I) is degradation with sulfuric or hydrochloric acids, which results in

sulfate esters or hydroxylated cellulosic surfaces ( – O – SO 3 H and – OH groups at the cellulose surface). Treatment of celluloses with acetic acid or TEMPO Ɵ radical with oxidant ClO , results in acetyl esters ( – O – COCH 3 ) or free carboxylic acid ( – COOH) groups on the cellulose sur face.  Surface functionalization via electrostatic adsorption of charged molecules (i.e. surfactants). The already surface functionalized celluloses with charged or Ɵ Ɵ ionizable groups (i.e. – O – SO 3 or – COO ) are capable to adsorb counterions + like surfactants (i.e. cetyltrimethylammonium bromide, CH 3 (CH 2 ) 15 N (CH 3 ) 3 ) or polyethyleneimines.  Chemical derivatization by covalent bonding of molecules (i.e. grafting of celluloses). The presence of several secondary and primary – OH in cellulose make it amenable to ch emical functionalization with sulfuric acid to obtain sulfate esters, with acyl chlorides to obtain esters, with anhydrides to obtain

esters with free carboxylic acid groups for further reactions, with epoxides to obtain ether linkages, with isocyanates to obtain urethanes, with TEMPO and ClO Ɵ to obtain free carboxylic acid groups on the surface, with chloroacetic acid in aqueous sodium hydroxide to obtain carboxymethyl groups on the cellulose surface, and with other chemicals to obtain other cellulose deri vatives. Following these approaches, it is possible to attach polymers, or reactive groups for polymerization with different monomers on the surface of cellulosic materials. 196,222 It is worth mentioning that the surface modification of celluloses with aci d halides introduces bromoesters for later Atom Transfer Radical Polymerization of styrene and other vinyl monomers in different solvents, and allows Single - Electron Transfer Living Radical Polymerization carried out on the cellulose surface. 196,222 Ether ification and esterification of celluloses with hydrophobic long chain aliphatic acids resulted in the formation of amphiphilic polymers that self - assembled in nanomicelles in aqueous medium. 189,222 The esterification of cellulose was carried out with diff erent aliphatic acid chlorides in LiCl/ N,N - dimethylacetamide solution, giving highly substituted cellulose esters. 189 On the other hand, water - soluble cellulose ethers like methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and (hydroxypropy l)methyl cellulose ( Figure 14 ) are easily produced via alkali cellulose treatment with methyl chloride, ethylene oxide, propylene oxide and a mixture of methyl chloride and propylene oxide, respectively. 190 In the case of mixed cellulose ethers like the (h ydroxypropyl)methyl cellulose, the ratio of methyl and hydroxypropyl substituents can be adjusted from the molar ratio of methyl chloride and propylene oxide reacting with cellulose at the same time or subsequently. 190 The degree of substitution of the cel lulose ethers and the molecular weight of the products determine the solubility in water and organic solvents. 190

Figure 14. Chemical structure of methyl cellulose (MC), hydroxyethyl cellulose (HEC) and (hydroxypropyl)methyl cellulose (HPMC).

Water - sol uble cellulose ethers (i.e. methyl cellulose (MC), hydroxyethyl cellulose (HEC), (hydroxypropyl)methyl cellulose (HPMC)) retain many of cellulose properties like biocompatibility, biodegradability, and some free hydroxyl groups still amenable to further mo dification to prepare nanomaterials for applications in medicine, cosmetics and food industry. 189,200,223,224 For example, MC hydrogels coated polystyrene Petri dishes are used to culture human embryonic stem cells. 200 Similarly, HPMC and Si -

HPMC hydrogels carrying Ca 3 PO 4 demonstrated good biocompatibility, osteogenic potential for bone regeneration and are potential materials for tissue engineering applications. 200 Methyl cellulose, hydroxyethyl cellulose and (hydroxypropyl)methyl cellulose are soluble in water and DMSO, stable at 2

1.6. Silk fibroin Silk fibroin (SF) is a natural protein produced by different organisms. Its primary structure and properties are determined by the source. 229 - 231 The m ain producers of SF are the spiders and the silkworms, with the domesticated mulberry Bombyx mori silkworm being the principal source of silk fibers used in the textile industry for more than 200 years. 229,230 A single Bombyx mori silkworm cocoon is made o f a 600 - 1500 m long and 10 - 25 µm thick single silk fiber. 231 - 233 The silk fiber in the cocoon is composed of two SF fibers held together or “glued” with sericin proteins. 232,234 SF consists of three

proteins: a heavy chain protein (~350 kDa), a light chain protein (~26 kDa) covalently linked via a single disulfide bond to the heavy chain at the C - terminus, and a glycoprotein (P25, ~25 kDa) non - covalently encapsulated in the heavy - light chain assembly at a ratio of 6:6:1 (heavy chain:light chain:P25). 231,234 ,235 The hydrophilic light chain adopts a globular conformation in SF fibers. 231 The heavy chain is the major component of SF. 235 The primary structure of the heavy chain of SF consists of 20 aminoacids with approximately 5000 residues ( Table 2 ). 236,237 It is an amphiphilic alterna ting block copolymer composed of hydrophobic Gly - Ala - Gly - Ala - Gly - Ser blocks (alanine rich sequences, ~94% of the heavy chain sequence) that self - assemble upon stimulus in a β - sheet structure responsible of SF crystallinity. 229,232,237 Glycine - rich hydrophi lic blocks form an amorphous matrix surrounding the β - sheets. 229,232 - 234,237 The β - sheet domains crosslink the SF via inter - and intramolecular hydrogen bonds and van der Waals interactions with the formation of nanocrystals of stacked β - sheet arrays. 234,2 37 The amorphous hydrophilic components of SF mostly adopt a random coil conformation, with minor contribution of α - helix, turn and bend conformations. 232 - 234,238 The most studied crystalline polymorphs of SF are the metastable silk I (pre - spinning struct ure, rich in β - turn conformers, without β - sheet conformers) and the more stable silk II (structure after spinning into a fiber, rich in β - sheet conformers that form β - sheet nanocrystals). 232,238,239

Table 2. Amino acid composition of SF hea vy chain. Adapted with permission from reference 237 . Amino acid Number of aminoacids Content (mol%) Ala 1593 30.3 Arg 14 0.3 Asn 20 0.4 Asp 25 0.5 Cys 5 0.1 Gln 10 0.2 Glu 30 0.6 Gly 2415 45.9 His 5 0.1 Ile 13 0.2 Leu 7 0.1 Lys 12 0.2 Met 4 0 .1 Phe 29 0.6 Pro 14 0.3 Ser 635 12.1 Thr 47 0.9 Trp 11 0.2 Tyr 277 5.3 Val 97 1.8

SF is obtained after removal of the accompanying sericin in silk fibers obtained from the silkworm cocoon, a process known as degumming. Exhaustive removal of serici n from SF is necessary to ensure obtaining SF materials that could be safely used in biomedical applications, without strong inflammatory physiological response upon implantation. 230,231,235,240 - 261 After several steps, the purified aqueous SF solution, kn own as reconstituted or regenerated SF, is ready for the preparation of different SF materials intended to medical or research uses. Alternatively, SF materials can be prepared from water - soluble SF hydrolysate obtained by acidic and/or enzymatic hydrolysi s of SF. 262 The extent of transition of random coil and β - turn into β - sheet structures during the preparation of the SF materials controls its properties. 233,235,237 This spontaneous self - assembly of SF is affected by temperature, pH, ionic strength, use o f organic solvents among other factors. 233,235,237

1.6.1. Silk fibroin properties and applications SF materials are comprehensively used because of their excellent biocompatibility, tunable degradability, low immunogenicity, lack of toxicity (lethal dose o f a SF peptide superior to 5g/kg in rats, median lethal dose of SF hydrolysate powder superior to 11.5 g/kg in mice), 236,263 easy processibility and self - assembly tendency, which allows the formation of material structures with tunable stability, mechanica l properties, physical and chemical properties. 240 - 244 SF materials prepared of unmodified SF are negatively charged in aqueous solution at pH over 5 (isoelectric point (pI) of SF is about 4.2 - 4.8), allowing a favorable interaction with positively charged drugs at physiological conditions (pH about 7.4) and formation of hydrogels at pH close to the pI of SF. 232,264 The biocompatibility, cytocompatibility, low immunogenicity and lack of toxicity of SF materials are exhaustively documented. 230,231,235,240 - 261 SF was approved as a biomaterial by the FDA in 1993. 245 One of the first reports on the evaluation of SF as biomaterial in 1995, unveiled the potential of SF scaffolds prepared from Bombyx Mori for growth and proliferation of fibroblasts. 245 Other human c ells that have been cultured in vitro in different SF materials showing good cytocompatibility includes osteoblasts, hepatocytes, fibroblasts, keratinocytes, endothelial, epithelial and mesenchymal stem cells among others. 230,235,237,265 The immunogenicity and inflammatory response to SF films evaluated in vitro on human mesenchymal stem cells, was found to be minimal and similar to the one provoked when using collagen or polylactic acid films. 235 However, superior mesenchymal stem cells proliferation (cyto compatibility) was observed on SF films when compared to polylactic acid or collagen matrices. 235 In this sense, Liu et al. demonstrated the good cytocompatibility (relative cell viability over 94% after 3 days with 1.47 wt% of SF) of prepared SF - based hyd rogel with extracellular matrix - like structure to L929 cells. 242 It must be noted that cell binding, growth and differentiation over SF materials can be adjusted with a variation of SF surface and/or hydrophilicity of SF matrix via chemical modification of SF with different compounds (i.e. polylactic acid, poly(ethylene glycol), lactose, others). 237,265 SF implants, scaffolds and hydrogels have also been studied in vivo to assess the biocompatibility, immunogenic and inflammatory response in rats, rabbits, pigs and humans. 231,235 These studies concluded that low to mild immune response was observed to SF scaffolds implanted in the animals after 2 to 52 weeks, with reduced inflammatory response as compared to collagen or polylactic acid and good biodegradatio n. 231,235 Furthermore, covalent binding of arginine - glycine - aspartic acid tripeptide (RGD) or bone morphogenetic protein (BMP - 2) to SF is found to promote cell binding, differentiation and proliferation in different SF tissue scaffold implants. 237 SF is

co nsidered to be compatible with blood, 245 and SF modified with sulfate groups or 2 - methacryloyloxyethyl phosphorylcholine exhibited anticoagulant properties, and reduced cell, platelet and protein adhesion as required for specific applications. 237 However, an exhaustive evaluation of cytocompatibility, long - term biocompatibility, immunogenicity and inflammatory response, toxicity, biodegradation and additional required parameters (i.e. mechanical and chemical stability, toxicity of metabolites) is usually ca rried out on every new SF material before its clinical use. 231,235,245 On the other hand, SF materials can be enzymatically degraded by α - chymotrypsin, protease XIV and collagenase IA in short length peptides and finally in absorbable aminoacids. 231,245 Re generated SF biomaterials are considered to degrade faster than silk fibers. 231 SF biodegradation time can be adjusted accordingly to required application of the SF material from less than 3 months for some injectable SF hydrogels in rats, to 6 - 12 months f or SF scaffolds in rats, or even to 18 - 36 months for SF sutures in goats. 235 The crystallinity, the relative content of silk I and silk II polymorphs, the ratio of intermolecular and intramolecular silk II, and the molecular weight of SF material affect it s degradation rate. In general, SF materials with reduced crystallinity, lower content of silk II (β - sheet structures) and/or lower molecular weight degrade faster. 235,245 The source of SF material and its concentration also influence the degradation rate. 235,245 For example, mulberry SF degrades faster than non - mulberry SF due to the lower crystallinity and α - helix and β - sheet contents in the former. 245 Similarly, SF hydrogels with lower SF concentration degraded faster. 235 SF fibers are water insoluble an d characterized by a high strength superior to the steel and harder than Kevlar, 231 good resistance to chemicals and low elasticity due to its crystalline structure. 232 On the other hand, most of the SF materials prepared from regenerated SF are considered weak and brittle. 231 However, mechanical and physical properties (i.e. compressive modulus, tensile strength, swelling, aqueous solubility) of regenerated SF materials are affected by the crystallinity and relative content of silk I and silk II polymorphs . 231,232,235 Higher crystallinity and relative content of silk II in SF materials are associated to superior mechanical properties, enhanced thermal stability, reduced aqueous solubility and lower swelling capacity. 232,235 It is reported the fabrication of a SF blood vessel with tensile strength of 2.42 MPa and SF scaffolds with compressive modulus of <50 kPa to 2.2 MPa and 13 MPa. 231,245 There are also reports of cryogel fabricated SF scaffolds with compressive modulus of 50 MPa, SF hydrogels with elastic modulus of 6.5 MPa and tensile stress of 0.7 MPa, and SF particles with elastic modulus of approximately 3 MPa and 0.8 GPa in the hydrated and dried states, respectively. 231,233,240,245

The major components of SF are the non - reactive aminoacids glycine (4 5.9 mol%) and alanine (30.3 mol%) ( Table 2 ). 233,237 It explains the relative chemical stability of SF scaffolds to proteolytic degradation (retained 50% of tensile strength 2 months after implantation), 232 70% EtOH in water or ethylene oxide (55 o C, 4 h) d uring autoclaving. 235 The main synthetic target points for modification or functionalization of the heavy SF chain are the serine (12.1 mol%), tyrosine (5.3 mol%), threonine, glutamic acid (0.6%) and aspartic acid (0.5%) components ( Table 2 ). 233,237,265,26 6 The tyrosine residues of SF chain are usually reacted with a cyanuric chloride derivative of different molecules (i.e. poly(ethylene glycol), lactose), 237,265,266 with chlorosulfonic acid to obtain sulfated derivatives of SF, 237 with diazonium salts to o btain azobenzene derivatives of SF, 237 and with mushroom tyrosinase for grafting of chitosans and other biopolymers on SF materials. 237 Serine aminoacids in SF have been modified with 2 - methacryloyloxyethyl isocyanate for radical polymerization to obtain a poly(methacrylate) derivative or a 2 - methacryloyloxyethyl phosphorylcholine derivative of SF. 237 Carbodiimide coupling of glutamic acid and aspartic acid aminoacids of SF with peptides or of lysine residues of SF with polylactic acid have also been carrie d out. 237 Some enzymes and peptides covalently linked to SF nanoparticles include superoxidase, glucose oxidase, L - aspariginase, and insulin among others. 267 The discussed properties and benefits of SF materials explain the interest and intense research on its applications in biomedicine and other fields. T here are scarce reports on the application of SF in agriculture, where it is mostly used for nitrogen mineralization of soils, immobilization of enzymes for detection of pesticides, coating of perishable foods for prolonged preservation and seeds coating for increased germination of some plants. 268 - 271 Murase and Yonebayashi reported that silk waste, composed mostly of sericin and SF, can be used as slow and long - term nitrogen fertilizer of soils, with a n et nitrogen mineralization of approximately 25% – 268 (quantified as nitrogen detected as NO 3 ) after 56 days. In another research, Xue et al. prepared an amperometric biosensor based on acetylcholinesterase immobilized on regenerated SF matrix capable to det ect organophosphate and carbamate pesticides, with preserved bioactivity of the enzyme that achieved detection limits of 0.5 µmol/L and 0.06 µmol/L for methyl parathion and carbaryl, respectively. 269 Marelli et al. report that SF coating of strawberry and bananas resulted in formation of micrometric - thin SF membranes around the fruits, capable to enhance fruits shelf life at room conditions over 7 days. 270 There were also significant less dehydration, more firmness and delayed fruits ripening of SF coated f ruits when compared with non - coated fruits. 270 In contrast, Zhang et al. have shown that lettuce seeds coated with SF (4 hours

submerged in 50 mg/L SF solution), exhibited increased germination under high temperature stress. 271 These authors also demonstra ted the protective effect of SF on lettuce seedlings under heat stress conditions, causing a reduced impact of oxidative stress evidenced in increased levels of antioxidant enzymes. 271 On the other hand, SF materials are widely applied in biomedicine and t herapeutic applications as injectable hydrogels and microgels, scaffolds, films, micro and nanoparticles, composites, micro and nanofibers and many other forms for tissue engineering, drug delivery, enzyme immobilization, vascular grafts, DNA preservation and others. 229,231,241 - 246,272 FDA approved silk materials include Seri Surgical Scaffold (Allergan), a resorbable RGD - modified SF scaffold for cruciate ligament repair, and several silk sutures such as Surusil (Suru) and Sofsilk (Covidien). 235,247 TymPaSi l (CG Bio Inc.) approved from the Ministry of Food and Drug Safety of South Korea and Sidaiyi (Suzhou Soho Biomaterial Science and Technology Co., Ltd) approved from the China Food and Drug Administration also are in use as a SF patch for healing of tympan ic membrane and a SF - silicone scaffold for skin wound treatments, respectively. 248 Additional promising applications of SF materials comprise SF films, membranes and scaffolds for eye regeneration via corneal endothelium cell’s proliferation instead of cor neal transplantation or amniotic membrane transplantation, which can avoid potential transmission of HIV, syphilis, hepatitis B and C associated to traditional transplant of donated corneal tissue or amniotic membrane. 230 Biodegradable SF hydrogels are als o proposed for controlled ocular delivery of bevacizumab, an anti - vascular endothelial growth factor used to treat age - related macular degeneration in eyes, with controlled drug release over 3 months, good biocompatibility and bioactivity of the drug. 249 P articularly interesting is the use of SF discs releasing antibodies and proteins for HIV prevention, with extended release of

HIV inhibitor 5P12 - RANTES during 31 days and sustained inhibitory levels over IC 50 in HIV - 1 YU.2 cultured on in vitro blood and ex vivo colorectal tissue after 31 days. 250 Furthermore, Yavuz et al. demonstrated that SF discs can stabilize 5P12 - RANTES and other HIV inhibitors for 1 year or longer at 50 o C, with observed sustained release of 5P12 - RANTES and in vivo distribution from SF is currently studied in macaques. 250 On the other hand, SF materials have been extensively prepared for delivery of different anticancer drugs like doxorubicin, 232,251,252,255 paclitaxel, 233,235,252 curcumin, 232,252,257 methotrexate, 232,252 vincristine, 25 3,255 floxuridine, 252 cis - dichlorodiamminoplatinum, 252 etoposide, 254 anastrozole. 256 In this sense, Totten et al. demonstrated the role of the lysosomal environment of MCF - 7 human breast cancer cells in doxorubicin release from SF nanoparticles. 251 These a uthors observed endocytosis and traffic of

doxorubicin - loaded SF nanoparticles to the lysosomes, where low pH and enzymatic degradation accelerated drug release and nuclear translocation within 5 hours of dosing. The authors also reported that an increase of lysosomal pH and enzyme inhibition significantly reduced in approximately 40% to 30% the nuclear accumulation of doxorubicin. 251 Furthermore, Totten et al. demonstrated that simulated in vitro studies of anticancer drug releases from polymeric nanoparti cles lead to a very rough approximation to in vivo lysosomal drug release, where low pH and several lysosomal enzymes drive the release rate and simultaneous degradation of polymeric nanoparticles that accelerate the drug delivery. 251 There are scarce repo rts on the design and study of SF materials for the release of campthotecin or its derivatives (i.e. topotecan, irinotecan) and epirubicin. 257,258 For example, Gou et al. used camptothecin as a model compound to study the release profile of chondroitin sul fate - functionalized

SF nanoparticles in an oxidative environment (10 µM, 100 µM or 1 mM H 2 O 2 aqueous solution) or a reductive environment (0.1 mM, 1 mM or 10 mM glutathione (GSH) aqueous solution) at different pH values (7.4, 6.5, 4.5) to mimic blood plasm a, early endosomes and lyposomes. 257 The authors found that camptothecin release is significantly faster and more quantitative in a more acidic medium and higher concentration of H 2 O 2 or GSH. The use of camptothecin as a model compound was necessary becaus e of the inherent instability of curcumin loaded in chondroitin sulfate - functionalized SF nanoparticles or in carboxymethyl cellulose - functionalized SF nanoparticles aimed to alleviation of ulcerative colitis. 257 Choi et al. prepared SF/heparin nanofilms f or controlled release of epirubicin. 258 The authors controlled the epirubicin loading efficiency and release profile from the SF/heparin nanofilms with the β - sheet contents of the nanofilms. 258 Furthermore, slight anticancer effect of SF hydrolysate is reported on MCF - 7 human breast cancer cell s, with relative cell viability of 78.7% and 63.9% after 3 days at 1 mg/mL and 2.5 mg/mL of SF, respectively. 273 In another study, Wang et al. found that SF inhibited the in vitro growth of human lung cancer A549 and lung cancer H460 xenograft mice cells w ith IC 50 values of 9.9 and 9.1 mg/mL, respectively. 274 These authors also showed that SF administered via intraperitoneal injection at 200 or 500 mg/kg/day for 40 days, stopped the growth of H460 xenograft tumor in mice. 274 These results motivated our grou p to research on hydrophobic modification or functionalization of SF and preparation of SF nanoparticles for the sustained release of diosgenin, agrochemicals and anticancer drug camptothecin.

1.6.2. Preparation of silk fibroin particles SF particles (mic ro and nanoparticles) are promising candidates for drug delivery of different molecules, genes, viruses, enzymes and other agents. Furthermore, the biocompatibility, tunable degradation and drug loading, as well as release rate, and possible targeted drug delivery of SF nanoparticles explain the interest and potential on their preparation and assessment. 232 The main techniques used for preparation of SF nanoparticles include salting out, electrospraying, supercritical fluid, microdot printing, ionic liquid processing, polyvinyl alcohol blending, and organic solvent desolvation, among others. 252 Zhao et al. reviewed and detailed the preparation of SF nanoparticles using these techniques: 252  Desolvation or simple coacervation. This method is based on the reduc ed solubility of SF upon the addition of a non - solvent to a SF solution. As a result of the continuous addition of the non - solvent, SF particles are formed (precipitation or coacervate formation) in the mixture of the solvent and the non - solvent, where SF is no longer soluble. Initially, stable SF particles are formed and further addition of the non - solvent (desolvation) increases the yield of SF particles precipitated. The pH of the SF solution can be adjusted according to the desired SF particle size and yield. It is considered that SF nanoparticles formation is related to a conversion of silk I to silk II, with the self - assembly of several SF chains. 259 SF solution is usually prepared in water as solvent. Methanol, ethanol, propanol, isopropanol, acetone, DMSO and polyvinyl alcohol are usually used as non - solvent or desolvating agents, with formation of SF nanoparticles of 35 to 170 nm or SF microparticles of 0.98 to 1500 µm. 252,260 This is the most frequently used method for the preparation of SF nanopart icles because of the mild conditions, simplicity and the small particle sizes attainable. However, SF particles prepared with this method show tendency to aggregation. Particular care has to be taken to remove residues of the organic desolvating agent and low drug loading efficiency is usually achieved when drug loading is carried out simultaneous with SF coacervation.  Salting out. It consists in SF precipitation or coacervation due to addition of a

salt (for instance, K 3 PO 4 ) to an SF aqueous solution. SF p rotein has hydrophilic and hydrophobic parts that interact differently with water molecules. Hydrophilic interaction of SF with the solvent (water) is reduced with increased salt concentration, and hydrophobic interactions among different parts of SF molec ule and different SF molecules are favored. Then, the SF molecules coacervate (precipitate) from the aqueous solution. SF particles prepared using

this method show sizes ranging from 486 to 1200 nm, with bigger particles

obtained when using more concentrat ed K 3 PO 4 (pH 8.0), and different drugs are loaded in the SF particles. This method is characterized by simplicity, high yield and reduced cost; but, particular care on removal of the salting out compounds must be taken.  Microemulsion method. In this method , micro or nanodroplets of one liquid are formed and stabilized in the second liquid (microemulsion) with the aid of a surfactant at the interface of the two liquids. There are several microemulsions:

water - in - oil (w/o), oil - in - water (o/w) and water - in - sup ercritical - CO 2 (w/sc - CO 2 ). SF nanoparticles of 167 to 169 nm have been prepared by a w/o microemulsion with Triton X - 100 used as surfactant and a mixture of MeOH and EtOH used to recover the SF particles and to remove the surfactant. This technique allows a precise control of particle size, but traces of surfactant and organic solvents (oil phase) can remain in the SF nanoparticles.

 Supercritical fluid technologies. In this method, supercritical CO 2 (anti - solvent) and SF aqueous solution are atomized under controlled temperature and pressure to form nanodroplets of supersaturated SF solution, with precipitation of SF nanoparticles. SF nanoparticles of 50 to 100 nm have been prepared using this method, and indomethacin was efficiently loaded using this techn ique. The particle size and morphology can be controlled with the concentration of the solute, flow rate of the solute and anti - solvent, and temperature. Additionally, no organic solvent residues are found in the nanoparticles prepared with this method and relatively high drug loading is attainable. However, high cost, complicated equipment and necessary further treatment of SF nanoparticles to ensure insolubility in aqueous medium are important disadvantages of this technique.  Electrospraying. It consists on the formation of nanoparticles by liquid atomization using an electrical field. SF aqueous solution is flowed out of a capillary nozzle at high voltage (10 - 30 kV). This procedure allowed formation of blank and cis - dichlorodiamminoplatinum - loaded SF nano particles of 59 to 75 nm. The nanoparticles formed using this method present high monodispersity and purity, their sizes can be controlled.  Capillary - microdot technique. In this technique, a water insoluble drug (i.e. curcumin) is dispersed in a SF aqueou s solution to form a suspension that is deposited on a glass slide using a microcapillary to form microdroplets. The glass slides with the SF microdroplets are frozen and lyophilized. The solid

drug - loaded SF microdots are transferred to MeOH for crystalli zation. The SF nanoparticles are separated by centrifugation and rinsed several times with water or PBS to afford less than 100 nm particles. The main advantage of this method is the simplicity, but residual MeOH or organic solvents can be found in the SF nanoparticles thus prepared. However, SF particles have shown a tendency to aggregate in biological media with high ionic strength. 246 That is why some cationic polymers (i.e. glycol chitosan, N , N , N - trimethyl chitosan, polyethylenimine and PEGylated polyet hylenimine) are used for coating SF nanoparticles via electrostatic interactions, to increase the stability in biological media. 246 In another work, Cheng et al. prepared 360 to 1225 nm SF microspheres via the desolvation method with EtOH as desolvating ag ent. 261 These microspheres were later used as surfactant of an emulsion of polycaprolactone droplets in CHCl 3 dispersed in water to form polycaprolactone capsules stabilized with an outer layer of SF. 261

1.7. Polyphosphazenes Polyphosphazenes are a group o f synthetic macromolecular polymers based on a phosphorus - nitrogen repeating unit, which form the backbone with alternating single and double bonds. 275 - 279 Pentavalent phosphorus atoms in the backbone are also di - substituted with side groups (frequently or ganic) to give poly(organo)phosphazenes. 275 - 279 Since the successful isolation of soluble poly(dichloro)phosphazene ([NPCl 2 ] n ) by Allcock et al. almost sixty years ago, 275 it remains as the most used polymer precursor to synthesize a vast array of poly(org ano)phosphazenes via substitution of the two chlorine atoms per repeat unit with organic substituents ( Figure 15 ). 275,276,279

Figure 15. General structure of the polymer precursor ([NPCl 2 ] n and the poly(organo)phosphazenes. Adapted with permission from references 275,279 .

The poly(dichloro)phosphazene backbone is highly flexible and the highly reactive chlorine atoms make it unstable, being crosslinked by intermolecular condensation and degraded in the presence of water. 275,279 The preparation of linear

poly(dichloro)phosphazene is carried out by two synthetic methods: ring - opening polymerization (ROP) of hexachlorocyclotriphosphazene, and living cationic polymerization of a chlorophosphoranimine ( Figure 16 ). 275,276,279 In the ROP of hexachlorocyclotriph osphazene, high molecular weight poly(dichloro)phosphazene is prepared by high temperature (~250 o C) induced heterolytic rupture of a P - Cl bond in a hexachlorophosphazene molecule and the nucleophilic attack of the N atom of another monomer molecule with s ubsequent ring opening and propagation of the chain reaction as long as the temperature is maintained and/or the monomer is consumed ( Figure 16I ). 275,279 The ROP method was the first approach used for the synthesis of the precursor poly(dichloro)phosphazen e. 275,276,279 Precise control of the temperature, purity of the monomer hexachlorophosphazene, moisture free reaction and high scale (>10 g) must be carefully taken to obtain high molecular weight [PCl 2 ] n (n~10000), but uncontrolled branching and crosslink ing of growing cationic polyphosphazenium intermediate results 275,279 in high polydispersity (M w /M n = 2 to 7). Some variants of ROP of hexachlorophosphazene include the use of Lewis acid catalysts (i.e. AlCl 3 , BCl 3 ) allowing to carry out the polymerization at lower temperatures (~200 o C). 275,279 In the second method, cationic living polymerization of trichlorophosphoranimine (Cl 3 P=NSiMe 3 ) in CH 2 Cl 2 solution undergoes a living chain growth polycondensation initiated by PCl 5 at room temperature, with occurren ce of some bidirectional chain growth ( Figure 16II ). 275,279 The cationic living polymerization of trichlorophosphoranimine can be also initiated with chlorinated phosphines (i.e.

Ph 3 PCl 2 ) in similar reaction conditions for a controlled unidirectional chain growth ( Figure 16II ). 275,279 The cationic living polymerization allows a better control of the molecular weight or chain length upon precise control of the monomer to initiator ratio, proceeds without branching and gives [NPCl 2 ] n with low polydispersity ( M w /M n = 1.01 - 1.4), but typically results in [NPCl 2 ] n with much lower molecular weights (n~100) than the ROP 275,279 route. It is also reported the direct synthesis of [NPCl 2 ] n from PCl 3 with higher polydispersity than when using trichlorophosphoranimine, bu t with lower polydispersity than observed in ROP of hexacyclophosphazene and good chances for up - scale. 275,279 The living cationic polymerization route allows the preparation of different block copolymers via addition of a second substituted phosphoranimin e (ClR 2 P=NSi(CH 3 ) 3 ) to the trichlorophosphoranimine, which give a vast group of possible copolymers. 275,279 Grafting polymers onto the organic side groups of the poly(organo)phosphazenes (i.e. thiol - yne addition of thiolactone to a poly(ethynyloxy)phosphaz ene) allowed obtaining

molecular brushes with high branching density. 277,279 In another approach, Henke et al. prepared dendritic poly(organo)phosphazenes by synthesis of a three - arms polymer, later substituted with a diphenylphosphine containing group for further polymerization giving highly branched polyphosphazenes with estimated degree of polymerization between 15000 - 30000 and polydispersity between 1.2 - 1.6. 278,279

Figure 16 . (I) Thermal and/or catalyzed ring opening polymerization (ROP) of hexach lorotricyclophosphazene to obtain [NPCl 2 ] n and proposed mechanism, (II) living cationic polymerization of trichlorophosphoranimine to obtain [NPCl 2 ] n initiated with

PCl 5 (a) or with a phosphine (Ph 3 PCl 2 ). Reproduced with permission from references 275 ,279 .

The nucleophilic substitution of the chlorine atoms in [NPCl 2 ] n with the selected organic substituents is usually named post - polymerization chemical functionalization or macromolecular substitution. 275,276,279 A single organic substituent or a combi nation of different substituents such as sodium alkoxydes and aryloxydes, amines,

organometallic substituents can be introduced in [NPCl 2 ] n , but particular care must be taken to ensure complete chlorine substitution from the backbone to avoid further uncon trolled side reactions and fast autocatalytic aqueous degradation due to generation of aqueous hydrochloric acid. 275,276,279 To this end, an excess of the substituent and a base (i.e. Et 3 N, NaOH) are added to a THF solution of [NPCl 2 ] n to perform the macro molecular substitution, which is driven to completion by precipitation 275,276,279 of Et 3 NHCl or NaCl. When mixed substitution of [NPCl 2 ] n is envisioned, the substituents are added sequentially (usually introducing first the bulkiest ones) and slowly to ach ieve a random distribution along the backbone of the obtained copolymers. 275,276,279

1.7.1. Polyphosphazenes properties and applications The flexible P - N backbone of polyphosphazenes allows obtaining high molecular weight polymers with highly tunable physi co - chemical properties determined by the composition and kind of substituents linked. Hydrophilic - hydrophobic balance and associated solubility in organic and aqueous solvents, chemical reactivity or stability, thermal properties, degradability, biocompati bility, mechanical properties and others, can be tailored by means of precise control of the post - polymerization functionalization of poly(dichloro)phosphazene. 279,280 Polyphosphazenes are characterized by a high functionality (P atom with two possible fun ctional groups per repeating unit) and conformational flexibility, with possible introduction of chirality and tailored properties upon smart selection of the proper substituents for post - polymerization functionalization and/or further chemical modificatio n of the groups covalently bonded to the polyphosphazene backbone. 281 In this sense, König et al. developed a series of phosphine - functionalized polyphosphazenes for efficient (~80 - 99% yields) and fast (~15 min) chlorination of different alcohols (primary, secondary, aromatic alcohols), with easy recovery or separation of the obtained phosphine oxide - bearing polyphosphazene, regeneration and used again with good efficiency in chlorination of alcohols. 281 Allcock and Fuller pioneered the synthesis of polypho sphazenes functionalized with different bioactive steroids (i.e. desoxoestrone, estrone, 17β - estradiol and estradiol derivatives) and co - substituted with methylamine, ethyl glycinate or n - butylamine for potential medical applications in 1980, after their p revious work on biodegradable and water - soluble polyphosphazenes based on amino acid substituents and methylamine. 282 Biocompatible and biodegradable polyphosphazenes containing some vitamins (i.e. O - linked tocopherol or pyridoxine, N - linked ethyl 2 - aminob enzoate) and aminoacids (i.e.

ethyl glycinate, phenylalanine ethyl ester) were synthesized by Allcock et al. to study the introduction of bulky substituents (tocopherol) in the polyphosphazene backbone via post - polymerization functionalization, the degrada tion and sensitivity of the polymers in aqueous medium at different pH. 283 The authors reported successful synthesis of polyphosphazenes with complete chlorine atoms replacement in poly(dichloro)phosphazene when post - polymerization functionalization was ca rried out with small molecules pyridoxine and ethyl 2 - aminobenzoate, but bulky tocopherol substituent was introduced up to a 47% when poly(dichloro)phosphazene was also functionalized with ethyl alkoxide. 283 The introduction of different aminoacids in thes e polyphosphazenes allowed to tune their aqueous solubility and degradation rate in aqueous medium with 10 - 100% weight loss during 6 weeks and obtained pH from 2.5 to 9, which allows different potential biomedical applications from drug delivery to short - or medium - term used scaffolds for tissue engineering. 283 It is well established that the complete or final degradation of polyphosphazenes in aqueous medium (in simulated physiological conditions and in vivo ) due to hydrolysis ends up in a mixture of phosp hates, ammonia and the side groups previously linked to the P - N backbone ( Figure 17 ). 279,280 Allcock et al. also demonstrated that the hydrolytic degradation of N - linked amino acid substituted poly(organo)phosphazenes occurs by bulk erosion. 284

Figure 1 7. Proposed hydrolytic degradation pathway of poly(organo)phosphazenes. Reproduced with permission from reference 279 .

The innocuity of phosphates and ammonia, a natural buffer found in the body with pH around 7.0, and a proper selection of non - toxic orga nic substituents attached to the polyphosphazene backbone, ensures the non - toxicity of poly(organo)phosphazenes and their degradation products for potential biomedical use. 279,280 As already mentioned, the degradation rate via hydrolysis shows to be highly dependent on the substituent nature. Based on that, the durability of these polymers can be adjusted from days when used glyceryl - based substituents, to weeks (aminoacids as substituents) or even years when fluoroalkoxy substituents are attached to the P - N

backbone of the polyphosphazenes. 279 Composition of mixed substituted poly(organo)phosphazenes, hydrophilic - hydrophobic balance of the final polymer, steric hindrance of the substituents and other factors affecting the access of water molecules to the P - N backbone also regulate the hydrolytic degradation rate. 279,280 Rothemund and Teasdale summarized the following factors affecting the hydrolysis rate of poly(organo)phosphazenes: 279  N - linked organic substituents attached to the P - N backbone (P - NH - R) are o ften hydrolyzed faster than O - linked organic substituents (P - O - R), due to stronger P - O bond.  More acidic aqueous media (lower than pH 7) accelerate the polyphosphazenes hydrolysis, while slight ly alkaline aqueous media (7

spacer and their degradation products, demonstrated non - cytotoxicity to HCT116, A2780 and Hep3B cells at 0.01 to 10 µmol/L in PBS (pH 7.4) and MES buffer (pH 6.0). 285 Based on the observed fast degradation of poly(glycine Jeffamine M - 1000)phosphazene, Iturmendi et al. prepared self - immolative poly(arylboronate glycine - co - Jeffamine M - 1000 glycine)phosphaze ne and poly(arylboronate glycine - co - 286 glycine ethyl ester)phosphazene with H 2 O 2 triggered degradation. These poly(organo)phosphazenes might have potential as biosensor for in vivo detection of 286 overexpressed H 2 O 2 . Biocompatibility and in vivo biodegrada tion studies of some poly(amino acid ester)phosphazenes used as implants for bone tissue engineering have shown primary rat osteoblasts adhesion comparable to PLGA, good proliferation of Swiss 3T3 and HepG2 cells, good in vivo biocompatibility and biodegra dation after 12 weeks with minimal inflammatory and immunogenic response to subcutaneously implanted polyphosphazenes in rats. 280 Good bone mineralization on these polyphosphazene - based implant on New Zealand white rabbits without inflammation is also desc ribed. 280 Significant biodegradation of poly(amino acid ester)phosphazenes implants for bone tissue regeneration in rats, with a reduction of molecular weights from 80% to 98% after 12 weeks is reported. 287 Some poly(amino acid ester)phosphazenes have show n comparable or even superior mechanical properties when compared to PLGA. 287 It is also possible to tailor the compressive strength, tensile strength and elasticity of the polyphosphazenes via changing small amino acid substituents by aromatic side groups . 287 Therefore, the preparation of polyphosphazene - based scaffolds, blends with other polymers and composites with different inorganic salts is actively investigated. 280,287 In this sense, Rothemund et al. reported the preparation of biocompatible and biod egradable 3D porous scaffolds based on photopolymerizable glycine - based polyphosphazenes. 288 The photopolymerization of poly(allyl glycine)phosphazene with adipic acid divinyl ester, glutathione and trithiol allowed to obtain interconnected porous scaffold s with pore sizes of 100 - 200 µm, and tunable degradation rates from non - degradable polymers for pure polyester to 85% weight loss at 120 days for the polymer with 51 wt% of poly (allyl glycine)phosphazene and 49 wt% of trithiol. 288 No cytotoxicity was obse rved for these scaffolds or their degradation products on adipose - derived stem cells, but less cell adhesion was observed on this scaffolds when compared to a commercial collagen - based scaffold (TissuFleece E of Baxter). 288

In another work, Allcock et al. synthesized poly(amino acid ethyl ester - co - ferulic acid)phosphazenes with different amino acid substituents (i.e. glycine, valine, alanine, phenylalanine ethyl esters) for later photocrosslinking of ferulic acid molecules, with formation of scaffolds for t issue engineering applications. 289 The photocrosslinked polyphosphazene - based scaffolds contained ~75 - 80 mol% of ferulic acid and showed no appreciable degradation in aqueous medium (5% to 0% weight loss) after 8 weeks. 289 Nerve, tendon and ligament tissue engineering applications of poly(organo)phosphazenes based on good conductive properties, enhanced cell biocompatibility and adjustable biodegradation rate of poly(glycine ethyl ester - co - aniline pentamer)phosphazenes and poly(amino acid ester)phosphazene - coated PCL fibrous mats. 287 Further developments of biodegradable polyphosphazenes with the introduction of dipeptide side groups capable to form miscible polyphosphazene - PLGA blends are currently performed by Allcock, Laurencin et al . 290 These polyphospha zene - PLGA blends are considered unique biomaterials because of the formation of interconnected porous structure upon in vivo degradation, with reduced inflammatory response of the implants when compared to pure PLGA implants in rats, tailored mechanical pr operties and excellent perspectives for regenerative engineering applications. 290 Linhardt et al. introduced a tetrapeptide (glycine - phenylalanine - leucine - glycine) side group as substituent in poly(organo)phosphazenes co - substituted with Jeffamine M - 1000 t o obtain water - soluble enzymatically biodegradable polyphosphazenes. 291 Interestingly, imiquimod (R837) bearing poly(glycine - phenylalanine - leucine - glycine - co - Jeffamine M - 1000 g lycine) p hosphazene self - assembled in aqueous medium to form 200 nm aggregates th at released 65% of imiquimod in citrate buffer (pH 6) and 100% of imiquimod in citrate buffer (pH 6) in presence of the enzyme papain after 14 days at 37 o C. 291 Furthermore, poly(glycine - phenylalanine - leucine - glycine)phosphazene showed no degradation in ac etate buffer (pH 5) and considerable degradation in acetate buffer (pH 5) with papain added after 5 weeks at 37 o C. 291 Another major application of poly(organo)phosphazenes in the medical field is for drug delivery in form of nano/microparticles or aggrega tes, scaffolds, hydrogels, composites, blend fibers and others. 279,280,283 - 285,287 - 292 For example, Theis et al. reported the synthesis of polyphosphazene - based ruthenium bearing supramolecular gels photocleavable with visible or near - infrared irradiation with potential for photothermal anticancer therapy and/or photothermal triggered delivery applications. 292 In another research, Iturmendi et al. synthesized coumarin carrying polyphosphazenes (i.e. poly(coumarin glycine - co - glycine ethyl ester)phosphazene a nd poly(coumarin glycine -

co - Jeffamine M - 1000)phosphazene), which undergo photocleavable coumarin release and fast degradation after visible - light irradiation. 293 Furthermore, polyphosphazene - based matrices have been prepared for the controlled delivery of insulin, progesterone, naproxen, colchicines, bovine serum albumin to promote neovascularization in tissue engineering applications. 280 The self - assembly behavio u r of amphiphilic poly(organo)phosphazenes 275,279 is widely exploited in the preparation of mic elles and polymersomes for drug loading and sustained delivery of doxorubicin, 294,295 diflunisal, 296 and other medicines. Another strategy to prepare polyphosphazene - based drug delivery systems is the covalent bonding of the drug or a prodrug to the P - N ba ckbone of poly(organo)phosphazenes. In this sense, it is reported the synthesis of biodegradable antibiotic (i.e. ciprofloxacin, norfloxacin) linked poly(amino acid ethyl esters)phosphazenes, 297 doxorubicin linked poly(amino acid esters)phosphazene, 280,287 paclitaxel linked poly(organo)phosphazenes, 280 Pt (II) - linked poly(organo)phosphazenes, 280 Pt (IV) prodrug - linked poly(organo)phosphazene, 298 epirubicin - linked poly(organo)phosphazenes with folic acid substituent for active drug targeting, 299 camptothecin - linked poly(organo)phosphazenes, 300 and multiple drug (i.e. curcumin, Ce6 photodynamic agent, bis( - (4 - hydroxyphenyl)disulfide)) - linked polyphosphazene for 301 coating Fe 3 O 4 nanoparticles for imaging - guided chemo - photodynamic therapy. Interestingly, macromo lecular Pt(IV) prodrug - linked poly(organo)phosphazenes synthesized by Henke et al. exhibited significantly higher in vitro A2780 ovarian cancer cell uptake and cytotoxicity on A2780 ovarian cancer cells, HCT116 colon carcinoma cells when compared to therap eutically used Pt(II) drugs. 298 Furthermore, Pt(II) drug - resistant A2780 and HTC116 sublines displayed less resistance to prepared Pt(IV) prodrug - linked poly(organo)phosphazenes. 298 Teasdale et al. reported the synthesis of pH - responsive epirubicin - linked poly(organo)phosphazenes with folic acid substituents for prospective active drug targeting delivery to cancer cells overexpressing folate receptors, with observed in vitro ~100% epirubicin released after 10 hours in acetate buffer (pH 5) and ~15% epirubic in released after at 24 hours in PBS (pH 7.4) at 37 o C. 299 Therefore, these epirubicin - linked poly(organo)phosphazenes seems promising for lysosomal delivery of their cargo after cancer cell uptake. 299 However, missing in vitro cytotoxicity and cancer cell uptake studies, as well as the lack of any evaluation of hydrodynamic sizes of the aqueous aggregates, or in vivo studies of antitumor activity of the epirubicin - linked poly(organo)phosphazenes makes it impossible to assess the potential of these drug del ivery system. 299 In another work, Cho et al. reported the synthesis of camptothecin - linked poly(isoleucine ethyl ester - co - glycine - glycine - co - α -

amino -  - methoxy - poly(ethylene)glycol M - 550)phosphazenes with in vitro stability observed beyond 2 weeks in PBS (p H 7.4) at 37 o C. 300 It was observed a quantitative camptothecin release (~100%) from 10 to 60 days as an inverse function of the hydrogel viscosity, and good cytotoxicity to four cancer cell lines (i.e.A549, DLD - 1, HCT116, HT - 29). 300 Camptothecin - linked po lymers exerted significantly superior antitumor activity on HT - 29 colon cancer cell xenograft tumor in mice, with observed significant tumor growth inhibition by camptothecin - linked poly(organo)phosphazenes when compared to parent camptothecin ( - 29% of tum or size growth for camptothecin - linked polymers treatment against +130% of tumor size growth for parent camptothecin treatment after 4 weeks). 300 These facts motivated us to investigate the synthesis of poly(tocopherol glycine - co - Jeffamine M - 1000)phosphaze nes and poly(testosterone glycine - co - Jeffamine M - 1000)phosphazene for epirubicin and camptothecin loading in micelle - like structures, to evaluate their drug delivery and in vitro cytotoxicity to MCF - 7 human breast cancer cells and HEK - 293 embryonic kidney cells. There are scarce reports on the literature about poly(organo)phosphazenes devoted to agrochemical applications. 302,303 For example, Krishnamoorthy and Rajiv reported the preparation of polyphosphazene blends with polyvinylpyrrolidone for seed coatin gs with direct N and P supply to the seeds due to slow biodegradation of the polyphosphazene component. 302,303 Therefore, the design and synthesis of poly(organo)phosphazenes for delivery of plant growth regulators (i.e. brassinosteroids) in agriculture is almost unexplored. Hereby, we decided to investigate on preparation of some brassinosteroid - linked poly(organo)phosphazenes for agrochemical applications.

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2. Cellulose - based carriers for delivery of agrochemicals and drugs

This chapter shows the function alization of water - soluble cellulose ethers ( methyl cellulose, hydroxyethyl cellulose, (hydroxypropyl)methyl cellulose ) via esterification with steroid hemisuccinates (diosgenin hemisuccinate, DI31 and S7 hemisuccinate, testosterone hemisuccinate) or with vitamin hemisuccinates (α - tocopherol hemissucinate, vitamin D2 hemisuccinate) using a carbodiimide - mediated coupling in homogeneous phase (celluloses dissolved in 10% LiCl in N,N - dimethylacetamide). The steroid - grafted cellulose ethers formed aggregates in aqueous media, with sustained drug release behaviour. Camptothecin was hydrophobically encapsulated in the core of the testosterone - , tocopherol - and vitamin D2 - grafted cellulose particles up to a 13 wt%, in spite of low degree of substitution of cellulos e ethers with testosterone and vitamins. It corresponds to 100 - fold increase of camptothecin solubility in water for a 1 mg/mL aqueous dispersion of camptothecin - loaded cellulose particles. In vitro agrochemical activity evaluation on radish cotyledons of agrochemical bearing cellulose particles demonstrated their potential application in agriculture, with observed slightly superior plant growth enhancer effect. On the other hand, the camptothecin - loaded cellulose particles maintained the antiproliferative effect of parent camptothecin on MCF - 7 human breast cancer cells. This chapter is based on the following papers:

2.1. Self - assembled cellulose particles for agrochemical applications.

2.2. Testosterone - and vitamin - grafted cellulose ethers for sustained re lease of camptothecin.

My contribution to the papers

I designed and conducted all the experimental work and related characterization, except the AFM and SEM. I interpreted the results and wrote the manuscripts.

2.1. Self - assembled cellulose particles fo r agrochemical applications

Water - soluble cellulose ethers ( methyl cellulose, hydroxyethyl cellulose, (hydroxypropyl)methyl cellulose ) were functionalized with bioactive steroids via esterification with the steroid hemisuccinates (diosgenin hemisuccinate (MSD), DI31 hemisuccinate (MSDI31), S7 hemisuccinate (MSS7)) as shown in the graphical abstract of the publication ( TOC 1 ). The synthesized steroid - grafted cellulose conjugates self - assembled in aqueous med ia as aggregates. TEM showed spherical particles o f steroid - cellulose conjugates in dried state ( TOC 1 ). Sustained steroid release was observed during 3 days ( TOC 1 ). DI31 - and S7 - grafted cellulose ethers exhibited good agrochemical activity evaluated on radish cotyledons growth.

TOC 1. Synthesis and structure s of steroid - grafted cellulose ethers, TEM micrographs of dried cellulose particles, in vitro steroid release profile in aqueous media and radish cotyledons used to evaluate the agrochemical activity. Figure reproduced with permission.

Self - assembled cellulose particles for agrochemical applications

Javier Pérez Quiñones , a * Cezarina Cela Mardare , b Achim Walter Hassel , b Oliver Brüggemann a

––––––––– a Johannes Kepler University Linz, Institute of Polymer Chemistry, Altenberger Straβe 69, 4040 Linz, Austria. b Johannes Kepler University Linz, Christian Doppler Laboratory for Combinatorial Oxide Chemistry (COMBOX) at Institute of Chemical Technology of I norganic Materials, Altenberger Straβe 69, 4040 Linz, Austria.

E - mail: [email protected]

–––––––––

European Polymer Journal 93 ( 2017) 706 – 716

Abstract

The present work focuses on the hydrophobic functionalization of water soluble cellu loses methyl cellulose, hydroxyethyl cellulose and (hydroxypropyl)methyl cellulose with the anticancer steroid diosgenin, and two synthetic brassinosteroids (DI31 and S7) used as agrochemicals. Fourier transform infrared (FT - IR) and nuclear magnetic resona nce (NMR) spectroscopies confirmed the cellulose modification. Prepared amphiphilic steroid - cellulose conjugates can self - assemble in water as stable and almost neutral particles with micelle - like structure, as depicted using dynamic light scattering. Wher eas scanning and transmission electron microscopies showed 50 – 300 nm almost spherical particles and aggregates in dried state, atomic force microscopy assessed particles aggregates with mean sizes of 220 – 355 nm. These cellulose particles showed sustained s teroid release in acidic aqueous medium over 72 h, and good stimulatory agrochemical activity in radish cotyledons assay. Thus, the outlined synthesis of steroid - cellulose conjugates, which would be capable to form self - assembled particles in water for con trolled release of agrochemicals, is envisioned as a promising strategy.

KEYWORDS: Self - assembled particles; Cellulose; Agrochemical; Sustained release; Steroids.

2.1.1. Introduction Cellulose, a natural linear polysaccharide based on repeating units of β( 1 → 4) linked D - glucose, is the most abundant biopolymer as the key structural component of plants (33% of vegetal material) . It combines biocompatibility, good biodegradability (glycoside hydrolases and cellulase enzymes in some ruminants, termites and fu ngi) and no toxicity, while exhibiting proper reactivity towards esterification [1], [2], [3]. Even, when cellulose itself is not soluble in water, several cellulose esters show proper aqueous solubility, or are able to form stable aqueous nanoparticulate or micelle dispersions after further functionalization [4], [5]. In this sense, methyl cellulose (MC), hydroxyethyl cellulose (HEC) and (hydroxypropyl)methyl cellulose (HPMC) are water - soluble cellulose esters widely used in food and pharmaceutical industr y, and envisioned as promising materials for novel smart medicines [6], [7], [8]. Particularly, HPMC based drug delivery systems are well established in medical applications due to the polymer matrix biocompatibility and swelling properties upon contact wi th biological fluids [9], [10], [11]. Thus, cellulose based micro/nanoparticles, hydrogels, fibers, films and composites have been

proposed for different medical applications (e.g. antibiotic and anticancer drug delivery) [12], [13], [14], [15], [16]. An a dded value of cellulose - based systems is their good biodegradability [17], [18], antimicrobial behaviour observed after cationization or when prepared as composites or other formulations against L. monocytogenes and E. coli [14], and low cytotoxicity [15], [19]. On the other hand, the self - assembly of stimuli - responsive amphiphilic celluloses as nanoparticulate systems for sustained release of different drugs is an active research area [5], [20]. However, these self - assembled systems are mostly devoted to r elease of loaded hydrophobic drugs [5], [20], instead of delivery of the covalently grafted compound. Furthermore, reports about preparation of agrochemical controlled delivery systems are not as common as the ones devoted to medicine. Our research extends previous work on the field of synthesis and assay of different polymer - based systems for sustained release of brassinosteroids for agriculture [21]. Diosgenin ((25R) - spirost - 5 - en - 3β - ol) is a steroidal sapogenin mostly obtained by basic hydrolysis of dioscin, the most available steroidal saponin. Both, dioscin and the derived diosgenin, exhibit antioxidant, anti - inflammatory, estrogenic activity, and cy totoxicity to some cancer cell lines [22], [23], [24]. Diosgenin is the main substrat e in chemical synthesis of some steroids (i.e. progesterone, corticosteroids, and contraceptives), due to the fact that the required backbone and stereochemistry are alrea dy present in diosgenin [24]. In this sense, diosgenin is the precursor of two Cuban synthetic analogues of brassinosteroids (DI31 and S7) [25] used as commercial agrochemicals over the last two decades (Biobras - 16). Biobras - 16 regulates plants growth and protects the crops of biotic and abiotic stress once applied, with increases in harvest of 5 – 25% [26], [27]. Nevertheless, the expected agrochemical benefits are not fully achieved in plants because the exogenous brassinosteroids are rapidly metabolized. C onsequently, up to two or three foliar spray applications are usually applied to crops, which increase economic cost of the Biobras - 16 application [26]. Moreover, the hydrophobicity of brassinosteroids DI31 and S7 limits their bioavailability to plants and commercial Biobras - 16 formulation includes plenty of ethanol, and some environmentally unfriendly additives (i.e. N,N - dimethylformamide, surfactants). Herein, it is proposed that synthesis of novel biodegradable conjugates of diosgenin, DI31 and S7, by co njugation to water soluble cellulose esters via hydrolysable ester bonds, should improve bioavailability of the parent steroids and provide their sustained release over time. In the present research, we synthesised steroid - cellulose conjugates functionaliz ed with three different steroids linked via ester bond, characterized them by attenuated total reflectance Fourier transform infrared

(ATR - FTIR), proton nuclear magnetic resonance ( 1 H NMR) and bi - dimensional nuclear magnetic resonance (2D - NMR) spectroscopi es, as well as assessed self - assembly of these conjugates by dynamic light scattering, atomic force microscopy, scanning and transmission electron microscopies. In vitro drug release of the steroids from conjugates was investigated in an acidic aqueous med ium. In vitro agrochemical activity of the prepared cellulose nanoparticles towards radish ( Raphanus sativus ) was also studied. To the best of our knowledge, this is the first approach to the preparation of cellulose self - assembled particulate - based system for the delivery of brassinosteroids as agrochemicals.

2.1.2. Experimental Materials Three water soluble celluloses named methyl cellulose (MC) (14 mPa s 2% in water at 20 °C, methoxyl content 30.2%, number - average molecular weight Mn ca. 14,000 g/mol), h ydroxyethyl cellulose (HEC) (178.6 mPa s 1% in water at 20 °C, Mn ca. 220,000 g/mol) or (hydroxypropyl)methyl cellulose (HPMC) (22.1 mPa s 2% in water at 25 °C, methoxyl content 28.8% and hydroxypropyl content 8.9%, Mn ca. 25,000 g/mol) (Sigma A.G.) were u sed to prepare the steroid - cellulose conjugates. Solvents and chemicals were employed as purchased from Sigma - Aldrich. The diosgenin and synthetic analogues of brassinosteroids (DI31 and S7) were supplied by the Center of Natural Products at University of Havana, Cuba. Hemisuccinates of diosgenin and two synthetic analogues of brassinosteroids with agrochemical activity (DI - 31 and S7) were synthesised by base - catalyzed traditional esterification in pyridine with succinic anhydride [28].

Synthesis of steroid - cellulose conjugates 100 mg (0.4 – 0.6 mmol monosaccharide units) of methyl cellulose (MC), hydroxyethyl cellulose (HEC) or (hydroxypropyl)methyl cellulose (HPMC) were stirred 48 h at room temperature with 20 mg (0.05 mmol) of diosgenin or two synthetic bra ssinosteroid DI31 and S7 hemisuccinates (MSD, MSDI31 and MSS7), with 20 mg (0.1 mmol) of 1 - ethyl - 3 - (3 ′ - dimethylamino)carbodiimide hydrochloride and 20 mg (0.16 mmol) of 4 - (dimethylamino)pyridine in 10% LiCl in N,N - dimethylacetamide. Products were dialyzed (Spectra/Por 6, MWCO 1 kDa, Spectrum Lab., USA) against methanol (1 time, 600 mL, 12 h) and bi - distilled water (2 times, 1 L, 24 h), and lyophilized affording white cotton wool like products. Dissolution of studied celluloses in 10% LiCl in N,N - dimethylace tamide prior to chemical reaction was conducted [29]. All studies were performed in triplicate for each sample.

Preparation of the self - assembled particles The synthesised steroid - cellulose conjugates were able to form nanoparticles in aqueous solution aft er stirring overnight. To this end, the cellulose conjugates (ca. 0.5 – 2.0 mg/mL) were stirred overnight at 100 rpm in bi - distilled water or phosphate buffered saline solution (PBS, pH 7.4).

Characterization The number - average molecular weight of celluloses and steroid - cellulose conjugates were determined with gel permeation chromatography (GPC) using a Viscotek GPCmax (Malvern, Germany) with a PFG column from PSS, 300 × 8 mm2 , 5 μm particle size. The samples (100 μL of injection volume, 2 mg/mL) were eluted with 0.01 mol/L LiBr in (N,N) - dimethylformamide at a flow rate of 0.75 mL/min at 60 °C. The cellulose solutions were filtered through a 0.22 μm microporous nylon film syrin ge filter (Macherey - Nagel, Germany). The molecular weights were determined with a Viscotek TDA 305 Triple Detector Array (Malvern, Germany) using a multidetector calibration with different polystyrene standards from PSS (Malvern, Germany). The steroid - cell ulose conjugates were characterized by ATR - FT - IR spectroscopy using a Perkin - Elmer Spectrum 100 FT - IR systems spectrophotometer (Perkin - Elmer Corporation, Norwalk, Connecticut, USA) with 32 scans and 4 cm − 1 resolution in the region of 4000 – 650 cm − 1 , and solid samples were measured directly after 2 days in desiccator. 1 H NMR and 2D - NMR (gradient heteronuclear single quantum correlation and gradient heteronuclear multiple bond correlation, g - HSQC and g - HMBC) spectra were recorded with a Varian Unity INOVA 600 MHz spectrometer, operating at 599.4 MHz for 1H at 70 °C with concentrations of approximately 25 – 30 mg/mL in deuterated water and d6 - DMSO and analysed with the VNMRJ software, version 2.2. Steroid - cellul ose particles were studied by dynamic light scattering (DLS) performed using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at 25 °C to obtain the particle size and zeta potential. Size and morphology of dried steroid - cellulose particles were studi ed by transmission electron microscopy (TEM) with a Jeol JEM - 2011 (Jeol Ltd., Tokyo, Japan) operating at 100 kV and images taken with a Gatan Slow - Scan CCD camera. Samples were stirred in bi - distilled water (ca. 1 mg/mL) for 12 h and a drop was deposited o n a carbon coated copper grid. The excess solution was removed, negatively stained with a drop of uranyl acetate solution (1 wt.%), and dried on air. Scanning electron microscopy (SEM) of steroid - cellulose particles was performed in a field emission Zeiss Gemini 1540 XB SEM (Zeiss, Germany) with an acceleration voltage of 3 kV and secondary electrons detector. SEM samples were prepared by

depositing a drop of steroid - cellulose particles suspension on a silicon wafer (0.5 × 0.2 cm2), room temperature evapora tion of the water and gold coating with a HUMMER X (Anatech Ltd., Alexandria, VA, USA) sputter coater system.

In vitro drug release studies In vitro drug release of diosgenin, DI31 or S7 from steroid - cellulose nanoparticles was studied by UV detection (Spe ctraMax M3 spectrometer microplate reader, Molecular Devices, CA, USA) of the delivered steroids (from 200 nm to 500 nm) at pH 5.0. For this purpose, ca. 5 mg of steroid - cellulose nanoparticles was dispersed in phosphate buffered saline (PBS) solutions (1. 5 mL) at pH 5.0 and the dispersions were placed in dialysis cups (Slide - A - Lyzer MINI Dialysis Devices, 3.5 KDa MWCO, 2 mL, ThermoSCIENTIFIC, IL, USA). The samples were dialyzed against the release media (PBS pH 5.0, 10 mL) at 25 °C with constant agitation at 100 rpm. The entire media was removed at determined time intervals, and replaced with the same volume of fresh media. 200 μl aliquots were analyzed in a 96 well plate at 25 °C. The amount of released steroids was determined from a previously obtained ca libration curve. These studies were conducted in triplicate for each sample. Linear fitting of release profiles of steroid - cellulose conjugates up to 8 h and growth - sigmoidal fitting to a SWeibull2 function (y = a − (a − b) ∗ exp( −(k ∗ x)^d) up to 72 h wer e conducted with the Origin 2015 software (Microcal Origin, OriginLab, Northampton, MA, USA).

Agrochemical activity The radish ( Raphanus sativus ) assay was employed in order to evaluate the plant growth activity. This bioassay consists of determining the i ncreased weight of the treated radish’s cotyledons (auxin type activity). To this end, radish seeds previously sterilized by sodium hypochlorite treatment were germinated over wet filter paper in dark at 25 °C for 3 days [30]. Cotyledons were separated fro m hypocotyls, weighted and treated with 5 mL of brassinosteroid - cellulose nanoparticles in water (10 − 1 – 10 − 7 mg/mL of particles, brassinosteroid content 10 − 5.4 – 10 − 11.6 mol/L of DI31 or 10 − 4.8 – 10 − 11.2 mol/L of S7), DI31 or S7 solutions (10 − 1 – 10 − 7 mg/mL, 10 − 3 .6 – 10 − 9.7 mol/L), or pure water (control). Parent brassinosteroid DI31 and S7 solutions were prepared at 1 mg/mL (10 − 2.6 mol/L) in ethanol, while required brassinosteroid concentrations were achieved upon dilution in water. After 72 h, cotyledons weights w ere measured. These studies were conducted in triplicate for each sample and concentration (10 cotyledons per each run).

2.1.3. Results and discussion Synthesis of cellulose conjugates involves mild and quantitative esterification (ca. 90% yields, referre d to starting cellulosic material) between the – OH group at C6 posit ion of commercial water - soluble celluloses and the steroid hemisuccinates ( Fig. 1 ). Preliminary synthesis optimization showed that steroid to cellu lose feed ratios higher than 20 wt.% lead to conjugates that precipitated in aqueous solutions. Table

1 shows substitution degree (SD mol%), as measured by proton NMR in DMSO - d 6 at 70 °C, steroid content (wt.%), nominal ratios of modifications defined by the steroid to saccharide unit feed molar ratios (R%), yield of reaction based on starting cellulose materials (%), and number - average molecular weight (Mn).

Fig. 1 . Structures and synthesis of steroid - cellulose conjugates.

Table 1 . Substitution degree (SD, mol%), steroid content (wt.%), nominal ratios of modifications of steroid to saccharide units (R%), yield of reaction (%), and number - average molecular weight(Mn, g/mol) of steroid - cellulose conjugates. SD/mol% wt.% R% yields/% Mn/(g/mol)

MC - MSD 1.9 4.6 30 92 13,547

HEC - MSD 1.5 3.0 1 5 87 221,458

HPMC - MSD 1.8 4.4 27 90 25,052

MC - MSDI31 0.6 1.6 8 93 14,249

HEC - MSDDI31 0.5 1.1 5 88 220,891

HPMC - MSDI31 0.7 1.9 9 94 26,105

MC - MSS7 3.0 7.7 41 91 15,084

HEC - MSS7 1.2 2.6 13 90 220,652

HPMC - MSS7 1.9 5.1 25 93 24,616

Characterization Th e ATR - FTIR spectra of the cellulose conjugates are dominated by the intense peaks of the saccharide moiety because the low substitution degrees on steroid (<5 mol%). Thus, the characteristic saccharide peaks observed in the methyl cellulose, hydroxyethyl c ellulose and (hydroxypropyl)methyl cellulose at 3460 or 3359 cm − 1 (O – H stretching band), 2898 cm − 1 and 2873 or 2835 cm − 1 (aliphatic C – H stretching bands), 1454 cm − 1 (methyl C – H vibrations), 1373 cm − 1 (methyl C – H vibrations) and 1054 cm − 1 (skeletal vibrations of C – O and C - C stretching, C – O – C bridge stretch) [31], [32] ( Fig. 2I ) are dominant in the spectra of related steroid - cellulose conjugates ( Fig. 2II – IV ). However, the C = O peak of the new ester linkage formed is still observed at 1735 – 1733 cm − 1 ( Fig. 2II – IV ), while the C=O peak of ketone related to S7 steroid is visi ble at 1713 – 1717 cm − 1 ( Fig. 2IV ). This confirms the functionalization of studied water - soluble celluloses with steroid hemisuccinates. The conjugates also exhibited an absorption peak at 1646 cm − 1 (water vapour from atmosphere adsorbed onto cellulose surfa ce) [33].

Fig. 2 . FT - IR spectra of cellulose derivatives and steroid - cellulose conjugates (I) (a) MC, (b) HEC, (c) HPMC; (II) (a) MC - MSD, (b) HEC - MSD, (c) HPMC - MSD; (III) MC - MSDI31, (b) HEC - MSDI31, (c) HPMC - MSDI31; (IV) (a) MC - MSS7, (b) HEC - MSS7, (c) HPMC - MSS7 (see Fig. 1 for structures).

1 H NMR spectra of the cellulose conjugates in D 2 O and DMSO - d 6 ( Fig. 3I and Fig. 1 of supplementary material) supports the formation of particles like - micelles in aqueous phase, with the grafted steroid moieties shielded in the hydrophobic core of the aggregates, and the hydrophilic cellulose chains forming the shell oriented to the continuous aqueous phase ( Fig. 3II (a) ). Thus, cellulose conjugates self - assembled as particles like - micelle or nanometric aggregates in D 2 O, making it not possible to observe the proton peaks of the steroid moieties ( 1 H NMR of cellulose conjugates in

D 2 O is too similar to related spectrum of MC, HEC and HPMC, Fig. 3I (a) – (b) and Fig. 1 of supplementary material). On the other hand the steroid peaks in cellulose 1 conjugates are visible by H NMR in DMSO - d 6 ( Fig. 3I (c) , Figs. 1 and 2 of supplementary material), then it was possible to determine the steroid content based on the intense characteristic peaks related to them. Theref ore, it is proposed that 1 cellulose conjugates stand as extended chains in DMSO - d 6 ( Fig. 3II (b) ). The H NMR peaks used to calculate substitution degrees were the H2 glucose proton at 2.83 – 2.84 ppm and the anomeric proton signal around 4.3 – 4.4 ppm of the c elluloses [34], and the methyl group peaks of the steroids around 1 ppm ( Fig. 3I (c) , Figs. 1 and 2 of supplementary material). Particularly, the cellulose derivatives MC, HEC and HPMC

showed the characteristic saccharide peaks at 2.83 – 2.84 ppm (H2 of gluc ose repeating units), a broadband ca. 3.40 ppm (H3, H4, H5, H6 sugar protons of glucose repeating units), and 4.34 – 4.38 ppm (Ha, anomeric sugar proton of glucose repeating units). In addition to these intense sugar peaks, the steroid - cellulose conjugates s howed their typical steroid peaks at 0.61 ─1.09 ppm (methyl groups, H18 + H19 + H21 + H27 of diosgenin and DI31 moieties; H18 + H19 + H21 + H26 + H27 + H29 of S7 moiety), 5.34 – 5.35 ppm (methylene proton, H6 of diosgenin). Furthermore, covalent modifica tion of celluloses is confirmed with shield ing of the hemisuccinate proton chemical shifts from characteri stic values in parent diosgenin and brassinosteroid hemisuccinates from ca. 2.5 ppm to observed 2.09 ppm (singlet) or 2.14 and 2.28 ppm (doublets). 13 C NMR spectra showed mostly the sugar peaks, while the steroid signals were not observed, due to the low substitution degree and a lower sensitivity of this technique (data not shown). 2D - NMR experiments (g - HSQC and g - HMBC) confirmed the presence of steroi d moieties in the cellulose co njugates, as the characteristic steroid signals at 15 ─ 21 p pm (methy l groups, C18 + C19 + C21 + C27 of diosgenin and DI31; C18 + C19 + C21 + C26 + C27 + C29 of S7), 109.9 – 111.4 ppm (C22 of diosgenin and DI31), 124.6 and 142.7 p pm (C = C, C5 + C6 of diosgenin), 174.6 – 175.6 ppm (C = O, ester bond steroid succinates), 181.3 ppm (C = O, formed ester bond of steroid - cellulose conjugates), 207.7 ppm (C = O, C6 ketone of DI31 and S7 moieties) ( Fig. 3 of supplementary material).

1 Fig. 3 . (I) H NMR spectra of (a) MC, (b) MC - MSD in D 2 O at 25 °C, (c) MC - MSD in

DMSO - d 6 at 70 °C; (II) scheme of cellulose conjugates (a) self - assembly as particles like - micelle or nanometric aggregates in D 2 O, (b) extended chains in DMSO - d 6 (see Fig. 1 for structure s).

Dynamic light scattering studies of synthesised cellulose conjugates conducted in bi - distilled water, confirmed the formation of particles smaller than 850 nm ( Table 2 ). HEC afforded the less substituted cellulose conjugates and significantly biggest particles (except for the HEC - MSD). It is due to the high molecular weight (ca. 220,000 g/mol) of studied HEC, which impedes the accessibility of hydroxyl groups to esterification with steroid hemisuccinates. In spite of the almost neutral zeta potential ( ca. 0 mV) of synthesised steroid - cellulose conjugates, particles remained stable in aqueous dispersion as observed by DLS after 1 month (similar hydrodynamic sizes and PDI values) (data not shown). The trends in the CMC values were related to the substitut ion degrees of different cellulose conjugates for each steroid. Thus, it was observed that the highest substitution degree in each serial of steroid - cellulose conjugates is related to the highest critical micelle concentration.

Table 2 . Hydrodynamic diame ters (d, nm), polydispersity index (PDI), zeta potential ( ζ, mV) and critical micelle concentration (CMC, mg/mL) of cellulose conjugates in bi - distilled water at 25 °C. d/nm PDI ζ/mV CMC/(mg/mL)

MC - MSD 152 ± 2 0.7 ± 0.1 − 1.83 ± 0.09 0.02

HEC - MSD 223 ± 4 0.31 ± 0.04 − 0.74 ± 0.09 0.01

HPMC - MSD 246 ± 5 0.67 ± 0.02 1.91 ± 0.07 0.01

MC - MSDI31 139.8 ± 0.9 0.567 ± 0.003 − 4.5 ± 0.2 0.04

HEC - MSDI31 500 ± 8 0.798 ± 0.001 − 3.3 ± 0.1 0.07

HPMC - MSDI31 127 ± 2 0.71 ± 0.02 − 3.3 ± 0.1 0.08

MC - MSS7 170 ± 3 0.59 ± 0. 03 − 2.5 ± 0.1 0.09

HEC - MSS7 842 ± 3 0.41 ± 0.02 − 1.36 ± 0.01 0.04

HPMC - MSS7 118 ± 2 0.681 ± 0.007 − 2.06 ± 0.07 0.03

Atomic force microscopy, scanning electron microscopy and transmission electron microscopy showed some aggregates and individual nanopart icles in dried state with almost spherical shapes and sizes from 50 to 300 nm. SEM showed some aggregates and almost spherical particles of ca. 100 – 300 nm ( Fig. 4 ). Additionally, the steroid - modified HEC particles appeared as the bigger particles, in good agreement with the dynamic light scattering determinations. On the other hand, TEM micrographs showed almost circular or disk shaped individual particles of ca. 55 – 160 nm, when dried ( Fig. 5 ). Finally, AFM showed rounded aggregates of particles with mean s izes ca. 220 – 355 nm ( Fig. 4 of supplementary material).

Fig. 4 . Scanning electron micrographs of steroid - cellulose conjugates (a) MC - MSD, (b) HEC - MSD, (c) HPMC - MSD, (d) MC - MSDI31, (e) HEC - MSDI31, (f) HPMC - MSDI31, (g) MC - MSS7, (h) HEC - MSS7 and (i) HPMC - MSS7 at 80,000× magnifications (see Fig. 1 for structures).

Fig. 5 . Transmission electron micrographs of steroid - cellulose conjugates (a) MC - MSD, (b) HEC - MSD, (c) HPMC - MSD, (d) MC - MSDI31, (e) HEC - MSDI31, (f) HPMC - MSDI31, (g) MC - MSS7, (h) HEC - MSS7 and ( i) HPMC - MSS7 at 100,000× magnifications (see Fig. 1 for structures).

Drug delivery In vitro drug delivery studies in PBS solution at pH 5.0 showed almost linear steroid release for the first 8 h ( Fig. 5 and Table 1 of supplementary material), sustained a nd almost quantitative for 3 days (up to 75 – 89%) ( Fig. 6I – III , fitting to a growth - sigmoidal function SWeibull2 with adjusted R - square from 0.9525 to 0.9984). In vitro drug releases were conducted at pH 5.0 because acidic conditions are needed to achieve t he hydrolysis of the ester linkage and the steroid delivery. A control steroid delivery experiment was also conducted in PBS at pH 7.0, when no release is expected, which resulted in less than 5% steroid delivered after 72 h (data shown only for diosgenin - celluloses, Fig. 6IV ). Then, deviation of the release profiles from Fick’s law arises from the kinetics of ester hydrolysis. Thus, releases in PBS at pH 5.0 achieved 82% in MC - MSD, 88% in HEC - MSD, 77% in HPMC - MSD, 88% in MC - MSDI31, 85% in HEC - MSDI31, 79% i n HPMC - MSDI31, 75% in MC - MS7, 87% in HEC - MSS7 and 80% in HPMC - MSS7. It was observed that releases rates and extension were affected by the substitution degree and particles sizes. As expected, for each serial (same steroid, different cellulose substituent) , the releases were generally faster and more quantitative with lower substitution degree and bigger particles.

Fig. 6 . In vitro release profiles of steroid - cellulose conjugates (I) (a) MC - MSD, (b) HEC - MSD, (c) HPMC - MSD; (II) (a) MC - MSDI31, (b) HEC - MSDI31, (c) HPMC - MSDI31; (III) (a) MC - MSS7, (b) HEC - MSS7, (c) HPMC - MSS7 in PBS (pH 5.0) at 25 °C; (IV) (a) MC - MSD, (b) HEC - MSD, (c) HPMC - MSD in PBS (pH 7.0) at 25 °C (see Fig. 1 for structures).

Agrochemical activity Finally, the in vitro agrochemical activities based in the radish cotyledon assay [30] of the synthetic brassinosteroids DI31 and S7, and brassinosteroid - modified cellulose conjugates are shown in Fig. 7 . Plant growth stimulator activities of the synthetic brassinosteroids DI31 and S7 are quite similar, showing best results at 10 − 3 and 10 − 4 mg/mL concentrations, but almost a doubled cotyledons weight was reached as compared to control (no treatment, water) with the lowest concentrations (10 − 6 and 10 − 7 mg/mL) ( Fig. 7I ). On the other hand, th e brassinosteroid - modified cellulose nanoparticles in aqueous solution showed very good stimulatory activities at studied concentration of particles (10 − 1 – 10 − 7 mg/mL, brassinosteroid concentrations of 10 − 5 – 10 − 11.5 mol/L), with cotyledons weight increased a lmost two - three times compared to control ( Fig. 7II – III ). The excellent stimulatory activity found at lowest concentration of particles (10 − 6 and 10 − 7 mg/mL, brassinosteroid concentrations of 10 − 9.8 – 10 − 11.6 mol/L) is particularly promising for potential ap plications in agriculture. Thus, the brassinosteroid - modified cellulose conjugates exhibited 1.5 – 3 times stimulatory effects compared to the parent brassinosteroids DI31 and S7, probably as a result of brassinosteroids controlled release and cellulose stim ulatory effect in the radish cotyledons. Furthermore, in vivo plant growth stimulator effect of these brassinosteroid - modified cellulose conjugates is expected to be more important than the already observed results with the in vitro radish cotyledon’s bioa ssay, as well known for commercial formulations of DI31 in different crops [35], [27].

Fig. 7 . Agrochemical activity of (I) : (a) Control (C), (b) DI31, (c) S7; (II) : (a) Control (C), (b) MC - MSDI31, (c) HEC - MSDI31, (d) HPMC - MSDI31; (III) : (a) Control (C) , (b) MC - MSS7, (c) HEC - MSS7, (d) HPMC - MSS7 at 25 °C. (*) Not measured because cotyledons died as result of high ethanol content. Data are the mean ± standard deviation ( n = 3) (see Fig. 1 for structures).

2.1.4. Conclusions Nine novel steroid - modified cel lulose conjugates were synthesised in good yields, which exhibited sustained release of covalently linked steroids for 72 h in acidic conditions. Successful steroid functionalization of studied cellulose derivatives was assessed with ATR - FTIR, 1 H NMR and 2 D - NMR spectroscopies. Synthesised cellulose conjugates self - assembled as almost neutral particles in aqueous medium and remained as stable dispersions over 30 days. These particles exhibited sustained release of studied steroids over 72 h. Brassinosteroid - modified cellulose particles showed to be more active as agrochemicals than parent DI31 and S7 in an in vitro radish cotyledons assay. Therefore, synthesised steroid - cellulose conjugates might find practical potential in agriculture, and they constitute a proof of concept about

preparation of self - assembled particles in water of hydrophobically modified natural polymers and their derivatives.

Acknowledgements The authors wish to thank the Erasmus Mundus for a research grant to Javier Pérez Quiñones. Günter Hesser is acknowledged for TEM training and help with TEM imaging of steroid - cellulose particles at ZONA facility of JKU Linz, Linz, Austria. Lisa Maria Uiberlacker of JKU Linz, Linz, Austria and Dr. Pavlo Gordiichuk of Zernike Institute of Advanced Materi als at University of Groningen, The Netherlands are acknowledged for AFM measurements. The financial support by the Austrian Federal Ministry of Science, Research and Economy and the National Foundation for Research, Technology and Development through the Christian Doppler Laboratory for Combinatorial Oxide Chemistry (COMBOX) is gratefully acknowledged by CCM and AWH.

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(hydroxypropyl)methyl cellulose and aloe vera as promising material for wound dressing, J. Appl. Polym. Sci. 124 (2012) 3520 - 3524. [12] Y. Zhang, C. Gao, X. Li, C. Xu, Y. Zhang, Z. Sun, Y. Liu, J. Gao, Thermosensitive methyl cellulose - based injectable hydrogels for post - operation anti - adhesion, Carbohyd. Polym. 101 (2014) 171 - 178. [13] C. Ding, M. Zhang, G. Li, Preparation and characterization of collagen/hydroxypropyl methylcellulose (HPMC) blend film, Carbohyd. Polym. 119 (2015) 194 - 201. [14] C. Bilbao ─Sainz, B. ─S. Chiou, D. Valenzuela ─Medina, W. ─X. Du, K. S. Gregorski, T. G.Williams, D. F. Woo d, G. M. Glenn, W. J. Orts, Solution blown spun poly(lactic acid)/hydroxypropyl methylcellulose nanofibers with antimicrobial properties , Eur. Polym. J. 54 (2014) 1 - 10. [15] M. T. Calejo, A. ─L. Kjoniksen, E. F. Marques, M. J. Araujo, S. A. Sande, B. Nystr öm, Interactions between ethyl(hydroxyethyl) cellulose and lysine - based surfactants in aqueous media, Eur. Polym. J. 48 (2012) 1622 - 1631. [16] M. T. Calejo, A. ─L. Kjoniksen, A. Pinazo, L. Perez, A. M. S. Cardoso, M. C. P. de Lima, A. S.Jurado, S. A. Sande, B. Nyström, Thermoresponsive hydrogels with low cytoxicity from mixtures of ethyl(hydroxyethyl) cellulose and arginine - based surfactants, Int. J. Pharm. 436 (2012) 454 - 462. [17] S. Rimdusit, S. Jingjid, S. Damrongsakkul, S. Tiptipakorn, T. Takeichi, Biode gradability and property characterizations of methyl cellulose: effect of nanocomposition and chemical crosslinking, Carbohyd. Polym. 72 (2008) 444 - 455. [18] Z. Liu, P. Yao, Injectable thermos - responsive hydrogel composed of xanthan gum and methylcellulose double networks with shear - thinning property, Carbohyd. Polym. 132 (2015) 490 - 498. [19] N. Lin, A. Dufresne, Nanocellulose in biomedicine: current status and future prospect , Eur. Polym. J. 59 (2014) 302 - 325. [20] D. Bielska, A. Karewicy, K. Kaminski, I. Kielkowicz , T. Lachowicz, K. Szczubialka , M. Nowakowska, Self - organized thermo - responsive hydroxypropyl cellulose nanoparticles for curcumin delivery, Eur. Polym. J. 49 (2013) 2485 - 2494. [21 ] J. Pérez - Quiñones, R. Szopko, C . Schmidt, C. Peniche - Covas, Nove l drug delivery systems: Chitosan conjugates covalently attached to steroids with potential anticancer and agrochemical activity, Carbohyd. Polym. 84 (2011) 858 - 864. [22] K. Patel, M. Gadewar, V. Tahilyani, D. K. Patel, A review on pharmacological and anal ytical aspects of diosgenin: a concise report, Nat. Prod. Bioprosp. 2 (2) (2012) 46 - 52. [23] P. Chen, Y. Shih, H. Huang, H. Cheng, Diosgenin, a steroidal saponin, inhibits migration and invasion of human prostate cancer PC - 3 cells by reducing matrix metall oproteinases expression, PLoS ONE 6 (5) (2011) p. e20164. [24] S. Selim, S. A. Jaouni, Anticancer and apoptotic effects on cell proliferation of diosgenin isolated from Costus speciosus (Koen.) Sm, BMC. Complem. Alt. Med. 15 (2015) 301 - 307. [25] D. G. Rive ra, F. León, F. Coll, G. P. Davison, Novel 5 β - hydroxyspirostan - 6 - ones ecdysteroid antagonists: Synthesis and biological testing, Steroids 71 (1) (2006) 1 - 11. [26] E. Terry, M. M. D. Armas, J. Ruiz, T. Tejeda, M. E. Zea, F. Camacho - Ferre, Effects of different bioactive products used as growth stimulators in lettuce crops ( Lactuca sativa L. ), J. Food Agric. Environ. 10 (2012) 386 - 389.

[27] Y. C. Serrano, R. R. Fernandez, F. R. Pineda, L. T. S. Pelegrin, D. G. Fernandez, M. C. G. Cepero, Synergistic effect of low doses of X - rays and Biobras - 16 o n yield and its components in tomato ( Solanum lycopersicum L. ) plants, Am. J. Biosci. Bioeng. 3 (2015) 197 - 202. [28] T. Abe, K. Hasunuma, M. Kurokawa. Vitamin E orotate and a method of producing the same. US: 3944550, 1976. [29] X. Yuan, G. Cheng, From cel lulose fibrils to single chains: understanding cellulose dissolution in ionic liquids, Phys. Chem. Chem. Phys. 17 (2015) 31592 - 31607. [30] E. Alonso ─Becerra, Y. Bernardo ─Otero, F. Coll ─Manchado, F. Guerra ─Martínez , G. Martínez ─Massanet, C. Pérez ─Martínez, Synthesis and biological activity of epoxy analogues of 3 - dehydroteasterone, J. Chem. Res. 5 (2007) 268 - 271. [31] A. Synytsya, M. Novak, Structural analysis of glucans, Ann. Transl. Med. 2 (2) (2014) 1 - 14. [32] H. Akinosho, S. Hawkins, L. Wicker, Hydroxypr opyl methylcellulose substituent analysis and rheological properties, Carbohyd. Polym. 98 (2013) 276 - 281. [33] C. Ye, S. T. Malak, K. Hu, W. Wu, V. V. Tsukruk, Cellulose nanocrystal microcapsules as tunable cages for nano - and microparticles, ACS Nano 9 (1 1) (2015) 10887 - 10895. [34] P. L. Nasatto, F. Pignon, J. L. M. Silveira, M. E. R. Duarte, M. D. Noseda, M. Rinaudo, Methylcellulose, a cellulose derivative with original physical properties and extended applications, Polymers 7 (2015) 777 - 803. [35] M. V. N uñez, C. Robaina, F. Coll, Synthesis and practical application of brassinosteroid analogs, S. Hayat, A. Ahmad (Eds.), Brassinosteroids: Bioactivity and Crop Productivity, Springer Science+Media Business, B. V.: The Netherlands (2003), pp. 100 - 111.

SUP P OR TING INFORMATION

Javier Pérez Quiñones, Cezarina Cela Mardare, Achim Walter Hassel, Oliver Brüggemann

Supporting information available online:

https://doi.org/10.1016/j.eurpolymj.2017.02.023

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2.2. Testosterone - and vitamin - grafted cellulose ethe rs for su stained release of camptothecin

Water - soluble cellulose ethers ( methyl cellulose, hydroxyethyl cellulose, (hydroxypropyl)methyl cellulose ) were esterified with testosterone, tocopherol and vitamin D2 to obtain nine cellulose conjugates (I to IX). The cellulose conjugates (I - IX) self - assembled in aqueous media as stable particles ( TOC 2 ). TEM showed small particles of testosterone - and vitamin - grafted celluloses in dried state ( TOC 2 ). Hydrophobic anticancer drug camptothecin (CPT) was successfully encapsulated in the cellulose particles I - IX via solvent exchange from DMSO to water ( TOC 2 ). CPT - loaded cellulose particles exhibited strong cytotoxicity to MCF - 7 human breast cancer cells. The anticancer activity of CPT - loaded cellulose particles is hyp othesized to occur via cell uptake of the particles and CPT release inside the cancer cells with partial metabolism of the nanoparticles ( TOC 2 ).

TOC 2. Self - assembly of testosterone - and vitamin - grafted cellulose ethers (I - IX) in aqueous media , TEM micr ographs of dried cellulose particles , CPT encapsulation in cellulose aggregates via DMSO/water exchange, hypothesized MCF - 7 cell uptake and partial metabolism of CPT - loaded cellulose particles that result in CPT release and cell apoptosis, as observed usin g relative cell viability assays. Figure reproduced with permission.

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Testosterone - and vitamin - grafted cellulose ethers for sustained release of camptothecin

Javier Pérez Quiñones , a * Cezarina Cela Mardare , b Achim Walter Hassel , b Oliver Brüggemann a

–– ––––––– a Johannes Kepler University Linz, Institute of Polymer Chemistry, Altenberger Straβe 69, 4040 Linz, Austria. b Johannes Kepler University Linz, Christian Doppler Laboratory for Combinatorial Oxide Chemistry (COMBOX) at Institute of Chemical Technology of I norganic Materials, Altenberger Straβe 69, 4040 Linz, Austria.

E - mail: [email protected]

–––––––––

Carbohydrate Polymers 206 ( 2019) 641 – 652

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Abstract

Camptothecin (CPT), a potent anticancer drug with known antiviral activity, is halted of clini cal use. Few drug delivery systems of CPT are approved for therapy. Hereby, we propose the encapsulation of hydrophobic CPT in the inner core of cellulose nanoaggregates for sustained release with retaining of antiproliferative activity. Cellulose conjugat es were synthesized by esterification of methyl cellulose, hydroxyethyl cellulose and (hydroxypropyl)methyl cellulose with testosterone, ergocalciferol and dl - α - tocopherol hemisuccinates. The degree of substitution attained ranged from 0.004 to 0.025 and no depolymerization was observed by size exclusion chromatography. ATR - FTIR and NMR spectroscopies confirmed grafting of testosterone and vitamins to cellulose s. According to dynamic light scattering, it resulted in their self - assembly in aqueous medium as stable and slightly negatively charged nanoaggregates of 213 to 731 nm. Nanoaggregates formation was also assessed using transmission electron and atomic forc e microscopies. CPT was encapsulated in the cellulose nanoaggregates, achieving a content of 1.7 – 13.0 wt %. Sustained release of camptothecin over 150 h was observed in simulated physiological conditions. CPT - loaded cellulose nanoparticles appeared to be p ossible candidates for chemotherapy, according to observed cytotoxicity against MCF - 7 cancer cells.

KEYWORDS: Camptothecin ; Cellulose; Self - assembled nanocarriers; Sustained release .

2.2.1. Introduction Cancer is one of the top five leading causes of death in the world, with over 100 types of cancers affecting humans. It was the cause for around 15% of deaths worldwide in 2015. 14.1 million new patients of cancers were diagnosed in 2012 and 23.6 million estimated new cases annually by 2030 (Krukiewicz & Zak , 2016; Syazwani et al. , 2017). The incidence and deceases associated to cancer increase every year, in spite of improvements in the medical treatment of different cancer and tumors ( i.e. tailored chemotherapy, radiotherapy and genetically designed medicin es). On the other side, traditional chemotherapy agents used in medicine for cancer treatment since the 60’s like doxorubicin, cisplatin or camptothecin derivatives exhibit negative side effects and cancer cells resistance develops at a medium rate (Casals , Gusta, Cobaleda - Siles, García - Sanz, & Puntes, 2017; Nurgali, Jagoe, & Abalo, 2018). Campt othecin (CPT), a plant alkaloid found in Camptotheca acuminate, is a potent antiproliferative agent against different tumors with demonstrated good antiviral

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activit y during in vitro and in vivo studies (Jiménez - Pardo et al. , 2015; Liu et al. , 2013; Wu & Chu, 2017). CPT cytotoxic activity was first reported in 1966. Its mechanism of action is nowadays well established (inhibition of DNA replication via formation of a complex with DNA and topoisomerase I). However, severe side effects together with reduced solubility, stability and in vivo efficacy at physiological conditions (aqueous medium, pH 7.4, 37 °C) of campthotecin halted further medical use (Li, Jiang, Li, & Ling, 2017; Liu et al. , 2013; Silva et al. , 2017). To bypass these limitations two approaches have been taken: (1) preparatio n of CPT derivatives with reduced side effects and better aqueous solubility ( i.e. Irinotecan and Topotecan), and (2) CPT encapsulation within some polymers (PEG, hyaluronic acid, chitosan). The CPT loading was attained via physical embedding into the hydr ophobic core or chemical functionalization and grafting into the polymer chains to obtain nanogels that enhanced the CPT solubility and bioavailability, with reduced toxicity (Chen et al. , 2018; Omar, Bardoogo, Corem - Salkmon, & Mizrahi, 2017; Quiñones et a l. , 2018; Silva et al., 2017). However, few formulations releasing camptothecin are available for medical treatments (Krukiewicz & Zak, 2016; Li, Jiang et al. , 2017). For that reason, CPT was encapsulated in hyaluronic acid - testosterone nanoaggregates (2.8 wt % of camptothecin), reaching similar antiproliferative effect on MCF - 7 cancer cells to the effect observed in parent camptothecin (Quiñones et al. , 2018). Testosterone was chosen as hydrophobic component in the inner core of the hyaluronic acid nanocar riers because of its benefits. It was demonstrated that a better survival rate and lower cancer relapse ratio can be achieved when breast tumors are treated with testosterone, an aromatase inhibitor (anastrozole, letrozole) and tamoxifen (Glaser, York, & D imitrakis, 2017). Vitamins E and D2 (α - tocopherol, ergocalciferol) are also suitable for functionalization of hydrophilic polysaccharides and later encapsulation of lipophilic camptothecin in the formed nanoassemblies. Due to known anticancer and cardiovascular - protective e ffects these vitamins might contribute to a better prognosis of cancers and mitigate the side effects of antitumor chemotherapy (Angulo - Molina, Reyes - Leyva, López - Malo, & Hernández, 2014; Atoum & Alzoughool, 2017; Bjelogrlic, Radic, Jovic, & Radulovic, 200 5). High levels of 25 - hydroxyvitamin D in plasma influenced via vitamin D supplementation and solar UVB exposure, can reduce incidence rates and mortality of breast and colorectal cancers (Autier & Gandini, 2007; Grant, 2015). The potential of cellulosic s elf - assembled systems in chemotherapy with the benefits of incorporating testosterone or vitamins E and D2, motivated us to propose the encapsulation of camptothecin in cellulose nanoparticles (NPs). These cellulose NPs might be

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prepared via esterification of water - soluble cellulose ethers with testosterone, vitamins D2 and E ligands. The CPT - loaded cellulose NPs are expected to efficiently encapsulate camptothecin, slowly release the anticancer drug with reduced side effects and to protect the CPT lactone ring for higher antiproliferative activity against tumors. The abundance of cellulose, considered a renewable resource, which derivatives are widely produced with tailored properties and versatile applications (Omura, Imagawa, Kono, Suzuki, & Minami, 2017) , also motivated the proposed research. Cellulose, a linear homopolymer consisting of β(1 → 4) linked D - glucose repeating units, is widely distributed on earth. It is derived from cell walls of plants, many algaes, oomycetes and bacterial biofilms (Cacicedo, Castro, Servetas, Bosnea, & Boura, 20 16; Yang & Li, 2018). Cellulose itself and its derivatives are proven to be biocompatible, biodegradable, harmless to mammals and suitable to esterification for applications in nanomedicine (Kondaveeti, Damato, Carmona - Ribeiro, Sierakowski, & Petri, 2017; Yang & Li, 2018; Yang, Guo, Sun, & Wang, 2016). Particularly, methyl cellulose (MC), hydroxyethyl cellulose (HEC) and (hydroxypropyl)methyl cellulose (HPMC) are used as inert fillers for medicines or dietary fiber. These cellulose ethers can self - assemble once functionalized, to form stable nanoaggregates in water with stimuli - responsive behavio u r (pH, redox potential, temperature) for biomedical and agrochemical uses (Javanbakht & Namazi, 2018; Nsor - Atindana et al. , 2017; Quiñones, Mardare, Hassel, & Brügg emann, 2017). In this study, different cellulose ethers were functionalized with testosterone, vitamins D2 and E to form self - assembled nanoaggregates capable to carry the anticancer drug camptothecin for medical applications. We discuss the chemical struc ture, particle properties and diffusion - mediated release of encapsulated CPT from the cellulose nanoaggregates. Particularly interesting resulted the high camptothecin loading achieved in spite of minor degree of substitution of cellulose ethers with the h ydrophobic modifiers and minor impact on their thermal and structural properties. The stability of formed particles was assessed using dynamic light scattering, while antiproliferative activity against MCF - 7 cancer cells of CPT - loaded cellulose nanoaggrega tes was also evaluated.

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2.2.2. Experimental Materials Methyl cellulose (MC) [14 mPa s at 2% in water at 20 °C, methoxyl DS (degree of substitution, number of hydroxyl positions substituted per monosaccharide) of 1.8 and number average molecular weight M n approx. 14 000 g mol − 1 ] was used as received from Sigma - Aldrich. Hydroxyethyl cellulose (HEC) [178.6 mPa s at 1% in water at 20 °C, hydroxyethyl DS of 1.6, hydroxyethyl MS (molar substitution, number of substituents per monosaccharide) of 3.0 according to 1 H NMR determination (Clemett, − 1 1973), M n approx. 220 000 g mo l ] and ( hydroxypropyl)methyl cellulose (HPMC) (22.1 mPa s at 2% in water at 25 °C, methoxyl DS of 1.9 and hydroxypropyl MS of 0.23, M n approx. 25 000 g mol − 1 ) were also purchased from Sigma - Aldrich and used as received. All analytical specifications of MC, HEC and HPMC were provided upon request by Sigma - Aldrich, except when indicated. The cellulose ethers were esterified with testosterone, ergocalciferol and tocopherol using a carbodiimide coupling route. Testosterone hemisuccinate, ergocalciferol hemisuccinate and D L - α - tocopherol hemisuccinate were synthesized via base - catalyzed esterification of testosterone and vitamins with succinic anhydride in pyridine ( Fig. 1I (a) ) (Abe et al. , 1976). Testosterone, ergocalciferol, DL - α - tocopherol, other chemicals and solvents we re acquired from Sigma - Aldrich and used as received. Regenerated cellulose dialysis membranes [Spectra/Por 6, 1 kDa molecular weight cut - off (MWCO)] and Slide - A - Lyzer MINI Dialysis Devices [3.5 kDa MWCO, 3 mL] were purchased from Spectrum Labs (Thermo Scie ntific, IL, USA). Deionized water used for preparation of nanoaggregates solutions (18.2 M Ω cm resistivity) was obtained from Milli - Q Reference A + System (EMD Millipore, Fisher Scientific, USA).

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Fig. 1 . (I) Synthesis and structures of (a) testoste rone and vitamin hemisuccinates, (b) cellulose - testosterone and cellulose - vitamin conjugates. (II) Schematic presentation of cellulose - testosterone and cellulose - vitamin conjugates (a) after dispersion in water or D 2 O followed by (b) their self - assembl y as nanoaggregates in the aqueous medium. (c) Uncoiled chains of cellulose conjugates after dissolution in DMSO or DMSO - d 6 followed by (d) encapsulation of anticancer drug CPT in the core of the self - assembled cellulose - testosterone and cellulose vitamin nanoaggregates during the solvent exchange to water by dialysis.

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Synthesis of cellulose - testosterone and cellulose - vitamin conjugates Cellulose ethers were dissolved in 10% LiCl in N,N - dimethylacetamide for a proper disruption of strong intermolecular hyd rogen bonding of cellulose chains, allowing further reaction of the hydroxyl groups (Yuan & Cheng, 2015). Esterification of water - soluble celluloses with testosterone or vitamin hemisuccinates was attained via carbodiimide mediated coupling with 1 - ethyl - 3 - (3 ′ - dimethylamino) carbodiimide hydrochloride (EDC) and 4 - (dimethylamino)pyridine (DMAP) (Quiñones et al. , 2017). Briefly, 100 mg (0.4 to 0.6 mmol of monosaccharide units) of cellulose ethers in 10% LiCl in N,N - dimethylacetamide were stirred 2 days at room temperature with 20 mg (0.1 mmol) of EDC, 20 mg (0.16 mmol) of DMAP and 20 mg (approx. 0.04 - 0.05 mmol) of testosterone or vitamin hemisuccinates. The mixtures were dialyzed (1 kDa MWCO) against methanol (1 time, 600 mL, 12 h) and deionized water (2 times, 1 L, 24 h). The dialyzed solutions were lyophilized affording the cellulose conjugates as white cotton wool like products (MC - Test (I), MC - VitD2 (II), MC - Toc (III), HEC - Test (IV), HEC - VitD2 (V), HEC - Toc (VI), HPMC - Test (VII), HPMC - VitD2 (VIII), HPMC - Toc ( IX), Fig. 1I ).

Preparation of cellulose - testosterone and cellulose - vitamin nanoaggregates Cellulose - testosterone and cellulose - vitamin conjugates were stirred overnight at 100 rpm in deionized water or PBS (pH 7.4), (concentration between 0.5 and 2.0 mg mL − 1 ). This procedure allowed the self - assembly of cellulose - grafted testosterone and cellulose - grafted vitamin molecules as aggregates in a micelle - like structure ( Fig. 1II ).

Preparation of CPT - Loaded cellulose - testosterone and cellulose - vitamin nanoaggrega tes CPT - loaded cellulose - testosterone and cellulose - vitamin NPs were prepared using a dialysis method consisting of a solvent exchange from DMSO to water, followed by lyophilization. This enabled obtaining the CPT loaded cellulose particles (Quiñones et al . , 2018). Briefly, 10 mg of cellulose conjugates and 0.5 or 1.5 mg of CPT were dissolved in 10 mL of DMSO. Mixtures were stirred for 30 min at room temperature. Removal of free CPT and formation of nanoparticles was conducted via dialysis (1 kDa MWCO) again st deionized water (1 time, 2 L, 4 h). White to slightly brownish powders were obtained after lyophilization (CPT - loaded cellulose - testosterone or cellulose - vitamin nanoparticles, 5%CPT - or 15%CPT - I, II - IX) ( Fig. 1II ).

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Size exclusion chromatography (SEC) A Viscotek GPCmax (Malvern, Germany) equipped with a PFG column (PSS, 300 × 8 mm 2 , 5 μm particle size) that was thermostated at 60 °C was used to determine the number average and weight average molecular weights of parent cellulose ethers and cellulose conj ugates. A Viscotek TDA 305 Triple Detector Array (Malvern, Germany) with integrated refractive index, viscometer and light scattering detectors was coupled with the SEC column. Multidetector calibration with polystyrene standards from PSS (Malvern, Germany ) allowed estimation of molecular weights. The cellulose - based samples (100 μL of injection volume at 2 mg mL − 1 ) were filtered through a 0.22 μm Nalgene syringe filter) and eluted with 0.01 M LiBr in N,N - dimethylformamide at a flow rate of 0.75 mL min − 1 .

C haracterization of cellulose conjugates Attenuated reflectance Fourier transform infrared (ATR - FTIR) spectroscopy was carried out on a Perkin - Elmer 1720 FTIR spectrophotometer (Perkin - Elmer Corporation, Norwalk, Connecticut, USA) with 32 scans and 4 cm − 1 r esolution in the region of 4000 – 650 cm − 1 . Solid samples were directly placed on the diamond. Thermal properties of prepared cellulose conjugates were evaluated with differential scanning calorimetry (DSC). Analyses were conducted using a Perkin - Elmer Diffe rential Scanning Calorimeter Pyris 1 (Perkin - Elmer Instrument Inc., Boston, MA, USA). Approximately 10 mg of sample were analyzed under nitrogen dynamic flow of 20 mL min − 1 and a heating - cooling rate of 10 °C min − 1 (Lai, Pitt, & Craig, 2010). Samples were deposited in aluminum capsules and hermetically sealed. Indium was used to calibrate the instrument. Enthalpy (ΔH in J g − 1 dry weight) and peak temperature were computed automatically. Samples were heated and cooled from 0 to 250 °C. The results were proce ssed with the Pyris 1 software (version 6.0.0.033). Wide - angle X - ray diffraction (WAXD) patterns of powdered cellulose samples were acquired using a Bruker D8 Venture diffractometer operated with Mo Kα radiation. Data were collected at 5° min − 1 , with a sca n angle from 4° to 50°. Nuclear magnetic resonance spectroscopy [ 1 H NMR and 2D NMR (gradient heteronuclear single quantum correlation g - HSQC and gradient heteronuclear multiple bond correlation g - HMBC)] was carried out on a Varian Unity INOVA 600 MHz spec trometer, operated at 599.4 MHz for 1 H at 70 °C. Samples were − 1 prepared at approximately 25 – 30 mg mL in D 2 O and DMSO - d 6 . Chemical shifts in ppm are referred to residual solvent peaks as internal standards (D 2 O 4.79 ppm, DMSO - d 6 2.50 ppm). NMR spectra were analyzed with the VNMRJ software, version 2.2.

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Particle size, zeta - potential and CMC of Nanoaggregates Dynamic light scattering (DLS) measurements of cellulose nanoaggregates were performed using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at 2 5 °C, equipped with a 4 mW He - Ne laser emitting at 633 nm for backscattering measurements (detection angle of 173°). The Stokes - Einstein and Smoluchowski equations were used to estimate the hydrodynamic parameters. Samples were prepared at 0.5 mg mL − 1 . Fol ded capillary cells DTS1070 were used to estimate the average hydrodynamic particle sizes in deionized water and PBS (pH 7.4), and the zeta - potential values in deionized water. Measurements were performed in triplicated, using automatic measurement setting s. Scanning electron microscopy (SEM) [field emission Zeiss Gemini 1540 XB SEM (Zeiss, Germany)] operated at acceleration voltage of 3 kV and secondary electron detector depicted the dried cellulose particles. To this purpose, a drop of cellulose particles suspension was deposited on a silicon wafer (0.5 × 0.2 cm 2 ) and the water was evaporated at room temperature. SEM samples were gold coated with a HUMMER X (Anatech Ltd., Alexandria, VA, USA) sputter coater before imaging. Transmission electron microscopy (TEM) of dried cellulose particles was performed using a Jeol JEM - 2011 instrument (Jeol Ltd., Tokyo, Japan) operated at 100 kV. TEM images were acquired with a Gatan Slow - Scan CCD camera. Samples preparation for TEM was conducted as explained. Briefly, cel luloses conjugates were stirred in deionized water (approx. 1 mg mL − 1 ) for 12 h and a drop was deposited on a carbon coated copper grid. The excess solution was removed, negatively stained with a drop of uranyl acetate solution (1 wt %), and dried on air. The critical micelle concentration (CMC) of prepared cellulose - testosterone and cellulose - vitamin nanoaggregates was determined in deionized water using the pyrene probe method (Ashjari, Khoee, Mahdavian, & Rahmatolahzadeh, 2012; Jeon, Shin, Kwon, & Lee, 2 018). The fluorescence spectra were recorded on a microplate reader SpectraMax M2 (Molecular Devices, LLC, USA) using standard 96 - well plates for fluorescence measurements, at 25 °C. Samples containing pyrene were excited at 337 nm and emission scan from 3 20 to 420 nm was recorded for different concentration of cellulose nanoaggregates. CMC was determined from the inflection point at the plot of I 372 /I 393 against concentration of cellulose conjugates.

CPT content and in vitro drug release studies The CPT co ntent of CPT - loaded cellulose - testosterone and cellulose - vitamin NPs was determined using fluorescence spectroscopy recorded on a SpectraMax M2 (Molecular

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Devices, LLC, USA) with quartz cuvettes (excitation at 370 nm and detection at 430 nm for camptotheci n). To this end, weighted samples were dissolved in 3.5 mL of DMSO at 25 °C and fluorescence due to camptothecin was measured. CPT content was determined based on a previously obtained calibration curve for parent CPT in DMSO. UV detection of absorbance (C ary 60 UV – vis spectrophotometer, Agilent, CA, USA) at 249 nm for testosterone, 265 nm for ergocalciferol, and 292 nm for D L - α - tocopherol in PBS at pH 7.4 was employed for studying the in vitro drug release of testosterone and vitamins from blank cellulose NPs. In vitro released camptothecin from CPT - loaded cellulose NPs was determined using fluorescence detection (excitation at 370 nm and detection at 430 nm for camptothecin) in PBS at pH 7.4. In vitro release experiments were carried as explained. Briefly, approximately 5 mg of CPT - loaded or blank cellulose NPs were dispersed in PBS solutions (2 mL) at pH 7.4 and the dispersions were placed in 3 mL dialysis cups (3.5 kDa MWCO). The samples were dialyzed against the release media (PBS pH 7.4, 10 mL) at 37 °C with constant agitation at 100 rpm. The entire media was removed at determined time intervals, and it was replaced with the same volume of fresh media. The amount of the released testosterone, ergocalciferol, D L - α - tocopherol and camptothecin were determin ed based on previously obtained calibration curves.

Cytotoxicity assay Cytotoxicity of the samples against MCF - 7 cells (human breast adenocarcinoma cell line) was evaluated using a XTT assay [Cell Proliferation Kit II (XTT), Sigma - Aldrich]. To this end, MC F - 7 cells were grown in RPMI (Invitrogen) media supplemented with 10% fetal bovine serum, 1% penicillin – streptomycin, 2 mM of ultraglutamine 1 and 1 nM of 17 - β - estradiol at 37 °C in 5% CO 2 and 100% humidity. Once achieved the required cells population, MCF - 7 cells in full growth medium were seeded in a 96 - well plate (1 × 10 4 cells/well). After the cells were suitably attached on the plate, the medium was changed to a serum free medium with camptothecin, blank or CPT - loaded cellulose NPs at different concent rations (with final CPT concentration changed from 0 to 13 μg mL − 1 ). MCF - 7 cells were incubated for additional 48 h. Then, the medium was substituted with full growth medium and 50 μL of activated XTT reagent was added to each well. Incubation was continue d for another 2 h at 37 °C in a humidified chamber with 5% CO 2 and 100% humidity. The absorbance was measured at 490 nm using a microplate reader SpectraMax M2. Experiments were conducted in triplicated. Results expressed as relative cell viability were no rmalized to control for each experiment.

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Statistics All experiments were performed in triplicate (three independent experiments, n = 3) and data is shown as arithmetic mean value ± standard deviation (SD). Statistical assessment was performed with Statgrap hics Plus 5.1, Professional edition. Results were evaluated using one - way analysis of variance (ANOVA), Tukey post - hoc test for between - group comparisons, multiple comparisons procedure and Kruskall - Wallis test at 95% confidence level (p = 0.05). Mean valu es without significant differences are presented with the same letter (p > 0.05). Significant differences exist between values marked with the same letter but different numbers added to the letter (p < 0.05). Arithmetic means with no letter at all stand fo r significantly different values (p < 0.05).

2.2.3. Results and discussion Synthesis of hydrophobically - modified cellulose conjugates A partial disruption of intermolecular hydrogen bonds between different cellulose chains is crucial for further reaction o f cellulosic ─ OH groups. Molecular weight of cellulose ethers and related chain lengths, degree of substitution of parent celluloses and the selected esterification route will determine the attained functionalization and properties of synthesized cellulose conjugates (Quiñones, Szopko, Schmidt, & Covas, 2011). In this sense, preliminary experiments were carried out on cellulose ethers to find the best conditions to obtain water dispersible cellulose conjugates. Following the acyl chloride approach, acyl chl oride of diosgenin - 3β - succinate was reacted homogeneously with MC in 10% LiCl in N,N - dimethylacetamide to obtain a cellulose - diosgenin conjugate with approximately 20 wt % of diosgenin. However, this conjugate of MC was too hydrophobic to be dispersed in w ater. In another approach, esterification of MC with diosgenin - 3β - succinate via carbodiimide mediated coupling in 10% LiCl in N,N - dimethylacetamide, allowed obtaining a cellulose - diosgenin conjugate with 4.6 wt % of diosgenin that formed stable nanoaggrega tes in deionized water (Quiñones et al. , 2017). Consequently, the carbodiimide route was employed to synthesize the cellulose conjugates ( Fig. 1I ). This route resulted in a low degree of substitution with the hydrophobic substituents and slight increase of M n ( Table 1 ). However, the yield and efficiency of esterification (expressed by dividing the amount of the esterified testosterone or vitamins per mass unit of the cellulose conjugate to the amount of testosterone or vitamins in feed) were moderate ( Table 1 ). The synthesis of cellulose - vitamin conjugates was significantly more efficient with the MC and HPMC derivatives. The lower M n and viscosity of parent MC and HPMC made more accessible their glucose units for esterification and facilitated the diffusion of EDC -

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activated vitamin hemisuccinates through shorter and more extended cellulose chains. The low reactivity of testosterone - 17β - succinate resulted in lower degree of substitution in the cellulose - testosterone conjugates. This behavio u r of testosterone - 17β - succinate was similarly observed towards amide bond formation with hyaluronic acid hydrazides (Quiñones et al. , 2018).

Table 1 . Degree of substitution (DS) with testoste rone and vitamins for cellulose conjugates, drug content (wt %), nominal ratios of modifications with testosterone and vitamin residues (R%), preparation yields (%) and number average molecular − 1 weight (M n , g mol ) of parent cellulose ethers and prepared cellulose conjugates. wt % R% 2 Y% M 3 Sample DS 1 n % % % g mol − 1

MC – – – – 13615

HE C – – – – 221650

HPMC – – – – 23484

I 0.004 a 0.7 e 4 g 87 h 13859

II 0.01 b 1.5 9 92 222178

III 0.004 a 0.7 e 4 g 89 i 24303

IV 0.021 c 4.7 31 91 15056

V 0.01 b 2.0 11 89 i 221581

VI 0.018 d 4.0 26 87 h 24585

VII 0.025 6.0 32 85 14141

VIII 0.02 c 4.2 f 22 87 h 22 2042

IX 0.018 d 4.3 f 27 90 25143 1 Degree of substitution calculated from 1 H NMR spectra. 2 Defined as percentage of testosterone or vitamin reagent in feed. 3 Determined based on SEC method.

Physico - chemical characterization of cellulose conjugates The e sterification of cellulose ethers with the testosterone and vitamin succinates was monitored using ATR - FTIR spectroscopy. Prepared cellulose conjugates exhibited typical absorption peak of esters at 1733 – 1750 cm − 1 (  (C = O), C = O stretching) in addition to sa ccharide IR absorptions ( Fig. 2 ) (Akinosho, Hawkins, & Wicker, 2013; Quiñones et al. , 2017). 2D NMR spectroscopy (gHSQC and gHMBC) further

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corroborated the functionalization of cellulose ethers with testosterone, vitamin D2 and tocopherol succinates. Typic al 13 C resonances of testosterone were observed at 15.8 and 19.5 ppm (C18 + C19 in testosterone), at 176 ppm (C = O of ester bond testosterone succinate), 182 ppm (C = O of formed ester bond in cellulose - testosterone conjugate) and 205 ppm (C = O, C3 ketone in testos terone) in the gHSQC and gHMBC spectra of cellulose - testosterone conjugates in DMSO - d 6 ( Fig. S1I (a) – (f ) in Supporting information). Cellulose - vitamin conjugates also showed characteristic aliphatic 13 C resonances at 22.5 and 35.5 ppm (aliphatic carbons of ergocalciferol moiety), 25.5 – 41.7 ppm (aliphatic carbons of α - tocopherol moiety), unsaturated 13 C resonances appeared at 133 and 138 ppm (alkene C22 and C23 of ergocalciferol) and at 120 – 150 ppm (aromatic carbons C5 to C9 of α - tocopherol) in the gHSQC and gHMBC spectra in DMSO - d 6 ( Fig. S1II and III in Supporting information).

Fig. 2 . ATR - FTIR spectra of parent celluloses and cellulose conjugates: (I) (a) MC, (b) HEC, (c) HPMC; (II) (a) I, (b) II, (c) III; (III) (a) IV, (b) V, (c) VI; (IV) (a) VII, (b) V III, (c) IX (see structures in Fig. 1I ).

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Structural study of cellulose conjugates Differential scanning calorimetry analysis of the synthesized cellulose conjugates was used to obtain an insight into the impact of celluloses functionalization on their str ucture and thermal properties. Parent celluloses exhibited an intense endothermic peak at 58.2 °C (MC, HPMC) and 64.9 °C (HEC) with related ΔH of 119.9 – 183.8 J g − 1 ( Fig. S2 and Table S1 of Supporting information), which are associated to moisture loss of c elluloses (Akinosho et al. , 2013; Ford, 1999). Moreover, some endothermic and exothermic peaks with very low thermal effect (ΔH) were also observed for MC and HPMC at 170.6 – 197.2 °C , which were related to partial dissociation of polymeric chains with melti ng of microcrystals and partial oxidative degradation (Lai et al. , 2010). On the other hand, cellulose conjugates revealed a quite similar thermal profile as for parent celluloses. Endothermic peaks due to dehydration of cellulose conjugates were observed at similar temperatures (50.2 – 71.9 °C). Some endothermic and exothermic peaks related to the partial depolymerization of cellulose conjugates also appeared in MC and HPMC derivatives at similar temperatures as observed for native celluloses. The grafting o f hydrophobic testosterone and vitamins on cellulose ethers seems to have a minor impact on their thermal properties, which might be due to the low degree of substitution achieved. X - ray diffraction studies were conducted to investigate the impact of hydro phobic modification on cellulose crystalline structure. Fig. 3 shows the X - ray diffraction pattern of parent cellulose ethers and synthesized cellulose conjugates. Cellulose derivatives MC and HPMC exhibited two broad peaks at 10.1 – 10.9° (halo of low van d er Waals) and 19.8 – 20.0° (van der Waals halo). Both peaks are attributed to the existence of regions with aggregates of segments of parallel chains (Oliveira et al. , 2015). The synthesized MC and HPMC conjugates (I, VII, III, VI, IX) also showed these char acteristic broad peaks, but a change of peak intensity (area and high) is observable for the van der Waals halo ( ∼ 20°) ( Fig. 3I, III ). This can be interpreted as a disruption of polymer chain packing of MC and HPMC due to hydrophobic grafting of celluloses, with the related reduction of crystallinity. Particularly, sample IV exhibited only a broad peak at 19° ( Fig. 3 I (c ) ). On the other hand, HEC showed only a broad peak at 20.5° (van der Waals halo) and two sharp peaks at 31.8° and 45.5° (poly(ethylene oxide) chains). The HEC conjugates showed the characteristic broad peak of HEC at 20.5° with no appreciable change a nd a slight shoulder at approx. 12° ( Fig. 3II ). Therefore, the grafting of the hydrophobic substituents on the celluloses affected the crystalline structures of cellulose ethers in solid state.

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Fig. 3 . WAXD of parent celluloses and cellulose conjugates: (I) (a) MC, (b) I, (c) IV, (d) VII; (II) (a) HEC, (b) II, (c) V, (d) VIII; (III) (a) HPMC, (b) III, (c) VI, (d) IX (see structures in Fig. 1I ). Study of cellulose - testosterone and cellulose - vitamin nanoaggregates 1 H NMR is often employed to assess substi tution or functionalization degree of polymers and macromolecular systems. It also allows the study of fast exchanging micellization or self - assembly processes. In this sense, the formation of cellulose nanoaggregates with a micelle - like structure in D 2 O, where the hydrophobic grafting groups are accommodated in the inner part ( Fig. 1II (b) ), provoke an electronic screening of testosterone and vitamin substituents. Contrariwise, new characteristic 1 H NMR peaks of testosterone and vitamins appeared or enhanc ed significantly its intensity when NMR spectra of cellulose conjugates were recorded in DMSO - d 6 ( Fig.

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4I (b) and (c) and II (a) and (b) ). Uncoiled polymer chain conformations predominant in

DMSO - d 6 solution ( Fig. 1II (c) ) allowed to estimate degree of su bstitution of celluloses.

Cellulose ethers (MC, HEC, HPMC) in DMSO - d 6 showed distinctive saccharide proton NMR resonances at 2.83 – 2.84 ppm (H2, glucose proton of monosaccharide units) and 4.3 – 4.4 ppm (H1, anomeric glucose proton of monosaccharide units) (F ig. 4). These two signals were used as reference peaks to calculate degree of substitution of synthesized cellulose conjugates (Nasatto et al. , 2015). Additionally, a broad peak at approx. 3.4 – 3.5 ppm (H3 – H6 of glucose monosaccharide units + protons of CH 3 O – ,

HO – (CH 2 CH 2 O) n – and H – (OCH(CH 3 )CH 2 ) n – groups of cellulose ethers) is observed in parent and hydrophobically - modified celluloses. Whereas, cellulose conjugates in

DMSO - d 6 also exhibited the characteristic resonances of methyl group protons at 0.81 – 1.09 pp m (H18 + H19 in testosterone, H18 + H21 + H26 + H27 + H28 in vitamin D2 and 4’CH 3 − + 8’CH 3 - + 12’CH 3 − in α - tocopherol) and 2.02 ppm (H2 + H5 + H7 + H8 in α - tocopherol) ( Figs. 4 and S3 in Supporting information). Some olefin protons of cellulose conjugates were observed at 5.62 ppm and 7.05 – 7.14 ppm (H4 of testosterone, H6 of vitamin D2), while the protons of succinate linker were visible at 2.09 – 2.22 ppm (4H of succinate linker in testosterone succinate) ( Figs. 4 and S3 in Supporting information).

1 Fig. 4 . H NMR spectra of: (I) (a) MC in D 2 O at 25 °C, (b) I in D 2 O at 25 °C, (c) I in

DMSO - d 6 at 70 °C; (II) (a) II in D 2 O at 25 °C, (b) II in DMSO - d 6 at 70 °C, (c) III in

DMSO - d 6 at 70 °C (see structures in Fig. 1I ).

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Hydrodynamic parameters, CMC and morpholog y of cellulose nanoaggregates Dynamic light scattering studies revealed the formation of cellulose nanoparticles with hydrodynamic sizes ranging from 213 to 731 nm in aqueous medium ( Table 2 ). The self - assembly of amphiphilic cellulose conjugates as nanoag gregates in aqueous medium, as proposed from 1 H NMR results, is verified. The blank cellulose nanoparticles I, IV, V, VII showed significantly smaller hydrodynamic diameters when dispersed in PBS, while II, III, VI, VIII and IX exhibited bigger hydrodynami c sizes in PBS. The HEC - based nanoaggregates appeared as the biggest particles in aqueous medium, which might be due to the higher number average molecular weight of HEC (approx. 222 000 g mo l − 1 ). All cellulose particles were slightly negatively charged with zeta - potentials of −0.44 to −6.2 mV. This is probably due to the presence of few molecules of testosterone and vitamin hemisuccinates adsorbed on the surface. However, blank cellulose particles appeared stable in aqueous dispersion over 1 month (no significantly different hydrodynamic diameters a nd PDI values were observed after 1 month) ( Table S2 of Supporting information). Camptothecin loading in cellulose - testosterone and cellulose - vitamin conjugates generally gave significantly bigger nanoparticles in PBS as compared to the blank cellulose nan oparticles. This finding is probably due to a bigger association number of cellulose molecules into a micelle - like assembly with the lipophilic camptothecin in the inner core ( Fig. 1II ). All cellulose conjugates showed unimodal distribution of the blank an d CPT - loaded cellulose nanoaggregates, with polydispersity index values of 0.21 – 0.62. Zeta - potential values of CPT - loaded cellulose nanoparticles in water ranged from −0.50 to −6.65 mV , similar to values observed in the respective blank cellulose nanoparti cles. On the other hand, the CMC of blank cellulose nanoaggregates in water was determined by fluorimetry using pyrene as molecular probe (Li, Zhou et al. , 2017). It was observed that the cellulose conjugates with highest degree of substitution in testoste rone or vitamins generally exhibited the highest CMC value in each group. Finally, CPT content in weight ranged from 1.7% to 13.0% and CPT encapsulation achieved a good yield of approx. 78 – 92%.

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Table 2 . Hydrodynamic diameters (nm), polydispersity i nd ex (PDI), zeta ─potential (ζ - pot., mV) and critical micelle concentrations (CMC, mg mL − 1 ) of cellulose conjugates at 25 °C; CPT contents in weight (wt %) of CPT - loaded cellulose nanoparticles. Samples diameter * , (PDI) diameter ** , (PDI) ζ - pot. * CMC *

335 ± 5 a1,b1 234 ± 4 I − 3.4 ± 0.5 j 0.01 (0.27 ± 0.08) (0.42 ± 0.02)

327 ± 4 b2 342 ± 7 a II − 6.2 ± 0.3 k1 0.03 m (0.30 ± 0.04) (0.40 ± 0.03)

213 ± 3 284 ± 3 III − 2.0 ± 0.5 l 0.02 (0.38 ± 0.06) (0.37 ± 0.02)

508 ± 8 c 427 ± 2 IV − 5.0 ± 0.2 0.06 n (0.27 ± 0.02) (0.30 ± 0.04)

731 ± 8 565 ± 2 V − 0.44 ± 0.01 0.04° (0.23 ± 0.02) (0.23 ± 0.03)

400 ± 8 d 435 ± 5 VI − 3.8 ± 0.1 j1 0.06 n (0.30 ± 0.05) (0.23 ± 0.01)

319 ± 3 e 263 ± 3 f1 VII − 2.42 ± 0.03 l1 0.04° (0.30 ± 0.05) (0.25 ± 0.02)

259 ± 4 f 381 ± 8 g VIII − 2.16 ± 0.01 l2 0.03 m (0.28 ± 0.04) (0.3 0 ± 0.01)

253 ± 3 f2 294 ± 3 h IX − 2.49 ± 0.08 l1 0.04° (0.22 ± 0.05) (0.23 ± 0.01)

Samples diameter ** , (PDI) ζ - pot. * wt % ***

328 ± 3 b 5%CPT - I − 2.1 ± 0.1 l2 2.0 (0.34 ± 0.02)

399 ± 4 d 5%CPT - II − 6.65 ± 0.02 k2 2.5 (0.42 ± 0.05)

388 ± 4 g 5%CPT - I II − 2.7 ± 0.3 l1 4.0 p (0.36 ± 0.04)

348 ± 1 a2 5%CPT - IV − 1.5 ± 0.1 l3 2.8 (0.44 ± 0.03)

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Samples diameter ** , (PDI) ζ - pot. * wt % ***

625 ± 4 5%CPT - V − 3.2 ± 0.2 j2 3.0 (0.23 ± 0.02)

337 ± 6 a1 5%CPT - VI − 1.65 ± 0.03 l4 4.2 (0.38 ± 0.01)

251 ± 5 f2 5%CPT - VII − 3.9 ± 0.2 j1 1.7 (0.21 ± 0.02)

412 ± 3 i 5%CPT - VIII − 3.1 ± 0.3 j2 3.7 q (0.32 ± 0.04)

313 ± 2 e 5%CPT - IX − 2.8 ± 0.1 j2 3.7 q (0.46 ± 0.05)

296 ± 7 h 15%CPT - I − 3.7 ± 0.3 j3 7.0 (0.29 ± 0.03)

413 ± 5 i 15%CPT - II − 6.5 ± 0.5 k 5.3 (0.28 ± 0.02)

336 ± 4 a1 15%CPT - III − 3.0 ± 0.1 j2 6.0 r (0.38 ± 0.02)

272 ± 5 15%CPT - IV − 1.9 ± 0.2 l2 10.0 s (0.36 ± 0.02)

552 ± 5 15%CPT - V − 0.50 ± 0.03 12.0 (0.48 ± 0.07)

507 ± 4 c 15%CPT - VI − 1.4 ± 0.2 l3 10.0 s (0.55 ± 0.02)

411 ± 4 i 15%CPT - VII − 3.9 ± 0.1 j1 4.0 p (0.62 ± 0.05)

453 ± 3 15%CPT - VI II − 3.7 ± 0.1 j1 13.0 (0.40 ± 0.03)

314 ± 1 e 15%CPT - IX − 2.2 ± 0.1 l2 6.0 r (0.36 ± 0.01) * Samples dispersed in deionized water. ** Samples dispersed in PBS. *** wt % = (weight anticancer drug in NPs/weight NPs) × 100.

TEM imaging revealed dried cel lulose nanoparticles with almost spherical shape and sizes from 60 to 165 nm ( Fig. 5 ). A significant shrinkage of blank cellulose nanoparticles upon drying was observed for all cellulose conjugates, which ranged

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from 37% to 82% ( Table S3 of Supporting info rmation). Similarly, shrinkage of particles during drying as evidenced by smaller particle sizes measured using TEM, SEM or AFM techniques has been described in other amphiphilic cellulose - base d particles and hyaluronic acid nanogels (Mayol et al. , 2014; Q uiñones et al. , 2017, 2018; Song, Zhang, Gan, Zhou, & Zhang, 2011). This result might be associated to the dehydration or water loss during drying that increases hydrophobic interactions in the inner core of the polymer aggregates with the subsequent shrin kage. Additionally, DLS technique estimate the hydrodynamic diameter of particles in a hydrated state measuring the laser radiation scattered from the hydrated or solvation outer layer of particles and aggregates. It is known that DLS overestimates the con tribution of larger particles or aggregates to the mean hydrodynamic diameters (Mayol et al. , 2014). It seems that hydrodynamic particle sizes were overestimated because the contribution of some bigger aggregates of cellulose - testosterone and cellulose - vit amins, which are depicted as small particles and few aggregates using TEM, SEM and AFM. HEC based particles generally showed bigger diameters according to TEM measurements, similarly as estimated in DLS determinations. Scanning electron microscopy confirme d the formation of 80 – 250 nm rounded cellulose particles with smooth surfaces ( Fig. S4 of Supporting information). Additionally, atomic force microscopy revealed cellulose aggregates and particles of 60 – 270 nm, in good agreement with particle sizes estimat ed using TEM ( Fig. S5 of Supporting information).

Fig. 5 . TEM micrographs of blank cellulose nanoparticles at 100,000× magnification: (a) I, (b) II, (c) III, (d) IV, (e) V, (f) VI, (g) VII, (h) VIII and (i) IX (see structures in Fig. 1I ).

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In vitro drug delivery studies In vitro release of testosterone and vitamins from the blank cellulose nanoparticles after hydrolysis of the ester bond was assessed in PBS (pH 7.4) at 37 °C. Almost no release of testosterone and vitamins from the cellulose conjugates was observed for 150 h ( Fig. S6 of Supporting information). This is probably caused by the negligible hydrolysis of the ester bond in these conditions. Approximately 5% of the bonded testosterone and vitamins were released after 150 h, with no significant difference among samples (p > 0.05). In contrast, CPT - loaded cellulose nanoparticles in same conditions (PBS, pH 7.4, 37 °C) showed almost linear release of encapsulated camptothecin for the first 8 h ( Fig. S7 and Table S4 of Supporting information), reaching release plateaus after 6 days. Camptothecin released after 188 h varied between 42% and 93% of the drug encapsulated in the cellulose nanoaggregates ( Fig. 6I – VI ). Interestingly, bigger nanoparticles with lower CPT contents and lower degree of substitution often exhibited faster a nd more quantitative CPT release ( i.e. 5%CPT - III > 5%CPT - I > 5%CPT - II). This effect might be due to weaker hydrophobic interactions between the testosterone and vitamin moieties in the core and the encapsulated CPT. In order to obtain an insight in the inte ractions that affect the CPT release mechanism, the log(Cumulative Release (%)) of CPT on the log(Time) was adjusted to a linear fitting of Eq. (1) , according to the Korsmeyer - Peppas model (Kondaveeti et al. , 2017) ( Fig. S8 and Table S5 of Supporting infor mation). The equilibrium condition was considered achieved at 148 h, due to a negligible change of CPT Cumulative Release (%) after that time. log(Cumulative Release (%)) = nlog(Time(hours) + logk (1)

The slope of the linear fitting (“ n” coefficient), known as diffusional coefficient is associated to the dominant release mechanism. The interaction of CPT with the polymer matrix was also studied based on the shifting of characteristic CPT IR adsorption of C = O at 1737 cm − 1 , observed in th e CPT - loaded cellulose particles ( Table S5 and Fig. S9 of Supporting information). Most of CPT releases appeared to follow an anomalous diffusion mechanism with “n” values of 0.6 to 0.7, except in particles 5%CPT - VIII, 15%CPT - I and 15%CPT - VII that obey a F ickian release mechanism ( Table S5 of Supporting information). However, the interactions between CPT and cellulose matrix (hydrophobic CPT/testosterone and CPT/vitamins interactions combined with polar CPT/cellulose interactions) exerted a shifting of IR  (C = O) adsorption from 1737 cm − 1 in parent CPT to 1740 – 1743 cm − 1 in the CPT - loaded cellulose nanoparticles. Interestingly, testosterone - cellulose particles 5%CPT - I

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Fig. 6 . In vitro release profiles of CPT - loaded cellulose nanoparticles: (I) (a) 5%CPT - I, (b) 5%CPT - II, (c) 5%CPT - III; (II) (a) 5%CPT - IV, (b) 5%CPT - V, (c) 5%CPT - VI; (III) (a) 5%CPT - VII, (b) 5%CPT - VIII, (c) 5%CPT - IX; (IV) (a) 15%CPT - I, (b) 15%CPT - II, (c) 15%CPT - III; (V) (a) 15%CPT - IV, (b) 15%CPT - V, (c) 15%CPT - VI; (VI) (a) 15%CPT - VII, (b) 15%CPT - VIII, (c) 15%CPT - IX in PBS (pH 7. 4) at 37 °C. Data represent the mean ± standard deviation, n = 3 (see structures in Fig. 1I ). and 15%CPT - II, with lower CPT content in each group, exhibited the lowest shifting of IR  (C = O) adsorption (1738 – 1739 cm − 1 ) ( Table S5 of Supporting information). It might be due to the absence of conjugated double bonds or aromatic rings in testosterone, which might facilitate the hydrophobic interactions of CPT with vitamins D2 and E in the other cellulose particles. However, it must be noted that in vitro releas e experiments conducted at pH 7.4 were mostly devoted to show the stability of cellulose nanoaggregates at pH 7.4 and slow release of CPT for the first 2 – 4 h, when approximately 10 – 15% of encapsulated CPT is released. The most promising therapeutic candida tes appear to be 15%CPT - I and 15%CPT - IV nanoaggregates with lowest hydrodynamic sizes of 267 to 303 nm, slightly negative surface charges, slow CPT release during the first 8 h and high CPT content of 7 – 10 wt %.

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In vitro cytotoxicity studies MCF - 7 cells (h uman breast adenocarcinoma cell line) were chosen to evaluate the cytotoxicity of CPT - loaded cellulose nanoaggregates using the XTT assay. A good in vitro anticancer activity of CPT - loaded cellulose nanoparticles (approx. 0.5 relative cell viability for CP T - loaded cellulose nanoparticles at 50 – 100 to 400 – 800 ng mL − 1 of CPT concentration) ( Fig. 7 ) was observed. The resulted cytotoxicity was comparable to that of free camptothecin at similar concentrations ( Fig. S10 of Supporting information). Blank cellulose nanoparticles (NP in “X” axis) appeared to be non - cytotoxic (relative cell viability ca. 0.7 – 1.2) towards MCF - 7 cells at a concentration of 0.1 mg mL − 1 . It must be noted that physical loading of CPT via hydrophobic interactions with the testosterone and v itamins in the core of cellulose nanoaggregates did not affect the CPT cytotoxicity. Furthermore, it appears that most of CPT loaded in nanoaggregates was assimilated for the MCF - 7 cancer cells after 2 days because similar cytotoxicity was observed as comp ared with the parent CPT. Therefore, the antiproliferative effect of CPT - loaded cellulose particles was due to the release of CPT cargo.

Fig. 7 . MCF - 7 relative cell viability assessed by XTT assay for control (C), blank cellulose nanoparticles at 0.1 mg mL − 1 concentration (NP), and CPT - loaded cellulose nanoparticles: (I) (a) 5%CPT - I, (b) 5%CPT - II, (c) 5%CPT - III; (II) (a) 5%CPT - IV, (b) 5%CPT - V, (c) 5%CPT - VI; (III) (a) 5%CPT - VII, (b) 5%CPT - VIII, (c) 5%CPT - IX; (IV) (a) 15%CPT - I, (b) 15%CPT - II, (c) 15%CPT - III; (V) (a) 15%CPT - IV, (b) 15%CPT - V, (c) 15%CPT - VI; (VI) (a) 15%CPT - VII, (b) 15%CPT - VIII, (c) 15%CPT - IX. Data represent the mean ± standard deviatio n, n = 3 (see structures in Fig. 1I ).

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The data reported demonstrated that the prepared cellulose - testosterone and cellulose - vitamin particles might be suitable carriers for in vivo delivery of camptothecin. It is hypothesized that synthesized CPT - loaded n anoaggregates might be directly injected in the tumor site or effectively accumulated in the interstitial space of cancer tissues via passive diffusion and “enhanced permeability and retention” EPR effect (Baetke, Lammers, & Kiessling, 2015; Kato et al. , 2 013; Krukiewicz & Zak, 2016; Prabhakar et al. , 2013). Long circulation times and reduced “reticuloendothelial system” RES clearance of our cellulose particles are expected because they are formed by soft nanogels (no cross - linked celluloses) self - assembled as 60 – 270 nm nanoaggregates and few larger aggregates (Zhang et al. , 2012; Zhao et al. , 2017). The almost neutral surface charge of CPT - loaded cellulose nanoaggregates with zeta potential values between −0.50 to −6.65 mV also contributes to extended circu lation times and reduced RES clearance (Ernsting, Murakami, Roy, & Li, 2013). It is also known that extracellular space of cancer tissues is slightly acidic (pH approx. 6.5) (Baetke et al. , 2015; Kato et al. , 2013). Consequently, CPT - loaded cellulose nanop articles might deliver the anticancer drug via diffusion of CPT from nanoparticle core and partial degradation of the cellulose nanoparticles due to acidic and enzyme catalyzed hydrolysis of the ester linkage bonding testosterone and vitamin to cellulose m atrix. However, in vivo studies are required for evaluation of biodistribution, pharmacokinetics, anticancer effects and toxicity of the prepared CPT - loaded cellulose nanoparticles.

2.2.4. Conclusions Cellulose - testosterone and cellulose - vitamin conjugates were produced in good yields and efficiently loaded with camptothecin for controlled release applications. ATR - FTIR and NMR spectroscopies, differential scanning calorimetry and X - ray diffraction allowed evaluating the impact of the hydrophobic grafting w ith testosterone and vitamins on cellulose ethers properties. The low degree of substitution achieved (from 0.004 to 0.025) explains the minor impact of hydrophobic modification on the thermal properties and crystallinity of celluloses. However, it was suf ficient to promote formation of cellulose nanoaggregates in water. 1 H NMR spectroscopy and DLS measurements confirmed that synthesized cellulose conjugates self - assembled in aqueous medium to form stable and slightly negatively charged aggregates of 213 – 73 1 nm and zeta - potentials of −0.50 to −6.65 mV. After drying, the cellulose aggregates appeared as roughly spherical sole particles and aggregates of 60 – 270 nm, depicted by electron and atomic force microscopy. Encapsulation of the anticancer drug in the

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ce llulose nanoparticles was accomplished with 1.7 – 13.0 wt % of CPT content. The CPT - loaded cellulose nanoparticles generally showed larger hydrodynamic diameters and similar zeta - potential values as compared to the blank nanoparticles. Sustained release of c amptothecin was observed during 188 h in simulated physiological conditions (PBS at pH 7.4, 37 °C). Cytotoxicity of CPT - loaded cellulose nanoparticles against MCF - 7 cancer cell line appeared similar to the observed cytotoxicity of the parent drug. Hence, e ncapsulation of CPT in the cellulose particles, later lyophilization, storage and re - dispersion in aqueous medium seemed to have no effect in the cytotoxic activity of the anticancer drug. CPT - loaded cellulose - testosterone and cellulose - vitamin nanoparticl es are promising candidates for chemotherapy during treatment of tumors.

Acknowledgements The authors thank Erasmus Mundus foundation for a research grant to Javier Pérez Quiñones. We acknowledge Günter Hesser for TEM training and help with TEM imaging of cellulose nanoparticles at ZONA facility of JKU Linz, Linz, Austria. Dr. Pavlo Gordiichuk of Zernike Institute of Advanced Materials at University of Groningen, The Netherlands is acknowledged for AFM measurements. Karolin Römhild at Clinical Applications of Bio - and Nanomaterials, Department of Polymer Chemistry and Bioengineering, Zernike Institute of Advanced Materials at University of Groningen, The Netherlands is thankfully acknowledged for training and help with the biological experiments. The financi al support by the Austrian Federal Ministry for Digital and Economic Affairs and the National Foundation for Research, Technology and Development through the Christian Doppler Laboratory for Combinatorial Oxide Chemistry (COMBOX) is gratefully acknowledged by AWH and CCM.

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SUP P ORTING INFORMATION

Javier Pérez Quiñones, Cezarina Cela Mardare, Achim Walter Hassel, Oliver Brüggemann

Supporting information available online:

https://doi.org/10.1016/ j.carbpol.2018.11.047

https://doi.org/10.1016/j.carbpol.2018.12.085

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3. Silk fibroin - based carriers for delivery of agrochemicals and drugs

This chapter describes the functionalization of a water - soluble silk fibroin hydrolysate via esterification of silk fibroin tyrosine residues with N - hydroxysuccinimide esters of the steroid and vitamin hemisuccinates for controlled release of diosgenin, DI31 and S7, or further encapsulation of camptothecin. The steroid - grafted silk fibroin conjugates formed aggregates in aqueous media, with sustained drug release behaviour. Camptothecin was hydrophobically encapsulated in the core of the testosterone - , tocopherol - and vitamin D2 - grafted cellulose particles, with a camptothecin content of 6.3 - 8.5 wt%. It corresponds to 5 0 - 70 fold increase of camptothecin solubility in water for a 1 mg/mL aqueous dispersion of camptothecin - loaded silk fibroin particles. In vitro agrochemical activity evaluation on radish cotyledons of agrochemical bearing cellulose particles showed superio r plant growth enhancer effect when compared to parent DI31 and S7. Camptothecin - loaded cellulose particles maintained the antiproliferative effect of parent camptothecin on MCF - 7 human breast cancer cells. It was observed good cell uptake of these particl es by MCF - 7 cells. This chapter is based on the following papers:

3.1. Steroid - grafted silk fibroin conjugates for drug and agrochemical delivery.

3.2. Self - assembled silk fibroin - based aggregates for delivery of camptothecin.

My contribution to the papers

I designed and conducted all the experimental work and related characterization, except the AFM, elemental analysis and cell - related assays. I interpreted the results and wrote the manuscripts.

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3.1. Steroid - grafted silk fibroin conjugates for drug and agrochemical delivery

A water - soluble silk fibroin hydrolysate was esterified with diosgenin, DI31 and S7 via esterification of the tyrosine residues to obtain steroid - grafted silk fibroin conjugates ( SF1 - SF3 ) , with structures shown in the graphical a bstract of the publication ( TOC 3 ). The steroid - grafted silk fibroin conjugates formed aggregates in water. A ggregates of smaller particles were depicted using TEM ( TOC 3 ). DI31 - and S7 - grafted cellulose ethers showed good agrochemical activity evaluated o n radish cotyledons test . Radish cotyledons are separated from hypocotyls after 3 days of germination, weighted and treated with the studied compounds ( TOC 3 ). The increase of radish cotyledons weight is measured after another 3 days, with weight increase associated to agrochemical activity ( TOC 3 ).

TOC 3. Structure s of steroid - tyrosine moieties in steroid - grafted silk fibroin conjugates synthesized via esterification of tyrosine, TEM micrograph of a dried steroid - grafted silk fibroin aggregate formed by smaller particles, radish cotyledons used to evaluate the agrochemical activity of DI31 - and S7 - grafted silk fibroin particles. Figure reproduced with permission.

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Steroid - grafted silk fibroin conjugates for drug and agrochemical delivery

Javier Pére z Quiñones , a * Cornelia Roschger , b Andreas Zierer , b Carlos Peniche , c Oliver Brüggemann a

––––––––– a Johannes Kepler University Linz, Institute of Polymer Chemistry, Altenberger Straβe 69, 4040 Linz, Austria. b University Clinic for Cardiac - , Vascular - and Thoracic Surgery, Medical Faculty, Johannes Kepler University Linz, Krankenhausstraβe 7a, 4020 Linz , Austria. c Facultad de Química, Universidad de La Habana, Zapata S/N entre G y Carlitos Aguirre, 10400 La Habana, Cuba.

E - mail: [email protected]

–––––––––

European Polymer Journal 119 ( 2019) 169 – 175

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Abstract

Steroid - grafted silk fibroin (S F) conjugates with a degree of substitution of 1.1 – 4.3 mol% were efficiently synthesised for controlled release of anticancer drug diosgenin and agrochemicals DI31 and S7. The esterification of tyrosine residues in SF conjugates was confirmed using element al analysis, ATR - FTIR and 1 H NMR spectroscopies. The thermal properties of SF conjugates analysed using DSC and TGA confirmed the ATR - FTIR findings on SF conjugates that β - sheet conformation was the predominant one in solid state. DLS revealed the impact o f hydrophobic modification of SF on its hydrodynamic parameters, which resulted in more than doubled hydrodynamic sizes and zeta potential values. SF conjugates were observed as 250 – 600 nm aggregates formed by nanoparticles of 25 – 41 nm, as depicted using T EM and AFM. Controlled release of 40 – 50% grafted steroids from SF conjugates at pH 6.0 was achieved after 5 days. Agrochemical - grafted SF conjugates showed better plant growth enhancing effect than parent DI31 and S7 when evaluated on radish plants. SF con jugates showed low cytotoxicity at concentrations below 0.025 mg/mL when applied on MCF - 7 and HEK - 293 cells. Due to these results, we envisage that the synthesised DI31 - and S7 - grafted SF conjugates are good candidates for agrochemical use. This work also contributes to progress the use of silk fibroin in sustainable agriculture.

KEYWORDS: Silk fibroin; Diosgenin; Agrochemical; Nanoparticles; Controlled release.

3.1.1. Introduction Silk fibroin (SF) materials have been widely used as injectable hydrogels, f ilms, scaffolds, resorbable surgical sutures, particles and other forms for tissue engineering and drug delivery applications because their good biocompatibility and biodegradability [1], [2], [3]. These materials are mostly made of SF filaments obtained f rom the cocoons of Bombyx mori and other silkworms after degumming (removal of sericin), which are formed by a long chain protein of approximately 350 – 370 kDa and two shorter proteins with 25 – 27 kDa [2]. The SF proteins are composed by 17 aminoacids, with glycine, alanine, serine and tyrosine as major components [4], [5]. In this sense, the high content of tyrosine (approximately 5 mol%) makes the esterification of SF an often exploited strategy for functionalisation of SF materials with different drugs [6] . Furthermore, SF materials are generally prepared from reconstituted silk fibroin solution (RSF), which is obtained after a tedious sequential process (degumming the Bombyx mori silk cocoons with aqueous Na 2 CO 3 , aqueous dialysis

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and lyophilisation to obta in pure silk fibroin fibers that are later dissolved in aqueous LiBr and exhaustively dialysed against water to give RSF) [2], [7]. On the other hand, some SF materials for biomedical applications are prepared from water - soluble SF hydrolysates that retain the characteristic biocompatibility and benefits of SF [8]. Additionally, SF has been also used in food industry as adsorbent to extract polyphenols from crude olive leaf extracts and edible coating for perishable food preservation [9], [10]. However, app lication of SF in agriculture and a proper evaluation of its impact on crops are scarce. In this sense, main uses of SF in agriculture are for immobilisation of enzymes for detection of some pesticides or nitrogen mineralisation of SF waste in soils [11], [12]. In this study, a commercial water - soluble hydrolysate of SF (approximately 30 kDa) was esterified with anticancer drug diosgenin and two synthetic analogues of brassinosteroids (DI31 and S7) for their sustained release and agrochemical applications. To the best of our knowledge, this is the first study on SF functionalisation for application in agriculture and preliminary assessment of the plant growth enhancer activity of unmodified SF hydrolysate and SF - brassinosteroid conjugates using radish cotyle don test. Diosgenin, a steroidal sapogenin, is widely used as precursor for the synthesis of several corticosteroids, other hormonal steroids such as progesterone and some synthetic analogues of brassinosteroids [13], [14], [15]. Diosgenin, some of its nat ural saponins (e.g. dioscin) and many of its synthetic ester derivatives have shown good biological activity as antioxidant, anti - inflammatory, anti - cancer, anti - diabetic and hypocholesterolemic. Particularly, glycosylated diosgenin derivatives appear capa ble to reduce cancer of colon and bone, benign prostatic hyperplasia and to inhibit metastasis related to prostate and breast cancers. In this sense, the saccharide moiety bonded to diosgenin plays a key role on observed anticancer activity [16]. We have u sed diosgenin as a model compound of synthetic analogues of brassinosteroids to optimise the synthesis of some polymer - brassinosteroid conjugates and to study the relation among structure, composition and properties [17], [18]. Diosgenin was also used as a model compound of DI31 in this research to optimise the esterification of SF and to investigate the drug release behaviour of similar steroids. DI31 (commercial agrochemical with trademark Biobras16), is a synthetic analogue of brassinosteroid prepared fr om diosgenin in three steps and shows a quite similar structure to the precursor. On the other hand, S7 is another synthetic analogue of brassinosteroid with agrochemical activity as plant growth regulator, which is synthesised from stigmasterol.

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Brassinos teroids, a naturally occurring phytohormone (content of 10 − 1 – 10 − 7 nmol/g), regulate plant growth and development, as well as plant response to biotic and abiotic stress [19], [20]. That is why natural and synthetic analogues of brassinosteroids when used in very low doses (5 – 100 mg/ha) as agrochemicals significan tly raise the efficiency of the crops [21]. When DI31 and S7 are used as agrochemicals at 10 – 30 mg/ha, a 5 – 30% of crops yields increase is observed [22]. However, low aqueous solubility and fast metabolism of DI31 and S7 limits their extensive application in agriculture. To overcome these difficulties, our group has been working in the synthesis of proper drug delivery systems with increased aqueous solubility and good agrochemical activity for delivery of DI31 and S7 [17], [18]. Herein, it is anticipated t hat SF hydrolysate efficiently esterified with diosgenin, DI31 and S7 hemisuccinates might allow obtaining steroid - grafted SF conjugates capable to slowly release diosgenin and the agrochemicals DI31 and S7 with good anticancer and plant growth enhancer ac tivity. The presented methodology is expected to contribute to a sustainable and more efficient use of other agrochemicals or plaguicides, as it will corroborate the potential and benefits of SF as excipient for sustained release of agrochemicals DI31 and S7. To this end, this research is aimed to synthesise and thoroughly characterise steroid - grafted SF conjugates capable to self - aggregate in aqueous medium, for delivery of diosgenin and agrochemicals. Furthermore, plant growth effect of DI31 - and S7 - graft ed SF conjugates will be assessed on radish cotyledons and cytotoxicity of all steroid - grafted SF conjugates will be evaluated on MCF - 7 breast cancer cells and HEK - 293 non cancer cells.

3.1.2. Experimental Materials The silk fibroin hydrolysate (94% purity , number average molecular weight M n approx. 30 000 g/mol) was purchased from Leap Labchem Co., Ltd, Hangzhou, China. SF hydrolysate was dialysed against distilled water for 2 days at 4 °C (2 L, 4 times, 48 h) using cellulose dialysis membrane with 12 – 14 kDa molecular weight cut off (Spectra Por 4, MWCO 12 – 14 kDa) prior to use. Diosgenin ((25 R ) - 5 - spirosten - 3 - β - ol), DI31 ((25 R ) - 3β,5α - dihydroxyspirostan - 6 - one) and S7 ((22 R ,23 R ) - 22,23 - epoxy - 3β,5α - dihydroxystigmastan - 6 - one) were kindly supplied by the Center of Natural Products of the University o f Havana, Cuba. Hemisuccinates of diosgenin, DI31 and S7 were synthesised using base - catalysed esterification in pyridine with succinic anhydride [23]. Steroid hemisuccinate N - hydroxysuccinimide esters required for SF modification were prepared as previous ly reported [17]. For example, diosgenin hemisuccinate (0.107 g,

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0.21 mmol), N,N' - dicyclohexylcarbodiimide (0.055 g, 0.27 mmol) and

N - hydroxysuccinimide (0.034 g, 0.30 mmol) were dissolved in 10 mL of CH 2 Cl 2 and stirred for 24 h at room temperature. The precipitated N , N' - dicyclohexylurea was removed by filtration with a 0.2 μm Nalgene syringe filter and the remaining solvent was evaporated under nitrogen flow. All other reagents and solvents were purchased from Sigma - Aldrich and used without further purification.

Synthesis of steroid - grafted silk fibroin conjugates SF - steroid conjugates were synthesised by esterification of tyrosine residues of SF with activated steroid hemisuccinate N - hydroxysuccinimide esters to avoid cross - link ing of SF and undesired reactions among SF residues [17]. Briefly, purified SF hydrolysate (0.200 g, approximately 0.13 mmol of tyrosine residues) was dissolved in 1 mL of distilled water. Then, 11 mL of N - methyl - 2 - pyrrolidone was added and the mixture was further stirred for 30 min. Later, a mixture of steroid hemisuccinate N - hydroxysuccinimide esters (0.21 mmol) and 4 - (dimethylamino)pyridine (0.020 g, 0.16 mmol) in 9 mL of N - methyl - 2 - pyrrolidone was added to the SF solution. The reaction mixture was stirr ed for 24 h at room temperature. Once finished the reaction, the SF conjugates were purified using dialysis (Spectra Por 3, MWCO 3.5 kDa) against DMSO (400 mL, 1 time, 8 h), a mixture of DMSO/water 50% (800 mL, 1 time, 12 h), 0.1 M NaCl in water (3 L, 1 ti me, 12 h) and distilled water (3 L, 3 times, 36 h). SF conjugates were obtained as white powders after lyophilisation (SF1, SF2 and SF3 conjugates with diosgenin, DI31 and S7 respectively are shown in Fig. 1 ) (additional characterisation data of SF conjuga tes is included in the supplementary material).

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Fig. 1 . Illustration of the synthesis and structures of SF - steroid conjugates SF1 - SF3. (a) Synthesis of steroid hemisuccinate N - hydroxysuccinimide esters; (b) esterification of tyrosine residues of SF.

Characterisation ATR - FTIR spectroscopic characterisation of SF conjugates was conducted on Perkin Elmer Spectrum 100 FT - IR spectrop hotometer equipped with an ATR accessory, with 4 cm − 1 resolution. UV – Vis spectra were acquired on a Perkin Elmer Lambda 25 UV/VIS 1 spectrophotometer. H NMR spectra were recorded at 25 °C in DMSO - d 6 ( δ = 2.51 ppm) [25], using a Bruker Avance 300 spectromete r operated at 300 MHz. Molecular weights of parent SF were estimated using a Viscotek GPCmax gel permeation chromatograph (GPC) equipped using a PFG column from PSS (Mainz, Germany) (300 mm × 8 mm, 5 μm particle size), equipped with a Viscotek TDA 305 Trip le Detector Array (Malvern, Germany). Multidetector calibration (refractive index, right angle light scattering and viscometer) against polystyrene standards from PSS was used. Raw SF sample was eluted with DMF containing 10 mM LiBr at a flow rate of 0.75 mL/min at 60 °C. Dynamic light scattering (DLS) studies were performed on a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, UK) with a detection angle of 173° and measurements done in triplicate at 25 °C. The samples were prepared at 0.5 mg/mL in water or PBS, filtered through a 0.45 μm nylon film syringe filter and a

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folded capillary cell was used for DLS determinations. Differential scanning calorimetry (DSC) studies of SF samples were performed on TA Instruments DSC Q2000, with a sample weight of app roximately 5 mg and a heating - cooling rate of 10 °C/min under nitrogen flow of 20 mL/min. Samples were cooled and heated from 0 °C to 300 °C in closed aluminium pans with holes to allow evaporation of bounded water. TGA Q5000 instrument was used for thermo gravimetric analyses (TGA) of approximately 5 mg SF samples on platinum pans. Heating from 40 °C to 800 °C at a rate of 10 °C/min under a nitrogen flow of 25 mL/min was set up for TGA. Transmission Electron Microscopy (TEM) micrographs were recorded with a Jeol JEM - 2011 FasTEM at 100 kV. A drop of SF conjugates at 1 mg/mL in water was placed on a Pioloform coated copper grid and negatively stained with 1% uranyl acetate aqueous solution before TEM imaging. Atomic Force Microscopy (AFM) images (2 μm × 2 μm) were depicted with MFP 3D - Stand Alone AFM (Asylum Research) with the cantilever OMCL - AC160TSA of Olympus, at a resonant frequency of 300 kHz and spring constant of 26 N/m, 50 – 70% set point and scan rate of 1 Hz. A droplet of SF conjugates at 1 mg/mL in wat er was deposited on a silicon wafer and spins coated at 40 Hz for 6 s.

Drug release studies The in vitro release studies of diosgenin, DI31 and S7 were performed in phosphate buffered saline solution (PBS, pH 6.0) at 25 °C. To this purpose, 2.0 mL of silk fibroin conjugates SF1, SF2 and SF3 (2.5 mg/mL) were placed in dialysis cups (MWCO 3.5 kDa, Slide – A – Lyzer Mini Dialysis Devices, ThermoScientific, USA) and immersed in 40 mL of the release medium at 25 °C and stirred at 100 rpm. The entire release media wa s replaced at every required time point, and analysed using UV spectroscopy

(diosgenin λ em = 280 nm, DI31 and S7 λ em = 300 nm) ( Fig. 1 of supplementary material). Experiments were conducted in triplicate. Calibration curves of steroids ( Fig. 1 of supplemen tary material) allowed quantification of the delivered drugs (diosgenin − 1 − 1 − 1 − 1 − 1 − 1 ε 280 = 688 M cm , DI31 ε 300 = 1693 M cm , S7 ε 300 = 1290 M cm ) ( Figs. 2 – 4 of supplementary material).

Agrochemical activity The agrochemical activity as plant growth enhancer of synthesised SF conjugates carrying the brassinosteroid analogues DI31 and S7 (SF2 and SF3) were evaluated in vitro on radish ( Raphanus sativus ) plants. Increased weight of radish cotyledons was used to evaluate the agrochemical activity. Radish seeds w ere sterilised with sodium hypochlorite solution and germinated on wet filter paper in dark for 3 days at room temperature [24]. Afterwards, hypocotyls were discarded and the cotyledons weighed

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and transferred to Petri dishes with 5 mL of SF2 or SF3 at 10 − 1 – 10 − 7 mg/mL aqueous solutions, or water (control). Cotyledons were allowed to growth for additional 3 days and weighed again. Similarly, agrochemical activity of parent DI31 and S7 was evaluated. To this end, 1 mg/ml stock solutions of DI31 and S7 in etha nol were prepared and proper aqueous dilutions carried out to achieve a final steroid concentration of 10 − 1 – 10 − 7 mg/ml. Experiments were performed in triplicate, with 10 cotyledons used for each sample.

Cell culture and cytotoxicity tests Cytotoxicity of t he samples against the human breast adenocarcinoma cell line MCF - 7 and the non - cancerous human embryonic kidney 293 cell line HEK - 293 was evaluated using the 2,3 - bis(2 - methoxy - 4 - nitro - 5 - sulfophenyl) - 2 H - tetrazolium - 5 - carboxanilide assay (XTT) (Cell Prolifer ation Kit II (XTT), Sigma - Aldrich). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) – high glucose supplemented with 10% fetal bovine serum, 1% penicillin – streptomycin and 1% L - glutamine. The media and supplements except L - glutamine (PAA lab oratories) were purchased from Sigma -

Aldrich. Cells were grown in a humidified atmosphere at 37 °C in 5% CO 2 . For all experiments, 70 – 80% confluent cells were used. Cells (1 × 10 4 cells/well) in complete growth medium were seeded into 96 - well culture plate s. The day after, the medium was changed to a serum free medium with 0.2 mg/ml of parent SF aqueous solution, diosgenin, DI31 and S7 aqueous solutions or SF1, SF2 and SF3 NPs dispersions at different concentrations. Cells were incubated for another 48 h, the medium was changed to full growth medium and 50 μL of XTT reagent was added to each well. Incubation was continued for another 3 h at 37 °C in a humidified chamber with 5%

CO 2 and 100% humidity. The absorbance was measured at 490 nm using a GloMax® Mu ltimode Microplate Reader (Promega). Three independent experiments (with each sample in triplicate) were performed, and the cell viability was normalised to the untreated control. The cell viability curves were analysed with Origin 2015 (Microcal Origin, O riginLab, MA, USA).

3.1.3. Results and discussion Synthesis of SF conjugates Fig. 1 shows the synthetic route followed to prepare the SF - steroid conjugates using esterification of SF tyrosine residues with activated steroid hemisuccinate N - hydroxysuccinimi de esters [17]. This approach is adopted to avoid cross - linking of SF via undesired reactions among SF residues with complementary functions

(i.e. ─ COOH groups Vs. ─ OH and ─ NH 2 groups), which are amenable to reaction if

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direct carbodiimide - mediated esterification is chosen. It must be noted that modification of SF tyrosine residues is a generally adopted strategy for functionalisation of SF [5], [6], because the phenolic ─ OH groups of tyrosine exhibit higher nucleophilicity than other suitable ─ OH groups of SF residues, which together with the abundance of tyrosine in SF (~5 mol%) make it a suitable synthetic target point. Therefore, esterification of SF was highly efficient, with yields between 80 - 93% that correspond to up to 94% of tyrosine residues esterified with the steroid hemisuccinates. It was observed a good agreement of degree of substitution (DS, in mol% expressed as molecules of steroid gra fted on 100 SF chains) calculated from elemental analyses and the values estimated from 1 H NMR ( Table 1 ). The impact of SF functionalisation with the hydrophobic steroids on the structure and properties of SF were assessed using ATR - FTIR and NMR spectrosco pies, DSC and TGA analyses, as well as DLS, TEM and AFM.

Table 1 . Composition, C/N ratio, degree of substitution (DS, mol%) and molecular weight of parent and SF conjugates SF1 - SF3.

1 2 3 4 4 Sample C/N DS DS M n M w /M n mol% mol% kg/mol

SF 2.6049 – – 27.2 1.22

SF1 4.0601 4.7 4.0 – –

SF2 2.9593 1.1 1.3 – –

SF3 4.0225 4.3 4.5 – – 1 C/N ratio determined from elemental analysis. 2 Degree of substitution calculated from the C/N ratio from elemental analysis. 3 Degree of substitution estimated using 1 H NMR. 4 Numbe r average molecular weight and polydispersity measured using GPC against polystyrene standards.

Structural characterisation and morphologies ATR - FTIR showed successful esterification of SF with the steroid hemisuccinates, as characteristic C = O absorption p eaks of ester related steroid hemisuccinate moieties were observed between 1731 and 1735 cm − 1 , while C = O absorption peaks of the new SF - steroid ester bonds appeared at 1698 cm − 1 ( Fig. 2a ). The FTIR spectrum of SF exhibited absorption peaks of characteristi c random coil and β - sheet conformations at 1645 cm − 1 and 1535 cm − 1 , 1625 cm − 1 and 1515 cm − 1 respectively [1], [27]. However, SF1 - SF3 conjugates showed only absorption peaks at 1625 cm − 1 and 1515 cm − 1 (silk II), related to a β - sheet conformation. Therefore, the hydrophobic modification of SF

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fibrils with steroids seems to determine β - sheet conformation to be the predominant structure in the prepared SF conjugates. Similarly, 1 H NMR spectroscopy of SF conjugates in DMSO - d 6 confirmed the SF functionalisation w ith steroid hemisuccinates, as observed in Fig. 2b . The aromatic protons of tyrosine residues in SF observed at 6.7 – 8.2 ppm were used as reference to calculate the DS of SF conjugates SF1 - SF3 based on the characteristic steroid peaks at 0.6 – 1.0 ppm (C H 3 ─ g roups C18, C19, C21 and C27 in diosgenin and DI31; C18, C19, C21, C26, C27 and C28 in S7) and at 5.35 ppm ( = C H ─ group C6 in diosgenin) ( Fig. 2b ; Figs. 5 – 7 of supplementary material). Eqs. ( 1 ), ( 2 ) were used to calculate the DS of SF1 - SF3 conjugates from th eir 1 H NMR spectra. ( ) ( % ) = . ∗ ( ∗ ) ( 1 ) ( ) # . ∗ ∗ ( # # ( % ) = ( 2 ) ∗ ( ) #

w here stands for the integration of the proton NMR signal at 5.35 ppm associated to ( = C H ─ ) proton of d iosgenin in SF1 conjugate ; stands for the integration of the aromatic protons of tyrosine residues at 6.7 – 8.2 ppm in SF1 spectra; SF# stands for

( ) SF1, SF2 or SF3 conjugates; # stands for the integration of C H 3 ─ proton s of steroids in SF conjugates at 0. 6 – 1.0 ppm; number (CH 3 ─ ) groups SF# stands for the number of methyl groups of steroids in SF1 - SF3 conjugates .

Fig. 2 . (a) FTIR spectra of parent SF and SF conjugates; (b) 1 H NMR spectrum of SF1 in DMSO - d 6 ; (c) DSC curves of parent SF and SF1 - SF3 conjugates; (d) TGA thermogram of SF3 with weight loss indicated (see structures in Fig. 1 ).

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The influence of hydrophobic grafting of SF with steroids on its thermal properties is shown in Fig. 2c and d . SF conjugates SF1 - SF3 pre sented an endothermic peak at 69 – 78 °C with related peak enthalpy ( ΔH) of 144 – 154 J/g, with associated weight loss of 2.5 – 3.4% ( Fig. 2d ; Figs. 8 and 9 of supplementary material). It can be attributed to loss of water bound to SF [26]. Similar behaviour was also observed in parent SF that exhibited an endothermic peak at 80 °C, associated enthalpy of 209.4 J/g and weight loss of 7.9% ( Figs. 2c and d ; Fig. 10 of supplementary material). On the other hand, the glass transition of parent SF was observed at 182 °C, in good agreement with reported value of 178 °C for pure non - crystalline SF [26], [27]. This glass transition is due to the transition of amorphous (random coil conformation) in SF [26], which was not observed in the SF conjugates SF1 - SF3 because they adopted mostly β - sheet conformations as evidenced by FTIR. The degradation of SF was observed in parent SF and SF1 - SF3 as intense endothermic peaks at approximately 266 – 280 °C with associated peak enthalpy of 43 J/g and 115 – 135 J/g respectively, observed a s a first step of pyrolysis on the TGA curves with a weight loss of 49 – 62% ( Fig. 2c and d ; Figs. 8 – 10 of supplementary material). The total weight loss of parent SF and SF1 - SF3 conjugates ranged from 97.9 to 98.8% when samples were heated until 800 °C. DLS studies allowed getting an insight on the impact of SF functionalisation with steroids on the size of SF agglomerates and their net surface charge in aqueous medium. As observed in Table 2 , hydrophobic modification of SF with the steroids lead to increase d hydrodynamic sizes and negative zeta potential values of SF aggregates, which almost doubled after steroids grafting on SF chains. Interestingly, SF1 - SF3 aggregates showed similar hydrodynamic diameters and net surface charges in aqueous medium, of appro ximately 510 – 563 nm and −32.1 to −34.3 mV. It is known that aqueous particles with zeta potential values around ±30 mV appear to be more stable in colloidal dispersion [28]. The stability of SF1 - SF3 aggregates in PBS was confirmed with the no significant c hange of their hydrodynamic diameters after 1 month ( Table 2 ).

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Table 2 . Hydrodynamic parameters in aqueous medium (hydrodynamic diameter d h , polydispersity index PDI and zeta potential ζ), average diameters by AFM of dried nanoaggregates of SF and SF1 - SF3.

1 2 3 1 Sample d h d h d h ζ d AFM nm nm nm mV nm (PDI) (PDI) (PDI)

SF 216 ± 7 260 ± 4 262 ± 3 − 14.8 ± 0.8 – (0.5) (0.45) (0.42)

SF1 563 ± 2* 543 ± 2 540 ± 4 − 34.2 ± 0.5* 25 ± 7* (0.37) (0.32) (0.36)

SF2 535 ± 4 559 ± 4 563 ± 6 − 34.3 ± 0.6* 27 ± 1* (0.21) (0.27) (0.30)

SF3 561 ± 3* 510 ± 1 514 ± 5 − 32.1 ± 0.6 41 ± 2 (0.35) (0.33) (0.35) 1 Samples dispersed in distilled water. 2 Samples dispersed in PBS. 3 Samples dispersed in PBS and measured after 1 month. * Stand for no significant difference of means in each colum at a 95% confidence level (p > 0.05).

TEM technique depicted the dried SF1 - SF3 conjugates as rounded or irregular flake - like aggregates of approximately 250 – 600 nm ( Fig. 3a ). Particularly interesting was to observe small rounded particles of a pproximately 40 – 80 nm that formed the 600 nm aggregate in SF3 ( Fig. 3 ). AFM confirmed the formation of small dried nanoparticles with approximately 25 to 41 nm sizes for SF1 - SF3 conjugates ( Table 2 and Fig. 3b ).

Fig. 3 . (a) TEM micrographs of SF1 - SF3 dr ied aggregates at 21,000× magnification; (b) AFM micrographs of SF1 - SF3 dried aggregates (see structures in Fig. 1 ).

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Drug delivery studies Fig. 4 shows the in vitro steroid release profiles of SF1 - SF3 conjugates at 25 °C in PBS (pH 6.0). The release exper iments were performed in acidic medium as a model of SF conjugates degradation suffered in the vegetal vacuoles, where the SF aggregates are kept after cell uptake via endocytosis [29]. The hydrolysis of ester bonds of SF conjugates is negligible at 25 °C in water, but significant ester hydrolysis occurs in acidic or alkaline conditions [30]. The release experiments were carried out until 5 days, when not significant drug release was further detected (plateau trend started after 3 – 5 days). All releases were almost linear during the first 8 h, with adjusted R 2 of 0.99 and most steroids released during this time ( Fig. 4 and Table 1 in supplementary material). It was observed that bigger SF aggregates carrying less steroid showed a faster drug release profile (S F2 > SF1 > SF3), which might be due to a less hydrophobic core of the micelle - like SF aggregates that facilitate the hydrolysis of SF - steroid ester bonds and diffusion of free steroid molecules to the outer release medium [30]. On the other hand, in vivo v acuolar metabolism in plants is enzyme mediated (i.e. esterases, phosphatases, others), with expected faster hydrolysis of SF2 and SF3 conjugates [31]. That is why a proper evaluation of agrochemical activity of synthesised SF conjugates is required.

Fi g. 4 . In vitro release profiles of SF1 - SF3 conjugates in PBS (pH 6.0) at 25 °C (inset shows the linear fittings of release profiles up to 8 h). Data represents the mean ± standard deviation (n = 3) (see structures in Fig. 1 ).

Agrochemical activity Fig. 5 shows the agrochemical activity of SF2 and SF3 conjugates assessed using increased weight of radish cotyledons ( Raphanus sativus ) to detect auxin type activity [32]. Plant growth enhancer effect of SF2 and SF3 conjugates appeared to be significantly superior at concentrations of 1 0 − 1 – 10 − 3 m g/mL, with almost three times

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increased radish cotyledons weight as compared to control (radish cotyledons treated with water). When SF2 and SF3 were applied to radish cotyledons at lower concentrations (10 − 4 – 10 − 7 mg/mL), radish cotyledons weight doubled a s compared to control. It was observed in all cases same stimulatory activity for SF2 and SF3 conjugates when evaluated at same concentrations. Interestingly, SF2 and SF3 conjugates exhibited very good agrochemical activity at 10 − 6 and 10 − 7 mg/mL, with a D I31 and S7 content of 10 − 7 – 10 − 8 mg/mL and 10 − 6.75 – 10 − 7.75 mg/mL respectively. Therefore, SF conjugates showed better agrochemical activity as compared to parent DI31 and S7 at same total concentration (brassinosteroids content of ca. 10 – 20%) ( Fig. 11 of su pplementary material). On the other hand, no stimulatory effect was observed for the raw SF at all concentrations ( Fig. 11 of supplementary material). Therefore, the superior agrochemical effect of SF2 and SF3 observed on radish cotyledons is probably due to the slow release of the DI31 and S7 contained within.

Fig. 5 . Agrochemical activity of SF2 and SF3 conjugates. Data represents the mean ± standard deviation (n = 3) (see structures in Fig. 1 ). Cytotoxicity studies Fig. 6 shows the relative cell viabilities of MCF - 7 and HEK - 293 cells after treatment with SF conjugat es. It must be noted that raw SF itself appeared no toxic to MCF - 7 and HEK - 293 cells at 0.2 mg/mL, with observed relative cell viabilities of (99 ± 5)% and (85 ± 6)%, respectively (data not shown).

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Fig. 6 . (a) Relative cell viability of (a) MCF - 7 breast cancer cells and (b) non - cancer HEK - 293 cells treated with SF conjugates. Data represents the mean ± standard deviation (n = 3) (see structures in Fig. 1 ).

SF1 and SF3 conjugates exhibited moderate anticancer activity towards MCF - 7 at maximal concentrati on of 0.1 mg/mL (relative cell viability of approximately 60% with a steroid content of approximately 0.02 mg/mL), but showed no cytotoxicity to lower concentrations. SF2 exerted a slight anticancer effect at maximal concentration of 0.1 mg/mL (relative ce ll viability of 77%). On the other hand, all SF conjugates seemed almost non - toxic to HEK - 293 cells at 0.05 mg/mL to 0.0015625 mg/mL (relative cell viability over 73% with steroids content from 0.01 mg/mL to 0.0003125 mg/mL). Similarly, DI31 and S7 showed non - toxic to non - cancerous cells HEK - 293 when applied below 0.01 mg/mL (relative cell viability over 90%) ( Fig. 12 in supplementary material). These results demonstrated that SF conjugates carrying brassinosteroids for agrochemical use are safe when used a t recommended concentrations of Biobras16 (DI31) in agriculture (10 − 4 – 10 − 6 mg/mL).

3.1.4. Conclusions Three steroid - grafted SF conjugates were efficiently synthesised via esterification of tyrosine residues for controlled release of diosgenin and agrochemi cals DI31 and S7.

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Functionalisation of SF was confirmed using elemental analysis, FTIR and 1 H NMR spectroscopies. DLS determinations revealed that the esterification of SF with the steroids significantly affected the hydrodynamic parameters of raw SF aggre gates in aqueous medium. Therefore, steroid - grafted SF conjugates formed 510 – 563 nm aggregates in aqueous medium with zeta potential values of −32.1 to −34.3 mV. These steroid - grafted aggregates showed sustained release of diosgenin, DI31 or S7 during 140 h in simulated vacuole conditions (pH 6.0, 25 °C). Finally, DI31 - and S7 - grafted SF conjugates showed better agrochemical activity than parent DI31 and S7 when evaluated on radish plants. The low cytotoxicity at concentrations below 0.025 mg/mL of prepared SF conjugates evidenced on MCF - 7 and HEK - 293 cells, together with a wide biomedical use of SF and application of DI31 in agriculture, demonstrate the potential of brassinosteroid - grafted SF conjugates in agriculture. On the other hand, the similar cytotox icity of diosgenin - grafted SF aggregates to MCF - 7 breast cancer cell and HEK - 293 non - cancer cells, discourage its direct application in biomedicine for chemotherapeutic treatments, and further studies and optimisations are required to this end.

Acknowledge ments Erasmus Mundus is acknowledged for a scholarship to Javier Pérez Quiñones. The authors thank Günter Hesser for training with TEM imaging of nanoparticles and Lisa M. Uiberlacker for AFM imaging of nanoparticles at JKU Linz, Linz, Austria. The access to NMR facilities of Upper Austrian – South Bohemian Research Infrastructure Center in JKU Linz, Linz, Austria, supported by the European Union (ETC Austria - Czech Republic 2007 - 2013, Project M00146) is also acknowledged.

3.1.5. References [1] F. Chen, S. L u, L. Zhu, Z. Tang, Q. Wang, G. Qin, J. Yang, G. Sun, Q. Zhang, Q. Chen, Conductive regenerated silk - fibroin - based hydrogels with integrated high mechanical performances, J. Mater. Chem. B 7 (2019) 1708 - 1715. [2] N. Kasoju, N. Hawkins, O. Pop - Georgievski, D. Kubies, F. Vollrath, Silk fibroin gelation via non - solvent induced phase separation, Biomater. Sci. 4 (2016) 460 - 473. [3] M. B. Elsner, H. M. Herold, S. Müller - Herrmann, H. Bargel, T. Scheibel, Enhanced cellular uptake of engineered spider silk particle s, Biomater. Sci. 3 (2015) 543 - 551. [4] A. R. Murphy, D. L. Kaplan, Biomedical applications of chemically - modified silk fibroin, J. Mater. Chem. 19 (2009) 6443 - 6450. [5] Y. Gotoh, M. Tsukada, N. Minoura, Chemical modification of silk fibroin with cyanuric chloride - activated poly(ethylene glycol): Analyses of reaction site by proton NMR spectroscopy and conformation of the conjugates, Bioconj. Chem. 4 (1993) 554 - 559. [6] Y. Gotoh, S. Niimi, T. Hayakawa, T. Miyashita, Preparation of lactose - silkfibroin conjug ates and their application as a scaffold for hepatocyte attachment, Biomaterials 25 (2004) 1131 - 1140.

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[7] Y. Liu, Z. Zheng, H. Gong, M. Liu, S. Guo, G. Li, X. Wang, D.L. Kaplan, DNA preservation in silk, Biomater. Sci. 5 (2017) 1279 - 1292. [8] J. - H. Yeo, K. - G. Lee, H. - Y. Kweon, S. - O. Woo, S. - M. Han, S. - S. Kim, M. Demura, Fractionation of a silk fibroin hydrolysate and its protective function of hydrogen peroxide toxicity, J. Appl. Polym. Sci. 102 (2006) 772 - 776. [9] E. Altiok, D. Baycin, O. Bayraktar, S. Ülk ü, Isolation of polyphenols from the extracts of olive leaves ( Olea europeae L.) by adsorption on silk fibroin, Sep. Purif. Technol. 62 (2008) 342 - 348. [10] B. Marelli, M.A. Brenckle, D.L. Kaplan, F.G. Omenetto, Silk fibroin as edible coating for perishab le Food Preservation, Sci. Rep. (2016) 25263. [11] R. Xue, T. - F. Kang, L. - P. Lu, S. - Y. Cheng, Immobilization of acetylcholinesterase via biocompatible interface of silk fibroin for detection of organophosphate and carbamate pesticides, Appl. Surf. Sci. 16 (2012) 6040 - 6045. [12] A. Murase, K. Yonebayashi, Nitrogen mineralization of silk waste applied to soil under aerobic conditions, Soil Sci. Plant Nutr. 47 (2001) 233 - 240. [13] I. Herráiz, Chemical pathways of corticosteroids, industrial synthesis from sapo genins, J. - L. Barredo, I. Herráiz (Eds.), Microbial Steroids: Methods and Protocols, Methods in Molecular Biology, Springer Science+Business Media LLC (2017) 15 - 27. [14] Q. Xu, X. Peng, W. Tian, A new strategy for synthesizing the steroids with side chains from steroidal sapogenins: synthesis of the aglycone of OSW - 1 by using the intact skeleton of diosgenin, Tetrahedr. Lett. 44 (2003) 9375 - 9377. [15] M. Jesus, A. P. J. Martins, E. Gallardo, S. Silvestre, Diosgenin: recent highlights on pharmacology and ana lytical methodology, J. Anal. Meth. Chem. (2016) 4156293. [16] A. Sethi, P. Singh, N. Yadav, P. Yadav, M. Banerjee, R. P. Singh, Greener approach for synthesis of novel steroidal prodrugs using ionic liquid, their DFT study and apoptosis activity in prosta te cancer cell line, J. Mol. Struct. 1180 (2019) 733 - 740. [17] J. P. Quiñones, O. Brüggemann, C. P. Covas, D. A. Ossipov, Self - assembled hyaluronic acid nanoparticles for controlled release of agrochemicals and diosgenin, Ca rbohydr. Polym. 173 (2017) 157 - 1 69. [18] J. P. Quiñones, C. C. Mardare, A. W. Hassel, O. Brüggemann, Self - assembled cellulose particles for agrochemical applications, Eur. Polym. J. 93 (2017) 706 - 716. [19] R.N. Furio, P.L. Albornoz, Y. Coll, G.M. Martínez ─Zamora, S.M. Salazar, G.G. Marto s, J.C. Díaz Ricci, Effect of natural and synthetic Brassinosteroids on strawberry immune response against Colletotrichum acutatum, Eur. J . Plant Pathol. 153 (2019) 167 - 181. [20] S.D. Clouse, Brassinosteroid signal transduction and action, P.J. Davies (Ed.) , In Plant Hormones, Springer (2010), pp. 427 - 450. [21] D. Holá, O. Rothová, M. Kocová, L. Kohout, M. Kvasnica, The effect of brassinosteroids on the morphology, development and yield of field - grown maize, Plant Growth Regul. 61 (2010) 29 - 43. [22] M. Serna , F. Hernández, F. Coll, A. Amorós , Brassinosteroid analogues effect on yield and quality parameters of field - grown lettuce ( Lactuca sativa L.), Sci. Hortic. 143 (2012) 29 - 37. [23] T. Abe, K. Hasunuma, M. Kurokawa, Vitamin E orotate and a method of produci ng the same, US: 3944550, 1976. [24] E. Alonso - Becerra, Y. Bernardo - Otero, F. Coll - Manchado, F. Guerra - Martínez, G.Martínez - Massanet, C. Pérez - Martínez, Synthesis and biological activity of epoxy analogues of 3 - dehydroteasterone, J. Chem. Res. 5 (2007) 268 - 271.

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SUPPORTING INFORMATION

Javier Pérez Qui ñones, Cornelia Roschger, Andreas Zierer, Carlos Peniche, Oliver Brüggemann

Supporting information available online:

https://doi.org/10.1016/j.eurpolymj.2019.07.025

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3.2. Self - assembled silk fibroin - based aggregat es for delivery of camptothecin

A w ater - soluble silk fibroin hydrolysate was esterified with testosterone, tocopherol and ergocalciferol via esterification of the tyrosine residues to obtain silk fibroin conjugates ( SF1 - SF3 ), with structures shown in the graphical abstract of the publicatio n ( TOC 4 ). The testosterone - and vitamin - grafted silk fibroin conjugates formed aggregates in water . Dried silk fibroin particles and aggregates were observed using AFM ( TOC 4 ). Almost quantitative and controlled release of camptothecin (CPT) was observed from CPT - loaded silk fibroin particles after 6 days. MCF - 7 human breast cancer cells showed good uptake of the CPT - loaded silk fibroin particles after 6 hours ( TOC 4 ), as evidenced with the strong blue fluorescence of the CPT - loaded particles inside the ce lls.

TOC 4. Structure s of testosterone - and vitamin - tyrosine moieties in functionalized silk fibroin prepared via esterification of tyrosine ( SF1 - SF3 ), AFM micrograph of dried ergocalciferol - grafted silk fibroin particles, sustained CPT release of CPT - loaded silk fibroin conjugates, MCF - 7 cell uptake of CPT - loaded silk fibroin particles with characteristic blue fluorescence of CPT visible. Figure reproduced with permission.

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Self - assembled silk fibroin - based aggregates for delivery of camptothec in

Javier Pérez Quiñones , a * Cornelia Roschger , b Andreas Zierer , b Carlos Peniche - Covas , c Oliver Brüggemann a

––––––––– a Institute of Polymer Chemistry (ICP), Johannes Kepler University Linz, Altenberger Str. 69, 4040 Linz, Austria. b Johannes Kepler Unive rsity Linz, Kepler Universit y Hospital GmbH, Department for Cardiac - , Vascular - and Thoracic Surgery, Altenberger Str. 69, 4040 Linz and Krankenhausstraβe 7a, 4020 Linz, Austria. c Facultad de Química, Universidad de La Habana, Zapata S/N entre G y Carlitos Aguirre, 10400 La Habana, Cuba.

E - mail: [email protected]

–––––––––

Submitted

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Abstract

A water - soluble hydrolysate of silk fibroin (SF) (~30 kDa) was esterified with tocopherol, ergocalciferol and testosterone to form SF aggregates for the controlled delivery of the a nticancer drug camptothecin (CPT). Grafting of vitamins and testosterone was evaluated by elemental analysis and 1 H NMR spectroscopy showing that the degree of substitution (DS) achieved was between 0.4 to 3.8 mol%. The yield of ergocalciferol - grafted SF, tocopherol - grafted - SF and testosterone - grafted SF were 71, 64 and 58%, respectively. CPT was efficiently incorporated into the lipophilic core of SF aggregates using a dialysis - precipitation method, achieving drug contents of 6.3 - 8.5 wt. %. The chemical st ructure, thermal and aggregates properties were assessed using ATR - FTIR, DS C, TGA, dynamic light scattering and TEM. FTIR spectra and DSC thermograms showed that tocopherol - and testosterone - grafted SF conjugates predominantly adopted a β - sheet conformation. After esterification of tyrosine residues on SF chains with the vitamins or testosterone the hydrodynamic diameters almost doubled or triplicated that of SF. The zeta potential values after esterification increased to about - 30 mV in the three cases, which favours the stability of aggregates in aqueous medium. Controlled and a lmost quantitative release of CPT was achieved after 6 days in PBS at 37 ºC, with almost linear release during the first 8 hours. CPT antiproliferative effect on MCF - 7 cancer cells seems unaltered after encapsulation in the SF aggregates . MCF - 7 cancer cell s exhibited good uptake of CPT - loaded SF aggregates after 6 hours. It is concluded that CPT - loaded SF aggregates are promising candidates for antitumor therapy.

3.2.1. Introduction Cancer is expected to rank as the leading cause of death worldwide in this century, with estimated 18.1 million new cancer diagnosed and 9.6 million cancer deaths in 2018. 1,2 Lung cancer is the most diagnosed and lethal cancer with 11.6% and 18.4% of the total cases and cancer - related deceases, followed by breast and prostate can cer, as well as colorectal cancer in terms of incidence and mortality. 1 Conventional chemotherapy is still widely used in medical treatment of most cancers and tumours usually in combination with surgery, radiotherapy and novel nanomedicines (i.e. genetica lly designed and personalized drugs, prodrugs and multiresponsive drug delivery systems, thermal phototherapy among others). 2 - 4 Among different anticancer drugs, irinotecan and topotecan, two camptothecin derivatives, are used to treat lung cancer, colon c ancer, metastatic or resistant breast cancer and ovarian cancer, while a liposome formulation of irinotecan (onivyde) was recently approved by FDA and EU

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regulatory agencies for treatment of metastatic pancreatic cancer. 5 - 8 The severe side effects of campt othecins, together with the short serum half - life of camptothecin lactones and to be the only marketed topoisomerase I inhibitors, 9,10 motivate intense research on preparation of different camptothecin analogues and novel drug delivery systems. 11,12 Howeve r, few camptothecin delivery systems are commercially available and approved for antitumor therapy in addition to onivyde. 13 In search of new nanomedicines also capable to tumour - targeted drug delivery, 14 we worked on camptothecin encapsulation in tocopher ol - , ergocalciferol - and testosterone - modified hyaluronic acid and cellulose nanogels (2 - 13 wt. % of camptothecin), with sustained camptothecin release and good cytotoxic activity on MCF - 7 cancer cells. 15,16 Tocopherol (vitamin E), ergocalciferol (vitamin D2) and testosterone were chosen as biocompatible modifiers of hydrophilic polymers for anticancer drug delivery because their antioxidant, cardiovascular - protective and anticancer effects. 17,18 These findings encourage us to synthesise tocopherol - , ergoca lciferol - and testosterone - grafted nanocarriers based on biocompatible silk fibroin for hydrophobic encapsulation of camptothecin and later controlled delivery, with good cytotoxicity against cancer cells and decreased side effects. The rational of using s ilk fibroin (SF) as polymer carrier of camptothecin was the exhaustive use of SF materials in diverse biomedical applications, 19,20 as well as straightforward SF functionalisation via tyrosine esterification . 21 SF, formed by a protein of 350 - 370 kDa and tw o shorter proteins of 25 - 27 kDa, is obtained after degumming of the Bombyx mori cocoons. 20 SF protein is composed by 17 aminoacids, with glycine, alanine, serine and tyrosine as major components. 22 On the other hand, marked biocompatible SF hydrolysates ar e sometimes preferred for medical use. 23 In this research, a water - soluble hydrolysate of SF ( ~ 30 kDa) was esterified with tocopherol, ergocalciferol and testosterone to form SF aggregates, which efficiently encapsulated and delivered camptothecin. The c hemical structure, thermal and aggregates properties, together with camptothecin controlled release behaviour and cytotoxicity of blank and camptothecin - loaded SF aggregates were assessed. It must be noted the high camptothecin content found in camptotheci n - loaded testosterone - grafted SF aggregates in spite of the significantly lowest degree of substitution of SF with testosterone, as well as the significant shrinkage of SF aggregates in aqueous medium once camptothecin was encapsulated in the hydrophobic c ore. Camptothecin antiproliferative effect on MCF - 7 cancer cells remained unaltered after camptothecin loading in the SF aggregates. Good uptake of camptothecin - loaded SF aggregates was

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observed on MCF - 7 cancer cells after 6 hours. To the best of our knowl edge, this is the first attempt to prepare camptothecin - loaded SF carriers for controlled delivery and anticancer applications.

3.2.2. Materials and methods Materials

The silk fibroin hydrolysate (98% purity, number average molecular weight M n approx. 30 0 00 g/mol) was purchased from Leap Labchem Co., Ltd, Hangzhou, China. It was further purified using dialysis membranes (Spectra/Por 4 cellulose dialysis membranes, molecular weight cut off (MWCO) 12 - 14 kDa, Spectrum Laboratories Inc., CA, USA) for 2 days at 4 o C (2L, 4 times, 48 hours), to obtain SF as a white cotton wool like solid after lyophilisation. ( S ) - (+) - Camptothecin (CPT) was purchased from Alfa Aesar. Testosterone, DL - α - tocopherol, ergocalciferol, chemicals and solvents were purchased from Sigma - Al drich and used as received without further purification. Testosterone hemisuccinate, tocopherol hemisuccinate and ergocalciferol hemisuccinate were synthesised using base - catalysed esterification in pyridine with succinic anhydride. 24 Testosterone and vita min hemisuccinate N - hydroxysuccinimide esters needed for SF esterification were synthesised as already reported. 25 To illustrate, ergocalciferol hemisuccinate (0.045 g, 0.09 mmol), N,N’ - dicyclohexylcarbodiimide (0.028 g, 0.14 mmol) and N - hydroxysuccinimide (0.017 g, 0.15 mmol) were dissolved in 10 mL of

CH 2 Cl 2 and stirred for 24 hours at room temperature. The precipitated N,N’ - dicyclohexylurea was filtered out with a 0.2 µm Nalgene syringe filter and the CH 2 Cl 2 was evaporated under nitrogen flow, to obtain the ergocalciferol hemisuccinate N - hydroxysuccinimide ester. Spectra/Por 3 cellulose dialysis membranes (Spectrum Laboratories Inc., CA, USA) with MWCO of 3.5 kDa were used for purification of the synthesised ergocalciferol - , tocopherol - and testosterone - g rafted SF conjugates.

Preparation of ergocalciferol - , tocopherol - and testosterone - grafted SF aggregates SF conjugates were prepared via esterification of tyrosine residues of SF with testosterone and vitamin hemisuccinate N - hydroxysuccinimide esters to pr event undesired side reactions among SF residues (i.e. cross - linking of SF) . The procedure is briefly described with ergocalciferol hemisuccinate N - hydroxysuccinimide ester : SF hydrolysate (0.100 g, approximately 0.07 mmol of tyrosine residues) was dissolv ed in 1 mL of distilled water. Then, 11 mL of N - methyl - 2 - pyrrolidone was added and the mixture was further stirred for 30 min. Subsequently, ergocalciferol hemisuccinate N - hydroxysuccinimide ester (0.09 mmol) and 4 - (dimethylamino)pyridine (0.010 g, 0.08

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mm ol) in 9 mL of N - methyl - 2 - pyrrolidone was added to the SF solution. The reaction mixture was stirred for 24 hours at room temperature. After that, the SF conjugates were dialysed against DMSO (400 mL, 1 time, 8 hours), a mixture of DMSO/water 1:1 (800 mL, 1 time, 12 hours), 0.1 M NaCl in water (3 L, 1 time, 12 hours) and distilled water (3 L, 3 times, 36 hours). Ergocalciferol - grafted SF ( SF1 ) was obtained as a brown powder, while tocopherol - grafted - SF ( SF2 ) and testosterone - grafted SF ( SF3 ) appeared as whi te powders after lyophilisation (characterisation data of SF1, SF2 and SF3 is included in the ESI † ). These SF conjugates formed aggregates of particles in aqueous medium when stirred overnight at 1 mg mL - 1 in water or phosphate buffer saline solution (PBS, pH 7.4).

CPT loading in SF aggregates Hydrophobic CPT was incorporated in the lipophilic core of ergocalciferol - , tocopherol - and testosterone - grafted SF aggregates using a dialysis - precipitation method, with lyophilisation. 16,26 To this end, 10 mg of SF 1 , SF2 or SF3 and 1 mg of CPT dissolved in 8 mL of DMSO were stirred overnight at room temperature in darkness. The formation of CPT - loaded SF aggregates and removal of CPT excess was carried out by dialysis against distilled water (2 L, 1 time, 5 hours). CPT - SF1 (slightly brown), CPT - SF2 and CPT - SF3 (white powders) were obtained after lyophilisation.

CPT content and drug release studies The content of CPT in CPT - loaded SF aggregates and related parameters (loading efficiency and yield of aggregates), as well as in vitro released CPT during drug release studies were determined by UV spectrophotometry, based on calibration curves of † - 1 - 1 camptothecin in DMSO and PBS (pH 7.4) (ES I ) (CPT = 21006 M cm , - 1 - 1 = 26930 M cm ). In vitro release studies of CPT were performed in phosphate buffered saline solution (PBS, pH 7.4) at 37 o C. To this end, 2.0 mL of CPT - loaded silk fibroin conjugates CPT - SF1 , CPT - SF2 and CPT - SF3 (2.5 mg mL - 1 ) in PBS at pH 7.4 were placed in dialysis cups (MWCO 3.5 kDa, Slide – A – Lyzer Mini Dialysis Devices, ThermoScientific, USA) and immersed in 10 mL of the release medium (PBS, pH 7.4) at 37 o C and stirred at 100 rpm. The enti re release media was replaced at every required time point, and analysed using UV spectroscopy. (CPT  emission = 370 nm).

Cytotoxicity tests Cytotoxicity of the samples against the human breast adenocarcinoma cell line MCF - 7 was evaluated using the 2,3 - bis (2 - methoxy - 4 - nitro - 5 - sulfophenyl) - 2 H - tetrazolium - 5 -

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carboxanilide assay (XTT) (Cell Proliferation Kit II (XTT), Sigma - Aldrich). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) – high glucose supplemented with 10% fetal bovine serum, 1% penici llin – streptomycin and 1% L - glutamine. The media and supplements except L - glutamine (PAA laboratories) were purchased from

Sigma - Aldrich. Cells were grown in a humidified atmosphere at 37 °C in 5% CO 2 . In all experiments, 70 - 80% confluent cells were used. C ells (1 × 10 4 cells/well) in complete growth medium were seeded into 96 - well culture plates. The day after, the medium was changed to a serum free medium with blank and CPT - loaded SF aggregates dispersions at different concentrations. Cells were incubated for another 48 hours, the medium was changed to full growth medium and 50 μL of XTT reagent was added to each well. Incubation was continued for another 3 hours at 37 °C in a humidified chamber with 5% CO 2 and 100% humidity. The absorbance was measured at 490 nm using a GloMax® Multimode Microplate Reader (Promega). Three independent experiments (with each sample in triplicate) were performed, and the cell viability was normalised to the untreated control. The cell viability curves were analysed with Origin 2015 (Microcal Origin, OriginLab, MA, USA).

Cell uptake MCF - 7 cancer cells were culture as described for cytotoxicity tests, but once medium was changed to a serum free medium (control) or CPT - loaded SF aggregates dispersions at 0.1 mg mL - 1 , cells were in cubated for 4 hours at 37 o C in a humidified chamber with 5% CO 2 and 100% humidity. Then, 50 nM of LysoTracker Yellow HCK - 123 (Invitrogen) were added and incubation continued for another 2 hours. Fluorescence imaging was performed on MCF - 7 cancer cells usi ng an Olympus IX73 inverted microscope with DAPI channel for the CPT - loaded SF aggregates (  excitation =

345 nm,  emission = 455 nm) and FITC channel for the LysoTracker Yellow HCK - 123

(  excitation = 494 nm,  emission = 518 nm).

Characterisation Molecula r weights of parent SF were determined using a Viscotek GPCmax gel permeation chromatograph (GPC) equipped using a PFG column from PSS (Mainz, Germany) (300 mm × 8 mm, 5 µm particle size), equipped with a Viscotek TDA 305 Triple Detector Array (Malvern, Ge rmany). Multidetector calibration (refractive index, right angle light scattering and viscometer) was performed using polystyrene standards from PSS. Raw SF sample was eluted with DMF containing 10 mM LiBr at a flow rate of 0.75 mL min - 1 at 60 o C. ATR - FTIR spectroscopic characterisation of SF conjugates was conducted on Perkin Elmer Spectrum 100 FT - IR spectrophotometer equipped with

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an ATR accessory, with 4 cm - 1 resolution. Differential scanning calorimetry (DSC) profiles of SF samples were obtained using a TA Instruments DSC Q2000, with approximately 5 mg samples and a heating - cooling rate of 10 o C min - 1 under nitrogen flow of 20 mL min - 1 . Samples were placed in closed aluminium pans with holes to allow evaporation of bounded water and were cooled and heate d from 0 o C to 300 o C. TGA Q5000 instrument was used for thermogravimetric analyses (TGA) of approximately 5 mg SF samples on platinum pans. Heating from 40 o C to 800 o C at a rate of 10 o C min - 1 under a nitrogen flow of 25 mL min - 1 was set up for TGA. 1 H N MR spectra were o 27 recorded at 25 C in DMSO - d 6 (  = 2.51 ppm), using a Bruker Avance 300 spectrometer operated at 300 MHz. UV - Vis spectra were acquired on a Perkin Elmer Lambda 25 UV/VIS spectrophotometer. Dynamic light scattering (DLS) studies were perfor med on a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, UK) with a detection angle of 173 o and measurements done in triplicate at 25 o C. The samples were prepared at 0.5 mg mL - 1 in water or PBS, filtered through a 0.45 µm nylon film syringe filter and a folded capillary cell was used for DLS determinations. Transmission Electron Microscopy (TEM) micrographs were recorded with a Jeol JEM - 2011 FasTEM at 100 kV. A drop of SF conjugates at 1 mg mL - 1 in water was placed on a Pioloform coated copper grid and negatively stained with 1% uranyl acetate aqueous solution before TEM imaging. Atomic Force Microscopy (AFM) images (2 µm × 2 µm) were depicted with MFP 3D - Stand Alone AFM (Asylum Research) with the cantilever OMCL - AC160TSA of Olympus, at a resonant frequ ency of 300 kHz and spring constant of 26 N m - 1 , 50 - 70% set point and scan rate of 1 Hz. A 70 µl droplet of SF conjugates at 1 mg mL - 1 in water was deposited on a silicon wafer and spins coated at 40 Hz for 6 s.

3.2.3. Results and discussion Synthesis and characterisation of SF conjugates Fig. 1 shows the structures of vitamins - and testosterone - grafted SF conjugates synthesised by esterification of SF tyrosine residues with vitamins and testosterone hemisuccinate N - hydroxysuccinimide esters. Tyrosine resid ues of SF were chosen as target point for the synthesis of SF conjugates because the higher nucleophilicity of its – OH groups and the high tyrosine content in SF (approx. 5 mol%). 22,28 Esterification of SF was moderately efficient, with yields of 58 - 71% th at correspond to 70 - 80% and 9% of tyrosine residues esterified with the vitamin hemisuccinates and testosterone hemisuccinate, respectively ( Table 1 ). Testosterone hemisuccinate was much less reactive than vitamin hemisuccinates in the esterification of SF , as previously observed in esterification of cellulose ethers. 16 DS values estimated from 1 H NMR for SF1 and

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SF2 were similar to the values calculated from elemental analyses. The effect of vitamins and testosterone hydrophobic functionalisation of SF on the SF structure and properties was studied using ATR - FTIR and NMR spectroscopies, DSC and TGA analyses, DLS, TEM and AFM.

Fig. 1 Synthesis and structures of vitamins and testosterone - SF conjugates SF1 - SF3 . (a) Synthesis of vitamin and testosterone hemi succinate N - hydroxysuccinimide esters; (b) esterification of tyrosine residues of SF.

Table 1 Composition, C/N ratio, degree of substitution (DS %), yield (Y %) and molecular weight of parent and SF conjugates SF1 - SF3 .

a b c d d Sample C/N DS DS Y M n M w /M n % % % kg/m ol SF 2.6049 - - 27.2 1.22 SF1 3.6218 3.2 3.0 71 - - SF2 3.8355 3.8 3.6 64 - - SF3 2.7061 0.4 - 58 - - a C/N ratio determined by elemental analysis. b Degree of substitution calculated from the C/N ratio from elemental analysis. c Degree of su bstitution estimated from the 1 H NMR. d Number average molecular weight and polydispersity measured by GPC against polystyrene standards.

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Structural characterisation and morphologies ATR - FTIR confirmed esterification of SF with the vitamins and testosteron e hemisuccinates, as C=O absorption peaks of ester associated to vitamins and testosterone hemisuccinate moieties were observed between 1732 - 1739 cm - 1 , while C=O absorption peaks of the new vitamin - and testosterone - grafted SF ester bonds showed at 1698 cm - 1 ( Fig. 2a ).

Fig. 2 (a) FTIR spectra of parent SF and SF conjugates; (b) DSC curves of parent SF and SF1 - SF3 conjugates; (c) TGA thermogram of SF1 with weight loss indicated; (d) 1 H NMR spectrum of SF1 in DMSO - d 6 .

The FTIR spectrum of SF and SF1 exhibited absorption peaks of characteristic random coil, β - turns and β - sheet conformations at 1645 cm - 1 and 1535 cm - 1 (silk I), 1625 cm - 1 and 1515 cm - 1 (silk II) respectively. 19 However, SF2 and SF3 conjugates showed only absorption peaks at 1625 cm - 1 an d 1515 cm - 1 (silk II), related to a β - sheet conformation. Esterification of SF fibrils with tocopherol or testosterone seems to determine β - sheet conformation to be the predominant structure in the prepared SF2 and SF3 conjugates. The effect of vitamins an d testosterone grafting of SF on its thermal properties is shown in Fig. 2b and 2c . SF1 - SF3 presented an endothermic peak at 66 - 71 o C with related peak enthalpy (   H) of 149 - 213 J g - 1 , with associated

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weight loss of 2.4 - 4.2 % ( Fig. 2b , 2c , Fig. SI - 4 and Fi g. SI - 5 in the ESI † ). It might be attributed to loss of water bound to SF. 29 Parent SF showed an endothermic peak at 80 o C, related enthalpy of 209.4 J g - 1 and weight loss of 7.9% ( Fig. 2b and Fig. SI - 6 in the ESI † ). The glass transition of parent SF was o bserved at 182 o C, near to reported value of 178 o C for non - crystalline SF. 29,30 This glass transition is due to the transition of amorphous random coil and β - turns conformations in SF (silk I), 29 which was not observed in the SF1 - SF3 . The degradation of SF was observed in parent SF and SF1 - SF3 as intense endothermic peaks at approx. 266 - 280 o C with related peak enthalpies of 43 J g - 1 and 100 - 133 J g - 1 respectively ( Fig. 2b ). The total pyrolysis of parent SF and SF1 - SF3 conjugates provoked a major weig ht loss of 91 - 96% ( Fig. 2c and Fig. SI - 4 to SI - 6 in the ESI † ). 1 H NMR spectroscopy of SF conjugates in DMSO - d 6 also confirmed the SF functionalisation with vitamins and testosterone hemisuccinates, as observed in Fig. 2d . The aromatic protons of tyrosine r esidues in SF observed at 6.7 - 8.2 ppm were used as reference to calculate the DS of SF conjugates SF1 and SF2 based on the characteristic vitamin peaks at 0.82 - 1.02 ppm (C H 3 - groups H18, H21, H26, H27 and

H28 in ergocalciferol; 4’C H 3 - , 8’C H 3 - and 12’C H 3 - i n tocopherol) and at 5.27 - 5.35 ppm (=C H - C H = group H6 and H7 in ergocalciferol) ( Fig. 2d , Fig. SI - 7 and Fig. SI - 8 in the ESI † ). The 1 H NMR spectra of SF3 also showed the characteristic testosterone peaks at 0.84 - 0.86 ppm (C H 3 - groups H18 and H19 in testoste rone) and at 5.34 ppm (O=CC - C H = group H4 in testosterone ) ( Fig. SI - 9 in the ESI † ). However, the high noise to signal ratio in the 1 H NMR spectra of SF3 resulted in underestimated integration of tyrosine aromatic protons and a proper DS estimation was not s uitable. DLS studies revealed the impact of SF esterification with vitamins and testosterone on the size of SF agglomerates and their net surface charge in aqueous dispersion. Table 2 shows that esterification of SF with the vitamins or testosterone lead to increased hydrodynamic sizes and negative zeta potential values of SF aggregates, which almost doubled or triplicated hydrodynamic diameters and doubled zeta potential values after esterification of tyrosine residues on SF chains.

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Table 2 Hydrodyna mic parameters in aqueous medium, average diameters by AFM of dried nanoaggregates of SF and SF1 - SF3 ; CPT contents in weight (wt. %) of CPT - loaded SF aggregates.

a b a Sample d h d h   d AFM wt. % nm nm mV nm (PDI) (PDI)  SF 216 ± 7 260 ± 4 - 14.8 ± 0.8 - - (0.5) (0.45) SF1 554 ± 3 471 ± 7 - 32.2 ± 0.4 75 ± 10 - (0.32) (0.37) SF2 653 ± 1 727 ± 4 - 28.3 ± 0.5 53 ± 9 - (0.45) (0.15) SF3 620 ± 3 824 ± 6 - 30.8 ± 0.3 45 ± 7 - (0.31) (0.28) CPT - SF1 - 420 ± 5 - - 8.3 (0.5) CPT - SF2 - 370 ± 7 - - 6.3 (0.51) CPT - SF3 - 340 ± 5 - - 6.8 (0.34) a Samples dispersed in distilled water. b Samples dispersed in PBS.

Interestingly, SF1 - SF3 aggregates showed hydrodynamic diameters and net surface charges in water, of 5 54 to 653 nm and - 28.3 to - 32.2 mV. It seems that intermolecular hydrophobic interaction of vitamins or testosterone groups grafted on SF chains caused stabilization of the SF aggregates in aqueous medium. It is known that aqueous particles with zeta poten tial values around ± 30 mV seems to be more stable in colloidal dispersion. 31 In general, the hydrodynamic sizes increased in PBS for raw SF and SF aggregates when compared to same aggregates dispersed in distilled water, except for SF1 aggregates. The sta bility of SF1 - SF3 aggregates in PBS was confirmed with the non - significant change of their hydrodynamic sizes after 1 month (data not shown). On the other hand, the hydrophobic loading of camptothecin in the inner core of SF aggregates resulted in signific ant hydrodynamic sizes reduction of 11% to 59%, probably due to stronger hydrophobic interactions of CPT molecules with vitamins and testosterone grafted on SF chains. The strong hydrophobic interactions between CPT and lipophilic vitamins and testosterone allowed achieving a high CPT content in the prepared CPT - SF1 to CPT - SF3 aggregates, with encapsulation efficiencies ranging from 60% to 84%.

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Particularly interesting was the high CPT content found in CPT - SF3 aggregates and associated high encapsulation ef ficiency achieved (67%) in spite the low testosterone content related to a DS of 0.4 mol% in SF3 conjugates. However, CPT - SF3 aggregates exhibited the lowest hydrodynamic diameters in aqueous medium. It might be due to additional − interactions between the phenyl groups of tyrosine residues in SF with the aromatic backbone of CPT. TEM technique depicted the dried SF1 - SF3 conjugates as rounded and irregular flake - like aggregates of approximately 50 - 800 nm ( Fig. 3a ). AFM showed the formation of small SF n anoaggregates with approximately 45 to 75 nm sizes for SF1 - SF3 conjugates ( Table 2 and Fig 3b ).

Fig. 3 (a) TEM micrographs of SF1 - SF3 dried aggregates at 21,000× magnification; (b) AFM micrographs of SF1 - SF3 dried aggregates.

Drug delivery studies

Fig . 4 shows the in vitro CPT release profiles of CPT - loaded SF aggregates in simulated physiological conditions (PBS at pH 7.4, 37 o C). All CPT releases appeared almost linear for the first 8 hours, with adjusted R 2 of 0.99 and slope ranging from 1.23 to 2.4 4 % h - 1 ( Fig. 4 and Table SI - 1 in the ESI † ). Camptothecin was almost quantitatively released after 6 days. CPT - SF2 and CPT - SF3 achieved a CPT release of approximately 97%, while the bigger CPT - SF1 aggregates carrying more CPT released 76% of the encapsulat ed anticancer drug. Furthermore, all CPT release profiles adjusted well to the Weibull model that describes the drug release from a matrix. 32,33 To this end, kinetics data was fitted following a SWeibull2 function (Cumulative Release (%) = a – (a – b)*exp( – (k*Time(hours)) d ), with adjusted R 2 ranged from 0.9760 to 0.9918 and d values of 1.1 and 1.2 associated to a complex drug release mechanism

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( Fig. 4 and Table SI - 2 in the ESI † ). 33 The CPT release kinetic data was also adjusted to the Korsmeyer - Peppas model (linear fitting of log(Cumulative Release(%)) = k*log(Time(hours) + m), to get an insight into the molecular interactions and factors dominant on the CPT release behaviour ( Table SI - 3 and Fig. SI - 10 in the ESI † ). 32,34 The slope of the linear fitting range d from 0.72 to 0.78, which is related to a non - Fickian drug release mechanism (anomalous diffusion mechanism). 34

Fig. 4 In vitro release profiles of CPT - loaded SF1 - SF3 aggregates in PBS (pH 7.4) at 37 o C, adjusted to a SWeibull2 function (inset shows th e linear fittings of release profiles up to 8 hours). Further evaluation of the interaction between the anticancer drug with the hydrophobically modified SF matrix was carried out based on the changes of characteristic IR adsorption peak of camptothecin a t 1737 cm - 1 observed in the CPT - loaded SF aggregates ( Table SI - 3 and Fig. SI - 11 in the ESI † ). To this end, the intensity of IR adsorption peaks at 1732 cm - 1 and 1739 cm - 1 in blank and CPT - loaded SF aggregates were normalized to characteristic 1515 cm - 1 ads orption peak in all spectra. The IR adsorption peaks at 1732 cm - 1 and 1739 cm - 1 of CPT - loaded SF aggregates exhibited a 0.4 to 1.6 increase of intensity when compared to blank SF1 - SF3 aggregates ( Fig. SI - 11 in the ESI † ). It might be due to overlap of exist ing C=O adsorption peak at 1732 cm - 1 and 1739 cm - 1 of blank SF1 and SF2 , SF3 aggregates with the intense CPT adsorption in the CPT - loaded aggregates. A significant shift of the CPT adsorption peak from 1737 cm - 1 in parent CPT to 1732 cm - 1 in CPT - SF1 aggreg ates was observed. It is probably due to slightly stronger interactions between CPT and the SF1 polymer matrix. On the other hand, CPT - SF2 and CPT - SF3 showed small shifting of CPT peak to 1739 cm - 1 .

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Cytotoxic activity and cell uptake Fig. 5 shows the relat ive cell viabilities of MCF - 7 cells after treatment with CPT - loaded SF conjugates or parent CPT. Parent SF appeared no toxic to MCF - 7 cells at 0.2 mg mL - 1 , with observed relative cell viability of (99 ± 5)% (data not shown). Similarly, SF1 - SF3 aggregates s howed not cytotoxic to MCF - 7 (89 - 94% relative cell viability) at 0.1 mg mL - 1 .

Fig. 5 Relative cell viability of MCF - 7 breast cancer cells treated with (a) CPT - loaded SF aggregates and (b) parent CPT. NP stands for blank SF aggregates at 0.1 mg mL - 1 . Dat a represents the mean ± standard deviation (n = 3).

CPT hydrophobically loaded in SF aggregates provoked a significant anticancer effect wich was not observed in SF conjugates. Particularly CPT - SF2 with a CPT concentration between 10 - 2.8 to 10 - 2.2 mg mL - 1 exhibited similar to slightly better anticancer activity than parent CPT evaluated at 10 - 2.6 to 10 - 2 mg mL - 1 . However, all CPT - loaded SF aggregates showed a moderate to slight anticancer effect (relative cell viability of 60% to 85%) when evaluated at CPT concentrations between 10 - 4 to 10 - 3 mg mL - 1 . These results demonstrated that synthesised SF - conjugates are good candidates for in vivo delivery of camptothecin. To this end, CPT - loaded SF aggregates in aqueous medium would be injected in the tumour or to selectively accumulate in the cancer tissues because combination of enhanced permeability and retention (EPR) effect and passive diffusion . 35,36 340 - 370 nm CPT - SF2 and CPT - SF3 aqueous soft aggregates formed by 45 - 75 nm particles might also exhibit extende d circulation times and moderated reticuloendothelial system (RES) clearance. 37 Once CPT - SF2 or CPT - SF3 aggregates are inside the cancer cells or in the slightly acidic cancer tissue, the CPT cargo might be delivered faster because enzyme - promoted and acid ic hydrolysis degradation of SF - tocopherol and SF - testosterone carriers. Additional in vivo

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assessment of bioavailability, biodistribution, antitumor activity, toxicity and pharmacokinetics of the CPT - loaded SF aggregates must be carried out before their m edical application. To evaluate the uptake and localization of the CPT - loaded SF aggregates on MCF - 7 cancer cells, fluorescence microscopy analysis were performed. Indeed, CPT - SF1 (Fig. 6), CPT - SF2 and CPT - SF3 ( Figs. SI - 12 and SI - 13 in the ESI † ) internali zed in cells (CPT associated blue fluorescence) and accumulated in the lysosomes (LysoTracker HCK - 123 associated green fluorescence). 38

Fig. 6 (a) MCF - 7 cells fluorescence images and slices (b) of cells without particles and LysoTracker (B), cells with 0.1 mg mL - 1 of CPT - SF1 aggregates, 50 nM of LysoTracker Yellow HCK - 123 and merged pictures (M), scale bars represents 20 µm. 3.2.4. Conclusions In this research, ergocalciferol - , tocopherol - and testosterone - grafted SF conjugates were synthesised via este rification of tyrosine residues and efficiently loaded with camptothecin for controlled release of the anticancer drug. Vitamins and testosterone grafting of SF attained a DS between 0.4 to 3.8 mol%, determined by elemental analysis and 1 H NMR spectroscopi es. FTIR spectra and DSC thermograms showed that tocopherol and testosterone - grafted SF conjugates predominantly adopted a β - sheet conformation. The hydrophobic functionalisation of SF with the vitamins and testosterone resulted in almost doubled hydrodyna mic sizes and zeta potentials of vitamins - and testosterone - grafted SF aggregates as compared to parent SF. TEM images showed 50 - 800 nm rounded and irregular flake - like SF aggregates, in good agreement with DLS results. The dialysis - precipitation method al lowed efficient CPT encapsulation in the SF aggregates, with a CPT content of 6.3 - 8.3 wt. %. A significant shrinkage of SF aggregates in PBS was observed once camptothecin was encapsulated in the hydrophobic core. Controlled and almost quantitative release of

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camptothecin was achieved after 6 days in PBS at 37 o C, with almost linear release during the first 8 hours. Camptothecin antiproliferative effect on MCF - 7 cancer cells seems unaltered after encapsulation in the SF aggregates. Particularly, CPT - loaded tocopherol - grafted SF aggregates showed slightly more cytotoxic to MCF - 7 cancer cells than parent camptothecin. Fluorescence microscopy imaging confirmed MCF - 7 cells uptake and lysosomal accumulation of CPT - loaded SF aggregates. Accordingly, camptothecin - l oaded SF aggregates are good candidates for antitumor therapy. However, further in vivo studies for a better evaluation of antitumor activity, toxicity, and other parameters are necessary.

Conflicts of interest There are no conflicts to declare.

Acknowl edgements Erasmus Mundus is acknowledged for a scholarship to JPQ. The authors thank Günter Hesser for training with TEM imaging of nanoparticles and Lisa M. Uiberlacker for AFM imaging of nanoparticles at JKU Linz, Linz, Austria. The access to NMR facilit ies of Upper Austrian – South Bohemian Research Infrastructure Center in JKU Linz, Linz, Austria, supported by the European Union (ETC Austria - Czech Republic 2007 - 2013, Project M00146) is also acknowledged.

3.2.5. Notes and references 1 F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A Torre and A. Jemal, CA Cancer J. Clin. , 2018, 68 , 394 – 424. 2 X. Jing, Z. Zhi, L. Jing, F. Wang, Y. Wu, D. Wang, K. Yan, Y. Shao and L. Meng, Nanoscale , 2019, 11 , 9457 – 9467. 3 H. Jin, X. - H. Dai, C. Wu, J. - M. Pan, X. - H. Wang, Y. S. Yan, D. - M. Liu and L. Sun, Eur. Polym. J. , 2015, 66 , 149 – 159. 4 Y. Peng, X. Zhu and L. Qiu, Biomaterials , 2016, 106 , 1 – 12. 5 P. J. Kuehl, M. J. Grimes, D. Dubose, M. Burje, D. A. Revelli, A. P. Gigliotti, S. A. Belinsky and M. Tessema, Drug Delivery , 20 18, 25 , 1127 – 1136. 6 H. Ishiguro, S. Saji, S. Nomura, S. Tanaka, T. Ueno, M. Onoue, H. Iwata, T. Yamanaka, Y. Sasaki and M. Toi, Cancer Med. , 2017, 6 , 2909 – 2917. 7 S. Keyvani - Ghamsari, A. Rabbani - Chadegani, J. Sargolzaei and M. Shahhoseini, Tumor Biol. , 2017, 1 – 10. 8 H. Zhang, OncoTargets Ther. , 2016, 9 , 3001 – 3007. 9 Y. Pommier, M. Cushman and J. H. Doroshow, Oncotarget , 2018, 9 , 37286 – 37288. 10 D. Kang, A. L. Liu, J. H. Wang and G. - H. Du, Camptothecin. In: Natural Small Molecules Drugs from Plants, G. - H. Du, Eds.; Sp ringer, Singapore, 2018, 491 – 496.

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11 J. Zou, S. Li, Z. Chen, Z. Lu, J. gao, J. Zou, X. Lin, Y. Li, C. Zhang and L. Shen, Cell Death Dis. , 2018, 9 , 661. 12 T. Deng, X. Mao, Y. Xiao, Z. Yang, X. Zheng and Z. - X. Jiang, Bioorg. Med. Chem. Lett. , 2019, 29 , 581 – 584. 13 F . Li, T. Jiang, Q. Li and X. Ling, Am. J. Cancer Res. , 2017, 7 , 2350 – 2394. 14 L. Liu, Q. Ye, M. Lu, Y. - C. Lo, Y. - H. Hsu, M. - C. Wei, Y. - H. Chen, S. - C. Lo, S. - J. Wang, D. J. Bain and C. Ho, Sci. Rep. , 2015, 5 , 10881. 15 J. P. Quiñones, J. Jokinen, S. Keinänen, C. P. Covas, O. Brüggemann and D. Ossipov, Eur. Polym. J. , 2018, 99 , 384 – 393. 16 J. P. Quiñones, C. C. Mardare, A. W. Hassel and O. Brüggemann, Carbohydr. Polym. , 2019, 206 , 641 – 652. 17 M. Atoum and F. Alzoughool, Breast Cancer: Basic Clin. Res. , 2017, 11 , 1 – 8. 18 R. Glaser, A. E. York and C. D. Dimitrakis, Menopause , 2017, 24 , 859 – 864. 19 F. Chen, S. Lu, L. Zhu, Z. Tang, Q. Wang, G. Qin, J. Yang, G. Sun, Q. Zhang and Q. Chen, J. Mater. Chem. B , 2019, 7 , 1708 – 1715. 20 N. Kasoju, N. Hawkins, O. Pop - Georgievski, D. Kubies and F. Vollrath, Biomater. Sci. , 2016, 4 , 460 – 473. 21 M. B. Elsner, H. M. Herold, S. Müller - Herrmann, H. Bargel and T. Scheibel, Biomater. Sci. , 2015, 3 , 543 – 551. 22 Y. Gotoh, S. Niimi, T. Hayakawa and T. Miyashita, Biomaterials , 2004, 25 , 1131 – 1140. 23 J. – H. Yeo, K. - G . Lee, H. - Y. Kweon, S. - O. Woo, S. - M. Han, S. - S. Kim and M. Demura, J. Appl. Polym. Sci. , 2006, 102 , 772 – 776. 24 T. Abe, K. Hasunuma and M. Kurokawa, Vitamin E orotate and a method of producing the same. U.S. Patent 3,944,550 A, 1976. 25 J. P. Quiñones, O. Brügge mann, C. P. Covas and D. A. Ossipov, Carbohydr. Polym. , 2017, 173 , 157 – 169. 26 Y. Peng, X. Zhu and L. Qiu, Biomaterials , 2016, 106 , 1 – 12. 27 H. E. Gotlieb, V. Kotlyar and A. Nudelman, J. Org. Chem. , 1997, 62 , 7512 – 7515. 28 A. R. Murphy and D. L. Kaplan, J. Mater. C hem. , 2009, 19 , 6443 – 6450. 29 X. Hu, D. Kaplan and P. Cebe, Thermochim. Acta, 2007, 461 , 137 – 144. 30 N. Jaramillo - Quiceno, C. Álvarez - López and A. Restrepo - Osorio, Procedia Eng. , 2017, 200 , 384 – 388. 31 S. Honary and F. Zahir, Trop. J. Pharm. Res. , 2013, 12 , 265 – 273 . 32 S. Dash, P. N. Murthy, L. Nath and P. Chowdury, Acta Pol. Pharm. , 2010, 67 , 217 – 223. 33 V. Papadopolou, K. Kosmidis, M. Vlachou and P. Macheras, Int. J. Pharm. , 2006, 309 , 44 – 50. 34 S. Kondaveeti, T. C. Damato, A. M. Carmona - Ribeiro, M. R. Sierakowski and D. F . S. Petri, Carbohydr. Polym. , 2017, 165 , 285 – 293. 35 K. Krukiewicz and J. K. Zak, Mater. Sci. Eng., C , 2016, 62 , 927 – 942. 36 U. Prabhakar, H. Maeda, R. K. Jain, E. M. Sevick - Muraca, W. Zamboni, O. C. Farokzhad, S. T. Barry, A. Gabizon, P. Grodzinski and. C. Bla key, Cancer Res. , 2013, 73 , 2412 – 2417. 37 L. Zhang, Z. Cao, Y. Li, J. - R. Ella - Menye, T. Bai and S. Jiang, ACS Nano , 2012, 6 , 6681 – 6686. 38 J. D. Totten, T. Wongpinyochit and F. P. Seib, J. Drug Targeting , 2017, 25 , 865 - 872.

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SUPPORTING INFORMATION

Javier Pérez Quiñones, Cornelia Roschger, Andreas Zierer, Carlos Peniche - Covas, Oliver Brüggemann

1. Supporting Tables Table SI - 1 . Linear fitting parameters of in vitro CPT release profiles of SF aggregates up to 8 hours (intercept 0, slope k, adjusted R - Square) in PBS (pH 7.4) at 37 o C Samples k Adjusted R - Square CPT - SF1 1.23 ± 0.04 0.9950 CPT - SF2 2.44 ± 0.02 0.9997 CPT - SF3 1.90 ± 0.02 0.9994

Table SI - 2 . SWeibull2 fitting parameters of Cumulative Release(%) Vs. Time(hours) of CPT - loaded SF aggregates up to 120 hours (Cumulative Release (%) = a – (a – b)*exp( – (k*Time(hours)) d ) in PBS (pH 7.4) at 37 o C Samples a b k d Adjusted R - Square CPT - SF1 124 ± 58 3 ± 2 0.007 ± 0.005 1.1 ± 0.2 0.9918 CPT - SF2 116 ± 25 6 ± 6 0.014 ± 0.005 1.1 ± 0.4 0.9760 CPT - SF3 138 ± 69 7 ± 4 0.008 ± 0.006 1.2 ± 0.4 0.9812

Table SI - 3 . Linear fitting parameters of log(Cumulative Release(%)) Vs. log(Time(hours)) of CPT - loaded SF conjugates up to 120 hours (intercept m, slope k, adjusted R - Square) in PBS (pH 7.4) at 37 o C, and wavenumber of CPT C=O ((  (C=O)) IR adsorption Samples m k R 2  (C=O) CPT - SF1 0.23 ± 0.03 0.78 ± 0.02 0.9939 1732 CPT - SF2 0.56 ± 0.05 0.72 ± 0.04 0.9825 1739 CPT - SF3 0.42 ± 0.05 0.75 ± 0.03 0.9837 1739

2. Characterisation data for parent SF and SF1 - SF3 conjugates SF: ATR - FTIR (solid)  max: 3291 (N – H), 3070 (C – H aromatic), 2937 (C – H aliphatic), 1645 (Amide I, silk I), 1535 (Silk I), 1515 (Silk II), 1453 (C – H bend), 1234 (C – N), 1169

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1 (C – H in - plane bending); H NMR (300 MHz, 298 K, CDCl 3 ):  = 0.84 (s, 1H, CH 3 - of valine ), 6.7 - 8.2 (m, 4.32H, aromatic protons of tyrosine and phenylalanine). GPC

(g/mol) M n = 15048, Mw = 22121. Elemental analysis (experimental): %C 44.83, %H 6.31, %N 17.21 SF1 : ATR - FTIR (solid)  max: 3284 (N – H), 3072 (C – H aromatic), 2937 (C – H aliphatic), 1733 (C=O, ergocalciferol - succinate bond), 1684 (C=O, SF - succinate bond), 1643 (Amide I, silk I), 1532 (Silk I), 1518 (Silk II), 1453 (C – H bend), 1240 (C – N), 1162 1 (C – H in - plane bending + C – O – C); H NMR (300 MHz, CDCl 3 ):  = 0.82 - 1.02 (d, 9.37H,

CH 3 – of val ine + H18 + H21 + H26 + H27 + H28), 5.27 - 5.35 (d, 0.82H, H6 + H7), 6.7 - 8.2 (m, 3.31H, aromatic protons of tyrosine and phenylalanine). Elemental analysis (experimental): %C 50.85, %H 6.60, %N 14.04 SF2 : ATR - FTIR (solid)  max: 3285 (N – H), 3075 (C – H aromati c), 2928 (C – H aliphatic), 1739 (C=O, tocopherol - succinate bond), 1698 (C=O, SF - succinate bond), 1624 (Amide II, silk II), 1514 (Silk II), 1448 (C – H bend), 1229 (C – N), 1155 (C – H in - plane bending + 1 C – O – C); H NMR (300 MHz, CDCl 3 ):  = 0.84 (s, 9.10H, CH 3 – of valine + 4’CH 3 – +

8’CH 3 – + 12’CH 3 – ), 6.7 - 8.2 (m, 3.84H, aromatic protons of tyrosine and phenylalanine). Elemental analysis (experimental): %C 52.70, %H 7.06, %N 13.74 SF3 : ATR - FTIR (solid)  max: 3282 (N – H), 3073 (C – H aromatic), 2935 (C – H aliphatic), 17 34 (C=O, testosterone - succinate bond), 1699 (C=O, SF - succinate bond), 1622 (Amide II, silk II), 1514 (Silk II), 1446 (C – H bend), 1230 (C – N), 1168 (C – H in - plane 1 bending + C – O – C); H NMR (300 MHz, CDCl 3 ):  = 0.84 - 0.86 (m, 1.38H, CH 3 – of valine + H18 + H19), 5.34 (s, 0.31, H4), 6.7 - 8.2 (m, 2.41H, aromatic protons of tyrosine and phenylalanine). Elemental analysis (experimental): %C 46.03, %H 6.28, %N 17.01 3. Supporting Figures

Figure SI - 1 UV spectra of CPT at 0.00503 mg mL - 1 , CPT - SF1 at 0.025 mg mL - 1 , CPT - SF2 at 0.0234 mg mL - 1 , CPT - SF3 at 0.0253 mg mL - 1 in DMSO.

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Figure SI - 2 Calibration curve of CPT in DMSO.

Figure SI - 3 Calibration curve of CPT in PBS (pH 7.4).

Fig. SI - 4 TGA curve of SF2 .

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Fig. SI - 5 TGA curve of SF3 .

Fig. SI - 6 TGA curve of parent SF.

1 Fig. SI - 7 H NMR spectrum of parent SF in DMSO - d 6 .

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1 Fig. SI - 8 H NMR spectrum of SF2 in DMSO - d 6 .

1 Fig. SI - 9 H NMR spectrum of SF3 in DMSO - d 6 .

Fig. SI - 10 Linear fitting of log(Cumulative Release (%)) Vs. log(Time(hours)) of CP T - loaded SF aggregates up to 120 hours in PBS (pH 7.4) at 37 o C.

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Fig. SI - 11 ATR - FTIR spectra of CPT and CPT - loaded SF aggregates.

Fig. SI - 12 (a) MCF - 7 cells confocal images and slices (b) of cells without particles and LysoTracker (B), cells with 0 .1 mg mL - 1 of CPT - SF1 aggregates, 50 nM of LysoTracker Yellow HCK - 123 and merged pictures (M), scale bars represents 20 µ m.

Fig. SI - 13 (a) MCF - 7 cells confocal images and slices (b) of cells without particles and LysoTracker (B), cells with 0.1 mg mL - 1 of CPT - SF3 aggregates, 50 nM of LysoTracker Yellow HCK - 123 and merged pictures (M), scale bars represents 20 µm.

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4. Polyphosphazene - based carriers for delivery of agrochemicals and drugs

This chapter describes the synthesis of poly(organo)phosphazenes s ubstituted with diosgenin, DI31, S7 glycinates and Jeffamine M - 1000 for controlled drug release and agrochemical applications, or with testosterone and tocopherol glycinates and Jeffamine M - 1000 for further camptothecin and epirubicin loading for sustained release and potential anticancer applications. All polyphosphazene - based carriers formed aggregates in aqueous media, with sustained drug release of diosgenin, agrochemicals, camptothecin and epirubicin. Camptothecin hydrophobically encapsulated in the co re of the testosterone and tocopherol bearing polyphosphazenes attained 10 - 13 wt% content. It corresponds to 77 - 100 fold increase of camptothecin solubility in water for a 1 mg/mL aqueous dispersion of camptothecin bearing polyphosphazenes. In vitro agroch emical activity evaluation of agrochemical bearing polyphosphazenes showed excellent plant growth enhancer effect. Additionally, strong cytotoxicity to MCF - 7 cells was observed for DI31 and S7 bearing polyphosphazenes. MCF - 7 cells showed good uptake of cam ptothecin and epirubicin bearing polyphosphazenes. This chapter is based on the following papers:

4.1. Polyphosphazene - based nanocarriers for the release of agrochemicals and potential anticancer drugs.

4.2. Polyphosphazene - based nanocarriers for the relea se of camptothecin and epirubicin.

My contribution to the papers

I designed and conducted all the experimental work and related characterization, except the AFM, SEM and cell - related assays. I interpreted the results and wrote the manuscripts.

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4.1. Polyp hosphazene - based nanocarriers for the release of agrochemicals and potential anticancer drugs

Six poly(organo)phosphazenes were prepared with diosgenin, DI31, S7 and Jeffamine M - 1000 ( P1 - P6 ) for agrochemical and potential medical applications ( TOC 5 ). DI3 1 and S7 bearing poly(organo)phosphazenes showed good agrochemical activity on the radish cotyledons test ( TOC 5 ), as well as strong cytotoxicity to MCF - 7 cancer cells.

TOC 5. Structure s of steroid bearing poly(organo)phosphazenes ( P1 - P5 ), and radish cot yledons used to evaluate the agrochemical activity. Figure reproduced with permission.

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Polyphosphazene - based nanocarriers for the release of agrochemicals and potential anticancer drugs

Javier Pérez Quiñones, a * Aitziber Iturmendi, a Helena Henke, a Cornelia Roschger, b Andreas Zierer b and Oliver Brüggemann a

––––––––– a Institute of Polymer Chemistry (ICP), Johannes Kepler University Linz, Altenberger Str. 69, 4040 Linz, Austria. b Johannes Kepler University Linz, Kepler Universi ty Hospital GmbH, Department for Cardiac - , Vascular - and Thoracic Surgery, Altenberger Str. 6 9, 4040 Linz and Krankenhausstraβe 7a, 4020 Linz, Austria.

E - mail: [email protected]

–––––––––

Journal of Materials Chemistry B (2019), in revision

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Abstract

The synthesis and characterisation of novel polyphosphazene nanocarriers, based on hyd rophilic polyalkylene oxide Jeffamine M1000 and hydrophobic steroids with a glycinate linker for pH - controlled release of diosgenin and two brassinosteroids (DI31 and S7) with agrochemical and potential anticancer activity, is hereby described. The influen ce of the Jeffamine M1000/steroid ratio during the synthesis of diosgenin - based polyphosphazene on the physico - chemical properties of the nanocarriers was demonstrated. Particularly, hydrophilicity of the polymers, hydrodynamic particle sizes and observed drug release rates were tailored by variation of the polyphosphazene composition with a diosgenin/Jeffamine M1000 ratio of 1:1 to 1:2. Six polyphosphazenes based on Jeffamine M1000 and three steroids (diosgenin, DI31 and S7), with different composition wer e prepared and thoroughly characterised. Polyphosphazenes carrying approximately 17 wt.% of DI31 or S7 self - assembled in water to form 120 - 150 nm nanoaggregates, which showed excellent plant growth effect on radish cotyledons due to a sustained delivery of the agrochemicals observed of approximately 30% after 4 days. Cytotoxic evaluation showed that all polymers carrying steroids and Jeffamine M1000 resulted more or less toxic to cancer MCF - 7 and non - cancer HEK - 293 cells at almost all concentrations evaluat ed, except at 0.00156 mg mL - 1 . Thus, DI31 and S7 bearing polymers applied at 10 - 4 to 10 - 6 mg mL - 1 for delivery of recommended DI31 or S7 quantities to the crops should be harmless to humans. Strong cytotoxic activity of DI31 and S7 bearing polymers to huma n breast adenocarcinoma cell line MCF - 7 at 0.1 to 0.00625 mg mL - 1 , proper hydrodynamic sizes between 100 - 200 nm, and slow sustained release of cytotoxic drugs (DI31, S7) might potentiate their accumulation in canc er tissues with good antitumour effect a nd minor side effects. These results demonstrated that prepara tion of polyphosphazenes carrying brassinosteroids is a promising strategy for the preparation of better agrochemicals with reduced pollutant impact on a sustainable a griculture and potential anticancer formulations based on analogues of brassinosteroids.

4.1.1. Introduction Brassinosteroids (BR), a naturally occurring steroid plant hormones group found in all vegetal organs, regulate plant growth and development by eli citing several physiological effects in combination with other phytohormones. 1,2 These phytohormones promote xylem differentiation, stem elongation, leaf bending, epinasty, biosynthesis of nucleic acids, proteins and ethylene, as well as to regulate assimi lation and allocation of carbohydrates, response mechanisms of plants to biotic and abiotic

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stress and to activate the photosynthesis. 3 - 5 The content of BR in plants range from 10 - 1 nmol g - 1 to 10 - 7 nmol g - 1 , being 100 times smaller than other phytohormo nes. 6,7 BR and some synthetic analogues of brassinosteroids are widely used at concentrations of 5 to 100 mg ha - 1 as agrochemicals and pesticides, with a significant improvement in the quality and efficiency of the crops. 8,9 Particularly promising results are observed when the synthetic analogues of brassinosteroids DI31 and S7 are applied at 10 - 20 mg ha - 1 as agrochemicals, with demonstrated increases of 5 - 30% of crops yields. 10,11 However, rapid metabolism of BR - based agrochemicals limits the efficacy of DI31 and S7 that result in periodic foliar applications required to achieve their known benefits. 11 The hydrophobicity of DI31 and S7 also complicate their application in agriculture, being necessary to prepare their commercial formulations as emulsions co ntaining 100 ppm of DI31 in 50 vol % water/ethanol and surfactants (Biobras - 16), with limited stability. 12,13 To avoid the rapid metabolism of DI31 or S7 and provide a constant supply of these compounds with increased solubility in water, they would be inc orporated in different polymer - based systems such as micelles, nanoparticles, vesicles, dendrimers or other carriers that protect them and assure a controlled release for a longer period of time. 14,15 However, relatively less drug delivery systems have bee n aimed to sustained release of agrochemicals than of pharmaceuticals. 16 In this sense, an ideal drug delivery system for application in agriculture should provide a sustained release of the agrochemical, achieving a high drug loading with retained activit y, good solubility and stability in water at normal usage conditions (pH 7.0, 25 o C) together with proper degradation and no toxicity of the polymer matrix and its metabolites in plants and animals. 17,18 Hence, polyphosphazenes, a group of degradable synth etic polymers with highly tuneable composition and properties, appear to be promising candidates to this end. On the other hand, diosgenin was chosen as a model - drug to assess the pH - promoted delivery of steroids from polyphosphazene nanocarriers. 19 This s teroid sapogenin is a cheap substrate widely used for synthesis of steroids, corticosteroids and the agrochemical DI31, 20 providing a right model for the bulky steroid substituents on polyphosphazenes. Diosgenin itself has shown good antidiabetic, hypochol esterolemic, antithrombotic and anticancer activity, but very low aqueous solubility and reduced oral bioavailability limit its medical applications. 19,21 Similarly, some brassinosteroids and synthetic analogues of brassinosteroids have displayed potent an tiproliferative effect on different cancer cells with reduced haemolysis and negative effects on non - cancer cells. 22,23 This is the reasoning behind designing an efficient polyphosphazene - based drug delivery system for diosgenin and brassinosteroids.

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Poly phosphazenes, also named as poly(organo)phosphazenes, are organic - inorganic hybrid polymers with a characteristic [ - R 2 P=N - ] repeating unit in which different nucleophiles attached to the phosphorus atom tailor the properties of the polymer. 24,25 Then, diff erent polyphosphazenes have been synthesised for wide diverse applications such as tissue engineering and drug delivery agents in biomedicine, 25 - 34 catalysts, 35 flame retardants, 36 high performance polymers 37 and other materials. 38,39 Polyphosphazenes are particularly attractive since their properties, architectures, functionality and degradability can be fine - tuned by precise control of the polymer composition and synthetic pathway (degree of polymerisation, distribution and ratio of hydrophobic/hydrophili c substituents, structure, etc.). 24 - 38 Another advantage of the application of polyphosphazene - based biomaterials is that they degrade to phosphates and ammonium salts alongside the organic substituents, thus being benign degradation products if innocuous substituents have been selected. 25,27,38 Therefore, smartly designed polyphosphazenes carrying brassinosteroids might fit the requirements to sustainably deliver the agrochemicals once applied to the plants with minimized environmental impact and increased plant growth effect. In this research, five polyphosphazene - based nanocarriers for delivery of diosgenin and two brassinosteroids (DI31 and S7), with agrochemical and potential anticancer activity were synthesised by living cationic polymerisati on of Cl 3 PNSiMe 3 to obtain a poly(dichloro)phosphazenes, which was further functionalised via post - polymerisation substitution with the steroids and hydrophilic Jeffamine M1000. Polyphosphazenes carrying diosgenin were synthesised with two degrees of polym erisation and steroid content to optimize the synthesis of the polymers, to study the effect of average chain length and chemical composition on the nanoaggregates hydrodynamic diameters and drug release behaviour. Sustained release of diosgenin from nanoc arriers in acidic medium via hydrolysis of ester bond encouraged us to prepare pH - labile polyphosphazenes with DI31 and S7 for agrochemical applications. Controlled delivery of covalently linked DI31 and S7 from their nanocarriers increased their plant gro wth stimulant activity as assessed using in vitro radish cotyledons bioassay. Further, cytotoxicity of all polymers to MCF - 7 cancer cells and HEK - 293 non - cancer cells was evaluated.

4.1.2. Materials and methods Materials

Amine capped polyetheramine copolym er (PEO — PPO — NH 2 ) with an M n of 1000 g mol - 1 and an ethylene oxide/propylene oxide ratio of 19/3, tradename Jeffamine

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M1000, was donated by Huntsman Performance Products and used as received. The diosgenin [(25 R ) - 5 - spirosten - 3β - ol] and synthetic analogues o f brassinosteroids (25 R ) - 3β,5α - dihydroxyspirostan - 6 - one (DI31) and (22 R ,23 R ) - 22,23 - epoxy - 3β,5α - dihydroxystigmastan - 6 - one (S7) were kindly supplied by University of Havana, Cuba. Triethylamine was distilled and stored over molecular sieves under argon. All other chemicals and solvents were purchased from Sigma - Aldrich and used without prior purification. All glassware was dried overnight in an oven at 120 o C prior to use. Spectra/Por 3 cellulose dialysis membranes (Spectrum Laboratories Inc., CA, USA) with m olecular weight cut off (MWCO) of 3.5 kDa were used for purification of the synthesised polymers.

Synthesis of steroid - Glycine - Boc

The synthesis of steroid - glycine - Boc and deprotected steroid - glycine - NH 2 (diosgenin - glycine - NH 2 ( 1 ), DI31 - glycine - NH 2 ( 2 ) and S7 - Glycine - NH 2 ( 3 )) were carried out as previously reported, with minor modifications. 26,27 The preparation of diosgenin glycinate is briefly described in the ESI † .

Synthesis of monomer trichlorophosphoranimine (Cl 3 P=N – Si(CH 3 ) 3 )

The monomer N - (trimethylsi lyl) - trichlorophosphoranimine (Cl 3 P=N – Si(CH 3 ) 3 ) was synthesised according to reported procedure with slight modifications ( ESI † ). 38,42

Synthesis of the polymers The synthesis of the poly(dichloro)phosphazene precursor was conducted via living cationic pol ymerisation of monomer trichlorophosphoranimine with triphenylphosphine dichloride. 24,28, 30,38 The synthesis of polymer P1 is described in detail in the ESI † . For polymers P2 - P6 the ratio of monomer to initiator, and the ratio of substituent steroid to Je ffamine M1000 were adjusted differently ( Table 1 ). All reactions were performed in the glovebox under inert atmosphere.

In vitro drug release studies The in vitro release studies of diosgenin, DI31 and S7 were performed in phosphate buffered saline (PBS, p H 6.0) solution at 25 o C. To this purpose, 2.0 mL of polymers P1 - P5 (2.5 mg mL - 1 ) in PBS at pH 6.0 were placed in dialysis cups (MWCO 3.5 kDa, Slide – A - Lyzer mini dialysis devices, ThermoScientific, USA) and immersed in 40 mL of the release medium (PBS, pH 6.0) at 25 o C and stirred at 150 rpm. The release medium was replaced every required time point, and analysed with UV spectroscopy

(diosgenin  em = 280 nm, DI31 and S7  em = 300 nm). Release experiments were conducted in triplicate. Calibration curves of steroids (ESI † ) allowed quantification of

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- 1 - 1 - 1 - 1 delivered drug (diosgenin  280 = 688 M cm , DI31  300 = 1693 M cm , S7  300 = 1290 M - 1 cm - 1 ). 43

In vitro agrochemical tests The agrochemical bioactivity as plant growth enhancer of synthesised polymers carryi ng the brassinosteroid analogues DI31 and S7 ( P4 and P5 ) were evaluated in vitro on radish ( Raphanus sativus ) plants. Increased weight of radish cotyledons was used to assess the agrochemical activity of tested compounds. Radish seeds were sterilised with sodium hypochlorite solution prior to germination on wet filter paper in dark at room temperature for 3 days. 44 Then, hypocotyls were discarded and the cotyledons weighed and transferred to Petri dishes with 5 mL of polymers P4 , P5 , DI31, S7, P6 (polyphosp hazene carrying only Jeffamine M1000) at 10 - 1 to 10 - 7 mg mL - 1 aqueous solutions, and water (control). Cotyledons were allowed to growth for another 3 days and weighed again. Experiments were performed in triplicate and 10 cotyledons were used for each samp le.

Cytotoxicity tests Cytotoxicity of the samples against the human breast adenocarcinoma cell line MCF - 7 and the non - cancerous human embryonic kidney 293 cell line HEK - 293 was evaluated using the 2,3 - bis(2 - methoxy - 4 - nitro - 5 - sulfophenyl) - 2 H - tetrazolium - 5 - carboxanilide assay (XTT) (Cell Proliferation Kit II (XTT), Sigma - Aldrich). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) – high glucose supplemented with 10% fetal bovine serum, 1% penicillin – streptomycin and 1% L - glutamine. The media and supplements except L - glutamine (PAA laboratories) were purchased from Sigma -

Aldrich. Cells were grown in a humidified 5% CO 2 atmosphere at 37 °C. In all experiments, 70 - 80% confluent cells were used. Cells (1 × 10 4 cells/well) in complete growth medium were seeded into 96 - well culture plates. The day after, the medium was changed to serum free medium with P1 - P6 aggregate disper sions at different concentrations. Cells were incubated for another 48 hours, the medium was changed to full growth medium and 50 μL of XTT reagent was added to each well. Incubation was continued for another 3 hours at 37 °C in a humidified atmosphere wit h 5% CO 2 and 100% humidity. The absorbance was measured at 490 nm using a GloMax® Multimode Microplate Reader (Promega). Three independent experiments (with each sample in triplicate) were performed, and the cell viability was normalised to the untreated c ontrol. The cell viability values were analysed with Origin 2015 (Microcal Origin, OriginLab, MA, USA).

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Characterisation Characterisation by ATR - FTIR spectroscopy was performed on a Perkin Elmer Spectrum 100 FT - IR spectrophotometer using an ATR accessory, with 32 scans and 4 cm - 1 resolution from 4000 to 650 cm - 1 . UV - Vis spectra were obtained with a Perkin Elmer Lambda 25 UV/VIS spectrophotometer using quartz cuvettes. 1 H, 13 C and 31 P{ 1 H} NMR spectra were recorded at 298 K using a Bruker Avance 300 spectrome ter operated at 300 MHz, 75 MHz and 121 MHz, respectively. CDCl 3 was used as an internal reference for 1 H and 13 C NMR measurements ( 1 H NMR signals were referenced to  = 7.26 ppm; 13 C NMR signals were referenced to 77.16 ppm), 40 while 85% phosphoric acid w as used as an external standard for 31 P{ 1 H} NMR spectra. Attached Proton Test (APT) 13 C NMR experiment was carried out to facilitate carbon assignment by separating carbons according to their number of attached protons (CH and CH 3 signals positive, C and CH 2 signals negative) with a single experiment, which is more sensitive than traditional 13 C { 1 H} NMR. TopSpin 3.5 pl 7 (Bruker BioSpin GmbH) software was used for NMR spectra processing. Molecular weights were estimated using a Viscotek GPCmax gel permeat ion chromatograph (GPC) equipped using a PFG column from PSS (Mainz, Germany) (300 mm × 8 mm, 5 µm particle size), equipped with a Viscotek TDA 305 Triple Detector Array (Malvern, Germany), and calibrated with polystyrene standards from PSS. Samples were e luted with DMF containing 10 mM LiBr at a flow rate of 0.75 mL min - 1 at 60 o C. Dynamic light scattering (DLS) determinations were carried out on a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, UK) with a detection angle of 173 o and a 4 mW He - Ne laser operated at 633 nm for backscattering measurements. The samples were prepared at 1 mg mL - 1 in deionised water, filtered through a 0.45 µ m nylon film syringe filter and determinations of hydrodynamic diameters ( d h ) were performed in triplicate using a fold ed capillary cell DTS1070 at 25 o C. Calorimetric studies of samples were conducted on TA Instrument Q10 differential scanning calorimeter (DSC) using aluminium pans, with a sample weight of approximately 5 mg and heating rate of 10 o C min - 1 under nitrogen flow of 20 mL min - 1 . Samples were cooled and heated from - 80 o C to 400 o C. Atomic Force Microscopy (AFM) images (10 µm × 10 µm and 2 µm × 2 µm) were taken with MFP 3D - Stand Alone AFM (Asylum Research) with the cantilever OMCL - AC160TSA of Olympus, at a resona nt frequency of 300 kHz and spring constant of 26 N m - 1 , 50 - 70% set point and scan rate of 1 Hz. 80 µL droplet of a 1 mg mL - 1 aqueous dispersion of the polymers was deposited on a silicon wafer spin coated at 40 Hz for 6 s. Transmission Electron Microscopy (TEM) micrographs were recorded with a Jeol JEM - 2011 FasTEM (Jeol Ltd, Tokyo, Japan) operated at 100 kV. A drop of polymer

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dispersions in deionized water (1 mg mL - 1 ) was placed on a Pioloform coated 300 Mesh Cu grids (Plano GmbH, Germany). Excess solution was eliminated with filter paper; samples were negatively stained with a drop of uranyl acetate aqueous solution at 1%, and dried several hours before measurements.

4.1.3. Results and discussion Synthesis and characterisation of polymers P1 - P6 Polyphospha zene with diosgenin as a model - drug and Jeffamine M1000 were first synthesised with a ratio of initiator to monomer of 1:25 and 1:50, and a ratio of substituents diosgenin to Jeffamine M1000 of 1:1 and 1:2 ( Table 1 ). These preliminary experiments permitted us to assess the efficiency of post - polymerisation functionalisation with steroid molecules (bulky groups) on the poly(dichloro)phosphazene backbone, the effect of hydrophilicity of synthesised polyphosphazene (based on Jeffamine M1000 content) on the agg regation in aqueous dispersions via hydrodynamic parameters and the effect of steroid content on size of dried nanoaggregates using AFM and TEM imaging. The synthetic approach followed in this work and the structures of obtained polymers are presented in Fig. 1 . Living cationic polymerisation of trichlorophosphoranimine initiated with triphenylphosphine dichloride in solution (CH 2 Cl 2 ) at room temperature allowed to obtain linear poly(dichloro)phosphazenes with controlled polymerisation degree (20 to 23 fo r P1, P2, P4, P5, P6 and 34 for P3 ) adjusted through initiator to monomer ratio, and low polydispersities of 1.37 - 1.50 for prepared polymers, consistent with previous reports ( Fig. 1b ). 24,28,38

Table 1 Composition, yield and molecular weight of polymers P1 - P6 . d e Y M n M n Sample I:M a DP b S:J c % kg kg Ð e mol - 1 mol - 1 P1 1:25 23 1:1 37 38 10 1.37 P2 1:25 22 1:2 38 42 15 1.47 P3 1:50 34 1:1 35 76 12 1.31 P4 1:25 20 1:2 34 43 14 1.35 P5 1:25 22 1:2 30 43 13 1.50 P6 1:25 23 0:2 49 51 11 1.44 a Initiator to monomer ratio feeding composition. b Degree of polymerisation estimated by 1 H NMR. c Steroid to Jeffamine M1000 ratio feeding composition. d Theoretical number average molecular. e Number average molecular weight and polydispersities measured by GPC against polystyrene standards.

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Fig. 1 Scheme of synthetic route and structures of prepared polyphosphazene P1 - P6 .

(a) Synthesis of steroid glycinates (steroid - glycine - NH 2 ), (b) polymerisation of trichlorophosphoranimine to obtain poly(d ichloro)phosphazene, (c) post - polymerisation functionalisation of chlorine atoms with steroid - glycine - NH 2 and Jeffamine M1000.

Different steroid - glycine - NH 2 and Jeffamine M1000 (PEO - PPO - NH 2 ) were later introduced via post - polymerisation functionalisation of the chlorine atoms in poly(dichloro)phosphazene, firstly introducing the bulky steroid groups and later the linear PEO - PPO - NH – substituent ( Fig. 1c ). Complete chlorine substitution of the poly(dichloro)phosphazene ([NPCl 2 ] n ), required to avoid later unc ontrolled polymer degradation and hydrolysis due to highly labile P - Cl bonds, 38 was confirmed using 31 P{ 1 H} NMR spectroscopy. Thus, 31 P{ 1 H} NMR spectra of polymers P1 - P6 only showed broad peaks at 0.5 - 1.0 ppm, associated to Jeffamine M1000 used as substitu ent, 24,26,28,33,38 while no characteristic peak of P - Cl bond was observed (ESI † ). 26 The presence of hydrophilic Jeffamine M1000 substituent in the synthesised polymers P1 - P6 make them suitable to be dispersed in aqueous solutions, allowing their later appl ication as carriers for controlled release of agrochemicals. The presence of characteristic aromatic protons of (C 6 H 5 ) 3 P=N – end group allowed the estimation of the attained degree of polymerisation (DP) for polymers P1 - P6 using 1 H NMR spectroscopy, which w as expected to be near to 25 or 50 from monomer to initiator feeding in the mixture reaction. The integration of aromatic protons (C 6 H 5 ) 3 P=N – at

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7.60 ppm of P1 - P6 was approximately 15/(20 - 23) or 15/(34) when referred to integration of – O – C H 2 – C H 2 – O – and C H 3 O – protons of Jeffamine M1000 at 3.62 - 3.63 and 3.36 ppm, respectively) once estimated the degree of functionalisation with the two † 28 substituents (steroid - glycine - NH 2 and Jeffamine M1000) (ESI ). It is observed in Table 1 , that DP of polyphosphazene with d iosgenin and Jeffamine M1000 were in good agreement with initiator to monomer ratio feeding composition for the reaction when polymers were prepared with 25 repeating units (polymers P1 , P2 , P6 ). However, polymer P3 prepared with an initiator to monomer ra tio of 1:50 only reached an estimated DP of 34, which is consistent with results of Wilfert et al . 24 when conducting phosphine - mediated polymerisations. The polyphosphazene carrying DI31 or S7 and Jeffamine M1000 ( P4 , P5 ) were synthesised with a initiator to monomer ratio of 1:25, allowing to attain a DP of 20 - 23. As observed from 1 H NMR data (ESI † ), the steroids to Jeffamine M1000 mole ratio in feed (1:2) and in obtained polymers were also in good agreement. The molecular weights of polymers P1 - P6 determin ed by GPC calibrated against linear polystyrene standards deviated by 2 - 4 orders of magnitude of the expected values calculated for the polymers, as previously reported and ascribed to the different hydrodynamic volume of branched polyphosphazenes and line ar polystyrene standards. 28 Then, GPC measurements were used in this research as a guide of molecular weights for similarly related polyphosphazenes and to check polydispersities of prepared polymers P1 - P6 . Amphiphilic co - substituted polyphosphazenes migh t self - assembly or self - aggregate at molecular and supramolecular levels in a micelle - like array with a core formed of hydrophobic moieties and a shield of hydrophilic groups in contact with water molecules, as a result of favoured intramolecular and inter molecular interactions of hydrophobic groups and van der Waals and hydrogen bond interactions of hydrophilic groups and water molecules of the solvent. 30,39,45,46 The average hydrodynamic diameters ( d h ) in water and PBS obtained using dynamic light scatter ing, sizes of dried nanoaggregates from AFM micrographs ( d AFM ) and TEM micrographs ( d TEM ) of polymers P1 - P6 are shown in Table 2 .

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Table 2 Hydrodynamic sizes, average diameters AFM and TEM of dried nanoaggregates of polymers P1 - P6 d a d b h h d c d d nm nm AFM TEM Sample nm nm (PDI) (PDI) 164 ± 2 160 ± 3 P1 58 ± 5 53 ± 9 (0.21) (0.29) 200 ± 5 216 ± 1 P2 88 ± 3 40 ± 7 (0.34) (0.27) 169 ± 2 162 ± 1 P3 61 ± 6 35 ± 8 (0.23) (0.26) 122 ± 1 101 ± 2 P4 36 ± 8 42 ± 5 (0.66) (0.70) 151 ± 2 163 ± 4 P5 52 ± 2 37 ± 8 (0.59) (0.63) 6.0 ± 0.2 6.5 ± 0.3 P6 - - (0.27) (0.40) a b Average hydrodynamic diameter (d h ) in water by DLS. Average hydrodynamic diameter (d h ) c d in PBS by DLS. Diameter of dried nanoaggregates by AFM (d AFM ). Diameter of dried nanoaggregates by TEM (d TEM ).

P6 showed average hydrodynamic diameter corresponding to predominant individual hydrated polymer chains in water, as expected because it is formed only for the hydrophilic Jeffamine M1000 bonded to the P - N backbone ( Table 2 and Fig. SI - 39 and SI - 45 in the ESI † ). P1 and P3 polymers with same ratio of hydrophilic to hydrophobic substituents (diosgenin to Jeffamine M1000 ratio of 1:1) exhibited similar average hydrodynamic diameters ca. 160 - 170 nm in water and PBS, even when DP of P3 was 1.4 t imes of the one obtained for P1 . Besides polymer P2 , with higher content of hydrophilic Jeffamine M1000, exhibited approximately 20% to 34% bigger average hydrodynamic diameter when compared to P1 and P3 in water and PBS respectively. However, polymers P4 and P5 with similar DP and steroid to Jeffamine M1000 ratio than P2 , displayed lower average hydrodynamic sizes than P2 . It is noteworthy that polymers P1 - P6 exhibited a bimodal size distribution by intensity or volume of their hydrodynamic diameters (ESI † ), related to the dynamic formation of aggregates with different sizes in water, probably due to the self - assembly process. Particularly, polymers P2, P4 and P5 showed unimodal distribution size by volume with sole peaks observed at 9 - 10 nm, which indicate the key contribution of single or few polymer chains to the self - assembly process of these amphiphilic polyphosphazene as small aggregates in water. However, further studies are required for a proper understanding

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of the factors controlling the self - assem bly mechanism and the hydrodynamic sizes ( d h ) of polyphosphazenes in water. Nevertheless, polymers P1 - P5 formed aggregates in aqueous medium with appropriated sizes (less than 200 nm) for possible medical or agrochemical applications. 18,47,48 Dried aggreg ates of P1 - P5 polymers appeared as aggregates and single rounded particles of approximately 35 to 90 nm when observed using AFM and TEM ( Fig. 2 , Fig. SI - 37 and SI - 38 in the ESI † ), with significant P1 - P5 shrinkage attributed to water removal during drying. 4 9 Particle sizes determined using AFM followed the same trend of hydrodynamic sizes estimated in water with DLS.

Fig. 2 (a) Hydrodynamic diameter by intensity distribution for polymers P1 - P3 in deionised water (concentration 1 mg mL - 1 ). Further DLS data can be seen in the ESI ( Fig. SI - 37 to SI - 45 in the ESI † ). Inserts show AFM micrographs of polymers P1 - P3 dispersed in deionised water (concentration 1 mg mL - 1 ). (b) TEM micrographs of polymers P1 - P3 dispersed in deionised water (concentration 1 mg mL - 1 ).

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Diosgenin and brassinosteroid release studies In this research, the influence of diosgenin content in weight (wt.%) of polymers P1 (26.5%) and P3 (26.6%) compared to P2 (16.2%), the hydrodynamic sizes of the polyphosphazenes P1 and P3 compared to P2 , and the DP of polymers P1 and P2 (n=25) compared to P3 (n=50) on the release profiles are studied. All in vitro release experiments were carried out at pH 6.0 and 25 o C to simulate the slightly acidic environment found inside the vacuoles in vegetal cells, wh ere the particles are stored once having entered the plant cells via endocytosis. 50,51 It must be noted that hydrolysis of the ester bond in the steroid glycinate moiety and degradation of the polyphosphazene backbone in aqueous medium at neutral pH and 25 o C is minimal or very slow, but significant hydrolysis occurs at acidic conditions. 25,26 However, in vivo vacuolar degradation of substances in plants is mostly enzyme - promoted and the loaded polymers P1 - P5 will suffer hydrolysis faster and more extensive ly due to esterases, phosphatases and other enzymes. 52 - 54 The release experiments were extended up to 106 h (approximately 4 days), because intended in vitro activity evaluation of agrochemical bearing polymers P4 and P5 will be performed 3 days after appl ication. The in vitro release profiles of diosgenin bearing polymers P1 - P3 in PBS (pH 6.0) at 25 o C are shown in Fig. 3a ). The observed trend of release rates was P2 > P3 ≈ P1 , with a cumulative diosgenin release of ca. 33% ( P2 ) and 21% ( P1 and P3 ) after 4 days. Releases appeared faster and almost linear during the first 8 h, with approximately half of total diosgenin released with slope ranged 1.34 to 2.5 % h - 1 , and adjus ted R - Square 0.98 - 0.99 ( Table SI - 1 in the ESI † ). The more hydrophilic and bigger particles of polymer P2 , with a lower content of hydrophobic diosgenin, exhibited a faster and significantly higher drug release after 4 days than polymers P1 and P3 , both wit h 26.5% of diosgenin. It was expected that the more hydrophilic polymer P2 with a diosgenin content in weight 40% lower than in P1 and P3, and with the capability to form particles with 34% bigger hydrodynamic diameters in PBS will occur with less compact and less dense hydrophobic cores ( Fig. 3c ), which are more suitable for ester hydrolysis and diosgenin release. On the other hand, polymers P1 and P3 with same diosgenin contents and quite similar hydrodynamic diameters of particles in aqueous medium showe d similar release profiles and no significant difference of total diosgenin released after 106 h at a 95% confidence level (Means comparison: t = - 2.236 at p - value = 0.0890; Variance comparison: F = 1.29132 at p - value = 0.8729. Statistical analyses conduct ed with Statgraphics Plus 5.1, licensed to JKU).

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Agrochemicals content in weight (wt.%) of polymers P4 and P5 was found to be 16.3 wt.% of DI31 ( P4 ) and 17.6 wt.% of S7 ( P5 ) respectively, as estimated using 1 H NMR data (ESI † ). Release profile of S7 bearing polymer P5 is characterised by an initial burst release up to 15.5% in first 2 h ( Fig. 3b ). It caused a fast S7 release rate during the first 8 h deviated of linear behaviour (adjusted R - Square 0.9132, Table SI - 1 in the ESI † ), followed with a slower relea se rate from 24 h to 106 h ( Fig. 3b ).

Fig. 3 (a) Cumulative diosgenin release from polymers P1 to P3 at 25 o C in PBS at pH 6.0; (b) Cumulative steroid release from polymers P4 and P5 at 25 o C in PBS at pH 6.0. The amount of diosgenin and steroids D I31 and S7 released were estimated using a calibration curve for the free drugs in PBS at pH 6.0. (c) Cross - section scheme of proposed structure of polyphosphazene nanoaggregates P1 - P3 and P5 in aqueous medium.

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The initial burst release of S7 from polymer P5 might be due to the mobility of lateral chain (carbons C20 to C29) in S7 combined with superior hydrophilicity of polymers P4 + and P5 , which aid to H 3 O and water uptake (required for generation of highly active acylium ion and further reaction with wat er during ester hydrolysis) 55 in a less hydrophobic core of P5 aggregates ( Fig. 3c ). 56 However, there is no significant difference of total agrochemicals released after 106 h for polymers P4 and P5 (30.9% of DI31 in polymer P4 or S7 in polymer P5 was rele ased) at 95% confidence level (Means comparison: t = - 2.589 at p - value = 0.0607; Variance comparison: F = 0.960533 at p - value = 0.9799). It is hypothesised that hydrolysis of ester bond between the steroids and glycinate linker attaching the steroids to th e polyphosphazene backbone determine the steroid release rates for polymers P1 - P5 , because drug releases of steroid - grafted celluloses and chitosans were also extended during 3 - 4 days when carried out in similar conditions. 15,46 On the other hand, glycine - Jeffamine M1000 bearing polyphosphazene is known to degrade slower, with approximately 10% of the polymer degraded after 5 days in acetate buffer (pH 5.0) at 37 o C. 26

In vitro agrochemical activity The results as plant growth enhancers of DI31 bearing p olymer P4 and S7 bearing polymer P5 , against radish ( Raphanus sativus ) plants are shown in Fig. 4 . The methodology employed to evaluate the agrochemical activity of studied compounds is based on detection of auxin type activity, 44,57 expressed as increased weight of radish cotyledons (Fig. 4a ). It was observed the same agrochemical activity for both polymers P4 and P5 when applied at same concentrations, except at 10 - 3 and 10 - 6 mg mL - 1 with P4 (DI31) significantly more effective as plant growth stimulator t han P5 (S7) ( Fig. 4b ). Stimulatory effect of polymers P4 and P5 showed maximal at 10 - 1 and 10 - 2 mg mL - 1 concentrations, with three times increased radish cotyledons weight as compared to radish cotyledons treated with water (control) ( Fig. 4b ). Further dil utions of P4 and P5 until 10 - 3 to 10 - 7 mg mL - 1 concentration of polymers still resulted in similar and good agrochemical activity, with twice radish cotyledon weight increase as compared to control experiment ( Fig. 4b ). Particularly attractive for further in vivo agrochemical application of polymers P4 and P5 is the superior plant growth enhancer effect observed at lowest polymer concentrations of 10 - 6 and 10 - 7 mg mL - 1 (content 10 - 6.8 and 10 - 7.8 mg mL - 1 of DI31 and S7) as compared to agrochemical effect of parent DI31 and S7 evaluated at 10 - 7 mg mL - 1 ( Fig. 4b and Fig. SI - 52 in the ESI † ). In the same way, polymers P4 and P5 displayed higher agrochemical activity at all concentrations as compared to DI31 and S7

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tested at same brassinosteroids concentration, ex cept when P4 and P5 were applied at 10 - 3 mg mL - 1 (content 10 - 3.8 mg mL - 1 of DI31 and S7) ( Fig. 4b and Fig. SI - 52 in the ESI † ). The agrochemical activity of parent DI31, S7 ( Fig. SI - 52 in the ESI † ) showed the same plant growth enhancer effect for both DI31 and S7 agrochemicals when evaluated at equal concentrations. The agrochemical activity of parent DI31 and S7 resulted diminished with dilutions, but DI31 and S7 still exhibited significant stimulatory activity when applied at 10 - 6 and 10 - 7 mg mL - 1 (nano molar concentration of synthetic analogues brassinosteroids and natural brassinosteroids are applied as agrochemicals in crops). 58,59 On the other hand, no stimulatory effect was observed for the Jeffamine M1000 bearing polymer P6 at all concentrations. Th erefore, the beneficial effects of DI37 bearing polymer P4 and S7 bearing polymer P5 , observed on radish plants might be due to the sustained release of the incorporated agrochemicals DI31 and S7.

Fig. 4 (a) Scheme of the process to evaluate the agroche mical activity of polymers P4 (DI31), P5 (S7), P6 (Jeffamine M1000), DI31 and S7 using radish cotyledons obtained from radish plants. (b) In vitro agrochemical activity expressed in terms of increased weight of 10 radish cotyledons as a function of concent ration of applied polymers P4 (DI31) and P5 (S7) (bottom axis) and of concentration of DI31 and S7 carried on the applied polymers P4 (DI31) and P5 (S7) (top axis), C refers to control (radish cotyledons treated with water). See the graph showing the agr ochemical activity of parent DI31, S7 and Jeffamine M1000 bearing polymer P6 in the ESI † ( Fig. SI - 52 in the ESI † ).

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Cytotoxic activity The relative cell viabilities of MCF - 7 and HEK - 293 cells are shown in Fig. 5 .

Fig. 5 Relative cell viability of (a) MC F - 7 breast cancer cells and (b) non - cancer HEK - 293 cells treated with polymers P1 - P5 .

The Jeffamine M1000 bearing polymer P6 appeared no toxic to MCF7 and HEK - 293 cells at 0.1 mg mL - 1 , with relative cell viabilities of (97 ± 4)% and (87 ± 8)%, respectivel y (Data not shown). The diosgenin bearing polymers P1 - P3 showed slight to moderate anticancer effect (relative cell viabilities of 60 - 80%) when evaluated on MCF - 7 at 0.1 to 0.025 mg mL - 1 (diosgenin content of 0.0266 to 0.00665 mg mL - 1 for P1 and P3 , and 0.0162 to 0.00405 mg mL - 1 for P2 ) ( Fig. 5a ), which resulted similar to the cytotoxic effect observed for parent diosgenin when evaluated on MCF - 7 at 0.01 to 0.005 mg mL - 1 ( Fig. SI - 53 in the ESI † ). These polymers also exhibited slight toxicity on non - cancer HEK - 293 cells at 0.1 to 0.025 mg mL - 1 , with observed relative cell viabilities of approximately 80% ( Fig. 5b ), which resulted slightly more cytotoxic than parent diosgenin at 0.01 to 0.005 mg mL - 1 (relative cell viability of 91 - 97%) ( Fig. SI - 54 in the ESI † ). Interestingly, DI31 bearing polymer P4 and S7 bearing polymer P5 exhibited

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strong to moderate toxicity to both cancer MCF - 7 cells and non - cancer HEK - 293 cells at 0.1 to 0.00625 mg mL - 1 (DI31 and S7 content of approximately 0.017 to 0.0010625 mg mL - 1 ), with observed relative cell viabilities of 50 - 70% ( Fig. 5 ). 101 nm P4 and 163 nm P5 might show extended circulation time and slow renal clearance, with accumulation in the cancer tissues due to passive diffusion and enhanced permeability and retention (EPR ) effect. 60,61 On the other hand, polymers P4 and P5 appeared no toxic to MCF - 7 and HEK - 293 cells (relative cell viabilities of approximately 94%) at lowest evaluated concentration of 0.00156 mg mL - 1 , related to DI31 and S7 contents of approximately 2.6 10 - 4 mg mL - 1 . Therefore, polymers P4 and P5 are safe to be used as agrochemicals at concentrations below 10 - 3 mg mL - 1 , for delivery of recommended 10 - 4 to 10 - 7 mg mL - 1 of DI31 or S7 to the crops. 11,13,58,59 The experimental results discussed herein demonstr ate that it is possible to tune the steroid release rate of polyphosphazene - based polymers with diosgenin and Jeffamine M1000 as co - substituents ( P1 - P3 ), as a function of hydrodynamic sizes and hydrophilicity of the synthesised polymers. Later post - polymer isation functionalisation together with the right selection of substituents allows obtaining polymers with different hydrophilicities that result in aqueous aggregates ranging from nanoaggregates to microaggregates. 28,29,31 The controlled and partial relea se of covalently linked DI31 from polymer P4 and S7 from polymer P5 , caused a very good in vitro agrochemical activity. Then, it is expected that polyphosphazenes P4 and P5 will exert an excellent and extended plant growth stimulator effect on crops using lower quantities than typical exogenous applications of DI31 and S7 when used as agrochemicals. 62,63 Additional benefits of polymers like P4 and P5 is the lower environmental impact associated to their use in agriculture, as they do not need incorporation of harmful additives to ensure colloidal stability and proper dispersion in water as the commercial formulations of DI31, 24 - epibrassinolide and other brassinosteroids on the market. 62,64 On the other hand, diosgenin bearing polymers P1 - P3 exhibited modera te cytotoxicity to MCF - 7 and HEK - 293 at medium to high concentration. DI31 bearing polymer P4 and S7 bearing polymer P5 showed strong to moderate cytotoxicity to MCF - 7 and HEK - 293 at almost all concentrations tested. Direct injection of P4 and P5 particles in solid tumours and/or their selective accumulation in cancer tissues via EPR effect and passive diffusion might enhance the therapeutic effect with a reduction of side effects. 60,61,65 Contrariwise, P4 and P5 polymers result in no toxicity to both cell lines when applied at 0.00156 mg mL - 1 . Thus, polymers P4 and P5 are safe to be used in

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agriculture at concentrations below 10 - 3 mg mL - 1 . Additionally, degradation of polyphosphazene backbones to ammonia and phosphates in aqueous medium, 38 a broad research about various polyphosphazenes in nanomedicine based on their biocompatibility, 66,67 no cytotoxic and inhibitory agrochemical effect observed for polymer P6 put forward a potential application of polymers P4 and P5 for chemotherapy treatments and in agricu lture.

4.1.4. Conclusions Six different polyphosphazene - based nanocarriers were prepared for sustained release of diosgenin and the agrochemicals DI31 and S7, via controlled living cationic polymerisation and later post - polymerisation functionalisation wit h the steroids and hydrophilic Jeffamine M1000, an amine capped polyetheramine copolymer. Exhaustive characterisation of synthesised polyphosphazenes P1 - P3 , carrying diosgenin as a model - drug, revealed that the hydrophilicity of the polymers, hydrodynamic particle sizes and observed drug release rates can be tailored as required upon precise stoichiometry control of post - polymerisation functionalisation. Polyphosphazenes P1 - P3 showed moderate toxicity to cancer MCF - 7 and non - cancer HEK - 293 cells when evalua ted at medium to high concentration. Further in vivo studies are required to evaluate the selectivity of these polymers to cancer tissues, the toxicity to non - cancer tissues, the pharmacokinetics and bioavailability, as well as other relevant parameters be fore a decision on their potential chemotherapeutic use can be done. Polyphosphazenes P4 and P5 carrying approximately 17 wt.% of DI31 ( P4 ) and S7 ( P5 ) respectively, formed aqueous nanoaggregates that exerted excellent stimulant plant growth effect on radi sh cotyledons due to the controlled delivery of the agrochemicals. Cytotoxic evaluation of polymers P4 and P5 showed that both polyphosphazenes resulted toxic to MCF - 7 and HEK - 293 at all concentrations evaluated, except at 0.00156 mg mL - 1 . Therefore, agroc hemical use of the polymers P4 and P5 at concentrations lower than 10 - 3 mg mL - 1 seems to be safe. P4 and P5 applied at 10 - 4 to 10 - 6 mg mL - 1 for delivery of recommended DI31 or S7 quantities to the plants should be harmless to humans. Particularly attractiv e for agricultural applications results the inherent degradability of the polyphosphazenes, which might be translated into greener agrochemical formulations. Additionally, strong antiproliferative activity of P4 and P5 on MCF - 7 breast cancer cells at a bro ad concentration range, together with optimal hydrodynamic sizes (101 nm and 163 nm) and slow sustained release of

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the cytotoxic DI31 and S7, are expected to provoke a good therapeutic effect on cancer tissues with reduced systemic side effects after admin istration. However, as indicated before for diosgenin bearing polymers, exhaustive in vivo evaluation of these potential anticancer polyphosphazenes must be performed before their clinical use as antitumoral agents. The results attained suggest that prepar ation of polyphosphazenes with attached brassinosteroids (i.e. P4 and P5 ) is a promising strategy for the synthesis of more efficient agrochemicals with reduced pollutant impact on a sustainable agriculture, and of potential anticancer formulations based o n analogues of brassinosteroids.

Acknowledgements Erasmus Mundus is acknowledged for a scholarship to JPQ. The authors would like to acknowledge Prof. Ian Teasdale for his valuable comments on the manuscript. The authors thank Günter Hesser for training wi th TEM imaging of nanoparticles and Lisa M. Uiberlacker for AFM imaging of nanoparticles at JKU Linz, Austria. The access to NMR facilities of Upper Austrian – South Bohemian Research Infrastructure Center in JKU Linz, Linz, Austria, supported by the Europ ean Union (ETC Austria - Czech Republic 2007 - 2013, Project M00146) is also acknowledged.

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SUPPORTING INFORMATION

Javier Pérez Quiñones, Aitziber Iturmendi, Helena Henke, Cornelia Roschger, Andreas Zierer and Oliver Brüggemann

1. Support ing Tables Table SI - 1 . Linear fitting parameters of in vitro release profiles of polymers P1 - P5 up to 8 h (intercept 0, slope k, adjusted R - Square) in PBS (pH 6.0) at 25 o C Samples k Adjusted R - Square P1 1.34 ± 0.09 0.9786 P2 2.5 ± 0.1 0.9833 P3 1.72 ± 0.06 0.9978 P4 1.65 ± 0.07 0.9899 P5 3.7 ± 0.5 0.9132

2. Synthesis of materials, monomer and polymers

Synthesis of diosgenin - glycine - NH 2 ( 1 ). Boc - protected amino acid Boc - Gly - OH (0.13 g, 0.72 mmol), 4 - (dimethylamino)pyridine (0.09 g, 0.72 mmol) and N, N’ - dicyclohexylcarbodiimide (0.19 g, 0.72 mmol) were dissolved in 20 mL CH 2 Cl 2 and stirred at room temperature for 2 h. The reaction mixture was added to a solution of diosgenin (0.30 g, 0.72 mmol) in 10 mL CH 2 Cl 2 and stirred for 48 h. The precipitated N,N ’ - dicyclohexylurea was removed by filtration and the filtrated was extracted with

10% NH 4 Cl aqueous solution (2 × 15 mL), with 5% NaHCO 3 aqueous solution (2 × 15 mL) and saturated NaCl solution (1 × 15 mL). The organic phase was dried over

MgSO 4 , filtered and removed under reduced pressure to yield diosgenin - glycine - Boc as a white solid (0.27 g, yield 65%). Then, diosgenin - glycine - Boc (0.27 g, 0.47 mmol) was dissolved in 10 mL of CH 2 Cl 2 . CF 3 COOH (1 mL, 13.10 mmol) was added dropwise to the diosgenin - glycine - Boc solution and stirred at room temperature overnight. The excess of CF 3 COOH and the solvent were removed under reduced pressure, CH 2 Cl 2 was added and removed under reduced pressure twice. The remnant solid was dissolved again in 30 mL of CH 2 Cl 2 and wash ed with 5% NaHCO 3 aqueous solution (2

× 15 mL), saturated NaCl solution (1 × 15 mL) and dried over MgSO 4 . The CH 2 Cl 2 was

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removed under reduced pressure, to obtain diosgenin - glycine - NH 2 ( 1 ) as a white powder (0.19 g, yield 85%).

Synthesis of monomer trichlo rophosphoranimine ( Cl 3 P=N – Si(CH 3 ) 3 ). Lithium bis(trimethylsilyl)amide (LiN(Si(CH 3 ) 3 ) 2 ) (25.00 g, 149.41 mmol) was dissolved in 500 o mL of anhydrous Et 2 O under Ar atmosphere, cooled to 0 - 4 C with ice bath and stirred for 0.5 h. 13.07 mL of PCl 3 (20.52 g, 14 9.41 mmol) were slowly added dropwise with a 20 mL syringe, while reaction mixture was stirred at 0 - 4 o C. Then, the reaction mixture was stirred at room temperature for 1 h. The solution was cooled to 0 - 4 o C with ice bath for a second time, 12.1 mL of SO 2 C l 2 (20.17 g, 149.41 mmol) were added dropwise and the mixture was stirred for 1 h at 0 - 4 o C. Then, the reaction mixture was fast filtered over Celite and Et 2 O was removed under reduced pressure at room temperature. The purification of the product was carri ed out via vacuum distillation with a Büchi glass oven (Büchi Labortechnik, Switzerland) at 40 o C under reduced pressure of 4 mbar to obtain Cl 3 P=N – Si(CH 3 ) 3 as a colourless liquid. The monomer was stored under Ar atmosphere at - 35 o C in the glovebox (18.0 0 g, yield 54%). 1 H NMR (300 31 1 MHz, CDCl 3 ):  = 0.16 (s, 9H) ppm; P{ H} NMR (121 MHz, CDCl 3 ):  = - 54.2 ppm.

Synthesis of the polymer P1 . (C 6 H 5 ) 3 PCl 2 (2.08 mg, 0.006 mmol) and Cl 3 P=N – Si(CH 3 ) 3

(35.00 mg, 0.15 mmol) were dissolved in 1 mL of anhydrous CH 2 Cl 2 and stirred at room temperature overnight. Then, the obtained poly(dichloro)phosphazene (yield quantitative) was transferred to another flask with diosgenin - glycine - NH 2 ( 1 ) (73.60 mg,

0.16 mmol) and an excess of Et 3 N (72.6 mg, 0.72 mmol) in 10 mL of anhyd rous THF, and stirred at room temperature for 24 h. Afterwards, the second post - polymerisation functionalisation was carried out with Jeffamine M1000. An excess of Jeffamine M1000

(0.22 g, 0.22 mmol) and Et 3 N (108.9 mg, 1.08 mmol) were added to the mixture and allowed to react another 24 h. Once the reaction was completed, the solvent was removed under reduced pressure and the product was purified by dialysis against deionised water (3 L, 1 time, 10 hours) and EtOH (750 mL, 10 times, 5 days). The

EtOH was r emoved with N 2 flow and the polymer was further dried under vacuum to give the polymer P1 as a colorless waxy solid (101.3 mg, yield 36%).

3. Characterisation data for materials and polymers P1 - P6

Diosgenin - glycine - NH 2 ( 1 ): ATR - FTIR (solid)  max: 2943 (C – H), 1726 (C=O), 1602 (N – H bending), 1557 (N – H bending), 1453 (C – H bending), 1212 (C – O – C) cm - 1 ; 1 H

NMR (300 MHz, 298 K, CDCl 3 ):  = 0.79 (t, 6H, J = 3.1 Hz, H18 + H27), 0.97 (d, 3H, J

= 6.9 Hz, H21), 1.03 (s, 3H, H19), 3.37 (t, 1H, J = 10.8 Hz, H26ax), 3.4 2 (s, 2H, NH 2 –

C H 2 – COO – ), 3.47 (m, 1H, H26eq), 4.41 (q, 1H, J = 7.4 Hz, H16α), 4.66 (m, 1H, H3α),

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13 5.38 (d, 1H, J = 4.7 Hz, H6) ppm; C NMR (75 MHz, CDCl 3 ):  = 14.7 (C21), 16.4 (C18), 17.3 (C27), 19.5 (C19), 21.0 (C11), 27.9 (C2), 29.0 (C24), 30.4 (C8 + C2 5), 32.0 (C23), 32.2 (C7), 34.1 (C15), 36.9 (C10), 37.1 (C1), 38.2 (C4), 39.9 (C12), 40.4 (C13), 41.8 (C20), 44.1 (NH2 – C H2 – COO – ), 50.1 (C9), 56.6 (C14), 62.2 (C17), 67.0 (C26), – 74.8 (C3), 80.9 (C16), 109.4 (C22), 122.7 (C6), 139.6 (C5), 156.8 (CF 3 C OO ), 17 3.4 ( C =O) ppm. 41 It must be noted the presence of characteristic 13 C chemical shifts of – CF 3 C OO related to obtaining steroid - glycine - NH 2 compounds as [steroid - glycine - + – NH 3 ][CF 3 COO ].

DI31 - glycine - NH 2 ( 2 ): ATR - FTIR (solid)  max: 3325 (N – H), 2929 (C – H), 173 6 (C=O), 1713 (C=O, ketone C6 of DI31), 1627 (N - H bend), 1578 (N - H bend), 1454 (C – H bend), - 1 1 1216 (C – O – C) cm ; H NMR (300 MHz, 298 K, CDCl 3 ):  = 0.75 (s, 3H, H18), 0.78 (d, 3H, J = 6.4 Hz, H27), 0.82 (s, 3H, H19), 0.96 (d, 3H, J = 6.7 Hz, H21), 2.77 (t, 1 H, J =

12.1 Hz, H7α), 3.35 (t, 1H, J = 10.7 Hz, H26 ax), 3.45 (m, 3H, H26 eq + NH 2 – C H 2 – 13 COO – ), 4.40 (m, 1H, H16α), 5.09 (m, 1H, H3α) ppm; C NMR (75 MHz, CDCl 3 ):  = 14.1 (C19), 14.6 (C21), 16.5 (C18), 17.3 (C27), 21.3 (C11), 26.4 (C2), 28.9 (C24), 29.6 (C1), 30.4 (C25), 31.5 (C23), 31.7 (C15), 32.5 (C4), 36.9 (C8), 39.7 (C12), 41.2 (C13), 41.7 (C20), 41.9 (C7), 42.6 (C10), 44.0 (NH2 – C H2 – COO – ), 44.4 (C9), 56.2 (C14), 62.2 – (C17), 67.0 (C26), 71.6 (C3), 80.2 (C5), 80.6 (C16), 109.4 (C22), 157.1 (CF 3 C OO ), 172.8 ( C =O, glycine), 212.2 ( C =O, C6) ppm.

S7 - glycine - NH 2 ( 3 ): ATR - FTIR (solid)  max: 3321 (N – H), 2934 (C – H), 1711 (C=O, ketone C6 of S7), 1625 (N - H bend), 1574 (N - H bend), 1448 (C – H bend), 1212 (C – O – - 1 1 C) cm ; H NMR (300 MHz, 298 K, CDCl 3 ):  = 0.64 (s, 3H, H1 8), 0.80 (s,3H, H19), 0.93 (m, 6H, H26 + H27), 0.98 (t, 3H, J = 6.6 Hz, H29), 1.00 (m, 3H, H21), 2.50 (m, 1H, 13 H22), 2.73 (m, 1H, H23), 3.40 (s, 2H, NH 2 – C H 2 – COO – ), 5.10 (m, 1H, H3α) ppm; C

NMR (75 MHz, CDCl 3 ):  = 12.2 (C18), 12.6 (C29), 14.1 (C19), 16.5 (C21), 19.2 (C27), 19.4 (C26), 21.5 (C11 + C28), 25.8 (C15), 26.4 (C2), 29.3 (C25), 29.4 (C16), 29.7 (C1), 32.6 (C4), 37.5 (C8), 39.7 (C12), 39.9 (C20), 41.9 (C7), 42.6 (C10), 43.3 (C13), 44.0 (NH2 – C H2 – COO – ), 44.4 (C9), 49.3 (C24), 56.0 (C17), 56.5 (C14), 62.6 (C22 + C23), – 71.8 (C3), 80.3 (C5), 156.9 (CF 3 C OO ), 173.8 ( C =O, glycine), 212.5 ( C =O, C6) ppm.

Polymer P1 : ATR - FTIR (solid)  max: 3339 (N – H), 2870 (C – H), 1741 (C=O), 1452 (C – - 1 1 H bending), 1100 (C – O – C), 1050 (P=N) cm ; H NMR (300 MHz, CDCl 3 ):  = 0.77 (s, 6H, H18 + H27), 0.95 (d, 3H, J = 6.5 Hz, H21), 0.99 (s, 3H, H19), 1.10 (br, 6H, – O –

CH 2 – CH(C H 3 ) – of Jeffamine M1000), 3.36 (s, 3H, C H 3 O – end groups of Jeffamine

M1000), 3.62 (m, 35H, polyalkylene oxide – C H 2 – of Jeffamine M1000), 4.39 (m, 1H,

H16α), 4.5 3 (m, 1H, H3α), 5.32 (s, 1H, H6), 7.60 (d, 0.65H, protons of (C 6 H 5 ) 3 P=N –

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31 1 13 end group) ppm; P{ H} NMR (121 MHz, CDCl 3 ):  = 0.6 ppm; C NMR (75 MHz,

CDCl 3 ):  = 14.7 (C21), 16.4 (C18), 17.3 (C27), 19.5 (C19), 20.9 (C11), 27.8 (C2), 28.9 (C24), 30.4 (C8 + C25), 32.0 (C23), 32.2 (C7), 33.8 (C15), 36.8 (C10), 38.1 (C4), 39.9

(C12), 40.4 (C13), 41.7 (C20), 45.2 ( – NH – C H 2 – COO – ), 50.1 (C9), 56.7 (C14), 59.1 ( –

O – CH 2 – C H(CH 3 ) – of Jeffamine M1000), 62.3 (C17), 66.9 (C26), 70.7 ( – O – C H 2 – C H 2 –

O – of Jeffamine M1000), 72.0 ( – O – C H 2 – CH(CH 3 ) – of Jeffamine M1000), 74.3 (C3), - 1 80.8 (C16), 109.3 (C22), 122.4 (C6), 139.8 (C5), 171.7 ( C =O) ppm. GPC (g mol ) M n = o 9586, M w = 13134. Glass transition temperature ( T g ) = - 17.2 C.

Polymer P2 : ATR - FTIR (solid)  max: 3312 (N – H), 2867 (C – H ), 1740 (C=O), 1453 (C – - 1 1 H bend), 1100 (C – O – C), 1052 (P=N) cm ; H NMR (300 MHz, 298 K, CDCl 3 ):  = 0.77 (s, 6H, H18 + H27), 0.95 (d, 3H, J = 6.3 Hz, H21), 1.00 (s, 3H, H19), 1.11 (br, 14H,

CH 3 – of PPO groups of Jeffamine M 1000), 3.36 (s, 6H, CH 3 O – end grou ps of

Jeffamine M 1000), 3.63 (m, 119H, polyalkylene oxide – CH 2 – ), 4.40 (m, 1H, H16α), 31 5.33 (s, 1H, H6), 7.61 (d, 0.68H, (C 6 H 5 ) 3 P=N – end group) ppm; P NMR (121 MHz, - 1 CDCl 3 ):  = 0.8 ppm. GPC (g mol ) M n = 15048, Mw = 22121. Glass transition o o temperature ( T g ) = - 13.5 C, melting temperature ( T m ) = 17.9 C.

Polymer P3 : ATR - FTIR (solid)  max: 3327 (N – H), 2869 (C – H), 1741 (C=O), 1453 (C – - 1 1 H bend), 1098 (C – O – C), 1051 (P=N) cm ; H NMR (300 MHz, CDCl 3 ):  = 0.77 (s, 6H,

H18 + H27), 0.95 (d, 3H, J = 6.3 Hz, H21), 0.99 (s, 3H, H19), 1.11 (br, 6H, CH 3 – of

PPO groups of Jeffam ine M 1000), 3.36 (s, 3H, CH 3 O – end groups of Jeffamine

M1000), 3.62 (m, 32H, polyalkylene oxide – CH 2 – ), 4.39 (m, 1H, H16α), 4.53 (m, 1H, 31 H3α ), 5.32 (s, 1H, H6), 7.61 (d, 0.44H, (C 6 H 5 ) 3 P=N – end group) ppm; P NMR (121 - 1 o MHz, CDCl 3 ):  = 0.8 ppm. GPC (g mol ) M n = 11926, Mw = 15624. T g = - 60.8 C.

Polymer P4 : ATR - FTIR (solid)  max: 3518 (O – H), 3316 (N – H), 2868 ( C – H), 1734 (C=O), 1714 (C=O of ketone), 1454 (C – H bend), 1104 (C – O – C), 1040 (P=N) cm - 1 ; 1 H

NMR (300 MHz, CDCl 3 ):  = 0.78 (s, 9H, H18 + H19 + H27), 0.94 (s, 3H, H21), 1.11

(br, 14H, CH 3 – of PPO groups of Jeffamine M 1000), 3.36 (s, 6H, CH 3 O – end groups of

J effamine M 1000), 3.63 (m, 108H, polyalkylene oxide – CH 2 – ), 4.38 (m, 1H, H16α), 31 7.60 (d, 0.75H, (C 6 H 5 ) 3 P=N – end group) ppm; P NMR (121 MHz, CDCl 3 ):  = 0.8 - 1 o o ppm. GPC (g mol ) M n = 14108, Mw = 19046. T g = - 14.1 C, T m = 20.9 C.

Polymer P5 : ATR - FTIR (solid)  max: 3303 (N – H), 2867 (C – H), 1734 (C=O), 1713 (C=O of ketone), 1455 (C – H bend), 1103 (C – O – C), 1038 (P=N) cm - 1 ; 1 H NMR (300

MHz, CDCl 3 ):  = 0.62 (s, 3H, H18), 0.77 (s, 3H, H19), 0.81 (d, 3H, J = 6.6 Hz, H21),

0.91 (m, 9H, H26 + H27 + H29), 1.11 (br, 15H, C H 3 – of PPO groups of Jeffamine

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M1000), 3.35 (s, 6H , C H 3 O – end groups of Jeffamine M 1000), 3.62 (m, 137H, polyalkylene oxide – C H 2 – ), 5.06 (s, 1H, H3α), 7.60 (d, 0.67H, (C 6 H 5 ) 3 P=N – end group) 31 - 1 ppm; P NMR (121 MHz, CDCl 3 ):  = 0.8 ppm. GPC (g mol ) M n = 13016, Mw = o o 19524. T g = - 13.6 C, T m = 17.6 C.

Polymer P6 : ATR - FTIR (solid)  max: 3300 (N – H), 2883 (C – H), 1459 (C – H bend), - 1 1 1108 ( C – O – C), 1041 (P=N) cm ; H NMR (300 MHz, CDCl 3 ):  = 1.12 (br, 14H, C H 3 – of PPO groups of Jeffamine M 1000), 3.36 (s, 6H, C H 3 O – end groups of Jeffamine

M1000), 3.63 (m, 148H, polyalkylene oxide – C H 2 – ), 7.60 (d, 0.66H, (C 6 H 5 ) 3 P=N – end 31 - 1 group) ppm; P NMR ( 121 MHz, CDCl 3 ):  = 1.0 ppm. GPC (g mol ) M n = 10783, Mw o o = 15489. T g = - 18.1 C, T m = 27.9 C. 4. Supporting Figures

Figure SI - 1 UV spectra of: (a) diosgenin at 0.056 mg mL - 1 , (b) DI31 at 0.057 mg mL - 1 , S7 at 0.058 mg mL - 1 in PBS (pH 6.0).

Fig ure SI - 2 Calibration curve of diosgenin.

202

Figure SI - 3 Calibration curve of DI31.

Figure SI - 4 Calibration curve of S7.

Figure SI - 5 FT - IR spectra of diosgenin - glycine - Boc and diosgenin - glycine - NH 2 ( 1 ).

203

Figure SI - 6 FT - IR spectra of DI31 - gly cine - Boc and DI31 - glycine - NH 2 ( 2 ).

Figure SI - 7 FT - IR spectra of S7 - glycine - Boc and S7 - glycine - NH 2 ( 3 ).

Figure SI - 8 FT - IR spectra of polymer P1 .

204

Figure SI - 9 FT - IR spectra of polymer P2 .

Figure SI - 10 FT - IR spectra of polymer P3 .

Figu re SI - 11 FT - IR spectra of polymer P4 .

205

Figure SI - 12 FT - IR spectra of polymer P5 .

Figure SI - 13 FT - IR spectra of polymer P6 .

1 Figure SI - 14 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of diosgenin - glycine -

NH 2 ( 1 ).

206

1 Figure SI - 15 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of DI31 - glycine - NH 2 ( 2 ).

1 Figure SI - 16 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of S7 - glycine - NH 2 ( 3 ).

1 Figure SI - 17 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of monomer trichlorophosphoranimine ( Cl 3 P=N – Si(CH 3 ) 3 ).

207

1 Figure SI - 18 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of polydichlorophosphazene.

1 Figure SI - 19 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of polymer P1 .

1 Figure SI - 20 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectr um of polymer P2 .

208

1 Figure SI - 21 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of polymer P3 .

1 Figure SI - 22 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of polymer P4 .

1 Figure SI - 23 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of polyme r P5 .

209

1 Figure SI - 24 Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of polymer P6 .

13 Figure SI - 25 Partial APT C NMR (75 MHz, 298 K, CDCl 3 ) spectrum of diosgenin - glycine - NH 2 ( 1 ).

13 Figure SI - 26 Partial APT C NMR (75 MHz, 298 K, CDCl 3 ) spectru m of DI31 - glycine -

NH 2 ( 2 ).

210

13 Figure SI - 27 Partial APT C NMR (75 MHz, 298 K, CDCl 3 ) spectrum of S7 - glycine -

NH 2 ( 3 ).

13 Figure SI - 28 Partial APT C NMR (75 MHz, 298 K, CDCl 3 ) spectrum of polymer P1 .

31 1 Figure SI - 29 Partial P{ H} NMR (121 MHz, 298 K, CDCl 3 ) spectrum of monomer trichlorophosphoranimine (Cl 3 P=N – Si(CH 3 ) 3 ).

211

31 1 Figure SI - 30 Partial P{ H} NMR (121 MHz, 298 K, CDCl 3 ) spectrum of polydichlorophosphazene.

31 1 Figure SI - 31 Partial P{ H} NMR (121 MHz, 298 K, CDCl 3 ) spectrum of polymer P1 .

31 1 Figure SI - 32 Partial P{ H} NMR (121 MHz, 298 K, CDCl 3 ) spectrum of polymer P2 .

212

31 1 Figure SI - 33 Partial P{ H} NMR (121 MHz, 298 K, CDCl 3 ) spectrum of polymer P3 .

31 1 Figure SI - 34 Partial P{ H} NMR (121 MHz, 298 K, CDCl 3 ) spectrum of polyme r P4 .

31 1 Figure SI - 35 Partial P{ H} NMR (121 MHz, 298 K, CDCl 3 ) spectrum of polymer P5 .

213

31 1 Figure SI - 36 Partial P{ H} NMR (121 MHz, 298 K, CDCl 3 ) spectrum of polymer P6 .

6 ) t 5 n e c r

e 4 P (

y t

i 3 s n e t 2 n

I 400 nm 1

0 0.1 1 10 100 1000 Size (d.nm) Figure SI - 37 Dynamic light scattering, size distribution by intensity o f polymer P4 . Insert: AFM image of P4 , scale bar = 400 nm.

8 ) t n e c

r 6 e P (

y t i

s 4 n e t 400 nm n I 2

0 0.1 1 10 100 1000 Size (d.nm) Figure SI - 38 Dynamic light scattering, size distribution by intensity of polymer P5 . Insert: AFM image of P5 , scale bar = 400 nm.

214

12 ) t 10 n e c r

e 8 P (

y t

i 6 s n e t 4 n I 2

0 0.1 1 10 100 1000 Size (d.nm) Figure SI - 39 Dynamic light scattering, size distribution by intensity of polymer P6 .

9 8

7 ) t

n 6 e c r

e 5 P (

e 4 m u l 3 o V 2 1

0 0.1 1 10 100 1000 Size (d.nm) Figure SI - 40 Dynamic light scattering, size distribution by volume of polymer P1 .

20 ) t n e

c 15 r e P (

m 10 u l o V 5

0 0.1 1 10 100 1000 Size (d.nm) Figure SI - 41 Dynamic light scattering, size distribution by volume of polymer P2 .

215

8 ) t n e

c 6 r e P (

m 4 u l o V 2

0 0.1 1 10 100 1000 Size (d.nm) Figure SI - 42 Dynamic light scattering, size dist ribution by volume of polymer P3 .

20 ) t n e

c 15 r e P (

m 10 u l o V 5

0 0.1 1 10 100 1000 Size (d.nm) Figure SI - 43 Dynamic light scattering, size distribution by volume of polymer P4 .

20 ) t n e

c 15 r e P (

m 10 u l o V 5

0 0.1 1 10 100 1000 Size (d.nm) Figure SI - 44 Dynamic light scattering, size distribution by volume of polymer P5 .

216

20 ) t n e

c 15 r e P (

m 10 u l o V 5

0 0.1 1 10 100 1000 Size (d.nm) Figure SI - 45 Dynamic light scattering, size distribution by volume of polymer P6 .

Figure SI - 46 Calorimetry differential scanning curve of polymer P1 .

Figure SI - 47 Calorimetry differential scanning curve of polymer P2 .

217

Figure SI - 48 Calorimetry differential scanning curve of polymer P3 .

Figure SI - 49 Calorimetry differential scanning curve of polymer P4 .

Figure SI - 50 Calorimetry differential scanning curve of polymer P5 .

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Figure SI - 51 Calorimetry differential scanning curve of polymer P6 .

Figure SI - 52 In vitro agroche mical activity expressed in terms of increased weight of 10 radish cotyledons as a function of concentration of applied polymer P6 , parent DI31 and S7, C refers to control (radish cotyledons treated with water). * Data not shown because radish cotyledons d ied as result of high ethanol content in DI31 and S7 solutions at 10 - 1 and 10 - 2 mg mL - 1 .

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Figure SI - 53 Relative cell viability of MCF - 7 breast cancer cells treated with parent steroids.

Figure SI - 54 Relative cell viability of non - cancer HEK - 293 ce lls treated with parent steroids.

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4.2. Polyphosphazene - based nanocarriers for the release of camptothecin and epirubicin

Three poly(organo)phosphazenes were prepared with tocopherol, testosterone and Jeffamine M - 1000 ( P1 - P 3 ) for camptothecin and epi rubicin loading ( TOC 6 ). The poly(organo)phosphazenes formed aggregates in aqueous media. Dried aggregates were observed as spherical nanoparticles using TEM and AFM ( TOC 6 ). Sustained release of CPT and EPI loaded in poly(organo)phosphazenes was observed during 6 days ( TOC 6 ). MCF - 7 cancer cells showed good uptake of CPT - and EPI - loaded poly(organo)phosphazenes after 6 hours ( TOC 6 ) .

+ EPI

MCF - 7

TOC 6. Structure s of tocopherol and testosterone bearing poly(organo)phosp hazenes ( P1 - P3 ), schematic representation of the formation of aggregates in aqueous media and CPT or EPI loading in P1 - P3 , TEM micrographs of P1 and P2 dried particles, MCF - 7 cells with characteristic fluorescence of EPI - loaded poly(organo)phosphazenes, an d EPI release from EPI - loaded P1 and P2 . Figure reproduced with permission.

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Polyphosphazene - based nanocarriers for the release of camptothecin and epirubicin

Javier P. Quiñones , a * Aitziber Iturmendi , a Helena Henke , a Cornelia Roschger , b Andreas F. Zier er , b Carlos Peniche - Covas , c Oliver Brüggemann a

––––––––– a Institute of Polymer Chemistry (ICP), Johannes Kepler University Linz, Altenberger Str. 69, 4040 Linz, Austria. b Johannes Kepler University Linz, Kepler University Hospital GmbH, Department for Cardiac - , Vascular - and Thoracic Surgery, Altenberger Str. 69, 4040 Linz and Krankenhausstraβe 7a, 4020 Linz, Austria. c Facultad de Química, Universidad de La Habana, Zapata S/N entre G y Carlitos Aguirre, 10400 La Habana, Cuba.

E - mail: [email protected] m

–––––––––

Submitted

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Abstract

Polyphosphazene nanocarriers are presented for the sustained release of the anticancer drugs camptothecin and epirubicin. The polymers are substituted with hydrophobic tocopherol or testosterone glycinate, and mix ed with hydrophilic polyalkylene oxide Jeffamine M1000, in a ratio of 1:1 or 1:3, respectively, to encapsulate the anticancer drugs. The encapsulation process is carried out via solvent exchange/precipitation, attaining camptothecin contents of 9.9 - 13.3 wt . %, and epirubicin contents of 0.3 - 2.3 wt. %. Camptothecin - loaded polyphosphazenes formed 140 - 200 nm aggregates at 1 mg/mL in simulated body physiological conditions (PBS, pH 7.4), for an 80 - 100 fold increase of camptothecin solubility. Similarly, epirubi cin - loaded polyphosphazenes formed 250 nm aggregates in aqueous media. Camptothecin and epirubicin release in simulated physiological conditions (PBS pH 7.4, 37 o C) was extended for 150 hours, being almost linear during the first 8 - 9 hours. Slow release of testosterone was also sustained for 150 hours in PBS (pH 7.4 and 6.0) at 37 o C, with potential in vivo co - delivery of testosterone or tocopherol and the anticancer drugs from anticancer drug loaded polyphosphazenes expected to exert synergic antitumor eff ect. MCF - 7 human breast cancer cells demonstrated good uptake of anticancer drug loaded polyphosphazene - based nanocarriers after 6 hours, shown by the intense fluorescence of the nanocarriers inside cells. Cytotoxic evaluation showed that all anticancer dr ug loaded polyphosphazenes showed similar toxicity to MCF - 7 cells than parent anticancer drugs at all concentrations, demonstrated that antitumor activity of camptothecin and epirubicin was maintained after loading in the nanocarriers and sustained release . Consequently, synthesised polyphosphazene - based nanocarriers might be potential nanomedicines for chemotherapy.

KEYWORDS: Camptothecin; Epirubicin; Polyphosphazene; Nanocarriers; Sustained release; Cell uptake.

4.2.1. Introduction Cancer is a major cause of death worldwide, with about 9.6 million cancer deaths in 2018 [1,2]. Lung, breast, prostate and colon cancer are the most common and fatal cancers with 11.6% to 6.1% of the new - diagnosed cases and 18.4% to 5.8% mortality [2]. Chemotherapy based on camp tothecin derivatives (i.e. topotecan, irinotecan) and epirubicin anticancer drugs occasionally fails in treatment of metastatic or resistant cancers, and patients relapse is observed in a moderated to low rate due to severe side effects of the antitumor dr ugs. Epirubicin, or more precisely epidoxorubicin, is an

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anthracycline antibiotic capable to inhibit topoisomerase II, leading to apoptosis of tumour cells due to its interference in the DNA replication process [3]. However, cardiotoxicity related to epiru bicin use is estimated to affect up to 5% of the patients after 15 years of the initial treatment [3]. On the other hand, camptothecins are the only available topoisomerase I inhibitors widely applied for treatment of several cancers despite the medium to severe side effects of this drug [4 - 6]. This drawback can be circumvented by the use of nanomedicines (i.e. the application of nanotechnology to medicine) [7]. The major focus of nanomedicine lies in new pharmacological and therapeutic release systems (dev elopment, bio - functionalisation and validation of nanoconjugates with therapeutic capacity, improvement of the biodistribution and controlled release of drugs, increase of their specificity, sensitivity and reduction of their pharmacological toxicity) [8]. The approval of nanoparticles for drug delivery to solid tumours, such as albumin - based paclitaxel nanoparticles has catalysed the field to develop second - generation nanoparticles for this purpose [9]. In search for nanomedicines for tumour - targeted drug delivery [10], we worked on camptothecin encapsulation in tocopherol - , ergocalciferol - and testosterone - modified cellulose nanogels (2 - 13 wt. % of camptothecin), with sustained camptothecin release and good cytotoxic activity on MCF - 7 cancer cells [11]. DL - α - Tocopherol (vitamin E) and testosterone were chosen as biocompatible substituents of amphiphilic polymers for anticancer drug delivery applications due to their hypocholesterolemic, antioxidant and anticancer effects [12 - 14]. These facts motivated us to synthesise tocopherol and testosterone substituted polyphosphazene nanocarriers for hydrophobic encapsulation of camptothecin or hydrophilic loading of epirubicin, and their controlled delivery with strong cytotoxicity against cancer cells and reduced sid e effects. Polyphosphazenes, inorganic - organic hybrid polymers, consisting of an inorganic backbone of alternating phosphorus and nitrogen atoms, and organic side groups, introduced during post - polymerisation substitution, are of great interest for biomed ical applications [15]. Living cationic polymerisation yields polymers with controlled molecular weights and narrow polydispersities, while the post - polymerisation substitution results in multifunctional polymers, with the polymer characteristics determine d by choice of substituents [16]. Their tuneable degradation rates [3] and, especially, their degradation to nontoxic degradation products phosphates and ammonia [17], make them highly advantageous for drug delivery applications. Polyphosphazenes have been investigated for anticancer drug delivery both in vitro and in vivo , ranging from doxorubicin loaded polymersomes proving to be less toxic while exhibiting a comparable effectiveness to standard delivery methods [18], to

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injectable hydrogels bearing coval ently linked camptothecin showing a better tumour growth inhibition of the intratumorally injected hydrogel compared to the drug being administered alone [19]. Macromolecular drugs based on polyphosphazenes bearing covalently linked epirubicin [20] are cla imed to inhibit medullary thyroid carcinoma and small intestinal neuroendocrine tumour cells proliferation coupled with a decrease in cell viability in vitro [21]. However, lack of quantitative cytotoxicity data of these polyphosphazenes bearing covalently bonded epirubicin, as well as missing comparison of their anticancer activity with the observed effect of free epirubicin, motivated us to keep investigating on synthesis of other polyphosphazenes carrying epirubicin for antitumor application. Further stu dies with macromolecular metal prodrugs based on polyphosphazenes bearing Pt(IV) prodrugs show a 30 - fold increase in drug uptake and promising results to overcome drug resistance in vitro with an improvement in loss of tumour volume in vivo [22]. Similarly , ruthenium bearing macromolecular drugs, providing an increase in solubility of the bound metal drugs, show an increase in tolerability with a decrease in tumour growth in vivo [23]. In this research, three polyphosphazene - based nanocarriers were synthes ised by living cationic polymerisation of Cl 3 PNSiMe 3 to obtain poly(dichloro)phosphazenes which were further functionalised via post - polymerisation substitution with tocopherol or testosterone glycinate and hydrophilic Jeffamine M1000. Synthesised polyphos phazenes were utilized to encapsulate and deliver the anticancer drugs camptothecin and epirubicin, respectively.

4.2.2. Materials and methods Materials

Amine capped polyetheramine copolymer (PEO — PPO — NH 2 ) with an M n of 1000 g/mol and an ethylene oxide/prop ylene oxide ratio of 19/3, tradename Jeffamine M1000, was donated by Huntsman Performance Products and used as received. All chemicals and solvents were purchased from Sigma - Aldrich and used as received without further purification, unless otherwise stated . ( S ) - (+) - Camptothecin (CPT) was purchased from Alfa Aesar. The human breast adenocarcinoma cell line MCF - 7 was donated by Prof. Dr. Barbara Krammer, University of Salzburg (Austria). Triethylamine was distilled and stored over molecular sieves under argon . All glassware was dried overnight in an oven at 100 o C prior to use. Spectra/Por 3 cellulose dialysis membranes (Spectrum Laboratories Inc., CA, USA) with molecular weight cut off (MWCO) of 3.5 kDa were used for purification of the synthesised polyphosph azenes. The synthesis and characterisation of tocopherol - glycine - Boc, testosterone - glycine - Boc

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and related deprotected tocopherol - glycine - NH 2 and testosterone - glycine - NH 2 is described in the supplementary material. The monomer N - (trimethylsilyl) - trichlorop hosphoranimine (Cl 3 P=N – Si(CH 3 ) 3 ) was synthesised according to literature procedure [20,24].

Methods Synthesis of the polymers The synthesis of the poly(dichloro)phosphazene precursor was conducted via living cationic polymerisation of monomer trichlorophos phoranimine (Cl 3 P=N – Si(CH 3 ) 3 ) with triphenylphosphine dichloride ((C 6 H 5 ) 3 PCl 2 ) [25,26]. The synthesis of polymer P1 is briefly described in the supplementary material. For polymers P2 - P3 the ratio of monomer to initiator was maintained, and the ratio of su bstituent tocopherol or testosterone to Jeffamine M1000 was adjusted differently ( Table 1 ). These syntheses were performed in a glovebox (MBRAUN) under argon. Polyphosphazenes P1 - P3 formed nanoaggregates in aqueous medium when stirred overnight at 1 mg/mL in water or phosphate buffer saline solution (PBS, pH 7.4).

CPT and EPI loading in polyphosphazene nanocarriers CPT or EPI were incorporated in the polyphosphazene - based nanocarriers using a solvent exchange/precipitation method, with lyophilisation [11] . To this end, approximately 10 mg of P1 , P2 or P3 and 1.2 - 1.5 mg of CPT or EPI dissolved in 10 mL of DMSO, were stirred overnight at room temperature in darkness. The formation of CPT - or EPI - loaded polyphosphazene nanoaggregates and removal of CPT and EP I excess was carried out by dialysis against distilled water (2 L, 1 time, 5 hours). CPT - loaded polyphosphazenes prepared with 1.5 mg of CPT and 10 mg of P1 ( 1.5CPT - P1 ), approximately 1.0 - 1.2 mg of CPT and 10 mg of P1 - P3 ( CPT - P1, CPT - P2 and CPT - P3 ) (slight ly brown waxy solids), and EPI - loaded polyphosphazenes prepared with 1 mg of EPI and 10 mg of P1 or P2 ( EPI - P1 and EPI - P2 ) (red waxy solids) were obtained after lyophilisation.

Anticancer drug content and drug release studies The content of CPT in CPT - load ed polyphosphazene nanocarriers ( Fig. SI - 1 in the supplementary material), and in vitro release of CPT during drug release studies were determined by UV - Vis spectrophotometry measurements, based on calibration curves of camptothecin in DMSO and PBS (pH 7.4 ) ( Fig. SI - 2 and SI - 3 in the supplementary material). Similarly, the content of EPI in EPI - loaded polyphosphazene nanocarriers ( Fig. SI - 4 in the supplementary material) and in vitro EPI released were determined by

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UV - Vis spectrophotometry, using calibratio n curves of epirubicin hydrochloride in DMSO and PBS (pH 7.4) ( Fig. SI - 5 and SI - 6 in the supplementary material). In vitro release studies of CPT and EPI, respectively, were performed in phosphate buffered saline solution (PBS, pH 7.4) at 37 o C. To this en d, 2.0 mL of CPT - or EPI - loaded polyphosphazenes 1.5CPT - P1 , CPT - P1 , CPT - P2 , CPT - P3, EPI - P1 and EPI - P2 (2.5 mg/mL) in PBS at pH 7.4 were placed in dialysis cups (MWCO 3.5 kDa, Slide – A – Lyzer Mini Dialysis Devices, ThermoScientific, USA) and immersed in 10 mL of the release medium stirred at 100 rpm. The entire release media was replaced at every required time point, and analysed using UV - Vis spectroscopy. (CPT  emission = 369 nm;

EPI  emission = 482 nm). Similarly, tocopherol or testosterone delivery of carri er polyphosphazene P1 - P3 in PBS (pH 7.4 and pH 6.0) at 37 o C, was evaluated using the same procedure than for CPT and EPI drug release studies, except that tocopherol and testosterone were determined using the calibration curves of tocopherol or testostero ne in PBS at pH 7.4 and 6.0 ( Fig. SI - 7 to SI - 10 in the supplementary material). All drug release data was fitted to a SWeibull2 model (Cumulative Release (%) = a – (a – b)*exp( – (k*Time(hours)) d ). The CPT and EPI release data were also adjusted to the Korsm eyer - Peppas model (linear fitting of log(Cumulative Release(%)) = k*log(Time(hours) + m).

Cell culture The MCF - 7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) – high glucose supplemented with 10% fetal bovine serum, 1% penicillin – streptomy cin and 1% L - glutamine. The media and supplements except L - glutamine (PAA laboratories) were purchased from Sigma - Aldrich. Cells were grown in a humidified atmosphere at 4 37 °C in 5% CO 2 . In all experiments, 70 - 80% confluent cells were used and 1 × 10 cells/well in 96 - well plates or 3 ×10 4 cells/well in 8 well glass bottom µ - Sli des (Ibidi) were seeded, respectively.

Cell uptake For the cell uptake, cells were grown overnight in 8 well glass bottom µ - Slides. The day after, the medium was changed to a serum free medium (control) or CPT - loaded polyphosphazene nanoaggregates dispersi ons at 0.1 mg/mL and incubated for 4 hours. Afterwards, 50 nM of LysoTracker Yellow HCK - 123 (Invitrogen) were added and incubation continued for another 2 hours. For the EPI - loaded polyphosphazene nanoaggregate dispersions, cells were incubated for 6 hours at a concentration of 0.1 mg/mL and subsequently counterstained with 1 µg/mL Hoechst 33342 (Fluka). Fluorescence imaging was performed on MCF - 7 cancer cells using an Olympus IX73

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inverted microscope with DAPI channel for the 1.5CPT - P1, CPT - P1 , CPT - P2 and CPT -

P3 nanoaggregates (  excitation = 345 nm,  emission = 455 nm) and FITC channel for the

LysoTracker Yellow HCK - 123 (  excitation = 494 nm,  emission = 518 nm). Similarly, EPI - P1 and EPI - P2 nanoaggregates (  excitation = 550 nm,  emission = 565 nm) were ana lysed using CY3 channel, while DAPI channel was used for the cell nuclei marker Hoechst

33342 (  excitation = 345 nm,  emission = 455 nm).

Cytotoxicity tests To determine the cytotoxic effects of the CPT - or EPI - loaded polyphosphazene - based nanocarriers , MC F - 7 cells were grown overnight in 96 - well plates. The day after, cells were treated with various concentrations of unloaded polymers and CPT - or EPI - loaded polyphosphazene nanoaggregate dispersions in serum free medium. After 48 hours the medium was change d to full growth medium and 50 μL of XTT (2,3 - bis(2 - methoxy - 4 - nitro - 5 - sulfophenyl) - 2 H tetrazolium - 5 - carboxanilide) reagent was added to each well for another 3 hours. The absorbance was measured at 490 nm using a GloMax® Multimode Microplate Reader (Promeg a). Three independent experiments (with each sample in triplicate) were performed. The cell viability was normalised to the untreated control and were analysed with Origin 2015 (Microcal Origin, OriginLab, MA, USA).

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The samples were prepared at 1 mg /mL in deionised water, filtered through a 0.45 µm nylon film syringe filter and determinations of hydrodynamic diameters ( d h ) were performed in triplicate using a folded capillary cell DTS1070 at 25 o C. Calorimetric studies of samples were conducted on TA Instrument Q10 differential scanning calorimeter (DSC) using aluminium pans, with a sample weight of approximately 5 mg and heating rate of 10 o C/min under nitrogen flow of 20 mL/min. Samples were cooled and heated from - 80 o C to 400 o C. TGA Q5000 instrumen t was used for thermogravimetric analyses (TGA) of approximately 5 mg samples on platinum pans. Heating from 40 o C to 900 o C at a rate of 10 o C/min under a nitrogen flow of 25 mL/min was set up for TGA. Transmission Electron Microscopy (TEM) micrographs we re recorded with a Jeol JEM - 2011 FasTEM (Jeol Ltd, Tokyo, Japan) operated at 100 kV. A drop of polymer dispersions in deionized water (1 mg/mL) was placed on a Pioloform coated 300 Mesh Cu grids (Plano GmbH, Germany). Excess solution was eliminated with fi lter paper; samples were negatively stained with a drop of uranyl acetate aqueous solution at 1%, and dried several hours before measurements. Atomic Force Microscopy (AFM) images (10 µm × 10 µm and 2 µm × 2 µm) were taken with MFP 3D - Stand Alone AFM (Asyl um Research) with the cantilever OMCL - AC160TSA of Olympus, at a resonant frequency of 300 kHz and spring constant of 26 N/m, 50 - 70% set point and scan rate of 1 Hz. 80 µL droplet of a 1 mg/mL aqueous dispersion of the polymers was deposited on a silicon wa fer spin coated at 40 Hz for 6 s. Scanning Electron Microscopy (SEM) micrographs were recorded with a field emission Zeiss Gemini 1540 XB SEM (Zeiss, Germany) operated at 7 - 8 kV, using a secondary electron detector. A drop of polymer dispersions in deioniz ed water (1 mg/mL) was deposited on a silicon wafer (0.35 × 0.35 cm 2 ), the water was evaporated overnight and samples were gold coated with a HUMMER X (Anatech Ltd., Alexandria, VA, USA) sputter coater before measurements.

Statistics Statistical evaluatio n of data was carried out using Statgraphics Plus 5.1, Professional Edition. Results were assessed using one - way analysis of variance (ANOVA), Tukey post - hoc test for between groups comparisons, multiple comparison and Kruskall - Wallis tests at 95% confiden ce level (p = 0.05). No significant different means are marked with * followed with the same number (p > 0.05).

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4.2.3. Results Synthesis and characterisation of polyphosphazenes P1 - P3

First, linear poly(dichloro)phosphazenes ([NPCl 2 ] n ) with polymerisati on degree (DP) near to 25 were synthesized via living cationic polymerisation of trichlorophosphoranimine initiated with (C 6 H 5 ) 3 PCl 2 in CH 2 Cl 2 at room temperature ( Fig.

1b ). Tocopherol - glycine - NH 2 or testosterone - glycine - NH 2 respectively ( Fig. 1a ), and

Jef famine M1000 (PEO - PPO - NH 2 ) were subsequently introduced via post - polymerisation functionalisation of the [NPCl 2 ] n , firstly introducing the bulky tocopheryl - or testosterone - moieties and later the linear PEO - PPO - NH 2 substituent ( Fig. 1c ).

Complete chlorine substitution of [NPCl 2 ] n , necessary to avoid later uncontrolled polyphosphazene degradation via hydrolysis due to highly labile P - Cl bonds, was ensured by using an excess of Jeffamine M1000 [25], and was confirmed by 31 P{ 1 H} NMR spectroscopy measurements with the disappearance of characteristic peaks of unsubstituted chlorine atoms of RClP=N ( Fig. SI - 18 in the supplementary material) [27]. Amphiphilic co - substituted polyphosphazenes might self - aggregate at molecular and supramolecular levels in a micelle - l ike array with a core formed of hydrophobic moieties and a shield of hydrophilic groups in contact with water molecules, as a result of favoured intramolecular and intermolecular interactions of hydrophobic groups and van der Waals and hydrogen bond intera ctions of hydrophilic groups and water molecules of the solvent ( Fig. 1d ) [29 - 31]. The micelle - like structure allows the hydrophobic encapsulation of lipophilic drugs in the core or the loading of hydrophilic drugs on the shell ( Fig. 1e ).

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Fig. 1. (a) Synthesis of tocopherol and testosterone glycinates (ToT - glycine - NH 2 ), (b) polymerisation of trichlorophosphoranimine to obtain poly(dichloro)phosphazene, (c) post - polymerisation functionalisation of chlorine atoms with ToT - glycine - NH 2 and Jeffamine M1000 ; schematic representation of self - aggregation of polyphosphazenes P1 - P3 to form nanoaggregates (d) and encapsulation of CPT and EPI (e) .

31 P{ 1 H} NMR spectra of polymers P1 - P3 showed only broad peaks at 0.0 to 1.0 ppm ( Fig. 2a ), characteristic of polyphos phazene backbone with Jeffamine M1000 used as

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substituent [20,26]. The hydrophilic Jeffamine M1000 substituent in the synthesised polymers P1 - P3 makes them suitable to be dispersed in aqueous solutions, allowing their later application as carriers for drug delivery of CPT and EPI. The integration of aromatic protons (C 6 H 5 ) 3 P=N – (7.60 ppm) of P1 - P3 was approximately 15/(20 to 24) when referred to integration of – O – C H 2 – C H 2 – O – (3.62 - 3.63 ppm) and C H 3 O – (3.36 ppm) protons of Jeffamine M1000, with a degree of po lymerisation (DP) near to 25 ( Table 1 ) ( Fig 2b , Fig. SI - 21 and SI - 22 in the supplementary material) [20]. GPC measurements reproduced the trend of calculated number average molecular weights of the polymers ( > > ), and showed low polydispersities (Ð from 1.28 - 1.48), consistent with previous reports [20,25,26]. ATR - FTIR spectroscopy also demonstrated the post - polymerisation functionalisation of the [NPCl 2 ] n with the tocophe rol and testosterone glycinate ( Fig. 2c ). Thus, C=O absorption peaks of tocopherol and testosterone glycinate were observed in the polymers P1 - P3 at 1768 cm - 1 and 1739 cm - 1 respectively. Additionally, C=O absorption peak of ketone due to testosterone subst ituent was observed at 1672 cm - 1 .

Table 1 Composition, yield and molecular weight of polymers P1 - P3 .

a b c d e Sample DP ToT:M1000 Yield M n M n Ð % kg/mol kg/mol P1 24 1:1 56 39 14 1.35 P2 20 1:3 44 45 19 1.28 P3 21 1:3 48 43 17 1.48 a Degree of polymerisat ion estimated by 1 H NMR. b Tocopherol or Testosterone (ToT) : Jeffamine M1000 ratio feeding composition. c Theoretical number average molecular weight. d Number average molecular weight and polydispersities measured by GPC against polystyrene standard s.

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31 1 1 Fig. 2. (a) P{ H} NMR spectra of P1 - P3 polyphosphazenes in CDCl 3 ; (b) H NMR spectrum of P1 in CDCl , DP was calculated as: = = ; (c) FTIR spectra of 3 P1 - P3 polyphosphazenes.

The average hydrodynamic diameters ( d h ) and the sizes of dried nanoaggregates

( d AFM ), as an indication of the self - assembly process, seem to be influenced by the hydrop hobicity of the synthesised polyphosphazenes ( Table 2 ). P1 , the most hydrophobic synthesised polyphosphazene with tocopherol to Jeffamine M1000 ratio of 1:1, showed the smaller hydrodynamic sizes in water and PBS ( ~ 22 nm) and size of dried nanoaggregates ( ~ 27 nm) among the three polymers. Polyphosphazenes P2 and P3 , with a tocopherol or testosterone to Jeffamine M1000 ratio of 1:3, formed aqueous nanoaggregates of 96 - 134 nm which upon drying resulted in 42 - 58 nm nanoparticles ( Table 2 ). However, the inclusi on of a hydrophobic drug (CPT) or a hydrophilic drug

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(EPI) during the self - assembly process via solvent exchange from DMSO to water, impacts significantly the hydrodynamic sizes of the aggregates formed by the polyphosphazenes P1 - P3 ( Fig 1e and Table 2 ). I n this sense, the CPT loading in P1 - P3 polymers resulted in a hydrodynamic sizes increase from 22 nm ( P1 ) to 198 nm for CPT - P1 and 142 nm for 1.5CPT - P1 , from 127 nm ( P2 ) to 158 nm for CPT - P2 , and from 96 nm ( P3 ) to 194 nm for CPT - P3 . The most hydrophobic p olymer P1 was capable to load the maximal CPT quantity ( CPT - P1 with 10.9 wt. % CPT content) when compared with CPT - P2 and CPT - P3 , all prepared using the same drug loading conditions (1.2 mg of CPT and 10 mg of each polymer ( P1 - P3 )). It might be due to the 50 mol% content of tocopherol in P1 , capable to stablish π - π interactions with the aromatic backbone of CPT in the core of the P1 aggregates. However, all CPT bearing polymers dispersed at 1 mg/mL in PBS (pH 7.4) are capable to carry 80 - 100 fold times the camptothecin soluble in water as pa rent drug (1.3 mg/L) [31]. Interestingly, EPI loading in the outer hydrophilic shell formed by Jeffamine M1000 chains of the P1 and P2 aggregates ( Fig. 1d ) was significantly less efficient than CPT encapsulation, attaining 0.3 - 2.3 wt. % EPI content. It res ulted in bigger hydrated sizes of approximately 250 nm in EPI - P1 and EPI - P2 . Electron microscopy techniques (TEM and SEM) and AFM allowed getting an insight on the morphology and particulate structure of dried P1 - P3 polyphosphazene - based nanocarriers. TEM technique depicted the dried P1 - P3 nanoaggregates as 20 - 100 nm rounded particles ( Fig. 3a ). AFM also showed the formation of small rounded nanoaggregates with average sizes of approximately 20 to 60 nm for polymers P1 - P3 ( Table 2 and Fig 3b ). Similarly, SE M displayed small nanoparticles of 20 - 50 nm and a few 100 - 250 nm aggregates for polymers P1 and P2 ( Fig. SI - 30 in the supplementary material ). The significant shrinkage of P2 and P3 aggregates upon drying ( ~ 60%) might be due to water loss and collapse of t he soft micelle - like structure.

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Table 2 Hydrodynamic sizes, average diameters AFM and TEM of dried nanoaggregates of polymers P1 - P3 , weight content percent of camptothecin or epirubicin (wt. %). d a d b h h d c nm nm AFM wt. % Sample nm (PDI) (PDI) 21 ± 1* 1 22 ± 3* 1 P1 27 ± 9 - (0.29) (0.30) 108 ± 8 127 ± 3 P2 42 ± 5 - (0.58) (0.42) 134 ± 2 96 ± 2 P3 58 ± 6 - (0.55) (0.61) 198 ± 11* 3 1.5CPT - P1 - - 13.3 (0.54) 142 ± 1 CPT - P1 - - 10.9 (0.69) 158 ± 3 CPT - P2 - - 9.9 (0.54) 194 ± 5* 3 CPT - P3 - - 10.2 (0.2 9) 250 ± 9* 4 EPI - P1 - - 2.3 (0.65) 253 ± 4* 4 EPI - P2 - - 0.3 (0.52) a b Average hydrodynamic diameter (d h ) in water by DLS. Average hydrodynamic diameter (d h ) c in PBS by DLS. Diameter of dried nanoaggregates by AFM (d AFM ). * Stands for no signifi cant different values when followed by same number (p > 0.05).

Fig. 3. (a) TEM micrographs of P1 - P3 dried nanoaggregates at 21,000× magnification; (b) AFM micrographs of P1 - P3 dried nanoaggregates.

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Therefore, soft CPT - loaded polyphosphazene - based aggre gates (d h 142 -

198 nm) and EPI - loaded polyphosphazene - based aggregates (d h ~ 250 nm) have the appropriate sizes (100 - 200 nm), deformability and number average molecular weight (<30 kDa) to allow an extended serum circulation with reduced renal clearance, whi ch allow selective accumulation of these anticancer drug - loaded nanocarriers in the cancer tissues via passive diffusion and aided by the enhanced permeability and retention (EPR) effect associated to the leaky vasculature and poor lymphatic drainage of tu mour tissues [33,34]. Once these CPT - and EPI - loaded nanocarriers deliver their cargo to the cancer cells, the polyphosphazene - based nanocarriers are expected to degrade into innocuous phosphates and ammonia at physiological conditions over time, avoiding harmful long - term accumulation in the body [17].

Drug delivery studies Fig. 4 shows the in vitro CPT and EPI release profiles of CPT - and EPI - loaded aggregates in simulated physiological conditions (PBS at pH 7.4, 37 o C). All CPT and EPI releases appeared almost linear for the first 8 - 9 hours, with adjusted R 2 over 0.98 and slope ranging from 1.06 to 1.60 %/h for CPT and 1.5 or 3.5 %/h for EPI ( Table SI - 1 and Fig. SI - 31 and SI - 32 in the supplementary material). Camptothecin release reached approximately 86 % of encapsulated CPT for 1.5CPT - P1 and CPT - P2 , 78% for CPT - P3 and approximately 69% for CPT - P1 after 6 days. Then, CPT - P1 with the smaller 142 nm aggregates in PBS and second higher CPT content, released less quantity of CPT. EPI release showed quite diff erent for 250 - 253 nm aggregates EPI - P1 and EPI - P2. EPI - P2 aggregates with only 0.3 wt. % content of EPI released quantitatively ( ~ 99%) on the sixth day and significantly faster from the first 9 hours (3.5 %/h) the loaded EPI, while the EPI - P1 aggregates wi th 2.3 wt. % content of EPI reached a 48% of the drug released on the sixth day ( Fig. 4 , Table SI - 1 in the supplementary material). Therefore, releases appeared to be controlled by the hydrodynamic size of the aggregates in PBS and the drug content. CPT an d EPI release profiles were adjusted to a Weibull model that describes the drug release from a matrix [35,36].

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(a)

(b)

Fig. 4. In vitro release profiles of (a) CPT - loaded aggregates and (b) EPI - loaded aggregates in PBS (pH 7.4) at 37 o C, adjusted to a SWeibull2 function.

In this sense, drug release data fitted well to a SWeibull2 function, with adjusted R 2 ranged from 0.90 to 0.99 and d values of 1.0 for all drug - loaded aggregates (except for EPI - P1 ), associated to a perfectly exponential drug relea se behaviour ( Fig. 4 and Table SI - 2 in the supplementary material) [35]. The anticancer drug release data was also adjusted to the Korsmeyer - Peppas model to better clarify the molecular interactions and factors leading to the anticancer drug release ( Table SI - 3 , Fig. SI - 33 and SI - 34 in the supplementary material) [36,37]. The slope of the linear fitting to the Korsmeyer - Peppas model for CPT releases of CPT - loaded aggregates ranged from 0.79 to 0.87, which is associated with an anomalous diffusion mechanism of release [37]. Similarly, EPI - P1 and EPI - P2 aggregates released EPI with a slope of 1.0 and 0.8 respectively, associated with an anomalous diffusion mechanism of release (slope >

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0.5) characteristic of weak interactions between the anticancer drugs and t he polymers [37]. Finally, degradation of polyphosphazene P1 - P3 in PBS (pH 7.4 and 6.0) at 37 o C via release of tocopherol and testosterone was also investigated ( Fig. SI - 35 to SI - 37 in the supplementary material). The hydrolysis of the ester bond in the t ocopherol glycinate moiety of the polyphosphazenes P1 and P2 resulted in the release of 4.5% and 10% of tocopherol incorporated in P1 after 1 day and 6 days in PBS (pH 7.4) at 37 o C, or 6% and 15% of tocopherol incorporated in P2 after 1 day and 6 days in similar conditions. In the same way, hydrolysis of the testosterone glycinate fragment of the polyphosphazene P3 in simulated physiological conditions (PBS at pH 7.4, 37 o C), resulted in the release of approximately 4% and 21% of testosterone incorporated in P3 after 1 day and 6 days respectively. Slightly acidic medium (PBS, pH 6.0) significantly catalyzed the hydrolysis of P1 - P3 polymers, with tocopherol and testosterone release increased until 8% and 16% of tocopherol released at 1 day and 6 days for P1 , 13% and 24% of tocopherol released at 1 day and 6 days for P2 , and 10% and 29% of testosterone delivered at day 1 and day 6 for P3 . Therefore, the anticancer drug - loaded polyphosphazenes P1 - P3 should deliver both the CPT ( 1.5CPT - P1 , CPT - P1 , CPT - P2 , CPT - P3 ) or EPI ( EPI - P1 , EPI - P2 ) and the testosterone ( P3 - based nanocarriers) or tocopherol ( P1 - and P2 - based nanocarriers) when administered to the patients, due to simultaneous slow diffusion of the anticancer drugs from the nanocarriers and hydrolysis of glyci nate linker with release of testosterone or tocopherol. Interestingly, the degradation of P1 - P3 and the CPT and EPI releases in almost neutral conditions (PBS pH 7.4) might progress slowly enough during the first 8 hours to allow the accumulation of the ag gregates in the slightly more acidic cancer tissues and the cancer cell uptake via passive diffusion and EPR. That is why in vitro cell uptake and cytotoxicity studies were carried out in a first approach to assess the potential application of the synthesi sed polyphosphazene - based nanocarriers for chemotherapy. However, further in vivo studies are required to evaluate the biodistribution, availability and enzyme - promoted delivery inside the cancer cells and lysosomes of the CPT and EPI cargo, as well as the degradation of P1 - P3 aggregates inside the living cells.

Cell uptake and cytotoxic activity The uptake and localization of the CPT - and EPI - loaded polyphosphazene nanoaggregates on MCF - 7 cancer cells was evaluated using fluorescence microscopy analysis. I ndeed, CPT - P1 ( Fig. 5a ), 1.5CPT - P1 , CPT - P2 and CPT - P3 ( Fig. SI - 38 to SI - 40 in the supplementary material) internalized in cells (CPT associated blue

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fluorescence) and accumulated in the lysosomes (LysoTracker HCK - 123 associated green fluorescence) [38]. Si milarly, EPI - P1 ( Fig. 5b ) and EPI - P2 ( Fig. SI - 41 in the supplementary material) internalized in MCF - 7 cells (EPI associated red fluorescence), and nuclei were counterstained with Hoechst 33342 associated blue fluorescence).

(a)

(b)

Fig. 5. MCF - 7 cell s fluorescence images and slices of cells without particles and LysoTracker or Hoechst (B). (a) Cells with 0.1 mg/mL of CPT - P1 aggregates, 50 nM of LysoTracker Yellow HCK - 123 and merged pictures (M); (b) Cells with 0.1 mg/mL of EPI - P1 aggregates, 1 µg/mL o f Hoechst 33342 and merged pictures (M), scale bars represents 20 µm.

Fig. 6 displays the relative cell viabilities of MCF - 7 cells after treatment with CPT - or EPI - loaded polyphosphazene nanoaggregates. Blank P1 - P3 polyphosphazene nanoaggregates seemed to exhibit no toxicity to MCF - 7 cells at 0.1 mg/mL, with observed relative cell viability of approximately 96%.

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(a)

(b)

Fig. 6. Relative cell viability of MCF - 7 breast cancer cells treated with (a) CPT - loaded polyphosphazene nanoaggrega tes and (b) EPI - loaded polyphosphazene nanoaggregates. NP stands for blank polyphosphazene nanoaggregates at 0.1 mg/mL. Data represents the mean ± standard deviation (n = 3).

1.5CPT - P1 and CPT - P1 with higher CPT contents (13.3% and 10.9%) showed slightly less cytotoxic to MCF - 7 human breast cancer cells at all evaluated concentrations than the parent CPT ( Fig. SI - 42 in the supplementary material). This might be due to incomplete CPT release from the nanoaggregates observed at day 3 ( Fig. 4a ), associated to hydrophobic interactions and π - π stacking of CPT with the tocopherol core in P1 nanoaggregates. CPT - P2 (9.9 wt. % of CPT) with relative cell viability of 25% to 65%, and CPT - P3 (10.2 wt. % of CPT) with relative cell viability of 28% to 66%, exhibited slig htly better anticancer activity than the parent CPT at all concentrations. On the other hand, EPI - loaded nanoaggregates EPI - P1 (2.3 wt. % of EPI) with relative cell viability from 50% to 90%, and EPI - P2 (0.3 wt. % of EPI) with relative cell viability from 56% to 84%, showed moderated to slight anticancer activity when evaluated between 0.1 mg/mL to 0.0015625 mg/mL of the nanocarriers in solution. Therefore, synthesised polyphosphazenes are promising candidates for in

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vivo tests for the delivery of camptothe cin and epirubicin. It is assumed that CPT - and EPI - loaded polyphosphazene nanoaggregates might selectively accumulate in the cancer tissues due to combination of passive diffusion and EPR effect [33,34,38]. 158 nm CPT - P2 and 194 nm CPT - P3 nanoaggregates fo rmed by approximately 40 - 60 nm particles might also exhibit extended circulation times and moderated reticuloendothelial system (RES) clearance [34]. Once CPT - P2 or CPT - P3 nanoaggregates are inside the cancer cells, a co - delivery of CPT cargo and tocophero l or testosterone might occur due to enzyme - promoted and acidic hydrolysis of ester bond in the tocopherol - or testosterone - glycine moiety.

4. 2 .4. Conclusions In this research, tocopherol and testosterone functionalised polyphosphazenes were synthesised vi a living cationic polymerisation of the monomer trichlorophosphoranimine

(Cl 3 P=N – Si(CH 3 ) 3 ) and subsequent post - polymerisation functionalisation. These polyphosphazenes self - aggregated in aqueous medium and were loaded with anticancer drugs camptothecin and epirubicin, respectively, for drug delivery applications. Camptothecin and epirubicin were slowly released in simulated physiological conditions (PBS, pH 7.4, 37 o C), with an almost constant release rate during the first 8 - 9 hours and sustained release tha t reached from 69 - 86% of encapsulated camptothecin and 48% to 99% for epirubicin loaded in the polyphosphazene - based aggregates. The broad margin of epirubicin released after 4 days ( EPI - P1 48%, EPI - P2 99%) is due to the eight times superior content of epi rubicin in EPI - P1 (2.3 wt. % of EPI) when compared to EPI - P2 (0.3 wt. % of EPI). Camptothecin - and epirubicin - loaded polyphosphazene aggregates resulted similarly cytotoxic to MCF - 7 human breast cancer cells as parent anticancer drugs, probably due to almo st complete camptothecin and epirubicin enzyme - mediated release inside the cancer cells. In this sense, camptothecin and epirubicin cytotoxic effect to MCF - 7 cancer cells was almost unaltered after inclusion in the polyphosphazene - based nanocarriers . There fore, polyphosphazenes appear to be proper carriers for the anticancer drugs, with the capacity to protect sensitive camptothecin from degradation and to favour the more active closed lactone ring conformation of camptothecin at in vitro cell culture condi tions. Particularly, CPT - loaded in more hydrophilic polyphosphazenes (tocopherol and testosterone to Jeffamine M1000 ratio 1:3) revealed to be slightly more cytotoxic to MCF - 7 cancer cells than parent camptothecin. Fluorescence microscopy imaging confirmed MCF - 7 cells uptake both camptothecin - and epirubicin - loaded nanoaggregates, as well as lysosomal accumulation of CPT -

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loaded nanoaggregates. It was demonstrated strong cytotoxic activity and good cell uptake with MCF - 7 human breast cancer cells of the anti cancer drug - loaded polyphosphazenes. Furthermore, appropriate hydrodynamic sizes of CPT - loaded polyphosphazene aggregates (between 100 - 200 nm) would allow extended blood circulation and slow renal clearance, allowing accumulation in cancer tissues via pass ive diffusion. In addition, slow sustained release of the anticancer drugs and co - delivery of testosterone or tocopherol with antioxidant and anticancer effects, make camptothecin - and epirubicin - loaded polyphosphazene - based nanocarriers good candidates fo r chemotherapy.

Conflicts of interest Declaration of interest: none.

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SUPPORTING INFORMATION

Javier P. Quiñones, Aitziber Iturmendi, Hel ena Henke, Cornelia Roschger, Andreas F. Zierer, Carlos Peniche - Covas, Oliver Brüggemann

1. Supporting Tables

Table SI - 1 Linear fitting parameters of in vitro CPT and EPI release profiles of CPT - and EPI - loaded polyphosphazene nanoaggregates up to 8 h ( intercept 0, slope k, adjusted R - Square) in PBS (pH 6.0) at 37 o C. Samples k Adjusted R - Square 1.5CPT - P1 1.06 ± 0.02 0.9977 CPT - P1 1.13 ± 0.02 0.9984 CPT - P2 1.47 ± 0.03 0.9978 CPT - P3 1.60 ± 0.04 0.9989 EPI - P1 1.5 ± 0.1 0.9814 EPI - P2 3.5 ± 0.1 0.9945

Table SI - 2 SWeibull2 fitting parameters of Cumulative release (%) Vs. Time (hours) of CPT - and EPI - loaded polyphosphazene nanoaggregates up to 150 hours (Cumulative release (%) = a – (a – b)*exp( – (k*Time(hours)) d ) in PBS (pH 7.4) at 37 o C. Samples a b k d Adjusted R - Square 1.5CPT - P1 110 ± 40 0.0 ± 0.7 0.009 ± 0.005 1.0 ± 0.1 0.9863 CPT - P1 72 ± 16 0.0 ± 0.7 0.014 ± 0.006 1.0 ± 0.1 0.9823 CPT - P2 84 ± 18 0.0 ± 0.9 0.014 ± 0.005 1.0 ± 0.1 0.9758 CPT - P3 82 ± 13 0 ± 1 0.016 ± 0.005 1.0 ± 0.1 0.9905 EPI - P1 43 ± 7 0 ± 3 0.05 ± 0.02 1.2 ± 0.5 0.8971 EPI - P2 103 ± 15 0 ± 11 0.03 ± 0.01 1.0 ± 0.4 0.9566

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Table SI - 3 Linear fitting parameters of log(Cumulative release(%)) Vs. log(Time(hours)) of CPT - and EPI - loaded polyphosphazene nanoaggregates up to 100 hours (intercept m, slope k, adjusted R - Square) in PBS (pH 7.4) at 37 o C. Samples m k R 2 1.5CPT - P1 0.08 ± 0.05 0.87 ± 0.03 0.9894 CPT - P1 0.14 ± 0.05 0.82 ± 0.04 0.9837 CPT - P2 0.25 ± 0.06 0.80 ± 0.04 0.9806 CPT - P3 0.30 ± 0.07 0.79 ± 0.05 0.970 7 EPI - P1 0.1 ± 0.2 1.0 ± 0.2 0.893 EPI - P2 0.6 ± 0.2 0.8 ± 0.2 0.8569

2. Synthesis of materials, monomer and polymers P1 - P3

Synthesis of tocopherol - glycine - NH 2 ( 1 ) and testosterone - glycine - NH 2 ( 2 ). Boc - protected amino acid Boc - Gly - OH (85 mg, 0.49 mmol), 4 - (dimethylamino)pyridine (59 mg, 0.48 mmol) and N,N’ - dicyclohexylcarbodiimide (124 mg, 0.60 mmol) were dissolved in 15 mL CH 2 Cl 2 and stirred at room temperature for 2 h. The reaction mixture was added to a solution of tocopherol (207 mg, 0.48 mmol) or tes tosterone

(139 mg, 0.48 mmol) in 10 mL CH 2 Cl 2 and stirred for 48 h. The precipitated N,N’ - dicyclohexylurea was removed by filtration and the filtrated was extracted with 10%

NH 4 Cl aqueous solution (2 × 15 mL), with 5% NaHCO 3 aqueous solution (2 × 15 mL) an d saturated NaCl solution (1 × 15 mL). The organic phase was dried over MgSO 4 , filtered and removed under reduced pressure to yield tocopherol - glycine - Boc as a white solid (212 mg, yield 75%) or testosterone - glycine - Boc as a slightly yellow solid (165 mg, yield 77%). Then, tocopherol - glycine - Boc (212 mg, 0.36 mmol) or testosterone - glycine - Boc (165 mg, 0.37 mmol) was dissolved in 10 mL of CH 2 Cl 2 .

CF 3 COOH (1 mL, 13.10 mmol) was added dropwise to the tocopherol - glycine - Boc solution and stirred at room temperat ure overnight. The excess of CF 3 COOH and the solvent were removed under reduced pressure, CH 2 Cl 2 was added and removed under reduced pressure twice. The remnant solid was dissolved again in 30 mL of CH 2 Cl 2 and washed with 5% NaHCO 3 aqueous solution (2 × 15 mL), saturated NaCl solution

(1 × 15 mL) and dried over MgSO 4 . The CH 2 Cl 2 was removed under reduced pressure, to obtain tocopherol - glycine - NH 2 ( 1 ) as a white powder (131 mg, yield 75%), or testosterone - glycine - NH 2 ( 2 ) as a yellow powder (74.4 mg, yield 58 %).

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for 0.5 h. 13.07 mL of PCl 3 (20.52 g, 149.41 mmol) were slowly added dropwise with a 20 mL syringe, while reaction mixture was stirred at 0 - 4 o C. Then, the reaction mixture was stirred at room temperature for 1 h. The solution was cooled to 0 - 4 o C with ice bath fo r a second time, 12.1 mL of SO 2 Cl 2 (20.17 g, 149.41 mmol) were added dropwise and the mixture was stirred for 1 h at 0 - 4 o C. Then, the reaction mixture was fast filtered over Celite and Et 2 O was removed under reduced pressure at room temperature. The purif ication of the product was carried out via vacuum distillation with a Büchi glass oven (Büchi Labortechnik, Switzerland) at 40 o C under reduced pressure of 4 mbar to obtain Cl 3 P=N – Si(CH 3 ) 3 as a colourless liquid. The monomer was stored under Ar atmosphere at - 35 o C in the glovebox (18.00 g, yield 54%). 1 H NMR (300 31 1 MHz, CDCl 3 ):  = 0.16 (s, 9H) ppm; P{ H} NMR (121 MHz, CDCl 3 ):  = - 54.2 ppm.

Synthesis of the polymer P1 . (C 6 H 5 ) 3 PCl 2 (3.27 mg, 0.01 mmol) and Cl 3 P=N – Si(CH 3 ) 3

(55.00 mg, 0.25 mmol) were dissol ved in 1 mL of anhydrous CH 2 Cl 2 and stirred at room temperature overnight. Then, the obtained poly(dichloro)phosphazene (yield quantitative) was transferred to another flask with tocopherol - glycine - NH 2 (1) (120.0 mg, 0.25 mmol) and excess of Et 3 N (72.6 mg, 0.72 mmol) in 10 mL of anhydrous THF, and stirred at room temperature for 24 h. Afterwards, the second post - polymerisation functionalisation was carried out with Jeffamine M1000. An excess of Jeffamine M1000

(344.0 mg, 0.34 mmol) and Et 3 N (108.9 mg, 1.08 mmol) were added to the mixture and allowed to react another 24 h. Afterwards, the solvent was removed under reduced pressure, dispersed in 15 mL of absolute EtOH and the product was purified by dialysis against deionised water (3 L, 2 times, 8 hours) and EtOH (750 mL, 8 times, 4 days).

The EtOH was removed with N 2 flow and the polymer was further dried under vacuum to give the polymer P1 as a slightly brown waxy solid (212.0 mg, yield 56%). Polymer P2 was obtained also as a slightly brown waxy solid, while polymer P3 appeared as a colorless waxy solid .

3. Characterisation data for materials and polymers P1 - P3

Tocopherol - glycine - NH 2 ( 1 ): ATR - FTIR (solid)  max: 3323 (N – H), 2927 (C – H), 1760 (C=O), 1624 (N - H bend), 1571 (N - H bend), 1449 (C – H bend), 1243 (C – O – C ) cm - 1 ; 1 H

NMR (300 MHz, 298 K, CDCl 3 ):  = 0.85 (t, 12H, 4’C H 3 - + 8’C H 3 - + 12’C H 3 - ), 1.96 - 2.11

(m, 9H, 5C H 3 - + 7C H 3 - + 8C H 3 - ), 2.59 (t, 2H, H4), 3.48 (m, 1H, NH2 – C H 2 – COO – ), 13 3.76 (s, 1H, NH2 – C H 2 – COO – ) ppm; C NMR (75 MHz, CDCl 3 ) [1]:  = 11.4 (5 C H 3 - ),

12 .0 (8 C H 3 - ), 12.3 (7 C H 3 - ), 19.8 (4’ C H 3 - ), 19.9 (8’ C H 3 - ), 20.7 (C4), 21.2 (C2’), 22.8

(12’C H 3 - ), 22.9 (12’C H 3 - ), 23.9 (2 C H 3 - ), 24.6 (C6’), 25.0 (C10’), 28.1 (C12’), 32.8 (C4’), 32.9 (C8’), 34.1 (C3), 37.4 (C7’), 37.5 (C5’), 37.6 (C9’), 37.7 (C3’), 39.5 (C1’ + C11’),

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43.9 (NH2 – C H2 – COO – ), 74.7 (C2), 117.6 (C9), 118.7 (C5), 121.3 (C7), 122.7 (C8), – 144.7 (C9), 145.7 (C10), 149.7 (C6), 156.9 (CF 3 C OO ), 173.1 ( C =O, glycine) ppm.

Testosterone - glycine - NH 2 ( 2 ): ATR - FTIR (solid)  max: 3324 (N – H), 2931 (C – H), 1733 (C=O ), 1671 (C=O, ketone C3 of testosterone), 1625 (N - H bend), 1574 (N - H bend), - 1 1 1448 (C – H bend), 1183 (C – O – C) cm ; H NMR (300 MHz, 298 K, CDCl 3 ) [2]:  = 0.82 (s, 3H, H18), 1.18 (s, 3H, H19), 3.41 - 3.87 (m, 2H, NH2 – C H 2 – COO – ), 5.72 (s, 1H, H4) 13 ppm; C NMR (75 MHz, CDCl 3 ):  = 12.2 (C18), 17.6 (C19), 20.7 (C11), 23.7 (C15), 27.7 (C16), 31.6 (C7), 32.9 (C6), 34.1 (C2), 35.5 (C8), 35.9 (C1), 36.8 (C12), 38.8 (C10), 42.7 (C13), 44.2 (NH2 – C H2 – COO – ), 50.4 (C14), 53.8 (C9), 83.1 (C17), 124.1 – (C4), 156.9 (CF 3 C OO ), 17 1.0 (C5), 174.4 ( C =O, glycine), 199.6 ( C =O, C3) ppm.

Polymer P1 : ATR - FTIR (solid)  max: 3270 (N – H), 2882 (C – H), 1768 (C=O), 1457 (C – - 1 1 H bend), 1108 (C – O – C), 1043 (P=N) cm ; H NMR (300 MHz, 298 K, CDCl 3 ):  = 0.83

(t, 12H, 4’C H 3 - + 8’C H 3 - + 12’C H 3 - ), 1.10 (br, 14H, CH 3 – of PPO groups of Jeffamine

M 1000), 3.35 (s, 6H, CH 3 O – end groups of Jeffamine M 1000), 3.62 (m, 139H, 13 polyalkylene oxide – CH 2 – ), 7.58 (d, 0.63H, (C 6 H 5 ) 3 P=N – end group) ppm; C NMR (75

MHz, CDCl 3 ) [1]:  = 11.4 (5 C H 3 - ), 11.9 (8 C H 3 - ), 12.4 (7 C H 3 - ), 17.4 (C+), 19.8 (4’ C H 3 -

+ 8’ C H 3 - ), 20.9 (C4), 21.2 (C2’), 22.7 (12’C H 3 - ), 22.8 (12’C H 3 - ), 23.9 (2 C H 3 - ), 24.6 (C6’), 25.0 (C10’), 28.1 (C12’), 32.9 (C4’ + C8’), 34.1 (C3), 37.4 - 37.5 (C3’ + C5’ + C7’ +

C9’), 39.5 (C1’ + C11’), 46.2 ( – NH – C H2 – COO – ), 59.1 ( – O – CH 2 – C H(CH 3 ) – of

Jeffamine M1000), 70.7 ( – O – C H 2 – C H 2 – O – of Jeffamine M1000), 72.0 ( – O – C H 2 –

CH(CH 3 ) – of Jeffamine M1000), 75.2 (C2), 117.6 (C9), 118.7 (C5), 121.3 (C7), 122.7 31 (C8), 144.7 (C9), 145.6 (C10), 149.7 (C6); P NMR (121 MHz, CDCl 3 ):  = 0.0 ppm . o GPC* (g/mol) M n = 14174, Mw = 19121. Glass transition temperature ( T g ) = - 15.0 C, o melting temperature ( T m ) = 26.7 C.

Polymer P2 : ATR - FTIR (solid)  max: 3265 (N – H), 2883 (C – H), 1768 (C=O), 1465 (C – - 1 1 H bend), 1108 (C – O – C), 1044 (P=N) cm ; H NMR (300 MHz , CDCl 3 ):  = 0.82 (t, 4H,

4’C H 3 - + 8’C H 3 - + 12’C H 3 - ), 1.10 (br, 14H, CH 3 – of PPO groups of Jeffamine M 1000),

3.35 (s, 6H, CH 3 O – end groups of Jeffamine M 1000), 3.61 (m, 136H, polyalkylene 31 oxide – CH 2 – ), 7.59 (d, 0.76H, (C 6 H 5 ) 3 P=N – end group) ppm; P NMR (121 MHz, o CDCl 3 ):  = 0.84 ppm. GPC* (g/mol) M n = 18775, Mw = 24068. T g = - 14.6 C, o T m = 28.4 C.

Polymer P3 : ATR - FTIR (solid)  max: 3289 (N – H), 2866 (C – H), 1739 (C=O), 1672 (C=O, ketone C3 of testosterone), 1454 (C – H bend), 1107 (C – O – C), 1041 (P=N) cm - 1 ; 1 H NMR (300 MHz, CDCl 3 ):  = 0.81 (s, 1H, H18), 1.11 (br, 14H, CH 3 – of PPO groups

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of Jeffami ne M 1000), 3.36 (s, 6H, CH 3 O – end groups of Jeffamine M 1000), 3.62 (m, 31 139H, polyalkylene oxide – CH 2 – ), 7.59 (d, 0.73H, (C 6 H 5 ) 3 P=N – end group) ppm; P

NMR (121 MHz, CDCl 3 ):  = 0.80 ppm. GPC* (g/mol) M n = 17117, Mw = 25382. o o T g = - 14.5 C, T m = 26.8 C.

* The molecular weights of polymers P1 - P3 determined by GPC measurements calibrated against linear polystyrene standards deviated from the valu es calculated for the polymers, due to the different hydrodynamic volume of branched polyphosphazenes and linear polystyrene standards [20].

4. Supporting Figures

Fig. SI - 1. UV spectra of CPT at 0.00503 mg/mL, 1.5CPT - P1 at 0.025 mg/mL, CPT - P1 at 0.025 mg/mL, CPT - P2 at 0.022 mg/mL, CPT - P3 at 0.018 mg/mL in DMSO.

- 1 - 1 Fig. SI - 2. Calibration curve of CPT in DMSO (CPT = 21145 M cm ).

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- 1 - 1 Fig. SI - 3. Calibration curve of CPT in PBS (pH 7.4) (CPT = 26927 M cm ).

Fig. SI - 4. UV spectra of EPI at 0.019 mg/mL, EPI - P1 at 0.583 mg/mL, EPI - P2 at 0.4 mg/mL in DMSO.

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- 1 - 1 Fig. SI - 5. Calibratio n curve of EPI in DMSO (EPI = 12238 M cm ).

- 1 - 1 Fig. SI - 6. Calibration curve of EPI in PBS (pH 7.4) (EPI = 10730 M cm ).

Fig. SI - 7. Calibration curve of testosterone in PBS (pH 7.4) (Testosterone = 17 594 M - 1 cm - 1 ).

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Fig. SI - 8. Calibration curve of testosterone in PBS (pH 6.0) (Testosterone = 16613 M - 1 cm - 1 ).

Fig. SI - 9. Calibration curve of DL - α - tocopherol in PBS (pH 7.4) (Tocopherol = 8960 M - 1 cm - 1 ).

Fig. SI - 10. Calibration curve of DL - α - tocopherol in PBS (pH 6.0) - 1 - 1 (Tocopherol = 10768 M cm ).

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Fig. SI - 11. FT - IR spectra of tocopherol - glycine - NH 2 ( 1 ).

Fig. SI - 12. FT - IR spectra of testosterone - glycine - NH 2 ( 2 ).

1 Fig. SI - 13. Partial H NMR (3 00 MHz, 298 K, CDCl 3 ) spectrum of tocopherol - glycine -

NH 2 ( 1 ).

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1 Fig. SI - 14. Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of testosterone - glycine -

NH 2 ( 2 ).

13 Fig. SI - 15. Partial APT C NMR (75 MHz, 298 K, CDCl 3 ) spectrum of tocopherol - glycine - NH 2 ( 1 ).

13 Fig. SI - 16. Partial APT C NMR (75 MHz, 298 K, CDCl 3 ) spectrum of testosterone - glycine - NH 2 ( 2 ).

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31 1 Fig. SI - 17. Partial P{ H} NMR (121 MHz, 298 K, CDCl 3 ) spectrum of monomer trichlorophosphoranimine (Cl 3 P=N – Si(CH 3 ) 3 ).

31 1 Fig. SI - 18. Partial P{ H} NMR (1 21 MHz, 298 K, CDCl 3 ) spectrum of polydichlorophosphazene.

1 Fig. SI - 19. Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of monomer trichlorophosphoranimine (Cl 3 P=N – Si(CH 3 ) 3 ).

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1 Fig. SI - 20. Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of polydichloroph osphazene.

1 Fig. SI - 21. Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of polymer P2 , DP was calculated as: DP = = .

1 Fig. SI - 22. Partial H NMR (300 MHz, 298 K, CDCl 3 ) spectrum of polymer P3 , DP was calculated as: DP = = .

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13 Fig. SI - 23. Partial APT C NMR (75 MHz, 298 K, CDCl 3 ) spectrum of polymer P1 .

Fig. SI - 24. Calorimetry diffe rential scanning curve of polymer P1 .

Fig. SI - 25. Calorimetry differential scanning curve of polymer P2 .

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Fig. SI - 26. Calorimetry differential scanning curve of polymer P3 .

Fig. SI - 27. TGA curve of P1 .

Fig. SI - 28. TGA curve of P2 .

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Fig. SI - 29. TGA curve of P3 .

Fig. SI - 30. SEM micrographs of P1 - P2 dried nanoaggregates at 20,000× magnification.

Fig. SI - 31. Linear fitting of in vitro CPT release profiles of CPT - loaded polyphosphazene nanoaggregates up to 8 hours in PBS (pH 7.4) at 3 7 o C.

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Fig. SI - 32. Linear fitting of in vitro EPI release profiles of EPI - loaded polyphosphazene nanoaggregates up to 9 hours in PBS (pH 7.4) at 37 o C.

Fig. SI - 33. Linear fitting of log(Cumulative Release (%)) Vs. log(Time(hours)) of CPT - loaded po lyphosphazene nanoaggregates up to 100 hours in PBS (pH 7.4) at 37 o C.

Fig. SI - 34. Linear fitting of log(Cumulative Release (%)) Vs. log(Time(hours)) of EPI - loaded polyphosphazene nanoaggregates up to 24 hours in PBS (pH 7.4) at 37 o C.

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Fig. SI - 35. In vitro release profiles of tocopherol from P1 aggregates in PBS (pH 7.4 and 6.0) at 37 o C, adjusted to a SWeibull2 function.

Fig. SI - 36. In vitro release profiles of testosterone from P2 aggregates in PBS (pH 7.4 and 6.0) at 37 o C, adjusted to a SW eibull2 function.

Fig. SI - 37. In vitro release profiles of testosterone from P3 aggregates in PBS (pH 7.4 and 6.0) at 37 o C, adjusted to a SWeibull2 function.

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Fig. SI - 38. MCF - 7 cells fluorescence images and slices of cells without particles and Ly soTracker (B). Cells with 0.1 mg/mL of 1.5CPT - P1 aggregates, 50 nM of LysoTracker Yellow HCK - 123 and merged pictures (M), scale bars represents 20 µm.

Fig. SI - 39. MCF - 7 cells fluorescence images and slices of cells without particles and LysoTracker (B ). (a) Cells with 0.1 mg/mL of CPT - P2 aggregates, 50 nM of LysoTracker Yellow HCK - 123 and merged pictures (M), scale bars represents 20 µm.

Fig. SI - 40. MCF - 7 cells fluorescence images and slices of cells without particles and LysoTracker (B). (a) Cell s with 0.1 mg/mL of CPT - P3 aggregates, 50 nM of LysoTracker Yellow HCK - 123 and merged pictures (M), scale bars represents 20 µm.

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Fig. SI - 41. MCF - 7 cells fluorescence images and slices of cells without particles and Hoechst (B). Cells with 0.1 mg/mL of EPI - P2 aggregates, 1 µg/mL of Hoechst 33342 and merged pictures (M), scale bars represents 20 µm.

Fig. SI - 42. Relative cell viability of MCF - 7 breast cancer cells treated with parent CPT or EPI. Data represents the mean ± standard deviation (n = 3).

References [1] P. P. Lankhorst, T. Netscher, A. L. L. Duchateau, A Simple 13 C NMR method for the discrimination of complex mixtures of stereoisomers: All eight stereoisomers of α - tocopherol resolved, Chirality 27 (2015) 850 - 855. Doi: 10.1002/chir.22535 [2] K. Hayamizu, T. Ishii, M. Yanagisawa, Complete assignments of the 1 H and 13 C NMR spectra of testosterone and 17α - methyltestosterone and the 1 H parameters obtained from the 600 MHz spectra, Magnetic Resonance in Chemistry 28 (1990) 250 - 256.

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5. Summary and Outlook

In this thesis, different polymer - based carriers of diosgenin, agrochemicals DI31 and S7, as well as anticancer drugs camptothecin and epirubicin have been presented with adjustable drug content, properties and drug delivery rates. Furthermore , it was observed a good biological activity as agrochemicals or anticancer drugs of the synthesized polymer - based carriers, similar and in some cases slightly superior to the parent drugs. First, water - soluble cellulose ethers were esterified with steroid s and vitamins to obtain water - dispersible cellulose - based particles with pH - promoted delivery of agrochemicals, or able to encapsulate camptothecin for later sustained release ( Chapter 2 ). All the cellulose - based carriers were thoroughly characterized, wi th particular emphasis on the study of the drug release behavio u r and biological activity. Interestingly, all cellulose - based carriers formed stable and almost neutral aggregates in aqueous media. The hydrolysis of the succinate linker bonding the steroids to the cellulose backbone controlled the diosgenin and agrochemical release rates, with almost quantitative drug release (75% - 88%) at 72 hours in PBS (pH 5.0). On the other hand, negligible diosgenin release was observed for diosgenin - grafted cellulose et hers after 72 hours in PBS (pH 7.0). In general, bigger particles in aqueous media, with lower camptothecin and drug content showed faster and more quantitative drug delivery. Agrochemical activity of DI31 - and S7 - grafted cellulose ethers evaluated using r adish cotyledons, appeared superior when compared to parent DI31 and S7. Particularly, agrochemical bearing celluloses showed 1.3 times plant growth enhancer activity compared to free DI31 and S7 at the lowest concentrations typically used in agriculture ( 10 - 6 to 10 - 7 mg/mL). Camptothecin - loaded cellulose particles maintained the characteristic cytotoxicity of parent camptothecin to MCF - 7 human breast cancer cells. Consequently, these cellulose - based carriers could be potentially used in agriculture and med icine. However, further in vitro and in vivo studies are necessary on the camptothecin - loaded celluloses to properly evaluate the pharmacokinetic parameters, toxicity, side effects and other relevant information before going into clinical trial studies. S econd, steroid - and vitamin - grafted silk fibroin conjugates have been presented ( Chapter 3 ). Particularly, diosgenin - , DI31 - and S7 - grafted silk fibroin conjugates were efficiently synthesized for drug release of agrochemicals. Testosterone - , tocopherol - a nd ergocalciferol - grafted silk fibroin conjugates were indeed prepared for later

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hydrophobic encapsulation of camptothecin and controlled delivery of this anticancer drug. The steroid - grafted silk fibroin aggregates exhibited a sustained release of diosgen in and agrochemicals DI31 and S7 that attained a 40 - 50% of linked steroid in PBS (pH 6.0) after 5 days. It was also observed that bigger silk fibroin aggregates with lower steroid contents showed a higher release rate with a higher quantity of released dru g. Good agrochemical activity and low cytotoxicity of DI31 - and S7 - grafted silk fibroin conjugates were observed when evaluated at low concentration on radish cotyledons, MCF - 7 and HEK - 293 cells, respectively. In contrast, camptothecin - loaded silk fibroin aggregates exhibited almost quantitative (76% - 97%) drug release after 6 days in simulated physiological conditions (PBS, pH 7.4, 37 o C). Additionally, camptothecin - loaded silk fibroin conjugates appeared to be highly cytotoxic to MCF - 7 and good cell uptake was observed after 6 hours. Therefore, the synthesized silk fibroin - based carriers seem potential candidates for agrochemical applications, as well as for chemotherapy treatment of metastatic tumors and cancers resistant to irinotecan. However, as previou sly mentioned, in vivo studies are needed for a proper assessment of the camptothecin - loaded silk fibroin particles. Finally, diferent poly(organo)phosphazenes carrying stero ids or vitamins and Jeffamine M - 1000 have been described ( Chapter 4 ). Diosgenin, DI31 and S7 glycinates were intro duced together with Jeffamine M - 1000 onto the polyphosphazene backbone for use in agriculture. Testosterone and tocopherol gly cinates, as well as Jeffamine M - 1000 were incorporated in the poly(organo)phosphazene backbone to ob tain amphiphilic polymers that self - aggregated in aqueous media, with the capacity to encapsulate lipophilic camptothecin or to be loaded with hydrophilic epirubicin. All the poly(organo)phosphazenes formed aggregates in aqueous media with dimensions from 20 - 250 nm. Sustained drug release of diosgenin, agrochemicals DI31 and S7, camptothecin and epirubicin was observed for the prepared poly(organo)phosphazenes. In vitro evaluation of DI31 and S7 bearing poly(organo)phosphazenes showed excellent agrochemical activity and strong cytotoxicity to MCF - 7 cells. Camptothecin and epirubicin bearing poly(organo)phosphazenes exhibited potent cytotoxicity to MCF - 7 cell line, similar to the one observed for parent camptothecin and epirubicin. Furthermore, MCF - 7 cells sh owed good uptake of camptothecin and epirubicin bearing polyphosphazenes. Consequently, the synthesized poly(organo)phosphazenes demonstrated good potential for fine tuning optimization of their properties (i.e. degradation rate,

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dispersi bility in water an d drug release rate). Furthermore, they are excellent candidates for use in agriculture as plant growth regulator and in medicine for treatment of tumors by one - or multi - drug delivery strategy (i.e. camptothecin and/or epirubicin).

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COMPLETE LIST OF P UBLICATIONS

. J. P. Quiñones , A. Iturmendi, H. Henke, C. Roschger, A. F. Zierer, C. Peniche - Covas, O. Brüggemann, Polyphosphazene - based nanocarriers for the release of camptothecin and epirubicin, 2019, submitted. . J. P. Quiñones , C. Roschger, A. Zierer, C. P eniche - Covas, O. Brüggemann, Self - assembled silk fibroin - based aggregates for delivery of camptothecin, 2019, submitted. . J. P. Quiñones , A. Iturmendi, H. Henke, C. Roschger, A. Zierer, O. Brüggemann, Polyphosphazene - based nanocarriers for the release of ag rochemicals and potential anticancer drugs, 2019, in correction of reviewers comments. . J. P. Quiñones , C. Roschger, A. Zierer, C. Peniche, O. Brüggemann, Steroid - grafted silk fibroin conjugates for drug and agrochemical delivery, European Polymer Journal 1 19 (2019) 169 - 175. Doi: 10.1016/j.eurpolymj.2019.07.025 . J. P. Quiñones , C. C. Mardare, A. W. Hassel, O. Brüggemann, Testosterone - and vitamin - grafted cellulose ethers for sustained release of camptothecin, Carbohydrate Polymers 206 (2019) 641 - 652. Doi: 10 .1016/j.carbpol.2018.11.047 . J. P. Quiñones , H. Peniche, C. Peniche, Chitosan based self - assembled nanoparticles in drug delivery, Polymers 10 (2018) 235. Doi: 10.3390/polym10030235 . J. P. Quiñones , O. Brüggemann, J. Kjem s, M. H. Shahavi, C. P. Covas, Novel brassinosteroid - modified polyethylene glycol micelles for controlled release of agrochemicals, Journal of Agricultural and Food Chemistry 66 (2018) 1612 - 1619. Doi: 10.1021/acs.jafc.7b05019 . J. P. Quiñones , J. Jokinen, S. Kein ä nen, C. P. Covas, O. Brüggemann, D. Ossipov, Self - assembled hyaluronic acid - testosterone nanocarriers for delivery of anticancer drugs, European Polymer Journal 99 (2018) 384 - 393. Doi: 10. 1016/j.eurpolymj.2017.12.043 . J. P. Quiñones , O. Brüggemann, C. P. Covas, Hydrophobically - modified chitosan microspheres for release of diosgenin, Internatinoal Journal of Nanoparticle Research 2 (2018) 0001 - 0009. . J. P. Quiñones , C. C. Mardare, A. W. Hassel , O. Brüggemann, Self - assembled cellulose particles for agrochemical applications, European Polymer Journal 93 (2017) 706 - 716. Doi: 10.1016/j.eurpolymj.2017.02.023 . J. P. Quiñones , J. Kjems, C. Yang, C. Peniche, O. Brüggemann, Self - assembled O6 - succinyl chi tosan nanoparticles for controlled release of diosgenin and agrochemicals, Nanomedicine & Nanotechnology Open Access 2 (2017) 000128.

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. J. P. Quiñones , O. Brüggemann, C. Peniche, Fructose chitosan based self - assembled nanoparticles for sustained release of v itamins and steroids, JSM Nanotechnology & Nanomedicine 5 (2017) 1056. . J. P. Quiñones , O. Brüggemann, C. P. Covas, D. A. Ossipov, Self - assembled hyaluronic acid nanoparticles for controlled release of agrochemicals and diosgenin. Carbohydrate Polymers 173 (2017) 157 - 169. Doi: 10.1016/j.carbpol.2017.05.048 . C. Becheran, L. Bocourt, J. P. Quiñones , C. Peniche, Chitosan in biomedicine. From gels to nanoparticles, Advances in Chitin Science 14 (2014) 217 - 224. . J. P. Quiñones , K. V. Gothelf, J. Kjems, A. Heras, C. Schmidt, C. Peniche, Novel self - assembled nanoparticles of testosterone - modified glycol chitosan and fructose chitosan for controlled release, Journal of Biomaterials and Tissue Engineering 3 (2013) 164 - 172. Doi: 10.1 166/jbt.2013.1071 . J. P. Quiñones , K. V. Gothelf, J. Kjems, C. Yang, A. M. H. Caballero, C. Schmidt, C. P. Covas, Self - assembled nanoparticles of modified - chitosan conjugates for the sustained release of D L - α - tocopherol, Carbohydrate Polymers 92 (2013) 856 - 864. Doi: 10.1016/j.carbpol.2012.10.005 . J. P. Quiñones , K. V. Gothelf, J. Kjems, C. Yang, A. M. H. Caballero, C. Schmidt, C. P. Covas, N, O6 - partially acetylated chitosan nanoparticles hydrophobically - modified for the controlled release of steroids and vita min E, Carbohydrate Polymers 91 (2013) 143 - 151. Doi: 10.1016/j.carbpol.2012.07.080 . J. P. Quiñones , K. V. Gothelf, J. Kjems, A. M. H. Caballero, C. Schmidt, C. P. Covas, Self - assembled nanoparticles of glycol chitosan - ergocalciferol succinate conjugate for controlled release, Carbohydrate Polymers 88 (2012) 1373 - 1377. Doi: 10.1016/j.carbpol.2012.02.039 . J. P. Quiñones , R. Szopko, C. Schmidt, C. P. Covas, Novel drug delivery systems: chitosan conjugates covalently attached steroids with potential anticancer a nd agrochemical activity, Carbohydrate Polymers 84 (2011) 858 - 864. Doi: 10.1016/j.carbpol.2010.12.007 . J. P. Quiñones , Y. C. García, H. Curiel, C. P. Covas, Microspheres of chitosan for controlled delivery of brassinosteroids with biological ac tivity as agrochemicals, Carbohydrate Polymers 80 (2010) 915 - 921. Doi: 10.1016/j.carbpol.2010.01.006 . R. Suardiaz, J. P. Quiñones , D. García, C. S. Pérez, A DFT study of the substitu ent induced shifts of a 5α - OH on ring A 13C chemical shifts of spirostanic sapogenins, Journal of Molecular Structure: THEOCHEM 769 (2006) 87 - 89. Doi: 10.1016/j.theochem.2006.04.027

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CONFERENCE CONTRIBUT IONS (LAST FIVE YEARS)

. J. P. Quiñones , A. Itur mendi, I. Teasdale, O. Brüggemann, Polyphosphazene - based nanoparticles for delivery of camptothecin , oral presentation at 4 th International Conference on Bio - Based Polymers and Composites ( BiPoCo 2018), Hungary, September 2018. . J. P. Quiñones , L. M. Uiberl acker , C. P. Covas, O. Brüggemann, Self - assembled silk fibroin - based particles for delivery of camptothecin, poster presentation at Symposium “Catalysis – A keyplayer in science” (LIKAT - INCA) , JKU University Linz, Austria, February 2018. . J. P. Quiñones , A. Iturmendi , I. Teasdale, C. C. Mardare, A. W. Hassel, O. Brüggemann, Polyphosphazene - based nanoparticles for delivery of diosgenin and agrochemicals, poster presentation at Chemietage 2017 Joint Meeting of the Swiss & Austrian Chemistry Societ ies, University of Slayburg, Austria, September 2017. . J. P. Quiñones , L. M. Uiberlacker, C. C. Mardare, A. W. Hassel, O. Brüggemann, Novel self - assembled steroid - silk fibroin particles for delivery of agrochemicals and diosgenin, oral presentation at ISPAC 2017 International Symposium on Polymer Analysis and Characterization, JKU University Linz, Austria, June 2017. . J. P. Quiñones , C. C. Mardare, A. W. Hassel, O. Brüggemann, Self - assembled cellulose particles for agrochemical applications, 3 rd Internationa l Conference on Bio - Based Polymers and Composites ( BiPoCo 2016), Hungary, September 2016 .

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