Luận án tiến sĩ: Synthesis and characterization of bola-type dendritic macromolecules for use in biomedical applications

Consequently, hydrogels based on macromolecules possessing esterlinkages were explored as sealants for corneal wounds, while scaffolds for cartilage repair were prepared from macromolecu

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Copyright 2006 byDegoricija, Lovorka

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First Reader ⁄⁄⁄.⁄

Mark W Grinstaff, Ph.D

Associate Professor of Chemistry and Biomedical Engineering

Second Reader ( U »Scott E Schaus, Ph.D

Assistant Professor of Chemistry

I would like to thank the Boston University Department of Chemistry I wouldespecially like to thank my committee members (Prof Mark W Grinstaff, Prof PinghuaLiu, Prof Scott E Schaus, Prof John K Snyder, and Prof Joyce Y Wong) for theiradvice and guidance Specifically, I would like to thank Mark Grinstaff for his guidanceand support over the last five years I appreciate your willingness to help, yourgenerosity (the ski trips were awesome), and your great concern for students.

I would like to thank all members of the Grinstaff group, past and present I

would also like to thank Chad Immoos and Michael Carnahan for their friendship and

crazy stories I thank Prashant Bansal for his friendship and the many late nights we

stayed in lab in order to finish a project I also thank Steve Meyers for all the

cytotoxicity studies he performed, Carla Prata for her advice, and Abby Oelker for her

help with FRAP studies

I would like to thank all the people I have collaborated with over the past five

years, especially Dr Starck Johnson for performing many hours of corneal laceration and

PKP experiments, and Dr Terry Kim for proof reading one of my papers

I would particularly like to thank Michel Wathier for his advice, patience, and

most importantly, his friendship You made coming to lab more interesting AaronBeeler, I thank you for your support during the last three years and your willingness to

help with any problem inside or outside of the laboratory For that, I am truly grateful

I cannot express how grateful I am to mom, dad, Ved and Em for their unconditional love

and support through tough times I would not have been able to make it this far without

iv

rewarding chapter in my life.

MACROMOLECULES FOR USE IN BIOMEDICAL APPLICATIONS

(Order No )LOVORKA DEGORICIJABoston University Graduate School of Arts and Sciences, 2007

Major Professor: Mark W Grinstaff, Associate Professor of Chemistry and Biomedical

Engineering

ABSTRACTDendrimers are well defined, highly branched macromolecules that consist of a core, internal branching units and a multitude of peripheral groups Dendrimers possess low viscosities, high solubilities, single molecular weights, and a globular shape in solution As a result, the architecture and physiochemical properties of dendrimers offerspecific advantages over linear polymers

Currently, millions of people in the Unites States seek treatment for the repair ofcorneal wounds The standard of care for repairing these wounds involves using sutures,which do not actively participate in healing of the surgical procedure Another commonmedical problem involves the degradation of cartilage due to osteoarthritis or trauma.Since cartilage has a limited capacity for self-repair, the current methods of treatmentinvolve the use of non-surgical and/or surgical techniques to reduce pain while

maintaining joint function Consequently, there is interest in synthesizing dendritic

macromolecules specifically for these biomedical applications

vi

divergent method in an iterative process of monomer unit coupling, followed by deprotection The design of these dendritic macromolecules was based on an ABA triblock architecture, wherein two dendritic arms, or A blocks, flanked a linear linker, or

B block, via ester linkages The dendritic arms consisted of biocompatible materials,while the linear linker was a non-immunogenic poly(ethylene glycol) of three differentmolecular weights Degradation of these macromolecules leads to natural metabolites,such as succinic acid and glycerol, thus they are termed biodendritic macromolecules.The same synthetic strategy was used to prepare biodendritic macromolecules containingcarbamate linkages, in addition to previous ester linkages, to obtain materials with adifferent set of physical properties

Further functionalization of these macromolecules was needed to preparephotocrosslinkable systems for the formation of hydrated networks, or hydrogels.Forming hydrogel networks from highly branched biodendritic macromers offers

advantages in preparing hydrogels at low polymer concentration, varying mechanical

properties, and developing in situ polymerizing systems for delivery to an irregularly

shaped wound site Consequently, hydrogels based on macromolecules possessing esterlinkages were explored as sealants for corneal wounds, while scaffolds for cartilage

repair were prepared from macromolecules containing carbamate linkages

vii

F.\Y0.)I9À140290605) 6001.0007077 434 1V.Y= ý ›Š- oiiiidddỎddiddddtddỎiỎởỎỞỎỐỐỎ - viTABLE OF CONTENTS :tdtdtdỶỖỒ VillLIST OF 00.0.0117 XLIST OF FIGUR.ES HH HH TH HH HH TH HH Hà HH Hàng xiLIST OF SCHEMES ng TT nh TT nh TH HT TH TH TT TH XIVLIST OF ABBREVIATIONS 4đađđaadỐẢẢẢỎ XVCHAPTER 1—Dendritic Macromolecules: From Synthesis to Biomedical EngineeringApplications TT e Ả 1Introduction to DenndrirmeTS§ - t1 vn ng HT TH nh TT HH TH TH nhện 1Adhesives for Sealing Corneal Wounnds - - «ch ng HH ykp 12

6 7/2-/z4//7/1 // /2800n00n00n0n8nẼn8Ẻ 16[272/18 /0.//1 5E nh ốố.ốốẦốẦẦỐ 17Chondroitin Sulfate Aldehyde (Š€QÏŒ1HÉS tk nh nh Hi gcr 19Biodendritic-based Hydrogel] Ádl€SÏVÉS sàng TH TH TH HH nhờ 19Scaffolds for Cartilage R.€DAIT ác TS vn SH HH1 ng Hy TH ng ng nêu 20Current Treatments—Non-surgical and SUrgicdl cccccccscccscssecsssscessssscesseseseneessenees 23Non-injectable SCQ[ƒỌdẲS ác vn TT TH TH TH ng Hà Hà TT v4 26Injectable SCQ[ŸỌ(Í nh HT TH TH TT TH TH Tà HT HH TH Hà Hiện 31Biodendritic-based Hydrogel Scaffolds c.ccccsccsssessseessesssesssccesseseveessesensssesssessssenens 35CHAPTER 2—Synthesis and Characterization of Bola-type Amphiphilic Dendritic

15011051000 ng ốố ‹ddd:‹::11Y 36Results and DiSCUSSION 0A nh aga 37SẠS)595428552)92v510I00PnẼnĐ hố 52Characterization of Hydrogels ccscsscscsscesssssssessssscsecssensesesssesecsessscseesessucascassaseasees 54SUMIMALY s.sccsscscssesessesesessesesesseeeseeseeeseesecesseessenessessecseesesseesseaseascssssecausnessassesarensenenes 67CHAPTER 3—([G1]-PGLSA-MA),-PEG Biodendritic Macromolecules as OphthalmicAdhesives for Central Lacerations and Penetrating Keratoplasties - .-.-: 69H000) 000008 69Experimental] Sef-Up «ch nàng HT Hà HH HH HT Hà TH Tà HT t0, 70Results and Discussion for the Central Laceration S†udy - :ccccccccccsretees 75Results and Discussion for the Penetrating Keratoplasty Study : -‹ccc c5: 78Results and Discussion for the India ink stUdy cv vtvereirrrrrtrererrrire 82SUMIMALY 0 ố ốốốố ốốốố.ốốốốốốốố 83CHAPTER 4—Synthesis of Carbamate-based Photocrosslinkable Biodendritic Scaffolds:Implications for Tissue EngIn€€rifiE s99 1111 1111111 1g HH eg 86IntrOdUCfIOTA - ng TT nh HH nh HH TH TT gà TH TH TT 01111101514 114 111 110g 86Results and D1SCUSSIOTI nh HH HH ng HH HT TT TT g0 11100 91Characterization of Macromolecules cccecsssessessscesessecesececsaseseesseseseessesseserssseeses 94Hydrogel PrepraftiOr - ch TT TH HH Hà KH Tà H1 11 1kg 102

R0 - 115

CHAPTER 5—Application of (G1]-PGLBA-MA);-PEG as a Resorbable Three-Dimensional Scaffold for Cartilage RegeneratiOn - án ng re 118 006/0): DEET 118

Cartilage SUructure HT TT TT TT TT TT TT TH HH kg 119 Results and Discussion for In vitro SfUd1©S c1 312v HH ty, 127 Preparation of Cell-Hydrogel COnStructs ch HH HH, 127 Characterization of Cell-Hydrogel COHISÍTIHCÍS ng iu 128 Results and Discussion for In vIvo SfUd1€§ ác tk Hy He, 138 Evaluation Of SCQƒƒỌỈS cá cLtEtkEt KH HT Ty KT TT TH ch ng HH 139 SUMMALY 00 1n 142

CHAPTER 6—Methods and Materials - Ác 1 121191100111 1110 1 ng gu 143 "0551 0 eeesesccsecesesscnscssesresscsecsecsecseesessessessessesssessseseseseseessassessassassesasensateasnes 143 InstrUI€TIfAfIOTI - HH HT Tà TT TT HT HT gà Hà TT Tà TH gà 0114 Hee 143 NGA ố 144

II09)2/9)0-9)/.1.1111ẺẺ 192

REFERENCES SẼ s 197

9814:3001060/01460.)2110100777 212

1X

Table 1.1: Performance characteristics of various corneal wound closures as compared to

biodendrimer based adhesives, 106 1001 eet g ga 0110111101111 711711120.) 22

Table 1.2: Summary of surgical techniques for OA pafienIs «seo 28Table 2.1: MALDI-TOF and SEC data for ({Gn]-PGLSA)2-PEG3400, 10000, and 20000

6i 1990/10I<91171- 28AA8Ẻ8Ẽ 48Table 2.2: MDSC data for ([Gn]-PGLSA)2-PEG3400, 10000, and 20000 Macromolecules 47Table 2.3: The critical aggregation concentrations (CAC) for each soluble generationwithin the three PEG macromolecules All CAC are reported as mM concentrations.beceeceuvaceseveuseccevsueecesuensccessasevesseeecusnasesseeassecseresevasereesesstsscesuescassuenesseueesccansuereessueecees 51Table 4.1: MALDI-TOF and SEC data for ester and carbamate-based biodendritic

MACTFOMOLECUIES . - G0 00111000011 111023 11119010 1 11001111101 1 E0 110 60959 99Table 4.2: MDSC data for carbamate-based biodendritic macromolecules 100Table 4.3: CAC values for carbamate and ester-based macromolecules 101

Figure 1.1: Basic dendrimer SỈTUCfUTC 5á nhìn Hàn HH HH tu 2Figure 1.2: Convergent approaches towards the synthesis of dendrons and dendrimers 3Figure 1.3: Divergent method for the synthesis of dendrimers ¿5c cc cà: 4Figure 1.4: Synthetic scheme of cascade poÏy1m€TS chớ 7Figure 1.5: Synthesis of PAMAM dendrimers nh Hàn He 8Figure 1.6: Synthesis of poly(aryl ether) dendrOTis - ĩc nhan 9Figure 1.7: Synthesis of poly(aryl ether) dendrimer .- ca cành 10Figure 1.8: Fourth generation poly(glycerol succinic acid) dendrimer 13Figure 1.9: ABA triblock architecure of biodendrirmers «cty 14Figure 1.10: Structure of the monomer unit in cyanoacrylate øÌues ‹-‹ 18Figure 1.11: Two-component adhesive composed of oxidized chondroitin sulfate and0.0 ốc 21Figure 1.12: Osteoarthritic cartilage in a knee JO]TIÍ - ĩ1 vn nhi, 24

Figure 1.13: Surgical procedure for state of the art treatment (Cartieel”*9 -.¿ 29

Figure 1.14: Mechanical properties of hyaline cartilage .- - Sen 30Figure 1.15: Non-injectable synthetic polymer scaffolds for cartilage repair 32Figure 1.16: Non-injectable natural polymer scaffolds for cartilage repa1r 33Figure 1.17: Synthetic polymer scaffolds for in situ cartilage repair that are (A) thermalactivated, or (B) photochemical actIvated ĩc LH nh Hye 34Figure 2.1: Schematic of the ABA architecture of (G4]-PGLSA)a-PEG bola-type

dendritic macromolecule consisting of succinic acid, glycerol, and

non-1mmunogenic PEC - - cv g1 019 TH Hà ng nh nàn Hà Hà H014 38Figure 2.2: 'H NMR spectra of ([G3]-PGLSA-bz1d);-PEGioooo (top) and ({G3]-PGLSA-

OH)2-PEGioo00 Macromolecules (DOffOrn) - c2 19T 9g y1 rời 46

Figure 2.3: 'H NMR of([G3]-PGLSA-MA);-PEGioooo macromolecule - 47

Figure 2.4: Formation of a (G1]-PGLSA-MA);-PEG hydrogel - ĩc ccxssx+: 53Figure 2.5: Normalized weight of the hydrogel samples at 5, 7.5, 10, and 20% w/v (n=3)for the (MA)2-PEG macromolecules containing 3400, 10000 and 20000 Mw PEG

995215 1 4 55Figure 2.6: Normalized weight of the hydrogel samples at 5, 7.5, 10, and 20% w/v (n=3)for the (G0]-PGLSA-MA);-PEG macromolecules containing 3400, 10000 and

20000 Mw PEG polyme?S nh 43 56Figure 2.7: Normalized weight of the hydrogel samples at 5, 7.5, 10, and 20% w/v (n=3)for the (G1]-PGLSA-MA);-PEG macromolecules containing 3400, 10000 and

20000 Mw PEG poÏyI€VS HH TH TT HT TH TH HH HH Hà TH TH Tự 57Figure 2.8: Compressive modulus for (MA)2-PEG3400, 10000, and 20000; ([GO] -PGLSA-MA);-

PEG3400, 10000, and 20000, and ([G1]-PGLSA-MA)2-PEG3a00, 10000, and 20000 dendritic

hydrogels at 5, 7.5, 10, and 20% w/v before swelling (top) and after swelling

(DOOM) cece 61Figure 2.9: Complex modulus for (MA)2-PEG3a00, 10000, and 20000; ([G0]-PGLSA-MA)¿-

PEG3400, 10000, and 20000, and ([G1]-PGLSA-MA) -PEG3400, 10000, and 20000 dendritic

xi

Figure 2.10: Loss angle for (MA)¿-PEGaaoo, 10000, and 20000, ([GO]-PGLSA-MA) 2-PEG3<99,

10000, and 20000» and ([G1]-PGLSA-MA);-PEG+4oo, 10000, and 20000 dendritic hydrogels at 5, 7.5, 10, and 20% w/v before swelling (top) and after swelling (bottom) 63

Figure 2.11: Diffusion coefficients of (MA)2-PEG3a00, 10000, and 200005 PEG3400, 10000, and 20000, and ([G1]-PGLSA-MA) -PEG3<00, 10000, and 20000 hdyrogels at four different concentration (5, 7.5, 10, and 20% W/V) ng HH 66 Figure 3.1: Cross sectional view of the human COFn€â cà che 71 Figure 3.2: First generation (G1) dendritic polymer, ([G1]-PGLSA-MA),-PEG,

(G0]-PGLSA-MA);-consisting of natural metabolites such as glycerol and succinic acid (triangle), and non-immunogenic poly(ethylene glycol) (rectang]e) - «ác nnhhnhey 72 Figure 3.3: Leaking pressures for the 4.1 mm central lacerations with three differenthydrogel adhesives at 10, 20, and 40% W/V cà kh HH Hà Hà Hà HH Hà nh 77Figure 3.4: Leaking pressures for PKP autografts sealed with either 8 or 16 10-0 nylonsutures and three different adhesive formulations at 20% W/V Hee 80Figure 3.5: Photograph of a secured autograft in an enucleated porcine eye after

placement of 16 interrupted 10-0 nylon sutures followed by application and

photocrosslinking of the hydrogel adhesIve -ó- nh He 81Figure 3.6: Clockwise from upper left picture: histological sections of India ink on anautograft with 16 sutures and no hydrogel adhesive, 8 sutures and no hydrogeladhesive, 8 sutures and hydrogel adhesive on top of the epithelium, and 16 sutureswith hydrogel adhesive on the epitheÌium nh HH Hệ, 84Figure 4.1: The monomer units selected for the synthesis of ester and carbamate

Containing macrornoÌe€CUÌ€S - - óc s3 1T HH nh 88Figure 4.2: The structures of (A) ([G1]-PGLSA)›;-PEG, (B) {G0]-PGLSA-[GI]-

PGLBA);-PEG, (C) ([G1]-PGLBA) -PEG, and (D) PEG maromol€CuÌ€§ <1 HT ng HH ch Hàng 89Figure 4.3: Schematic of the ([G0]-PGLBA-[G1]-PGLSA).-PEG macromolecule

([G0]-PGLBA-[G1]-PGLSA),-consisting of natural metabolites (succinic acid, glycerol), amino acid derivative(beta-alanine), and non-immunogenic PEG, where 71 18 77 seo 90Figure 4.4: Normalized weight of the hydrogel samples at 5, 7.5, 10, and 20% w/v (n=3), stored in PBS at 37 °C and 5% CO; as a function of tiMe ‹ .- 104Figure 4.5: Compressive modulus of the four hydrogel formulations (n = 3) at a range ofconcentrations (5, 7.5, 10, and 20% w/v) before (BS) and after swelling (AS) 109Figure 4.6: Complex modulus of the four hydrogel formulations (n = 3) at a range ofconcentrations (5, 7.5, 10, and 20% w/v) before (BS) and after swelling (AS) 110Figure 4.7: Loss angle measurements of the four hydrogel formulations (n = 3) at a range

of concentrations (5, 7.5, 10, and 20% w/v) before (BS) and after swelling (AS) 111Figure 4.8: FRAP measurements of the four hydrogel formulations (n = 3) at a range ofconcentrations (5, 7.5, 10 and 20% WV) LH ng nà kh nh kh 113Figure 4.9: Picture of the custom designed servomotor-actuator system for the simulation

of rabbit knee kinermaf1CS óc nh TH TH TH HH HH ng th nh nh nh 114

xii

integrated in the defect site after repeated loading with a seven pound dead-weight.

— 116Figure 5.1: Schematic representation and photomicrograph of the collagen fibril

gu nh 121Figure 5.2: Amino acids that compose the repeat Glycine-X-Y unit of collagen, where X

is often proline and Y is often hydroxyproline .c.cccccssesseseseesesteseesestereeseeseaees 122

Figure 5.3: Glucosaminoglycan stTUCfUT€S LH HH ng ng gu 124Figure 5.4: Collagen-proteoglycan matrix in cartiÌaỹ© càng ra 125Figure 5.5: Cartilage uÏtrAaS{TUCÍUT€ 9S HT TH HT ng ng hy 126Figure 5.6: Compressive modulus of all cell-hydrogel constructs at two, four, and eight

<1 66 (.(. đđ 4 130Figure 5.7: Complex modulus of all cell-hydrogel constructs at two, four, and eight2< Ầ 131Figure 5.8: Loss angle of all cell-hydrogel constructs at two, four, and eight weeks 132Figure 5.9: Histological sections of the cellular and acellular hydrogel constructs afterTWO WEEKS INCUDALION 0 ee ccecteetecseeseeecserseeeseceseeescssecscssctaseesessesesaecsseasenseases 134Figure 5.10: Histological sections of the cellular and acellular hydrogel constructs aftereight weeks InCUbafIOT Án v 9 19119111 1311111 11 1 Hà ng 0T TT Hà Ta 135Figure 5.11: Calcium (top) and alkaline phosphatase (bottom) assays of the osteoblastcell-hydrogel constructs at two, four, and eight WEEKS .cccccsessetssseessessessesees 136Figure 5.12: In vivo MR images of a rabbit knee model in an axial plane 140Figure 5.13: Ex vivo MR images of rabbit condyles immediately after sacrifice taken inthe sagittal DÏaT€ HH HH HH TH gà ng To TT TH TH HH Hà Hu 141

XIHI

Scheme 2.1: Divergent synthesis of (G2]-PGLSA-OH);-PEG bola-type dendritic

macromolecule Scheme 1 depicts the general synthetic route using the 3400,

10000, and 20000 Mw PEG polymers, where n is 77, 227, and 455, respectively 41Scheme 2.2: Divergent synthesis of ([G4]-PGLSA-OH)2-PEG macromolecule from(G2]-PGLSA-ORH);-PEG Scheme depicts the synthetic route using 3400, 10000,and 20000 Mw PEG, where n is 77, 227, and 445, respectvely 42Scheme 4.1: Scheme shows the synthetic route for the isolation of the glycerol betaalanine MONOMET UNIt .- G1191 1 9v 1v ng ng kg kiện 92Scheme 4.2: Scheme shows the divergent synthesis of (G0]-PGLSA-[G1]-PGLBA-MA)›-PEG, 24, where n = 77 for a PEG of MW 3400 nen Heeu 95Scheme 4.3: Scheme shows the divergent synthesis of ([GO]-PGLBA-[G1]-PGLSA-MA);-PEG, 29, where n = 77 for a PEG of Mw 3400 nhe 96Scheme 4.4: Scheme shows the divergent synthesis of (G1]-PGLBA-MA);-PEG, 32where n = 77 for a PEG Of Mw 340Ô cv HH nh Hà Hà gà Hà tệ, 97

XIV

D diffusion coefficient (cm”/sec)

ỗ loss angle (degrees)

DSC differential scanning calorimetry

E compressive modulus (kPa)

EY eosin Y

FAB fast atom bombardment

FDA US food and drug administration

XV

magnetic resonance imaging

molecular weight

nuclear magnetic resonancedeprotected hydroxy group

Xvi

poly(ethylene glycol)

poly(glycolic acid)

poly(glycerol beta alanin)

poly(glycerol succinic acid)

poly(lactic acid)

poly(lactic-co-glycolic acid)poly(N-isopropylacrylamide)

parts per million

room temperature

singletsize exclusion chromatography

spin-lattice relaxation time

triethanolaminethermogravimetric analysis

glass-rubber transition temperature

solid-liquid transition temperature

tetrahydrofuran

microliter

xvi

UV ultraviolet

VP 1-vinyl-2-pyrrolidinonewiv weight per volume

XVill

Engineering Applications

Introduction to Dendrimers

Dendrimers are polymers that have an architecture resembling the branches of atree are actively being investigated in academic and industrial settings (Figure 1.1).Dendrimers are well-defined, single molecular weight, globular polymers containing a

central core, branching layers, and numerous end groups.'* Compared to linear polymers,

dendrimers possess single molecular weights, low viscosity, high solubility andmiscibility, and a large number of end groups for functionalization Two synthetic

approaches are used to prepare dendritic macromolecules In the convergent method,synthesis starts from what will become the exterior portion of the dendrimer and proceedsinwards by coupling the end groups to each branch of the monomer to create dendriticarms, or dendrons Once the desired size or generation of these dendrons has been

achieved, coupling to a polyfunctional core affords the final dendrimer.”” In the

divergent method, synthesis is initiated form a core and proceeds outward by an iterativeprocess of coupling and activation to form the next higher generation, corresponding to

the addition of more monmer units (Figures 1.2 and 1.3).'”'“ Buhleier reported the first synthesis of cascade polymers in the late 70’s.'’ These systems were synthesized in an

iterative process by the Michael addition of acrylonitrile to benzylamine, followed by

reduction to double the number of peripheral amine groups (Figure 1.4)

Figure 1.1: Basic dendrimer structure.

` Eigure 1.2: Convergent approaches towards the synthesis of dendrons and dendrimers.

Figure 1.3: Divergent method for the synthesis of dendrimers

Tomalia and Newkome,*”?*8 which included the complete synthesis of poly(amidoamine) dendrimers (PAMAMs) These dendrimers where synthesized using thedivergent strategy by successive of coupling methyl acrylate to an ammonia corefollowed by exhaustive amidation with ethylenediamine (Figure 1.5) This approach was

a significant advance in overcoming problems of purity and low yields in the preparation

of dendritic marcromolecules.'Š'® Soon after, Hawker and Fréchet synthesized dendrimers

using 3,5-dihyroxybenzy! alcohol as the monomer unit and performed high yielding

Williamson ether synthesis and bromination reactions to produce produce poly(aryl ether)

dendrimers (Figures 1.6 and 1.7).°° Since these reports, dendrimers based on PAMAMsS and poly(aryl ether) have been actively investigated for a wide range of industrial” and

medical applications In the medical field, these macromolecules are currently being

explored as vehicles for drug delivery,°® cationic agents for gene transfection,”

75 carriers for boron neutron capture therapy,” and

inhibitors of influenza viruses,

contrast agents for magnetic resonance imaging Unfortunatley, PAMAM and poly(aryl ether) dendrimers are limited by their lack of biocompatibility.” Consequently, there is

interest in the synthesis and characterization of dendritic macromolecules that possess

improved biocompatibility for in vivo and in vitro evaluation

Dendrimers possess multiple end groups that can be modified to form crosslinked

polymer networks.'*'*'°*°*’ This highly branched architecture offers specific advantages

in preparing crosslinked materials at low polymer concentration and consequently a high

water content.

Í Now CN NH NH

io on N ÔN

NC~ NC~ *CNẺCN CL NH> NH» NH> NH»

Reagents and conditions: a) acrylonitrile, acetic acid; b) Co(II, NaBH,, CH;OH

Figure 1.4: Synthetic scheme of cascade polymers

Reagents and conditions: a) ethylene diamine; b) methyl acrylate.

Figure 1.5: Synthesis of PAMAM dendrimers

Reagents and conditions: a)K,CO,, 18-crown-6, acetone; b) PPH,, CBr,, THF; c)

3,5-dihydroxybenzyl alcohol, K,CO,, 18-crown-6, acetone

Figure 1.6: Synthesis of poly(aryl ether) dendrons

Reagents and conditions: a) K,CO,, 18-crown-6, acetone.Figure 1.7: Synthesis of poly(aryl ether) dendrimers.

This allows for varying mechanical properties, and development of in situ polymerizingsystems for delivery to an irregular shaped wound site.

Most recently, crosslinked polymer networks possessing a high water content,known as hydrogels, have been evaluated for medical applications in areas such as drug

delivery, tissue engineering, and more recently, as tissue adhesives.”” Specifically,

there is great interest in the formation of hydrogels from photocrosslinkable polymers for

medical applications, where in situ photopolymerization of a polymer affords a temporary

cell scaffold that can aid in tissue repair.*”

The principles of a branched architecture, which lead to multiple end groups, andthe ability to modify the periphery with photocrosslinkable groups, were applied todendritic macromolecules synthesized in our lab Previously, our laboratory had used thedivergent method to synthesize poly(glycerol succinic acid) dendrimers, PGLSA, based

on biocompatible materials that can degrade to natural metabolites, such as succinic acid,and glycerol, which are used in biosynthesis (Figure 1.8) Thus, these dendrimers were

termed biodendrimers.5 Photocrosslinkable systems were afforded by modifying the

hydroxy periphery with methacrylate groups in order to generate three-dimensional gelscaffolds for the potential use in the medical and biotechnological fields Gel scaffoldswere prepared from PGLSA dendrimers in dichloromethane with 2,2-dimethoxy-2-phenylacetophenone as the initiator and cured under UV light (Amax = 375 nm) Althoughthese systems are biocompatible and water-soluble with hydroxyls around the periphery,they become insoluble in aqueous solutions with the addition of hydrophobic

methacrylate groups

To alleviate this problem of insolubility with methacrylate PGLSA biodendrimers, a hydrophilic poly(ethylene glycol) (PEG) linker was incorporated between two dendritic arms, to form an ABA triblock architecture (Figure 1.9) The dendritic arms are composed of the same biocompatible materials previously used in the

PGLSA dendrimers”, and the addition of the PEG linker imparts greater water solubility

of the macromolecules at higher generations These triblock macromolecules can bemodified to include carbamate linkages, in addition to the previous ester linkages, for thepreparation of macromolecules with a different set of physical properties From thesemacromolecules, photocrosslinkable systems were prepared by further modification of the hydroxy periphery with methacrylate groups, wherein both sets of bola-type macromolecules can be readily dissolved and photocrosslinked in aqueous solutions The end result is a three-dimensional hydrogel that can be specifically used as an adhesive in sealing corneal wounds, which was explored with the macromolecules possessing ester linkages, and as a temporary scaffold for cartilage repair, which was prepared from themacromolecules containing carbamate linkages

Adhesives for Sealing Corneal Wounds

Each year, more than 2.5 million individuals in the United States seek treatmentfor the repair of ocular wounds There are a number of ophthalmic conditions andprocedures that result in corneal wounds, including corneal ulcers, lacerations,perforations, transplants, incisions for cataract removal and IOL implantation, and LASIK flap complications.””!Ở Today, the standard procedure for repairing cornealinjuries involves using nylon sutures, and depending on the pattern and extent

Figure 1.8: Fourth generation poly(glycerol succinic acid) dendrimer The core anddendritic arms are composed of natural metabolites, such as glycerol and succinic acid.

A block PEG—B block

A block

Figure 1.9: ABA triblock architecure of biodendrimers These biodendrimers areellipsoidal at lower generations and become increasingly spherical at higher generations

of injury, multiple sutures are often needed to restore the structural integrity of thecornea Unfortunately, sutures do not actively participate in healing, reduce the time, orinvasiveness of the surgical procedure Consequently, there are several drawbacks in

using standard 10-0 nylon sutures, which include: (1) inflicting additional trauma to

corneal tissue, especially when multiple passes are needed; (2) acting as a nidus for

infection and inciting corneal inflammation and vascularization,’ which increases the

propensity for corneal scarring; (3) yielding uneven healing, which results in a regular or

irregular astigmatism;'” (4) becoming loose and/or broken postoperatively, and requiring

additional attention for prompt removal; and (5) requiring removal often several months

after surgery, which inflicts additional trauma and opens the opportunity for new

infections Therefore, the need for alternative methods to repair ocular wounds is of

current interest Of the methods being explored, the use of polymeric adhesives or

sealants had attracted significant attention

Polymers that adhere to tissue'TM are of clinical value for applications where

surgical procedures using sutures are not efficacious It is for this reason that tissuesealants are an attractive alternative to sutures It is hypothesized that the negative effects

largely associated with sutures can be minimized by the use of tissue sealants In fact,

there is a precedent for their use in ophthalmology, particularly in the management of

corneal perforations

The goal of tissue sealant therapy is to provide immediate restoration of structural

integrity and occasionally to prevent further corneal thinning An effective polymer

sealant for repairing corneal perforations must meet a number of requirements Ideally, it

should: (1) be biocompatible; (2) adhere to the moist corneal surface and polymerize toseal the corneal wound in a controlled manner; (3) quickly restore the intraocularpressure; (4) possess solute diffusion properties favorable for normal corneal healing; (5)

be more elastic than corneal tissue so as to disfavor formation of an astigmatism duringhealing; and (6) be bioabsorbed on a time scale consistent with tissue regeneration

Previous ophthalmic sealants based on synthetic and/or natural polymers such ascyanoacrylates, or superglues, fibrin, and a modified chondroitin sulfate-aldehyde systemhave been explored for sealing small corneal wounds consisting of 1-3 mm linearlacerations Unfortunately, these sealants failed due to a lack of biocompatibility,degradation, expensive costs, limitations in sealing only small ocular wounds, and/orcomplex methods of application

Cyanoacrylate Sealants

Of the previous sealant, cyanoacrylates (Figure 1.10) have received the most

attention Cyanoacrylate glues were initially presented in 1959 by Coover et đi.,'” and

were subsequently used in the 1960's for the repair of corneal perforations as reported by

Webster et al.'° Cyanoacrylate sealants have proven to be an effective therapeutic option

in certain ophthalmic settings such as sealing small corneal perforations and preemptive

treatment of progressive corneal thinning disorders.”!°'!°"* However, they have limitations with regard to their ease of application and effectiveness,°"!#!2 which

include immediate polymerization on contact with wet surfaces, the generation of heat,

and low patient tolerance, such as abrasiveness Furthermore, several complications with

these corneal sealants have been reported and include cataract formation, corneal

infiltration, granulomatous keratitis, glaucoma, and even retinal toxicity."*'"" Currently,

cyanoacrylate glues are not approved by the FDA for ophthalmic use, and their use on theeye is considered off-label Table 1.1 summarizes the disadvantages of cyanacrylateglues as tissue sealants

The methods of applying cyanoacrylates vary from direct application with atuberculin syringe to initial placement of sealant on a piece of sterile draping Thesemethods can be cumbersome, often requiring adept and delicate application of a preciseamount of sealant on a dry environment in order to facilitate sealant solidification.Excessive amounts of glue can cause premature dislodgement of the sealant, generallycaused by normal eyelid movement, and may result in a foreign-body sensation.Following a procedure using cyanoacrylates, a bandage contact lens is frequently placedover the glue in order to reduce patient discomfort

ulcers.!⁄4!!3' This adhesive hemostat meets some of the requirements for a tissue

adhesive, but these autologous products carry the disadvantages of post-surgical

tisk of transmitting viral infections.!”°!3? The advantages and disadvantages of this tissuesealant are also summarized in Table 1.1 Consequently, the potential for these sealants

to be used in the clinic is limited and prompted the generation of new sealant systems

Chondroitin Sulfate Aldehyde Sealants

Recently, a chondroitin sulfate aldehyde adhesive has been reported forsealing 3 mm corneal incisions.'*? This two-component adhesive is composed of anoxidized chondroitin sulfate (CS) and polyvinyl alcohol covinyl amine (PVA-A) polymer(Figure 1.11) This adhesive forms as a result of the reaction between the amine of PVA-

A and the aldehyde of CS forming a reversible Schiff base linkage First, the PVA-Acomponent of the adhesive was applied on the surface of the incision and in the internalwound flap This was followed by the application the CS aldehyde over the first layer,which sealed the two layers of tissue Although this sealant is relatively new and hasshown promising results, it is limited to small corneal incisions This adhesive has notundergone extensive in vivo studies to determine the toxicity, degradation, mechanicalproperties, and post-surgical complications associated with this material Table 1.1summarizes only the preliminary characteristics of this adhesive

Biodendritic-based Hydrogel Adhesives

Biodendritic bola-type (Figure 1.7) hydrogel adhesives are of particular

interest in the ophthalmic field, since they have been evaluated as adhesives for large and

small corneal wounds, which will be discussed in Chapter 3 Biodendritic-based

hydrogel adhesives may offer unique advantages for the restoration of corneal tissuefunction over previous ocular sealants since they meet all the requirements for thepreparation of a successful tissue sealant, as previously discussed More importantly, theuse of biodendritic hdyrogels as adhesives for ocular wounds in the clinic would have a

significant impact on the repair of traumatic corneal wounds, since they have the

potential to eliminate many of the drawbacks inherent with the use of sutures

Scaffolds for Cartilage Repair

Cartilage repair involves the use of either surgical or, in the near future, tissueengineering techniques for the regenerating of cartilage tissue Trauma, disease brought

on by osteoarthritis, or a lifetime of wear-and-tear have prompted more than one million

surgical procedures, involving the knee, hip, and shoulder joint, annually in the U.S."

Osteoarthritis, or OA, is the most common form of arthritis that causes the breakdown

and eventual loss of cartilage Cartilage degenerates by either flaking or forming tiny

crevasses (Figure 1.12), and in advanced cases there is complete loss of cartilage This

loss of cartilage causes friction between the long bones, leading to extreme pain andlimited joint mobility Since articular cartilage tissue is predominantly avascular innature, with a very low cell density, it has a limited capacity for self-repair following

injury or with OA

Cartilage is dense connective tissue that covers the ends of long bones This

tissue, which is typically 1 mm thick, provides a smooth surface for the movement ofarticulating bones

HN lo

H HC

OH C

Polyvinyl alcohol co-vinylamine (PVA-A)

Figure 1.11: Two-component adhesive composed of oxidized chondroitin sulfate and

PVA-A

Table 1.1: Performance characteristics of various corneal wound closures as compared

to biodendrimer based adhesives 106 101,115-122,125,132,133

Performance Nylon Sutures Cyanoacrylate Fibrin Glue Characteristic Glues AldehydeRepairs small wounds < YES YES YES YES

Soft and flexible NO NO NO Toxic degradation NO YES NO -

-products

Transparent NO NO NO YESPreparation time prior to 1 MIN 1 MIN 20-30 MIN 1 MIN