polymers

Article Photo- and Acid-Degradable Polyacylhydrazone– Doxorubicin Conjugates

Maria Psarrou 1, Martha Georgia Kothri 2 and Maria Vamvakaki 1,3,*

1 Department of Materials Science and Technology, University of Crete, Vasilika Vouton, 700 13 Heraklion, Crete, Greece; [email protected] 2 School of Medicine, University of Crete, Vasilika Vouton, 700 13 Heraklion, Crete, Greece; [email protected] 3 Institute of Electronic Structure and Laser, Foundation for Research and Technology-Hellas, Vasilika Vouton, 700 13 Heraklion, Crete, Greece * Correspondence: [email protected]; Tel.: +30-2810-545019

Abstract: Light-mediated polymer degradation has attracted considerable attention in various applications, including photo-patterning, tissue engineering and photo-triggered drug delivery. In this study, we report the synthesis and characterization of a new, linear, main-chain photo- and acid- degradable copolymer based on acylhydrazone linkages. The polymer was synthesized via a step- growth copolymerization of adipic acid dihydrazide with a bifunctional poly(ethylene glycol) bearing benzaldehyde end-groups, under mild acidic conditions, to afford a hydrophilic PEG-alt-adipic acid (PEG-alt-AA) alternating copolymer. The synthesized polymer was characterized by size exclusion chromatography, proton nuclear magnetic resonance and attenuated total reflection-Fourier transform



 infrared spectroscopies. The main-chain photo- and acid-induced degradation of the copolymer in dimethylsulfoxide and water, respectively, was verified by UV-vis spectroscopy at light intensities Citation: Psarrou, M.; Kothri, as low as 0.1 mW cm−2 at λ = 254 nm. Next, a model anticancer drug, doxorubicin (DOX), was M.G.; Vamvakaki, M. Photo- and Acid-Degradable Polyacylhydrazone– chemically linked to the polymer chain end(s) via acylhydrazone bond(s), resulting in amphiphilic Doxorubicin Conjugates. Polymers PEG-alt-adipic acid-DOX (PEG-alt-AA-DOX) polymer–drug conjugates. The conjugates were self- 2021, 13, 2461. https://doi.org/ assembled in water to form spherical nanoparticles, as evidenced by scanning and transmission 10.3390/polym13152461 electron microscopies. The irradiation of the self-assembled PEG-alt-AA-DOX conjugates with UV light and the decrease of the solution pH resulted in the disruption of the assemblies due to the Academic Editors: Asterios photolysis and acidolysis of the acylhydrazone bonds, and the release of the therapeutic cargo. (Stergios) Pispas, Spiros H. Anastasiadis and Hermis Iatrou Keywords: polyacylhydrazones; main-chain cleavage; photo-degradable polymers; acid-degradable polymers; prodrugs; doxorubicin Received: 6 May 2021 Accepted: 22 July 2021 Published: 27 July 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Stimuli-degradable polymers (SDPs) have gained increasing attention due of their published maps and institutional affil- wide range of potential applications in various fields, including nano- and bio-technology iations. as drug carriers and actuators, and in bio-patterning, etc [1,2]. Recently, SDPs have also attracted significant scientific and technological interest in the design of new recyclable and sustainable materials to eliminate the concerns of global environmental pollution from the non-degradable plastics [3,4]. Among the various chemical and physical stimuli (e.g., pH, temperature, enzymes, Copyright: © 2021 by the authors. ultrasound) employed to trigger the degradation of the polymer chains, light-degradable Licensee MDPI, Basel, Switzerland. This article is an open access article polymers have attracted considerable interest. Light, as an external stimulus, offers unique distributed under the terms and advantages such as spatiotemporal control, as well as the tunability of the irradiation conditions of the Creative Commons wavelength and intensity [5]. So far, the vast majority of photodegradable polymers have Attribution (CC BY) license (https:// relied on the light-induced cleavage of specific chemical bonds, such as ester, triazine, creativecommons.org/licenses/by/ ketal/acetal and urethane, which are accompanied by certain photo-absorbing moieties, 4.0/). namely o-nitro benzyl (ONB) groups, coumarin derivatives or other aromatic units. The

Polymers 2021, 13, 2461. https://doi.org/10.3390/polym13152461 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 2461 2 of 20

photolabile bonds are usually located either in the pendant groups of the polymer chain or as a single photocleavable bond as a junction point of two long polymer chains. How- ever, the degradation of such materials results in the production of high molecular weight photo-products, which hinder their use in biomedical applications (drug delivery or tissue engineering) in which the removal of the by-products from the living organisms is crucial. On the other hand, photodegradable polymers bearing multiple photo-cleavable groups along their main-chain, to produce small molecule photo-products upon irradiation, are limited [2,6]. In 2011, our group introduced, for the first time, a new class of photodegrad- able polymers based on poly(ketals/acetals), exhibiting main-chain scission at incredibly low irradiation doses (10 mJ cm−2) compared to other polymeric materials [7,8]. More recently, Kowollik and co-workers reported the synthesis of main-chain photodegradable polyurethanes by introducing ONB moieties along the backbone of a polyurethane chain, and studied the degradation profiles of these materials both in solution and on thin films using a nanosecond pulsed laser at 340 nm [9]. SDPs have been extensively explored as nanocarriers for the controlled and/or pro- grammable delivery of drugs and other active biomolecules, exhibiting very promising results. Polymer micelles, polymersomes, nanogels and polymer–drug conjugates are typical nanostructures employed in drug delivery applications [1,10–17]. Among them, polymer–drug conjugates, also known as prodrugs, offer specific advantages over other drug delivery systems. First, the drug is covalently bound either along or at the end of the polymer chain, thus preventing its toxic side effects. Second, the covalent attachment of the drug onto the polymer prevents the passive diffusion of the drug and its leakage from the carrier during blood circulation, which is a common problem in physically entrapped drugs within nanocarriers, resulting in a lower drug accumulation at the tissue of interest. Last, the incorporation of stimuli-degradable drug–polymer linkages allows site-specific drug release and increases its therapeutic efficacy [18–23]. Even though there are a few studies on stimuli-activatable prodrugs, polymer–drug conjugates based on a main-chain photodegradation process have been scarcely reported in the literature [24]. Linear, main-chain polyhydrazones and polyacylhydrazones belong to a polymer family that has barely been studied in the literature because of their low solubility in both polar and non-polar solvent media [25,26]. Hydrazones and acylhydrazones are acid-sensitive bonds, typically formed by the reaction of a carbonyl moiety (aldehyde or ketone) with a hydrazine or hydrazide group, respectively, under mild conditions. The hydrazone bond has been extensively employed in the development of acid-degradable polymer-drug conjugates, polymer micelles, hydrogels and other materials [27–32]. It is well-known that the pH varies in the different tissues and cellular compartments. In cancer tissues, the microenvironment is slightly acidic, 0.5–1.0 pH lower compared to the normal tissues, due to the increased glycolysis in cancer cells. Moreover, a larger decrease in the pH is observed in the intracellular organelles, ranging from pH 6.3 in the early endosome to pH 4.7 in the lysosome [33]. Another impressive feature of the (acyl)hydrazone bonds is their dynamic character. The (acyl)hydrazone bond is characterized as a dynamic covalent bond because of its ability to reversibly form and break under mild conditions. The cleavage of the (acyl)hydrazone bond produces the initial molecules without the generation of any toxic by-products. Due to this unique property, several research groups have used these covalent bonds in the development of dynamic materials, including self-healing polymers, adaptable polymer networks, shape-memory materials and others [30,34–36]. In this study, we designed a linear, main-chain, light-cleavable polyacylhydrazone which was used in the development of photo-degradable polymer-drug conjugates and pro- drug nanoparticles. The polymer was synthesized via the step-growth copolymerization of adipic acid dihydrazide with dibenzaldehyde terminated poly(ethylene glycol) (PEGald), under mild acidic conditions, to afford a hydrophilic PEG-alt-adipic acid (PEG-alt-AA) alternating copolymer (Scheme1b). The PEG ald macromonomer was prepared by the esterification of HO-PEG-OH with 4-carboxybenzaldehyde (Scheme1a). The main-chain photo-degradation of the resulting copolymer, upon irradiation at λ = 254 nm with a very Polymers 2021, 13, x 3 of 19

Polymers 2021, 13, 2461 alt-AA) alternating copolymer (Scheme 1b). The PEGald macromonomer was prepared3 of 20by the esterification of HO-PEG-OH with 4-carboxybenzaldehyde (Scheme 1a). The main- chain photo-degradation of the resulting copolymer, upon irradiation at λ = 254 nm with a very low power density, 0.1 mW cm–2, in dimethylsulfoxide (DMSO) and water, was low power density, 0.1 mW cm−2, in dimethylsulfoxide (DMSO) and water, was inves- tigated.investigated. Copolymers Copolymers with awith hydrazide a hydrazide end-group end-group were were prepared prepared using using an excess an excess of the of adipicthe adipic acid acid dihydrazide dihydrazide comonomer comonomer during duri theng the polymerization. polymerization. Next, Next, the the hydrophobic hydropho- modelbic model anticancer anticancer drug, drug, doxorubicin doxorubicin (DOX), (DOX was), was linked linked to the to the hydrazide hydrazide end-groups end-groups of theof the polymer polymer via acylhydrazonevia acylhydrazone linkages, linkages, to produce to produce amphiphilic amphiphilic PEG- altPEG--adipicalt-adipic acid-DOX acid- (PEG-DOX alt(PEG--AA-DOX)alt-AA-DOX) drug conjugates drug conjugates (Scheme (Scheme1c). The 1c conjugates). The conjugates self-assembled self-assembled in water in towater form to spherical form spherical nanoparticles nanoparticles evidenced eviden byced scanning by scanning and transmission and transmission electron electron micro- scopies.microscopies. Following Following the irradiation the irradiation of the of nanoparticles the nanoparticles with UV with light, UV thelight, assemblies the assemblies were disruptedwere disrupted due to due the to photolysis the photolysis of the of acylhydrazone the acylhydrazone bonds bonds along along the polymer the polymer backbone, back- andbone, the and drug the molecules drug molecules were released.were released. The synergistic The synergistic action action of the of two the stimuli, two stimuli, pH and pH lightand light irradiation, irradiation, augmented augmented the disruption the disrupti ofon the of self-assembled the self-assembled prodrug prodrug nanostructures nanostruc- andtures the and drug therelease. drug release.

SchemeScheme 1. 1.Synthetic Synthetic routesroutes for for the the preparation preparation of of the the ( a()a PEG) PEGaldaldmacromonomer, macromonomer,( b(b)) PEG- PEG-altalt-AA-AA alternating alternating copolymer copolymer andand ( c()c) PEG- PEG-altalt-AA-DOX-AA-DOX drug drug conjugate. conjugate. (d )(d Schematic) Schematic representation representation of the of self-assemblythe self-assembly process process of the of PEG- the altPEG--AA-DOXalt-AA- conjugatesDOX conjugates and the and pH- the and/or pH- and/or light-induced light-induced disruption disruption of the of assemblies. the assemblies.

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2. Experimental 2.1. Materials and Methods The 4-carboxybenzaldehyde (98%) and triethylamine (TEA, 99+%) were purchased from Alfa Aesar (Kandel, Germany). The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, ≥97%), 4-(dimethylamino)pyridine (DMAP, 99%), adipic acid dihydrazide (≥98%) −1 and poly(ethylene glycol) (Mn = 4000 g mol ) were obtained from Sigma Aldrich (Stein- heim, Germany) and the doxorubicin hydrochloride salt (DOX·HCl, >99%) was obtained from LC Laboratories (Woburn, MA, USA). The diethyl ether, petroleum ether, n-hexane (96%) and dichloromethane (DCM) were purchased from Scharlau (Sentmenat, Spain). The DCM was dried over calcium hydride. The DMSO (99.9%) was obtained from Fisher Chemical (Loughborough, UK) and the ethanol (≥98%) from Honeywell (Seetze, Germany). Milli-Q water, with a resistivity of 18.2 MΩ·cm at 298 K, was obtained from a Millipore apparatus, and was used for the preparation of all of the samples.

−1 2.2. Synthesis of the Dibenzaldehyde Terminated Poly(ethylene glycol) (Mn = 4000 g mol ) Macromonomer First, 4-carboxybenzaldehyde (0.3 g, 2 mmol) and EDC (0.23 g, 1.5 mmol) were dissolved in 20 mL anhydrous DCM, and were stirred at 40 ◦C for 30 min, followed by the addition of DMAP (0.36 g, 3 mmol) and HO–PEG–OH (2 g, 0.5 mmol). The reaction was stirred at 40 ◦C for 24 h. Next, the product was precipitated in diethyl ether, washed 10 times with petroleum ether and dried overnight under vacuum at room temperature (RT). The dried product was dissolved in chloroform and filtered through a 0.45 µm PTFE filter to remove the unreacted 4-carboxybenzaldehyde. Finally, it was precipitated again in diethyl ether and dried overnight under vacuum to obtain PEGald at 82.5% yield.

2.3. Synthesis of the PEG-alt-AA Alternating Copolymer Adipic acid dihydrazide (25 mg, 0.14 mmol) was dissolved in 2 mL milli-Q water. The monomer solution was placed in a sonication bath until its complete dissolution. Next, PEGald (0.5 g, 0.12 mmol) was added to the solution, followed by the addition of 2–3 drops of 0.1 N HCl. The reaction was left under stirring for 24 h at 25 ◦C. The next day, the solvent was evaporated using a rotary evaporator, and the reaction was dried under vacuum at RT. The white solid was dissolved in chloroform, and was purified by fractional precipitation ◦ in hexane. The final copolymer was stored under a N2 atmosphere at 4 C until use.

2.4. Synthesis of a Small-Molecule Acylhydrazone Analogue Adipic acid dihydrazide (0.1 g, 0.57 mmol) was dissolved in 4 mL ethanol. The solution was first sonicated for 10 min and then stirred for 10 min in a water bath at 40 ◦C until its complete dissolution. Next, a solution of 4-carboxybenzaldehyde (0.17 g, 1.14 mmol) in 2 mL ethanol was prepared. The two solutions were mixed and a white solid precipitated instantly. The reaction was stirred for another 3 h before filtering off the white solid product and washing it with a large amount of ethanol and water. The final product was dried overnight under reduced pressure at RT.

2.5. Synthesis of the PEG-alt-AA-DOX Drug Conjugate First, DOX·HCl (2 mg, 0.003445 mmol) was dissolved in DMSO (2.5 mL), and TEA (3 µL) was added to neutralize the HCl. After 20 h stirring at RT, hydrophobic DOX was obtained. Next, the PEG-alt-AA copolymer (0.05 g, 0.00179 mmol) was added to the DOX solution, and the reaction mixture was stirred for 20 h before being transferred to a dialysis membrane with a molecular weight cut-off of 3500 Daltons. The mixture was dialyzed against DMSO to remove the unreacted DOX molecules. The conjugated DOX molecules Polymers 2021, 13, 2461 5 of 20

were quantified by fluorescence spectroscopy using a standard calibration curve of DOX in DMSO, and the drug loading was calculated using Equation (1).

conjugated DOX (gr) % Drug loading = × 100 (1) weight of polymer (gr)

2.6. Self-Assembly of the Amphiphilic PEG-alt-AA-DOX Drug Conjugates in Water A solution of the PEG-alt-AA-DOX conjugate (5 mg mL−1) in 5 mL DMSO was prepared. Next, 15 mL water (pH 7.4) was added to the solution under vigorous stirring at a rate of 0.1 mL min−1 using a syringe pump. Afterwards, the solution was dialyzed against water using a dialysis membrane with a molecular weight cut-off of 3500 Daltons to remove the organic solvent. The final solution was filtered through a 0.45 µm pore size cellulose acetate syringe filter, and was kept at 4 ◦C until use.

2.7. Characterization Methods A Waters size exclusion chromatography (SEC) instrument comprising a Waters 515 isocratic HPLC pump, two Mixed-D and Mixed-E (Polymer labs) columns (Agilent Tech- nologies, Santa Clara, CA, USA, a Waters 2745 Dual absorbance detector and a Waters 410 refractive index (RI) detector (Waters, Milford, MA, USA) were used for the determina- tion of the molecular weight (Mn) and the molecular weight distribution (Mw/Mn) of the polymers. THF (containing 2 v/v% triethylamine) was used as the eluent at a flow rate of 1 mL min−1. The calibration curve was based on six narrow poly(methyl methacry- late) (PMMA) standards with molecular weights ranging from 625 to 138,000 g mol−1. In order to prepare the samples for SEC, the polymers were dissolved in THF at a concen- tration of 15 mg mL−1; the polymer solutions were then filtered through 0.45 µm pore size PTFE syringe filters and injected into the system. 1H Nuclear Magnetic Resonance (1H NMR) spectra were recorded on two Brucker 300 MHz and 500 MHz spectrometers (Bruker, Rheinstetten, Germany) using CDCl3 or DMSO-d6 as the solvent. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra were recorded on a Nicolet 6700 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). For each spectrum, 64 scans were collected in the 500–4000 cm−1 range. The ultraviolet–visible (UV-vis) ab- sorption spectra were recorded on a Shimadzu UV-2600 spectrophotometer (Kyoto, Japan) in the wavelength range of 200–600 nm. The samples were prepared by the dissolution of the polymers or polymer–DOX conjugates in DMSO or H2O (pH 7.4) at a concentra- tion of 10−3 mg mL−1. A Malvern Zetasizer Nano ZS instrument (Grovewood Road, UK) equipped with a He–Ne laser (λ = 633nm), at a 90◦ scattering angle, was used for the dynamic light scattering (DLS) measurements. Before each measurement, the sample was filtered through a 0.45 µm pore size hydrophilic, cellulose acetate syringe filter. The measurements were conducted at 25 ◦C. The size and the morphology of the self-assembled structures were characterized by field emission-scanning electron microscopy (FE-SEM) us- ing a JEOL JSM-7000F microscope (Tokyo, Japan), and by transmission electron microscopy (TEM) using a JEOL JEM-2100 microscope (Tokyo, Japan). The samples were prepared by the deposition of a drop of a dilute polymer solution on a glass substrate for SEM or a carbon coated copper grid for TEM, and were dried overnight at 25 ◦C. The fluores- cence spectra were recorded on a Lumina Fluorescence Spectrometer (Thermo Scientific) (Waltham, MA, USA) equipped with a 150 W CW Xenon-arc lamp. The excitation and emission slits were set at 10 nm and 20 nm, respectively. The fluorescence spectra of the DOX and the PEG-alt-AA-DOX conjugate were recorded from 500 nm to 900 nm upon excitation at 488 nm, whereas the respective spectrum of the copolymer was recorded upon excitation at 302 nm. All of the samples were measured at room temperature. Polymers 2021, 13, 2461 6 of 20

2.8. Photoirradiation Experiments All of the photoirradiation experiments were performed using a UV lamp at 254 nm and a power density of 0.1 mW cm−2. The samples were transferred to a quartz cuvette, and were placed at a distance of 1 cm from the lamp. The photodegradation percentage of the small molecule acylhydrazone, the PEG-alt-AA alternating copolymer and the self- assembled PEG-alt-AA-DOX drug conjugate were calculated from the recorded UV-visible spectra by monitoring the decay of the maximum absorption band. For the calculations, the following equation was used:

A − A % Photodegradation = 0 t × 100 (2) A0

where A0 and At are the maximum absorbance of the acylhydrazone (λ = 302 nm), the PEG- alt-AA alternating copolymer (λ = 302 nm) or the PEG-alt-AA-DOX conjugate (λ = 297 nm), before and after irradiation for the different time intervals (t), respectively.

2.9. Acidic Hydrolysis of the PEG-alt-AA Alternating Copolymer A 0.2 mg/mL PEG-alt-AA alternating copolymer solution in water was prepared; its pH was adjusted at 5.2 using HCl, and the solution was kept under stirring at 37 ◦C. The hydrolysis of the polymer was monitored by measuring the absorption of the polymer solution at predesigned time intervals by UV-vis spectroscopy.

2.10. In Vitro Acid- and Photo-Triggered Drug Release Aqueous solutions of the self-assembled PEG-alt-AA-DOX conjugate (2.5 mg mL−1, 3 mL) at pH 7.4, 5.2 and 2.0 were prepared; each solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 Daltons, and was immersed in 10 mL H2O at pH 7.4, 5.2 and 2.0, respectively, under gentle stirring (100 rpm) at 37 ◦C. Next, the samples were irradiated at 254 nm for 5 min at predesigned time intervals. After each irradiation cycle, the solutions were stirred for 15 min, and then the aqueous media were collected and replaced with 10 mL fresh medium of the respective pH value. An aqueous solution of the self-assembled PEG-alt-AA-DOX conjugate (2.5 mg mL−1, 3 mL) at pH 2.0 was also prepared as a control sample, and the medium was collected and replaced as described above at the predesigned time intervals without prior irradiation. The collected samples were dried, first using the rotary evaporator and then under vacuum for 20 h. The dry samples were dissolved in DMSO (2.5 mL) and their fluorescence spectra were recorded. The concentration of DOX in each sample was determined using a standard calibration curve of DOX in DMSO. The DOX percentage released was calculated using the following equation: C + C − % DOX release = t t 1 × 100 (3) Ctotal

where, Ct and Ct−1 are the concentrations of DOX at two successive irradiation time intervals, and Ctotal is the initial concentration of DOX in the self-assembled conjugate.

3. Results and Discussion 3.1. Synthesis and Characterization of the PEG-alt-AA Alternating Copolymer The main-chain polyacylhydrazones were synthesized via a typical step-growth copolymerization of a dihydrazide and a dialdehyde. Adipic acid dihydrazide was chosen because it is a small, hydrophilic molecule that has previously been used in the biomedical field [37,38]. The second comonomer was a macromonomer, poly(ethylene glycol) (PEG) −1 (Mn = 4000 gr mol ), which is hydrophilic, non-toxic and FDA approved. Moreover, it is well known for its stealth properties, and has been used extensively in drug delivery [39,40]. In order to introduce the photosensitive moieties and the aldehyde end-groups required to react with the hydrazide functionalities of adipic acid, dihydroxy-functional PEG (HO- PEG-OH) was esterified with 4-carboxybenzaldehyde. The synthesized PEGald was char- Polymers 2021, 13, 2461 7 of 20 Polymers 2021, 13, x 7 of 19

1 1 Polymers 2021, 13, x acterized by H NMR and ATR-FTIR spectroscopies and SEC. The H NMR spectrum7 of of19 assigned to the methylene protons of the monomer repeat units of PEG, and two peaks at PEGald (Figure1) showed an intense peak at δ 3.67 ppm (H1), which was assigned to the δ 3.87methylene ppm and protons δ 4.53 of theppm monomer (H2 and repeat H3) unitsassigned of PEG, to andthe methylene two peaks at protonsδ 3.87 ppm next and to the newlyδ 4.53 formed ppm (H2 ester and bonds, H3) assigned verifying to the the methylene successful protons derivatization next to the newly of the formed polymer ester end- assigned to the methylene protons of the monomer repeat units of PEG, and two peaks at groups.bonds, In verifying addition, the successfulthe presence derivatization of a peak of at the δ polymer10.13 ppm, end-groups. which corresponds In addition, the to the δ 3.87 ppm and δ 4.53 ppm (H2 and H3) assigned to the methylene protons next to the presence of a peak at δ 10.13 ppm, which corresponds to the aldehyde protons (Ha), and aldehydenewly formedprotons ester (Ha), bonds, and theverifying peaks the at δsuccessful 8.0 and 8.24derivatization ppm, attributed of the polymer to the aromatic end- the peaks at δ 8.0 and 8.24 ppm, attributed to the aromatic protons (Hb and Hc) of the protonsgroups. (Hb In andaddition, Hc) of the the presence benzaldehyde of a peak group, at δ 10.13confirmed ppm, thewhich presence corresponds of the toaromatic the benzaldehyde group, confirmed the presence of the aromatic aldehyde functionalities at aldehydealdehyde functionalities protons (Ha), atand the the two peaks polymer at δ 8.0 chain and 8.24ends. ppm, The attributedATR–FTIR to spectrumthe aromatic of the the two polymer chain ends. The ATR–FTIR spectrum of the product (Figure2a) showed product (Figure 2a) showed the characteristic band of the carbonyl bond (1711 cm–1) theprotons characteristic (Hb and bandHc) of of the the benzaldehyde carbonyl bond group, (1711 confirmed cm−1) attributed the presence to the of aldehyde the aromatic and attributed to the aldehyde and ester groups introduced at the polymer chain ends and esteraldehyde groups functionalities introduced atat thethe polymertwo polymer chain chain ends ends. and supportedThe ATR–FTIR the NMR spectrum results. of the Fi- –1 supportednally,product the the(Figure SEC NMR traces 2a) results. of showed the PEG Finally, the macromonomer characterist the SECic traces (Figureband ofof2b), thethe before PEGcarbonyl and macromonomer after bond esterification (1711 cm (Figure) 2b),withattributed before 4-carboxybenzaldehyde, andto the after aldehyde esterification and demonstrated ester groupswith the 4-carboxybenzaldehyde,introduced absence of at chain the polymer degradation demonstratedchain during ends theand the absenceend-groupsupported of chain modificationthe NMRdegradation results. reaction. duringFinally, thethe end-groupSEC traces ofmodification the PEG macromonomer reaction. (Figure 2b), before and after esterification with 4-carboxybenzaldehyde, demonstrated the absence of chain degradation during the end-group modification reaction.

11 ald 3 FigureFigure 1. 1. HH1 NMR NMR spectrum ofof PEGPEG in in CDCl CDCl3. . Figure 1. H NMR spectrum of PEGaldald in CDCl3.

. . FigureFigure 2.2.ATR–FTIR ATR–FTIR spectraspectra ( a(a)) and and SEC SEC traces traces ( (bb)) of of PEGPEG beforebefore (black(black solidsolid line)line) andand afterafter (red(red dasheddashed line)line) esterificationesterification Figurewithwith 2. 4-carboxybenzaldehyde.ATR–FTIR 4-carboxybenzaldehyde. spectra (a) and SEC traces (b) of PEG before (black solid line) and after (red dashed line) esterification with 4-carboxybenzaldehyde. For the synthesis of the alternating polyacylhydrazone copolymers (PEG-alt-AA), the twoFor comonomers—PEG the synthesis of theald alternatingand adipic acidpolyacylhydrazone dihydrazide—were copolymers reacted at (PEG- a 1:1.2alt -AA),molar the ratio under mildly acidic conditions. The SEC analysis of the polyacylhydrazone polymer two comonomers—PEGald and adipic acid dihydrazide—were reacted at a 1:1.2 molar ratioprepared under mildly(Figure acidic3) showed conditions. a peak which The SEC was analysisshifted to of lower the polyacylhydrazoneelution times compared polymer to prepared (Figure 3) showed a peak which was shifted to lower elution times compared to

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the PEG macromonomer, verifying the synthesis of a copolymer with Mn = 28,000 gr mol– For the synthesis of the alternating polyacylhydrazone copolymers (PEG-alt-AA), the 1 and Mw/Mn = 1.21. The molar mass of the copolymer is quite high and its molecular two comonomers—PEGald and adipic acid dihydrazide—were reacted at a 1:1.2 molar ratioweight under distribution mildly acidic conditions. is relatively The SEC analysisnarrow, of the given polyacylhydrazone the nature polymer of the condensation preparedpolymerization. (Figure3) showed Moreover, a peak whichthe 1H was NMR shifted spectrum to lower elution of the times product compared (Figure to 4) showed peaks −1 theattributed PEG macromonomer, to the protons verifying of theboth synthesis the PEG of a copolymerand adipic with acidMn =comonomer 28,000 g mol repeat units, as well and Mw/Mn = 1.21. The molar mass of the copolymer is quite high and its molecular weight distributionas the characteristic is relatively narrow,peak of given the the hydrazone nature of the protons condensation at δ 11.42–11.52 polymerization. ppm (Ha), and con- Moreover,firmed the the SEC1H NMR results spectrum discussed of the product above. (Figure It should4) showed be noted peaks attributedthat the absence to of the peak of the protons of both the PEG and adipic acid comonomer repeat units, as well as the the aldehyde proton of PEGald at δ 10.13 ppm suggests that the quantitative reaction of characteristic peak of the hydrazone protons at δ 11.42–11.52 ppm (Ha), and confirmed thePEG SECald resultswith the discussed dihydrazide above. It shouldcomonomer be noted was that thein excess absence in of the peak reaction. of the Figure 5 shows the aldehydeATR–FTIR proton spectra of PEGald ofat adipicδ 10.13 ppmacid suggests dihydrazide, that the quantitative the PEG reactionald macromonomer of PEGald and the PEG- withalt-AA the dihydrazidealternating comonomer copolymer. was inThe excess characteristic in the reaction. vibration Figure5 shows bands the ATR– at 2869 cm−1, 1694 cm−1 FTIR spectra of adipic acid dihydrazide, the PEG macromonomer and the PEG-alt-AA and 1095 cm−1—assigned to the N–H, C=Oald and C–O stretching vibrations, respectively,— alternating copolymer. The characteristic vibration bands at 2869 cm−1, 1694 cm−1 and 1095were cm observed−1—assigned in to the the N–H,spectrum C=O and of C–O the stretching alternating vibrations, copolymer. respectively,—were More importantly though, observedthe presence in the spectrumof the characteristic of the alternating vibratio copolymer.n band More of importantly the acylhydrazone though, the bond at 1600 cm−1 −1 presencein the spectrum of the characteristic of the vibration copolymer, band of which the acylhydrazone is absent bond in atthe 1600 ATR–FTIR cm in the spectra of the two spectrum of the copolymer, which is absent in the ATR–FTIR spectra of the two precursor comonomers,precursor comonomers, verified the successful verified synthesis the ofsuccessful the polymeric synthesis product. of the polymeric product.

Figure 3. 3.SEC SEC traces traces of the of PEG-the altPEG--AAalt alternating-AA alternating copolymer (blackcopolymer dashed (black line), the dashed PEGald line), the PEGald pre- precursorcursor macromonomer macromonomer (wine (win solide solid line) and line) the and PEG- altthe-AA PEG- copolymeralt-AA after copolymer 1 h irradiation after at 1 h irradiation at 254 254nm nm (red (red dotted dotted line).line).

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the PEG macromonomer, verifying the synthesis of a copolymer with Mn = 28,000 gr mol– 1 and Mw/Mn = 1.21. The molar mass of the copolymer is quite high and its molecular weight distribution is relatively narrow, given the nature of the condensation polymerization. Moreover, the 1H NMR spectrum of the product (Figure 4) showed peaks attributed to the protons of both the PEG and adipic acid comonomer repeat units, as well as the characteristic peak of the hydrazone protons at δ 11.42–11.52 ppm (Ha), and con- firmed the SEC results discussed above. It should be noted that the absence of the peak of the aldehyde proton of PEGald at δ 10.13 ppm suggests that the quantitative reaction of PEGald with the dihydrazide comonomer was in excess in the reaction. Figure 5 shows the ATR–FTIR spectra of adipic acid dihydrazide, the PEGald macromonomer and the PEG- alt-AA alternating copolymer. The characteristic vibration bands at 2869 cm−1, 1694 cm−1 and 1095 cm−1—assigned to the N–H, C=O and C–O stretching vibrations, respectively,— were observed in the spectrum of the alternating copolymer. More importantly though, the presence of the characteristic vibration band of the acylhydrazone bond at 1600 cm−1 in the spectrum of the copolymer, which is absent in the ATR–FTIR spectra of the two precursor comonomers, verified the successful synthesis of the polymeric product.

Figure 3. SEC traces of the PEG-alt-AA alternating copolymer (black dashed line), the PEGald pre- Polymers 2021, 13, 2461 9 of 20 cursor macromonomer (wine solid line) and the PEG-alt-AA copolymer after 1 h irradiation at 254 nm (red dotted line).

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1 Figure 4.Figure1H NMR 4. spectrum H NMR of the spectrum PEG-alt-AA of alternating the PEG- copolymeralt-AA inalternating DMSO-d6. copolymer in DMSO-d6.

FigureFigure 5. ATR-FTIR5. ATR-FTIR spectra ofspectra adipic acid of dihydrazide adipic acid (black dihy dasheddrazide dotted line), (black PEGald dashed(red dashed dotted line), PEGald (red dashedline) and theline) PEG- andalt-AA the alternating PEG-alt copolymer-AA alternating (wine solid copolymer line). (wine solid line). 3.2. Synthesis and Photodegradation of a Small Acylhydrazone Molecule 3.2. BeforeSynthesis investigating and Phot theodegradation photodegradation of behaviora Small of Acylhydrazone the PEG-alt-AA copolymer, Molecule the photosensitivity of the aromatic acylhydrazone bonds was examined using a small dia- cylhydrazoneBefore molecule investigating in a proof-of-concept the photodeg study.radation For this, 4-carboxybenzaldehyde behavior of the wasPEG-alt-AA copolymer, thereacted photosensitivity with adipic acid dihydrazide of the aromatic to prepare aacyl diacylhydrazonehydrazone analogue bonds (Figure was S1aexamined). using a small 1 diacylhydrazoneThe H NMR spectrum molecule of the product in showeda proof-of-conce a characteristicpt peak study. at δ 11.37–11.51 For this, ppm, 4-carboxybenzaldehyde was reacted with adipic acid dihydrazide to prepare a diacylhydrazone analogue (Figure S1a). The 1H NMR spectrum of the product showed a characteristic peak at δ 11.37–11.51 ppm, which was attributed to the acylhydrazone protons, verifying the successful synthe- sis of the diacylhydrazone derivative (Figure S1b). Next, a solution of the diacylhydrazone molecule in DMSO was subjected to light irradiation (254 nm, 0.1 mW cm–2) and its degradation profile was monitored by UV-vis spectroscopy. Before the irradiation, the diacylhydrazone molecule exhibited a broad ab- sorption band centered at ~302 nm (Figure 6a). After the irradiation, the absorbance at 302 nm gradually decreased and a new peak at 254 nm eventually appeared, which coincided with the absorption band of 4-carboxybenzaldehyde, verifying the photo-induced cleav- age of the acylhydrazone bonds. The kinetics of the photodegradation process were de- termined from the decrease in the absorption intensity at 302 nm as a function of the irra- diation time (Figure 6b), and showed that ~45% of the acylhydrazone bonds were cleaved after only 10 minutes of irradiation, whereas the degradation of the bonds was slowed down for longer time intervals, reaching ~65% cleavage after ~2 h irradiation.

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which was attributed to the acylhydrazone protons, verifying the successful synthesis of the diacylhydrazone derivative (Figure S1b). Next, a solution of the diacylhydrazone molecule in DMSO was subjected to light irradiation (254 nm, 0.1 mW cm−2) and its degradation profile was monitored by UV-vis spectroscopy. Before the irradiation, the diacylhydrazone molecule exhibited a broad absorption band centered at ~302 nm (Figure6a). After the irradiation, the absorbance at 302 nm gradually decreased and a new peak at 254 nm eventually appeared, which coin- cided with the absorption band of 4-carboxybenzaldehyde, verifying the photo-induced cleavage of the acylhydrazone bonds. The kinetics of the photodegradation process were determined from the decrease in the absorption intensity at 302 nm as a function of the Polymers 2021, 13, x 10 of 19 irradiation time (Figure6b), and showed that ~45% of the acylhydrazone bonds were cleaved after only 10 min of irradiation, whereas the degradation of the bonds was slowed down for longer time intervals, reaching ~65% cleavage after ~2 h irradiation.

Figure 6. (a) UV-visFigure 6. (absorptiona) UV-vis absorption spectra spectra of a of0.02 a 0.02 mg mg ml mL–1− 1solutionsolution of of the the small small diacylhydrazone diacylhydrazone molecule inmolecule DMSO upon in DMSO upon irradiation atirradiation 254 nm, at and 254 nm, (b) and the (b )photodegradation the photodegradation percentagepercentage of the of diacylhydrazone the diacylhydrazone as a function as of thea function irradiation time.of the irradiation time. 3.3. Photodegradation of the PEG-alt-AA Alternating Copolymer Having established the photosensitivity of the aromatic acylhydrazone bonds, the 3.3. Photodegradationphoto-induced degradation of the PEG-alt-AA of the PEG- altAlternating-AA alternating Copolymer copolymer was investigated Havingusing SEC established to determine the changes photosensitivity in the polymer of molar the mass aromatic upon light acylhydrazone irradiation. As bonds, the photo-inducedobserved in degradation Figure3, the copolymer of the peak PEG- decreasedalt-AA significantly, alternating and copolymer a new peak, which was investigated coincides with that of the precursor PEGald macromonomer, appeared in the SEC trace of using SECthe polymer to determine after 1 h irradiationthe changes at 254 in nm. the In polymer addition, molar a new peak mass appeared upon atlight much irradiation. As observedhigher in elutionFigure times, 3, the indicating copolymer the formation peak ofdecreased a low-molecular-weight significantly, byproduct, and a which new peak, which was attributed to adipic acid dihydrazide; however, its size was beyond the analytical range coincides with that of the precursor PEGald macromonomer, appeared in the SEC trace of of SEC. These results strongly support the photo-induced cleavage of the acylhydrazone the polymerbonds along after the 1 main h irradiation chain of the PEG-at 254alt-AA nm. alternating In addition, copolymer a new to produce peak theappeared two at much higherprecursor elution comonomers, times, indicating PEGald and the adipic formation acid dihydrazide. of a low-molecular-weight byproduct, which wasThe attributed light-mediated to adipic degradation acid of dihydrazide; the acylhydrazone-based however, copolymer its size was was further beyond the ana- verified by UV-vis spectroscopy. Figure7a shows the absorption spectra of a 0.2 mg mL−1 lytical PEG-rangealt- AAof copolymerSEC. These solution results in DMSO. strongly Before support irradiation, the the polymerphoto-induced peak was cen-cleavage of the acylhydrazonetered at ~302 bonds nm, whereas along afterthe exposuremain chain to UV of light, the the PEG- peakalt intensity-AA alternating gradually de- copolymer to producecreased, the two and twoprecursor new peaks comonomers, at 260 nm and PEG 288 nmald and emerged, adipic which acid correspond dihydrazide. to the absorption bands of the PEGald macromonomer and support the GPC results discussed Theabove. light-mediated Figure7c shows thedegradation kinetics of the of photodegradation the acylhydrazone-based process of the copolymer copolymer in was further verifiedDMSO, by UV-vis determined spectroscopy. by the UV-vis spectra. Figure An 7a almost-linear shows the degradation absorption profile spectra was found of a 0.2 mg ml–1 PEG-alt-forAA the first copolymer 60 min of irradiation, solution corresponding in DMSO. toBefore the ~55% irradiation, cleavage of the the polymer polymer bonds, peak was cen- tered at ~302 nm, whereas after exposure to UV light, the peak intensity gradually de- creased, and two new peaks at 260 nm and 288 nm emerged, which correspond to the absorption bands of the PEGald macromonomer and support the GPC results discussed above. Figure 7c shows the kinetics of the photodegradation process of the copolymer in DMSO, determined by the UV-vis spectra. An almost-linear degradation profile was found for the first 60 min of irradiation, corresponding to the ~55% cleavage of the poly- mer bonds, whereas for longer times the degradation kinetics were slowed down and reached 75% cleavage after 130 min irradiation. Similar results were obtained for the pho- todegradation process of the copolymer in water. The irradiation of an aqueous copoly- mer solution at pH 7.4, to prevent the acidolysis of the acylhydrazone bonds, led to the decrease of the absorbance at 302 nm, and simultaneously to the appearance of a new peak at 260 nm, which corresponded to the PEGald macromonomer (Figure 7b). The rate of pho- todegradation in water was almost linear for more than 2 h of irradiation time (Figure 7d), albeit significantly slower than that found in DMSO, with ~32% photocleavage within the first 1 h of exposure. It is remarkable to mention that despite the fact that the irradiation was performed with UV illumination at 254 nm, which is considered to be a harmful wavelength for biological applications, the irradiation dose used for the complete degra- dation of the polymer was extremely low (720 mJ cm–2) compared to other photodegrada- ble polymers reported in the literature [41,42].

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whereas for longer times the degradation kinetics were slowed down and reached 75% cleavage after 130 min irradiation. Similar results were obtained for the photodegradation process of the copolymer in water. The irradiation of an aqueous copolymer solution at pH 7.4, to prevent the acidolysis of the acylhydrazone bonds, led to the decrease of the ab- sorbance at 302 nm, and simultaneously to the appearance of a new peak at 260 nm, which corresponded to the PEGald macromonomer (Figure7b). The rate of photodegradation in water was almost linear for more than 2 h of irradiation time (Figure7d), albeit significantly slower than that found in DMSO, with ~32% photocleavage within the first 1 h of exposure. It is remarkable to mention that despite the fact that the irradiation was performed with UV illumination at 254 nm, which is considered to be a harmful wavelength for biological applications, the irradiation dose used for the complete degradation of the polymer was Polymers 2021, 13, x −2 11 of 19 extremely low (720 mJ cm ) compared to other photodegradable polymers reported in the literature [41,42].

FigureFigure 7.7. UV-visUV-vis absorptionabsorption spectra spectra of of (a ()a a) a 0.2 0.2 mg mg mL ml−–11 copolymercopolymer solutionsolution in in DMSO, DMSO, and and ( b(b) a) a 0.02 0.02 mg mg mL ml−–11 copolymer 2 solutionsolution inin HH2OO atat pHpH 7.4,7.4, uponupon irradiationirradiation at at 254 254 nm. nm. The The photodegradation photodegradation percentage percentage of of the the PEG- PEG-altalt-AA-AA copolymer copolymer as as a a function of irradiation time in (c) DMSO and (d) H2O at pH 7.4. function of irradiation time in (c) DMSO and (d)H2O at pH 7.4.

As discussed above, poly(acyl)hydrazones have been studied in the literature as dy- namic covalent polymers, and have been proposed for the development of self-healing materials and adaptable networks due to the reversible nature of the (acyl)hydrazone bonds, which can be cleaved and reformed upon the application of a stimulus, such as acidic media or temperature [34,43,44]. In order to investigate the dynamic nature of the acylhydrazone bonds of the PEG-alt-AA copolymer prepared in this work, a solution of the polymer in H2O was first irradiated for 2 h at 254 nm to cleave the acylhydrazone bonds, and was afterwards left under stirring for 24 h in the dark. The UV-vis absorption spectra of the copolymer after irradiation and following 24 h in the dark were compared (Figure 8), verifying that no changes in the adsorption took place in the dark, suggesting that the copolymer was not reformed. An explanation for this result could be the sensitiv- ity of the aldehyde groups formed at the PEG chain ends, following the photocleavage of the acylhydrazone bonds by the light stimulus. Aromatic and aliphatic aldehydes (or ke- tones) are well known to undergo several photochemical reactions upon UV light irradi- ation [45]. More specifically, the interaction of light with aromatic aldehyde moieties can lead to the formation of different radical species (Scheme 2). In order to further support our hypothesis, 1H NMR spectroscopy was used to investigate the fate of the aldehyde end-groups of PEGald upon UV irradiation. Figure 9 shows that the characteristic peak of the aldehyde protons at δ 10.11 ppm decreased significantly after 1 h of irradiation of the

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As discussed above, poly(acyl)hydrazones have been studied in the literature as dynamic covalent polymers, and have been proposed for the development of self-healing materials and adaptable networks due to the reversible nature of the (acyl)hydrazone bonds, which can be cleaved and reformed upon the application of a stimulus, such as acidic media or temperature [34,43,44]. In order to investigate the dynamic nature of the acylhydrazone bonds of the PEG-alt-AA copolymer prepared in this work, a solution of the polymer in H2O was first irradiated for 2 h at 254 nm to cleave the acylhydrazone bonds, and was afterwards left under stirring for 24 h in the dark. The UV-vis absorption spectra of the copolymer after irradiation and following 24 h in the dark were compared (Figure8 ), verifying that no changes in the adsorption took place in the dark, suggesting that the copolymer was not reformed. An explanation for this result could be the sensitivity of the aldehyde groups formed at the PEG chain ends, following the photocleavage of the acylhydrazone bonds by the light stimulus. Aromatic and aliphatic aldehydes (or ketones) are well known to undergo several photochemical reactions upon UV light irradiation [45]. Polymers 2021, 13, x 12 of 19 More specifically, the interaction of light with aromatic aldehyde moieties can lead to the formation of different radical species (Scheme2). In order to further support our hypothesis, 1H NMR spectroscopy was used to investigate the fate of the aldehyde end- groups of PEGald upon UV irradiation. Figure9 shows that the characteristic peak of the aldehydePEGald macromonomer protons at δ 10.11 ppm at 254 decreased nm, and significantly disappeared after 1 h completely of irradiation after of the 2 h exposure to UV PEGlight.ald macromonomerMoreover, the at 254peaks nm, andattributed disappeared to the completely aromatic after (7.99 2 h exposure and 8.24 to UV ppm) and ester (4.53 light.ppm) Moreover, protons the changed peaks attributed and shifted to the aromaticto lower (δ ppm,7.99 and whereasδ 8.24 ppm) two and new ester peaks appeared at 2.2 (δ 4.53 ppm) protons changed and shifted to lower ppm, whereas two new peaks appeared atppmδ 2.2 and ppm 9.8 and ppm,δ 9.8 ppm, which which were were attributed attributed to to the the formation formation of acetaldehyde of acetaldehyde as a as a by-product by-productof the photodegradation of the photodegradation reaction. reaction.

FigureFigure 8. UV-vis8. UV-vis absorption absorption spectra of spectra a 0.02 mg of mL a− 0.021 PEG- mgralt-AA ml alternating–1 PEG-alt copolymer-AA alternating solution copolymer solution in H O before (black solid line) and after (red dashed line) irradiation for 2 h at 254 nm, and in H2 2O before (black solid line) and after (red dashed line) irradiation for 2 h at 254 nm, and subse- subsequently,quently, after after stirring stirring for for 24 24 h in h the in darkthe (bluedark dotted (blue line). dotted line).

Scheme 2. Proposed photodissociation pathways of PEGald upon irradiation with UV light.

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PEGald macromonomer at 254 nm, and disappeared completely after 2 h exposure to UV light. Moreover, the peaks attributed to the aromatic (7.99 and 8.24 ppm) and ester (4.53 ppm) protons changed and shifted to lower ppm, whereas two new peaks appeared at 2.2 ppm and 9.8 ppm, which were attributed to the formation of acetaldehyde as a by-product of the photodegradation reaction.

Figure 8. UV-vis absorption spectra of a 0.02 mgr ml–1 PEG-alt-AA alternating copolymer solution Polymers 2021, 13, 2461 13 of 20 in H2O before (black solid line) and after (red dashed line) irradiation for 2 h at 254 nm, and subse- quently, after stirring for 24 h in the dark (blue dotted line).

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Scheme 2. ProposedScheme 2.photodissociationProposed photodissociation pathways pathways of PEG of PEGaldald uponupon irradiationirradiation with with UV light. UV light.

1 1 Figure Figure9. H 9.NMRH NMR spectra spectra of of PEG PEGaldald( A(A) before,) before, (B) after(B) 1after h and 1 ( Ch) and after 2(C h) irradiation after 2 h at irradiation 254 nm in CDCl at3 .254 nm in CDCl3. 3.4. Synthesis and Self-Assembly of the PEG-alt-AA-DOX Drug Conjugates

3.4. Synthesis and Self-AssemblyThe PEG- ofalt the-AA PEG-alt-AA-DOX alternating copolymer Drug was used Conjugates for the synthesis of the PEG-alt-AA- DOX drug conjugates. For the conjugation of DOX at the ends of the polyacylhydrazone The PEG-alt-AA chains,alternating the copolymer copolymer and DOX was were used mixed for at the a 1:2 synthesis mole ratio inof DMSO the PEG- and werealt-AA- reacted DOX drug conjugates.for For 24 h,the followed conjugation by dialysis of against DOX DMSOat the to ends eliminate of the the polyacylhydrazone unreacted DOX molecules. Figure 10a shows the UV-vis absorption spectrum of the PEG-alt-AA-DOX conjugate chains, the copolymerin and DMSO, DOX in whichwere themixed polymer at a peak1:2 mole at 302 ratio nm is in clearly DMSO observed, and were whereas reacted the inset for 24 h, followed by showsdialysis the magnifiedagainst DMSO spectrum to in eliminate the 340–700 the nm range,unreacted where theDOX absorption molecules. peak of Figure 10a shows theDOX UV-vis is evident, absorption indicating spectrum the successful of the conjugation PEG-alt of-AA-DOX the drug at theconjugate polymer chainin ends. Figure 10b shows the fluorescence emission spectra of the polymer, DOX and the DMSO, in which the polymerPEG-alt-AA-DOX peak at conjugate 302 nm in is DMSO. clearly As observed, expected, the whereas copolymer the does inset not shows emit light the magnified spectrumin the in 500–800 the 340–700 nm range, nm whereas range, DOX where and thethe PEG- absorptionalt-AA-DOX peak conjugate of DOX both emitis evident, indicating theat successful 600 nm, verifying conjugation the conjugation of the of drug the drug at the at the polymer polymer chain ends. ends. The Fig- drug ure 10b shows the fluorescenceloading of DOX emission on the copolymer spectra chains of the was polymer, quantified fromDOX the and fluorescent the PEG- intensityalt- of the PEG-alt-AA-DOX conjugate using a calibration curve of DOX in DMSO (Figure S2), AA-DOX conjugate inand DMSO. was found As toexpected, be 2.7 × 10 the−3 g copolymer DOX/g polymer. does not emit light in the 500– 800 nm range, whereas DOX and the PEG-alt-AA-DOX conjugate both emit at 600 nm, verifying the conjugation of the drug at the polymer chain ends. The drug loading of DOX on the copolymer chains was quantified from the fluorescent intensity of the PEG-alt-AA- DOX conjugate using a calibration curve of DOX in DMSO (Figure S2), and was found to be 2.7 x 10−3 g DOX/g polymer.

Figure 10. (a) UV-vis absorption spectrum of the PEG-alt-AA-DOX conjugate in DMSO (inset: mag- nified spectrum in the 340–700 nm range) and (b) fluorescence spectra of the PEG-alt-AA copolymer (black solid line), DOX (blue dashed line) and the PEG-alt-AA-DOX conjugate (red dashed dotted line).

Amphiphilic molecules containing a hydrophilic and a hydrophobic part can self- assemble into various morphological structures (micelles, vesicles, cylinders, etc.) in wa- ter. We envisaged that the conjugation of the hydrophobic DOX drug moieties at one or

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Figure 9. 1H NMR spectra of PEGald (A) before, (B) after 1 h and (C) after 2 h irradiation at 254 nm in CDCl3.

3.4. Synthesis and Self-Assembly of the PEG-alt-AA-DOX Drug Conjugates The PEG-alt-AA alternating copolymer was used for the synthesis of the PEG-alt-AA- DOX drug conjugates. For the conjugation of DOX at the ends of the polyacylhydrazone chains, the copolymer and DOX were mixed at a 1:2 mole ratio in DMSO and were reacted for 24 h, followed by dialysis against DMSO to eliminate the unreacted DOX molecules. Figure 10a shows the UV-vis absorption spectrum of the PEG-alt-AA-DOX conjugate in DMSO, in which the polymer peak at 302 nm is clearly observed, whereas the inset shows the magnified spectrum in the 340–700 nm range, where the absorption peak of DOX is evident, indicating the successful conjugation of the drug at the polymer chain ends. Fig- ure 10b shows the fluorescence emission spectra of the polymer, DOX and the PEG-alt- AA-DOX conjugate in DMSO. As expected, the copolymer does not emit light in the 500– 800 nm range, whereas DOX and the PEG-alt-AA-DOX conjugate both emit at 600 nm, verifying the conjugation of the drug at the polymer chain ends. The drug loading of DOX on the copolymer chains was quantified from the fluorescent intensity of the PEG-alt-AA-

PolymersDOX2021 conjugate, 13, 2461 using a calibration curve of DOX in DMSO (Figure S2), and was found14 to of 20 be 2.7 x 10−3 g DOX/g polymer.

FigureFigure 10. 10. ((a)) UV-visUV-vis absorption absorption spectrum spectrum of the PEG- of altthe-AA-DOX PEG-alt conjugate-AA-DOX in DMSO conjugate (inset: magnified in DMSO spectrum (inset: in mag- the nified340–700 spectrum nm range) in and the (b )340–700 fluorescence nm spectra range) of theand PEG- (balt) fluorescence-AA copolymer (blackspectra solid of line), the DOXPEG- (bluealt-AA dashed copolymer line) and (blackthe PEG- solidalt-AA-DOX line), DOX conjugate (blue (red dashed dashed dottedline) and line). the PEG-alt-AA-DOX conjugate (red dashed dotted line). Amphiphilic molecules containing a hydrophilic and a hydrophobic part can self- assemble into various morphological structures (micelles, vesicles, cylinders, etc.) in Amphiphilic moleculeswater. We containing envisaged that a thehydrop conjugationhilic ofand the hydrophobica hydrophobic DOX drugpart moieties can self- at one assemble into variousor morphological both ends of the hydrophilicstructures PEG- (micelles,alt-AA copolymer vesicles, chains, cylinders, to produce etc.) amphiphile in wa- drug conjugates, could drive their self-assembly into nanostructures with hydrophilic ter. We envisaged thatand the hydrophobic conjugation compartments. of the hydrophobic In order to test DOX this hypothesis, drug moieties the PEG- atalt one-AA-DOX or conjugates were self-assembled in water using the dialysis method. First, the polymer-drug conjugates were dissolved in DMSO, which is a good solvent for both the copolymer and the DOX drug, and next, an excess of water was added dropwise to the organic solution, followed by the removal of the DMSO by dialysis. The morphology of the PEG-alt-AA-DOX assemblies in water was examined by SEM and TEM microscopies. Interestingly, spherical nanoparticles with an average diameter of ~300 nm were observed by SEM (Figure 11a), and a similar size (~300 nm) was found by TEM (Figure 11b). The hydrodynamic size of the nanoparticles in water was found to be 250 nm by DLS (Figure 12), in good agreement with the SEM and TEM results. The photo-mediated disruption of the self-assembled PEG-alt-AA-DOX nanoparticles and the release of DOX, upon the cleavage of the acylhydrazone bonds with UV light, was investigated by SEM, TEM and DLS. Figure 11c shows an SEM image of the self-assembled PEG-alt-AA-DOX conjugates in water (pH 7.4) following irradiation with UV light for 2 h. The absence of the PEG-alt-AA-DOX assemblies after irradiation is evident, indicating the main-chain cleavage of the polyacylhydrazone backbone. Smaller nanoparticles, 25–30 nm in size, were observed in the SEM image after light irradiation, which were attributed to aggregates formed by the released hydrophobic DOX molecules. The results were also confirmed by TEM (Figure 11d), which showed the absence of the spherical PEG-alt-AA- DOX assemblies, and the presence of small, dark-colored nanoparticles (D~15–20 nm) attributed to the drug aggregates. Finally, the disruption of the self-assembled PEG-alt- AA-DOX nanostructures, upon exposure to UV light, was verified by DLS (Figure 12). The large particles disappeared and a hydrodynamic diameter of ~6.5 nm was measured, which was attributed to the constituent PEG macromonomer chains that remained in the solution after the degradation of the polymer backbone. Polymers 2021, 13, x 14 of 19

both ends of the hydrophilic PEG-alt-AA copolymer chains, to produce amphiphile drug conjugates, could drive their self-assembly into nanostructures with hydrophilic and hy- drophobic compartments. In order to test this hypothesis, the PEG-alt-AA-DOX conju- gates were self-assembled in water using the dialysis method. First, the polymer-drug conjugates were dissolved in DMSO, which is a good solvent for both the copolymer and the DOX drug, and next, an excess of water was added dropwise to the organic solution, followed by the removal of the DMSO by dialysis. The morphology of the PEG-alt-AA- DOX assemblies in water was examined by SEM and TEM microscopies. Interestingly, spherical nanoparticles with an average diameter of ~300 nm were observed by SEM (Fig- ure 11a), and a similar size (~300 nm) was found by TEM (Figure 11b). The hydrodynamic size of the nanoparticles in water was found to be 250 nm by DLS (Figure 12), in good agreement with the SEM and TEM results. The photo-mediated disruption of the self-assembled PEG-alt-AA-DOX nanoparti- cles and the release of DOX, upon the cleavage of the acylhydrazone bonds with UV light, was investigated by SEM, TEM and DLS. Figure 11c shows an SEM image of the self- assembled PEG-alt-AA-DOX conjugates in water (pH 7.4) following irradiation with UV light for 2 h. The absence of the PEG-alt-AA-DOX assemblies after irradiation is evident, indicating the main-chain cleavage of the polyacylhydrazone backbone. Smaller nanopar- ticles, 25–30 nm in size, were observed in the SEM image after light irradiation, which were attributed to aggregates formed by the released hydrophobic DOX molecules. The results were also confirmed by TEM (Figure 11d), which showed the absence of the spher- ical PEG-alt-AA-DOX assemblies, and the presence of small, dark-colored nanoparticles (D ~ 15–20 nm) attributed to the drug aggregates. Finally, the disruption of the self-assem- bled PEG-alt-AA-DOX nanostructures, upon exposure to UV light, was verified by DLS (Figure 12). The large particles disappeared and a hydrodynamic diameter of ~6.5 nm was Polymers 2021, 13, 2461 15 of 20 measured, which was attributed to the constituent PEG macromonomer chains that re- mained in the solution after the degradation of the polymer backbone.

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FigureFigure 11. 11.SEM SEM (a ,(ca), andc) and TEM TEM (b,d ()b images,d) images of the of self-assembled the self-assembled PEG-alt-AA-DOX PEG-alt-AA-DOX drug conjugates drug conjugates beforebefore ( a(,ab,)b and) and after after (c,d ()c irradiation,d) irradiation for 2 hfor at 2 254 h at nm. 254 nm.

FigureFigure 12. 12.Hydrodynamic Hydrodynamic diameter for diameter the PEG-alt for-AA-DOX the PEG- assembliesalt-AA-DOX in water before assemblies (black solid in water before (black solid line) and after (red dashed line) irradiation for 2 h at 254 nm. line) and after (red dashed line) irradiation for 2 h at 254 nm.

3.5. Photo- and Acid-Induced Disruption of the Self-Assembled PEG-alt-AA-DOX Nanoparticles and the Release of the Drug Finally, the effect of light and a low solution pH on the disruption of the PEG-alt-AA- DOX assemblies was studied. Figure 13a shows the UV-vis absorption spectra of an aque- ous PEG-alt-AA-DOX solution at pH 7.4, for different irradiation times at 254 nm (0.1 mW cm–2). As discussed above, the characteristic absorption band of the polymer at ~302 nm decreased with the irradiation time, while a new band at ~260 nm emerged, indicating the main-chain cleavage of the polymer chains and the formation of the precursor PEG mac- romonomer. A similar degradation process was found for the PEG-alt-AA-DOX assem- blies upon the irradiation of a polymer solution adjusted to pH 5.2 (Figure 13b), whilst, when comparing the degradation rates at the two pH values, a slightly faster degradation of the polymer—and thus disruption of the assemblies—was found at the low solution pH. In particular, 75% of the polymer was cleaved after 2 h irradiation at pH 7.4, whereas the degree of degradation reached 90% for the same irradiation time at pH 5.2 (Figure 13c), suggesting the synergistic photo- and acidolysis of the labile bonds of the polymer. It is noted that the low DOX content of the PEG-alt-AA-DOX assemblies prohibited the quantification of the photo-induced drug release by UV-vis spectroscopy, and fluores- cence spectroscopy was used instead. For this, an aqueous PEG-alt-AA-DOX solution was first adjusted to pH 5.2 to simulate the acidic environment of a tumor tissue, and was next irradiated with UV light for different irradiation intervals, up to 2 h total irradiation time. The solution was dialyzed against water (pH 5.2) during the irradiation; the dialysate was collected after each irradiation cycle, and its DOX content was analyzed by fluorescence spectroscopy. In parallel, another PEG-alt-AA-DOX solution at neutral pH 7.4 was irradi- ated with UV light, and the release of the cargo in the dialysate was followed by fluores- cence spectroscopy. As shown in Figure 14, the release kinetics of the conjugated DOX molecules upon irradiation at pH 5.2 and pH 7.4 were similar (60–65% released DOX at 2 h), denoting that the release of the drug is dominated by the photoinduced cleavage of the acylhydrazone bonds, whereas the solution pH, in this range, does not play a significant role in the drug release profile at short irradiation times (2 h). To support our results, we investigated the degradation rate of the polymer at pH 5.2 as a function of time by UV-vis spectroscopy (Figure S3), and found that the polymer hydrolysis is rather low in the first 2 h (Figure S3 inset) and becomes significant only after 23 h. The slow acidolysis of the hydrazone bonds and the low amount of released cargo at pH 5.0 and 5.5 has also been reported in the literature [22,27,46]. In order to speed up the release kinetics of DOX from the PEG-alt-AA-DOX assemblies, an aqueous PEG-alt-AA-DOX solution at pH 2.0 was

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3.5. Photo- and Acid-Induced Disruption of the Self-Assembled PEG-alt-AA-DOX Nanoparticles and the Release of the Drug Finally, the effect of light and a low solution pH on the disruption of the PEG-alt- AA-DOX assemblies was studied. Figure 13a shows the UV-vis absorption spectra of an aqueous PEG-alt-AA-DOX solution at pH 7.4, for different irradiation times at 254 nm (0.1 mW cm−2). As discussed above, the characteristic absorption band of the polymer at ~302 nm decreased with the irradiation time, while a new band at ~260 nm emerged, indicating the main-chain cleavage of the polymer chains and the formation of the precursor PEG macromonomer. A similar degradation process was found for the PEG-alt-AA-DOX assemblies upon the irradiation of a polymer solution adjusted to pH 5.2 (Figure 13b), whilst, when comparing the degradation rates at the two pH values, a slightly faster degradation of the polymer—and thus disruption of the assemblies—was found at the low solution pH. In particular, 75% of the polymer was cleaved after 2 h irradiation at pH 7.4, whereas the degree of degradation reached 90% for the same irradiation time at pH 5.2 (Figure 13c), suggesting the synergistic photo- and acidolysis of the labile bonds of the polymer. It is noted that the low DOX content of the PEG-alt-AA-DOX assemblies prohibited the quantification of the photo-induced drug release by UV-vis spectroscopy, and fluorescence spectroscopy was used instead. For this, an aqueous PEG-alt-AA-DOX solution was first adjusted to pH 5.2 to simulate the acidic environment of a tumor tissue, and was next irradiated with UV light for different irradiation intervals, up to 2 h total irradiation time. The solution was dialyzed against water (pH 5.2) during the irradiation; the dialysate was collected after each irradiation cycle, and its DOX content was analyzed by fluorescence spectroscopy. In parallel, another PEG-alt-AA-DOX solution at neutral pH 7.4 was irradiated with UV light, and the release of the cargo in the dialysate was followed by fluorescence spectroscopy. As shown in Figure 14, the release kinetics of the conjugated DOX molecules upon irradiation at pH 5.2 and pH 7.4 were similar (60–65% released DOX at 2 h), denoting that the release of the drug is dominated by the photoinduced cleavage of the acylhydrazone bonds, whereas the solution pH, in this range, does not play a significant role in the drug release profile at short irradiation times (2 h). To support our results, we investigated the degradation rate of the polymer at pH 5.2 as a function of time by UV-vis spectroscopy (Figure S3), and found that the polymer hydrolysis is rather low in the first 2 h (Figure S3 inset) and becomes significant only after 23 h. The slow acidolysis of the hydrazone bonds and the low amount of released cargo at pH 5.0 and 5.5 has also been reported in the literature [22,27,46]. In order to speed up the release kinetics of DOX from the PEG-alt-AA-DOX assemblies, an aqueous PEG-alt-AA-DOX solution at pH 2.0 was irradiated with UV light for 2 h. Indeed, a faster drug release profile was found (Figure 14, red squares), with 64% of the DOX being released after 1 h irradiation and reaching 82% free drug after 2 h irradiation. On the contrary, the solution pH alone (pH 2 without light irradiation) resulted in only 35% drug release in 2 h (black circles). Based on the above results, light irradiation is a convenient and remote stimulus, allowing to control the drug release from the PEG-alt-AA-DOX assemblies in short times, whereas the solution pH enables the prolonged release of the cargo, except for very low pH values (pH 2.0), at which a synergistic photolysis and acidolysis of the prodrug leads to faster release kinetics. Therefore, the proposed drug delivery system could be used for the effective photo-triggered drug release at specific sites/tissues, in which the pH ranges from neutral (normal tissues) to acidic (cancer tissues), providing an additional level of control over the drug release profile. Further studies are underway to investigate the in vitro effectiveness of the proposed polymer–drug conjugates towards cancer cells. Polymers 2021, 13, x 16 of 19

irradiated with UV light for 2 h. Indeed, a faster drug release profile was found (Figure 14, red squares), with 64% of the DOX being released after 1 h irradiation and reaching 82% free drug after 2 h irradiation. On the contrary, the solution pH alone (pH 2 without light irradiation) resulted in only 35% drug release in 2 h (black circles). Based on the above results, light irradiation is a convenient and remote stimulus, allowing to control the drug release from the PEG-alt-AA-DOX assemblies in short times, whereas the solu- tion pH enables the prolonged release of the cargo, except for very low pH values (pH 2.0), at which a synergistic photolysis and acidolysis of the prodrug leads to faster release kinetics. Therefore, the proposed drug delivery system could be used for the effective photo-triggered drug release at specific sites/tissues, in which the pH ranges from neutral Polymers 2021, 13, 2461 (normal tissues) to acidic (cancer tissues), providing an additional level of control over the 17 of 20 drug release profile. Further studies are underway to investigate the in vitro effectiveness of the proposed polymer–drug conjugates towards cancer cells.

Figure 13. UV-vis absorption spectra of an aqueous PEG-alt-AA-DOX solution at (a) pH 7.4 and (b) pH 5.2 for different Figure 13. UV-vis absorption spectra of an aqueous PEG-alt-AA-DOX solution at (a) pH 7.4 and (b) pH 5.2 for different irradiation intervals at 254 nm. (c) Photodegradation percentage of the PEG-alt-AA-DOX nanoparticles as a function of Polymersirradiation 2021irradiation, 13 intervals, x time at at 254pH 7.4 nm. (red (c )dots) Photodegradation and pH 5.2 (black percentage squares). of the PEG-alt-AA-DOX nanoparticles as a17 functionof 19 of

irradiation time at pH 7.4 (red dots) and pH 5.2 (black squares).

FigureFigure 14. 14. Time-dependantTime-dependant release release profile profile of DOX of from DOX the fromPEG-alt the-AA-DOX PEG-alt assemblies-AA-DOX upon assemblies ir- upon –2 radiation (254 nm, 0.1 mW cm ) at −pH2 7.4 (blue triangles), pH 5.2 (green triangles) and pH 2 (red irradiationspuares), and (254 the nm,control 0.1 sample mW cm (without) at pHirradiation) 7.4 (blue at triangles),pH 2 (black pHcircles). 5.2 (green triangles) and pH 2 (red spuares), and the control sample (without irradiation) at pH 2 (black circles). 4. Conclusions In summary, a new, main-chain photo- and acid-degradable, amphiphilic polymer– drug conjugate, based on a hydrophilic polyacylhydrazone chain and a hydrophobic model anticancer drug, DOX, was proposed. The alternating polyacylhydrazone copoly- mer was synthesized via a step-growth polymerization reaction of adipic acid dihydra- zide with dibenzaldehyde-terminated PEG. The photodegradation profile of the copoly- mer—PEG-alt-AA—in DMSO and water, upon irradiation at 254 nm (0.1 mW cm–2), was studied, and the cleavage of the hydrazone bonds along the polymer main-chain to pro- duce the two precursor comonomers was verified at very low irradiation doses (720 mJ/cm−1). Next, amphiphilic PEG-alt-AA-DOX conjugates were prepared by the conden- sation of DOX molecules at the polymer chain end(s), and the conjugates were self-assem- bled in water to form spherical nanoparticles. At neutral and slightly acidic pH values (pH 5.2), the PEG-alt-AA-DOX assemblies were disrupted, and the conjugated drug was released at shorter irradiation times and a low total irradiation dose, whereas the drug- release kinetics were accelerated upon the synergistic effect of light irradiation and a low solution pH (2.0), denoting the remotely controlled drug release nanosystem. We envisage that such photo- and acid-degradable polymers could be used for the conjugation of both hydrophilic and hydrophobic drug molecules, vitamins or other bioactive molecules bear- ing an aldehyde or ketone group, and are thus attractive for the development of diverse drug carriers. Current studies are underway to investigate the effectiveness of the poly- mer–drug conjugates towards cancer cells, and to red-shift the irradiation wavelength re- quired for the photodegradation of the polymer acylhydrazone bonds and therefore im- prove their potential in the biomedical field.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Figure S1: (a) Schematic representation of the synthetic procedure followed for the preparation of the small diacylhydrazone molecule, and (b) 1H NMR spectrum of the product in DMSO-d6. Figure S2: (a) Fluorescence emission spectra of DOX as a function of the drug concentration in DMSO, and (b) standard calibration curve of DOX. Figure S3: UV-vis absorption spectra of an aqueous PEG-alt-AA solution at pH 5.2 at different time intervals.

Polymers 2021, 13, 2461 18 of 20

4. Conclusions In summary, a new, main-chain photo- and acid-degradable, amphiphilic polymer– drug conjugate, based on a hydrophilic polyacylhydrazone chain and a hydrophobic model anticancer drug, DOX, was proposed. The alternating polyacylhydrazone copolymer was synthesized via a step-growth polymerization reaction of adipic acid dihydrazide with dibenzaldehyde-terminated PEG. The photodegradation profile of the copolymer—PEG- alt-AA—in DMSO and water, upon irradiation at 254 nm (0.1 mW cm−2), was studied, and the cleavage of the hydrazone bonds along the polymer main-chain to produce the two precursor comonomers was verified at very low irradiation doses (720 mJ/cm−1). Next, amphiphilic PEG-alt-AA-DOX conjugates were prepared by the condensation of DOX molecules at the polymer chain end(s), and the conjugates were self-assembled in water to form spherical nanoparticles. At neutral and slightly acidic pH values (pH 5.2), the PEG-alt-AA-DOX assemblies were disrupted, and the conjugated drug was released at shorter irradiation times and a low total irradiation dose, whereas the drug-release kinetics were accelerated upon the synergistic effect of light irradiation and a low solution pH (2.0), denoting the remotely controlled drug release nanosystem. We envisage that such photo- and acid-degradable polymers could be used for the conjugation of both hy- drophilic and hydrophobic drug molecules, vitamins or other bioactive molecules bearing an aldehyde or ketone group, and are thus attractive for the development of diverse drug carriers. Current studies are underway to investigate the effectiveness of the polymer–drug conjugates towards cancer cells, and to red-shift the irradiation wavelength required for the photodegradation of the polymer acylhydrazone bonds and therefore improve their potential in the biomedical field.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/polym13152461/s1. Figure S1: (a) Schematic representation of the synthetic procedure followed for the preparation of the small diacylhydrazone molecule, and (b) 1H NMR spectrum of the product in DMSO-d6. Figure S2: (a) Fluorescence emission spectra of DOX as a function of the drug concentration in DMSO, and (b) standard calibration curve of DOX. Figure S3: UV-vis absorption spectra of an aqueous PEG-alt-AA solution at pH 5.2 at different time intervals. Author Contributions: Supervision, M.V.; methodology, M.P., M.G.K. and M.V.; experiments, M.P. and M.G.K.; writing—original draft preparation, M.P. and M.G.K.; writing—review and editing, M.V.; M.P. and M.G.K. contributed equally to this work. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors would like to kindly acknowledge Aleka Manousaki and Dimitrios Theodoridis for their technical assistance with the SEM and TEM measurements, respectively. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Hu, X.; Zhang, Y.; Xie, Z.; Jing, X.; Bellotti, A.; Gu, Z. Stimuli-responsive polymersomes for biomedical applications. Biomacro- molecules 2017, 18, 649–673. [CrossRef] 2. Manouras, T.; Vamvakaki, M. Field responsive materials: Photo-, electro-, magnetic- and ultrasound-sensitive polymers. Polym. Chem. 2017, 8, 74–96. [CrossRef] 3. Seo, W.; Phillips, S.T.; Pennsyl, T.; State, V.; Uni, V.; Park, U.V.; Pennsyl, V. Patterned plastics that change physical structure in response to applied chemical signals. J. Am. Chem. Soc. 2010, 16802, 9234–9235. [CrossRef][PubMed] 4. Chen, T.; Wang, H.; Chu, Y.; Boyer, C.; Liu, J.; Xu, J. Photo-induced depolymerisation: Recent advances and future challenges. Chem. Photo. Chem. 2019, 3, 1059–1076. [CrossRef] Polymers 2021, 13, 2461 19 of 20

5. Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A. Light-controlled tools. Angew. Chem. Int. Ed. 2012, 51, 8446–8476. [CrossRef] 6. Pasparakis, G.; Manouras, T.; Argitis, P.; Vamvakaki, M. Photodegradable polymers for biotechnological applications. Macromol. Rapid Commun. 2012, 33, 183–198. [CrossRef][PubMed] 7. Pasparakis, G.; Manouras, T.; Selimis, A.; Vamvakaki, M. Laser-Induced cell detachment and patterning with photodegradable polymer substrates. Angew. Chem Int. Ed. 2011, 50, 4142–4145. [CrossRef] 8. Pasparakis, G.; Manouras, T.; Vamvakaki, M.; Argitis, P. Harnessing photochemical internalization with dual degradable nanoparticles for combinatorial photo-chemotherapy. Nat. Commun. 2014, 5, 3623. [CrossRef] 9. Petit, C.; Bachmann, J.; Michalek, L.; Catel, Y.; Blasco, E.; Blinco, P.J.; Unterriener, N.A.; Barner-Kowollik, C. UV-Induced photolysis of polyurethanes. Chem. Comm. 2021, 57, 2911–2914. [CrossRef] 10. Tong, R.; Xia, H.; Lu, X. Fast release behavior of block copolymer micelles under high intensity focused ultrasound/redox combined stimulus. J. Mater. Chem. B 2013, 1, 886–894. [CrossRef] 11. Hu, Q.; Katti, P.S.; Gu, Z. Enzyme-responsive nanomaterials for controlled drug delivery. Nanoscale 2014, 6, 12273–12286. [CrossRef] 12. Zhang, Q.; Re Ko, N.; Kwon Oh, J. Recent advances in stimuli-responsive degradable block copolymer micelles: Synthesis and controlled drug delivery applications. Chem. Commun. 2012, 48, 7542. [CrossRef][PubMed] 13. Duncan, R. Polymer conjugates as anticancer nanomedicines. Nat. Rev. Cancer 2006, 6, 688–701. [CrossRef][PubMed] 14. Seifu, M.F.; Nath, L.K.; Seifu, M.F.; Nath, L.K. Polymer-Drug conjugates: Novel Carriers for Cancer Chemotherapy. Polym. Plast. Technol. Eng. 2018, 58, 158–171. [CrossRef] 15. Li, J.; Dirisala, A.; Ge, Z.; Wang, Y.; Yin, W.; Ke, W.; Toh, K.; Xie, J.; Matsumoto, Y.; Anraku, Y.; et al. Therapeutic vesicular nanoreactors with tumor-specific activation and self-destruction for synergistic tumor ablation. Angew. Chem. Int. Ed. 2017, 56, 14025–14030. [CrossRef] 16. Li, J.; Anraku, Y.; Kataoka, K. Self-boosting catalytic nanoreactors integrated with triggerable crosslinking membrane networks for initiation of immunogenic cell death by pyroptosis. Angew. Chem. Int. Ed. 2020, 59, 13526–13530. [CrossRef] 17. Li, J.; Kataoka, K. Chemo-physical Strategies to Advance the in vivo functionality of targeted nanomedicine: The next generation. J. Am. Chem. Soc. 2021, 143, 538–559. [CrossRef] 18. Wei, X.; Luo, Q.; Sun, L.; Li, X.; Zhu, H.; Guan, P.; Wu, M.; Luo, K.; Gong, Q. Enzyme- and pH-sensitive branched polymer- doxorubicin conjugate-based nanoscale drug delivery system for cancer therapy. ACS Appl. Mater. Interfaces 2016, 8, 11765–11778. [CrossRef] 19. Seetharaman, G.; Kallar, A.R.; Vijayan, V.M.; Muthu, J.; Selvam, S. Design, preparation and characterization of pH-responsive prodrug micelles with hydrolyzable anhydride linkages for controlled drug delivery. J. Colloid Interface Sci. 2016, 492, 61–72. [CrossRef][PubMed] 20. Tomlinson, R.; Heller, J.; Brocchini, S.; Duncan, R. Polyacetal-doxorubicin conjugates designed for pH-dependent degradation. Bioconjug. Chem. 2003, 14, 1096–1106. [CrossRef][PubMed] 21. Samanta, S.; De Silva, C.; Leophairatana, P.; Koberstein, J. Main-chain polyacetal conjugates with HIF-1 inhibitors: Temperature- responsive, pH-degradable drug delivery vehicles. J. Mater. Chem. B 2018, 6, 666–674. [CrossRef] 22. Dong, S.; Sun, Y.; Liu, J.; Li, L.; He, J.; Zhang, M.; Ni, P. Multifunctional polymeric prodrug with simultaneous conjugating camptothecin and doxorubicin for pH/reduction dual-responsive drug delivery. ACS Appl. Mater. Interfaces 2019, 11, 8740–8748. [CrossRef][PubMed] 23. Li, J.; Li, Y.; Wang, Y.; Ke, W.; Chen, W.; Wang, W.; Ge, Z. Polymer prodrug-based nanoreactors activated by tumor acidity for orchestrated oxidation/chemotherapy. Nano Lett. 2017, 17, 6983–6990. [CrossRef][PubMed] 24. Zhang, Y.; Yin, Q.; Yin, L.; Ma, L.; Tang, L.; Cheng, J. Chain-shattering polymeric therapeutics with on-demand drug-release capability. Angew. Chem. Int. Ed. 2013, 52, 6435–6439. [CrossRef][PubMed] 25. Skene, W.G.; Lehn, J.M.P. Dynamers: Polyacylhydrazone reversible covalent polymers, component exchange, and constitutional diversity. Proc. Natl. Acad. Sci. USA 2004, 101, 8270–8275. [CrossRef][PubMed] 26. Michel, R.H.; Murphey, W.A. Polymers from the condensation of dihydrazides with dialdehydes and diketones. J. Appl. Polym. Sci. 1963, 7, 617–624. [CrossRef] 27. Guo, X.; Shi, C.; Wang, J.; Di, S.; Zhou, S. PH-triggered intracellular release from actively targeting polymer micelles. Biomaterials 2013, 34, 4544–4554. [CrossRef][PubMed] 28. Zhou, L.; Yu, L.; Ding, M.; Li, J.; Tan, H.; Wang, Z.; Fu, Q. Synthesis and characterization of pH-sensitive biodegradable polyurethane for potential drug delivery applications. Macromolecules 2011, 44, 857–864. [CrossRef] 29. Binauld, S.; Stenzel, M.H. Acid-degradable polymers for drug delivery: A decade of innovation. Chem. Commun. 2013, 49, 2082. [CrossRef] 30. Deng, G.; Tang, C.; Li, F.; Jiang, H.; Chen, Y. Covalent cross-linked polymer gels with reversible sol-gel transition and self-healing properties. Macromolecules 2010, 43, 1191–1194. [CrossRef] 31. Ossipov, D.A.; Yang, X.; Varghese, O.; Kootala, S.; Hilborn, J. Modular approach to functional hyaluronic acid hydrogels using orthogonal chemical reactions. Chem. Commun. 2010, 46, 8368–8370. [CrossRef] Polymers 2021, 13, 2461 20 of 20

32. Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Design of environment-sensitive supramolecular assemblies for intracellular drug delivery: Polymeric micelles that are responsive to intracellular pH change. Angew. Chem. Int. Ed. 2003, 42, 4640–4643. [CrossRef] [PubMed] 33. Casey, J.R.; Grinstein, S.; Orlowski, J. Sensors and regulators of intracellular pH. Nat. Rev. Mol. Cell Biol. 2009, 11, 50–61. [CrossRef][PubMed] 34. Kuhl, N.; Bode, S.; Bose, R.K.; Vitz, J.; Seifert, A.; Hoeppener, S.; Garcia, S.J.; Spange, S.; Zwaag, S.; Van Der Hager, M.D.; et al. Acylhydrazones as reversible covalent crosslinkers for self-healing polymers. Adv. Funct. Mater. 2015, 25, 3295–3301. [CrossRef] 35. Chakma, P.; Konkolewicz, D. Dynamic covalent bonds in polymeric materials. Angew. Chem. 2019, 131, 9784–9797. [CrossRef] 36. Winne, J.M.; Leibler, L.; Du Prez, F.E. Dynamic covalent chemistry in polymer networks: A mechanistic perspective. Polym. Chem. 2019, 10, 6091–6108. [CrossRef] 37. Su, W.Y.; Chen, Y.C.; Lin, F.H. Injectable oxidized hyaluronic acid/adipic acid dihydrazide hydrogel for nucleus pulposus regeneration. Acta Biomater. 2010, 6, 3044–3055. [CrossRef] 38. Braschler, T.; Wu, S.; Wildhaber, F.; Bencherif, S.A.; Mooney, D.J. Soft nanofluidics governing minority ion exclusion in charged hydrogels. Soft Matter 2015, 11, 4081–4090. [CrossRef][PubMed] 39. Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U.S. Poly(ethylene glycol) in drug delivery: Pros and cons as well as potential alternatives. Angew. Chem. Int. Ed. 2010, 49, 6288–6308. [CrossRef] 40. Davis, M.E.; Chen, Z.; Shin, D.M. Nanoparticle therapeutics: An emerging treatment modality for cancer. Nat. Rev. Drug Discov. 2008, 7, 771–782. [CrossRef] 41. Fan, W.; Tong, X.; Yan, Q.; Fu, S.; Zhao, Y. Photodegradable and size-tunable single-chain nanoparticles prepared from a single main-chain coumarin-containing polymer precursor. Chem. Commun. 2014, 50, 13492–13494. [CrossRef][PubMed] 42. Jin, Q.; Wang, Y.; Cai, T.; Wang, H.; Ji, J. Bioinspired photo-degradable amphiphilic hyperbranched poly(amino ester)s: Facile synthesis and intracellular drug delivery. Polymer 2014, 55, 4641–4650. [CrossRef] 43. Folmer-Andersen, J.F.; Lehn, J. Thermoresponsive dynamers: Thermally induced, reversible chain elongation of amphiphilic poly(acylhydrazones). J. Am. Chem. Soc. 2011, 133, 10966–10973. [CrossRef] 44. Xu, X.; Ma, S.; Wang, S.; Wu, J.; Li, Q.; Lu, N.; Liu, Y.; Yang, J.; Feng, J.; Zhu, J. Dihydrazone-based dynamic covalent epoxy networks with high creep resistance, controlled degradability, and intrinsic antibacterial properties from bioresources. J. Mater. Chem. A 2020, 8, 11261–11274. [CrossRef] 45. Theodoropoulou, M.A.; Nikitas, N.F.; Kokotos, C.G. Aldehydes as powerful initiators for photochemical transformations. Beilstein J. Org. Chem. 2020, 16, 833–857. [CrossRef][PubMed] 46. Du, J.; Du, X.; Mao, C.; Wang, J. Tailor-made dual pH-sensitive polymer à doxorubicin nanoparticles. J. Am. Chem. Soc. 2011, 133, 17560–17563. [CrossRef]