Luận án tiến sĩ: Tổng hợp dendrimers bola cho ứng dụng y sinh

BOSTON UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES Dissertation SYNTHESIS AND CHARACTERIZATION OF BOLA-TYPE DENDRITIC MACROMOLECULES FOR USE IN BIOMEDICAL APPLICATIONS by LOVORKA DEGORICIJA B., Santa Clara University, 2001 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2007 UMI Number: 3240617 Copyright 2006 by Degoricija, Lovorka All rights reserved. INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted.

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ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 © Copyright by LOVORKA DEGORICHA 2006 Approved by First Reader ⁄⁄⁄. Associate Professor of Chemistry and Biomedical Engineering Second Reader ( U » Scott E. Assistant Professor of Chemistry ACKNOWLEDGEMENTS I would like to thank the Boston University Department of Chemistry.

I would especially like to thank my committee members (Prof. Pinghua Liu, Prof. Snyder, and Prof. Wong) for their advice and guidance.

Specifically, I would like to thank Mark Grinstaff for his guidance and support over the last five years. I appreciate your willingness to help, your generosity (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. Aaron Beeler, 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 your guidance and love. Again, I thank everybody for helping me finish this very rewarding chapter in my life.

SYNTHESIS AND CHARACTERIZATION OF BOLA-TYPE DENDRITIC MACROMOLECULES FOR USE IN BIOMEDICAL APPLICATIONS (Order No. ) LOVORKA DEGORICIJA Boston University Graduate School of Arts and Sciences, 2007 Major Professor: Mark W. Grinstaff, Associate Professor of Chemistry and Biomedical Engineering ABSTRACT Dendrimers 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 offer specific advantages over linear polymers. Currently, millions of people in the Unites States seek treatment for the repair of corneal wounds. The standard of care for repairing these wounds involves using sutures, which do not actively participate in healing of the surgical procedure. Another common medical problem involves the degradation of cartilage due to osteoarthritis or trauma.

Since cartilage has a limited capacity for self-repair, the current methods of treatment involve 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 Fourth generation, bola-type dendritic macromolecules were synthesized by the 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 different molecular 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 containing carbamate linkages, in addition to previous ester linkages, to obtain materials with a different set of physical properties. Further functionalization of these macromolecules was needed to prepare photocrosslinkable 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 ester linkages were explored as sealants for corneal wounds, while scaffolds for cartilage repair were prepared from macromolecules containing carbamate linkages. vii TABLE OF CONTENTS F. vi TABLE OF CONTENTS.

Vill LIST OF 00. X LIST OF FIGUR. HH HH TH HH HH TH HH Hà HH Hàng xi LIST OF SCHEMES. ng TT nh TT nh TH HT TH TH TT TH XIV LIST OF ABBREVIATIONS.

XV CHAPTER 1—Dendritic Macromolecules: From Synthesis to Biomedical Engineering Applications TT. 1 Introduction to DenndrirmeTS§.- t1 vn ng HT TH nh TT HH TH TH nhện 1 Adhesives for Sealing Corneal Wounnds. --- - «ch ng HH ykp 12 67/2-/z4//7/1. 17 Chondroitin Sulfate Aldehyde (Š€QÏŒ1HÉS.

tk nh nh Hi gcr 19 Biodendritic-based Hydrogel] Ádl€SÏVÉS. sàng TH TH TH HH nhờ 19 Scaffolds for Cartilage R. ác TS vn SH HH1 ng Hy TH ng ng nêu 20 Current Treatments—Non-surgical and SUrgicdl .cccccccscccscssecsssscessssscesseseseneessenees 23 Non-injectable SCQ[ƒOÏdẲS. ác vn TT TH TH TH ng Hà Hà TT v4 26 Injectable SCQ[ŸOÏ(Í.

nh HT TH TH TT TH TH Tà HT HH TH Hà Hiện 31 Biodendritic-based Hydrogel Scaffolds .ccccsccsssessseessesssesssccesseseveessesensssesssessssenens 35 CHAPTER 2—Synthesis and Characterization of Bola-type Amphiphilic Dendritic II 242010101 An. ‹ddd:‹::11Y 36 Results and DiSCUSSION 0A nh aga. 37 SẠS)595428552)92v510I00PnẼnĐ hố. 52 Characterization of Hydrogels .sccsscscssesessesesessesesesseeeseeseeeseesecesseessenessessecseesesseesseaseascssssecausnessassesarensenenes 67 CHAPTER 3—([G1]-PGLSA-MA),-PEG Biodendritic Macromolecules as Ophthalmic Adhesives for Central Lacerations and Penetrating Keratoplasties.

69 Experimental] Sef-Up. «ch nàng HT Hà HH HH HT Hà TH Tà HT t0, 70 Results and Discussion for the Central Laceration S†udy.- :ccccccccccsretees 75 Results and Discussion for the Penetrating Keratoplasty Study.:---‹ccc c5: 78 Results and Discussion for the India ink stUdy. cv vtvereirrrrrtrererrrire 82 SUMIMALY 0 ố ốốốố ốốốố. 83 CHAPTER 4—Synthesis of Carbamate-based Photocrosslinkable Biodendritic Scaffolds: Implications for Tissue EngIn€€rifiE.

1g HH eg 86 IntrOdUCfIOTA. - ng TT nh HH nh HH TH TT gà TH TH TT 01111101514 114 111 110g 86 Results and D1SCUSSIOTI. nh HH HH ng HH HT TT TT g0 11100 91 Characterization of Macromolecules .cccecsssessessscesessecesececsaseseesseseseessesseserssseeses 94 Hydrogel PrepraftiOr. TT TH HH Hà KH Tà H1 11 1kg 102 Characterization of HydrogeÌS.- - c1 v39 TH ng HH nh Hi Hiệp 103 Ex Vivo MRI Integration SŠfudy.

ác HH nh HH HH TT nh HH tiệt 112 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. 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. ngiu 128 Results and Discussion for In vIvo SfUd1€§. tk Hy He, 138 Evaluation Of SCQƒƒOÏỈS. cá cLtEtkEt KH HT Ty KT TT TH ch ng HH 139 SUMMALY 00.

142 CHAPTER 6—Methods and Materials. - Ác 1 121191100111 1110 1 ng gu 143 "0551 0 eeesesccsecesesscnscssesresscsecsecsecseesessessessessesssessseseseseseessassessassassesasensateasnes 143 InstrUI€TIfAfIOTI. HT Tà TT TT HT HT gà Hà TT Tà TH gà 0114 Hee 143 NGA. 212 1X LIST OF TABLES Table 1.1: Performance characteristics of various corneal wound closures as compared to biodendrimer based adhesives, 106 1001 eetg ga 0110111101111 711711120.2: Summary of surgical techniques for OA pafienIs.1: MALDI-TOF and SEC data for ({Gn]-PGLSA)2-PEG3400, 10000, and 20000 6i1990/10I<91171- 28AA8Ẻ8Ẽ.2: MDSC data for ([Gn]-PGLSA)2-PEG3400, 10000, and 20000 Macromolecules.3: The critical aggregation concentrations (CAC) for each soluble generation within the three PEG macromolecules.

All CAC are reported as mM concentrations.1: MALDI-TOF and SEC data for ester and carbamate-based biodendritic MACTFOMOLECUIES.2: MDSC data for carbamate-based biodendritic macromolecules.3: CAC values for carbamate and ester-based macromolecules. 101 LIST OF FIGURES Figure 1.1: Basic dendrimer SỈTUCfUTC. -- 5á nhìn Hàn HH HH tu 2 Figure 1.2: Convergent approaches towards the synthesis of dendrons and dendrimers.3: Divergent method for the synthesis of dendrimers .-- -- ¿5c cc cà: 4 Figure 1.4: Synthetic scheme of cascade poÏy1m€TS.5: Synthesis of PAMAM dendrimers.-- nh Hàn He 8 Figure 1.6: Synthesis of poly(aryl ether) dendrOTis.7: Synthesis of poly(aryl ether) dendrimer.- -- ca cành 10 Figure 1.8: Fourth generation poly(glycerol succinic acid) dendrimer.9: ABA triblock architecure of biodendrirmers.10: Structure of the monomer unit in cyanoacrylate øÌues.11: Two-component adhesive composed of oxidized chondroitin sulfate and 0.12: Osteoarthritic cartilage in a knee JO]TIÍ.- ó1 vn nhi, 24 Figure 1.13: Surgical procedure for state of the art treatment (Cartieel”*9.14: Mechanical properties of hyaline cartilage.15: Non-injectable synthetic polymer scaffolds for cartilage repair.16: Non-injectable natural polymer scaffolds for cartilage repa1r.17: Synthetic polymer scaffolds for in situ cartilage repair that are (A) thermal activated, or (B) photochemical actIvated. óc LH nh Hye 34 Figure 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 38 Figure 2.2: 'H NMR spectra of ([G3]-PGLSA-bz1d);-PEGioooo (top) and ({G3]-PGLSA- OH)2-PEGioo00 Macromolecules (DOffOrn).3: 'H NMR of([G3]-PGLSA-MA);-PEGioooo macromolecule.4: Formation of a (G1]-PGLSA-MA);-PEG hydrogel.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 1.6: 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.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 57 Figure 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).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 hydrogels at 5, 7.5, 10, and 20% w/v before swelling (top) and after swelling (ĐOffOfm). nh HH HH HH HH ng HH 011111211110 11 1410111117111101110 62 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).11: Diffusion coefficients of (MA)2-PEG3a00, 10000, and 200005 (G0]-PGLSA-MA);- PEG3400, 10000, and 20000, and ([G1]-PGLSA-MA) -PEG3<00, 10000, and 20000 hdyrogels at four different concentration (5, 7. ng HH 66 Figure 3.1: Cross sectional view of the human COFn€â.-- cà che 71 Figure 3.3: Leaking pressures for the 4.1 mm central lacerations with three different hydrogel adhesives at 10, 20, and 40% W/V.

cà kh HH Hà Hà Hà HH Hà nh 77 Figure 3.4: Leaking pressures for PKP autografts sealed with either 8 or 16 10-0 nylon sutures and three different adhesive formulations at 20% W/V.