Yewon Joanna Pak Email

In-vitro Efficacy and Intracellular Mechanism of Riboflavin- Conjugated PEGylated Poly- L-Lysine Dendrimer

Item Type dissertation

Authors Pak, Yewon

Publication Date 2017

Abstract Chemotherapeutic drugs have advanced using different drug delivery methods to treat breast cancer specifically. This development has arisen because many classical drugs exhibit physicochemical limitations including solubility, specificity, stability,...

Keywords Dendrimers; Doxorubicin; Drug Delivery Systems; Riboflavin

Download date 24/09/2021 01:49:54

Link to Item http://hdl.handle.net/10713/7049 Curriculum Vitae

Contact Information:

Yewon Joanna Pak

Email: [email protected] Education Ph.D Candidate in Pharmaceutical Sciences, Aug 2011-July 2017 University of Maryland-Baltimore, Baltimore, MD

Bachelor of Sciences in Chemistry and Bachelor of Arts in Biochemistry, September 2006-June 2011 University of Washington, Seattle, WA

Research Experience Graduate Research Assistant (Ph.D Candidate), August 2011-July 2017 University of Maryland, Baltimore, Department of Pharmaceutical Sciences Advisor: Dr. Peter W. Swaan, Ph.D Investigated synthesizing and formulating a nanoparticle/polymer drug delivery system, dendrimer, with a drug compound to efficiently deliver the drug to its targeting tumor cells.

Undergraduate Research Lab Assistant, February 2010-June2011 University of Washington, Department of Pharmaceutics Investigated functional characterization of mouse PMAT (Plasma Membrane Monoamine Transporter). Experimented with mouse PMAT on transport uptake of certain radioactive compounds like MPP+, Dopamine, and 5-HT.

Student Assistant, July2007-April 2008 University of Washington, Department of Chemistry, Center on Materials and Devices for Information Technology Research Daily works including filings, using Excel, and handling projects. Other jobs consisted of preparing for conference by making pamphlets, sending out mails and organizing the website. Helped organizing conference preparations including Friday Harbor Conference 2007 and Retreat/Expo Survey 2007.

Lab Assistant, April 2008-June 2008 University of Washington, Department of Environmental Science Filtered urine for eliminating substances that was not needed, cleaned instruments, and managed certain bottles of urine by separating them.

Skills Acquired Cell culture, Calorimetric Assays, Atomic Microscopy, Mass Spectrometry, Chromatography- analytical HPLC, UPLC, Size Exclusion Chromatography (SEC), Reverse Phase Chromatography, Liquid chromatography–mass spectrometry (LC-MS), Confocal microscopy imaging, Inverted microscpy imaging, UV-Vis Spectroscopy, Fluorescence Anisotropy, Zetasizer/DLS, Cytotoxicity assays, Purification methods, antibody staining, and Pharmacokinetic modeling with Pharsight (NONMEM and WinNonlin).

Leadership and Activities American Association of Pharmaceutical Scientist (AAPS) Student Chapter Secretary, October 2014-October 2015 University of Maryland, Baltimore Responsibilities include writing minutes during chapter meetings, and organizing AAPS student chapter events including seminars. Have worked diligently and proficiently in dynamic team settings with people from different research background.

American Association of Pharmaceutical Scientist (AAPS) Student Chapter Treasurer, September 2012-October 2014 University of Maryland, Baltimore Demonstrated interpersonal skills by recruiting speakers from different places and interacting with researchers. Responsibilities included managing AAPS student account, filing expenses, and organizing AAPS student chapter events including seminars.

Research Translator, September 2013-September 2014 Heajin Jung Law Firm LLC: Life Science Tech Transfer Translated research papers from Korean to English. Also, translated and made brochures and presentationsfor different pharmaceutical companies.

President's Leadership Student Institute (PSLI), September 2012-May 2013 University of Maryland, Baltimore Participated in attending different leadership seminars and volunteered for more than 20 hours to help Baltimore community.

US-Korea Conference on Science, Technology, and Entrepreneurship (UKC) Conference 2010 volunteer, August 2010 Korean-American Scientists and Engineers Association (KSEA) Took notes on the speakers from different areas of study including pharmaceutical science and chemistry. Also, participated in organizing registration, and assisted with events during the conference.

Honors and Award Rho Chi Honors Society, University of Maryland-Baltimore, February 2016- August 2017 Dr Gerald P and Margaret M Polli Graduate Student Travel Awards, October 2015 CBI Training Grant, July 2014-July 2016 USA Funds Access to Education Scholar, September 2006-June 2010

Special Programs and Certifications Chemistry and Biology Interface Program NIH supported program, which prepared students to pursue cross-disciplinary training in the chemical and biological sciences. Includes presenting a seminar in front of peers two times a year and participating in research at another discipline.

Tablets & Capsules Short Course Participated in lectures and hands-on laboratory sessions for learning process of formulation and manufacturing multi-particulate beads, capsules and tablets.

Abstracts/ Poster Presentations Yewon Pak and P. Swaan. "Anti-Cancer Efficacy of PEGylated Poly-L-Lysine Dendrimer with Doxorubicin." Annual Nanomedicine and Drug Delivery Symposium. September 15, 2015. Baltimore, MD. Y. J. Pak and P. Swaan. "Synthesis and Characterization of Native and PEGylated Poly-L-Lysine Dendrimers." Graduate Research Conference. March 23, 2016. Baltimore, MD. Y. J. Pak and P. Swaan. "Synthesis and Characterization of Native and PEGylated Poly-L-Lysine Dendrimers." American Association of Pharmaceutical Scientists Annual Conference 2015. October 25, 2015. Orlando, FL. Y. J. Pak and P. Swaan. "Synthesis and Characterization of Native and PEGylated Poly-L-Lysine Dendrimers." Globalization of Pharmaceutics Education Network Conference 2014. August 28, 2014. Helsinki, Finland.

Publications Y. J. Pak*, B. Avaritt*, P. Swaan. “Dendrimers: Origins, Architectures, and Applications” Journal of Pharmaceutical Research, Submitted on July 6, 2017 Y.Shirasaka, N. Lee, H. Duan, and J Pak.“Interspecies Comparison of the Functional Characteristics of Plasma Membrane Monoamine Transporter (PMAT) between Human, Rat and Mouse. “ Journal of Chemical Neuroanatomy. Accepted 14 Sept. 2016. Y. J. Pak, P. Swaan.“In-vitro Efficacy of Riboflavin Conjugated PEGylated Poly-L- Lysine Dendrimer” in preparation for submission Y. J. Pak, P. Swaan.“Intracellular Trafficking of Riboflavin Conjugated PEGylated Poly-L-Lysine Dendrimer” in preparation for submission

Abstract

Title of Dissertation: In-vitro Efficacy and Intracellular Mechanism of Riboflavin- conjugated PEGylated Poly-L-Lysine Dendrimer

Yewon Joanna Pak, Doctor of Philosophy, 2017

Dissertation Directed by: Peter Swaan, Ph.D. Associate Dean for Research and Graduate Education Director of the Center for Nanobiotechnology Department of Pharmaceutical Sciences School of Pharmacy University of Maryland, Baltimore, MD, USA

Chemotherapeutic drugshave advanced using different drug delivery methodsto treat breast cancer specifically. This development has arisen because many classical drugs exhibit physicochemical limitations including solubility, specificity, stability, biodistribution, and therapeutic efficacy. There were numerous adverse effectsassociated with these limitations because chemotherapeutic drugs enter normal tissues. In order to eliminate off-target side effect, nanoparticles were developed to target anticancer drugs to a specific carcinogenic area. As one of developing nanomedicines, dendrimers possess ability to be utilized in different administration routes and has potential to stay in the blood circulation longer while showing increased accumulation in tumor cells. Commercially available poly (amidoamine)

(PAMAM) dendrimers have the potential to cause toxicity in-vivodue to lack of biodegradation at sites of accumulation. Poly-L-Lysine (PLL) dendrimers are an alternative class of dendrimers that possess a biodegradable structure. PEGylated poly-l-lysine (PLL) dendrimers are known to be more favorable due to lessened cytotoxicity manifested by masking of cationic charges and avoiding uptake by

Reticulo Endothelial System (RES). Using this biodegradable dendrimer, we sought to examine the effect of PEGylation as well as delivering anti-cancer drug,

Doxorubicin (DOX), to a targeted internalization pathway in human breast cancercells effectively.

PEGylated PLL dendrimers also have their limitation, in which some tumor cells are not dependent upon enhanced permeability and retention (EPR) effect. As a result, riboflavin receptor, which is found to be upregulated in the exterior of breast and ovarian cancer cells, was utilized by attaching a riboflavin ligand to PEGylated

PLL dendrimers in order to be actively uptaken by breast cancer cells. To target chemotherapeutic drug selectively and efficaciously, riboflavin (RF) conjugated PLL dendrimers were assessed in-vitro by investigating cytotoxicity, uptake accumulation, and intracellular colocalization. Further investigation on the endocytosis mechanism and detailed intracellular trafficking in different compartments of the cells were analyzed in order to fully understand the machinery behind delivering chemotherapeutic drugs successfully.

In-vitro Efficacy and Intracellular Mechanism of Riboflavin-conjugated PEGylated Poly-L-Lysine Dendrimer

by Yewon Joanna Pak

Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2017

Table of Contents

Chapter 1 Introduction ...... 1

1.1 Introduction ...... 1

1.1.1 Chemotherapy ...... 1

1.1.2 Macromolecules ...... 1

1.1.3 Dendrimer ...... 2

1.1.4 Targeting ...... 3

1.2 Specific Aims ...... 4

1.3 Scope and Organization ...... 5

Chapter 2 Dendrimers: Origins, Architectures, and Applications [13]1 ...... 6

2.1 Introduction ...... 6

2.2 History ...... 6

2.3 Structure and Properties ...... 8

2.4 Dendrimer Synthesis ...... 9

2.4.1 Divergent ...... 10

2.4.2 Convergent ...... 10

2.4.3 Mixed Methods ...... 11

2.5 Types of Dendrimers ...... 14

2.5.1 Poly(propyleneimine) (PPI)Dendrimers ...... 14

2.5.2 Poly(amidoamine) (PAMAM)Dendrimers ...... 15

2.5.3 Polyether and Polyester Dendrimers ...... 16

2.5.4 Heteroatom (Si and P)-based Dendrimers ...... 16

2.5.5 Peptide-based Dendrimers ...... 17

2.5.6 Notable Dendrimer Hybrids ...... 18

2.6 Applications ...... 19 iii 2.6.1 Therapeutic ...... 20

2.6.2 Emerging Administration Routes ...... 38

2.6.3 Diagnostics ...... 40

2.7 Future Directions and Conclusions ...... 45

Chapter 3 Synthesis and Characterization of Native and PEGylated Poly-L-

Lysine Dendrimers ...... 46

3.1 Introduction ...... 46

3.2 Materials and Methods ...... 49

3.2.1 Synthesis of G3 PLL Dendrimers ...... 49

3.2.2 Addition of PEG ...... 50

3.2.3 Characterization ...... 50

3.2.4 Cell culture ...... 51

3.2.5 Cytotoxicity ...... 51

3.3 Results ...... 52

3.3.1 Synthesis and Characterization ...... 52

3.3.2 Cytotoxicity ...... 58

3.4 Discussion ...... 60

3.5 Conclusion ...... 62

Chapter 4 In-vitro Efficacy of Riboflavin-Targeted Doxorubicin-

Conjugated PEGylated Poly-L-Lysine Dendrimers ...... 63

4.1 Introduction ...... 63

4.2 Materials and Methods ...... 67

4.2.1 Materials: ...... 67

4.2.2 Synthesis of PEGylatedDendrimers ...... 67

4.2.3 RF- and DOX-conjugated PEGylated PLL dendrimer synthesis ...... 68 iv 4.2.4 Characterization ...... 68

4.2.5 Cell Culture ...... 68

4.2.6 Drug release study ...... 69

4.2.7 Cell cytotoxicity ...... 69

4.2.8 Cellular uptake ...... 70

4.2.9 Confocal microscopy ...... 70

4.3 Results ...... 71

4.3.1 Synthesis of PEGylated Dendrimers ...... 71

4.3.2 PH dependent release of DOX ...... 71

4.3.3 Cytotoxicity ...... 77

4.3.4 Cellular trafficking using immunofluorescence ...... 77

4.3.5 Cellular Uptake ...... 80

4.4 Discussion ...... 82

4.5 Conclusion ...... 86

Chapter 5 Intracellular Trafficking of Riboflavin-Conjugated PEGylated Poly-

L-Lysine Dendrimers ...... 88

5.1 Introduction ...... 88

5.2 Materials/Methods ...... 91

5.2.1 Materials ...... 91

5.2.2 Synthesis of RF Dendrimer Conjugates ...... 91

5.2.3 Characterization ...... 92

5.2.4 Cell culture ...... 93

5.2.5 Cellular Colocolization at Early Endosome and Lysosome ...... 93

5.2.6 Cellular Uptake ...... 94

5.3 Results ...... 95

v 5.3.1 Synthesis of RF- and DOX- conjugated PEGylated PLL dendrimers ...... 95

5.3.2 Characterization of RF targeted and non-

targeted PEGylated PLL dendrimers with DOX ...... 95

5.3.3 Cellular Colocolization at Early Endosome and Lysosome ...... 98

5.3.4 Intracellular Uptake ...... 101

5.4 Discussions ...... 102

5.5 Conclusion ...... 105

Chapter 6 Conclusions and Future Directions ...... 107

6.1 Conclusions ...... 107

6.2 Future Direction ...... 110

References ...... 112

vi List of Tables

Table 3.1 Characterization of PEGylated PLL Dendrimers ...... 57

Table 4.1 Polydispersity Index of G3 PLL Dendrimers, PEGylated PLL Dendrimers with DOX, and PEGylated PLL Dendrimers with RF and DOX ...... 74

Table 5.1 Polydispersity Index and Size of Dendrimer Conjugates ...... 97

vii List of Figures

Figure 3.1 Structure of Poly-L-Lysine (PLL) dendrimer ...... 48

Figure 3.2 Synthesis scheme of G3 PEGylated PLL dendrimers ...... 54

Figure 3.3 Mass Spectrometry Results of G3 PLL dendrimers and PEGylated

G3 PLL dendrimers ...... 55

Figure 3.4 NMR analysis of PEGylated G3 PLL dendrimers ...... 56

Figure 3.5Cell viability of G3 PLL dendrimers, G3 half-PEGylated PLL dendrimers, and G3 PEGylatePLL dendrimers ...... 59

Figure 4.1 Scheme of EPR effect ...... 66

Figure 4.2 Synthesis of RF- and DOX-conjugated G3 PEGylated PLL dendrimers ...... 73

Figure 4.3 Characterization of G3 PEGylated PLL dendrimers with RF and

DOX ...... 75

Figure 4.4 PH-dependent release of DOX ...... 76

Figure 4.5 Cell cytotoxicrty of dendrimer conjugates after 48hr incubation………………………………………………………………………..78

Figure 4.6 Confocal microscopy images of dendrimer conjugates after 3hr incubation...... 79

Figure 4.7 Uptake of DOX, PEGylated PLL DM with DOX, and PEGylated PLL dendrimers with RF and DOX ...... 81

Figure 5.1 Endocytosis pathway for macromolecules ...... 90

Figure 5.2 Cellular trafficking of dendrimer conjugates in the early endosome and the lysosome ...... 99

viii Figure 5.3 Uptake of dendrimer conjugates in presence of endocytosis inhibitors

...... 100

ix List of Abbreviations

5-ALA 5-Aminolevulinic acid Aβ β-Amyloid ANOVA Analysis of variance AuNP Gold nanoparticles AuNR Gold nanorods bis-MPA 2,2-Bis(methylol)propionic acid BNCT Boron neutron capture therapy Boc tert-Butyloxycarbonyl BPA Boronophenylalanine BSA Bovine serum albumin BSH Sodium borocaptate COX-2 Cyclooxygenase-2 CRET Chemiluminescence resonance energy transfer CuAAC Copper(I)-catalyzed alkyne-azide cycloaddition COX-2 Cyclooxygenase-2 CRET Chemiluminescence resonance energy transfer Cy5 Indodicarbocyanine DAG Diacylglycerol DAPI 4',6'-Diamidino-2-phenylindole DCM Dichloromethane DM Dendrimer DMEM Dulbecco's modified Eagle's DMF Dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DOX Doxorubicin DPBS Dulbecco's phosphate buffered saline DYN Dynasore EDA Ethylenediamine

EDC 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide EDTA Ethylenediamine tetraacetic acid EEA1 Early endosome antigen 1 EGFR Epidermal growth factor receptor FA Folic acid FBS Fetal bovine serum FDA Food and Drug Administration FIL Filipin x FITC Fluorescein isothiocyanate FRET Fluorescence resonance energy transfer G Generation GEN Genistein GTA Glutaraldehyde GTPase Guanosine triphosphate hydrolytic enzyme HBSS Hank's balanced salt solution HEPES 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid HIV Human immunodeficiency virus HPLC High performance liquid chromatography HPMA N-(2- hydroxypropyl)methacryla mide HSV Herpes simplex virus IgG Immunoglobulin G IP3 Inositol 1,4,5-triphosphate LAMP1 Lysosomal-associated membrane protein 1 LHRH Luteinizing hormone- releasing hormone LPS Lipopolysaccharide MALDI-TOF Matrix-assisted laser desorption/ionization- time of flight MAP Multiple antigen peptide MDC Monodansyl cadaverine MRA Magnetic resonance angiography MRI Magnetic resonance imaging NIR Near infrared PAMAM Poly(amidoamine) PAO Phenylarsine oxide Papp Apparent permeability PCI Photochemical internalization PDT Photodynamic therapy PEG Polyethylene glycol PEI Poly (ethyleneimine) PGLSA Poly (glycerol succinic acid) PIC Polyion complex PIP2 Phophatidyl inositol (4,5)- xi bisphosphate PLC Phospholipase C PLL Poly-L-lysine PPI Poly (propyleneimine) PpIX Protoporphyrin IX PI Propidium iodide PrP Prion protein PTT Photothermal therapy RF Riboflavin RGD Arginylglycylaspartic acid RI Refractive index RIPA Radio-immunoprecipitation assay SD Standard deviation SEC Size exclusion chromatography siRNA Small interferring ribonucleic acid SPAAC Strain-promoted azide- alkyne cycloaddition TBS Tris-buffered saline TEA Triethylamine TEC Thiol-ene coupling TFA Trifluoroacetic acid TYC Thiol-yne coupling VEGF Vascular endothelial growth factor WORT Wortmannin WST-1 Water soluble tetrazolium salt 1 Z Carboxybenzyl

xii Chapter 1 Introduction

1.1 Introduction

1.1.1 Chemotherapy

Breast cancer is one of the most widespread cancers in women, accounting for 22.9% of all cancers [9]. Although treatment options such as surgery or radiation therapy have shown promise, these methods are invasive and cannot completely eliminate all of the cancerous tissues. In order to complement these options, chemotherapy regimens are given post-surgery or radiation. Chemotherapy consists of drugs that kill fast dividingtumor cells.. However, since other healthy cells like bone marrow cells and the lining of large intestinesdivide quickly, the chemotherapeutic drug canalso target non-tumorigenic cancer cells. This phenomenon creates both short- term and long-term side effect for the patients during the use of chemotherapy.

Moreover, to achieve the maximum therapeutic effect in the cancer tissue, a high dose of chemotherapeutic drug needs to be administered, creating even higher toxicity level.

In order to selectively target tumor cells while crossing biological membranes, macromolecular delivery system has been extensively studied for chemotherapeutic delivery [1].

1.1.2 Macromolecules

Macromolecules are nanoparticles delivering drugs to the site of tumor cells more precisely than a classical chemotherapeutic drug. Many studies have been conducted using macromolecule-conjugated chemotherapeutic drug and results showed decrease in systemic toxicity. Due to leaky vasculature of tumore ndothelial cells during the process of obtaining nutrients and oxygen, certain macromolecules with a wide range 1 of sizes can become more permeable, enabling entrance into the tumor cell. Tumor cells also lack lymphatic excretion; therefore macromoleculescan accumulate more drugs inside the tumor cell with less amount of free drug in the blood circulation, ultimately leading to fewer side-effect [2, 3]. Also, covalent conjugation or entrapment of chemotherapeutic drugs to macromolecules haveshown to increase solubility for non-soluble compound and ameliorate pharmacokinetic properties [4].

As a result, macromolecule drug delivery shows a promising potential for targeted and efficacious delivery of chemotherapeutic drugs [5].

1.1.3 Dendrimer

Dendrimersare macromolecule utilized as one of the polymeric nanodelivery system.

They are characterized by nanosized branched structures used for different purposes.

Dendrimers can be synthesized with architectural variation, homogeny in size, variety in branching length and shape. As the generation of dendrimer increases, the diameter increases linearly and at the same time, the surface groups increase exponentially, generating increased surface area for variety of places for drugs, imaging agents, and targeting moieties to be conjugated [6]. Compared with other nanocarrier drug delivery system, dendrimer-conjugated chemotherapeutics showed drugs to be present in blood circulation for prolonged time with enhanced tumor accumulation and longer retention [7]. Dendrimers also have advantage of having higher biocompatibility and monodispersity. Although PAMAM dendrimers have been more widely studied, they are not biodegradable, causing toxicity and imposing risks to patients. Recent works showed that poly-l-lysine (PLL) dendrimers could be more advantageous due to their biodegradable structures, which are composed of L- lysines. Such biodegradable PLL dendrimers are easier for enzymatic degradation and excretion of low molecular weight degradation products [8, 9]. 2 1.1.4 Targeting

There are two routes in which polymer nanoparticles, like dendrimer, can enter the cancer cells: passive or active targeting. Passive targeting consists of drug entering the cell passively in a form of pro-drug and become active inside the tumor cell due to leaky vasculature of tumor tissue also known as enhanced permeation and retention (EPR) effect. Active targeting entails conjugating nanoparticles to the drug and directly targeting the cancer cell through carbohydrate-directed targeting or receptor-mediated targeting in addition to EPR effect [6,8]. Certain tumor tissues have upregulation of cell-surface receptors where high affinity ligand can bind to the corresponding receptor. This method can be an excellentway to target a chemotherapeutic drug as ligand-receptor complex with nanoparticle because the drug conjugate will invaginate into the cell and fuse with endosome to release the active drug as the pH become acidic to about 5.5 (since drug will be attached with a pH sensitive hydrolyzable bond) [9].

A targeting moietyutilized for ligand-receptor complex for dendrimer and drug-conjugated delivery is riboflavin (RF). Riboflavin is a water-soluble vitamin

(B2) that is absorbed into the eukaryotic cell [6]. As an essential micronutrient, riboflavin plays a key role in maintaining regulation of cellular growth and development during cellular metabolism utilizing its cofactors flavin mononucleotide

(FMN) and flavinadenine dinucleotide (FAD) [10].

A previous study identifiedthat while RF serum levels decreased, riboflavin carrier proteins, which transport RF into the cell, are upregulated in MCF-7 breast cancer cells [6]. Moreover, cellular trafficking of RF in human breast cancer cells

(MCF-7) indicated increased internalization of RF due to over-expression of RF receptors on the surface of breast cancer cells [11, 12]. Combining riboflavin and

3 dendrimer as a ligand-based drug delivery system, dendrimer conjugates will further advance to more specific targeting of cancer cells. Therefore, RF-conjugated PLL dendrimers have potential to improve overall clinical efficacy with minimal toxicity to healthy cells and investigation on RF-targeted PLL dendrimers are needed to examinetheir potential as a drug delivery system.

1.2 Specific Aims

The research presented in this dissertation elucidates the potential use of

PEGylated PLL dendrimers as an efficient drug delivery system and explores the efficacy of RF-targeted PEGylated PLL dendrimers, establishing mechanisms of both targeted and non-targeted PEGylated PLL dendrimers with DOX. This study also presents toxicity and internalization of RF-targeted PEGylated PLL dendrimersconjugated with DOX with the eventual aim of using receptor-targeted dendrimers to improve chemotherapy. To achieve this goal, the following Specific Aims were developed:

1. To synthesize and characterize a DOX-conjugated PEGylated PLL dendrimer to target specifically to riboflavin (RF) receptors in breast cancer cells

2. To investigate drug release and in-vitro anticancer efficacy of PEGylated

PLL dendrimers with RF and DOX in a human breast cancer cell model (MCF-7)

3. To investigate the mechanisms of intracellular internalization and trafficking of PEGylated G3 PLL dendrimers with DOX and RF-conjugated

PEGylated G3 PLL dendrimers with DOX.

4 These Specific Aims are designed to test the following hypotheses:

1. Functionalized riboflavin-attached to the Poly-L-Lysine (PLL) dendrimers will serve as a target moiety for delivery of chemotherapeutic drug.

2. RF-targeted PEGylated Poly-L-lysine dendrimers with DOX show similar cytotoxicity level and higher accumulation in MCF-7 cells compared to PEGylated

PLL dendrimer with DOX or free DOX.

3. RF-targeted PEGylated PLL dendrimers with DOX will be internalized by macromolecule endocytosis pathways as well as non-specific macropinocytosis.

1.3 Scope and Organization

In Chapter 2 of the dissertation entails a brief history and structural characteristics as well as synthesis method of various dendrimer types. Detailed literature review shows recent biomedical applications of dendrimers including use in different disease states and administration routes. Chapter 3 provides overview on synthesis and characterization of PEGylated PLL dendrimers and emphasis on efficacy of using PEGylated PLL dendrimers as a potential drug delivery system. In

Chapter 4, PEGylated PLL dendrimers with RF and DOX are assessed for their prospective as areceptor targeted drug delivery vehicle. Not only synthesis but mechanisms on drug release, cytotoxicity, and intracellular trafficking are explored in this chapter. Going further investigation, Chapter 5 explores the mechanism of RF targeted PEGylated PLL DM and non-targeted PEGylated PLL DM endocytosis as well as cell trafficking in the early endosome and lysosome. Lastly, Chapter 6 summarizes conclusions from each chapter in this dissertation and future directions for the research described in this dissertation.

Chapter 2 Dendrimers: Origins, Architectures, and Applications [13]1

2.1 Introduction

Dendrimers are highly branched, nanosized polymers named from the Greek

“dendra” meaning tree. The tree-like structure of dendrimer creates unique properties that are considerably different from traditional linear polymers, many of which can be exploited for biomedical applications. The controlled synthesis of dendrimers allows for the size, surface chemistry, and topology to be manipulated to suit myriad applications.

In this section of the chapter, we provide an overview of the history and properties of dendrimers. The synthetic approaches and different types of dendrimer are also described. Lastly, the therapeutic and diagnostic applications of untargeted and targeted dendrimers are shown with recent results both in vitro and in vivo and an emphasis on emerging administration routes.

2.2 History

While Flory conceived the theory of branched polymers in the 1940s [14-16], the first published reports of dendrimers did not appear until 1978 when multiple groups independently worked to develop new synthetic strategies to obtain monodisperse macromolecules. Vögtle and co-workers published the first report of the divergent synthesis of poly (propyleneimine) (PPI) dendrimers [17]. Quickly after, the synthesis of an asymmetrical lysine dendrimer was patented by Denkewalter and colleagues [18].

------1. Yewon J Pak, Brittany R. Avaritt, Peter W. Swaan. Submitted to Journal of Pharmaceutical Research, July 6, 2017

Within seven years, two more types of dendrimers, poly(amidoamine)

(PAMAM) and arborols, had been developed by Tomalia at Dow Chemical Company and Newkome at Louisiana State University, respectively [19, 20]. While Vögtle’s original cascade synthesis suffered from low yields and purity, the synthetic scheme developed in the Dow laboratories overcame these challenges and is still the preferred methodology used for commercially available PAMAM dendrimers today. In 1993, one group at Freiburg University and the other at the Dutch company DSM, enhanced

Vögtle’s original synthesis to create a viable commercial route for synthesizing PPI dendrimers [21, 22].

Until 1990, all reported dendrimer syntheses utilized a divergent approach.

While this type of synthesis works well for PAMAM dendrimers, many other types of dendrimers synthesized in this manner suffer from stunted growth or structural defects. In an effort to design a more controlled synthetic scheme, the laboratory of

Fréchet at Cornell University developed a convergent method for synthesizing dendrimers[23-25]. By growing dendrimers through building blocks, dendrons were later attached to a single reactive focal point; the number of novel architectures that could now be achieved was enormous. The first dendrimers utilizing convergent synthesis were polyethers derived from 3,5-dihydroxybenzyl alcohol [25].

When first introduced to the scientific community, dendrimer research was met with much criticism. Tomalia reported that many scientists believed dendrimers would crosslink and behave similarly to microgels or latex with no unique properties and that dendrimers were not discrete chemical structures but should be classified as materials [26]. Publications were often delayed because reviewers could not believe such precise molecular control could be achieved by the syntheses described.

Advancements in characterization technologies, particularly the development of

matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, verified the structural precision of dendrimers and led to the eventual acceptance of this new class of polymers [27-32]. Since then, the number of dendrimer publications has grown exponentially. Today PAMAM, PPI, and phosphorus dendrimers (phenoxymethyl

(methylhydrazono), PMMH), as well as polyester dendrons (2,2-bis(hydroxylmethyl) propionic acid,bis-MPA), are commercially available, and a number of products containing dendrimer technology are on the market.

2.3 Structure and Properties

Dendrimers, unlike other polymers, have a well-defined, controllable structure consisting of three main sections: the core, branching groups emanating from the core, and terminal groups presenting at the surface [33]. The core, also known as the initiator, can affect the eventual size and shape of the dendrimer. The branches or dendronsare a region of amplification that can form internal cavities, and each new level of branch points within a dendron defines the dendrimer generation. The dendrimer periphery consists of reactive terminal groups to which new branches, targeting, imaging, or therapeutics moieties can be added. Core (Nc) and branch (Nb) multiplicities determine the number of terminal groups (Z) as a function of generation

G (G) through the equation Z=NcNb , allowing the surface group amplification to be predicted mathematically. As the generation increases, the number of terminal groups increases exponentially while the mass and radius increases linearly.

Dendrimers can be synthesized in discrete units, resulting in monodisperse structures

[34].

The molecular shape of dendrimers is generation-dependent. As the generation increases, dendrimers go from a flat and floppy conformation to ellipsoidal to nearly spherical [27, 35-38]. The generations at which these shape changes occur are 8

related to the core multiplicity. For example, PAMAM dendrimers with Nc=3 achieve molecular morphogenesis a generation later than PAMAM dendrimers with Nc=4

[39]. These morphogeneses are the result of so-called “de Gennes” dense packing that occurs as terminal groups increase exponentially while the radius only increases linearly [40]. Once reached, the dense-packed state can hinder further synthesis resulting in defects in dendrimers synthesized by divergent methods and decreased yields for dendrimers synthesized convergently [41].

In addition to precise and predictable structural control, dendrimers possess properties that are distinctly different from linear and branched polymers. Compared to linear polymers, dendrimers have a lower transition temperature, solubility that increases with increased molecular weight, lower viscosities that exhibit a maximum, no shear sensitivity, and no reputation [29, 42-47]. Dendrimers also have unique container-like properties [48]. As the dendrimer generation increases, an accessible interior is formed between the core and surface groups. Once de Gennes dense packing is reached, the interior becomes inaccessible allowing for encapsulation of guest molecules. These unique properties along with the nanoscale dimensions similar to many biological entities has led to much interest in utilizing dendrimers in various biological and diagnostic applications, many of which will be reviewed in subsequent sections.

2.4 Dendrimer Synthesis

Dendrimers are synthesized primarily through two different routes: divergent or convergent; however, both methods require an iterative approach. Unlike traditional polymers that are polydisperse because of statistical chain growth, dendrimers’ stepwise growth results in theoretically monodisperse polymers [34, 49].

This monodispersity limits synthetic and experimental variability making dendrimers ideal for a variety of applications.

2.4.1 Divergent

The earliest dendrimer syntheses utilized a divergent approach. In this method, dendrimers are constructed from the inside out where branches are attached to an initiator core, and each new propagation of branches results in a new generation.

Examples of dendrimers synthesized via divergent synthesis include Vögtle’s original cascade molecules [17], Newkome’sarborol structures [19], and the PAMAM dendrimers developed at Dow Chemical [20]. The divergent synthesis introduced the first commercial route to dendrimers and possesses several synthetic advantages.

The first advantage is that direct growth from the core allows for a wide range of compounds and materials to be used as cores, and a separate anchoring step, which can be sterically hindered at larger generations, is not required. Second, divergently synthesized dendrimers are amenable to scale-up in the multi-kilogram range using low-cost, readily available monomers such as acrylates, acrylonitriles, and alkyleneamines. Finally, this method can be used to prepare high generation dendrimersthat exceed the de Gennes dense-packed state [26]. While larger dendrimers can be synthesized, defects, or incomplete conjugations, are obtained once generations begin to approach and exceed the dense-packed state. Divergent approaches are, however, limited in the types of dendrimers that can be successfully synthesized.

2.4.2 Convergent

Convergent approaches to dendrimer synthesis have provided a useful alternative to the original divergent method. Convergent synthesis is an outside-in

approach that involves the synthesis of reactive dendrons, which are then attached to the core. First introduced by Hawker and Fréchet [23, 25], polyether dendrimers were the first type of dendrimers to be synthesized via the convergent route. Since then the convergent method has been used to prepare a wide variety of dendrimers with many different functionalities. Convergent methods have several advantages over divergent approaches. Unlike the exponential number of coupling steps required for each iteration of a divergent synthesis, convergent growth only involves the coupling of two dendrons to a monomer, limiting the amount of excess reagent needed and simplifying purification [25]. Dendrimers with multiple functionalities can be synthesized by attaching different dendrons to a single focal point, allowing for a versatility not afforded by the divergent method [24]. This method is also more amenable to creating dendrimer hybrids in which dendrons are combined with other types of polymers or scaffolds [50-52]. While convergent approaches allow for more versatility in the types of dendrimers that can be synthesized, these dendrimers are limited in size due to steric hinderance at the dendron attachment focal point.

2.4.3 Mixed Methods

Traditional divergent and convergent dendrimer synthesis methods both require multiple steps to achieve a single generation increase; therefore, several other methods have been developed in an effort to accelerate dendrimer synthesis. The double stage convergent, or hypercore, method involves attaching dendrons to a core to create a hypercore, which is then reacted with the dendrons again to obtain a higher generation dendrimer. This method was used by Fréchet and colleagues to obtain

G7 dendrimers with different internal and external branching groups creating a layer- block construction [53]. Using this method overcomes the typical steric hindrance encountered in traditional convergent synthesis, but no improvement in synthesis time 11

is achieved. A similar approach utilizing hypermonomers, or monomers with a higher than conventional number of functional groups, has been used to create dendrimers with the same number of surface groups in half the number of synthetic steps as traditional synthesis; using these monomers, a new generation is created in each step

[54]. Unfortunately, hypermonomers are typically lower generation dendrons thereby eliminating any reduction in synthetic steps. Another method utilizing an orthogonally protected monomer, known as the double-exponential method, has been used to synthesize a wide variety of dendrimers [55-60]. This method requires three synthetic steps for each doubling of dendron generation, which leads to a reduced number of overall steps. While this reduction is minimal for smaller generations, the result can be significant for larger dendrimers. The orthogonal coupling method combines two different monomers with chemo-selective functional groups such that the focal group on each monomer will react with the peripheral groups on the other monomer [61, 62]. By eliminating the need for deprotection steps, dendrimer synthesis is reduced by half. This method typically creates a layer-block assembly because of the two different monomers; however, Yu and co-workers created homogeneous dendrons using orthogonal synthesis [63]. A unique method combining the orthogonal approach with hyperbranched monomers termed ‘lego’ chemistry has been used to synthesize phosphorus dendrimers in a one-pot synthesis

[64-66]. Through this method, dendrimers were grown from 48 to 250 terminal groups in a single reaction with by-products of only nitrogen and water. Although the orthogonal approach greatly reduces the synthetic steps, the complex synthetic requirements and limited number of surface functionalities that can be incorporated are a limiting factor in the use of this method.

Another approach utilizing click chemistry, a concept first introduced by

Sharplessand colleagues in 2001, has found many application in dendrimer synthesis

[67]. Several types of click chemistries have been used in dendrimer synthesis:

Cu(I)-catalyzed alkyne cycloaddition (CuAAC) [68], Diels-Alder cycloaddition (DA)

[69, 70], thiol-ene and thiol-yne coupling (TEC and TYC, respectively) [71, 72], and

Michael addition. Combining convergent strategies with CuAAC, triazole-based dendrimers were successfully synthesized in aqueous conditions with only NaCl as the major by-product, and up to generation 3 was isolated without chromatographic separation in the first use of click chemistry for dendrimer synthesis [73]. This approach was also used to obtain Fréchet-type dendrimers via a divergent strategy

[74]. CuAAC has been used to synthesize a wide range of dendrimers including bis-

MPA [75], PAMAM [76], and poly(benzylether) [76], as well as to functionalize dendrimers with a variety of peptides, carbohydrates, targeting moieties, and imaging agents [77-80]. Although CuAA Creactions are highly useful for obtaining unsymmetrical and bifunctional dendrimers that would be difficult to synthesize by other means [81], the removal of the copper catalyst is extremely challenging.

Traces of the cytotoxic copper limit the use of dendrimers synthesized via this approach in biomedical applications. To overcome this limitation, strain-promoted azide-alkyne cycloaddition (SPAAC) has been investigated as an alternative synthetic approach [82].

While CuAAC has been the most researched click chemistry method applied to dendrimers, additional strategies have been investigated. DA is useful in the synthesis of thermo-responsivedendrimers because of the reversible nature of the reaction [83, 84]. Thiol-based reactions have gained interest for their potential to provide a greener synthesis. Thiol-ene coupling (TEC) has been used to synthesize

poly(thioester) and bis-MPA dendrimers in half the number of steps compared to traditional synthesis [85, 86]. TYC has also been used to synthesize G3 dendrimers with 192 terminal hydroxyl groups in six steps [87]. Synthesis of G4 polyester dendrimers has been demonstrated utilizing Michael addition combined with chemoselective monomers [88]. New approaches combining multiple types of click chemistry such as CuAAC-DA [89, 90], CuAAC-TEC [91], and TYC-Michael addition [92], can further accelerate dendrimer synthesis. In recent years, several dendrimersynthesis schemes showed that one-pot and one-step syntheses are the ideal synthetic strategy. Peptide-peptoidic dendrimers and tri-block dendrimers with 1,3- propanediol dendrons have been successfully synthesized utilizing a multicomponent approach [93, 94]. Although many techniques for synthesizing dendrimers already exist [95], the field of dendrimer chemistry will continue to grow as the applications for dendrimers-based therapeutics become more advanced.

2.5 Types of Dendrimers

With advances in dendrimer chemistry, the types of dendrimers have grown exponentially. We will briefly review commonly used dendrimers and notable dendrimer hybrids. Biological applications of these dendrimers will be reviewed in- depth in later sections.

2.5.1 Poly(propyleneimine) (PPI)Dendrimers

First developed by Vögtle in 1978 [17], PPI dendrimers had little success until researchers at Freiberg University and DSM modified the original approach to obtain higher generations in large quantities [21, 22]. PPI dendrimers are synthesized by a two-step iterative approach in which Michael addition of acrylonitrile is followed by heterogeneous hydrogenation of the terminal nitrile groups resulting in an amine-

terminated dendrimer. Typically, 1,4-diaminobutane is used as the core. These dendrimers are marketed as DAB-AM and sold up to generation 5 with 64 terminal amines. Because the surface groups are primary amines, PPI dendrimers are water soluble, basic compounds with properties similar to PAMAM dendrimer [96]. PPI dendrimers have been used in a variety of applications including as drug and gene delivery vehicles as well as antimicrobial agents [97].

2.5.2 Poly(amidoamine) (PAMAM)Dendrimers

PAMAM dendrimers were first developed by Tomalia and co-workers in 1984 and have become the most widely used dendrimers today [20]. Using a divergent approach, PAMAM dendrimers have been synthesized up to generation 12. Similar to PPI dendrimers, PAMAM dendrimers are synthesized in a two-step iterative process involving alkylation with methylacrylate followed by amidation with ethylenediamine.

Stopping the synthesis after alkylation produces ester-terminated dendrimers termed half generations. PAMAM dendrimers can be synthesized with a variety of cores but are traditionally alkyl diamines of various lengths. Commercially produced by Dendritech as Starburst® dendrimers, PAMAM dendrimers are available with five different cores and ten different surface functionalities in generations 0-

10.Amine-terminated PAMAM dendrimers have similar properties as PPI dendrimers, and, therefore, can be used in similar applications [97]. Additional surface functionalities have made PAMAM dendrimers a versatile polymer for both biological and physical applications [98-100].

2.5.3 Polyether and Polyester Dendrimers

Introduced in 1989, polyether, or Fréchet-type, dendrimers were the first dendrimers to be synthesized using a convergent approach [25]. The original polyether dendrimers were synthesized by creating dendrons based on 3,5- dihydroxybenzyl alcohols using activation/coupling iteration. Once the dendrons were synthesized, the same coupling method Williamson ether synthesis was used to attach the dendrons to a polyfunctional core to obtain polyether dendrimers up to G6.

Polyester dendrimers, developed by Ihre and colleagues are based on bis-MPA and synthesized via double stage convergence in which the acetonide-protected fourth generation dendrons are coupled to a core, deprotected, and coupled to various acid chlorides to obtain dendrimers [101]. Bis-MPA dendrons are commercially available up to generation 5 with a variety of focal group functionalities. Polyether and polyester dendrimers have different properties making them useful for different applications. The chemical stability of polyether dendrimers makes them more applicable for use in nanodevices [48], whereas the readily hydrolyzed polyester dendrimers are more biocompatible with potential use in medical applications [102].

2.5.4 Heteroatom (Si and P)-based Dendrimers

Heteroatom-based dendrimers offer unique properties when compared to their traditional organic counterparts. Silicon-based dendrimers were the first heteroatom-containing dendrimers synthesized. These dendrimers can have siloxane, carbosilane, or silane groups at branching points with the carbosilane dendrimers being the most utilized silicon dendrimer [103]. These dendrimers are synthesized by alternating alkenylation with Grignard reagents and hydrosilylation producing high yields [104]. Silicon dendrimers have primarily been used for catalysis [105] but are finding more uses in biological applications [106, 107]. 16

Phosphorus dendrimers were first reported by Rengan and Engal in 1990 [108] using phosphonium ions as branching points and popularized by Majoral and

Caminade[103].The most common synthesis involves nucleophilic substitution of Cl with 4-hydroxybenzaldehyde followed by condensation with a phosphohydrazide and this process is repeated until the desired generation is obtained. Commercially available, PMMH have either thiophosphoryl chloride or hexachlorocyclotriphosphazene cores with aldehyde or dichlorophosphinothioyl surface groups. With high thermal stability and large dipole moments, phosphorus dendrimers are finding use in chemical, biological, and materials science applications

[109].

2.5.5 Peptide-based Dendrimers

While PPI and PAMAM dendrimers are commonly decorated with proteins and peptides, these moieties, along with the amino acid building blocks, can also be used to form dendrimers. Peptide dendrimers can be divided into three main architectures. The first are branching polyamino acids. Denkewalter first patented what are now known as poly-l-lysine (PLL) dendrimers in 1979 [18]. Although PLL dendrimers are asymetrically branched, molecular dynamics simulations suggest that all the terminal groups are still accessible by water and the general structural characteristics of traditional symmetric dendrimers are still retained [110]. In addition to PLL dendrimers, lysine can be used as a branching core decorated with other amino acids [111]. Unlike PAMAM dendrimers, which are commercially available, chemically synthesized lysine based dendrimers offer the advantage of having biodegradability. This property of polylysine dendrimers makes the use of dendrimers more desirable due to enhanced enzymatic degradation and renal excretion of low molecular weight products from degradation [112]. Moreover, due 17

to its biodegradability, this particular dendrimer is widely studied for drug delivery of chemotherapeutic drugs. Attaching a pH labile linker and chemotherapeutic drug with PEGylated polylysine dendrimers had shown increased uptake into tumor tissue compared to a free drug [112]. With its great drug delivery potential, polylysine dendrimers have also been used as contrast agents for magnetic resonance imaging

(MRI) [113].

Polyamide dendrimers are typically the smallest of the peptide dendrimers.

The other two types of peptide-based dendrimers are grafted and peptide dendrimers.

Grafted peptide dendrimers contain an organic core, such as a PAMAM dendrimer, conjugated at the surface with amino acids [114], peptides [115], or sugars [116].True peptide dendrimers contain an amino acid core with peptidyl chains at the surface such as multiple antigen peptides (MAPs) [117].

2.5.6 Notable Dendrimer Hybrids

In addition to the types of dendrimers described above, many different dendrimer hybrids have been investigated for biological uses. Dendritic-dendritic block copolymers can be composed of dendrons of either different generations or different functionalities. Bow-tie dendrimers utilizing polyester or polyether dendrons of different generations have been investigated for chemotherapeutic applications [118]. By orthogonally protecting the dendrons, one side can be selectively PEGylated, and altering the generation of the dendron to be PEGylated affects the number of PEG groups that can be attached [119]. Using dendrons with different functionalities results in so-called Janus dendrimers, which provide even more options for functionalization [120]. Amphiphilic Janus dendrimers can self- assemble into dendrimersomes with uniform size as well as other complex architectures [121]. These dendrimers can be used to mimic biological membranes. 18

In addition to combining different types of dendrons, dendrons can also be combined with different types of polymers. Dendronized polymers consist of a linear polymer backbone with dendrons attached at every monomer repeat, creating rigid-rod architecture. As the dendron generation increases, the rigidity of the structure also increases [122]. Dendronized polymers applied to drug delivery have shown that adding dendrons to the linear polymer increased the drug loading capacity while maintaining a long circulation time [123]. Dendrimers can also be attached to other dendrimers to create tecto-dendrimers. By attaching smaller generation dendrimers to the periphery of a larger generation dendrimer the synthetic drawbacks of high generation dendrimers can be overcome [124]. Like the other dendrimer hybrids, tecto-dendrimers are finding use in biological applications [125]. Dendrimers and dendrons can also be used to encapsulate or coat inorganic nanoparticles such as gold and superparamagnetic iron oxide, which improves the stability, biocompatibility, and cellular delivery of the nanoparticles [126, 127]. Moreover, lipid-dendrimer hybrids have also shown to increase therapeutic efficacy of drug delivery by combining advantages of multifunctional delivery system. Dendrimer-nanoparticle hybrids are capable of further increasing the therapeutic applications of dendrimers.

2.6 Applications

Dendrimers have been extensively investigated for a wide variety of biomedical applications [128]. This review will focus primarily on the use of dendrimers in therapeutic applications and the emerging administration routes for which dendrimers are being investigated. We will also provide a brief overview of the current clinical uses of dendrimers as diagnostic tools for disease. While dendrimers can have therapeutic activity on their own, they are commonly used in conjunction with other therapeutic agents [129]. Drugs can be encapsulated, 19

complexed, or conjugated to dendrimers[130] and targeting moieties [131] and imaging agents [132] can also be incorporated. Each of these dendrimer constructs will be addressed for each therapeutic and diagnostic application where applicable.

2.6.1 Therapeutic

2.6.1.1 Antimicrobial Applications of Dendrimers

The multivalency of dendrimers makes them ideal candidates for the treatment of infectious diseases. Dendrimers have been shown to have both antibacterial and antiviral properties. For the treatment of bacterial infections, dendrimers can serve as antibacterial agents, bacteriophobic coatings, or as antibacterial drug delivery systems[133]. Cationic dendrimers act as antibacterial agents by disrupting and permeating bacterial cell membranes [134]. Quaternized PPI dendrimers have shown potency against E. coli, with activity dependent on dendrimer generation and the length of a single hydrophobic chain conjugated to the dendrimer [135]. G5

PAMAM dendrimers with and without polyethylene glycol (PEG) chains were investigated for antibacterial activity against laboratory and clinical strains of P. aeruginosa [136]. Optimization of G3 PAMAM dendrimers with 6% PEG led to more potent activity against P. aeruginosa with minimal cytotoxicity against other cells [137]. An in vivo guinea pig model of chorioamnionitis was used to illustrate the ability of hydroxyl-terminated G4 PAMAM dendrimers to prevent E. coli contamination of the amniotic fluid [138]. Cytokine levels in the animals were shown to be similar between the dendrimer-treated and infection-free control animals, indicating that the dendrimers did not cause an immune response. A recent study also displayed the effectiveness of low generation cationic PAMAM dendrimers against

Gram-negative and Gram-positive bacteria including drug resistant strains [139]. G2

PAMAM dendrimers were effective antibacterial agents without inducing drug resistance or causing toxicity. While anionic dendrimers have shown activity comparable to gentamycin in Gram-positive and Gram-negative bacteria, cationic dendrimers were more potent against both bacteria [140]. Additionally, viologen- conjugated phosphorus dendrimers have shown promising preliminary results against

S. aureus, E. coli, and P. vulgaris[141].

Additionally, dendrimers decorated with carbohydrates have shown exceptional antibacterial properties through either blocking uptake of bacterial toxins into eukaryotic cells or preventing bacterial cells from binding to human erythrocytes

[133]. Bacterial cells attach to eukaryotic cells via carbohydrate-protein interactions, and bacterial toxins are internalized though the same interactions [142].

Glycodendrimers can have carbohydrate groups specific to either the bacterium or the toxin, preventing binding and uptake. Both PPI and PAMAM dendrimers functionalized with a specific ganglioside attached have been used to inhibit cholera toxin B and heat-labile enterotoxin from binding to cells [143]. Because of their multivalency, toxins bind to the dendrimers at over a 1000-fold lower concentration than is needed for binding to the free glycoprotein. To prevent the binding of bacterial cells to human erythrocytes, glycodendrimers containing galabiose were designed to inhibit the meningitis-causing S. sues [144]. Octavalent-dendrimers were able to achieve minimum inhibitory concentrations in the nano- and picomolar range. Therefore, glycodendrimers have the potential to serve as antibacterial agents with high specificity.

Another type of dendrimer designed for antibacterial activity uses a peptide- coating. In this approach, dendrimers are decorated with peptides that mimic antimicrobial peptides that participate in innate immune response [145]. Tam and

colleagues attached tetra- or octapeptides to the surface of small PLL dendrimers and evaluated the antimicrobial activity of the constructs [146]. The peptide-dendrimers had broad-spectrum activity comparable to the linear peptide but with better solubility and stability and less toxicity to human cells. Other PLL dendrimers with tetrapeptides have been shown to display activity against multidrug resistant strains of

E. coli and S. aureus eliciting lower levels of resistance compared to traditional antibiotics like ciprofloxacin and gentamicin [147].

Dendrimers can also be used to treat and prevent biofilms, a group of bacterial cells often resistant to antibiotics. Glycodendrimers with fucose groups, a ligand for

LecB on P. aeruginosa, displayed complete inhibition of biofilm formation and complete dispersion of established biofilms [148]. Altering the dendrimer to have

D-amino acids instead of the original L-amino acids resulted in retained inhibition of biofilm formation with higher stability against proteolysis [149]. A tetravalent peptide dendrimer has also shown promise in treating biofilms by killing E. coli persister cells that are tolerant to antibiotic stress [150]. Dendrimers can also be used to coat metal implants to prevent biofilm formation. PLL dendrimers were appended with titanium-binding peptides and a single PEG chain and used to coat titanium beads [151]. Coated beads had a 90% reduction in S. aureus biofilm formation compared to uncoated beads, indicating the potential of dendrimers to inhibit infections from orthopedic implants.

In addition to potent antibacterial potential, dendrimers also possess antiviral activity. While cationic dendrimers are more potent for antibacterial activity, anionic surface groups are more effective against viruses. Dendrimers interact with the virus, preventing virus binding to the target cell [152]. Dendrimers have been investigated for use against influenza [153], respiratory syncytial virus

[154], and herpes simplex virus (HSV) [155]; however, activity against human immunodeficiency virus (HIV) has been the most reported. Dendrimers that have been evaluated for HIV prevention include anionic dendrimers, glycodendrimers, and targeted dendrimer-drug complexes [156]. The first dendrimer product to enter clinical trials was SPL7013, or VivaGel®, a G4 PLL dendrimer with terminal naphthalene sulfonic acid groups designed for vaginal delivery. SPL7013 had broad spectrum antiviral activity against HIV, HSV, and human papillomavirus [155].

After showing efficacy and safety in preclinical studies [157], Phase I clinical trials were performed to establish the tolerability of VivaGel® in women [158], and studies found that SPL7013 does not enter systemic circulation [159]. Phase II studies also displayed the effectiveness of SPL7013 in the prevention of HIV and HSV-2 transmission [160]. Although Phase III clinical trials with VivaGel® for the treatment of bacterial vaginosis failed to meet the clinical cure endpoint, the success of SPL7013 as an antiviral led to the approval of VivaGel®-coated condoms in Japan and Australia [161].

2.6.1.2 Application in Neurodegenerative Diseases

The cationic nature of dendrimers can be exploited for the prevention and treatment of neurodegenerative diseases. Many neurodegenerative diseases are the result of protein aggregation. In prion diseases, conformational changes in the prion protein (PrPC) can result in an aggregation-prone, protease-resistant scrapie protein

(PrPSc) that causes spongiform encephalopathy [162]. While prion diseases affect several different species, the human form is called Creutzfeldt-Jakob disease. The ability of dendrimers to treat prion-infected cells was serendipitously discovered by

Supattapone and colleagues, after attempting to transfect scrapie-infected ScN2a neuroblastoma cells with Superfect™, a PAMAM dendrimer-based transfection agent 23

[163]. After transfection, the cells were cleared of all PrPSc proteins. Other cationic dendrimers such as PPI and poly(ethyleneimine) (PEI) have also been shown to clear scrapie-infected cells without being cytotoxic [164, 165]. Typical treatments for prion diseases only stop the conversion of PrPC to PrPSc; however, dendrimers are the first compounds to result in the removal of previously formed PrPSc, which cannot be efficiently removed by endogenous mechanisms [163]. Phosphorous dendrimers have also been investigated for use against prion diseases and displayed the ability to clear PrPSc from the spleen of mice injected with the protein [166]. Interestingly, the spectrum of prion strains susceptible to PPI or phosphorus dendrimers is unique for each dendrimer [165, 166]. Guanidino- and urea-modified G2 PPI dendrimers were investigated for prion clearing ability also, and while the charged dendrimers were more potent, the neutral urea-modified dendrimers were still able to clear the infection while causing less cytotoxicity [167]. With the ability to treat prion diseases by both preventing PrPC conversion and solubilizing preexisting PrPSc aggregates, dendrimers are promising candidates for the treatment of prion diseases [168, 169].

The ability of dendrimers to disaggregate PrPSc by interrupting the protein β- sheets has led to the investigation of their use in another neurodegenerative disease caused by protein aggregation, Alzheimer’s disease [170, 171]. The development and onset of Alzheimer’s disease is believed to be related to the aggregation of β- amyloid (Aβ) peptides as described in the amyloid cascade hypothesis [172]. The

Aβ peptides first form soluble, neurotoxic oligomers before forming insoluble, nontoxic fibrils that lead to plaques. Therefore, treatments can prevent Aβ from aggregating, break up the already formed aggregates, or accelerate formation of the nontoxic fibrils. Early work showed that G3 PAMAM dendrimers were capable of modulating Aβ 1-28 peptide aggregation: low concentrations affected the fibril

nucleation rate increasing nontoxic fibril formation and high concentrations affected the elongation rate of fibrils preventing aggregate formation [171]. Phosphorus and gallic acid-triethylene glycol dendrimers conjugated to morpholine as well as lysine dendrimers also reduced the toxicity of Aβ 1-28 by modifying peptide aggregation in a similar manner while decreasing the cytotoxicity caused by the peptide [173-175].

In a study utilizing electron paramagnetic resonance to investigate dendrimer-peptide interactions, researchers found that the dipolar interactions between the dendrimer and the Alzheimer’s peptide Aβ 1-28 were stronger than the hydrophilic-hydrophobic interactions between the dendrimer and the prion peptide PrP 185-208 [176].

PAMAM dendrimers also showed a stronger interaction than both PPI and phosphorusdendrimers. The ability of PAMAM dendrimers to influence amyloid aggregation is generation dependent, with G5 PAMAM dendrimers showing a larger degree of aggregation inhibition and more effective disruption of preexisting fibrils than G3 and G4 [177]. G2-4 PAMAM dendrimers decorated with sialic acid, a molecule on cell surface glycoproteins with which Aβ binds to exert cellular toxicity, were found to attenuate Aβ toxicity at much lower concentrations than free sialic acid

[178]; however, the mechanisms of this attenuation could not be conclusively defined

[179]. More recent studies utilizing maltose-conjugated PPI dendrimers have shown promise in the amelioration of Aβ toxicity as these dendrimers also caused amyloid fibrils to clump and become nontoxic [180]. In vivo studies conducted in a transgenic mouse model of Alzheimer’s disease showed that neutral G4 PPI-maltose dendrimers were capable of reaching the brain and increasing the Aβ fibrillar content after intranasal delivery [181]. Unfortunately, these dendrimers did not improve the memory impairment of the mice, emphasizing the difficulty of translating molecular events to clinically meaningful outcomes. PPI dendrimers with maltotriose have also

been shown to modestly cross the blood brain barrier after intraperitoneal administration, taking dendrimers one step closer to realizing their potential in neurodegenerative diseases [182].

In addition to impacting Aβ fibrillation, dendrimers have also been investigated for the ability to inhibit the aggregation of α-synuclein. As the primary component of Lewy bodies, α-synuclein fibril formation and aggregation is implicated in several neurodegenerative diseases such as Parkinson’s disease [183]. Initial studies conducted with G3-5 PAMAM dendrimers indicated the ability of these dendrimers to both inhibit fibril formation and disassociate preexisting fibrils in a concentration- and generation-dependent manner [184]. The effects of cationic and anionic PAMAM dendrimers on α-synuclein aggregation have also been compared

[185]. While G4 PAMAM dendrimers directly interacted with the protein to prevent fibrillation, the anionic dendrimer had no interaction or effect on aggregation. Low concentrations of native and viologen-conjugated phosphorus dendrimers also inhibited fibril formation [186, 187]. Taken together, studies on the ability of dendrimers to impact protein aggregation, an important process in neurodegenerative diseases, exemplify the unique ways in which dendrimers may impact the treatment of a multitude of diseases.

2.6.1.3 Inflammatory Diseases

Inflammation underlies many chronic conditions including arthritis, asthma, retinal degeneration, and neurodegenerative disorders. While dendrimers have shown use in the solubilization, and in some cases improved the permeability of several anti-inflammatory drugs, dendrimers also have displayed intrinsic anti- inflammatory properties [188]. Testing the anti-inflammatory properties of

PAMAM dendrimer-indomethacin conjugates in an arthritic rat model, Chen and co- 26

workers discovered that although the conjugates preferentially accumulated within the inflamed regions to a greater extent than free drug, the dendrimer alone also exhibited an anti-inflammatory effect [189]. Therefore, the researchers investigated the use of

PAMAM dendrimers with different surface charge (G4, G4OH, G4.5) in multiple models of inflammation in rats. After intraperitoneal injection, the dendrimers displayed higher activity than indomethacin, with G4 exhibiting the highest activity

[190]. The anti-inflammatory activity of G4 and G4OH PAMAM dendrimers was determined to be produced from selective inhibition of cyclooxygenase-2 (COX-2).

Other enzyme inhibitor and molecular modeling studies have also indicated that smaller hydroxyl-terminated dendrimers are also capable of interacting with and inhibiting COX-2 and inducible nitric oxide synthase [191]. To specifically act upon arthritis-associated inflammation, targeting dendrimers to monocytesis is one potential therapeutic strategy [192]. Phosphorus dendrimers capped with azabisphosphonate were shown to target monocytes and direct them towards an anti-inflammatory response [193]. In a rheumatoid arthritis mouse model, the dendrimers reduced inflammatory cytokines and prevented cartilage damage and bone erosion after intravenous administration. The anti-osteoclastic activity of the phosphorus dendrimers was further corroborated by in vitro results. Moreover, folate-targeted and methotrexate-conjugated dendrimers were used to target macrophages to successfully reduce arthritis-induced parameters of inflammation in a collagen- induced arthritis rat model [194]. Arthritis can also be caused by infection, and

PAMAM G4-folic acid (FA) conjugates were again capable of targeting inflammatory sites in Chlamydia-induced reactive arthritis in mice [195].

Pulmonary inflammation has been another area of interest for dendrimer- targeted delivery. G4OH PAMAM-methylprednisolone conjugates have been shown

to decrease the number of eosinophils in the lung to a greater extent than free methylprednisolone after transnasal delivery in a mouse model of inhaled ovalbumin- induced pulmonary inflammation [196]. The improved treatment compared to free drug was due to the increased residence time of the dendrimer-drug conjugate in the lungs. Additionally, mannose-grafted poly(phosphorhydrazone) dendrimers were tested in a lipopolysaccharide (LPS) inhalation-induced murine inflammation model

[197]. Dendrimer treatment was discovered to reduce neutrophil influx to the lungs after LPS inhalation, and inhibition of pro-inflammatory cytokine activation was shown in vitro.

In another in vitro study, G4OH PAMAM dendrimers complexed with celastrol, a potent anti-inflammatory compound, improved the solubility of the drug and suppressed the LPS-mediated release of pro-inflammatory mediators without decreasing viability in microglial cells [198]. G4OH PAMAM dendrimersconjugated to fluocinaloneacetonide was shown to accumulate in retinal microglia after intravitreal injection in two different rat models of retinal degeneration, but not in healthy control rats [199]. Dendrimer-drug conjugates were able to arrest the retinal degeneration and preserve photoreceptor outer nuclear cell counts thereby showing promise in the treatment of retinal degeneration.

2.6.1.4 Applications of Dendrimers in Tissue Repair and Wound Healing

Collagen is an integral component of fibrous tissues, and an important scaffold for tissue engineering. Unfortunately, the weak mechanical properties and rapid biodegradation limit the use of natural collagen. Therefore, dendrimers have been investigated for use as collagen mimetics. Small anionic PAMAM dendrimers conjugated to peptides have been shown to achieve a triple helical structure similar to collagen but with better mechanical properties [200]. By adding additional peptides 28

to mediate cell adhesion and enzymatic crosslinking, PAMAM dendrimer collagen mimetics could form focal adhesions with cells comparable to natural collagen [201].

For tissue engineering, collagen is commonly crosslinked with 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) or glutaraldehyde (GTA); however, the gels formed do not have sufficient crosslinking to provide proper mechanical strength and residual crosslinking agent can be toxic limiting biocompatibility

[202].Cholecyst-derived extracellular matrix crosslinked with G1 PAMAM dendrimershad improved mechanical and degradation properties compared to traditionally crosslinked matrices [203]. Using G2 PAMAM dendrimers in conjunction with EDC or GTA crosslinking resulted in more biologically stable gels compared to the crosslinking agents alone, and inclusion of dendrimer with EDC resulted in a significant increase in fibroblast proliferation [202]. G2 and G3 PPI dendrimers both successfully crosslinked collagen to provide more stable gels as a result of a higher amount of crosslinking compared to EDC and GTA [204]. G2 PPI dendrimer-crosslinked collagen was further studied as a corneal tissue engineering scaffold [205]. The gel was found to have better optical transparency and glucose permeability than traditional collagen scaffolds with no toxicity. Utilizing the excess amines present from dendrimer crosslinking, Duan and colleagues conjugated a cell adhesion peptide to the scaffold [206]. The stable scaffold promoted human corneal epithelial cell adhesion and proliferation as well as neurite extension from dorsal root ganglia.

In addition to dendrimers as collagen mimetics and crosslinking agents, linear- dendritic block copolymers have found use as sealants in corneal tissue repair.

Poly(glycerol succinic acid) (PGLSA) functionalized with methacrylate (MA) groups attached to a PEG core form a photocrosslinkable polymer containing polyester-ether

bonds with properties amenable to ocular applications [207]. Grinstaff and colleagues have shown that (PGLSA-MA)2-PEG dendrimers can seal corneal lacerations with higher leaking pressures compared to sutures [208, 209].

Additionally, the gel formed has a smoother texture than currently used adhesives, and may be better at preventing postoperative infections compared to sutures because the wound is completely sealed [210]. In a chicken model, corneal lacerations were repaired with either the polymer adhesive or sutures [211]. Comparing the two methods, the adhesive was five times faster to apply than sutures, andafter 28 days of healing, adhesive use resulted in less inflammation and scarring. While the dendrimer-based adhesive showed promising results, the argon laser needed to cure the gel prompted researchers to develop a self-curing gel to make application easier.

Replacing the PGLSA dendrons with lysine dendrons capped with cysteine resulted in a self-gelling polymer when mixed with PEG functionalized with dialdehyde [212].

The polymer formed a hydrogel within minutes and was capable of increasing the leaking pressure that could be withstood with sutures [213]. Used in conjunction with sutures, the adhesive could reduce the number of sutures required for corneal transplant while still meeting the demands of intraocular pressure. In enucleated human eyes, the lysine-based adhesive showed wound sealing sufficient to prevent leakage both out of and into the corneal wound [214]. By modifying the PEG functional groups, Oelker et al. created an adhesive, which is able to secure ex vivo rabbit corneal wounds and withstand greater intraocular pressure than sutures [215].

The preclinical success of dendrimer-based sealants for corneal wounds has translated to OcuSeal®, a PEI-PEG-based liquid ocular bandage marketed in Europe. In patients, use of OcuSeal® resulted in lower rates of surgical-induced astigmatism and foreign-body sensation compared to sutures while promoting wound closure better

than unsutured incisions [216]. The same platform has been used to create the dural sealant Adherus® which provides a watertight closure with antibacterial properties and no neurotoxicity [217]. Marketed in Europe, Adherus® recently completed a

Phase III clinical trial in the U.S. to test safety and effectiveness.

In recent years, dendrimers have been used to deliver genes for tissue regeneration. To overcome the inherent instability of gene delivery using viral vector and other gene transfer techniques, uniquely modified dendrimers were investigated for application. PAMAM dendrimer conjugated with histidine and arginine residues showed potential to successfully transfect human mesenchymal stem cells, which comes from adult bone marrow [218]. Similarly, low concentration of amine terminated PAMAM dendrimer exhibited improved gene transfection in human embryonic stem cell (hSECs) [219]. Furthermore, conjugating a high binding affinity peptide specific for mesenchymal stem cell membrane to PAMAM dendrimer showed even more enhanced gene transfection capability [220]. These studies revealed potential for dendrimer to be used for cell-based therapy using tissue engineering since modified PAMAM dendrimers exhibited low cytotoxicity and successful gene delivery without eliciting stem cells to differentiate into multiple lineages or pluripotency.

2.6.1.5 Applications in Cancer

Dendrimers are well established as drug delivery carriers for the treatment of cancer, and the use of dendrimer-drug conjugates and complexes has been previously reviewed [221]. Recently Starpharma’s DEP™ drug delivery platform has entered clinical trials in Australia (starpharma.com). Conjugated to docetaxel, preclinical studies have indicated significantly improved efficacy compared to the currently marketed docetaxel formulation. In addition to successful drug delivery, dendrimers 31

have also been studied as anticancer vaccines [222]. In this review we will primarily focus on the use of dendrimers in novel therapeutic strategies.

2.6.1.5.1 Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is a highly selective form of radiation therapy utilizing epithermal neutrons to irradiate boron-10 to yield high energy α particles and lithium-7 [223]. The ionization energy produced from the reaction occurs in an area approximately equal to the diameter of a single cell; therefore, therapy only damages cells containing boron. BNCT has completed clinical trials for the treatment of high-gradegliomas and recurrent head and neck tumors resistant to conventional therapy [224-226]. For BNCT to be effective, a boron-containing agent must preferentially localize in tumor tissue. To deliver the boron, boronophenylalanine (BPA) and sodium borocaptate (BSH) are currently used.

However, neither of these compounds achieves sufficient targeting to realize the full potential of BNCT.Dendrimers have been investigated as boron delivery vehicles to decrease the normal tissue to tumor ratio. Boronated dendrimers conjugated to a monoclonal antibody targeted to melanoma have shown promising tumor accumulation but also accumulated in the liver and spleen [227]. Utilizing anti-

EGFR (epidermal growth factor receptor), boronated G5 PAMAM dendrimers have been used to target glioblastomas [228]. When used in combination with BPA or with a mixture of monoclonal antibodies towards wild-type EGFR and mutant

EGFRvIII, a significant increase in mean survival time was achieved compared to traditional radiation therapy in a rat glioma model [229]. Vascular endothelial growth factor (VEGF)-containing boronated dendrimers have also been investigated for targeting the tumor neovasculature of solid tumors [230]. In addition to

PAMAM dendrimers, bis-MPA dendrons have been investigated for use in

BNCT.Conjugating galactose groups to the dendron focal point resulted in improved cellular uptake and cytotoxicity in HepG2 human liver carcinoma cells compared to

BSH [231]. While BNCT has shown promising results for the treatment of malignancies with no other effective treatment, the limited number of facilities with an appropriate neutron source is both location and cost prohibitive.

2.6.1.5.2 Photodynamic Therapy

Photodynamic therapy (PDT) is another potentially selective therapy for the treatment of cancer. PDT uses a photosensitizer that, when exposed to a specific wavelength of light, produces a highly reactive singlet oxygen and induces cell death

[232]. By illuminating a tumor in which a photosensitizer has accumulated, the damage to healthy cells can be minimized. Currently, porfimer sodium (Photofrin®) is FDA approved for the treatment of esophageal and non-small cell lung cancers.

Because the wavelength of light needed for currently used photosensitizers can only penetrate less than 1 cm through tissue, PDT is limited to small tumors near the surface of the skin such as melanoma or accessible by endoscopy such as lung cancer

[233]. To advance PDT as an effective treatment option, tumor-targeted photosensitizers activated by light capable of deeper penetration must be developed.

Dendrimers have been used as carriers of photosensitizers to achieve one or both of these goals. The first dendrimer constructed for PDT contained 5-aminolevulinic acid (5-ALA), a commonly used precursor of protoporphyrin IX (PpIX) [234].

Dendritic 5-ALA displayed improved uptake in keratinocytes and epidermoid carcinoma cells and had more efficient porphyrin synthesis compared to free 5-ALA

[235]. In a mouse mammary adenocarcinoma xenograft, attaching 5-ALA to the

dendrimer prolonged porphyrin production in the tumor which could eventually allow for low level, extended irradiation and apoptosis of target cells instead of necrosis

[236]. In addition to 5-ALA-containing dendrimers, PEGylated PAMAM and PPI dendrimers have both been used to encapsulate other photosensitizers. PpIX and rose bengal (RB) were encapsulated in either PEGylated PAMAM or PPI dendrimers

[237]. While fewer PpIX molecules were encapsulated than RB, the PpIX- containing dendrimers were more stable, and both PPI dendrimers were more stable than the PAMAM dendrimers. PPI-PpIX dendrimers were shown to have more efficient toxicity after irradiation than both free PpIX and PAMAM-PpIX in vitro.

Recently, dendrimers have been combined with other nanomaterials to create targeted photosensitizers with long wavelength activation. Modifying 5-ALA containing-multi-walled carbon nanotubes with PAMAM dendrimers allowed for a significant increase in 5-ALA tumor accumulation [238]. PAMAM-grafted porous hollow silica nanoparticles loaded with a phthalocyanine-based photosensitizer could be activated with 670 nm light and displayed better light toxicity than free photosensitizer [239]. Folic acid-conjugated hydroxyl PAMAM dendrimers bound to graphene oxide were activated by 780 nm and showed minimal dark toxicity with

ROS generation after near infrared (NIR) irradiation [240]. Additionally, Zhang et al. have created self-activating polymer dots for targeted PDT and imaging [241].

The polymer dots consisted of a photosensitizer with a fluorescent, semiconducting polymer coated by Janus dendrimers conjugated to FA and horseradish peroxidase.

The polymer dots were shown to preferentially accumulate in cancerous cell lines with minimal uptake in a non-cancerous cell line, and toxicity was observed after photoirradiation. Interestingly, in the presence of chemiluminescence substrates, the photosensitizer can be activated through both chemiluminescence resonance energy

transfer (CRET) directly and fluorescence resonance energy transfer (FRET) initiated by CRET. Therefore, no light source is needed to initiate photodamage. This platform has the potential to overcome the limitations inherent with needing an external light source while simultaneously allowing for tumor imaging.

2.6.1.5.3 Photothermal Therapy

Photothermal therapy (PTT) involves heating a tumor to temperatures greater than 45°C to kill cellsdirectly or to 39-42°C in combination with other therapeutic strategies [242]. PTT is traditionally an invasive technique with non-uniform results.

The advent of inorganic nanomaterials efficient at converting an extrinsic energy source into heat has made PTT a more viable therapeutic option. Various nanomaterials are capable of using magnetic, light, radiofrequency, microwave, or other energy sources to generate heat. In combination with tumor targeting and cellular internalization, these nanomaterials are capable of inducing hyperthermia from within the tumor to provide a targeted therapy. Dendrimers combined with gold nanoparticles (AuNPs) for PTT are a recent addition to the anti-cancer arsenal.

AuNPs have been entrapped in PAMAM dendrimers conjugated to FA and fluorescein isothiocyanate (FITC) [243]. The constructs were water soluble, stable, biocompatible, and exhibited specific binding and internalization in cells that overexpressed the FA receptor. Comparing acetylated FA- and FITC-conjugated dendrimers with and without AuNP entrapped, Shi et al. discovered that the AuNPs had no effect on the targeting and internalization kinetics of the conjugates [244].

By adding PEG groups to the dendrimer, colloidal stability and solubility were improved, especially after freeze-drying [245]. After irradiation with visible light, the complexes generated heat comparable to AuNPs alone. RGD, a peptide used to

target the over-expressed integrin on tumor neovasculature, has also been studied as a targeting moiety for dendrimer-AuNP complexes. RGD-conjugated PAMAM dendrimers containing AuNPswere shown to only bind and internalize in integrin- expressing endothelial cells [246]. The peptide has also been used as a targeting moiety for PAMAM dendrimer-coated gold nanorods (AuNRs) [247]. The conjugates had significant tumor accumulation in a mouse model of melanoma, and after NIR irradiation tumor growth was significantly slowed. RGD-targeting improved the efficacy of the conjugates, and researchers saw complete tumor regression in 4 out of

10 mice. PAMAM-AuNR complexes have also been conjugated to DOX via an acid- labile linker for combined thermo-chemotherapy [248]. In vitro, the conjugates showed DOX release within the cell lysosomes. In vivo, significant tumor accumulation occurred in mouse colon carcinoma xenografts, and the conjugates were more potent than either DOX or PAMAM-AuNRs alone. More recently, efforts have been made to synthesize PAMAM-PEG AuNR complexes with the AuNRs within the dendrimer core [249]. The complexes had no cytotoxicity in HeLa cells and displayed excellent heat generation after NIR irradiation. Advances in dendrimer-AuNP construct show promise in the noninvasive thermal treatment of cancer.

2.6.1.5.4 Gene Silencing

Dendrimers have already been established as efficient transfection agents in vitro, exemplified by the marketed product, Superfect®. While efficient in vivo gene delivery is challenging to accomplish, recent work has achieved promising results in the treatment of cancer in animal models. One of the challenges of dendrimer gene delivery is the accumulation in organs of the reticuloendothelial system such as the

liver; therefore, one of the first successful dendrimer-nucleic acid complexes, or dendriplexes, was targeted to the liver by attaching galactose residues to PPI dendrimers[250]. Liver targeting was confirmed by dose-dependent decreases in liver luciferase expression after a ligand for the glycoprotein wasinjected in mice.

More recently, the preferential liver accumulation of dendriplexes has been exploited to deliver short-activating RNAs for the enhancement of albumin production in cirrhotic rat livers while reducing hepatocellular carcinoma tumor growth [251].

Lactose-bearing PAMAM dendrimers with α-cyclodextrin have also been shown to induce high gene transfer activity within the liver with lower splenic activity than non-targeted dendrimer[252]. By attaching mannose to the PAMAM-α-cyclodextrin constructs, targeting was shifted to the kidneys [253].

Targeting strategies have also been developed for gene delivery across the blood-brain barrier. PAMAM-PEG dendriplexes have shown successful brain targeting with both transferrin [254] and rabies virus glycoprotein (RVG29) conjugates [255]. In both cases, targeting ligands provided more efficient gene delivery compared to untargeted dendriplexes. Angiopep-conjugated PAMAM-PEG dendriplexes have also been shown to successfully cross the blood-brain barrier.

The conjugates showed parenchymal accumulation and high efficiency gene expression in mice brains [256].

Dendriplexes can also be used in combination with chemotherapeutic drugs to achieve greater reduction of tumor cells. PAMAM-PEG dendrimers have been used to co-deliver a gene for human tumor necrosis factor-related apoptosis-inducing ligand and DOX [257]. Dendriplex-drug conjugates displayed efficient tumor accumulation in a liver cancer xenograft with a significant reduction in tumor volume.PPI dendriplexes with paclitaxel have been investigated for the treatment of

ovarian cancer [258]. By targeting the conjugates with a LHRH peptide analog, siRNA for CD44 and paclitaxel were successfully delivered to human ovarian carcinoma xenografts in mice resulting in suppression of CD44 expression and decreased tumor volume. Triethanolamine cored-PAMAM dendrimers have also been studied for the treatment of ovarian cancer [259]. In conjunction with paclitaxel, AktsiRNA was delivered to ovarian cancer xenografts and was capable of reducing the tumor volume better than dendrimer-drug conjugates or dendriplexes alone.

2.6.2 Emerging Administration Routes

In addition to traditional intravenous administration, dendrimers have been extensively investigated for their ability to traverse barriers that drugs alone cannot cross. In this review we will focus on the use of dendrimers in ocular, oral, and pulmonary therapy.

2.6.2.1 Ocular

Delivery of drugs to the posterior segment of the eye is challenging but necessary for the treatment of ocular diseases such as glaucoma. Dendrimers have been used in combination with drugs for the treatment of glaucoma. Encapsulating pilocarpine and tropicamide resulted in sustained drug release at the site of action and improved miotic and mydriatic activity in rabbits after administration via eye drops

[260]. Conjugating glucosamine and glucosamine 6-sulfate to G3.5 PAMAM dendrimersprevented inflammation and neoangiogenesis in rabbits after glaucoma filtration surgery [261].

Subconjunctival injection of the dendrimer conjugates also prevented scar tissue formation. Carteolol-complexes with phosphorus dendrimers for the treatment

of glaucoma were shown to release the anti-hypertensive drug into the aqueous humor of rabbits in concentrations greater than those achieved with free drug while causing no irritation [262]. Incorporation of antiglaucoma drugs in dendrimer-based hydrogels allowed for increased drug uptake and transport [263]. Additionally the hydrogel formulation was capable of achieving a sustained reduction in intraocular pressure in rabbits [264].

2.6.2.2 Oral

First studied by Wiwattanapatapeeand colleagues, PAMAM dendrimers were shown to translocate across inverted rat intestinal sacs showing potential as oral drug delivery carriers [265]. Since then, PAMAM dendrimers have been extensively investigated for their ability to traverse the gut epithelium. Propranolol-PAMAM conjugates increased the flux of the drug across Caco-2 monolayers and successfully prevented P-glycoprotein efflux of propranolol [266]. Naproxen-PAMAM conjugates also displayed a significant enhancement in solubility and permeability compared to free drug [267]. Dendrimer-chemotherapy conjugates have also been studied for their ability to improve the transport of poorly soluble drugs with low bioavailability. PAMAM-SN38 complexes [268] and conjugates [269] have both been shown to increase the solubility, transport, and uptake of the drug regardless of dendrimer surface charge. PAMAM-DOX complexes exhibited significantly improved oral bioavailability in rat intestinal sections compared to free DOX [270], further suggesting the potential of dendrimers as oral drug delivery carriers.

More recently dendrimers have been investigated in vivo for the ability to increase drug oral bioavailability. Complexation of silybin with G2 PAMAM dendrimersincreased the bioavailability of the drug 2-fold after oral gavage in rats

[271]. Both anionic and cationic PAMAM dendrimers have also been shown to 39

increase the oral absorption of camptothecin 2-3 fold when co-administered in mice

[272]. As more in vivo studies are conducted, dendrimer-drug constructs can be optimized to achieve improved oral bioavailability.

2.6.2.3 Pulmonary

In addition to the pulmonary delivery for inflammation previously discussed, dendrimers can also be utilized for other pulmonary applications. For example,

PAMAM dendrimer-enoxaparin complexes were shown to be effective at preventing deep vein thrombosis in the lungs of rats after pulmonary administration [273].

Dendrimers have also shown promise for the delivery of a poorly soluble asthma drug via nebulization [274]. PEGylated PLL dendrimers have been investigated for use in both local delivery to the lungs and systemic delivery. The size of PLL-PEG conjugates impacted absorption from the lungs with larger conjugates being retained in the lungs and smaller conjugates being absorbed into systemic circulation [275].

DOX was conjugated to PEGylated PLL dendrimers for the treatment of breast cancer metastases in the lung [276]. In a mouse model, dendrimer-DOX conjugates were administered via intratracheal instillation or intravenously. Pulmonary administration of the conjugates significantly reduced lung tumor compared to conjugates administered intravenously, and free drug caused extensive toxicity.

Therefore, dendrimers have the potential to provide both local and systemic delivery of drugs for the noninvasive treatment of cancer and other diseases.

2.6.3 Diagnostics

2.6.3.1 Immunoassays

Apart from delivering drugs to the body, dendrimers have been used to detect diseases using disease-related proteins (biomarkers). Immunoassays using antigen-

antibody reaction have been a major tool for detecting biomarkers and especially electrochemical immunoassay exhibited promising advantages like high specificity, instant detection and cost efficiency [277]. Dendrimer was utilized because of its of its direct binding to the target and its high specificity toward amplifying signal in the electrochemical immunoassay. For example, carbon nanofibers fabricated polyamidoamine (PAMAM) dendrimer was conjugated with both Au@Ag nanorods and anti-human immunoglobulin G (IgG) inorder to detect IgG [278]. PAMAM dendrimer attached immunosensor was shown to be effective since peak current was similarto the concentration of IgG and detection limit was low. Abundance of conjugation sites at the exterior of the dendrimer promotes detection of the biomarker and hydrophilicity of the dendrimer helps dissolution of the gold nanoparticle. This was shown in immunoassay based on nanogold-penetrated PAMAM dendrimer

(AuNP-PAMAM) with anti-myoglobin antibody to detect myoglobin as a biomarker for acute myocardial infarction [277]. Moreover, dendrimer conjugated AuNP displayed higher voltammetric response due to higher specificity for its target and facilitated dissolution. Dendrimers have also shown high stability and biocompatibility in electrochemical immunoassay. A study using AuNP-PAMAM dendrimer functionalized wired ethyleneamine-viologen showed reproducibility of its specificity and sensitivity towards a cancer biomarker, AFP in human serum [113].

This suggests that dendrimer-based immunoassay can detect biomarker proteins in biological fluids such as the bloodstream. Therefore, dendrimers have the potential toserve as an immunoassay tool in order to enhance detection of different diseases.

2.6.3.2 Contrast agents

Contrast agents have been used widely to visualize internal body structures 41

such as blood vessels and the gastrointestinal tract. Traditionally, gadolinium (Gd) complexes are utilized for contrast agents. However, the major disadvantages of those complexes are nephrotic systemic fibrosis and low sensitivity due to minimal accumulation at the target site. In recent years, nanomaterials gained more attention as contrast agents because nanoscaled materialgives increased surface area for enhanced optical and mechanical properties [279]. Especially, dendrimers show advantage over traditional contrast agents, including good bioavailability, longer half- life of the particle, and higher accumulation at the tumor site. Therefore, many studies changed their gears to using dendrimers as a component in magnetic resonance imaging (MRI). For example, dendrimer-loaded magnetic nanoparticles functionalized with folic acid were used to conduct experiments to target image

Dalton's Lymphoma Ascites (DLA) cancer, and results showed that dendrimer conjugate can affect both T1 and T2 relaxation properties, improving the differentiation between normal and tumor cells [112]. Similarly, dendrimers show enhanced capability to detect tumor when PAMAM dendrimers with multiple ACPP and Gd-DOTA molecules were employed as a MRI agent instead of Gd [280].

PAMAM dendrimers along with attached chelate have been shown to optimize biosensing ability of biosensor imaging of redundant deviation in shifts (BIRDS), which is another form of MRI [281]. These studies suggest that dendrimers have high potential as a contrast agent, especially MRI.

In recent years, dendrimers have been examined for other types of contrast agents. For instance, dendrimers were used as a building block for positron emission tomography (PET) by incorporating LyP-1, a cyclic peptide, to recognize tumor cells and macrophages for atherosclerosis [282]. This PET imaging study demonstrated that the dendrimer building block increased recognition of the targeted tumor cells

and higher visualization of the aorta for inflammation. Dendrimers not only contributed to enhancement of PET imaging but also computed tomography (CT) for a powerful disease diagnostic tool. In vivo and in vitro studies involving G2

PAMAM dendrimers conjugated with both AuNP and folic acid showed tumor imaging enhancement while having cytocompatibility in various environments [279].

Additionally, PAMAM dendrimers with targeting ligand chlorotoxin and HPAO have been exploredfor radionuclide-based imaging like single photon emission computed tomography (SPECT) [283]. This study revealed that targeting conjugation-based dendrimers effectively accumulate dendrimers within liver compared toother organs for imaging. Thus, this nanoplatform carries the potential to overcome restrictions in the originalcontrast agents, allowing for enhanced disease diagnostics and stability in vivo.

2.6.3.3 Dendrimers as Molecular Probes

In order to understand biological features at the molecular level, molecular probes have been introduced to the scientific field within past several years.

Different molecular imaging probes were used for their specific use. However, past molecular probes faced critical challenges with limited availability of photostable fluorescent probes and small sized molecular probes with high affinity. Therefore, many studies started to utilize dendrimers due to their small size and accessibility of numerous conjugating sites for fluorescent tags. Fluorescent imaging with DNA dendrimers to carry functional nucleic acids (FNAs) for in situ monitoring of biological molecules in living cells showed successful imaging ability at recognizing function of aptamer while maintaining biocompatibility [284]. This study demonstrates that dendrimers overcome the previous limitations of low stability and 43

low affinity for biomolecules of interest. Further, in a different fluorescence imaging study, G5 and G6 PAMAM dendrimers with cyanine dye were combined to assay single-molecule nucleic acid hybridization and image microtubules in cytoskeletal networks [285]. Dendrimer molecules served as a molecular scaffold to achieve brighter fluorescence emission for imaging microtubules due to higher cyanine dye conjugation. Moreover, the dendrimer scaffold exhibited enhanced photostability with longer photobleaching lifetime and specifically revealed true location of the nucleic acid hybridization. Similar to previously mentioned studies, a perylene-based dendrimers also displayed brighter fluorescence and more water- solubility when attached with chromophoric phosphoramidites [286]. This outcome was due to long and flexible arms of dendrimers that give the highest coupling efficiency compared to other carriers. Dendrimers were used not only for fluorescent imaging, but also to improve the molecular probe of other types of imaging techniques. In order for electrochemical sensors to enhance their ability to probe carbohydrate-lectin recognition, G0-G2 PAMAM dendrimer functionalized with mannopyranosylferrocenyl moieties was investigated [287]. The results showed that PAMAM-based dendrimers with a moiety had enhanced affinity for lectin ConcanavalinA (Con A), thus enabling target imaging. Also, the multivalent binding reaction and multi-electron exchange from dendrimer based moieties show improvement in the redox sensing ability in electrochemical sensors. Therefore, applying dendrimers to molecular probes looks promising as dendrimer-based probes show increases in fluorescence and binding affinity, enabling efficient biological studies.

2.7 Future Directions and Conclusions

Dendrimers have shown great promise in the treatment of many disease states.

Unlike other polymers, within a relatively short time, dendrimers have emerged in marketed products for clinical applications as a wound sealant as well as for disease diagnosis and detection. Dendrimers are also moving forward in the clinic as

Starpharma conducts clinical trials for the prevention of bacterial vaginosis and the treatment of solid tumors. To improve upon the already diverse dendrimer applications, researchers can continue to search for unmet clinical needs in which dendrimers may create a new therapy or improve upon existing therapies. The application of dendrimers to non-invasive routes of administration is another way dendrimers can improve upon the current therapies for many diseases, especially cancer. As more in vivo studies are conducted, mechanistic studies still remain critical to establishing the impact of dendrimer design on safety and efficacy. With the scientific foundation already laid, dendrimers are poised to continue making a significant impact on human health.

Chapter 3 Synthesis and Characterization of Native and PEGylated Poly-L-Lysine Dendrimers

3.1 Introduction

Nanoparticles consist of macromolecules delivering drugs to the site of tumor cells more precisely than classical drugs [288]. Due to large pore sizes of tumor endothelial cells formed during the process of obtaining nutrients and oxygen, certain macromolecules with a wide range of sizes can enter into the tumor cell. Tumor cells also lack lymphatic drainage [4]; therefore drug delivery using nanoparticles can accumulate more drugs inside the tumor cell.

Dendrimersare one of the common polymeric nanodelivery systems used for different purposes, including drug delivery, gene delivery, and disease-detecting agents [33]. There are various types of dendrimers, including polyamidoamine dendrimers (PAMAM), poly-propyleneimine (PPI), polyether, and polyester dendrimers [25, 289]. As the generation of dendrimer increases, the diameter increases linearly and the surface groups increase exponentially, generating an increased surface area for conjugating drugs, imaging agents, and targeting moieties.

Although polyamidoamine dendrimers are available for commercial purchases, they are not biodegradable, creating potential for toxicity when administered to the human body. Polyester dendrimers are biodegradable, but they have the potential for stability hindrance by rapid hydrolysis [290].

Poly-l-lysine (PLL) dendrimers are biodegradable polymers that are easily degraded enzymatically and excreted as low molecular weight degradation products.

Because PLL dendrimers are more applicable for biological uses due to less toxicity exerted by their biodegradability, it is important to utilize PLL dendrimers for

chemotherapeutic drug delivery. The structure of PLL dendrimers consists of continuous branching of L-lysine amino acids as shown in figure 3.1.

H3N O

H3N

H3N NH

NH O

NH3 NH O O NH NH

NH3 O

H3N NH O O

H3N O HN

HN O NH3

O NH3

O O NH3 NH O

H3N HN NH NH

H2N O

NH3 NH O O HN

H3N H3N

H3N

Figure 3.1 Poly-L-Lysine (PLL) dendrimer with cationic amine terminal

Although PLL dendrimersare favorable for clinical use, cationic charges fromamine surface groupscausetoxicity to cell membranes [291]. Also, at a lower generation of

PLL dendrimers, they are small in hydrodynamic size, which leads to rapid renal clearance[292]. Poly ethylene glycol (PEG) is a polymer derived from ethylene oxide and it has been commonly used for drug excipients due to its low toxicity fromits neutral charge and flexibility [293]. Therefore, attachment of PEG to cationiccharged PLL dendrimers will reducecytotoxicity to cells, while at the same timeincreasing molecular weight to bypass the reticulo endothelial system.

In this work, half and fully PEGylated PLL dendrimers have been prepared through convergent synthesis of PLL dendrimers and PEG was added through amide formation. In order to accurately half- PEGylate PLL dendrimers, Boc-protected and

Z-protected L-lysines were added after the generation 3 dendrimer synthesis. All synthesized compounds will be evaluated using characterization tools to confirm a successful synthesis, molecular weight, size, and monodispersity. To show

PEGylation reduces PLL dendrimer toxicity in cells, a breast cancer cell line, MCF-7, will be used as a cell model for an in-vitro cytotoxicity study.

3.2 Materials and Methods

3.2.1 Synthesis of G3 PLL Dendrimers

Generation 3 (G3) Poly-L-Lysine (PLL) dendrimers were synthesized as described previously (PLL dendrimer trafficking). Briefly, PLL dendrimers were synthesized by adding L-lysine methyl ester with BOC-L-Lysine-ONP in anhydrous dimethylsulfoxide (DMSO) and triethylamine under argon at 75°C for 3hr. When the reaction was completed, the product was purified using liquid-liquid extraction in dichloromethane (DCM) then dried over magnesium sulfate. Final solid compound of Boc protected dendrimer was achieved by rotary evaporation. G1 PLL 49

dendrimers were deprotected by adding 1:1 anhydrous DCM and trifluoroacidic acid

(TFA) for 1hr. Then, solvents were removed to obtain G1 PLL dendrimer compounds. These steps were repeated to achieve deprotected G2 Poly-L-Lysine compound.

3.2.2 Addition of PEG

During synthesis of half-PEGylated G3 Poly-L-Lysine dendrimers, BOC and Z protected L-Lysine-ONP was used instead and 0.1 N sodium bicarbonate was used rather than liquid-liquid extraction. The product was filtered with acetonitrile and dried over vacuum for Z- and Boc-protected G3 PLL dendrimer products. After removing BOC protection with previously mentioned method, Poly ethyl glycol (PEG)

2000 Da was added with dimethylformamide (DMF) and DCM. This mixture was sealed with argon and reacted overnight. When reaction was complete, ethylenediamine was added to quench remaining starting material and acetic anhydride was added afterwards. Solvent products were filtered into diethyl ether, precipitated, and dried under high vacuum. After getting half-PEGylated PLL dendrimers, Z-groups were deprotected through hydrolysis and dried under high vacuum. For fully PEGylated PLL DM, Boc-protected ONP activated L-Lysine was added instead of Boc- and Z-protected L-Lysine. Rest of the reaction was carried out in similar manner to what is described before.

3.2.3 Characterization

All of the intermediate and final products were confirmed using Ion trap Mass spectrometry for molecular weight <1000Da and MALDI-TOF (BrukerAutoflex,

Billerica, MA) for molecular weight >1000Da. Structure of G3 PLL dendrimers and

PEGylated PLL dendrimers were confirmed using 2D Heteronuclear Multiple Bond

Correlation (HMBC) Nuclear Magnetic Resonance (NMR) by looking at bond formation between carbon and proton. Size exclusion chromatography with HPLC was used to measure differently conjugated dendrimer polydispersity with 0.05mM

LiBr containing Dimethylformamide (DMF) mobile phase. The hydrodynamic radius of each synthesized dendrimer conjugates were measured using a Malvern

(Westborough, MA) Zetasizer Nano ZS. G3 PLL dendrimers, half-PEGylated PLL dendrimers, and fully-PEGylated PLL dendrimers were solubilized in Hank's

Balanced Salt Solution (HBSS) at concentration of 1mg/mL and measured in triplicate.

Measurements were achieved with a refractive index of 1.33 and viscosity of 0.88 at

25◦C.

3.2.4 Cell culture

MCF-7 cells, a human breast cancer cell line, were obtained from the

American Type Culture Collection (Manssas, VA). MCF-7 cells were grown at 37°

C with 5% CO2 and 95% relative humidity. The cells were cultured with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 U/ml penicillin, 1% nonessential amino acids and 10,000 μg/ml streptomycin. Media was changed every two days and cells were passaged with 0.25% trypsin at approximately

80-90% confluency.

3.2.5 Cytotoxicity

PEGylated PLL dendrimersand non-PEGylated dendrimers cytotoxicity was measured using Water-soluble tetrazolium salt (WST-1) cytotoxicity assay.

Cells were first seeded with 50,000 cell density per each well in 96 well microplates and cells were grown over 48hr. Then, the cell viability of MCF-7 cells were quantified by treating microplate (96wells) cultured cells with 0.1, 1, 10, 100, 300,

500, 1000 and 2000µM of G3 PLL dendrimers, half-PEGylated PLL dendrimers, and

PEGylated PLL dendrimers. After treating cells for 2 hr, the cells were washed and

110µl of WST-1 reagent was added to each treated wells. Absorbance of the samples was measured using a microplate (ELISA) reader at 420-480 nm with reference wavelength of 600nm and cytotoxicity was analyzed.

3.3 Results

3.3.1 Synthesis and Characterization

G3 PLL dendrimers and PEGylated PLL dendrimers were synthesized

(Fig 3.2) to determine the efficacy of PEGylation on masking cytotoxicity of PLL dendrimers compared to non-PEGylated PLL dendrimers . Successful synthesis of

G3 PLL dendrimers and PEGylated PLL dendrimers are described in figure 3.1. The first step in the process of synthesizing PEGylated PLL dendrimers was to synthesize

G3 PLL dendrimers. Molecular weight of G3 PLL dendrimers was confirmed with

MALDI-TOF spectra shown in figure 3.3, in which exact mass isat 1954 Da. As Z and BOC-protected L-lysines were added to available amine groups, molecular weight shift occurred from 1954 to 3850 Da. When BOC was deprotected, PEG added, and hydrolyzed for deprotection of Z, a broad peak around 19kDa occurred as PEG increased molecular weight of PLL dendrimers by about 10 fold. Heteronuclear single quantum coherence (HSQC) NMR in figure 3.4a shows NMR results from correlation between 1H and 13C peaks, which indicate successful conjugation of PLL groups to G2 PLL dendrimers. In HSQC, protons coincide with carbons next to amine (38ppm) and alkyl carbons (52ppm) next to carbonyl carbon. Additionally,

PEGylated dendrimers were prepared by reacting G3 PLL dendrimers with PEG2000 and dissolved in dimethyl sulfoxide (DMSO) to be analyzed in NMR. Heteronuclear

Multiple Bond Correlation (HMBC) NMR showed successful conjugation of PEG 52

2000 to amine groups in G3 PLL dendrimers by creating an amide bond (Fig 3.4b).

Especially, second and third carbonyl carbons from PEGylated PLL dendrimers were confirmed by coupling between 1H at 3.5ppm and both 162ppm and 174ppm of 13C.

Polydispersity of each dendrimer compounds were determined using size exclusion chromatography attached to HPLC. Polydispersity of each conjugates were 1.04, 1.05, and 1.07for PLL dendrimers, half-PEGylated PLL dendrimers, and fully-PEGylated PLL dendrimers respectively (table 3.1). This shows that even though polydispersity has increased as dendrimers become larger in size, it still remains monodisperse with PDI around 1.

Figure 3.2 Synthesis schemes of G3 PEGylated PLL dendrimers from methyl L-

Lysine.

19215.806 B Intens. [a.u.] . 5000

4000

3000

2000

1000

0 10000 20000 30000 40000 50000 60000 m/z

Figure 3.3Mass spectrometry analysis of G3 PLL dendrimer (A) confirms the presence of the product. [M+Na]+ m/z=1976 shows sodium adduct of G3 PLL dendrimer. Half-PEGylated PLL dendrimer molecular weight (B) found m/z= 19.21kDa.

Figure 3.4 Assessment of G3 PLL dendrimer synthesis completion was done by HSQC spectroscopy (A), which detects correlation between nuclei of two different types and in this case, both 1H and 13C NMR. G3 PEGylated PLL dendrimers were analyzed using HMBS spectroscopy in similar manner (B).

Synthesized Mw Mn Poly Dispersity Size (nm)

Compounds Index (PDI)

(Mw/Mn)

G3 PLL 2500.52 2404.35 1.04 2.04±0.05

Dendrimer

Half PEGylated 18755.32 17800.24 1.05 7.97±1.23

G3 PLL

Dendrimers

G3 PEGylated 25323.91 23676.56 1.07 11.15±3.65

PLL Dendrimer

Table 3.1 Polydispersity index and size of G3 PLL dendrimers, half-PEGylated dendrimers and fully PEGylated PLL dendrimers using size exclusion chromatography and Zetasizer

Hydrodynamic radius of PLL dendrimer conjugates were analyzed using dynamic light scattering (DLS) and as PEG was conjugated to half of G3 PLL dendrimers, radius increased from 2.04±0.05 to 7.97±1.23. As G3 PLL dendrimers were fully

PEGylated, hydrodynamic size increased over 5 fold, which is at 11.15±3.65nm.

3.3.2 Cytotoxicity

Cell cytotoxicity of conjugated PEGylated PLL dendrimers and control dendrimers were determined using Water-soluble tetrazolium (WST)-1 assay. G3

PLL dendrimers were assessed in order to see if dendrimer itself has any cytotoxic effect on cells. G3 PLL dendrimers had a significant cell cytotoxicity after about

1mM concentration as shown in figure 3.5 and as PEG were added to half of the PLL dendrimers, percent of viable cells increased. When PLL dendrimers were fully

PEGylated, no cytotoxicity was displayed even at 2mM concentration of PLL dendrimers. This shows that the dendrimer itself has negligible cytotoxicity to MCF-7 cells because its IC50 was around 1mM concentration but with PEGylation, existing cytotoxicity is diminished so PEGylated dendrimers have better biocompatibility towards cells.

G3#PLL#Dendrimers 200 Half%PEGylated%G3%PLL%DM

% PEGylated%G3%PLL%DM y 150 t i l

i non3PEGylated%G3%PLL%DM b a i v

% 100 l l e c %

% 50

0 0 1 2 3 4 Log%concentra7on%(µM)

Figure 3.5Cytotoxicity of non-PEGylated G3 PLL dendrimers and half-PEGylated G3 PLL dendrimers and fully-PEGylated G3 PLL dendrimers using WST-1 assay. Mean ± standard deviation (SD) (n=3)

3.4 Discussion

Macromolecules are large molecules commonly created by polymerization of smaller monomers [294]. Recently, macromolecules have been explored for many medical reasons including drug delivery. This is because macromolecules deliver drugs to the site of tumor cells more precisely than a classical drug alone while having longer circulating half-life [288]. Dendrimers are a class of macromolecules and they're "tree-like" polymers, which can be used as drug delivery systems forimprovedtargetingtosolid tumors [288, 295]. Commercially available poly

(amidoamine) (PAMAM) dendrimers have the potential to cause toxicity in vivo duetoalack of biodegradation at sites of accumulation. Poly-L-Lysine (PLL) dendrimers are an alternative class of dendrimers that possess a biodegradable structure. Adding PEG to Poly-L-Lysine (PLL) dendrimers enhances their ability to act as adrug delivery vehicle for improved targeting to solid tumors. PEGylated PLL dendrimers minimize acute toxicity and improve longer retention in the blood circulation because of their higher molecular weight and neutral surface charge.

In this study, we sought to deliver the classic anti-cancer drugs through a targeted internalization pathway in human breast cancer cell using PEGylated PLL dendrimers. In order to effectively deliver anti-cancer drugs, we investigated if

PEGylated PLL dendrimers have minimal inherent toxicity to cells as compared to non-PEGylated PLL dendrimers. This investigation is a crucial step before using

PLL dendrimers as a drug delivery vehicle because it can be toxic to normal cells.

G3 PLL dendrimers were successfully synthesized and surface amine groups of PLL dendrimers were half and fully PEGylated.. All three batches of reacted compounds were examined for molecular weight, formation of bonds, polydispersity, and size. From MALDI-TOF results, it was clear that G3 PLL dendrimers were

accurately synthesized by showing an accurate mass at 1954 Da. However, with half-PEGylation, molecular weight increased by 10-fold to 19kDa, which is close to the predicted mass of 18.7 kDa. The peak of PEGylated PLL dendrimers' molecular weight had a broad peak due to increased polydispersity as molecular weight became more than 10kDa. NMR results also showed differences in structure as amine terminated G3 PLL dendrimers were conjugated with carboxylic acid terminated PEG.

An important result to note is that while PLL dendrimers had carbonpeaks from carbonsnext to amine (38ppm) and alkyl carbons (52ppm) next to carbonyl carbon,

PEGylated PLL dendrimers had peaks around 162 ppm, indicating formation of carbonyl carbons from an amide bond between G3 PLL dendrimer and PEG.

PDI was analyzed for non-PEGylated PLL dendrimers, half-PEGylated, PLL dendrimers, and fully PEGylated PLL dendrimers. As more PEG was added to PLL dendrimers, PDI increased because of significant increase in molecular weight, suggesting increase in polydispersity. However, PDI is still within 1, indicating both non-PEGylated and PEGylated PLL dendrimers have narrow range of size distribution although they have high molecular weight. It is important to note that

PLL dendrimers have narrow range of size distribution because this means when PLL dendrimers are used for chemotherapeutic drug delivery, it will have more consistent therapy with similar sized dendrimers.

Cytotoxicity of PEGylated PLL dendrimers validated biocompatibility of

PEGylated PLL dendrimersby showing no cytotoxicity to MCF-7 cells at a high concentration with even 2mM in concentration. Half PEGylation of dendrimers also increased cell viability significantly compared to non-PEGylated PLL dendrimers.

Although PLL dendrimers without PEGylation showed cytotoxicity at ~1mM concentration, this concentration is negligible concentration for further study

becausethe concentration is too high to be administered for clinical use.

3.5 Conclusion

The results from this study show that PEGylated PLL dendrimers have excellent potential as a drug delivery system, with low cytotoxicity and monodispersity, suggesting longer retention in blood stream. Dendrimers are known to have many advantages for enhanced chemotherapeutic drug delivery due to presence of many surface groups for conjugating ligands or drugs, biocompatibility, controlled synthesis, and specific delivery of drugs to targeting site [296, 297]. PLL dendrimers, even though biodegradable, was investigated due to cytotoxicity exertedby cationic charges from their surface groups. Successful synthesis from this study shows addition of PEG masks cationic charges present on surface of PLL dendrimers. Present study showed that PLL dendrimers are monodisperse even with

PEGylation, which suggests PEGylated PLL dendrimers will have discrete particle size cutoff. Through cytotoxicity study, PEGylated PLL dendrimers displayed almost no cytotoxicity to cells, showing that they are compatible to be used for delivering chemotherapeutics drugs because dendrimer itself will not createcytotoxicity to tumor cells or normal cells. Therefore, PEGylated PLL dendrimers show potential for targeted drug delivery using a biodegradable dendrimer-based chemotherapy especially with properties favorable for potential clinical application.

Chapter 4 In-vitro Efficacy of Riboflavin-Targeted Doxorubicin- Conjugated PEGylated Poly-L-Lysine Dendrimers

4.1 Introduction

Breast cancer isthemost prevalent cancer among women worldwide [298].

Many treatment options are available for breast cancer patients such as surgery and radiation therapy. Although these treatment choices have shown promise in the elimination of cancer cells to some extent, theyare invasive and cancer cells cannot be completely eliminated. Therefore, chemotherapy treatments are given to eliminate the remainder of the tumor cells. However, many chemotherapeutic drugs exhibit physicochemical limitations including solubility, specificity, stability, biodistribution, and therapeutic efficacy. Significantly, limitations due to low specificity towards target cells result in both short-term and long-term side effects for the patients. In order to selectively target tumor cells while crossing biological membranes, a macromolecular delivery system has been extensively studied for chemotherapeutic delivery[1, 299]. While macromolecular drug delivery is limited to endocytic internalization, chemotherapeutic drug conjugates are able to retain in blood circulation for a longer period of time due to enhanced permeability of neovasculature and also lack of lymphatic drainage [300-302]. Also, conjugating chemotherapeutic drugs to a carrier enhances tumor accumulation while avoiding drug-resistance mechanisms [303].

Dendrimers are a class of macromolecular nanodelivery system, derived from hyperbranched polymers used for target drug delivery. Dendrimers can be synthesized with architectural variations; as the generation of dendrimers increases, the surface groups increase exponentially, generating increased surface area for

conjugation of drugs, imaging agents, and targeting moieties [288]. Compared with other nanocarrier drug delivery systems, dendrimer conjugated chemotherapeutics have been shown in blood circulation for a prolonged time with enhanced tumor accumulation and longer retention [7]. Poly-L-Lysine (PLL) dendrimers are biodegradable dendrimers. They are more desirable than other kinds of dendrimers due to enhanced enzymatic degradation and renal excretion of low molecular weight products from degradation [218].

There are two ways in which macromolecules, like dendrimers, can enter the cancer cells: the enhanced permeability and retention (EPR) effect [300] shown in figure 4.1 or by active targeting using receptor-mediated endocytosis (RME).

Without a targeting ligand, dendrimers use the EPR effect for entry into the tumor cells, but previous study showed persisting dendrimer conjugates accumulation in organs other than the tumor cells. Active targeting entails conjugating macromolecules to the drug and directly internalizing them into the cancer cells through receptor targeting, in addition to the EPR effect [8, 288]. Most living cells require vitamins to survive, especially cancer cells, which are rapidly dividing cells.

They need even more vitamins to proliferate; thus many vitamin receptors are over- expressed on the surface of tumor cells. Particularly, vitamin B12, folic acid, and riboflavin have been indicated to have higher uptake in tumor cells since they are essential vitamins vital for cell division and tumor growth [304]. Therefore, these essential vitamins have been used to actively target tumor cells with high affinity specificity for the corresponding receptors .

Receptor expressions and ligand selectivity differ between different cancer cells. In various breast cancer cells, riboflavin receptors had much higher over- expression in contrast to folic acid receptors [305]. One of the vitamin B groups,

riboflavin (RF) is a water-soluble vitamin (B2) and plays a key role in maintaining regulation of cellular growth and development during cellular metabolism, utilizing its cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) [306].

Past studies have shown that riboflavin receptors are over-expressed on the surface of breast cancer cells and demonstrated the RME of riboflavin. Moreover, cellular trafficking of riboflavin in human breast cancer cells (MCF-7) indicated increased internalization of riboflavin [7, 307].

As a result, to target chemotherapeutic drugs selectively and efficaciously, riboflavin-conjugated PEGylated PLL dendrimers will be assessed in this study.

Doxorubicin (DOX), which is a topoisomerase inhibitor used widely for breast cancer chemotherapy, will be a model drug attached to riboflavin conjugated PEGylated PLL dendrimers. In order to determine its efficacy, RF- and DOX-conjugated PEGylated

PLL dendrimers will be synthesized, characterized and investigated in-vitro, evaluating drug release in acidic pH conditions mimicking lysosome, cell cytotoxicity, and cellular internalization.

Figure 4.1 Scheme of EPR effect. This phenomenon involves large molecules like macromolecules entering into the tumor cells and accumulate due to "leaky" nature and lack of lymphatic excretion.

4.2 Materials and Methods

4.2.1 Materials:

Boc-Lys-ONp was obtained from ChemImpex (Wood Dale, IL) and Poly ethyl glycol (PEG) was obtained from PolySciences, Inc. (Warrington, PA). Cell culture supplies including Dulbecco’s Modified Eagle Medium (DMEM), Phosphate

Buffered Saline (PBS), Hanks' Balanced Salt Solution (HBSS) and Prolong Gold with

4', 6-diamino-2-phenylindole (DAPI) were purchased from Life Technologies (Grand

Island, NY).

4.2.2 Synthesis of PEGylatedDendrimers

Generation 3 (G3) PLL dendrimers were synthesized as described previously

(Boyd). Briefly, Poly-L-lysine dendrimers were synthesized by adding L-Lysine methyl ester with BOC-L-Lysine-ONP in anhydrous dimethylsulfoxide (DMSO) and triethylamine under argon at 75°C for 3h. When reaction was completed, the product was purified using liquid-liquid extraction in dichloromethane (DCM) then dried over magnesium sulfate. Final solid compound of Boc protected dendrimer was achieved by rotary evaporation. G1 PLL dendrimer was deprotected by adding

1:1 anhydrous DCM and trifluoroacidic acid (TFA) for 1hr. Then the solvents were removed to obtain G1 PLL dendrimers. These steps were repeated to achieve deprotected G2 PLL compound. During synthesis of G3 PLL dendrimer, BOC- and

Z-protected L-Lysine-ONP was used instead and 0.1 N sodium bicarbonate was used rather than liquid-liquid extraction. The product was filtered with acetonitrile and dried over vacuum for Z and Boc protected G3 Poly-L-lysine product.

After removing BOC protection with previously mentioned method, Poly ethyl glycol (PEG) 2000 Da was added with dimethylformamide (DMF) and DCM.

This mixture was sealed with argon and reacted overnight. When reaction was

complete, ethylenediamine was added to quench remaining starting material and acetic anhydride was added afterwards. Solvent products were filtered into diethyl ether, precipitated, and dried under high vacuum. After getting half-PEGylated PLL dendrimers, Z-groups were deprotected through hydrolysis and dried under high vacuum.

4.2.3 RF- and DOX-conjugated PEGylated PLL dendrimer synthesis

Riboflavin (RF) and 4-hydrazino benzoic acid (HSBA) functionalized

Doxorubicin (DOX) was conjugated to half-PEGylated PLL dendrimers through addition of EDC bond. Equal amounts of RF and DOX were mixed in 0.1M MES buffer and PEGylated PLL dendrimers were added to the reaction. 10mg of EDC was dissolved in the MES buffer and immediately 50uL was transferred to previous mixture. The reaction ran for 2hr and smaller molecular weight compounds were separated using dialysis with 10KDa cutoff. Final products were lyophilized to gain a solid compound of RF- and DOX-conjugated PEGylated G3 PLL dendrimers.

4.2.4 Characterization

All of the intermediate and final products were confirmed using Ion-trap

Mass spectrometry for molecular weight <1000Da and MALDI-TOF (BrukerAutoflex,

Billerica, MA) for molecular weight >1000Da. Size exclusion chromatography with

HPLC was used to measure differently conjugated dendrimer polydispersity with

0.05mM LiBr containing Dimethylformamide (DMF) mobile phase.

4.2.5 Cell Culture

MCF-7 cells, a human breast carcinoma cell line, were obtained from

American Type Culture Collection (Manassas, VA). Cells were grown at 37°C with

5% CO2 and 95% relative humidity. Cells were cultured in Dulbecco's modified

Eagle's medium (DMEM) with 10% fetal bovine serum, 1% nonessential amino acids,

100 unit/mL penicillin, and 10,000ug/ml streptomycin. Medium was changed every other day and cells were passaged with 0.25% trypsine at around 70-80% confluency.

4.2.6 Drug release study

DOX release from both targeted and non-targeted PEGylated PLL dendrimers upon different pH conditions was measured over 3 days in pH 7.4 to pH 4.0 HBSS.

Both RF-conjugated and non-targeted PEGylated PLL dendrimers were solubilised to

1mg/ml in HBSS with different pH and incubated over 1,3,6, 9, 12, 24, 36, 48 hr at

37°C. At each time points, 100uL of the media was withdrawn and suspended with

200ul of HBSS in Nanosep tube with 10K cutoff. Centrifugating at 5000g for 10min, released DOX was removed and analyzed in fluorescence spectrometer at ex: 471nm and em: 593 nm.

4.2.7 Cell cytotoxicity

RF- and DOX-conjugated PEGylated PLL dendrimer cytotoxicity was measured using Water-soluble tetrazolium salt (WST-1) cytotoxicity assay. Cells were first seeded with 50,000 cell density per each well in 96 well microplates and cells were grown over 48hr. The cell viability of MCF-7 cells were quantified by treating microplate (96wells) cultured cells with 0.1, 1, 10, 50, 100, 200, and 500µM of DOX equivalent concentration in RF-targeted PEGylated PLL dendrimers with

DOX, non-targeted PEGylated PLL dendrimers with DOX, and free DOX. After each treatment, the cells were incubated for 48 hr and washed using HBSS buffer.

Accordingly, 110µl of WST-1 reagent was administered to each well. Absorbance of the samples was measured using a microplate (ELISA) reader at 420-480 nm with reference wavelength of 600nm.

4.2.8 Cellular uptake

Uptake of different dendrimer conjugates and free DOX were determined in

MCF-7 cells. Cells were seeded at 50,000 cells/well and 30µM dendrimer conjugates and free DOX were treated for 4 different time points (3hr, 6hr, 9hr, 12hr).

Cells were washed with buffered HBSS after treatment and DOX fluorescence was measured at ex: 470 and em: 590 cutoff. Consequently, cells were lysed with 1M

NaOH followed by 1M HCl. A Bradford assay was utilized to measure the protein content for each treatment. DOX standard curves were also made to determine concentration of DOX. Uptake results from control DOX, non-targeted PEGylated

PLL dendrimers, and RF-targeted PEGylated PLL dendrimers are shown as the amount of dendrimer over the amount of protein content in each well ±SD.

4.2.9 Confocal microscopy

A comparative cellular uptake study of free DOX, non-targeted DOX conjugated PEGylated PLL dendrimers, and RF-targeted DOX-conjugated PEGylated

PLL dendrimers were performed in MCF-7 cells using confocal microscopy (CLMS).

Cells were seeded with 20,000 cell density per each well in 4-well chamber slides and cells were grown over 24 hr. After 24 hr incubation, each chamber was seeded with

30uM of free DOX, DOX-conjugated PEGylated PLL dendrimers, and RF- and DOX- conjugated PEGylated PLL dendrimers. Each treatment was incubated for 3 and 6 hr and wells were washed with HBSS. Then, cells were fixed with methanol and stained with DAPI and fluoromount. After curing slide for 24 hr, the slides were imaged under confocal microscopy at excitation of 485nm and emission of 520nm.

4.3 Results

4.3.1 Synthesis of PEGylated Dendrimers

DOX conjugated PEGylated PLL dendrimers and RF and DOX co- conjugated PEGylated PLL dendrimers were synthesized to determine the efficacy of targeted PEGylated PLL dendrimers compared to non-targeted PEGylated PLL dendrimers and free DOX. Successful synthesis of both DOX conjugated PEGylated

PLL dendrimers and RF- and DOX-conjugated PEGylated PLL dendrimers are described in figure 4.2.

The first step in the process of synthesizing RF- and DOX-conjugated PEGylated PLL dendrimers was to synthesize G3 PLL dendrimers. Synthesis of final G3 PLL dendrimers was confirmed with MALDI-TOF spectra (Fig 4.3), in which there was the molecular weight shift to 1954 Da. Additionally, reacting G3 PLL dendrimers with PEG 2000 prepared PEGylated dendrimers. HMBC NMR showed successful conjugation of PEG 2000 to amine groups in G3 PLL dendrimers by creating an amide bond. Polydispersity of each dendrimer compounds were determined using size exclusion chromatography attached to HPLC. Polydispersity of each conjugates were 1.04, 1.07, and 1.09 for PLL dendrimers, PEGylated PLL dendrimers, and RF- and DOX-conjugated PEGylated PLL dendrimers respectively (Table 4.1).

4.3.2 PH dependent release of DOX

As the pH decreased from pH 5 to pH 4, more DOX was released 85% to 95% at the end of 24 hr incubation. By 48 hr after incubation in both pH 4 and 5 conditions, both G3 PEGylated PLL dendrimers and RF- and DOX-conjugated

PEGylated PLL dendrimers had 100% release of DOX from the polymer. To compare its release in neutral pH condition, both compounds had only about ~15%

DOX release even after 48hr. The release profile of both non-targeted PLL

dendrimers and targeted PLL dendrimers showed similar release, suggesting that the release rate is not influenced by conjugation of targeting ligand.

Figure 4.2. Structures of half PEGylated G3 PLL dendrimers (A.) attached with functionalized RF (R*) and HSBA linked DOX. Dendrimers have 16 primary amines.

Synthesized Mw Mn Poly Dispersity Index

Compounds (PDI)

G3 PLL 2500.52 2404.35 1.04

Dendrimers

G3 PEGylated PLL 26441.76 24483.11 1.08

Dendrimer with

DOX

G3 PEGylated PLL 25648.85 23531.06 1.09

Dendrimer with RF

and DOX

Table 4.1 Synthesis of G3 PLL dendrimers, half G3 PEGylated PLL dendrimers and G3 PEGylated PLL dendrimers with DOX were characterized by size exclusion chromatography. Polydispersity index (PDI) was determined from size exclusion chromatography with HPLC.

Figure 4.3 Assessment of G3 PLL dendrimer with RF and DOX synthesis completion was analyzed by MALDI-TOF (a). Peak at m/z=19.3 kDa was present, which is close to the theoretical MW, which is at 22kDa. Structure of final compound was confirmed by HMBC spectroscopy (b), which detects correlation between nuclei of two different types and in this case, both 1H and 13C NMR.

A G 3 P E G y la te d P L L D M w / D O X

1 5 0 p H 5 p H 4

e s

a 1 0 0 p H 7 .4

e l e

X O

D 5 0

0 0 2 0 4 0 6 0 T im e (h r)

B G 3 P E G y la te d P L L D M w / R F a n d D O X 1 5 0 p H 4

e p H 5 s

e 1 0 0 l p H 7 .4 e R

X O 5 0 D

0 0 2 0 4 0 6 0 T im e (h r)

Figure 4.4 Proportion of DOX released from G3 PEGylated PLL dendrimers with DOX (A) and G3 PEGylated dendrimers with RF and DOX (B) in 50 mM HBSS (37 °C) at pH 4, pH5, and pH 7.4. Total DOX was determined by incubating at pH 4 for 48 hr. Values are represented as mean±s.d. (n=3).

4.3.3 Cytotoxicity

Cell cytotoxicity of DOX induced cell cytotoxicity was measured with free

DOX, PEGylated PLL dendrimers with DOX, and RF- and DOX-conjugated

PEGylated PLL dendrimers using Water-soluble tetrazolium (WST)-1 assay (Fig 4.5).

For all three groups, high cytotoxicity was exhibited but free DOX exhibited highest cytotoxicity compare to targeted PEGylated PLL dendrimers. This might be due to different endocytosis pathway that dendrimer driven DOX enters the cell compare to

DOX itself where dendrimer, goes through endosomes and lysosomal release in order to get into the cells[308]. Non-targeted PEGylated PLL dendrimers showed least cytotoxic effect in MCF-7 cells.

4.3.4 Cellular trafficking using immunofluorescence

In order to determine colocalization of DOX in each compound, cells with treatment of free DOX and DOX-conjugated PEGylated PLL dendrimers were visualized with DAPI fluorescence for nuclear compartment and the inherent fluorescence of DOX over 3 and 6 hr periods. Transmitted light was also used to outline the boundaries of nuclei. After 3 hr oftreatment, free DOX was accumulated throughout the entire cell while DOX-conjugated with G3 PEGylated PLL dendrimers were primarily accumulated specifically inside nuclei and cytoplasm. Compared to non-targeted G3 PEGylated PLL dendrimers, RF-targeted PEGylated PLL dendrimerswere more accumulated inside the cells and more specifically colocalized near the nucleus. Also, there were some uneven agglomerates formed for non- targeted PEGylated PLL dendrimers while RF-targeted PEGylated PLL dendrimers were evenly dispersed DOX fluorescence present throughout cells.

MCF-7 Cytotoxicity with Treatments

150 G3 PEGylated PLL DM RF-DOX G3 PEGylated PLL DM DOX y t i l i 100 Free DOX b a i v

l l e c 50 %

0 -3 -2 -1 0 1 2 3 Log concentration (µM)

Figure 4.5 MCF-7 cell viability after 48hr incubation with RF- and DOX-conjugated G3 PEGylated PLL dendrimers, G3 PEGylated PLL dendrimers with DOX, and free DOX. Mean ± standard deviation (SD) (n=3).

Figure 4.6 Confocal laser scanning microscopy images of MCF-7 cells after 3hr incubation with DOX (A-D), G3 PEGylated PLL dendrimers conjugated with DOX (E-H), and G3 PEGylated PLL dendrimers with RF and DOX (I-L). The scale bars correspond to 25 µm in all images. Blue=nucleus, red=DOX.

4.3.5 Cellular Uptake

In order to determine the amount of intracellular accumulation of DOX in the cells, Free DOX, non-targeted PEGylated PLL dendrimers with DOX-conjugated and

RF-targeted PEGylated PLL dendrimers with DOX were treated for uptake measurement for 3, 6, 9, 12 hr treatment with 30μM equivalent concentration. The final results showed that RF-targeted and non-targeted PEGylated PLL dendrimers had the highest accumulation inside the cell compare to free DOX in first 3hr after treatment. In the first 3 hr, targeted PEGylated PLL dendrimers had the highest uptake out of all three treatments with 147.39 µmol/mg of protein. The difference in initial uptake between RF-targeted PEGylated PLL dendrimers and non-targeted

PEGylated PLL dendrimers show that targeted PEGylated PLL dendrimers were accumulated faster due to active endocytosis through RF-mediated endocytosis.

However, after 6 hr treatment, significantly higher uptake was evident in free DOX with 345-μmol/mg-protein compare to dendrimer with DOX conjugates. Also, while free DOX diffused through the cells continuously with constant increase in uptake, both targeted and non-targeted PEGylated PLL dendrimers with DOX show uptake level off after 9 hr treatment. This is possibly due to slower release from early endosome to lysosome from early endosome sorting [309] that can create accumulation of DOX-conjugated compounds so less DOX conjugated dendrimers can enter the cell. Uptake values for dendrimer conjugates were compared by one-way

ANOVA followed by Tukey's posttest for significant variation.

D O X U p ta k e

) 1 0 0 0 n i e t

o 8 0 0

r p

g 6 0 0

m / l

m 4 0 0 (

k 2 0 0 a t

U 0 0 5 1 0 1 5 T im e (h r)

D O X

G 3 P E G y la te d P L L D M -D O X

G 3 P E G y la te d P L L D M R F -D O X

Figure 4.7. Uptake of free DOX, G3 PEGylated PLL dendrimers with DOX, and RF- targeted PEGylated PLL dendrimers with DOX in MCF-7 after 3, 6, 9, and 12hr treatment. Results are shown as the mean ± SD (n=3).

4.4 Discussion

Dendrimers have been investigated many times as a novel drug delivery system to deliver chemotherapeutic drugs to solid tumors more effectively than the classical chemotherapeutic drug [310]. With drug conjugation to dendrimers, development of drug resistance from drug leakage can be prevented and allow selective tumor targeting through the EPR effect, characterized by leaky vasculature in solid tumors [311]. Particularly, in-vivo studies using lysine-based dendrimers have shown improved pharmacokinetic properties due to their biodegradability [9].

However, in-vivo studies have shown that even with PLL dendrimers, eliminating chemotherapeutic drugs from offsite targets wasinevitable [9]. Also, not all of the tumor tissues have EPR effect properties, which can result in macromolecules being unable to endocytose into the cell. Therefore, utilizing PLL dendrimers with a ligand that binds to the receptor over-expressed on the surface of the tumor tissues allows more specific delivery of chemotherapeutic drug at tumor site. Many vitamin receptors have shown to be over-expressed on the membrane of certain cancer cells.Studies have investigated these receptors for clinical use in chemotherapy [304].

In this study, a novel targeting drugdelivery system using RF and biodegradable dendrimers was used to optimize specific delivery of a chemotherapeutic drug to breast cancer tissue, where the RF receptor is highly expressed on the surface of the breast cancer cellmembrane [6, 312, 313]. Particularly, the MCF-7 cell line, which is a human breast cancer cell line, was shown to internalize RF-conjugates in a high affinity manner compared to SKBR-3, which is also a breast cancer cell line [305,

314]. Therefore, MCF-7 cell can be used to analyze macromolecule conjugates with

RF for enhanced targeting drug delivery.

A biodegradable and biocompatible PLL dendrimer was used as a drug 82

delivery vehicle so that a chemotherapeutic drug could be delivered to tumor sites efficaciously. Clinically, there is a great advantage if a drug conjugate is biodegradable because the product can be degraded and byproducts can be excreted from the body through metabolic pathway [4, 315]. Also, a macromolecule conjugated drug needs to be able to sustain during blood circulation without releasing the drug from the carrier. Since solid tumor cells have acidic extracellular pH due to anaerobic metabolism, attaching a chemotherapeutic drug to an acid labile linker provides a way to specifically release the drug inside tumor cells. In this study, RF was stably conjugated to the PLL dendrimer with a stable linker known as the EDC linker so that the ligand did not fall apart from the carrier. A chemotherapeutic drug,

DOX, was attached toan acid-labile linker, HSBA linker, so it could be only released at a low pH environment such as lysosome and early endosome in the cell.

In order to investigate the specific release of DOX in an acidic environment, both the RF-conjugated dendrimer and the non-targeted dendrimer were examined in various pH environments from 4.0-7.0 (Fig 4.4). There was a significant difference in therelease profile of DOX from 1 hr to 48 hr incubation. Results showed that both

RF targeted and non-targeted dendrimers had a significant increase in fluorescence from DOX. Especially at pH 4, most of DOX was released at 12 hr after incubation compare to physiological pH, which hardly any DOX were released. In the physiological condition, with lysosomal enzyme cathepsin B and esterases present inside the cells, cleavage of DOX from dendrimers will be much faster at acidic lysosomal pH. A similar release profile was seen in the intracellular trafficking of both non-targeted and RF-targeted PEGylated PLL dendrimers with DOX.

Particularly, DOX that was attached to RF-targeted PEGylated PLL dendrimers was not quickly released intothe nucleus but maintained attachment to conjugates

throughout the endocytosis pathway until it was released into the nucleus. Therefore, results fromthe pH dependent drug release study and in-vitro cellular trafficking of

RF-attached PEGylated PLL dendrimers show that attachment of DOX with an acid labile linker and RF ligand supports the controlled release of DOX from the dendrimer conjugate.

To further investigate RF-targeted PEGylated PLL dendrimer efficacy, in- vitro analysis of the drug conjugate was studied through cellular experiments using

MCF-7 cells, as mentioned previously. DOX was incorporated in the dendrimer conjugate as a chemotherapeutic drug as a proof-of-concept drug because it is a widely used topoisomerase inhibitor administered for chemotherapy. Although it is a consistently utilized chemotherapeutic drug for breast cancer, DOX has a major side effect, in which DOX diffuses through healthy cells, causing heart failure and typhilitis. Unlike this low molecular weight drug, macromolecule assisted chemotherapeutic drugs enter the cell through macromolecule endocytosis internalization using the EPR effect, eliminating pinocytosis and rapid diffusion.

Compared to non-targeted macromolecules, RF receptor-targeting macromolecules enter RF receptor over-expressed breast cancer cells with higher sensitivity and specificity through receptor-mediated endocytosis with high affinity substrate binding.

A cellular uptake study of free DOX, non-targeted PEGylated PLL dendrimers with

DOX, and RF-targeted PEGylated PLL dendrimers with DOX was conducted to measureand compare cellular accumulation of DOX after 3 and 6 hr of each treatment.

At 3 hr after treatment, RF-targeted PEGylated PLL dendrimers showed enhanced accumulation of DOX inside the breast cancer cell compared to both free DOX and non-targeted PEGylated PLL dendrimers. Non-targeted PEGylated PLL dendrimers also exhibited a higher accumulation of DOX compared to free DOX, but

RF-targeted PEGylated PLL dendrimers showed the highest accumulation (Fig 4.7).

This might be due to theactive internalization of RF-conjugated PEGylated

PLL dendrimers. The RF ligand specifically binds to the RF receptor and endocytosed into the cell, rather than simple passive diffusion or non-targeted endocytosis. Therefore, even though the endocytosis process is slower than diffusion, high-specificity binding of RF-conjugated dendrimers leads to faster internalization. Similar trend was shown with different macromolecules-attached chemotherapeutic drugs using different cell lines.

As thetreatment time extended, endosomal acidification separates ligand- receptor binding for receptor recycling inside the cell. The cytosolic build-up of ligand-receptor complexes causes slower availability of recycled receptors on the surface of the cancer tissue [6]. This occurrence was prominent after 6 hr treatment of RF-targeted PEGylated PLL dendrimers, in which less accumulation RF-targeted compounds were present in comparison to free DOX and non-targeted PEGylated

PLL dendrimers.

To look more closely at the intracellular trafficking of DOX-conjugated

PEGylated PLL dendrimer compounds, confocal microscopy was implemented for cellular trafficking study (Fig 4.6) and quantified for DOX residence within thenuclear compartment. Both non-targeted and RF-targeted PEGylated PLL dendrimers showed similar trafficking results, in which both compounds seem to be within endolysosomal compartments within 3hr after treatment while free DOX diffused quickly throughout the nucleus. This confirms that both RF-targeted

PEGylated PLL dendrimers and non-targeted PEGylated PLL dendrimers enter the breast cancer cells through macromolecule endocytosis pathway, so this process takes longer period of time for the drug to reach the nucleus. Both PEGylated PLL

8 5

dendrimer conjugates were found to have more nuclear colocalization from DOX release through lysosomal cleavage after 6 hr treatement. However, RF-attached

PEGylated PLL dendrimers showed more DOX colocalization inside and on the surface of the cell nucleus by the end of 6hr treatment compare to non-targeted

PEGylated PLL dendrimers. Further study is needed to specifically look at endosomal and lysosomal colocalization of non-targeted and RF-targeted PEGylated

PLL dendrimers in order to confirm a precise colocalization of DOX at different time points.

Investigation of non-targeted PEGylated PLL dendrimers for its efficacy showed similar cytotoxicity results compared to previous cytotoxicity studies using different macromolecules [316]. Notably, RF-conjugated PEGylated PLL dendrimers showed similar cytotoxicity compared to free DOX, suggesting similar efficacy of the drug conjugates even though RF and dendrimers are attached.

However, free DOX showed lowest cell viability compared to dendrimer-conjugated

DOX but it is important to understand that in-vivo, free DOX will show cytotoxicity to off-target tissues as well. Therefore, RF-targeted PEGylated PLL dendrimers with

DOX seems to an optimal compound for breast cancer tissues with high potency as well as eliminating cytotoxicity to off-target tissues.

4.5 Conclusion

Biodegradable drug delivery systems have shown potential to deliver chemotherapeutic drugs to target thetumor sites with less toxicity and more biocompatibility. Specifically, this study demonstrated the potential of ligand- targeted biodegradable dendrimers as a tumor-targeting delivery system using DOX.

Utility of RF as a targeting agent along with PEGylated PLL dendrimer has not only enhanced specific internalization of the dendrimer-DOX conjugate but also created an 86

effective and biocompatible therapy. In future studies, further investigations on endolysosomal internalization and intracellular endocytosis pathway including RF- specific recognition will be elucidated. Also, to take the study into thenext step, we would examine in-vivo pharmacokinetics and biodistribution of RF-targeted PLL dendrimer in order to see its biodegradability, efficacy, and distribution in different organs of an animal model.

Chapter 5 Intracellular Trafficking of Riboflavin-Conjugated PEGylated Poly-L-Lysine Dendrimers

5.1 Introduction

Dendrimers are tree-like nanosized macromolecules created from branches of polymers [295]. Although they are large molecules, dendrimers possess properties that distinguish themselves from other macromolecules. These properties includeenhanced solubility, monodispersity, and controlled synthesis [317]. With these unique physicochemical properties, dendrimers have been utilized for drug delivery systemwith various compounds such as DNA, chemotherapeutic drugs, anddetecting agents [318]. Specifically, many studies have focused on examining dendrimer use in delivering chemotherapeutic drugs to exact tumor sites in-vitro and in-vivo [319-321]. These studies have shown chemotherapeutic drug delivery with dendrimers has increased drug bioavailability, enhanced anti-tumor efficacy, and improved drug biodistribution. Although use of dendrimers as drug delivery systeme liminated off-target biodistribution compared to a free chemotherapeutic drug, there was still high accumulation of the drug in the liver and kidney. A possible reason for higher accumulation is that large non-degradable dendrimers, such as

PAMAM dendrimers, could not go through urinary elimination [322]. Therefore, a biodegradable dendrimer, polylysine dendrimer, was implemented to conduct an in- vivo chemotherapeutic biodistribution study. Use of chemotherapeutic drugs with biodegradable dendrimers reduced accumulation in the liver and kidneys; however, there was still significant amount of the drug delivered to these organs [4, 9].

Receptor-specific targeting dendrimers have been considered to bypass non-

specific accumulation by entering cells through receptor-mediated endocytosis pathway. For example, a folate receptor, which is a vitamin receptor over-expressed in ovarian and breast cancer cells, that, when conjugated to PAMAM dendrimer and methotrexate, exhibited higher efficacy and enhanced cellular binding and uptake[323]. Also, receptor-targeted chemotherapeutic studies have been conductedusing macromolecules besides dendrimers, such as riboflavin (RF)-targeted

HPMA with MMC [305]. These studies showed enhanced efficacy as well as drug accumulation inside specific cancer cells compare to chemotherapeutic drugs alone.

Even though receptor conjugated dendrimers were evaluated for their efficacy, mechanistic analysis of intracellular uptake and trafficking in the cells has yet to be elucidated. Therefore, in this study, we explored the mechanisms behind internalization and colocalization of RF ligand attached PEGylated PLL dendrimer by investigating influence of endocytosis inhibitors on conjugate internalization as well as looking at specific colocalization in lysosome and endosome as shown in fig 5.1.

Doxorubicin (DOX), which is a widely used topoisomerase inhibitor, was used as a model chemotherapeutic drug and conjugated to the dendrimer complex. Because

DOX emits fluorescence at 470nm and 590nm, uptake and colocalization was measured with fluorescence from doxorubicin. We compared transport, the endocytosis mechanism, and cellular trafficking of DOX-conjugated RF-targeted

PEGylated PLL dendrimers and non-targeted PEGylated PLL dendrimers with free

DOX. These studies willprovide more information about how RF-targeted

PEGylated PLL dendrimers enters the cell compare to small molecular chemotherapeutic drug while maintaining advantageous properties of targeted biodegradable dendrimers.

Figure 5.1

Routes of clathrin- and caveolin-mediated endocytosis as well as macropinocytosis.

Inhibitors for each endocytosis and non-specific macropinocytosis pathways are indicated.

5.2 Materials/Methods

5.2.1 Materials

Boc-Lys-ONP was obtained from ChemImpex (Wood Dale, IL) and Poly ethyl glycol (PEG) was obtained from PolySciences, Inc. (Warrington, PA). All cell culture supplies, including Dulbecco’s Modified Eagle Medium (DMEM),

Phosphate Buffered Saline (PBS), Hanks' Balanced Salt Solution (HBSS) and Prolong

Gold with 4', 6-diamino-2-phenylindole (DAPI) were purchased from Life

Technologies (Grand Island, NY). Early endosome antigen antibody (EEA-1) and lysosome-associated membrane protein antibody (LAMP-1) were obtained from Cell

Signaling Technologies (Danvers, MA). Endocytosis inhibitors and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

5.2.2 Synthesis of RF Dendrimer Conjugates

PLL dendrimers were synthesized in a same manner as described in Chapter 3.

To describe briefly, L-lysine methyl ester was reacted with Boc-protected L-lysine conjugated p-nitrophenol activated compound in dimethyl sulfoxide (DMSO) with distilled (TEA) at 75° C. After completion of reaction was confirmed, reacted compounds were extracted in dichloromethane (DCM) with weak base washes including ammonium chloride, sodium bisulfate, and brine one after the other. Final product was dried with magnesium sulfate and rotary vacuum. In order to deprotect

Boc protection, anhydrous DCM and trifluoroacetic acid (TFA) were added 1:1 ratio and solvents were evaporated using high-vacuum. By repeatedly adding Boc- protected L-lysine functionalized p-nitrophenol and deprotecting the Boc groups, all dendrimers were synthesized to generation 3 of PLL dendrimers. Then, surface amine groups were reacted with Z and Boc protected L-Lysine-p-nitrophenol compound. The reacted product was filtered with acetonitrile and dried over high-

vacuum. Boc-protection was first removed using DCM and TFA with 1:1 ratio and after drying them into solid products, poly ethyl glycol (PEG) 2000 was added to available amine-terminated groups. The product was precipitated with diethyl ether and dried under high vacuum. Z-protected groups were removed using hydrolysis with palladium and hydrolyzer and final product was dried under high vacuum again after removing excess palladium.

For non-targeted PEGylated PLL dendrimers, 4-hydrazino benzoic acid

(HSBA) functionalized DOX were added in DMF, diisopropylethylamine, followed by benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP).

The reaction ran overnight and high molecular weight was pooled with dialysis bag.

For conjugating both RF and DOX to PEGylated PLL dendrimers, RF and HSBA functionalized DOX were added into half-PEGylated PLL dendrimers dissolved in

0.1M MES buffer adjusted to pH 5. Quickly, 10mg of EDC dissolved in MES buffer was added to combine PEGylated PLL dendrimer solution and the reaction ran for 2 hr. Unreacted small molecules were separated utilizing dialysis bag with 10kDa cutoff. Solid product was achieved by evaporating 0.1M MES buffer using lyophilizer. All synthesized final products were stored in -20°C.

5.2.3 Characterization

In order to confirm successful synthesis of non-targeted and RF-targeted dendrimer conjugates, MALDI-TOF (Bruker Autoflex, Billerica, MA) as well as Ion- trap (Agilent Technologies, Santa Barbara, CA) was used for all intermediates and final products. The DOX content in each conjugates was measured by fluorescence standard curve using a Gemini XPS fluorescence microplate reader (Molecular

Devices) at ex=490nm and em=590nm. Drug loading (DL%) in PEGylated dendrimer was determined by weight ratio of DOX conjugated to PEGylated PLL 92

dendrimers and released DOX with PEGylated PLL dendrimers. Size Exclusion

Chromatography with a DMF/LiBr 0.2% mobile phase was used to investigate polydispersity of dendrimer conjugates and reaasure elimination of any small molecules or impurities. The hydrodynamic radius of each synthesized dendrimer conjugates were measured using a Malvern (Westborough, MA) Zetasizer Nano ZS.

Dendrimer batches were dissolved in Hank's Balanced Salt Solution (HBSS) at concentration of 1mg/mL and measured in triplicate. Measurements were achieved with a refractive index of 1.33 and viscosity of 0.88 at 25◦C.

5.2.4 Cell culture

MCF-7 cells, a human breast cancer cell line, were obtained from the

American Type Culture Collection (Manssas, VA). MCF-7 cells were grown at 37°

80-90% confluency.

5.2.5 Cellular Colocolization at Early Endosome and Lysosome

RF-targeted and non-targeted PEGylated PLL dendrimer colocalization in macromolecule endocytosis was measured in MCF-7 cells. MCF-7 cells were seeded in 4-chamber microscope slides at 20,000 per well and incubated for cells to adhere for 24 hr. After incubation, cells were washed two times with HBSS and treatments consisting of 30μM non-targeted PEGylated PLL dendrimersand RF- targeted PEGylated PLL dendrimers were added. DOX was used as a comparison.

After 1 and 2 hr incubation with treatment, cells were washed twice with ice-cold 93

HBSS and cells were fixed with ice-cold methanol at -20°C for 15min. Fixed cells were washed with Dulbecco's phosphate-buffered saline (DPBS) for three times and permeabilized and blocked with 0.2% (v/v) Triton X-100 along with 3% (w/v) bovine serum albumin (BSA) dissolved in DBS for 1 hr. Permeabilized cells were incubated with primary antibodies in blocking solution which consist of 2μg/mL rabbit anti-EEA1 or rabbit anti-LAMP1 for 1 hr at 37°C. Then, cells were washed again with DPBS three times and stained with secondary antibodies (1:400 Cy5 goat anti-rabbit IgG) in blocking solution for 1hr at room temperature followed by DBPS washes. Consequently, cells were excised and mounted on glass slides with Prolong

Gold containing DAPI stain. The mounting medium was cured for 24 hr at room temperature and slides were sealed and stored at 4°C.

Images were obtained using confocal microscope and DAPI, DOX, Cy5 were excited with 404, 561, 641nm lasers. 450/50, 525/50, and 595/50 filter blocks were used. Three images were analyzed for each treatments using the following parameters:

Plan apo VC 20x objective, 18.2µM pinhole, 4.6 µs pixel dwell, 2x line average, 4x optical zoom, and 512x512 image size.

5.2.6 Cellular Uptake

Uptake of different dendrimer conjugates and free DOX were determined in

MCF-7 cells. Cells were seeded at 50,000 cells/well and pretreated with inhibitors, which consisted of 300µM monodansyl cadaverine (MDC), 1µM phenylarsine oxide

(PAO), 4µM filipin (FIL), 50µM genistein (GEN), 50µM dynasore (DYN), or 0.1µM wortmannin (WORT) for 1hr. Then, mixture of dendrimer conjugates and free DOX with inhibitors were treated for another 1hr. Cells were washed with buffered HBSS after treatment and DOX fluorescence was measured at ex: 470 and em: 590 cutoff.

Consequently, cells were lysed with 1M NaOH followed by 1M HCl. A Bradford assay was utilized to measure the protein content for each treatment. DOX standard curves were also made to determine concentration of DOX. Cytotoxicity of inhibitors showed >87% cell viability at the specific concentrations utilized for uptake experiments. Uptake results from control DOX, non-targeted PEGylated PLL dendrimers, and RF-targeted PEGylated PLL dendrimers are shown as the amount of dendrimer over the amount of protein content in each well ±SD. Statistically significant decreases between dendrimer conjugate uptake and uptake with inhibitor treatments were analyzed by two-way ANOVA with Bonferroni’s posttest.

5.3 Results

5.3.1 Synthesis of RF- and DOX- conjugated PEGylated PLL dendrimers

Both RF targeted PEGylated PLL dendrimers and PEGylated PLL dendrimerswith DOX were synthesized by two different methods and DOX was conjugated with an acid-labile linker, HSBA, while RF was stably linked with succinic anhydride in order to create primary carboxylic acid group for amide bond formation. Synthesized conjugates were used to determine cellular trafficking and intracellular uptake of RF-targeted and non-targeted PEGylated PLL dendrimers in order to investigate intracellular mechanism behind two differently targeted attachments of PLL dendrimers to DOX compare to DOX alone.

5.3.2 Characterization of RF targeted and non-targeted PEGylated PLL dendrimers

with DOX

Ion-trap was utilized to confirm functionalization of carboxylic linker to RF and HSBA linker conjugation to DOX. Ion-trap was used for both functionalized RF and DOX because they possess molecular weight smaller than 1000 Da. MALDI- 95

TOF was utilized to confirm molecular weights of G3 PLL DM, PEGylated PLL dendrimers, and both RF- and DOX-conjugated PEGylated PLL dendrimers due to their higher molecular weights. The drug load for each compound was determined using fluorescence spectroscopy. The drug conjugation efficiency was about 27.5% wt/wt showing approximately 6 drug molecules attached to each dendrimers. Size exclusion chromatography analysis showed polydispersity of dendrimer conjugates polydispersity index (PDI) of half-PEGylated dendrimers showed 1.05 while DOX- conjugated PEGylated PLL dendrimers were determined to be 1.08 and RF-targeted

PEGylated PLL dendrimers with 1.09. Hydrodynamic radiuses of PLL dendrimer conjugates were analyzed using dynamic light scattering (DLS) and shown in table

5.1. As DOX and RF was conjugated to PEGylated PLL dendrimers, radius increased from 7.97±1.23 to 8.64±1.97 for RF- and DOX- conjugated PEGylated

PLL dendrimers and 9.88±2.02 for only DOX-conjugated PEGylated PLL dendrimers.

Synthesized Mw Poly Dispersity Index Size (nm)

Compounds (PDI)

G3 PLL Dendrimers 2500.52 1.04 2.04±0.05

G3 PEGylated PLL 26441.76 1.08 9.88±2.02

Dendrimer with DOX

G3 PEGylated PLL 25648.85 1.09 8.64±1.97

Dendrimer with RF

and DOX

Table 5.1 Polydispersity index and size of G3 PLL dendrimers, G3 PEGylated PLL dendrimers with DOX and G3 PEGylated PLL dendrimers with RF and DOX using size exclusion chromatography and Zetasizer.

5.3.3 Cellular Colocolization at Early Endosome and Lysosome

In order to achieve a clearer picture of endolysosomal internalization of RF- and DOX-conjugated PEGylated PLL dendrimers, intracellular trafficking of dendrimers was analyzed using specific cellular compartment antibodies. After staining cells with either early endosome antibody (EEA-1) for 1hr or lysosome

(LAMP-1) antibody for 2hr, both RF-targeted PEGylated PLL dendrimers and non- targeted PEGylated PLL dendrimers specifically colocalized in early endosome and lysosome(Fig 5.2). Free DOX showed rapid uptake into the cells' nucleus compared to PEGylated PLL dendrimer conjugates. As incubation time increased, both RF targeted PEGylated PLL dendrimers and non-targeted PEGylated PLL dendrimers showed change in colocalization, in which conjugates moved from the early endosome to thelysosome. RF-targeted PEGylated PLL dendrimers with DOX showed higher colocalization in the lysosome compared to non-targeted PEGylated

PLL dendrimers while early endosome colocalization for both compounds were displayed in a similar quantity. After 2 hr of incubation, some of the non-targeted

PEGylated PLL dendrimers and RF-targeted PEGylated PLL dendrimers exhibited nucleus colocalization as DOX is quickly released from lysosome whenHSBA linker cleaves off from DOX. These results shows that RF-conjugated and non-targeted

PEGylated PLL DM follows macromolecule endocytosis pathway [324] and conjugates move slower from early endosome to lysosome compare to DOX release from lysosome into the nucleus.

Figure 5.2 Visualization of DOX, G3PEGylated PLL dendrimers with DOX, and G3 PEGylated PLL dendrimers with RF and DOX trafficking over time in MCF-7 cells by confocal microscopy. Images are representative of the 1hr and 2hr time points for the early endosome (eea-1 antibody) and lysosome (lamp-1 antibody) respectively used to calculate colocalization. Blue=nucleus, red=DOX, purple=endosome/lysosome.

Figure 5.3 Uptake of free DOX, G3 PEGylated PLL dendrimers with DOX, and RF-targeted, DOX-conjugated PEGylated PLL dendrimers in MCF-7 cells with endocytosis inhibitors. Results are shown as the mean ± SD (n=3).*, **, and *** indicate a statistically significant decrease in uptake compared to dendrimer conjugates/free DOX uptake without any inhibitors with p<0.05, p<0.01, and p<0.001.

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5.3.4 Intracellular Uptake

Cellular uptake of RF-targeted PEGylated PLL dendrimers and non-targeted

PEGylated PLL dendrimers with DOX were investigated using inhibitors that blocks clatherin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis as well as macropinocytosis. Free DOX was used in comparison to analyze different uptake mechanism between small molecule and macromolecule. MDC and PAO were utilized to inhibit clatherin-mediated endocytosis. MDC increases clatherin polymerization making stabilized pits disabled from forming vesicles [325, 326].

PAO is known to inhibit clatherin-coated pit internalization as well by causing a depletion of DAG, a small signaling lipid that affects vesicle scission [327]. There were some MDC and PAO inhibition seen in non-targeted PEGylated PLL dendrimers but more inhibition was displayed with RF PEGylated PLL dendrimers (Fig 5.3).

This shows that non-PEGylated PLL dendrimers might be partially inhibited by MDC and PAO but RF-targeted PEGylated PLL dendrimers utilize clathrin-coated pit endocytosis. Caveaolin-mediated endocytosis was investigated using FIL and GEN inhibitors. FIL inhibits caveolin-mediated endocytosis by binding with cholesterol in non-coated membrane invaginations carrying abundance of cholesterol and interrupt vesicle formation [328]. GEN disrupts caveolin-mediated endocytosis through interfering with tyrosine kinases needed for actin cytoskeletal reorganization and bringing dynamin 2, which is a crucial part in caveolae-mediated endocytosis

[329]. Both FIL and GEN inhibited RF-targeted and non-targeted PEGylated PLL dendrimers but more non-targeted PEGylated PLL dendrimers were inhibited compare to RF-targeted PEGylated PLL dendrimers. DOX only showed negligible amount of inhibition after FIL and GEN administration, suggesting no involvement in caveolin-mediated endocytosis. This shows that non-targeted PEGylated PLL

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dendrimers utilize caveolae-mediated endocytosis for internalization pathway while

RF-targeted PEGylated PLL dendrimers partially use this method of internalization.

We also studied inhibitors thatimpede all macromolecule endocytosis mechanisms and macropinocytosis inhibitor. We first investigated administration of

DYN, a dynamin inhibitor [330], on affecting both endocytosis mechanisms.

Dynamin 2 is a GTPase that supports vesicle fission in both coat-depenedent and coat- independent internalization [331]. Blocking dynamin 2 resulted a significant uptake decrease in both RF-targeted and non-targeted PEGylated PLL dendrimers, indicating that both endocytosis mechanisms are involved in RF-targeted and non-targeted

PEGylated PLL dendrimers. WORT was treated for disrupting macropinocytosis by preventing phosphoinositide 3-kinase, a protein involved in shutting down macropinosomes. Both dendrimer conjugates as well as free DOX uptake was decreased significantly by WORT treatment. This indicates that macropinocytosis is also involved in the internalization pathway in addition to clathrin- and caveolin- mediated endocytosis.

5.4 Discussions

Dendrimers have been shown to deliver chemotherapeutic drugs more effectively than the drug alone [332]. In order to understand internalization and trafficking of chemotherapeutic drug attached dendrimers, PLL dendrimer uptake and intracellular trafficking has been previouslystudied [308]. However, the uptakemechanism and intracellular traffickingof PEGylated PLL dendrimers with chemotherapeutic drug has not been explored. Colocalization of high molecular weight PEGylated dendrimers can have significant effects in drug toxicity and release

[4, 333]. Thus, investigating the mechanism behind entry of PEGylated dendrimers with chemotherapeutic drugs is crucial. The current study has explored the uptake 102

and internalization of doxorubicin-conjugated PEGylated PLL dendrimers as well as

RF-targeted PEGylated PLL dendrimers in order to elucidate an endocytosis pathway of PEGylated dendrimer when attached to a chemotherapeutic drug. We compared both dendrimer-drug conjugates to free DOX to clarify difference in the internalization mechanisms between large macromolecules and small molecule drugs.

DOX is a topoisomerase inhibitor, used as a chemotherapeutic drug to treat cancers including breast cancer. Because DOX is fluorescent with ex=470nm and em=590nm, it was an ideal drug candidate to be used for studying internalization of

PEGylated PLL dendrimers with chemotherapeutic drugs. The RF receptor, which is over-expressed in breast cancer cells, was investigated using riboflavin ligand attached to the PEGylated PLL dendrimers in order to see how dendrimer internalization is also affected by a ligand attachment. Synthesis of both non- targeted PEGylated PLL dendrimers and RF-targeted PEGylated PLL dendrimers were confirmed successfully using mass spectrometry and a zetasizer. A consistent trend of significant increase in molecular weight after addition of PEG groups coincided with anincrease in the hydrodynamic radius. This indicates major enlargement in the overall size of the dendrimers, which is an important property for enhanced retention in blood circulation [334].

After successful synthesis of dendrimer conjugates, we investigated intracellular trafficking of both conjugates to elucidate which compartmental organelles dendrimer conjugates bypass during internalization. A previous study in

Chapter 4 showed different colocalization of non-targeted PEGylated PLL dendrimers and RF-targeted PEGylated PLL dendrimers, in which both conjugates were mostly within a different compartment other than the nucleus. Because DOX is conjugated to an acid-labile linker, HSBA, determination of colocalization in early endosome to

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lysosome is crucial in order for drug release to occurin the lysosome [9, 335].

PAMAM dendrimers with different chemotherapeutic drugs have been shown to colocalize in the nucleus [336]; however, the trafficking of ligand and chemotherapeutic drugs attachedto PEGylated PLL dendrimers have to be more closely investigated. G3 PEGylated PLL dendrimers with DOX and RF-targeted

PEGylated PLL dendrimers with DOX showed colocalization in the early endosome in first hour followed by rapid localization in lysosome. Both dendrimer conjugates showed slower localization in the early endosome, indicated by less fluorescence emitted by DOX, while higher colocalization inthe lysosome occurred with more fluorescence present from the dendrimer and DOX complex.

In order to further determine how internalization mechanisms are impacted by

RF ligand and drug delivery vehicle (PLL dendrimers), we studied uptake of dendrimer conjugates with inhibitors of various endocytosis mechanisms. The results showed that both dendrimer conjugates internalized through specific (clathrin- or caveolin-mediated) and nonspecific mechanisms. RF-targeted PEGylated PLL dendrimers hadthe most impact from inhibitors of a clathrin-mediated endocytosis pathway while non-targeted PEGylated PLL dendrimers were most influenced by inhibitors of acaveolin-mediated endocytosis pathway. The fact that RF-targeted

PEGylated PLL dendrimers are most hindered by inhibitors of the clathrin-mediated endocytosis coincides with a previous finding of RF receptor-mediated endocytosis where RF is endocytosed into the cell through clathrin-coated pits [6]. Also, free

DOX did not show much inhibition in uptake with both inhibitors of clathrin- and caveolin-mediated endocytosis, suggesting internalization of free DOX occurs through a different mechanism. Aside from specific inhibitors, use of a dynamin- mediated endocytosis inhibitor exhibited both dendrimer conjugates to have

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significant reduction in uptake, indicating caveolae and lipid rafts play arole in PLL dendrimer conjugate uptake [313]. In thepresence of WORT, both dendrimer conjugates as well as free-DOX had reduction in cell internalization, which shows that targeted and non-targeted PLL dendrimers go through multiple endocytosis pathways, including a non-specific endocytosis pathway.

Results from this study show similar yet different internalization mechanisms that are involved in both RF-targeted PEGylated PLL dendrimers and non-targeted

PEGylated PLL dendrimers. Having RF ligand attached to PLL dendrimer has changed the rate of entering different compartments in the cell, resulting higher accumulation of DOX in the lysosome. Both uptake and cellular trafficking studies showed themechanism behind higher uptake of RF-targeted PEGylated PLL dendrimers, in which thetargeted PLL dendrimer conjugate internalized through both clathrin- and caveolin mediated pathway while non-targeted PEGylated PLL dendrimers were mostly internalized through caveolin mediated pathway only.

5.5 Conclusion

In this study, we investigated detailed intracellular colocolization of both RF- targeted PEGyated PLL dendrimers and non-targeted PEGylated PLL dendrimers and compared in-depth mechanisms behind endocytosis pathways. We determined that

RF targeted PEGylated PLL dendrimers primarily engaged clathrin and dynamin mediated endocytosis with some caveolin-mediated endocytosis. Non-targeted

PEGylated PLL dendrimers were involved less in both specific endocytosis pathways compared to RF-targeted PLL dendrimers, but the most engagement was seen with caveolin and dynamin mediated endocytosis pathways. Both dendrimer conjugates were also actively taken up by nonspecific macropinocytosis. Both dendrimer conjugates localized in theearly endosomes and the lysosomes, but higher intensity 105

and more specific colocalization was seen with RF-targeted PEGylated PLL dendrimers. Detailed knowledge from this study will help determine the appropriate use of receptor-targeted dendrimers and more effectively achieve target therapeutic results. Further studies with in-vivo works can incorporate the findings from this study so that specific drug release and delivery routes can be determined.

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Chapter 6 Conclusions and Future Directions

6.1 Conclusions

Dendrimers are multi-purpose polymers with different therapeutic applications for variety of disease states. Additionally, dendrimers have significant potential for use as a drug delivery vehicle, disease detecting agents, and gene delivery system, improving targeting efficacy and minimizing side effects.

Particularly, dendrimers have potential to enhance chemotherapy for cancer patients, increasing biocompatibility and specificity of classical chemotherapeutic drug towards cancer cells. Dendrimers are able to enter tumor cells more effectively than classical drugs by going through enhanced permeability and retention (EPR) effect.

Poly (amidoamine) (PAMAM) dendrimers have been used widely as a tumor targeting drug carrier and studied for their efficacy usingdifferent types of chemotherapeutic drugs. However, one important aspect that PAMAM dendrimers lack is non-biodegradability. Biodegradability is a critical issue if multiple doses are required because non-biodegradable molecules can accumulate inside the body.

Therefore, our lab conducted a study with biodegradable PLL dendrimers and investigated for its transport and mechanism. In this dissertation, we explored amore specific application of PLL dendrimers as a chemotherapeutic drug delivery vehicle and established active-targeting PLL dendrimers as an optimal drug delivery system.

In Chapter 3, we investigated theeffect of PEGylation on optimizing the inherent cell cytotoxicity caused by PLL dendrimers themselves. This study was also conducted to examine whether PEGylated PLL dendrimers have properties for effective chemotherapeutic drug delivery. PEGylated PLL dendrimers were

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synthesized successfully through controlled step-by-step synthesis and both, non-

PEGylated PLL dendrimers and PEGylated PLL dendrimers, were characterized using

MALDI-TOF, Nuclear Magnetic Resonance (NMR), Size Exclusion Chromatography

(SEC), and Zetasizer. Even with higher molecular weight resulting from addition of

PEG 2000Da to G3 PLL dendrimers, only a small range of size distribution was shown. This suggests a concise chemotherapy bypassing reticulo endothelial system if used for chemotherapy with a chemotherapeutic drug. PEGylated PLL dendrimers had only minimal cell cytotoxicity at a very high concentration of PLL dendrimers compared to non-PEGylated PLL dendrimers. This shows that the addition of PEG to PLL dendrimers likely covers the cationic charges of amines on PLL dendrimers and increasing cell viability. Because PEGylated PLL dendrimers without any drugs show almost no cytotoxicity to tumor cells, they cannnot cause toxicity to non- tumorigenic cells. Even with half-PEGylated PLL dendrimers, minimal decrease in cell viability only showed after 1mM of PLL dendrimer concentration, therefore, some cationic charges on dendrimers do not impact cell viability so much.

Therefore, PEGylated PLL dendrimers can be used as a great drug delivery vehicle with no inherent cytotoxicity and wide range of molecular weights.

Although PEGylated PLL dendrimers show great promise as a drug delivery system, some cancer cells do not depend on EPR effect, suggesting that administering

PLL dendrimers alone can possibly be ineffective. Therefore, in Chapter 4, we investigated the use of RF ligand with PEGylated PLL dendrimers to actively target breast cancer cells through receptor-mediated pathway. In comparison to non- targeted PEGylated PLL dendrimer with DOX, RF targeted PEGylated PLL dendrimers were similarly capable of decreasing breast cancer cell viability with lower concentration of DOX. Due to non-targeted and RF-targeted PEGylated PLL

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dendrimer drug conjugates enter cells in different ways, however, higher accumulation of DOX was apparent in initial hours after RF-targeted PEGylated PLL dendrimer treatment. Additionally, more evenly distributed RF-targeted PEGylated

PLL dendrimer with DOX was present in intracellular colocalization study and faster accumulation towards nucleus was found in the study.

To further establish RF-targeted PEGylated PLL dendrimers as a chemotherapeutic drug delivery vehicle, we studied the internalization mechanism as well as cellular trafficking inside different compartments other than nucleus. In

Chapter 5, we successfully used synthesized RF-targeted and non-targeted PEGylated

PLL dendrimers with DOX to examine internalization mechanism and intracellular colocalization in early endosome and lysosome. By utilizing fluorescence from

DOX, we colocalized both RF-targeted and non-targeted PEGylated PLL dendrimers and measure uptakes. Both, RF-targeted and non-targeted PEGylated PLL dendrimers were colocalized in early endosome after 1hr and moved to the lysosome after 2 hr incubation. While less dendrimer conjugates were present in the early endosome, more dendrimer conjugates were present in lysosome, possibly due to faster sorting from the early endosome to the lysosome compared to the rate that the dendrimer conjugates endocytose into the cells. Also, more fluorescence was found with RF-targeted PEGylated PLL dendrimers, indicating that receptor-targeted delivery promotes higher accumulation of dendrimer conjugates into the cell.

Looking at uptake study with inhibitors, a ligand-targeted PEGylated PLL dendrimers entered cells through primarily clathrin-, caveolin-, and dynamin- mediated endocytosis, while non-targeted PEGylated PLL dendrimers entered through caveolin- and dynamin-mediated endocytosis. Both conjugates also followed macropinocytosis, suggesting that multiple endocytosis mechanisms are used by

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ligand-targeted and non-targeted PEGylated PLL dendrimers. These studies illustrated that RF-targeted PEGylated PLL dendrimers were the optimal drug delivery option, promoting higher accumulation inside breast cancer cells while off- limiting side effects by less-targeted drug delivery.

Summing up all the results, this dissertation shows the importance of using

RF-targeted PEGylated PLL dendrimers as a potential drug delivery system to specifically targeted tumor cells. To use the distinct properties of PEGylated PLL dendrimers, detailed mechanisms behind dendrimer properties need to be understood.

PLL dendrimers, with their cationic properties, show potential to chemotherapeutic drug delivery vehicle when PEGylated. Adding active targeting ligand, RF, to PLL dendrimers, increases the chance of specifically delivering chemotherapeutic drugs.

6.2 Future Direction

Even though detailed mechanisms underlying clathrin-and caveolin-mediated endocytosis have been identified, further study needs to be conducted to find key structures/players governing such uptake mechanisms. In-depth work on receptor- mediated endocytosis using PLL dendrimers is also required to specifically understand mechanistic properties involved in ligand and drug delivery vehicle combination.

We have knowledge that PLL dendrimers are biodegradable dendrimers, but more study needs to be conducted involving biodegradability in-vivo. It will be important to understand if PLL dendrimers are not degraded until DOX is released from dendrimer complex. In addition to biodegradability study, biodistribution study needs to be conducted to determine how effective RF-targeted PEGylated PLL dendrimers are specifically targeting tumor cells while bypassing other organs.

Further in-vivo studies, including pharmacokinetics of RF-targeted PEGylated PLL 110

dendrimers, are essential to understand absorption, distribution, metabolism, and elimination of PLL dendrimers in the animal model. The final results from this work as well as future works should expandmore dendrimer-based chemotherapeutic drugs onto the market asresearchers realize the immense potential that dendrimers have in treating cancer patients.

111

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