Design and Synthesis of Multifunctional Polyesters With

DESIGN AND SYNTHESIS OF MULTIFUNCTIONAL POLYESTERS WITH

'PEPTIDE-LIKE' PENDANT GROUPS

A Dissertation

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

Ying Xu

May, 2016 DESIGN AND SYNTHESIS OF MULTIFUNCTIONAL POLYESTERS WITH

'PEPTIDE-LIKE' PENDANT GROUPS

Ying Xu

Dissertation

Approved: Accepted:

______Advisor Department Chair Dr. Abraham Joy Dr. Coleen Pugh

______Committee Member Dean of the College Dr. Matthew L. Becker Dr. Eric J. Amis

______Committee Member Dean of the Graduate School Dr. Coleen Pugh Dr. Chand Midha

______Committee Member Date Dr. Ali Dhinojwala

______Committee Member Dr. Younjin Min

ii ABSTRACT

Current biomaterials including polylactic acid have good mechanical and biodegradable properties.1 But they are devoid of functional groups that enable integration with the cellular environment. We have designed a platform of modular multifunctional polyesters with pendant functional groups that address the lack of functional cues in current biomaterials.2 The polyesters were synthesized at room temperature by carbodiimide- mediated polymerization of pendant functionalized diols and succinic acid.3 The pendant groups were designed to mimic the side chains of peptides. It was shown that the physical properties of the polyesters can be modulated over a wide range by the selection of pendant groups. Orthogonal functionalization of the pendant groups with ligands such as fluorophores, poly (ethylene glycol) (PEG) or Arg-Gly-Asp (RGD) was shown.

One specific application of functional polyester was the design of mussel inspired adhesives by incorporation of catechol groups into side chain of such polyesters. The first generation adhesive polyester showed the effect of 3,4-dihydroxyphenylalanine (DOPA) groups, but the adhesion strength on aluminum substrate decreased in wet conditions. The second generation adhesive polyester was a copolymer with soybean oil based monomer, coumarin and DOPA monomer. It showed good adhesion under both dry and wet conditions.

Adhesion tests on porcine skin were also performed and the results demonstrated that our polymer had higher adhesion strength than the commercial fibrin glue.

iii A second application was the fabrication of nanofiber mats through electrospinnin for extended dual release of model drugs. Two types of electrospun fiber mats were made.

For one of them, two dyes (Rhodamine B and coumarin dye) were non-covalently encapsulated within the polymer fibers. For the other one, rhodamine B was covalently attached to the fibers, while coumarin dye was physically entrapped. For the fibers with non-covalently encapsulated dyes, the release of dyes over 90 days showed that the coumarin dye had a faster release profile compared to the rhodamine B dye. For the fibers where coumarin dye was encapsulated and rhodamine B was tethered, the release of coumarin dye was similar to the first one. The oxime bond of the covalently tethered rhodamine B was stable over 90 days, and there was no release of rhodamine B in

1×phosphate buffer saline(PBS) (pH = 7.4) which was similar to the studies by Raines and coworkers.4

Another project was to study the differentiation of stem cell into osteoblasts. Three polymers with 40% of carboxylic (COOH), amine (NH2), or hydroxyl (OH) pendant groups, were synthesized. The three polymers were used for examining the differentiation of mouse pre-osteoblast cell lines (MC3T3) into osteoblasts. Alkaline Phosphatase (ALP) staining and ALP acitivity of MC3T3 differentiated for 14 days were performed. From the ALP staining images, it was seen that the ALP production increased as COOH > blank > NH2 >

TCPS (Tissue Culture treated Polystyrene) > OH. Alizarin Red staining and von Kossa staining of the MC3T3 differentiated for 21 days were performed. From Alizarin Red staining, polymer with COOH group demonstrated the largest influence on differentiation, it was seen that the polymers with COOH or NH2 provided the most differentiation.

iv ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor, Professor Abraham Joy, for all his support, encouragement, and guidance during the past few years. I have benefited tremendously from his passion for science, profound knowledge and creative ideas. I would like to thank Professor Ali Dhinojwala for the collaboration and his valuable discussions and suggestions. I would like to take this opportunity to thank my dissertation committee members: Professor Coleen Pugh, Professor Matthew Becker, Professor Ali Dhinojwala and Professor Younjin Min for their valuable time and suggestions. I would like to thank

Dharamdeep Jain for the collaboration. I appreciate the help from students in College of

Polymer Science and Engineering, in particular Erin Childers, Dr. Shiwang Chen, Dr. Hao

Sun, Qianhui Liu, Dr. Shuangyi Sun, Dr. Sachin Gokhale, John Swanson, Chao Peng and all my group members. Their kind friendship, support and encouragement have helped me to go through this special journey in my life. Finally, I would like to give my special thanks to my husband Jinjun Zhou, my sister and my parents for their sincere love and understanding, and support during my Ph.D. studies.

v TABLE OF CONTENTS

Page

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

LIST OF SCHEMES ...... xvii

CHAPTER

I. INTRODUCTION ...... 1

1.1 Evolution of Biomaterials ...... 1

1.2 Synthesis of Functional Aliphatic Polyesters ...... 3

1.2.1 Functional aliphatic polyesters synthesized via condensation ...... 4

1.2.2 Functional aliphatic polyesters made by ROP ...... 5

II. A LIBRARY OF MULTIFUNCTIONAL POLYESTERS WITH ‘PEPTIDE-LIKE’ PENDANT FUNCTIONAL GROUPS ...... 17

2.1 Abstract ...... 17

2.2 Introduction ...... 17

2.3 Experimental Section ...... 19

2.3.1 Materials and instrumentation ...... 19

2.3.2 Synthesis of functional diols with different pendant Groups ...... 20

2.3.3 Synthesis of functional polyesters ...... 33

2.3.4 1H NMR of polyesters ...... 34

2.3.5 Conjugation of dyes to p(mLys0.5-co-Propargyl0.5) ...... 39

vi t 2.3.6 Functionalization of p(mAla0.4-co-mAsp Bu0.4-co-Propargyl0.2) with PEG and RGD ...... 40

2.4 Results and Discussion ...... 43

2.4.1 Synthesis of functional polyesters and modulation of physical properties ...... 43

2.4.2 Conjugations of two dyes to functional polyester ...... 49

2.4.3 Conjugation of RGD and PEG to functional polyester and cell adhesion study ...... 51

2.5 Conclusion ...... 54

III. MUSSEL INSPIRED ADHESIVES BASED ON ‘PEPTIDE-LIKE’ FUNCTIONAL POLYESTER ...... 55

3.1 Abstract ...... 55

3.2 Introduction ...... 56

3.3 Experimental Section ...... 59

3.3.1 Materials and Instrumentation ...... 59

3.3.2 Synthesis and characterization of monomers ...... 60

3.3.3 Synthesis of polymers ...... 64

3.3.4 Water contact angle measurement ...... 68

3.3.5 Lap shear adhesion test of catechol containing polymers and control polymers ...... 69

3.3.6 End to end adhesion test of p(SCD) and p(SCP) on porcine skin ...... 70

3.4 Results and Discussion ...... 71

3.5 Conclusion ...... 81

IV. ELECTROSPUN FIBER MAT FOR SUSTAINABLE DRUG RELEASE ...... 83

4.1 Abstract...... 83

4.2 Introduction ...... 83

4.3 Experimental Section ...... 85

vii 4.3.1 Materials and instrumentation ...... 85

4.3.2 Synthesis of rhodamine B derivative ...... 86

4.3.3 Synthesis of p(mPhe-Keto-BocGlu) ...... 90

4.3.4 Conjugation of rhodamine B alkoxyamine derivative with p(mPhe-Keto- BocGlu) ...... 91

4.3.5 Electrospinning of p(mPhe-Keto-BocGlu), 7-(diethylamino)coumarin-3- carboxylic acid, and rhodamine B ...... 92

4.3.6 Electrospinning of p(mPhe-Keto-BocGlu)-RB and 7-(diethylamino)coumarin- 3-carboxylic acid ...... 92

4.3.7 UV-Vis standard curve of rhodamine B in PBS ...... 93

4.3.8 UV-Vis standard curve of 7-(diethylamino)coumarin-3-carboxylic acid in PBS ...... 94

4.3.9 Release study of two dyes ...... 94

4.3.10 Degradation study of the fiber mat ...... 95

4.4 Results and Discussion ...... 95

4.5 Conclusion ...... 102

V. FUNCTIONAL POLYESTERS FOR OSTEOBLAST DIFFERENTIATION ...... 103

5.1 Abstract ...... 103

5.2 Experimental Section ...... 105

5.2.1 Materials and instrumentation ...... 105

5.2.2 Synthesis of polyesters with carboxylic, amine, and hydroxyl group ...... 106

5.3 Results and Discussion ...... 114

5.4 Conclusion ...... 124

VI. SUMMARY ...... 125

6.1 The Platform of Functional Polyesters with Peptide-Like Pendant Groups ...... 125

6.2 Mussel Inspired Adhesives Based on ‘Peptide-Like’ Functional Polyester ...... 128

viii 6.3 Electrospun Fiber Mat for Sustainable Drug Release ...... 131

6.4 Functional Polyesters for Osteoblast Differentiation ...... 133

REFERENCES ...... 135

APPENDIX ...... 149

ix LIST OF TABLES

Table Page

2.1 Characterization data summary for the ‘peptide-like’ polyesters …………………38

4.1 Molecular weights of p(mPhe-Keto-BocGLU)-RB fiber mats immersed in 1x PBS at 37 °C for different time…………………………………………………………101

5.1 Characterization of the polymers………………………………………………….115

5.2 Characterization of polymers……………………………………………………..122

x LIST OF FIGURES

Figure Page

1.1 Alizarin Red S staining (A, B; positive stain is red) and von Kossa staining (C, D; positive stain is black) of osteoblasts cultured on PSeD (A, C) and PLGA (B, D). More mineralized ECM was observed for osteoblasts seeded onto PSeD than those seeded onto PLGA (B, D) after 28 days in culture (magnification 40×, scale bar = 100 µm)…………………………………………………………………………………11

1.2 A simple synthetic strategy produced functionalizable PFM with two steps. Three functional groups, alkenyl, hydroxyl, and carboxyl, enabled versatile modifications of the physical, thermal, mechanical, and biological properties of the polymer…….12

1.3 Stress-strain curve of PFM. PFM showed strain dependent moduli with a compression stress and modulus in the range of those in native bones (130 - 180 MPa)…...... 13

1.4 Fluorescence images of DIBO - PCL nanofibers showing the covalent coupling of 9- methylene azidoanthracene. The scale bars are 20 mm (A) and 10 mm (B) respectively………………………………………………………………………...14

1.5 Fluorescence image of GRGDS modified nanofibers (A); a control experiment showing no fluorescence for non-GRGDS-treated nanofibers (B); UV−visible absorbance spectra of dopamine-functionalized nanofibers demonstrating about 16% of the available ketone groups reacted with NH2O-dopamine by comparing the 288 nm peak to a NH2O-dopamine solution of known concentration (C). A fluorescence image of BMP-2 peptide functionalized nanofibers (D) and a control experiment showing no fluorescence confirm the orthogonal nature of the reaction (E). The scale bar is 50 μm for all fluorescence images……………………………………………16

2.1 Representative examples of polyesters having mono, di and tri-functional pendant groups………………………………………………………………………………45

2.2 Variation of contact angles with copolymer composition…………………………46

2.3 Variation of Tg with polymer composition and its comparison to calculated values from Fox equation…………………………………………………………………..47

xi 2.4 Plot of plateau modulus vs mPhe content for copolymers of p(mPhe-co- mAsptBu)…………………………………………………………………………..48

2.5 SEC traces of the degradation of p(mAla) in PBS buffer (pH 7.4)…………………48

1 2.6 H NMR Spectrum of 10 % AA conjugated p(mLysBoc0.5-co-Propargyl0.5)………49

2.7 a) RI and UV response from SEC of p(mLysBoc0.5-co-Propargyl0.5),b) AA tagged p(mLysBoc0.5-co-Propargyl0.5. UV monitored at 350 nm…………………………50

2.8 IR spectrum of p(mLysBoc0.5-co-Propargyl0.5) and 10% AA tagged p(mLysBoc0.5- co-Propargyl0.5)…………………………………………………………………....50

2.9 a) Polyester tethered with AA and FITC b) UV absorbance spectrum of FITC-AA conjugated polyester (in water) and fluorescence spectrum of FITC-AA conjugated polyester (at 370 nm excitation) in DMSO-water (90:10)…………………………51

2.10 A) polyesters with PEG, RGD or PEG+RGD used for smooth muscle cell spreading studies b) cell area (over 50 cells) on glass coverslips (control), the base polyester (B), PEG conjugated polyester (BP), PEG and RGD conjugated polyester (BPR) and RGD conjugated polyester (BR). Images of cell spreading on the above samples: control (c); base polyester, B (d); BP (e); BPR (f); BR (g); scale bar = 25 µm (c-g)…………53

3.1 Pendant functionalized polyesters p(mAla0.8-co-mDOPA0.2) (A) and with p(mAla0.8- co-mPhe0.2) (B) serve to investigate the role of DOPA and hydrophilicity in adhesion between polymer and substrate……………………………………………………..72

3.2 Evolution of water contact angle and droplet width on spin-coated polyester films of p(mAla0.8-co-mDOPA0.2)(catechol polymer) (A) and with p(mAla0.8-co-mPhe0.2) (control polymer)………………………………………………………………….73

3.3 Lap shear geometry to test the adhesive strength of the polyesters (left). Lap shear strength of the p(mAla0.8-co-mDOPA0.2) (catechol) and p(mAla0.8-co-mPhe0.2) (control) polyesters under dry and wet conditions (right)………………………….74

3.4 Rheological properties of the SCD polyester at varying frequencies before and after crosslinking (A) and their corresponding viscosities at varying shear rates (B)……78

3.5 Evolution of contact angles for DOPA containing polyester, p(SCD) and the control polyester, p(SCP)…………………………………………………………………..78

3.6 Lap shear measurements of p(SCD) and p(SCP) under dry conditions (A) and wet conditions (B)………………………………………………………………………80

3.7 End to end test adhesion test geometry on porcine skin (A), adhesion strength for p(SCD), p(SCP), and fibrin glue (B)………………………………………………81

xii 4.1 UV-Vis standard curve of rhodamine B in PBS……….……………………………93

4.2 UV-Vis standard curve of 7-(Diethylamino)coumarin-3-carboxylic acid in PBS..…94

4.3 1H NMR of p(mPhe-Keto-BocGLU)…………………………….………………95

4.4 (A) SEM image of p(phe-keto-BocGLU)/1% Coumarin dye/ 0.5% Rhodamine B electrospun mat, (B) Diameter distribution of image A, (C) SEM image of p(phe- keto-BocGLU)-RB/1% Coumarin dye electrospum mat, (D) Diameter distribution of image C………...………………………………………………………………..97

4.5 (A), (C) Fluorescence image of p(phe-keto-BocGLU)-RB/1% Coumarin dye electrospun mat excited at 345 nm. (B), (D) Fluorescence image of p(phe-keto- BocGLU)-RB/1% Coumarin dye electrospun mat excited at 555 nm……………98

4.6 Cumulative release of coumarin dye and rhodamine B from fiber mat of p(mPhe- Keto-BocGLU) mixed with rhodamine B and 7-(Diethylamino)coumarin-3- carboxylic acid……………………………………………………………………99

4.7 Cumulative release of coumarin dye and rhodamine B from fiber mat of p(mPhe- Keto-BocGLU)-RB mixed with 7-(Diethylamino)coumarin-3-carboxylic acid…100

4.8 Plot of molecular weights vs days of immersing in 1x PBS……………………….101

4.9 GPC traces of degraded polymers…………………………………………………102

5.1 Water contact angle of polymers with COOH, NH2, and OH functional groups…115

5.2 Vinculin Immunofluorescent Staining of the MC3T3 cultured for 24 hours on A) Blank coverslip, coverslips coated with COOH (B), OH (C), NH2 (D) functionalized polymers…………………………………………………………………………..116

5.3 Alkaline Phosphatase (ALP) Staining of the MC3T3 differentiated for 14 days on A) Tissue Culture treated Poly(styrene) (TCPS), B) Blank coverslip, coverslips coated with COOH(C), OH(D), NH2(E) functionalized polymers………………………..117

5.4 Alkaline phosphatase activity……………………………………………………..118

5.5 Alizarin Red staining of the MC3T3 differentiated for 21 days on A) TCPS, B) Blank coverslip, coverslips coated with COOH(C), OH(D), NH2(E) functionalized polymers…………………………………………………………………………..119

5.6 von Kossa staining of the MC3T3 differentiated for 21 days on A) Tissue Culture treated Poly(styrene) (TCPS), B) Blank coverslip, coverslips coated with COOH(C), OH(D), NH2(E) functionalized polymers…………………………………………120

5.7 Water contact angle of polymers…………………………………………………..123

xiii 5.8 Cumulative release of OA peptide from electrospun fiber………………………123

6.1 Variation of contact angles with copolymer composition…………………………126

6.2 Variation of Tg with polymer composition and its comparison to calculated values from Fox equation………………………………………………………………126

6.3 a) Polyester tethered with AA and FITC b) UV absorbance spectrum of FITC-AA conjugated polyester (in water) and fluorescence spectrum of FITC-AA conjugated polyester (at 370 nm excitation) in DMSO-water (90:10)…………………………127

6.4 Lap shear geometry to test the adhesive strength of the polyesters (left). Lap shear strength of the p(mAla0.8-co-mDOPA0.2) (catechol) and p(mAla0.8-co-mPhe0.2) (control) polyesters under dry and wet conditions (right)………………………129

6.5 Lap shear measurements of p(SCD) and p(SCP) under dry conditions (A) and wet conditions (B)……………………………………………………………………..130

6.6 End to end test adhesion test geometry on porcine skin (A), adhesion strength for p(SCD), p(SCP), and fibrin glue (B)……………………………………………131

6.7 Cumulative release of coumarin dye and rhodamine B from fiber mat of p(mPhe- Keto-BocGlu) mixed with rhodamine B and 7-(Diethylamino)coumarin-3- carboxylic acid……………………………………………………………………132

6.8 Cumulative release of coumarin dye and rhodamine B from fiber mat of p(mPhe- Keto-BocGLU)-RB mixed with 7-(Diethylamino)coumarin-3-carboxylic acid…132

6.9 Cumulative release of OA peptide from electrospun fiber………………………133

A.1 1H NMR (DMSO-d6) of mTrp monomer…………………………………………149

1 A.2 H NMR (CDCl3) of mTyrBn monomer………………………………………..149

1 A.3 H NMR (CDCl3) of Keto monomer……………………………………………150

1 A.4 H NMR (CDCl3) of Azide monomer……………………………………………..150

1 A.5 H NMR (CDCl3) of mPhe monomer……………………………………………..151

1 A.6 H NMR (CDCl3) of mAla monomer………………………………...…………...151

1 t A.7 H NMR (CDCl3) of mAsp Bu monomer…………………………………………152

1 A.8 H NMR (CDCl3) of mSerTBDMS monomer……………………………………152

1 A.9 H NMR (CDCl3) of mLysBoc monomer…………………………………………153

xiv 1 A.10 H NMR (CDCl3) of mGluBn monomer………………………………………..153

1 A.11 H NMR (CDCl3) of Cat monomer……………………………………………..154

1 A.12 H NMR (CDCl3) of Cou monomer…………………………………………….154

1 A.13 H NMR (CDCl3) of SBO monomer……………………………………………155

1 A.14 H NMR (CDCl3) of p(mAla)…………………………………………………..155

1 A.15 H NMR (CDCl3) of p(mPhe)……………………………………………………156

1 A.16 H NMR (CDCl3) of p(mLysBoc)………………………………………………156

1 A.17 H NMR (CDCl3) of p(mSerTBDMS)………………………………………….157

A.18 1H NMR (DMSO-d6) of p(mSer)………………………………………………157

1 A.19 H NMR (CDCl3) of p(mGluBn)……………………………………………….158

1 A.20 H NMR (CDCl3) of p(N3)……………………………………………………...158

1 A.21 H NMR (CDCl3) of p(mTyrBn)………………………………………………..159

A.22 1H NMR (DMSO-d6) of p(mTrp)……………………………………………....159

1 A.23 H NMR (CDCl3) of p(mAla-co-mPhe)0.66:0.34…………………………………...160

1 A.24 H NMR (CDCl3) of p(mAla-co-mPhe)0.5:0.5……………………………………160

1 A.25 H NMR (CDCl3) of p(mAla-co-mPhe)0.25:0.75………………………………….161

1 A.26 H NMR (CDCl3) of p(mPhe-co-mGluBn)0.87:0.13………………………………161

1 A.27 H NMR (CDCl3) of p(mPhe-co-mGluBn)0.73:0.27……………………………...162

1 A.28 H NMR (CDCl3) of p(mPhe-co-mGluBn)0.5:0.5…………………………………162

1 t A.29 H NMR (CDCl3) of p(mPhe-co-mAsp Bu)0.9:0.1…….………………………....163

1 t A.30 H NMR (CDCl3) of p(mPhe-co-mAsp Bu)0.75:0.25……………………………..163

1 t A.31 H NMR (CDCl3) of p(mPhe-co-mAsp Bu)0.6:0.4…………………………….....164

1 A.32 H NMR (CDCl3) of p(mSerTBDMS-co-mPhe)0.75:0.25…………………………164

1 A.33 H NMR (CDCl3) of p(mSer-co-mPhe)0.75:0.25………………………………….165

1 A.34 H NMR (CDCl3) of p(mSerTBDMS-co-mPhe)0.5:0.5…………………………..165 xv 1 A.35 H NMR (CDCl3) of p(mSer-co-mPhe)0.5:0.5……………………………………166

1 A.36 H NMR (CDCl3) of p(mSerTBDMS-co-mPhe)0.25:0.75………………………...166

1 A.37 H NMR (CDCl3) of p(mSer-co-mPhe)0.25:0.75………………………………….167

1 A.38 H NMR (CDCl3) of p(mLysBoc-co-Propargyl)0.5:0.5……………………………167

1 t A.39 H NMR (CDCl3) of p(mAla-co-mAsp Bu-co-Propargyl)0.4:0.4:0.2………...... …168

A.40 1H NMR (DMSO-d6) of p(mAla-co-mAsp-co-Propargyl)……………………..168

1 A.41 H NMR (CDCl3) of p(mAla-co-mPhe)0.8:0.2……….…………………………...169

1 A.42 H NMR (CD3OD) of Rhodamine B-Base………………………………………169

1 A.43 H NMR (CD3OD) of Rhodamine B Piperazine amide……………………………170

1 A.44 H NMR (CD3OD) of Rhodamine B NHBoc……………………………………...170

1 A.45 H NMR (CDCl3) of p(mPhe-keto-BocGLU)0.8:0.2………………………………171

1 t A.46 H NMR (CDCl3) of p(mPhe-mAla-mAsp Bu-But)……………………………171

1 A.47 H NMR (CDCl3) of p(mPhe-mAla-mLysBoc-But)……………………………172

1 A.48 H NMR (CDCl3) of p(mPhe-mAla-mSerTBS-But)……………………………172

1 t A.49 H NMR (CDCl3) of p(mPhe0.1-mAla0.4-mAsp Bu0.3-Keto0.2)………………….173

1 A.50 H NMR (CDCl3) of p(mPhe0.1-mAla0.4-mAsp0.3-Keto0.2)……………………..173

1 t A.51 H NMR (CDCl3) of p(mPhe0.4-mAla0.1-mAsp Bu0.3-Keto0.2)………………….174

1 A.52 H NMR (CDCl3) of p(mPhe0.4-mAla0.1-mAsp-Keto0.2)………………………174

xvi LIST OF SCHEMES

Scheme Page

1.1 The synthesis of MPPD-based PESu and PBAD by a two-stage melt polycondensation. MPPD: 2-methyl-2-propargyl-1,3-propanediol. PESu: Poly- (ethylene succinate) PBAD: poly(butyleneadipate)……………….....……………4

1.2 Functionalized lactones suitable for ROP……………………….………………...... 5

1.3 Synthesis of protected hydroxyl and dihydroxyl monomers. Reaction conditions: (a) NaH, benzyl bromide, 25 °C, 16 h; (b) 2,2’-bis(phenyldioxymethyl)-propionyl chloride, Et3N, 25 °C, 24 h; (d) m-CPBA (60%), 24 h…………...... …………………6

1.4 Synthesis of protected carboxyl monomers. Reaction conditions: (a) PCC, 45 °C, 7 h; (b) H2SO4 (2%), 115 °C, 4 h; (c) K2CO3, benzyl bromide, 60 °C, 3 h; (d) (1) oxalyl chloride, DMF, 24 h (2) t-BuOH, methylamide, 25 °C, 1.5 h; (e) m-CPBA (60%), 65 °C, 2h………………..…………………………………………………………....6

1.5 Synthesis of protected amine monomer. Reaction conditions : (a) trifluoroacetic anhydride, 60 °C, 3 h; (b) p-TSA, 80 °C, 48 h; (c) m-CPBA (60%), 25 °C, 14 h……7

1.6 Synthesis of functionalized polycaprolactones and deprotection of protected groups.7

1.7 Synthesis of polycaprolactones with substituted amines in the backbone …………...8

1.8 Functional lactide and glycolide………………………………………..……………9

1.9 The synthetic strategy of PSeD leads to a biodegradable polymer that retains the biocompatibility of PGS but with a more defined structure that is advantageous for subsequent functionalization………………………………………….……………10

1.10 A model reaction for the conjugation of biomolecules to PSeD……………………11

1.11 Random copolymers of poly(ε-caprolactone-co-2-oxepane-1,5-dione) were prepared by ROP of caprolactone and 1,4,8-Trioxaspiro[4.6]-9-undecanone, followed by the deprotection of the ketone groups by triphenylcarbenium tetrafluoroborate……….13

1.12 The polymerization of Ɛ-caprolactone is initiated by 4-dibenzyocyclooctynol using normal Sn-based catalytic conditions………………………………………………14

xvii 1.13 Synthetic route for desired polymers with DIBO, ketone, alkyne, and alkene functional groups…………………………………………………………………...15

2.1 Synthesis of tryptophan mimic monomer mTry...... ……..…………...... ……..…20

2.2 Synthesis of tyrosine mimic monomer mTyrBn………………….……………..…21

2.3 Synthesis of Keto monomer……………...………………………………………...22

2.4 Synthesis of azide monomer………………………………………………………..23

2.5 Synthesis of phenylalanine mimic monomer mPhe……………..…………………24

2.6 Synthesis of alanine mimic monomer mAla………………………………………..25

2.7 Synthesis of aspartic acid mimic monomer mAsptBu…………………………...... 26

2.8 Synthesis of serine mimic monomer mSerTBDMS………………………………..28

2.9 Synthesis of lysine mimic monomer mLysBoc………………………………….....30

2.10 Synthesis of Glutamic acid mimic monomer mGluBn……………………………31

2.11 Polyesterification of functional diol(s) with succinic acid………………………….33

2.12 Deprotection of benzyl group of p(mTyrBn)…...………………………………….33

2.13 Conjugation of dyes to p(mLys0.5-co-Propargyl0.5)……………………………….39

2.14 Post-polymerization functionalization of p(mAla0.4-co-mAsp0.4-co-Propargyl0.2) with PEGNH2 and RGDN3…………………………………………………………41

3.1 Synthesis of DOPA mimic monomer mDOPApr……………………...……………60

3.2 Synthesis of coumarin monomer Cou……………………………………………...62

3.3 Synthesis of soybean oil monomer SBO…………………………………………...63

3.4 Synthesis of p(mAla-co- mDOPA) and p(mAla-co-mPhe)………………………65

3.5 Synthesis of viscoelastic polyester p(SCD) from pendant functionalized diols of long

alkyl chain (S), coumarin (C) and DOPA (D) units…………………………………67

4.1 Synthesis of rhodamine B derivative……………………………………………….87

4.2 Synthesis of p(mPhe-Keto-BocGLU)……………………………………………..90

xviii 4.3 Conjugation of rhodamine B alkoxyamine derivative with p(mphe-keto-

BocGLU)…………………………………………………………………………..91

5.1 Synthesis of polyester with carboxylic pendant group…………………………….106

5.2 Synthesis of polyester with amine pendant group…………………………………107

5.3 Synthesis of polyester with hydroxyl pendant group……………………………108

5.4 Synthesis of polymer for carrier of OA peptide…………………………………....110

6.1 Polyesterification of functional diol(s) with succinic acid………………………125

6.2 Synthesis of viscoelastic polyester p(SCD) from pendant functionalized diols of long alkyl chain (S), coumarin (C) and DOPA (D) units………………………………..130

xix CHAPTER I

INTRODUCTION

1.1 Evolution of Biomaterials

Biomaterials have been defined as any substance (other than a drug) or combination of substances, synthetic or natural in origin, which can be used for any period of time, as a whole or a part of a system which treats, augments or replaces any tissue, organ or function of the body.5 The first generation of biomaterials were created to have physical properties matching the replaced tissue and generate minimal immune response. Additionally, they are usually biologically inert and have a long lifetime within the body. Over time, second generation biomaterials were developed and became widely used. The second generation of biomaterials differed from the first in that they are bioactive which means they are able to interact with tissue in a controlled manner. For example, bioactive glasses, ceramics, glass-ceramics, and composites have been widely used in orthopedic and dental applications.6 Bio-resorbable polymers which hydrolytically or enzymatically degrade and clear from the body also belong to the second generation. For example, biodegradable sutures made from polyesters such as polyglycolic (PGA) and polylactide (PLA) have been widely used. Third generation biomaterials were designed to stimulate specific cellular responses at molecular level.6 For example, Atala et al. designed a biodegradable urinary bladder made from collagen and polyglycollic acid. This was then seeded with autologous

1 urothelial and muscle cells and was cultured for 7 weeks, the autologous engineered bladder constructs were implanted.7 Biodegradable materials can be divided into naturally derived materials and synthetic biodegradable polymers. Naturally derived materials include proteins such as silk, collagen, elastin, and fibrin; and polysaccharides which include chitin, cellulose, and chitosan. The advantages of these materials are their bioactivity and biocompatibility. However they also have some disadvantages such as immunogenic responses (especially for protein based materials), batch to batch variation, and elaborate and complicated manipulations. Synthetic polymers include polyesters, poly(ester-ethers), poly(ester-amides), and poly(amino acids). Compared to naturally derived materials, synthetic polymers have tunable properties and batch-to-batch reproducibility. Furthermore, there are no concerns about immunogenicity. However, one of the drawbacks of current synthetic materials are the lack of functional groups and their typically hydrophobic nature.8

Biological processes are well-coordinated events that rely on the presentation of various functional cues, signaling molecules, growth factors etc. with exquisite temporal and spatial coordination.9-10 For example, neovascularization proceeds from initiation of angiogenesis to neovessel formation, adaptation to tissue need, and then to maturation.

Each stage is controlled by a different set of growth factors.11-13 Similarly, wound healing is a complex orchestration of action by the extracellular matrix (ECM), cells, growth factors and cytokines over several days and weeks.14-16 Such ‘recognition and action’ events are the norm in all biological processes. The challenge in synthetic systems, whether they are pharmaceuticals or biomaterials, is to design them such that they are able to emulate their biological counterparts.17-18

2 Recent efforts in the field of biomaterials are beginning to address the need for such precisely designed materials to stimulate complex biological processes.6, 19-22 This approach is a significant evolution from the early stages of biomaterials research which focused on inert materials such as poly(methyl methacrylate) (PMMA), and those inert materials do not interact with physiological environment.23 Although such materials are still useful for applications such as tooth implants and hip joints replacement, biodegradable polymers such as PLA and polycaprolactone (PCL) were designed to be resorbed in the hydrolytic and enzymatic physiological environment.8, 24-25 These materials have been used in several applications and the FDA has approved numerous devices made from such biodegradable polymers. However, these materials do not meet the criteria that are required for promoting specific biological processes such as angiogenesis, where multiple signaling cues are needed from the synthetic polymer.

1.2 Synthesis of Functional Aliphatic Polyesters

Polyesters can be synthesized by two routes. One is step-growth polymerization from hydroxy acid or diacid and diol, known as polycondensation. The main drawback of this method is the harsh reaction conditions, such as high temperature, which may cause some deleterious side effects. Additionally, molecular weight of the polyester depends strongly on monomer stoichiometry and conversion, and as such it is difficult to obtain high molecular weight (Mn< 30 kDa) and narrow polydispersities (Đ).26 The other polyester synthetic method is ring opening polymerization (ROP) of cyclic monomers.

Compared to polycondensation, ROP has more control of the molecular weight, molecular weight distribution. In addition, with ROP, block copolymers can be synthesized.27

3 1.2.1 Functional aliphatic polyesters synthesized via condensation

Billiet et al. synthesized a diol containing a propargyl group which was reacted with succinic acid or adipic acid through a two stage melt polycondensation process. The obtained polyesters were post functionalized with BzN3 or α-methoxy- ω-zido-PEG

28 (PEG550N3) by copper catalyzed azide alkyne Huisgen cycloaddition (Scheme 1.1).

Scheme 1.1. The synthesis of MPPD-based PESu and PBAD by a two-stage melt polycondensation. MPPD: 2-methyl-2-propargyl-1, 3-propanediol. PESu: Poly(ethylene succinate) PBAD: poly(butyleneadipate).28 Scheme produced with permission from Ref.

Yosuke and Akibori reported a one-step synthesis of a polyester bearing hydroxyl groups by the polycondensation of diols such as glycerol and sorbitol with succinic acid, and diacids such as tartaric acid and malic acid with various diol. The reactions were

4 performed at low temperature (60-80 °C) and catalyzed by rare-earth scandium trifluoromethane-sulfonate (Sc(OTf)3). The resulting polymer (Mn ~ 3-22 kDa) with pendant hydroxyl group could be further modified by glycosidation.29 Ji et al. synthesized functional polyesters with side-chain hydroxyl and vinyl groups using the Baylis-Hillman reaction which involves the condensation of an aldehyde and an acrylate with base catalysis.

The authors used 1,3-butanediol acrylate and 2,6-pyridinecarbox-aldehyde as monomers and 1,4-diazabicyclo[2.2.2]octane (DABCO) as catalyst. This route afforded polyesters

(Mn ~ 9 kDa) bearing hydroxyl and vinyl functionality which could be quantitatively modified using phenyl isocyanate and methyl-3-mercaptopropionate, respectively.30

1.2.2 Functional aliphatic polyesters made by ROP

Polyesters bearing various functional groups such as chloride,31-32 bromide,6 iodide,33 alkene,34 alkyne,35 hydroxyl,36-38 carboxyl,39-41 and ketone42 have been synthesized from functional lactones with or without protection through ROP (Scheme 1.2).

Scheme 1.2. Functionalized lactones suitable for ROP.6, 31-42 5

Scheme 1.3. Synthesis of protected hydroxyl and dihydroxyl monomers. Reaction conditions: (a) NaH, benzyl bromide, 25 °C, 16 h; (b) 2,2’-bis(phenyldioxymethyl)-

38 propionyl chloride, Et3N, 25 °C, 24 h; (d) m-CPBA (60%), 24 h.

Scheme 1.4. Synthesis of protected carboxyl monomers. Reaction conditions: (a) PCC,

45 °C, 7 h; (b) H2SO4 (2%), 115 °C, 4 h; (c) K2CO3, benzyl bromide, 60 °C, 3 h; (d) (1) oxalyl chloride, DMF, 24 h (2) t-BuOH, methylamide, 25 °C, 1.5 h; (e) m-CPBA (60%),

65 °C, 2h.38 6

Scheme 1.5. Synthesis of protected amine monomer. Reaction conditions : (a) trifluoroacetic anhydride, 60 °C, 3 h; (b) p-TSA, 80 °C, 48 h; (c) m-CPBA (60%), 25 °C,

14 h.38

Scheme 1.6. Synthesis of functionalized polycaprolactones and deprotection of protected groups.38 7

Scheme 1.7. Synthesis of polycaprolactones with substituted amines in the backbone.38

Hedrick and coworkers38 synthesized lactones with protected functional groups such as hydroxyl, bishydroxyl, amino, and carboxyl through Baeyer-Villiger oxidation of the corresponding precursors (Scheme 1.3-1.5). The polymerizations were performed either in bulk at 110 °C with a benzyl 2, 2’-bis(hydroxymethyl)-propionate initiator and

i stanneous 2-ethyl hexanoate (Sn(Oct)2) catalyst or in toluene at 0 °C catalyzed by Al(O Pr)3.

The benzyl protection groups were readily removed by hydrogenation over Pd/C and the tert-butyl group was removed under acidic condition (Scheme 1.6). The trifluoroacetyl group was removed by NaBH4 reduction (Scheme 1.7). Molecular weights (Mn) ranged from 5 to 33 kDa with relatively low polydispersity (1.2~1.5).

Similar to functional caprolactones, functional lactides have been synthesized and the corresponding polymers have been obtained as well. Several groups have made important contributions to this area.43-48 For example, Weck and coworkers46 synthesized

8 homo- and copolymers of PLA which contained amine, hydroxyl, and carboxylic acid functionalities through ring opening of functional lactide monomers. Kimura and coworkers reported the synthesis of poly(glycolic acid) with pendant carboxylic groups.47

Baker, Smith and coworkers synthesized a propargyl containing glycolide which was subsequently polymerized to obtain a poly(propargyl glycolide) homopolyester or copolymer with lactide. The propargyl group was further reacted with alkyl azide or PEG azide by copper(I) catalyzed Huisgen 1,3-dipolar cycloaddition.48 Scheme 1.8 shows some examples of functional lactide and glycolide.

Scheme 1.8. Functional lactide and glycolide.46, 48

Wang et al. reported a polyester bearing a hydroxyl group prepared from epoxide ring opening polymerization as shown in Scheme 1.9.49 The traditional polycondensation of sebacic acid and glycerol produced a branched polyester poly(glycerol sebacate) (PGS).

Poly(sebacoyl diglyceride) (PSeD) generated by ROP of sebacic acid and diglycidy sebacate was shown to have a more defined structure and linear backbone, more free

9 hydroxyl groups, higher molecular weight, and lower polydispersity than PGS.

Crosslinking PSeD with sebacic acid yielded a polymer five times tougher and more elastic than cured PGS. The hydroxyl group on PSeD could be functionalized using Boc protected glycine with high efficiency as shown in Scheme 1.10. Additionally, the growth of osteoblast and human mesenchymal stem cells on PSed was studied.50 As shown in Figure

1.1, PSeD promoted increased mineralization of extracellular matrix secreted by human mesenchymal stem cells and rat osteoblasts as compared to poly(lactic-co-glycolic acid)

(PLGA). PSeD showed in vitro osteocompatibility and in vivo biocompatibility that matched or surpassed that of PLGA, demonstrating the potential of PSeD for use in bone regeneration.

Scheme 1.9. The synthetic strategy of PSeD leads to a biodegradable polymer that retains the biocompatibility of PGS but with a more defined structure that is advantageous for subsequent functionalization.49 Scheme 1.9 produced with permission from Ref. (49)

Scheme 1.10. A model reaction for the conjugation of biomolecules to PSeD.49 Scheme

Figure 1.1. Alizarin Red S staining (A, B; positive stain is red) and von Kossa staining (C,

D; positive stain is black) of osteoblasts cultured on PSeD (A, C) and PLGA (B, D). More mineralized ECM was observed for osteoblasts seeded onto PSeD than those seeded onto

PLGA (B, D) after 28 days in culture (magnification 40×, scale bar = 100 µm).50 Figure

11 Wang’s group also made a functionalizable polymer, poly(fumaroyl bioxirane) maleate (PFM), bearing three functional groups: hydroxyl, carboxyl and alkenyl.51 They investigated the performance of PFM for bone tissue engineering. PFM was readily synthesized in two steps as shown in Figure 1.2. PFM showed strain-dependent moduli with mechanical strength approaching native bones as shown in Figure 1.3. PFM supported the adhesion, spreading, proliferation, and maturity of rat calvarial osteoblasts. The alkaline phosphatase activity of osteoblasts on PFM was significantly higher than that on tissue- culture-treated polystyrene in vitro. The physical, mechanical, and biological properties of

PFM could be modulated by various functionalizations to explore methods to improve bone tissue engineering and regenerative medicine in general.

Becker et al. synthesized a ketone functionalized poly(caprolactone) (PCL). The ketone group was post functionalized with clickable alkyn, azide, and methyl acrylate groups by oxime ligation (Scheme 1.11).52 They further conjugated the RGD peptide onto

PCL thin films by click reactions. The conjugation reaction was monitored by quartz crystal microbalance (QCM).

Figure 1.2. A simple synthetic strategy produced functionalizable PFM with two steps.

Three functional groups, alkenyl, hydroxyl, and carboxyl, enabled versatile modifications of the physical, thermal, mechanical, and biological properties of the polymer.51 Figure 1.2 produced with permission from Ref. (51) (Copyright © 2011 Acta Materialia Inc.).

Figure 1.3. Stress-strain curve of PFM. PFM showed strain dependent moduli with a compression stress and modulus in the range of those in native bones (130 - 180 MPa).51

Inc.).

Scheme 1.11. Random copolymers of poly(ε-caprolactone-co-2-oxepane-1,5-dione) were prepared by ROP of caprolactone and 1,4,8-Trioxaspiro[4.6]-9-undecanone, followed by the deprotection of the ketone groups by triphenylcarbenium tetrafluoroborate.52 52

Chemical Society). 13 The Becker lab also made end functionalized PCL by using 4-dibenzocyclooctynol

(DIBO) as an initiator for the ring-opening polymerization of Ɛ-caprolactone (Scheme

1.12).53 The resulting polymer was electrospun into nanofibers. The DIBO group was post functionalized with fluorescent 9-methylene azidoanthracene by strain-promoted azide alkyne cycloaddition (SPAAC). Figure 1.4 shows the florescence images of fiber conjugate with 9-methylene azidoanthracene.

Scheme 1.12. The polymerization of Ɛ-caprolactone is initiated by 4-dibenzyocyclooctynol using normal Sn-based catalytic conditions.53

Figure 1.4. Fluorescence images of DIBO - PCL nanofibers showing the covalent coupling of 9-methylene azidoanthracene. The scale bars are 20 µm (A) and 10 µm (B) respectively.53 Figure 1.4 produced with permission from Ref. (53) (Copyright © 2013,

Royal Society of Chemistry).

Scheme 1.13. Synthetic route for desired polymers with DIBO, ketone, alkyne, and alkene functional groups.54

By using 4-dibenzocyclooctynol (DIBO) as an initiator for the ring-opening copolymerization of Ɛ-caprolactone and 1,4,8-trioxaspiro[4.6]-9-undecanone (TOSUO),

Becker et al. made DIBO end functionalized copolymers with protected ketone groups

(Scheme 1.13).54 Following deprotection of the protected group, the free ketone functional groups were post functionalized with aminooxyl-containing compounds via oxime ligation.

Electrospun mixtures of keto- and alkyne- containing polymers were prepared. Strain- promoted azide alkyne cycloaddition(SPAAC), oxime ligation, and copper-catalyzed azide alkyne cycloaddition (CuAAC) were used to sequentially functionalize the nanofibers first with fluorescent dyes and then with bioactive Gly-Arg-Gly-Asp-Ser (GRGDS), BMP-2 peptide, and dopamine (Figure 1.5). The polymer containing end functionalized DIBO and ketone groups was electrospun into random and aligned nanofibers.55 GRGDS and YIGSR peptides were then coupled to the random and aligned nanofiber networks using metal-free

15 click chemistry and oxime condensation methodologies. Schwann cell attachment and proliferation were examined on the peptide tethered fibers. The inclusion of YIGSR with

GRGDS alters the expression of the receptor for YIGSR. Aligned nanofibers acted as a potential guidance cue by increasing the aspect ratio and aligning the actin filaments.

Figure 1.5. Fluorescence image of GRGDS modified nanofibers (A); a control experiment showing no fluorescence for non-GRGDS-treated nanofibers (B); UV−visible absorbance spectra of dopamine-functionalized nanofibers demonstrating about 16% of the available ketone groups reacted with NH2O-dopamine by comparing the 288 nm peak to a NH2O- dopamine solution of known concentration (C). A fluorescence image of BMP-2 peptide functionalized nanofibers (D) and a control experiment showing no fluorescence confirm the orthogonal nature of the reaction (E). The scale bar is 50 μm for all fluorescence images.54 Figure 1.5 produced with permission from Ref. (54) (Copyright © 2015,

American Chemical Society).

16 CHAPTER II

A LIBRARY OF MULTIFUNCTIONAL POLYESTERS WITH ‘PEPTIDE-LIKE’

PENDANT FUNCTIONAL GROUPS

2.1 Abstract

The synthesis and characterization of a library of modular multifunctional polyesters with pendant functional groups is described. The polyesters were synthesized at room temperature by carbodiimide mediated polymerization of pendant functionalized diols and succinic acid. The pendant groups are designed to mimic the side chains of peptides and it is shown that the physical properties of the polyesters can be modulated over a wide range by the choice of pendant groups. We also show that the pendant groups can be orthogonally functionalized with fluorophores, PEG or RGD.

2.2 Introduction

The design of biomaterials has seen an evolution from first generation inert materials such as stainless steel and poly(methyl methacrylate) to current biomaterials that interface with the body.23, 56 Subsequent to the development of inert materials, degradable polymers such as polyesters, polyanhydrides and polycarbonates were developed.57-58 In addition to this requirement for biodegradability, there is a crucial need for biomaterials that are appropriately functionalized to enable interaction with and integration into the

17 cellular environment.57, 59-60 Natural materials such as collagen provide optimum cell – extracellular matrix (ECM) interactions and oftentimes outperform synthetic materials in biomedical applications.61 However, due to their advantages of reproducibility and scale up, well designed synthetic polymers that provide optimal biological cues will find increasing applications in tissue engineering.

It is this design challenge that we sought to address in the present work. Our goal was to design a hydrophilic degradable polyester system wherein multiple pendant functional groups could be incorporated in a modular fashion. The pendant groups were chosen to mimic the functional repertoire of peptides and to provide orthogonal functional groups. It was hypothesized that similar to peptoids and peptides, the pendant groups would modulate the physical and biological properties of the polyesters. The repeating unit of the polyester was designed such that it would have an appreciably high level of hydrophilicity in order to improve interactions with the cellular environment and enable cells to ‘sense’ the functional groups on the polyester. In this chapter, we will report synthesis, characterization and properties of this novel class of biodegradable multivalent polyesters with ‘peptide-like’ pendant functional groups.

Non-degradable polymers with a diverse variety of multifunctional pendant groups have been synthesized.62-69 However, in several biomedical applications non-degradable systems are not the preferred choice. Degradable polymers such as poly(lactic acid) and polycaprolactone are commonly used in many tissue engineering applications, but they lack appropriate functional groups to enable interactions within a biological environment.43

Several recent reports related to the design of polyesters, polyesteramides and polyurethanes have addressed this lack of functionality.70-73 Hydroxyl functionalized

18 polyesters have been prepared by polymerization of diepoxides with diacids or by the polymerization of protected tartaric acids with diols.49, 74-76 Several functionalized polyesters have been prepared via ring opening polymerization of functionalized cyclic lactones.44, 77-82 Functionalized polyesteramides have also been synthesized and their post- polymerization modifications have been demonstrated.83-84 The inspiration for our current work stems from peptoids and peptides, wherein the repeat structure is shorter and has several nitrogen and oxygen atoms in the structure, which increase their hydrophilicity.85-

2.3 Experimental Section

Experimental section describes the chemicals needed for synthesis of the targeted products, the method of synthesizing monomers and polymers, and the characterization and testing of materials.

2.3.1 Materials and instrumentation

Materials: All the reagents were purchased from Sigma Aldrich or Alfa Aesar and used without further purification unless otherwise noted. Dichloromethane was dried by distilling over anhydrous CaH2 and the DMF was dried by distilling over anhydrous CaH2.

Instrumentation: Polymer molecular weights were analyzed on TOSOH EcoSec

HLC-8320 GPC, with two PSS Gram Analytical GPC Columns in series, using 10 mM

LiBr in DMF as eluent at a flow rate of 0.8 mL/min. The column and detector temperatures were maintained at 50 °C, the molecular weights were reported relative to polystyrene standard. NMR spectra of the monomers and polymers were obtained on Varian

19 MERCURY 300 MHz or Varian NMRS 500 MHz spectrometers. ESI/MS was performed on Bruker HTC ultra QIT. Thermal transitions were analyzed using TA differential calorimeter Q2000 with a liquid N2 cooling unit using cooling cycle of 10 °C/ min and heating rate also at 10 °C/ min. TA Q500 thermogravimetric analyzer was used to collect

5% decomposition temperature data from 25 °C to 600 °C at a heating rate of 10 °C/ min in a N2 atmosphere. UV-Vis spectra and fluorescence spectra were recorded using Synergy

TM MX plate reader from BioTek. Small amplitude oscillatory shear experiments were done using an ARES-LS rotational rheometer, with two 8 mm diameter parallel plates.

Frequency sweeps from 100 to 0.1 Hz were conducted to measure elastic modulus (G’) and viscous modulus (G”) at 37 °C.

Scheme 2.1. Synthesis of tryptophan mimic monomer mTrp.

2.3.2 Synthesis of functional diols with different pendant Groups

Methyl 3-indol-3-ylpropanoate (M2.1): 3-Indolepropionoic acid (3.0 g, 15.8 mmol) was dissolved in anhydrous MeOH (50 mL). Thionyl chloride (3.0 mL, 2.5 eq.) was added to the solution dropwise with an addition funnel under ice bath. Ice bath was removed after

30 min. The reaction was stirred at room temperature overnight. Solvent was removed by

1 rotary evaporation and dried with high vacuum. H NMR (300 MHz, CDCl3): 훿� 7.95 (br. 20 s., 1H), 7.57 - 7.68 (m, 1H), 7.33 - 7.43 (m, 1H), 7.09 - 7.26 (m, 2H), 6.99 - 7.07 (m, 1H),

3.59 - 3.74 (m, 3H), 3.12 (t, J = 7.9 Hz, 2H), 2.74 (m, J = 7.9 Hz, 2H).

Synthesis of mTrp: M2.1 (3.24 g, 15.8 mmol) and diethanol amine (3.33 g, 31.6 mmol) were taken into a 100 mL round bottom flask. The mixture was heated to 80 °C and stirred overnight. The reaction was purified by column with 5% MeOH/DCM. A yellow

1 viscous oil was obtained (3.5 g, 80%). H NMR (300 MHz, DMSO-d6): 훿� 10.74 (br. s.,

1H), 7.49 (d, J = 7.61 Hz, 1H), 7.31 (dd, J = 0.88, 7.90 Hz, 1H), 7.09 - 7.16 (m, 1H), 7.04

(dt, J = 1.17, 7.47 Hz, 1H), 6.90 - 7.00 (m, 1H), 4.79 (br. s., 1H), 4.65 (br. s., 1H), 3.47 (br. s., 4H), 3.33 - 3.43 (m, 4H), 2.89 (t, J = 7.9Hz, 2H), 2.68 (t, J = 7.9Hz, 2H).

Scheme 2.2. Synthesis of tyrosine mimic monomer mTyrBn.

Methyl 3-(4-hydroxyphenyl)propanoate (M2.2): 3-(4-hydroxyphenyl)propanoic acid (4.38 g, 26.4 mmol) was dissolved in anhydrous MeOH (50 mL), thionyl chloride (4.0 mL, 2.0 eq.) was added to the solution dropwise with an addition funnel under ice bath. Ice bath was removed after 30 min. The reaction was stirred at RT overnight. Solvent was

1 removed by rotary evaporation and dried under vacuum. H NMR (300 MHz, CDCl3): 훿�

7.03 - 7.13 (m, 2H), 6.73 - 6.82 (m, 2H), 3.68 (s, 3H), 2.89 (t, J = 7.9Hz, 2H), 2.60 (t, J =

7.9Hz, 2H).

21 Methyl3-(4-(benzyloxy)phenyl)propanoate (M2.3): M2.2 (4.0 g, 22.2 mmol) was dissolved in anhydrous DMF (30 mL). K2CO3 (18.3 g, 132 mmol) and benzyl chloride (3.0 mL, 26 mmol) were added to the solution and the reaction was stirred at 120 °C overnight.

The mixture was poured into ice water (200 mL) and concentrated HCl was added to adjust pH = 1. The solid product was removed by filtration and dried under vacuum at 40 °C

1 overnight. A white powder was obtained (5.7 g, 95%). H NMR (500 MHz, CDCl3): 훿� 7.30

- 7.52 (m, 5H), 7.08 - 7.18 (m, 2H), 6.87 - 6.97 (m, 2H), 5.05 (s, 2H), 3.68 (s, 3H), 2.91 (t,

J = 7.83 Hz, 2H), 2.61 (t, J = 7.83 Hz, 2H).

Synthesis of mTyrBn: M2.3 (1 g, 3.7 mmol) and diethanol amine (1.5 g, 14.3 mmol) were taken into a round bottom flask. DMF (5 mL) was added. The mixture was stirred at

80 °C overnight. TLC showed low conversion. CH3ONa (50 uL, 5.4 M) was added and the reaction was monitored with TLC. Solvent was then removed by rotary evaporation. The reaction was purified by column with eluent from DCM to 5% MeOH/DCM. A white

1 powder was obtained (700 mg, 55%). H NMR (300 MHz, CDCl3): 훿� 7.31 - 7.48 (m, 5H),

7.09 - 7.18 (m, 2H), 6.84 - 6.97 (m, 2H), 5.05 (s, 2H), 3.86 (t, J = 4.83 Hz, 2H), 3.70 (t, J

= 5.27 Hz, 2H), 3.56 (t, J = 4.98 Hz, 2H), 3.41 (t, J = 5.27 Hz, 2H), 2.86 - 2.98 (m, 3H),

2.60 - 2.74 (m, 2H).

Scheme 2.3. Synthesis of Keto monomer.

22 Synthesis of Keto monomer: Diethanol amine (10.5 g, 100 mmol) and methyl 4- oxopentanoate (6.5 g, 50 mmol) were taken in a 100 mL round bottom flask. A condenser was added on the flask and the mixture was stirred at 80 °C for 1 h. The reaction was purified by column with eluent from DCM to 4% MeOH/DCM. A viscous liquid was

1 obtained (7.0 g, 68.9%). H NMR (300 MHz, CDCl3): 훿� 3.82-3.84 (m, 4H), 3.74 (br. s.,

1H), 3.51 - 3.65 (m, 4H), 3.45 (br. s., 1H), 2.84 (t, J = 6.0 Hz, 2H), 2.67 (t, J = 6.0 Hz, 2H),

2.21 (s, 3H).

Scheme 2.4. Synthesis of azide monomer.

Methyl 6-bomohexanoate (M2.4): 6-Bromohexanoic acid (5.0 g, 25.6 mmol) was dissolved in anhydrous MeOH (50 mL) and cooled with an ice bath. Thionyl chloride (4.0 mL, 55 mmol) was added dropwise via addition funnel. The reaction was stirred at room temperature for 24 h. Methanol was removed by rotary evaporation. The crude product was purified by column with gradient elution system from pure hexane to 15% EtOAc/hexane.

1 A colorless liquid was obtained (4.1 g, 76.6%). H NMR (300 MHz, CDCl3): 훿� 3.68 (s,

3H), 3.41 (t, J = 6.73 Hz, 2H), 2.34 (t, J = 7.47 Hz, 2H), 1.82 - 1.95 (m, 2H), 1.59 - 1.73

(m, 2H), 1.40 - 1.55 (m, 2H).

Methyl 6-azidehexanoate (M2.5): M2.4 (6.0 g, 24 mmol) and sodium azide (4.7 g,

72 mmol) were stirred in DMF (20 mL). The reaction mixture was heated to 80 °C

23 overnight. Salts were seen to form and were removed by filtration. The filtrate was poured into DCM (100 mL), and washed with water (4x150 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated by rotary evaporation. After drying under high vacuum, the product was obtained as a yellow liquid (4 g, 98%). 1H NMR (300 MHz,

CDCl3): 훿� 3.68 (s, 3H), 3.28 (t, J = 6.88 Hz, 2H), 2.33 (t, J = 7.47 Hz, 2H), 1.53 - 1.76 (m,

4H), 1.31 - 1.50 (m, 2H).

Synthesis of azide monomer: M2.5 (4.0 g, 23.3 mmol) and diethanol amine (5.0 g,

47.6 mmol) were taken into a 100 mL round bottom flask. The mixture was stirred at 80

ºC overnight. The crude product was purified by column chromatography with gradient eluent from pure DCM to 4% MeOH/DCM. The product was obtained as a pale yellow oil

1 (3.5 g, 62.5%). H NMR (300 MHz, CDCl3): 훿� 3.72 - 3.95 (m, 4H), 3.50-3.59 (m, 4H),

3.29 (t, J = 6.88 Hz, 2H), 3.20 (br. s., 1H), 3.05 - 3.16 (m, 1H), 2.43 (t, J = 7.46 Hz, 2H),

1.55 - 1.79 (m, 4H), 1.31 - 1.52 (m, 2H).

Scheme 2.5. Synthesis of phenylalanine mimic monomer mPhe.

Methyl 3-phenylpropanoate (M2.6): 3-phenylpropionic acid (10.93 g, 72.8 mmol) was added into a 250 mL round-bottom flask with a magnetic stir bar and anhydrous MeOH

(120 mL) was added into flask via a cannula under N2 environment. After 3-

24 phenylpropionic acid was completely dissolved in MeOH, the solution was cooled with an ice bath. Thionyl chloride (12 mL) was added dropwise by addition funnel. The ice bath was removed after 30 min, and the reaction mixture was stirred at room temperature overnight. Solvent was removed via rotational evaporation and dried with high vacuum to

1 get quantitative product. H NMR (300 MHz, CDCl3): 훿� 7.14 - 7.40 (m, 5H), 3.68 (s, 3H),

2.97 (t, J = 7.76 Hz, 2H), 2.65 (t, J = 7.76 Hz, 2H).

Synthesis of mPhe: Diethanol amine (18.99 g, 180.7 mmol) and M2.6 (10.93 g,

66.59 mmol) were taken into a 100 mL round bottom flask. The mixture was stirred at 75

ºC overnight. The crude product was purified by column chromatograph with gradient eluent from pure DCM to 5% MeOH/DCM. The product was dried by high vacuum to get

1 a white solid (11.8 g, 75%). H NMR (300 MHz, CDCl3): 훿� 7.12 - 7.40 (m, 5H), 3.79 -

3.92 (m, 2H), 3.64 - 3.77 (m, 2H), 3.56 (t, J = 4.68 Hz, 2H), 3.42 (t, J = 4.98 Hz, 2H), 2.98

(t, J = 7.76 Hz, 2H), 2.72 (t, J = 7.90 Hz, 2H), 2.50 (br. s., 2H).

Scheme 2.6. Synthesis of alanine mimic monomer mAla.

Synthesis of mAla: Ethyl propionate (10.22 g, 100 mmol), diethanol amine (22.08 g, 200 mmol) and a stir bar were added into a round-bottom flask. The mixture was stirred at 80 °C overnight. The crude product was purified by column chromatography with gradient eluent from 2% MeOH/DCM to 5% MeOH/DCM and dried on high vacuum to

25 1 get a white solid (10.5 g, 65%). H NMR (300 MHz, CDCl3): 훿� 3.72 - 3.99 (m, 4H), 3.55-

3.59 (m, 4H), 3.26 (br. s., 1H), 3.11 (br. s., 1H), 2.44 (q, J = 7.51 Hz, 2H), 1.16 (t, J = 7.47

Hz, 3H).

Scheme 2.7. Synthesis of aspartic acid mimic monomer mAsptBu.

Mono-tert-butyl succinate (M2.7): Succinic anhydride (10 g, 100 mmol), N- hydroxysuccinimide(NHS) (3.45 g, 30 mmol) and 4-dimethylaminopyridine(DMAP) (1.8 g, 15 mmol) were dissolved in anhydrous toluene (150 mL). Dry tert-butyl alcohol (17 mL) and dry Et3N (4.2 mL, 30 mmol) were added to the above solution. Reaction was refluxed at 110 °C for 24 hours. After cooling, reaction mixture was diluted with EtOAc (100 mL) and washed with 10% citric acid (2x100 mL) and brine. The organic fractions were combined and dried with anhydrous Na2SO4. The crude product was concentrated and purified via silica gel column chromatography with 40% EtOAc in hexane. A white solid

1 was obtained (12 g, 69%). H NMR (300 MHz, CDCl3): 훿� 2.59 - 2.68 (m, 2H), 2.48 - 2.59

(m, 2H), 1.45 (s, 9H).

26 Bis (2-((tert-butyldimethylsilyl)oxy)ethyl)amine (M2.8): Diethanol amine (3.15 g,

30 mmol), imidazole (8.16 g, 120 mmol) were dissolved in anhydrous DMF (15 mL). The solution was cooled to 0 °C with an ice bath. Tert-butyldimethylchlorosilane (10.85 g, 72 mmol) was added in portions with N2 protection. Reaction mixture became solid-like after

30 min. DCM (100 mL) was added into the flask and water (50 mL) was added. The solution was extracted with DCM (3x50 mL), washed with water (4x100 mL) and brine

(100 mL). The organic layers were combined and dried with anhydrous Na2SO4. The crude product was purified by silica gel column chromatography with gradient solution from pure

DCM to 3% MeOH/DCM. Liquid product was obtained. Calculated via NMR spectrum,

1 yield: 24.6 mmol 82.0%. H NMR (300 MHz, CDCl3): 훿� 3.74 (t, J = 5.42 Hz, 4H), 2.74 (t,

J = 5.27 Hz, 4H), 0.79 - 1.01 (m, 18H), 0.01 - 0.15 (m, 12H).

Synthesis of M2.9: M2.7 (6.8 g, 39 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (7.5 g, 48.3 mmol) were added into a 250 mL round bottom flask.

Anhydrous DMF (30 mL) was added to the flask. The flask was cooled with an ice bath and stirred for 10 min. M2.8 (10 g, 30 mmol) dissolved in anhydrous DMF (10 mL) was added into flask with N2 protection. The ice bath and N2 were removed after 0.5 hour and the reaction mixture was stirred at RT overnight. DMF was removed by rotary evaporation, then EtOAc (150 mL) and water (50 mL) were added into the flask. The reaction mixture was extracted with EtOAc (2x50 mL), washed with saturated NaHCO3 solution (50 mL), water and brine. The combined organic phase was dried with anhydrous Na2SO4. The product was purified by silica gel column chromatography with 15% EtOAc/Hexane. A

1 light yellow oil was obtained (12 g, 81.7%). H NMR (300 MHz, CDCl3): 훿� 3.74 (t, J =

27 5.71 Hz, 4H), 3.56 (t, J = 5.86 Hz, 2H), 3.48 (t, J = 5.71 Hz, 2H), 2.65 - 2.69 (m, 2H), 2.53

- 2.58 (m, 2H), 1.45 (s, 9H), 0.88 (s, 18H), 0.05 (s, 12H).

Synthesis of mAsptBu: M2.9 (10 g, 21.8 mmol) was dissolved in MeOH (15 mL), iodine (20% of the weight of product) and a stir bar were added into the flask. The reaction mixture was stirred overnight. Saturated Na2S2O3 solution was added dropwise into the flask until solution became colorless. Solvent was removed via rotational evaporation, then brine (50 mL) was added to the flask. The reaction mixture was extracted with DCM (3x50 mL). Organic fractions were combined and dried with anhydrous Na2SO4. The crude product was purified via silica gel column chromatography with gradient eluent from pure

DCM to 5% MeOH/DCM. Light yellow oil was obtained (3.47 g, 61%). 1H NMR (300

MHz, CDCl3): 훿� 4.31 (br. s., 1H), 4.12 (br. s., 1H), 3.73 (br. s., 4H), 3.49 (br. s., 4H), 2.56

- 2.65 (m, 2H), 2.47 - 2.56 (m, 2H), 1.35 - 1.47 (m, 9H).

Scheme 2.8. Synthesis of serine mimic monomer mSerTBDMS.

28 Methyl 4-hydroxybutanoate (M2.10): Butyrolactone (3.04 mL, 40 mmol), MeOH

(100 mL) and triethylamine (34 mL, 240 mmol) were added into a 250 mL round bottom flask. The reaction mixture was stirred at 60 °C for 18 hours. Then solvent was removed by rotary evaporation. The residual MeOH was removed azeotropically with hexane, dried on high vacuum. Colorless liquid was obtained (4.0 g). 1H NMR Showed 80% was product,

1 20% was starting material butyrolactone, yield: 67.8%. H NMR (300 MHz, CDCl3): 훿�

4.36 (t, J = 7.02 Hz, 0.55H), 3.58 - 3.80 (m, 5H), 2.37 - 2.59 (m, 2.56H), 2.18 - 2.35 (m,

0.55H), 1.86-1.94 (m, 2H).

Methyl 4-((tert-butyldimethylsilyl)oxy)butanoate (M2.11): M2.10 (4.0 g, 33.9 mmol), anhydrous DCM (60 mL), anhydrous triethylamine (10.4 mL, 74.7 mmol) were added into a round bottom flask. The solution was cooled with ice bath. Tert- butyldimethylchlorosilane (5.4 g, 35.8 mmol) was added into the flask. The ice bath was removed after 30 min. The reaction mixture was stirred at RT overnight. Some solid precipitate formed, and the color of the reaction mixture turned to be purple. The reaction was diluted with DCM (50 mL). Water (50 mL) was added, and extracted with DCM (2x

50ml). The organic fractions were combined and washed with water (50 mL), saturated sodium bicarbonate solution (50 mL), brine (50 mL) and dried with anhydrous Na2SO4.

The product was purified by silica gel column chromatography with 10% EtOAc/hexane, and dried under high vacuum. A colorless liquid was obtained (6.0 g, 95%). 1H NMR (300

MHz, CDCl3): 훿� 3.62 - 3.68 (m, 5H), 2.40 (t, J = 7.32 Hz, 2H), 1.74 - 1.94 (m, 2H), 0.89

(s, 9H), 0.04 (s, 6H).

Synthesis of mSerTBDMS: M2.11 (15.6 g, 67.1 mmol) and diethanol amine (15.0 g, 142.7 mmol) were added into a round bottom flask. The mixture was magnetically stirred

29 at 80 °C overnight. The product was purified by silica gel column chromatography with 5%

MeOH/DCM and dried under high vacuum. A white solid was obtained (14.6 g, 71%). 1H

NMR (500 MHz, CDCl3): 훿� 3.78 - 3.87 (m, 4H), 3.65 - 3.68 (m, 2H), 3.53 - 3.58 (m, 2H),

3.48 - 3.51 (m, 2H), 2.45 - 2.54 (m, 2H), 1.79 - 1.95 (m, 2H), 0.90 (s, 9H), 0.06 (s, 6H).

Scheme 2.9. Synthesis of lysine mimic monomer mLysBoc.

Synthesis of M2.13: Compound M2.12 (5.5 g, 37.6 mmol) was added to a 250 mL round bottom flask. H2O (30 mL) and dioxane (30 mL) were added to the flask. Di-tert- butyl dicarbonate (9.85 g, 45.1 mmol) was added. The mixture was cooled with ice bath.

Triethylamine (12.6 mL, 90.24 mmol) was added dropwise via addition funnel. The ice bath was removed after 30 min. The reaction mixture was stirred at RT overnight. Solvent was removed by rotary evaporation. The product was purified by silica gel column chromatography with gradient elution system from 10% EtOAc/Hexane to 20% EtOAc to

1 get a white solid (8.2 g, 89%). H NMR (300 MHz, CDCl3): 훿� 3.67 (s, 3H), 3.03 - 3.20 (m,

2H), 2.32 (t, J = 7.32 Hz, 2H), 1.60 - 1.73 (m, 2H), 1.41 - 1.57 (m, 11H), 1.26 - 1.41 (m,

2H).

30 Synthesis of mLysBoc: Compound M2.13 (8.2 g, 33.4 mmol) and diethanol amine

(14 g, 133.2 mmol) were added to a 100 ml round bottom flask. The mixture was heated to

75 °C for two days. The product was purified by silica gel column chromatography with gradient eluent from 2% to 5% MeOH/DCM. A white solid was obtained as product (8.0

1 g, 75.5%). H NMR (500 MHz, CDCl3): 훿� 4.78 (br. s., 1H), 4.20 (br. s., 1H), 4.12 (br. s.,

1H), 3.69 - 3.85 (m, 4H), 3.43 - 3.59 (m, 4H), 3.08 (q, J = 6.52 Hz, 2H), 2.39 (t, J = 7.46

Hz, 2H), 1.64 (quin, J = 7.52 Hz, 2H), 1.44 - 1.53 (m, 2H), 1.42 (s, 9H), 1.26 - 1.38 (m,

2H).

Scheme 2.10. Synthesis of Glutamic acid mimic monomer mGluBn.

6-(benzyloxy)-6-oxohexanoic acid (M2.14): Adipic acid (7.3 g, 50 mmol), benzyl alcohol (7.76 mL, 75 mmol), p-toluenesulfonic acid (95 mg, 0.5 mmol) and toluene (40 mL) were added into a 250 mL flask equipped with Dean-Stark trap and heated to reflux overnight. The reaction was cooled, water (40 mL) was added. The pH was adjusted to 8.0

31 with NaOH (6N). The aqueous layer was separated and washed with ether (2x20 mL), fresh ether (40 mL) was added, and the pH was adjusted to 2.0 with HCl (6N). The ether layer was separated, dried over anhydrous NaSO4 and concentrated to yield product as a white

1 solid (5.15 g, 53.6%). H NMR (300 MHz, CDCl3): 훿� 7.36 (s, 5H), 5.13 (s, 2H), 2.25 -

2.49 (m, 4H), 1.54 - 1.84 (m, 4H).

Synthesis of M2.15: M2.14 (1.934 g, 8.18 mmol) and EDC (1.59 g, 8.8 mmol) were taken in a 100 mL round-bottom flask. Anhydrous DMF (15 mL) was added to the flask.

The suspension was stirred for 10 min with the cooling of ice bath, then M2.8 (2.1 g, 6.29 mmol) which was dissolved in anhydrous DMF (5 mL) was added to the flask. The reaction was stirred overnight at room temperature. DMF was removed by rotary evaporation. The residue was dissolved in EtOAc and washed with saturated sodium bicarbonate solution, water, and brine. The combined organic phase was concentrated and dried on anhydrous

1 Na2SO4. EtOAc was evaporated to yield yellow oil (3.0 g, 93.5%). H NMR (300 MHz,

CDCl3): 훿� 7.6 (s, 5H), 5.12 (s, 2H), 3.63 - 3.80 (m, 4H), 3.39 - 3.58 (m, 4H), 2.40 (br. s.,

4H), 1.63 - 1.75 (m, 4H), 0.88 (m, 18H), 0.04 (m, 12H).

Synthesis of mGluBn: M2.15 (3.4 g, 6.1 mmol) was dissolved in anhydrous MeOH

(15 mL), and iodine (520 mg, 2 mmol) was added to the solution. The reaction was stirred overnight. Saturated Na2S2O3 solution was added until the color of the reaction solution became colorless. Methanol was removed by rotary evaporation. The residue was extracted with DCM (4x50 mL), washed with H2O (2x50 mL), and brine (50 mL). The organic phase was dried with anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with gradient eluent from pure

DCM to 5% MeOH/DCM. An oily product was obtained (1.6 g, 80%).1H NMR (300 MHz,

32 CDCl3): 훿� 7.36 (s, 5H), 5.11 (s, 2H), 3.68 - 3.90 (m, 4H), 3.41 - 3.61 (m, 4H), 2.28 - 2.50

(m, 4H), 1.69 (td, J = 3.51, 7.03 Hz, 4H).

2.3.3 Synthesis of functional polyesters

General procedure for the polyesterification of pendant functionalized diol(s) and succinic acid was described as following.

Scheme 2.11. Polyesterification of functional diol(s) with succinic acid.

Scheme 2.12. Deprotection of benzyl group of p(mTyrBn).

Pendant functionalized diol (1 equiv.) and succinic acid (1 equiv.), DPTS (0.4 equiv.) were suspended in dry CH2Cl2 (2 mL for 1 mmol of diacid). This mixture was warmed up

33 to 40 °C for 1-2 min to homogenize the reaction mixture. The reaction mixture was then cooled to 0 °C and DIC (3 equiv) was added dropwise by syringe. Then the reaction mixture was warmed to RT and stirred for 24-48 h. The polymer was precipitated from cold iPrOH/MeOH and dried under vacuum. The polymer was reprecipitated multiple times from cold iPrOH/MeOH to obtain polymer with low polydispersity.

Synthesis of p(mTyr): p(mTyrBn) (160 mg) was dissolved in DMF (5 mL), Pd/C

(40 mg) was added to the solution. Hydrogen gas was added and the reaction was reacted under hydrogen at room temperature for 24 h. Pd/C was filtered and the solvent was concentrated with reduced pressure. The product was precipitated from diethyl ether quantitatively.

General procedure for deprotection of TBDMS, Boc, tBu group: Polymer (1 g) was taken in CH2Cl2 (5 mL) and triisopropylsilyl (TIPS) (0.5 mL) was added to it. After cooling the reaction mixture to 0°C, trifluoroacetic acid (TFA) (5 mL) was added and the reaction was stirred at room temperature for 2 h. Then TFA was removed under reduced pressure and the polymer was precipitated from cold diethyl ether and dried.

2.3.4 1H NMR of polyesters

1 p(mAla): Yield = 70%, H NMR (300 MHz, CDCl3, TMS): δ 1.15(t, J= 7.46 Hz,

3H), 2.39(d, J = 7.46 Hz, 2H), 2.62(br, 4H), 3.60-3.70(m, 4H), 4.22-4.26(m, 4H); Mn=58 kg/mol, PDI=1.3.

1 p(mPhe): Yield = 61.7%, H NMR (300 MHz, CDCl3, TMS): δ 2.51-2.67(m, 6H),

2.93-2.99(m, 2H), 3.49-3.60(m, 4H), 4.10-4.23(m, 4H), 7.18-7.30(m, 5H); Mn=69 kg/mol,

PDI=1.9.

34 1 p(mLysBoc): Yield = 37.5%, H NMR (300 MHz, CDCl3): δ 1.33-1.68(m, 15H),

2.36(t, J = 7.46 Hz, 2H), 2.61-2.63(m, 4H), 3.05-3.14(m, 2H), 3.58-3.64(m, 4H), 4.22(br,

4H), 4.77(br, 1H); Mn=144 kg/mol, PDI=1.5.

1 p(mSerTBDMS): Yield = 40%, H NMR (300 MHz, CDCl3): δ 0.06 ( s, 6H), 0.89

(s, 9H), 2.58-2.63(m, 6H), 3.60-3.71(m, 4H), 3.93-3.97(m, 2H), 4.24( m, 4H); Mn=20 kg/mol, PDI=1.3.

1 p(mSer): yield = 65%, H NMR (300 MHz, DMSO-d6): δ 2.50-2.61(m, 6H), 3.50-

3.65(m, 6H), 4.10-4.28(m, 6H); Mn=4.9 kg/mol, PDI=1.9.

1 p(mGluBn): Yield = 45.1%, H NMR (300 MHz, CDCl3): δ 1.63-1.67 (m, 4H),

2.36-2.38 (m, 4H), 3.56-3.60(m, 4H), 4.19-4.21 (m, 4H), 5.10 (s, 2H), 7.36 (br, 5H); Mn=46 kg/mol, PDI=2.0.

1 p(N3): Yield = 69.8%, H NMR (300 MHz, CDCl3): δ 1.38-1.48( m, 2H), 1.59-

1.70(m, 4H), 2.36-2.41(m, 2H), 2.62(m, 4H), 3.27-3.32(m, 2H), 3.61-3.63(m, 4H), 4.23(m,

4H); Mn=16 kg/mol, PDI=2.2.

1 p(mAla0.66-co-mPhe0.33) : Yield = 64.3%, H NMR (300 MHz, CDCl3): δ 1.12-

1.17(m, 2H), 2.35-2.40(m, 2H), 2.53-2.69(m, 6H), 2.95-3.00(m, 1H), 3.54-3.61(m, 5H),

4.14-4.23(m, 5H), 7.17-7.37(m, 2H); Mn=53 kg/mol, PDI=1.5.

1 p(mAla0.5-co-mPhe0.5) : Yield = 59.1%, H NMR (300 MHz, CDCl3): δ 1.12-

1.17(m, 3H), 2.35-2.40(m, 2H), 2.53-2.69(m, 10H), 2.95-3.00(m, 2H), 3.54-3.60(m, 8H),

4.14-4.23(m, 8H), 7.16-7.30(m, 6H); Mn=57 kg/mol, PDI=1.7.

1 p(mAla0.25-co-mPhe0.75): Yield = 67.8%, H NMR (300 MHz, CDCl3): δ 1.11-

1.16(m, 1H), 2.33-2.40(m, 1H), 2.51-2.68(m, 11H), 2.94-2.99(m, 3H), 3.52-3.68(m, 8H),

4.11-4.22(m, 8H), 7.10-7.30(m, 8H); Mn=52 kg/mol, PDI=1.6.

35 1 p(mPhe0.9-co-mAsptBu0.1): Yield = 69.4%, H NMR (300 MHz, CDCl3): δ 1.44(s,

4H), 2.52-2.68(m, 30H), 2.95-3.00(m, 9H), 3.51-3.61(m, 20H), 4.12-4.24(m, 20H), 7.17-

7.29(m, 23H); Mn=106 kg/mol, PDI=1.2.

1 p(mPhe0.75-co-mAsptBu0.25): Yield = 66.5%, H NMR (500 MHz, CDCl3): δ1.44(s,

9H), 2.52-2.68(m, 26H), 2.96-2.99(m, 6H), 3.49-3.65(m, 16H), 4.14-4.22(m, 16H), 7.17-

7.29(m, 15H); Mn=98 kg/mol, PDI=1.7.

1 p(mPhe0.6-co-mAsptBu0.4): Yield = 66.7%, H NMR (300 MHz, CDCl3): δ 1.44(s,

9H), 2.52-2.69(m, 17H), 2.95-3.00(m, 3H), 3.54-3.65(m, 10H), 4.14-4.24(m, 10H), 7.20-

7.28(m, 7H); Mn=83 kg/mol, PDI=1.8.

1 p(mSerTBDMS0.75-co-mPhe0.25): Yield = 72%, H NMR (300 MHz, CDCl3): δ

0.06(s, 4H), 0.89(s, 7H), 2.58-2.70(m, 7H), 2.96-3.01(m, 1H), 3.54-3.69(m, 4H), 3.93-

3.97(m, 2H), 4.10-4.24(m, 4H), 7.22-7.34(m, 2H); Mn=21 kg/mol, PDI=1.4.

1 p(mSer0.75-co-mPhe0.25): Yield = 65%, H NMR (300 MHz, DMSO-d6): δ 2.5-

2.88(m, DMSO, 3H), 3.51-3.64(m, 13H), 4.10-4017(m, 5H), 4.59(br, 1H), 7.17-7.23(m,

2H); Mn=13 kg/mol, PDI=2.1.

1 p(mSerTBDMS0.5-co-mPhe0.5): Yield = 50 %, H NMR (300 MHz, CDCl3): δ

0.08(s, 6H), 0.88 (s, 9H), 2.53-2.67(m, 12H), 2.45-3.00(m, 2H), 3.54-3.88(m, 8H), 3.93-

3.97(m, 2H), 4.14-4.23(m, 8H), 7.19-7.34(m, 5H); Mn=22 kg/mol, PDI=1.6.

1 p(mSer0.5-co-mPhe0.5): Yield= 80%, H NMR (300 MHz, CDCl3): δ2.53-2.99(m,

14H0, 3.53-3.62(m, 8H), 4.14-4.24(m, 8H), 4.68(m, 2H), 7.20-7.27(m, 5H); Mn=31 kg/mol,

PDI=1.6.

1 p(mPhe0.87-co-mGluBn0.13): Yield = 77.6%, H NMR (500 MHz, CDCl3): δ 1.68-

1.69(m, 2H), 2.36-2.39(m, 2H), 2.52-2.58(m, 16H), 2.66(t, J= 7.70 Hz, 7H), 2.9(t, J = 7.70

36 Hz, 7H), 3.50-3.60(m, 16H), 4.12-4.23(m, 16H), 5.11 (s, 1H), 7.17-7.36(m, 20H); Mn=116 kg/mol, PDI=1.7.

1 p(mPhe0.73-co-mGluBn0.27): Yield = 72.6%, H NMR (500 MHz, CDCl3): δ 1.68-

1.69(m, 2H), 2.35-2.41(m, 2H), 2.52-2.58(m, 7H), 2.66(t, J = 7.70 Hz, 3H), 2.9(t, J = 7.70

Hz, 3H), 3.50-3.60(m, 7H), 4.12-4.23(m, 7H), 5.11 (s, 1H), 7.17-7.36(m, 8H); Mn=115 kg/mol, PDI=1.8.

1 p(mPhe0.5-co-mGluBn0.5): Yield = 67.3%, H NMR (500 MHz, CDCl3): δ 1.68-

1.69(m, 2H), 2.35-2.41(m, 2H), 2.52-2.58(m, 4H), 2.66(t, J = 7.70 Hz, 1H), 2.9(t, J= 7.70

Hz, 1H), 3.52-3.60(m, 4H), 4.13-4.23(m, 4H), 5.11 (s, 1H), 7.17-7.36(m, 5H); Mn=125 kg/mol, PDI=1.8.

1 p(mSerTBDMS0.25-co-mPhe0.75): Yield= 72% , H NMR (300 MHz, CDCl3): δ

0.05(s, 1H), 0.88(s, 2H), 2.52-2.66(m,6H), 2.95-3.00(m, 2H), 3.53-3.68(m, 4H), 3.92-

3.96(m, 1H), 4.13-4.22(m, 4H), 7.14-7.30(m, 5H); Mn=22 kg/mol, PDI=1.7.

1 p(mSer0.25-co-mPhe0.75): Yield = 93%, H NMR (300 MHz, CDCl3): δ 2.52-2.70(m,

6H), 2.94-2.99(m, 2H), 3.53-3.68(m, 4H), 4.14-4.22(m, 4H), 4.68(br, 1H), 7.20-7.37(m,

4H); Mn=18 kg/mol, PDI=1.6.

1 p(mLysBoc0.5-co-Propargyl0.5): Yield = 65%, H NMR (300 MHz, CDCl3): δ

1.22-1.26(m, 1H), 1.38-1.68(m, 15H), 2.37(m, 2H), 2.56-2.74(m, 12H), 3.10-3.12(m, 2H),

3.62-3.67(m, 8H), 4.02(m, 2H), 4.24(m, 8H); Mn=30 kg/mol, PDI=2.0.

1 p(mAla0.4-co-mAsptBu0.4-co-Propargyl0.2): Yield = 78%, H NMR (300 MHz,

CDCl3): δ 1.21-1.23(m, 2.5H), 1.44(s, 9H), 2.36-2.73(m, 18H), 3.61-3.75(m, 10H), 4.01-

4.03(m, 10H); Mn=21 kg/mol, PDI=1.6.

37 1 p(mAla0.4-co-mAsp0.4-co-Propargyl0.2): Yield = 90% , H NMR (300 MHz,

DMSO-d6): δ 1.05-1.16(m, 2.5H), 2.32-2.56(m, 18H, DMSO), 3.50-3.60(m, 10H), 3.83(m,

1H), 4.10-4.18(m, 10H), 8.23(m, 0.5H).

Table 2.1. Characterization data summary for the ‘peptide-like’ polyesters.

Contact Mn Mw Tg Td Polymer Entry PDI Angle (kDa) (kDa) (°C) (°C) (o) p(mAla) 58 75 1.29 5.2 281.6 27±1 p(mPhe) 69 129 1.87 18.9 299.5 87±2

p(N3) 16 34 2.21 -33.0 217.1 75±4 p(mLysBoc) 144 215 1.49 6.7 219.3

p(mAla0.67-co-mPhe0.33) 53 81 1.53 11.2 280.8 49±2

p(mAla0.5-co-nPhe0.5) 57 94 1.65 12.8 287.5 53±2

p(mAla0.25-co-mPhe0.75) 52 82 1.58 17.3 289.6 80±1

p(mPhe0.88-co-mGluBn0.12) 116 197 1.70 16.0 292.9 92±6

p(mPhe0.73-co-mGluBn0.27) 115 202 1.76 12.2 286.3 89±2

p(mPhe0.5-co-mGluBn0.5) 125 230 1.84 6.6 293.8 91±2 p(mGluBn) 46 92 2.01 -6.7 302.7 98±1

t p(mPhe0.9-co-mAsp Bu0.1) 106 126 1.19 17.1 225.7 93±1

t p(mPhe0.75-co-mAsp Bu0.25) 98 167 1.70 19.6 223.7 92±2

t p(mPhe0.6-co-mAsp Bu0.4) 83 146 1.75 18.6 221.5 92±0

p(mPhe0.9-co-mAsp0.1) 80 111 1.39 17.7 283.0 83±3

p(mPhe0.75-co-mAsp0.25) 136 178 1.31 19.0 187.5 82±1

p(mPhe0.6-co-mAsp0.4) 125 164 1.31 19.7 171.2 85±2 OTBDMS 20 26 1.29 -2.0 93±2 P(mSer) OH 4.9 9.2 1.87 -14.0 22±3 OTBDMS 21 29 1.37 -1.1 90±2

38 p(mSer-co- nPhe)(m:n OH 13 26 2.06 1.4 38±2 = 0.75:0.25) p(mSer-co- OTBDMS 22 35 1.58 -6.3 90±2 nPhe)(m:n = 0.5:0.5) OH 31 49 1.61 6.0 77±2 p(mSer-co- OTBDMS 22 38 1.73 5.1 92±1 nPhe)(m:n = 0.25:0.75) OH 18 28 1.57 3.52 81±1 p(mLys-co-nGlu) 88 102 1.1 6 m:n= 0.5:0.5

p(mLys0.5-co-ropargyl0.5) 30 58 1.95 16 151.6

t p(mAla0.4-co-Asp Bu0.4-co- 21 33 1.59 -7.7 197.4 48±3 Propargyl0.2)

Scheme 2.13. Conjugation of dyes to p(mLys0.5-co-Propargyl0.5).

2.3.5 Conjugation of dyes to p(mLys0.5-co-Propargyl0.5)

Conjugation of azido anthracene (AA) to p(mLysBoc0.5-co-Propargyl0.5): p(mLysBoc0.5-co-Propargyl0.5) (0.1 g) was taken in of THF (2 mL). To this solution CuBr

(cat.) and 9-azidomethylanthracene (3.9 mg corresponding to 12% of propargyl group on polymer) were added and reaction was stirred for 4 h. The resulting conjugate was precipitated from diethyl ether. This polymer conjugate was redissolved in THF (1 mL)

39 and this solution was added dropwise into EDTA solution (20 mL, 20 mmol) to remove

1 copper salts. H NMR (500 MHz, DMSO-d6): δ1.23-1.47(m, 13H), 2.29-2.55(m, 10 H),

2.88-3.03(m, 2H), 3.49-3.59(m, 8H), 3.83(m, 1H), 4.09-4.18(m, 8H), 5.75(s, 0.2H), 6.5-

6.88(m, 1H), 7.55-7.22(m, 1H), 8.13-8.21(m, 1H), 8.37-8.71(m, 0.6H).

Conjugation of FITC to AA tethered p(mLysBoc0.5-co-Propargyl0.5-AA): The above polymer (0.1 g) was taken into CH2Cl2 (2 mL) and triisopropyl silane (TIPS) (50 µL) was added as scavenger. This solution was cooled to 0 °C, TFA (2 mL) was added and stirred for 2 h at RT. The TFA–CH2Cl2 mixture was removed under reduced pressure and the polymer was precipitated from diethyl ether and dried. FITC (corresponding to 1% of amine groups of the polymer, from 0.5 mg/mL FITC stock solution) was added to AA tethered p(mLys0.5-co-Propargyl0.5) solution dissolved in 1 mL DMSO and DIPEA (50

µL) and the reaction was stirred for 2 h. Following this the polymer was precipitated from diethyl ether and dried.

t 2.3.6 Functionalization of p(mAla0.4-co-mAsp Bu0.4-co-Propargyl0.2) with PEG and RGD

t Polymer p(mAla0.4-co-mAsp Bu0.4-co-Propargyl0.2) was functionalized with PEG and RGD. The synthetic procedures were described in details.

2.3.6.1 Deprotection of the tert-butyl protected carboxylic acid group

t p(mAla0.4-co-mAsp Bu0.4-co-Propargyl0.2) (1 g) was taken in CH2Cl2 (5 mL) and

TIPS (0.5 mL) was added. After cooling to 0 °C, TFA (5 mL) was added and the reaction was stirred at room temperature for 2 h. Then TFA was removed under reduced pressure and the polymer was precipitated from cold diethyl ether and dried. Yield = 90%.

Scheme 2.14. Post-polymerization functionalization of p(mAla0.4-co-mAsp0.4-co-

Propargyl0.2) with PEGNH2 and RGDN3.

2.3.6.2 Coupling of 2-[2-(2-Methoxyethoxy)ethoxy]ethyl amine(PEGNH2) to carboxyl groups of p(mAla0.4-co-mAsp0.4-co-Propargyl0.2)

p(mAla0.4-co-mAsp0.4-co-Propargyl0.2) (0.1g) was taken in mixture of CHCl3-

DMF (1 mL, 10:1) and DIC (12 µL, corresponding to 55% of COOH group) was added.

After 15 min, PEGNH2 (11.3 mg, corresponding 50% of COOH groups) was added. The reaction was stirred for 2 h and then DIC (6 µL, corresponding to 22% of COOH group) was added again for further activation. To this activated reaction mixture 2- phenylethylamine (4.3 µL, 20% of COOH groups) was added and further stirred for 2 h.

The functionalized polymer was precipitated from cold iPrOH, centrifuged and dried.

2.3.6.3 Coupling of 2-phenylethylamine to carboxyl group of p(mAla0.4-co-mAsp0.4-co-

Propargyl0.2)

Similar procedure was followed as above for the functionalization of polymer p(mAla0.4-co-mAsp0.4-co-Propargyl0.2).

41 2.3.6.4 Spin coating of polymers on glass cover slips

Cover slips were cleaned by sonicating in EtOH, washed with distilled water and dried under strong stream of air making sure the cover slips were spot and debris free. The polymers were spin coated from 2% (w/v) polymer solution in CHCl3: DMF (10:1) mixture.

Spin coating of polymers were carried out at 2500 rpm for 1 min. The slides were then vacuum dried and used for further functionalization and contact angle measurements.

2.3.6.5 Surface functionalization

The surface propargyl groups were reacted with azide terminated RGD peptide (N3-

(CH2)5CONHGRGDSCO2H) by Cu catalyzed azide-alkyne cycloaddition (Click reaction).

The reagent was prepared by mixing CuSO4 (0.5 mL, 5 mM), sodium ascorbate (0.5 mL,

10 mM), and (N3-(CH2)5CONHGRGDSCO2H) (0.25 mL, 2 mM). The propargyl functionalized cover slips were inverted over a solution of the Click reagent (50 μL) and allowed to react for 4 h. Then these coverslips were washed with deionized water followed by EDTA solution (20 mM), and deionized water and then dried under vacuum.

2.3.6.6 Cell Adhesion Studies

Cover slips with spin-coated polymer film or surface functionalized film or blank coverslips (control) were rinsed with 70% ethanol followed by phosphate buffer saline wash (PBS, Fisher). A-10 cells were seeded on coated slides at a cell density of 20,000 cells/cm2 in 24-well plates and cultured in DMEM medium (GIBCO, Invitrogen) for 9 h in a 37 °C, 5% CO2 incubator. Cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.5% TritonX-100 for 9 min. 0.05% sodium borohydride in PBS was

42 added to each well for 10 min to remove excess formaldehyde. Non-specific binding was blocked by adding 5% donkey serum in PBS for 20 min. The actin filaments of the cytoskeleton were stained with rhodamine phalloidin (1:200 dilution in PBS; Invitrogen) for 1 h. Then the samples were rinsed three times with PBS. Alexa fluor 488 C5-maleimide

(1:200 dilution in PBS; Invitrogen) was added for 1 h. Then the samples were rinsed three times with PBS and the nucleus was stained with DAPI (1:1000 dilution in PBS; Invitrogen) for 1 h and rinsed four times with PBS. Finally samples were mounted onto glass slides with mounting medium (Vector Laboratories) and were viewed with IX 81 microscope

(Olympus, Center Valley, PA) using 10X and 40X objectives. Images were analyzed with the Image J software to determine average cell number and cell area.

2.4 Results and Discussion

In this section, the synthesis of functional polyesters, modulation of their physical properties, and the characterization results will be discussed.

2.4.1 Synthesis of functional polyesters and modulation of physical properties

We designed functionalized diols with pendant functional groups similar to those found in amino acids. The functionalized diols were prepared by the reaction of diethanolamine with an ester derivative of the required pendant functional unit (Scheme

2.1-2.6). Alternatively, functionalized diols were also synthesized by coupling of carbodiimide activated acid with TBDMS protected diethanolamine (Scheme 2.7).

As demonstrated by Stupp, and recently by Meyer, carbodiimide mediated coupling of diols and diacids (or hydroxyacids) is an efficient method for the synthesis of polyesters

43 under mild conditions.89-90 By the same methodology, functionalized polyesters were synthesized via diisopropylcarbodiimide (DIC) mediated coupling of a functionalized diol with succinic acid (Scheme 2.11). As shown in Figure 2.1, a variety of polyesters containing diverse pendant groups have been synthesized using this methodology. Homo- and statistical copolymers with two or more functional groups were obtained in high yield

(60-70%) with relatively low polydispersity (1.3-2.2) by DIC mediated room temperature polymerization. The low polydispersity was obtained by multiple precipitations. The nomenclature of the polyesters describes their similarity to the side chain of the corresponding amino acid or to the non-natural functional group. For example, p(mAla) refers to a polyester that is a mimic of alanine and p(N3) refers to a polyester with an azide pendant group. In the case of a protected functional group, such as the polyester with N- tert-butoxycarbonyl protected lysine p(mLysBoc), the nomenclature describes the protected functional group (Figure 2.1).

As characterized by 1H NMR, the obtained polyester composition closely matched the feed ratio (appendix). The number average molecular weight (Mn) and weight average molecular weight (Mw) of the polyesters were measured by size exclusion chromatography

(SEC) with DMF as mobile phase. Physical properties of the polyesters, such as contact angle, glass transition temperature (Tg), decomposition temperature (Td) and shear modulus were seen to be influenced by the pendant groups. Although there are prior examples of modulation of the physical properties of polyesters by varying the monomer ratio,91-92 our polyesters cover a wider range of physical properties through the choice of various functional groups. For example, different pendant groups provided polyesters with a spectrum of surface energies, from hydrophobic polymers such as the phenylalanine mimic

44 p(mPhe) (contact angle 87°) to hydrophilic polymers such as the serine mimic p(mSer) and the alanine mimic p(mAla) (contact angles of 22° and 27° respectively). Copolymers

Figure 2.1. Representative examples of polyesters having mono, di and tri-functional pendant groups.

Figure 2.2. Variation of contact angles with copolymer composition.

of p(mPhe-co-mAla) and p(mPhe-co-mSer) show a correlation between contact angles and the amount of mPhe in the copolymer (Figure 2.2).

The pendant groups also influence the Tg, which varied from 4 °C for p(mGluBn) to 20 °C for p(mPhe). The Tg of p(mGluBn-co-mPhe) copolymers and p(mAla-co-mPhe) copolymers increase with an increase in the ratio of mPhe (Figure 2.3). Interestingly, in the above examples the relationship between composition and Tg correlates well with theoretical predictions determined from the Fox equation.79, 93-94 This correlation is not observed in all copolymer compositions and deviation from the predicted value may indicate associative processes between the pendant functional groups or between the functional groups and the backbone. For example, the Tg of p(mSer-co-mPhe) copolymers deviate significantly from the theoretical values, indicating strong interactions when

46 pendant groups such as hydroxyl groups are present. The plateau storage modulus of the homopolymers and copolymers was also influenced by the pendant group and varies from

0.2 MPa for p(mGluBn-co-mPhe) to 0.58 MPa for p(mAla). A linear correlation of the plateau modulus of the copolymer p(mAsptBu-co-mPhe) as a function of composition is observed (Figure 2.4). Polyesters are hydrolytically degradable and preliminary degradation studies were carried out by incubation of p(mAla) in phosphate buffered saline

(PBS, pH 7.4). SEC analysis showed a steady decrease in polymer molecular weight as a function of time (Figure 2.5). It is expected that the identity of the pendant group will influence the rate of degradation.

Figure 2.3. Variation of Tg with polymer composition and its comparison to calculated values from Fox equation.

Figure 2.4. Plot of plateau modulus vs mPhe content for copolymers of p(mPhe-co- mAsptBu).

1.6 day 0, Mn=63.3 kDa, PDI=1.49 day 2, Mn=55.9 kDa, 1.2 PDI=1.50 day 4, Mn=47.3 kDa, PDI=1.53 day 7, Mn=41.4 kDa, 0.8 PDI=1.53

0.4

0 16 21 26

Figure 2.5. SEC traces of the degradation of p(mAla) in PBS buffer (pH 7.4).

1 Figure 2.6. H NMR Spectrum of 10 % AA conjugated p(mLysBoc0.5-co-Propargyl0.5).

2.4.2 Conjugations of two dyes to functional polyester

One of the advantages of these polyesters is their ability to tag multiple ligands

(dyes, drugs, growth factors etc.) via orthogonal pendant functional groups. Tethering of various ligands is an effective method for presentation of imaging, therapeutic and signaling moieties.95-97 As a proof of concept, we aimed to functionalize 10% of the propargyl groups of a p(mNHBoc-co-propargyl) polyester with azido anthracene (AA).

1H NMR characterization of the conjugate showed that 9% of the propargyl groups were functionalized (Figure 2.6). This AA conjugate was also characterized by SEC analysis which showed the conjugate had UV absorption at 350 nm and a higher molecular weight,

Figure 2.7. a) RI and UV response from SEC of p(mLysBoc0.5-co-Propargyl0.5),b) AA tagged p(mLysBoc0.5-co-Propargyl0.5. UV monitored at 350 nm.

Figure 2.8. IR spectrum of p(mLysBoc0.5-co-Propargyl0.5) and 10% AA tagged p(mLysBoc0.5-co-Propargyl0.5). 50 indicating successful AA tethering (Figure 2.7). Furthermore, IR characterization showed a decreased intensity of the C≡C stretching peak upon AA conjugation. Additionally, the peak corresponding to the azide of azido anthracene was not present in the IR spectrum of the AA conjugate, indicating the absence of non-covalently adsorbed AA (Figure 2.8).

Subsequently we deprotected the amine groups of p(mNHBoc-co-propargyl-AA) and covalently attached fluorescein isothiocyanate (FITC) to 1% of amine groups. As seen in

Figure 2.9, the polyester exhibits the absorbance and fluorescence of both dyes, proving the orthogonal conjugation.

Figure 2.9. a) Polyester tethered with AA and FITC b) UV absorbance spectrum of FITC-

AA conjugated polyester (in water) and fluorescence spectrum of FITC-AA conjugated polyester (at 370 nm excitation) in DMSO-water (90:10).

2.4.3 Conjugation of RGD and PEG to functional polyester and cell adhesion study

Several biomaterials have been designed to direct cellular outcomes such as stem cell differentiation, proliferation, and cell mobility.98-100 We envisioned that cell adhesion could be modulated by tethering appropriate ligands. This was examined using a polyester

51 with mAla, mAsp and propargyl pendant groups in a ratio of 2:2:1. To improve adhesion of the polymer to glass substrates, 10% of the COOH groups of mAsp were functionalized with 2-phenylethylamine and is denoted as the base polymer (B). A short PEG chain

[CH3O-(CH2CH2O)2-CH2CH2NH2], which has been shown to decrease cell attachment, was covalently tethered to 20% of the COOH groups and is denoted as BP.101-102 The resulting functionalized polymer was spin coated on glass coverslips. Then the propargyl groups on the polymer surface were functionalized with the cell attachment peptide, N3-

(CH2)5CONHGRGDSCO2H via Cu catalysed azide-alkyne cycloaddition. The RGD tripeptide has been shown to be a sufficient motif for enhancing cell attachment and proliferation.103-104 This process provided coverslips with both PEG and RGD and is denoted as BPR. Similarly, coverslips were made with either PEG (BP) or RGD (BR). The contact angles of spin coated polymer films on glass coverslips were influenced by the functionalization and varied as follows: Base polymer (24.3°), base polymer with PEG

(22.9°), base polymer with PEG and RGD (39.6°), base polymer with RGD (41.6°). The increase in contact angle with RGD is likely due to the addition of a five carbon spacer and a triazole ring upon cycloaddition of the N3-(CH2)5CONHGRGDSCO2H moiety.

These functionalized coverslips were plated with smooth muscle cells and the attachment and spreading were analyzed on the base polyester and compared to the same polyester functionalized with RGD or PEG or PEG+RGD. The results showed that the base polymer induced high cell attachment and spreading relative to the glass control (Figure

2.10). The RGD functionalized polyester showed increased cell attachment and spreading relative to the base polymer. However the lower than expected increase in cell spreading with the RGD functionalized polymer may be due to the already high cell attachment of

Figure 2.10. A) polyesters with PEG, RGD or PEG+RGD used for smooth muscle cell spreading studies b) cell area (over 50 cells) on glass coverslips (control), the base polyester

(B), PEG conjugated polyester (BP), PEG and RGD conjugated polyester (BPR) and RGD conjugated polyester (BR). Images of cell spreading on the above samples: control (c); base polyester, B (d); BP (e); BPR (f); BR (g); scale bar = 25 µm (c-g).

the base polymer. A decrease in cell attachment was observed with the PEG derivatized polymer. The polymer with both PEG and RGD showed an intermediate response. It was interesting that although these polymers had low contact angles, they exhibited high cell attachment which was similar to that seen with fibronectin or laminin. Usually synthetic 53 polymers with low contact angles (e.g. HEMA, PEG) show decreased cell attachment and those with high contact angles (e.g. polystyrene, PLGA) show higher cell attachment.105-

109

2.5 Conclusion

In summary, we have developed a versatile and modular polyester system that enables the efficient incorporation of multiple functional groups along the polymer. The polyesters were synthesized at room temperature by carbodiimide mediated polymerization with high yields. The polymerization is scalable to provide gram quantities of functionalized polyesters. The repeat unit of the polymer creates hydrophilic degradable polyesters with ‘peptide-like’ pendant groups. Similar to a peptide scaffold, the physical properties, such as water solubility, surface energy, Tg and modulus of the reported polyesters vary over a wide range and are modulated by the pendant groups. These polymers combine the breadth of properties (by choice of pendant groups) of acrylate type polymers and the degradable nature of conventional polyesters such as poly(lactic acid). In addition, these polymers have the functional group repertoire of peptides but with the scale- up and reproducibility of synthetic polymers. This multivalent ‘peptide-like’ polyester platform will enable the covalent attachment of various growth factors, therapeutics, imaging agents, or other ligands and has potential utility in several applications where simultaneous presentation of multiple functional cues is necessary.

54 CHAPTER III

MUSSEL INSPIRED ADHESIVES BASED ON ‘PEPTIDE-LIKE’ FUNCTIONAL

POLYESTER

3.1 Abstract

In the ocean, mussels secrete protein holdfasts that are capable of securing themselves on a variety of wet surfaces, namely all kinds of inorganic and organic surfaces.

A unique amino acid, L-3,4-dihydroxyphenylalanine (DOPA), were found in at least five adhesive protein subtypes of the widely studied blue mussel, Mytilus edulis. Inspired by mussel foot protein adhesives, DOPA groups were incorporated into side chain of functional polyesters. The first generation adhesive polyester contains 20% DOPA and 80% of alanine mimic monomer. It showed the increasing adhesion effect of DOPA groups under dry condition, but the strength decreased in wet conditions. The second generation adhesive polyester was a copolymer with soybean oil based monomer, coumarin and DOPA monomer. The coumarin monomer was added to have the spatial controlled UV irradiation curation. This polyester was a viscous polymer with glass transition temperature about -

50 °C. It showed good adhesion under both dry and wet conditions after 5 min UV irradiation. Adhesion tests on porcine skin was also performed and the results demonstrated that our polymer had higher adhesion strength than the commercial available fibrin glue after 5 min UV irradiation.

55 3.2 Introduction

Natural adhesives such as those secreted by mussels are remarkable products of nature. Mussel adhesives are capable of forming strong bonds to the substrate, even under conditions such as strong sea waves and high salinity.110 Mussels use bundles of adhesive threads, called byssus, to attach themselves to solid substrates.111, 112 These threads end in adhesive plaques and in these plaques are secreted a collection of adhesive proteins which enable mussel adhesion. There are several different proteins present in the adhesive plaques, however all of these proteins show the presence of the post-translationaly modified amino

113 acid L-3,4-dihydroxyphenylalanine (DOPA) in their composition. In these proteins

DOPA can vary from 2-30% of the composition.111 DOPA is a very efficient promotor of interfacial adhesion because it can chelate strongly with metal ions through its dihydroxy functionality.114-116 It can also undergo oxidation to the quinone form that can then react with strong nucleophiles such as amines present in the substrate. Furthermore, the quinone form can initiate inter-strand polymer crosslinking to enhance cohesive forces in the adhesive.117 Therefore the DOPA moiety is an optimum post-translational modification that mussels utilize to increase adhesive and cohesive components of adhesion.

Due to their exceptional adhesive performance in wet environments and the difficulty in extracting uncross-linked soluble mussel adhesive proteins, several synthetic polymers incorporating the DOPA unit have been designed as mimics of mussel adhesives.

Wilker et al. incorporated DOPA as pendant group for polystyrene and studied the effect of the amount of DOPA group, different oxidant, and the type of substrates on adhesion strength.118-120 They also studied the influences of molecular weight of the polymer on the adhesion strength.121 Incorporating ammonia group into the DOPA functionalized

56 polystyrene, Wilker et al. studied the dry and wet adhesion.122 Israelachvili et al. synthesized polypeptide with DOPA side chain and investigated the role of DOPA by using surface forces apparatus (SFA) to measure the force-distance profiles and adhesion and

123 cohesion energies of the films to mica and TiO2. Messersmith et al. reported the modification of four arm poly(ethylene glycol) (PEG) hydrogel with DOPA at the end and studied the role of DOPA for adhesion strength.124 They also synthesized zero- or negative- swelling polymer based on catechol-modified amphiphilic poly(propylene oxide)- poly(ethylene oxide) block copolymers.125 DOPA bearing polymer based on polyesters,126 poly(amino ester),127 polypeptides,123 poly(ethylene glycol),128-131 polyacrylates,132-135 polystyrene,118, 120, 136 etc. were synthesized to investigate their adhesion properties and potential as biomedical adhesives.

In addition to the role of DOPA, mussels also maximize the effect of flanking amino acids to generate hydrophobic, ionic and hydrogen bonding interactions which compensate for the oxidation of DOPA residues at higher pH;137-139 or synergistically enhance the adhesive interactions with the surface.140 Similar to several other research groups, we are intrigued by the exquisite ability of mussels to adhere to wet surfaces even under the conditions of high salinity and strong sea waves. Understanding the effect of various parameters that affect the adhesive and cohesive interactions of synthetic mimics of mussel adhesives can provide a blueprint for optimizing the parameters for effective wet adhesives.

Our group has developed a modular ‘peptide-like’ polyester platform that enables the synthesis of polyesters with a functional group at each repeat unit.2 Polyesters with a diverse set of properties can be synthesized by a choice of functionalized monomers. The modular nature of our polyesters enables the incorporation of DOPA mimicking monomer

57 as pendant functional groups in the polyesters. In this regard we have developed a series of polyesters with pendant catechol moieties (mimics of dihydroxyphenylalanine, DOPA) and herein we describe the rationale for their design and their performance characteristics in both dry and wet environments.

In order to understand the effect of catechol in adhesion, we designed and synthesized a copolymer p(mAla0.8-co-mDOPA0.2) from a DOPA mimicking monomer (20 mol%) and a alanine mimicking monomer (80 mol%) and succinic acid via diisopropylcarbodiimide (DIC) mediated coupling at room temperature. Subsequently, the polyester p(mAla0.8-co-mPhe0.2) as such by changing catechol group to phenyl group was synthesized and tested as the control. These polyester based control and catechol adhesives were tested in lap shear geometry in presence and absence of the crosslinking agent, tris(acetylacetonato) iron (III) under both dry and wet condition. Our results show that presence of catechol group significantly increases the adhesive properties as compared to control polyester. Additionally, the adhesive properties of the catechol polyester in wet conditions decreases as compared to that in dry due to the hydrophilic nature of the backbone thus explaining the importance of backbone properties in achieving better adhesion in wet conditions. The study provides important insights for developing new degradable mussel-inspired adhesives.

Besides this hydrophilic mussel inspired adhesive, we made another polymer which is more hydrophobic compared with the first generation of adhesive. It contains 80% soybean oil derived monomer, 15% catechol monomer, and 5% coumarin monomer. The coumarin can undergo crosslink under 350 nm irradiation which gives another method for curing of adhesive.141 Soybean is a sustainable resource and is the second largest field crop

58 (after corn) in the USA, which currently covers about 80 million acres7 over a total of

280,000 farms.8 It would be highly advantageous for soybean farmers to convert low cost readily available sustainable resources to high value added products.

3.3 Experimental Section

Experimental section describes the chemicals needed for synthesis of targeted product, the method of making monomers and polymers and the characterization and tests of materials.

3.3.1 Materials and Instrumentation

Materials: All the reagents were purchased from Sigma Aldrich or Alfa Aesar and used without further purification unless otherwise noted. N,N'-diisopropylcarbodiimide

(DIC, 99%) was purchased from Oakwood Chemical and used as received. 4-

(dimethylamino) pyridinium 4-toluene sulfonate (DPTS) was prepared according to

89 literature methods. Dichloromethane was dried by distilling over anhydrous CaH2 and the DMF was dried by distilling over anhydrous CaH2. Silica gel (40-63 μm, 230 x 400 mesh) for flash chromatography was purchased from Sorbent Technologies, Inc.

Instrumentation: All 1H and 13C NMR spectra of the monomers and polyesters were recorded on either a Varian Mercury 300 MHz or 500 MHz spectrometer. Chemical shifts were recorded in ppm (δ) relative to solvent signals. Polyester molecular weights were analyzed on a TOSOH EcoSec HLC-8320 GPC equipped with a refractive index detector

(RI) and UV detector. Separation occurred over two PSS Gram Analytical GPC Columns in series using 25 mM LiBr in DMF as eluent at a flow rate of 0.8 mL/min. The column

59 and detector temperatures were maintained at 50 °C. Molecular weights were obtained relative to PS standards using the RI signal. Thermogravimetric analysis (TGA) measurements were performed with TA Q500 over an interval of ambient temperature to

600 °C at a heating rate of 10 °C/min under a N2 atmosphere. Differential scanning calorimetry (DSC) were performed on TA Q2000 DSC with a liquid N2 cooling unit and a heating/cooling rate of 10 °C/min. ESI MS was performed on Bruker HTC ultra QIT.

3.3.2 Synthesis and characterization of monomers

In this section, synthesis of monomers with different pendant groups and characterization of obtained monomers are described.

3.3.2.1 Synthesis of DOPA mimic monomer mDOPApr

Scheme 3.1. Synthesis of DOPA mimic monomer mDOPApr.

Synthesis of Compound M3.1: 3,4-Dihydroxyphenylpropanoic acid (12.5 g, 68.6 mmol) was dissolved in 100 mL anhydrous MeOH. Thionyl chloride (13 mL, 2.5 eq.) was added dropwise under ice bath. Ice bath was removed after 30 min. The reaction was stirred at RT overnight. Solvent was removed by rotary evaporation and dried with high vacuum.

60 The dark blue viscous liquid solidified when put in freezer. 13.4 g (100%) solid product

1 was obtained. H NMR (300 MHz, CDCl3): 훿� 6.78 (d, J = 8.20 Hz, 1H), 6.72 (d, J = 2.05

Hz, 1H), 6.63 ( dd, J = 2.05, 8.20 Hz 1H), 5.37 (br. s., 1H), 5.23 (br. s., 1H), 3.63 - 3.73 (m,

3H), 2.85 (t, J = 7.76, 2H), 2.60 (t, J = 7.76, 2H).

Synthesis of compound M3.2: Compound M3.1 (4.18 g, 21.3 mmol), 2,2- dimethoxypropane (10.5 mL, 4 eq.), and anhydrous benzene (200 mL) were added to a three-neck 250 mL flask. One neck of the flask was fitted with a Soxhlet extractor, the thimble of which was filled with granular anhydrous CaCl2, another neck of the flask was sealed with a septum for sampling purpose. After the system was degassed with N2 for 5 min and then heated to reflux for another 5 min, p-toluenesulfonic acid monohydrate (203 mg, 0.05 eq.) was added. The reaction progress was monitored by FeCl3 test. After 3 h, negative test result was achieved, the reflux was stopped. After cooling, the mixture was filtered through a short silica-gel column, washed with DCM. The combined filtrate and washings were concentrated and run column with 2% DCM/hexane. When the first spot was flushed out, the eluent system was changed to gradient from 2% to 4% EtOAc/hexane,

1 4.2 g (83%) light yellow solid was obtained. H NMR (300 MHz, CDCl3): 훿� 6.59 - 6.66

(m, 2H), 3.68 (s, 3H), 2.86 (t, J = 7.76Hz, 2H), 2.59 (t, J = 7.76Hz, 2H), 1.67 (s, 6H).

Synthesis of DOPA mimic monomer mDOPApr: Diethanol amine (14.6 g, 138.9 mmol) and compound 3 (6.8 g, 34.7 mmol) were taken in a flask and heated at 75 °C overnight. Then this reaction mixture was purified by column chromatography with 5%

1 MeOH in CH2Cl2 as the gradient solvent system to yield 7.6 g light yellow solid (71%). H

NMR (300 MHz, CDCl3): 훿� 6.57 - 6.72 (m, 3H), 3.88 (q, J = 5.27 Hz, 2H), 3.72 (q, J =

5.27 Hz, 2H), 3.58 (t, J = 4.98 Hz, 2H), 3.44 (t, J = 4.98 Hz, 2H), 2.97 (t, J = 4.39 Hz, 1H),

61 2.89 (t, J = 7.76 Hz, 2H), 2.76 (t, J = 5.71 Hz, 1H), 2.66 (t, J = 7.76 Hz, 2H), 1.66 (s, 6H).

13 C NMR (75 MHz, CDCl3): 훿� 174.6, 147.4, 145.8, 134.2, 120.5, 117.6, 108.7, 108.0, 61.5,

60.8, 52.1, 50.6, 35.7, 31.2, 25.8.

3.3.2.2 Synthesis of coumarin monomer Cou

Scheme 3.2. Synthesis of coumarin monomer Cou.

Synthesis of compound M3.3: 7-Hydroxycoumarin (3.0 g, 18.5 mmol), potassium carbonate (5.12 g, 37.0 mmol) and 18-Crown-6 (244 mg, 0.924 mmol) were taken in a 100 mL round bottom flask. 20 mL anhydrous DMF was added. Methyl 4-bromobutyrate (4.7 mL, 37.2 mmol) was added by syringe. The mixture was stirred at 50 °C for 24h. The flask was covered with aluminum foil. The reaction mixture was filtered. Solvent was removed.

30 mL water was added and extracted with EtOAc (3x30 mL) and DCM (3x30 mL). The organic layer was combined and dried over Na2SO4. The solution was concentrated and washed with hexane. Light yellow solid was obtained (4.84g, 99.8%). 1H NMR (500 MHz,

CDCl3): 훿� 7.63 (d, J = 9.54 Hz, 1H), 7.37 (d, J = 8.56 Hz, 1H), 6.70 - 6.91 (m, 2H), 6.26

(d, J = 9.54 Hz, 1H), 4.09 (t, J = 6.11 Hz, 2H), 3.71 (br. s., 3H), 2.55 (t, J = 7.21 Hz, 2H),

2.16 (quin, J = 6.66 Hz, 2H).

62 Synthesis of coumarin monomer Cou: M3.3 (4.94 g, 18.8 mmol) and diethanol amine (8.0 g, 76 mmol) were mix in a round bottom flask, heated to 80 °C with microwave.

Run flash silica gel column with 10% MeOH/DCM. Then recrystallized from

1 CHCl3/Hexane. Coumarin monomer Cou was obtained as white solid. H NMR (300 MHz,

CDCl3): 훿� 7.63 (d, J = 9.37 Hz, 1H), 7.36 (d, J = 8.49 Hz, 1H), 6.72 - 6.95 (m, 2H), 6.24

(d, J = 9.37 Hz, 1H), 4.10 (t, J = 6.00 Hz, 2H), 3.73 - 3.97 (m, 4H), 3.47 - 3.67 (m, 4H),

3.16 (br. s., 2H), 2.64 (t, J = 7.03 Hz, 2H), 2.04 - 2.31 (m, 2H).

Scheme 3.3. Synthesis of soybean oil monomer SBO.

3.3.2.3 Synthesis of soybean oil monomer SBO

Synthesis of soybean oil monomer SBO: Diethanol amine (31.5g, 0.3 mol) was taken in a 500 mL round bottom flask. NaOCH3 (0.8 g, 14.8 mmol) was added. The mixture was heated to 110 °C and stirred until NaOCH3 was dissolved. Soybean oil (43.4 g) was added through addition funnel in 30 min. After addition, vacuum was applied. The reaction

63 mixture was stirred for another 1 h at 110 °C. Diluted with EtOAc, washed with 15wt%

1 NaCl solution. Run flush column with 5% MeOH/DCM. H NMR (500 MHz, CDCl3): 훿�

5.30 - 5.41 (m, 2.88H), 3.76 - 3.85 (m, 5.5H), 3.49 - 3.85 (m, 4H), 2.76 - 2.81 (m, 1.29H),

2.37-2.40 (t, J = 7.70 Hz, 2.09H), 2.00 - 2.07 (m, 3.38H), 1.63 (br. s., 2.11H), 1.26 - 1.1.39

(m, 18H), 0.87 - 0.99 (m, 3H).

3.3.3 Synthesis of polymers

Synthesis of p(mAla-co-mDOPApr): mAla monomer (2.5792 g, 16 mmol, 0.8 eq.),

DOPA mimic monomer mDOPApr (1.2374 g, 4 mmol, 0.2 eq.), succinic acid (2.3618 g, 20 mmol, 1.0 eq.) and DPTS (2.3384 g, 8 mmol, 0.4 eq.) were added to a 100 mL Schleck flask. 15 ml dry DCM was added to the flask under N2. This mixture was warmed up to

40 °C for 1-2 min and then cooled with ice bath. To this cooled mixture DIC (9.5 mL, 60 mmol, 3.0 eq.) was added dropwise and reaction mixture was stirred at room temperature for 52 h. Polymer was precipitated twice from methanol and dried under vacuum to yield

1 4.5 g polymer (82.4%). H NMR (300 MHz, CDCl3): 훿� 6.62 (br. s., 0.6H), 4.17 - 4.26 (m,

4H), 3.57 - 3.62 (m, 4H), 2.87 (q, J = 7.62 Hz, 0.4H), 2.59-2.62 (m, 4.4H), 2.39 (q, J =

7.32 Hz, 1.6H), 1.66 (s, 1.2H), 1.14 (t, J = 7.32 Hz, 2.4H).

Synthesis of p(mAla-co-mDOPA): p(mAla-co-mDOPApr) (650 mg) was dissolved in 6 mL DCM (degassed with N2 for 15 min). 6 mL TFA and 50 µL TIPS was added under N2. The solution was stirred at RT for 2 h. Solvent was evaporated by rotary evaporation and the polymer was precipitated twice from diethyl ether and dried under

1 vacuum to yield 598 mg polymer. H NMR (300 MHz, CDCl3): 훿� 6.74 (br. s., 0.6H), 6.56

64 (br. s., 4.4H), 4.14 - 4.25 (m, 4H), 3.48 - 3.62 (m, 4H), 2.83 (br. s., 0.4H), 2.62 (br. s., 4.4H),

2.41 (q, J = 7.03 Hz, 1.6H), 1.15 (t, J = 7.32 Hz, 2.4H).

Scheme 3.4. Synthesis of p(mAla-co-mDOPA) and p(mAla-co-mPhe)

Synthesis of control polymer p(mAla-co-mPhe): mAla monomer (2.0634 g, 12.8 mmol, 0.8 eq.), mPhe monomer (0.7593 g, 3.2 mmol, 0.2 eq.), succinic acid (1.8894 g, 16 mmol, 1.0 eq.) and DPTS (1.8707 g, 6.4 mmol, 0.4 eq.) were added to a 100 mL Schleck flask. 12 ml dry DCM was added to the flask under N2. This mixture was warmed up to

40 °C for 1-2 min and then cooled with ice bath. To this cooled mixture DIC (7.5 mL, 48 mmol, 3.0 eq.) was added dropwise and reaction mixture was stirred at room temperature for 52 h. Polymer was precipitated twice from methanol and dried under vacuum to yield

65 1 2.5 g polymer (60.4%). H NMR (300 MHz, CDCl3): 훿� 7.13 - 7.35 (m, 1.68H), 4.15 – 4.24

(m, 4H), 3.49 - 3.62 (m, 4H), 2.97 (d, J = 7.46 Hz, 0.4H), 2.53 - 2.70 (m, 4.4H), 2.39 (q, J

= 7.32 Hz, 1.6H), 1.14 (t, J = 7.32 Hz, 2.4H).

Synthesis of p(SCDpr) (scheme 3.5): Soybean oil monomer SBO (5.39g, 14.62 mmol, 0.8 eq.), protected DOPA mimic monomer mDOPApr(848 mg, 2.74 mmol, 0.15 eq.), coumarin monomer Cou (306.4 mg, 0.91 mmol, 0.05 eq.), sebacic acid (3.696 g, 18.27 mmol, 1.0 eq.) and DPTS (2.137 g, 7.31 mmol, 0.4 eq.) were added to a 100 mL Schlenk flask. 20 mL anhydrous DCM was added to the flask under N2. This mixture was warmed up to 40 °C for 1-2 min and then cooled with ice bath. To this cooled mixture DIC (8.7 mL,

54.8 mmol, 3.0 eq.) was added dropwise and reaction mixture was stirred at room temperature for 48 h. Polymer was precipitated twice from methanol and dried under

1 vacuum oven. H NMR (500 MHz, CDCl3): 훿� 7.63 (d, J = 9.29 Hz, 0.05H), 7.36 (d, J =

8.56 Hz, 0.05H), 6.82 - 6.85 (m, 0.10H), 6.59 - 6.63 (m, 0.43H), 6.24 (d, J = 9.54 Hz,

0.05H), 5.30 - 5.40 (m, 2.26H), 4.09 - 4.21 (m, 4H), 3.52 – 3.66 (m, 4H), 2.86 (t, J = 7.83

Hz, 0.32H), 2.75 - 2.80 (m, 1.03H), 2.57 - 2.62 (m, 0.42H), 2.24 - 2.35 (m, 5.65H), 2.14 -

2.20 (m, 0.19H), 2.00 - 2.09 (m, 2.68H), 1.59 - 1.65 (m, 6.81H), 1.25 - 1.38 (m, 22.25H),

0.97 (t, J = 7.46 Hz, 0.19H), 0.86 - 0.90 (m, 2.18H).

Synthesis of p(SCD): p(SCDpr) (800 mg) and triisopropylsilane (50 µL) were dissolved in 5 mL DCM (degassed for 30 min). 5 mL TFA was added and the solution was stirred at RT for 2 h under N2. Solvent was removed by rotary evaporation. The polymer

1 was precipitated from MeOH and dried under vacuum oven. H NMR (500 MHz, CDCl3):

훿� 7.64 (d, J = 9.54 Hz, 0.05H), 7.37 (d, J = 8.56 Hz, 0.05H), 6.73 - 6.85 (m, 0.39H), 6.58

(d, J = 7.34 Hz, 0.14H), 6.25 (d, J = 9.29 Hz, 0.05H), 5.30 - 5.41 (m, 2.24H), 4.09 - 4.22

66 (m, 4H), 3.52 – 3.67 (m, 4H), 2.76 - 2.85 (m, 1.26H), 2.57 - 2.62 (m, 0.39H), 2.24 - 2.37

(m, 5.76H), 2.14 - 2.20 (m, 0.16H), 2.01 - 2.07 (m, 2.60H), 1.60 (s, br, 5.93H), 1.25 - 1.38

(m, 23.63H), 0.97 (t, J = 7.46 Hz, 0.17H), 0.86 - 0.90 (m, 2.25H).

Scheme 3.5. Synthesis of viscoelastic polyester p(SCD) from pendant functionalized

diols of long alkyl chain (S), coumarin (C) and DOPA (D) units.

Synthesis of p(SCP): Soybean oil monomer SBO (5.8303g, 15.81 mmol, 0.8 eq.), mPhe monomer (703.6 mg, 2.97 mmol, 0.15 eq.), coumarin monomer Cou (331.4 mg,

0.99 mmol, 0.05 eq.), sebacic acid (3.998 g, 19.77 mmol, 1.0 eq.) and DPTS (2.311 g, 7.91 mmol, 0.4 eq.) were added to a 100 mL Schlenk flask. 20 ml anhydrous DCM was added to the flask under N2. This mixture was warmed up to 40 °C for 1-2 min and then cooled

67 with ice bath. To this cooled mixture DIC (9.3 ml, 59.31 mmol, 3.0 eq.) was added dropwise and reaction mixture was stirred at room temperature for 39 h. Polymer was precipitated

1 twice from methanol and dried under vacuum oven. H NMR (500 MHz, CDCl3): 훿� 7.63

(d, J = 9.29 Hz, 0.05H), 7.37 (d, J = 8.31 Hz, 0.05H), 7.18 - 7.30 (m, 1.11H),6.82 - 6.85

(m, 0.10H), 6.24 (d, J = 9.29 Hz, 0.05H), 5.30 - 5.41 (m, 2.21H), 4.09 - 4.22 (m, 4H), 3.51

- 3.67 (m, 4H), 2.98 (t, J = 7.71 Hz, 0.29H), 2.76 - 2.81 (m, 0.99H), 2.67 (t, J = 7.71 Hz,

0.29H), 2.59 (t, J = 7.09 Hz, 0.10H), 2.17 - 2.36 (m, 5.70H), 2.01 - 2.10 (m, 2.59H), 1.61

(d, J = 6.85 Hz, 2.68H), 1.26 - 1.39 (m, 21.93H), 0.98 (t, J = 7.58 Hz, 0.16H), 0.87 - 0.91

(m, 2.13H).

3.3.4 Water contact angle measurement

Glass cover slides were cleaned by sonicating in ethanol, washed with distilled water and dried under stream of air to make sure the cover slips were spot and debris free.

The polymers were spin coated from 2% (w/v) polymer solution in CHCl3. Spin coating of polymers were carried out at 2500 rpm for 1 min. The slides were then vacuum dried at room temperature and used for contact angle measurements. Water contact angle was measured by contact angle measure machine Rame-Hart. Each polymer coated cover slide was loaded onto sample stage and one drop (~10 µL) of ultrapure water was deposited onto the surface. Images were take every 10 second in 2 minutes. Contact angle was analyzed via DROP image advanced software which is determined by a contour fitting algorithm and the profile coordinates were used to calculate contact angles. 10 droplets were measured for each sample.

68 3.3.5 Lap shear adhesion test of catechol containing polymers and control polymers

For p(mAla-co-mDOPA) and p(mAla-co-mPhe), lap shear adhesion testing

(based on ASTM D1002) was carried out using Instron 5567 (Load cell: 1000 N) and custom built force rig (Load cells: 10 N and 50 N, Shimpo).The cross head speed was fixed at 15 mm/min. Pre-cleaned mirror polished aluminum substrates (8 cm x 1.2 cm, McMaster) were used as adherends. Modifications in the ASTM procedure were done as per experimental limitations. For application, polymers p(mAla-co-mPhe) (control polymer) or p(mAla-co-mDOPA) (DOPA based polymer) were dissolved in chloroform (25% w/v in chloroform). The oxidant solution was prepared by dissolving tris(acetylacetonato) iron

(III) (0.3 or 0.6 equivalents) in CHCl3. About 30 µL of the polymer solution was placed on one of the aluminum substrate (bonded area: 1.5-1.8 cm x 1.2 cm), applied with a wedge applicator (50 µm thickness) and bonded with the other substrate. When testing with oxidant, about 5 µL of oxidant was placed along with the polymer solution and mixed homogeneously with Teflon bar before adhering it to the other substrate. After bonding, the two substrates were clamped together with a paper clip to keep them intact until testing.

The specimens were cured by placing them in air for an hour and then exposing to vacuum for 22 hours. For dry environment testing, the cured specimens were loaded on the machine to record the force. For wet environment testing, water was sprayed over the bonded area and kept for one hour before loading on the machine. Force versus time data was recorded and Lap Shear Strength (MPa) was calculated as Maximum force recorded (N)/Bonded area (mm2). The data represented were from set of five measurements ± standard deviation.

The adhesion measurements were carried at room temperature (26 °C).

69 For p(SCD) and p(SCP), lap shear adhesion testing were carried out on glass adherends. When using glass as adherend, the testing method was based on ASTM D1002 and was carried out using Instron 5567 (Load cell: 1000 N).The cross head speed was fixed at 1.3 mm/min. The micro slides (25×75 mm, 1.0 mm thick, VWR) were cleaned by immersed in base bath for 30 min, rinsed with deionized water, dried with compressed air.

The viscous polymer was applied directly onto one end of substrate with a spatula, then another substrate was together in a lap figure. The bonded area was 12.7 × 25.4 mm. After bonding, the adhesive was cured with UV irradiation for certain amount of time. After irradiation, the two substrates were clamped together with a paper clip to keep them intact until testing. For dry environment testing, the cured samples were kept at atmosphere for

30 min before testing. For wet environment testing, the cured samples were immersed in water for 24 hours before testing. Force versus extension data was recorded and lap shear strength (kPa) was calculated as maximum force recorded (N) devided by bonded area

(mm2).

3.3.6 End to end adhesion test of p(SCD) and p(SCP) on porcine skin

Porcine skin was purchased from grocery store and the fat was removed with razor as much as possible. The skin was cut into strips which were about 30 mm long and 15 mm wide. The thickness of skin was from 2.5 mm to 3 mm. Polymer was deposited on the top cut, and irradiated with UV light (Dymax) for certain amount of time. For the first one minute, the two skin was twisted and wiggled by hands to let polymer go inside the cut.

The skin except the cut area was covered with gauze which was immersed in 1x PBS in order to avoid the dehydration of the skin during curing process. 30 min after cure, the

70 samples were tested on TA XT Plus Texture Analyzer (load cell: 50 N) with cross head speed at 10 mm/min. Force versus extension data was recorded the adhesion strength (kPa) was calculated as Maximum force recorded (N) / Bonded area (mm2).

Tisseel, a commercially available fibrin adhesive used as comparison to our adhesives. The fibrin glue was applied on one end of the cut and then the other end put together. After 30 min, the sample was tested with cross head speed at 10 mm/min. Force versus extension data was recorded. The adhesion strength (kPa) was calculated as

Maximum force recorded (N) / Bonded area (mm2).

3.4 Results and Discussion

Polyesters with pendant groups that mimic dihydroxyphenylalanine (DOPA) and alanine:

Our group has previously shown the synthesis of a modular polyester platform that can efficiently incorporate functional groups as pendant groups - thereby providing a platform to mimic the pendant groups of mussel adhesive peptides. A cursory view of the amino acid sequence of mussel adhesive proteins shows that the DOPA units are flanked by various hydrophilic amino acids. We therefore designed a mussel protein mimicking polyester containing pendant catechol and hydrophilic alanine-like groups p(mAla0.8-co- mDOPA0.2). This statistical copolymer was compared to a control polymer p(mAla0.8-co- mPhe0.2) which differed only in the catechol units (dihydroxyphenylalanine) being replaced by phenylalanine analogs (Figure 3.1). Thereby this provided an opportunity to probe whether i) the phenolic groups of catechol play a role in adhesion and ii) if hydrophilic units are able to preserve adhesive interactions in wet conditions.

71 A B

Figure 3.1. Pendant functionalized polyesters p(mAla0.8-co-mDOPA0.2) (A) and with p(mAla0.8-co-mPhe0.2) (B) serve to investigate the role of DOPA and hydrophilicity in adhesion between polymer and substrate.

The above polyesters were synthesized by carbodiimide mediated room temperature polyesterification of the corresponding diols with succinic acid. The structures of the two copolymers were confirmed by 1H NMR and it was confirmed that the compositions as determined by 1H NMR were close to the feed ratio of the two monomers.

The protected catechol polyester had a number average molecular weight (Mn) of 68 kDa and polydispersity index (PDI) of 1.62 whereas the control polyester had Mn of 50 kDa and

PDI of 1.50 as determined by size exclusion chromatography (SEC).

The adhesive strength of polymers is influenced by their surface energy and hence it was instructive to examine their contact angles for spin coated films of the two polyesters.

The hydrophilicity of the polyesters was characterized by the evolution of their water contact angle on spin coated polyester films during wetting over 120 s at room temperature.

Figure 3.2 shows contact angles and droplet width as a function of droplet interaction time.

The two polyester films have similar response to wetting. The initial contact angles for the

DOPA polyester and the control polyester were 78 ± 4° and 88 ± 4° respectively. However,

72 the contact angles decrease dramatically (by 20°) during the first 40s after contact with water and afterwards show a slower decrease to 43 ± 3° and 58 ± 2° over 120 s (Figure

3.2). Meanwhile, the water droplet width increases indicating spreading of water on the polyester film. The surface of film transforms from being hydrophobic in air to hydrophilic with liquid water due to wetting. This can be postulated to indicate rearrangement of polymer chains at the polyester surface, and it may also be facilitated by the glass transition temperatures of both polyesters being below room temperature.

Figure 3.2. Evolution of water contact angle and droplet width on spin-coated polyester films of p(mAla0.8-co-mDOPA0.2) (catechol polymer) (A) and with p(mAla0.8-co- mPhe0.2) (control polymer).

DOPA units in mussel proteins serve to increase both adhesive with the substrate and cohesive interactions between the protein chains. DOPA increases adhesive strength by complexation with metal ions through the phenolic groups. In addition, oxidation of the 73 phenolic groups to hydroquinone initiates crosslinking with adjacent oxidized hydroquinone groups resulting in increase of cohesive strength through crosslinked hydroquinones. Therefore optimization of both the adhesive and cohesive interactions will enable superior bonding in both dry and wet environments. To assess the adhesive strength of p(mAla0.8-co-mDOPA0.2) and p(mAla0.8-co-mPhe0.2) polyesters, lap shear tests were conducted using mirror polished aluminum plates as adherends. The DOPA containing polyester p(mAla0.8-co-mDOPA0.2) was dissolved in minimum amount of CHCl3 and spread uniformly on a mirror polished aluminum plate. To oxidize the DOPA units, Fe3+ was mixed with the polyester prior to coating the aluminum plate. Subsequently the second plate was pressed onto the first plate and allowed to cure in vacuum oven at room temperature for 22 h before testing. The control polyester was tested under similar conditions, without and with the oxidant. The adhesion was also tested under wet

Figure 3.3. Lap shear geometry to test the adhesive strength of the polyesters (left). Lap shear strength of the p(mAla0.8-co-mDOPA0.2) (catechol) and p(mAla0.8-co-mPhe0.2)

(control) polyesters under dry and wet conditions (right).

74 conditions. To simulate wet conditions, water was sprayed over the two bonded aluminum plates and allowed to seep into the bonded area over 60 min. Figure 3.3 shows the results of the lap shear test for both the polyesters under different conditions.

Under dry conditions, both p(mAla0.8-co-mDOPA0.2) and p(mAla0.8-co-mPhe0.2) showed similar lap shear strengths of 0.09±0.01 and 0.06±0.02 MPa, respectively in the absence of any oxidant. Upon failure, the polymer was present on both the adherends, indicating cohesive failure. After oxidation and crosslinking of the DOPA units of p(mAla0.8-co-mDOPA0.2) with tris(acetylacetonato) iron (III) (0.3 and 0.6 equivalents), the lap shear strength increased significantly to 0.35±0.11 MPa (0.3 eq. Fe3+) and to 0.52±0.18

MPa (0.6 eq. Fe3+). In both instances, the failure was determined to be a cohesive failure.

Oxidation of the control polyester with Fe3+ did not result in an increase of the adhesive

- strength. Various oxidants such as [IO4] have been used to crosslink the DOPA and the use

- of [IO4] as an oxidant in the current experiments were not successful as the oxidation was too fast to enable effective application of the adhesive onto the substrates.

The polyesters described here are hydrophilic as determined by their water contact angle and they rearrange in the presence of water. Their adhesive strength was significantly affected in presence of water (Figure 3.3). After water was allowed to interact with the adhesive for 60 min, the adhesion strength of p(mAla0.8-co-mDOPA0.2) cured with 0.3 and

0.6 equivalents of Fe3+ was 0.06±0.03 MPa and 0.13±0.08 MPa (cohesive failure) respectively. The adhesive force for control polyester p(mAla0.8-co-mPhe0.2) could not be recorded as the samples failed on loading onto the force rig. The interaction of water with the control polymer led to delamination of the adhesive film from the substrate leading to adhesive failure in specimens.

75 Although the above DOPA containing polyester failed under wet conditions, the above experiments were instructive. These experiments clearly showed that after oxidation

DOPA units are involved in increasing adhesive and cohesive interactions between the polymer and substrate. This is inferred from the results of lap shear strength of the control polymer, which is similar in all respects except for substituting DOPA units with phenylalanine units, to the DOPA containing polyester. The p(mAla0.8-co-mDOPA0.2) polyester upon oxidation with 0.6 eq. of Fe3+ showed a strength of 0.52±0.18 MPa, while the control polymer, p(mAla0.8-co-mPhe0.2) had less than 0.1 MPa before and after oxidation. The above experiments also showed that residues such as the mimic of alanine side chain used here, increase the overall hydrophilicity and water infiltration into the bonded area resulting in significantly decreasing of the overall strength of the adhesive.

However compared to the control polyester, the DOPA containing polyester was more effective in wet conditions, pointing to the role of DOPA units in adhesion. Moreover, in the DOPA containing polyester, loss of adhesion was seen to be a result of cohesive failure indicating that the DOPA units were involved in the adhesive interactions with the aluminum plates. On the other hand, in the control polymer under wet conditions, the adhesive delaminated from the aluminum plates resulting in an adhesive failure. In addition to the above interpretations, it was also inferred that in biomedical applications, the adhesive cannot be applied with any organic solvent.

Polyesters with pendant dihydroxyphenylalanine (DOPA), coumarin and alkyl chains:

Therefore a polyester was designed to be a viscoelastic melt at room temperature to enable application without any solvent. In addition, the polyester was designed to be

76 hydrophobic to keep out water from the interface, and furthermore to gain better control of the crosslinking process, a coumarin unit was introduced to enable photochemical crosslinking of the polymer units. This polyester had the ideal characteristics of room temperature application without any solvent, excellent adhesion in both dry and wet environments, and spatial and temporal control of the crosslinking mechanism.

The viscoelastic polyester contains three monomer units: a long alkyl chain that is derived from soybean oil, a DOPA pendant group and a coumarin pendant group. The long alkyl chain containing diol (S) is synthesized by the transamidation of diethanolamine with soybean oil. The polyester was synthesized by DIC mediated polyesterification of the alkyl chain containing diol (S), coumarin diol (C), and DOPA diol (D) with sebacic acid (Scheme

3.5). Sebacic acid increases the hydrophobic nature of the polymer and helps to decrease the modulus of the resulting polyester. As a control, a polyester was synthesized with a phenylalanine unit (P) in place of the DOPA unit. The ratio of S:C:D and S:C:P was maintained at 0.8:0.05:0.15. The molecular weights of both polyesters were maintained in the same range.

The coumarin units undergo [2+2] photocycloaddition upon irradiation at 350 nm, leading to crosslinking between polymer chains and therefore eliminate the need for Fe3+ in this system. The polyesters are viscoelastic at room temperature and the loss modulus is greater than the storage modulus between frequencies of 0.1-100 Hz (Figure 3.4A). Upon irradiation, the polyesters exhibit increasing elastomeric behavior with increasing irradiation, and the storage modulus becomes greater than the loss modulus at all frequencies after 16 min of irradiation. The crosslinked polyesters exhibit shear thinning behavior with increasing shear rates (Figure 3.4B).

Figure 3.4. Rheological properties of the SCD polyester at varying frequencies before and after crosslinking (A) and their corresponding viscosities at different shear rates (B).

Figure 3.5. Evolution of contact angles for DOPA containing polyester, p(SCD) and the control polyester, p(SCP).

To determine the behavior of the polyesters in presence of water, their water contact angles were tested on spin coated cover slips. The contact angles of the DOPA containing polyester p(SCD) and the corresponding control polyester p(SCP) were substantially 78 higher than the previous generation of polyesters. The initial contact angles of p(SCD) and p(SCP) were 87±3° and 86±1° respectively and over a period of 120 s, the contact angles decreased to 66±1° and 78±1° respectively (Figure 3.5). Similar to the first generation of polymers, the surface groups appear to rearrange in presence of water leading to a decrease in their contact angles. Due to the presence of phenolic groups, p(SCD) polyesters exhibited lower contact angles compared to p(SCP).

The adhesive behavior of the p(SCD) and p(SCP) polyesters was evaluated in both dry and wet conditions. The polymers were coated onto a fixed area (2.54 x 1.27 cm) of a

2.54 x 7.62 cm micro slides. After ensuring a uniform coating, 1.27 cm of the second plate was brought into contact with the coated area of the first plate and pressed together to ensure full contact. The plates were irradiated at 320-450 nm from the top of glass slides.

It has been shown in literature that the coumarin chromophore can also undergo crosslinking with alkene units and therefore it is expected that the unsaturated units of the soybean oil alkyl units also participate in the crosslinking process. Following irradiation, the samples were kept in room temperature for 30 min and then their adhesion was determined by lap shear tests. Under dry conditions, in the absence of irradiation, the DOPA containing polyester, p(SCD), does not register any force. However, after irradiation for 5 min and 10 min, p(SCD) and p(SCP) showed lap shear strength of 0.9 and 0.5 MPa respectively, indicating that in p(SCD), both cohesive interactions from inter-strand crosslinking and adhesive interactions from DOPA units binding to glass are contributing to the strength of the adhesive (Figure 3.6A). The adhesive strength of the polyesters were subsequently tested under wet conditions. As described above, the polymer adhesive was spread uniformly onto a glass slide, adhered onto a second slide and allowed to achieve

Figure 3.6. Lap shear measurements of p(SCD) and p(SCP) under dry conditions (A) and wet conditions (B).

uniform contact and irradiated for 5 min. Following this, the plates were immersed in tap water for 24 h, taken out and patted dry. Immediately following this, lap shear measurements were taken of both the p(SCD) and p(SCP) polyester bonded glass slides.

As shown in Figure 3.6B, there is no loss of adhesive strength of either the p(SCD) or p(SCP) polymers after exposing to water and have the same strength as their corresponding dry state. The alkyl chains are able to provide a hydrophobic environment that keeps away the water. Although p(SCD) is more hydrophilic relative to p(SCP), it appears that in both cases the viscoelastic nature of the adhesive forms good contact and prevents water from penetrating the adhesive.

The end to end test geometry by using porcine skin as substrate is shown in Figure

3.7A. p(SCD) or p(SCP) filled the cut and UV light (320-450 nm, Dymax) was shined on the adhesives. Fibrin glue was also tested under the same experiment condition. Figure

3.7B shows the adhesion strength for both polymers at different conditions. When there is no UV irradiation, both polymers show similar average strength of about 2 kPa.

Figure 3.7. End to end test adhesion test geometry on porcine skin (A), adhesion strength for p(SCD), p(SCP), and fibrin glue (B).

The strength for the two polymers increased to about 5 kPa after 3 min irradiation.

When the samples were irradiated for 5 min, the adhesion strength increased to 8 kPa. For the commercial available fibrin glue, the adhesion strength is around 5 kPa. In this set of experiment, we did not see much differences for polymer with or without DOPA mimic groups. One reason could be that both polymer are UV curable and the only difference we should see is the adhesion of polymer with substrates. What’s more, since the cut of skin area is very small, the differences may not be able to show from the macroscopic test.

3.5 Conclusion

In conclusion we have developed an adhesive that mimics the functionality of mussel adhesive proteins. The modular nature of the polyester platform enables incorporation of three different units that play key roles in adhesive interactions. The long alkyl chain units derived from soybean oil provide room temperature viscoelasticity that 81 enables application of the adhesive without any solvents. The DOPA units provide adhesive interactions with the substrate and the coumarin units provide sites for inter-strand crosslinking which provide cohesive strength for the adhesive. These key features will enable the p(SCD) adhesive to be utilized in surgical applications, especially where sutures cannot be used such as in cardiac surgery.

82 CHAPTER IV

ELECTROSPUN FIBER MAT FOR SUSTAINABLE DRUG RELEASE

4.1 Abstract

Electrospinning is a widely used method to produce nanofibers from a variety of materials. Electrospun nanofiber mats were fabricated from pendant functionalized polyesters. These electrospun mats contained dyes that were either conjugated or encapsulated within the polymer matrix. The fibers were smooth and relatively uniform.

The release of the dyes was evaluated over 90 days. The cumulative release profile of mixed dyes showed that the release of each dye had three phases. The coumarin dye exhibited a burst release over days 0-7, followed by a decreased release rate and then an accelerated rate after day 55. When Rhodamine B was encapsulated, it demonstrated similar kinetics as coumarin, but with lower cumulative release. However when the rhodamine B was conjugated to the polyester through oxime coupling to a pendant ketone functionality, the dye did not release even after 90 days, as the oxime bond is stable in

1×PBS (pH = 7.4).

4.2 Introduction

Compared to conventional dosage forms, drug delivery systems based on polymeric material have many advantages, such as reduced toxicity, improved therapeutic effect,

83 convenience, and so on.142 However, there are still some problems of this strategy, for example, burst release of drugs at the beginning and low efficiency to prepare nano- or microparticles for local or in situ chemotherapy.142 Methods that can constantly release therapeutic drug amounts over a period of multiple months are preferred.143-144 It was found that drugs can be encapsulated into electrospun nanofibers and those systems showed nearly zero-order kinetics of drug release.142 Devices based on electrspun fibers are promising for drug delivery applications.145

In the past few years, scaffolds based on polymeric nanofibers prepared via electrospinning technology have been used for tissue engineering.146 One unique property of these nanofiber materials is that they have physical structure that mimics the native extracellular matrix (ECM).146-149 The nanofiber-based scaffolds provide an environment for cells to remodel the cell-scaffold system since the fibers are dimensionally small and physically weak.146 In tissue engineering applications, in addition to cells and scaffolds, it would be advantageous to deliver appropriate growth factors in a controlled manner.

Growth factors need to be delivered in a controlled and sustained manner while maintaining their bioactivity.146 Thus, if controlled delivery of growth factors can be achieved from nanofiber scaffolds, they would be efficacious in various tissue engineering applications.

146, 150-151

Although electrospun fibers have been used for drug release, many of those studies adopted the method of simply mixing drugs and carrier polymers. This blending procedure usually results in a burst release phenomenon.144, 146, 152 In addition, non-degradable polymers are adopted which requires surgical retrieval at the end of the treatment.143, 153 To address these limitations, degradable polymers have been used for drug delivery systems

84 or tissue engineering scaffolds to eliminate the need for removal of the device.143, 152, 154

Zhang et al. encapsulated a model protein, fluorescein isothiocyanate-conjugated bovine serum albumin (fitcBSA), with a water-soluble polymer, poly(ethylene glycol) (PEG), within the biodegradable poly(ε-caprolactone) (PCL) nanofibers using a coaxial electrospinning technique.146 They showed that the core-sheath nanofibers alleviated the initial burst release for higher protein loading and gave better sustainability compared to blend type nanofibers.146 These studies have shown enhanced drug release profile, however, the natural tissue repair process involves multiple growth factors and signaling molecules.150 Consequently, the delivery system should optimally be capable of delivery multiple drugs or growth factors in a controlled fashion.147

In this contribution, electrospun fiber mats based on a degradable polyester with

‘peptide-like’ pendant groups were prepared for dual release studies. As a proof of concept, rhodamine B and coumarin dyes were used as drug models. The dyes were either blended into the fiber mats or conjugated to the polymer pendant groups via keto-oxime linkages.4,

155 The dual release of dyes were studied and compared.

4.3 Experimental Section

The experimental section describes the chemicals needed for synthesis of targeted product, the method of making monomers and polymers, and the characterization.

4.3.1 Materials and instrumentation

Materials: All the reagents were purchased from Sigma Aldrich or Alfa Aesar and used without further purification unless otherwise noted. N,N'-diisopropylcarbodiimide

85 (DIC, 99%) was purchased from Oakwood Chemical and used as received. 4-

(dimethylamino) pyridinium 4-toluene sulfonate (DPTS) was prepared according to

89 literature methods. Dichloromethane was dried by distillation over anhydrous CaH2 and the DMF was dried by distillation over anhydrous CaH2. Silica gel (40-63 μm, 230 x 400 mesh) for flash chromatography was purchased from Sorbent Technologies, Inc.

(RI) and UV detector. Separation occurred over two PSS Gram Analytical GPC Columns in series using 25 mM LiBr in dimethylformamide (DMF) as eluent at a flow rate of 0.8 mL/min. The column and detector temperatures were maintained at 50 °C. Molecular weights were obtained relative to PS standards using the RI signal. Thermogravimetric analysis (TGA) was performed with TA Q500 over an interval of ambient temperature to

600 °C at a heating rate of 10 °C/min under a N2 atmosphere. Differential scanning calorimetry (DSC) was performed on TA Q2000 DSC with a liquid N2 cooling unit and a heating/cooling rate of 10 °C/min. ESI MS was performed on Bruker HTC ultra QIT.

4.3.2 Synthesis of rhodamine B derivative

Synthesis of M4.1: tert-butyl-N-hydroxycarbamate (2.0 g, 15 mmol) was added to a 100 ml round bottom flask. 10 mL anhydrous DMF was added to the flask. 1,8-

Diazabicyclo [5.4.0]undec-7-ene (DBU) (2.23 mL, 15mmol) dissolved in 5 mL was added to the above solution. 1,6-dibromohexane (4.61 mL, 30 mmol) dissolved in 5 mL

Scheme 4.1. Synthesis of rhodamine B derivative.

anhydrous DMF was added to the above solution. After 3 h of stirring at RT, an additional

1 mL of 1, 8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added. After an additional 2 h of stirring at RT, the temperature was increased to 50 °C and allowed to stir for 24 h. The reaction mixture was then concentrated by rotary evaporation. The residue was dissolved in 9:1 ethyl acetate:methanol and filtered through a plug of silica gel to remove the DBU

HBr salt. The filtrate was collected and the solvent was evaporated by rotary evaporation.

The crude oil was purified by silica gel column chromatography with a 0-10% hexane in ethyl acetate gradient eluting system. The product was dried through rotary evaporation

87 1 and high vacuum and characterized via H NMR (500 MHz, CDCl3): δ 7.08 (s, 1H), 3.86

(t, J = 6.60 Hz, 2H), 3.42 (t, J = 6.85 Hz, 2H), 1.82 - 1.94 (m, 2H), 1.59 - 1.71 (m, 2H),

1.50 (s, 9H).

Synthesis of M4.2: Rhodamine B (1.0 g) was dissolved in 100 ml 1 M sodium hydroxide solution and stirred for 2 h. The solution was partitioned with DCM (100 mL).

The organic layer was isolated and the aqueous layer was extracted twice with DCM.

Combined organic layers were washed once with 1 M sodium hydroxide and brine. The organic solution was dried with anhydrous Na2SO4, filtered, concentrated with rotary evaporation and dried under high vacuum to yield 0.8 g of Rhodamine B base as a pink

1 foam. H NMR (300 MHz, CD3OD): δ 8.04 - 8.18 (m, 1H), 7.54 - 7.75 (m, 2H), 7.20 -

7.40 (m, 3H), 6.99 (dd, J = 2.49, 9.51 Hz, 2H), 6.91 (d, J = 2.34 Hz, 2H), 3.66 (q, J = 7.03

Hz, 8H), 1.30 (t, J = 7.03 Hz, 12H).

Synthesis of M4.3: A two neck flask and addition funnel were flame dried before use. Piperazine (2.32 g, 27 mmol) was added into the flask and vacuum back-filled with

N2 three times. 12 mL anhydrous DCM was added. Trimethylaluminium (6.7 mL 2M in toluene, 13.5 mmol) was added to the flask dropwise through an additional funnel and the reaction was stirred for 1 h to yield a white precipitate. A solution of rhodamine B base

(2.98g, 6.7 mmol) in 10 mL of anhydrous DCM was added dropwise to the heterogeneous solution. The mixture was refluxed for 24 h. A 0.1 M aqueous solution of HCl was added dropwise until gas evolution ceased. The heterogeneous solution was filtered and the retained solids were rinsed with DCM and a 4:1 DCM/MeOH solution. The combined filtrates were concentrated. The residue was dissolved in DCM, filtered to remove insoluble salts, and concentrated again. The resulting solid was then partitioned between

88 dilute aqueous NaHCO3 (150 mL) and EtOAc (100 mL). After isolation, the aqueous layer was washed with three additional portions of EtOAc (100 mL) to remove residual starting material. The retained aqueous layer was saturated with NaCl, acidified with 1 M aqueous

HCl, and then extracted with multiple portions of 2:1 isopropanol/dichloromethane until a faint pink color persisted. The combined organic layers were then dried over Na2SO4, filtered, and concentrated by rotary evaporation. The purple solid was dissolved in a minimum amount of MeOH and precipitated by dropwise addition to a large volume of diethyl ether. The product was collected by filtration as a dark purple solid (3.0 g, 87%).

1 H NMR (500 MHz, CD3OD): δ 7.73 - 7.86 (m, 3H), 7.48 - 7.58 (m, 1H), 7.27 (d, J = 9.54

Hz, 2H), 7.11 (dd, J = 2.20, 9.54 Hz, 2H), 6.98 (d, J = 2.20 Hz, 2H), 3.58 - 3.80 (m, 12H),

3.13 (br. s., 4H), 1.97 - 2.05 (m, 1H), 1.32 (t, J = 7.09 Hz, 12H).

Synthesis of M4.4: Compound M4.3 (113 mg, 0.206 mmol) and 500 µL anhydrous

DMF was added to a 20 mL scintillation vial with a magnetic stir bar. Compound M4.1

(76.4 mg, 0.258 mmol), N, N-diisopropylethylamine (DIPEA) (62.5 µL, 0.368 mmol), and an additional portion of anhydrous DMF (500 µL) were added to the vial. The reaction mixture was stirred at rt. for 20 h. An additional aliquot of compound M4.1 (76.4 mg, 0.258 mmol) and DIPEA (63 µL, 0.368 mmol) and anhydrous DMF (300 µL) was then added.

An additional aliquot of compound M4.1 (76.4 mg), DIPEA (63 µL), and DMF (180 µL) was then added after another 18 h. After an additional 18 h, the DMF was removed under reduced pressure, and the remaining crude mixture was partitioned between ethyl acetate and saturated sodium bicarbonate. The aqueous layer was extracted with a 1:3 mixture of isopropanol and dichloromethane until colorless, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated to afford 68 mg (46%) of dark

89 1 purple solid. H NMR (500 MHz, CD3OD): δ 7.70 - 7.81 (m, 2H), 7.59 - 7.70 (m, 1H), 7.45

- 7.56 (m, 1H), 7.23 - 7.32 (m, 2H), 7.07 (d, J = 9.54 Hz, 2H), 6.98 (br. s., 2H), 3.65 - 3.87

(m, 10H), 3.33 - 3.50 (m, 8H), 2.15 - 2.29 (m, 3H), 1.54 - 1.67 (m, 2H), 1.37 - 1.48 (m,

11H), 1.23 - 1.37 (m, 16H).

Synthesis of M4.5: Compound M4.4 (10.5 mg, 0.0144 mmol) was added to a 20mL scintillation vial. A mixture of 1 to 1 ratio of trifluoroacetic acid (TFA) and dichloromethane (1 mL) was added at room temperature. The mixture was allowed to stand for 2 min. The solvent was then evaporated under a stream of nitrogen gas. The following procedure was repeated three times. The crude residue was dissolved in 1 mL of dichloromethane and concentrated under a stream of nitrogen to yield 7.3 mg (80%) of dark purple solid. MS ESI: M+ calculated: 626.4, found: 626.3.

Scheme 4.2. Synthesis of p(mPhe-Keto-BocGlu).

4.3.3 Synthesis of p(mPhe-Keto-BocGlu)

mPhe (4.2234 g, 17.8 mmol), Keto monomer (0.9043 g, 4.45 mmol), Boc-L-

Glutamic acid (5.5006 g, 22.2 mmol), and DPTS (2.6011g, 8.9mmol) were added to a

Schlenk flask and vacuum backfilled with N2 three times. 20 mL anhydrous DCM was added to the flask by syringe. The reaction mixture warmed up to 40 °C for 1-2 min, and then cooled to 0 °C. DIC (11 mL, 70 mmol) was added dropwise by syringe and reaction

Scheme 4.3. Conjugation of rhodamine B alkoxyamine derivative with p(mPhe-Keto-

BocGlu).

mixture was warmed to RT and stirred for 72 h. Then polymer was precipitated twice from methanol and dried under vacuum. 4.2 g (42.9%) polymer was obtained. 1H NMR (500

MHz, CDCl3): δ 7.22 (br. s., 4H), 5.30 (br. s., 0.84H), 4.12 - 4.32 (m, 5H), 3.54 - 3.61 (m,

4H), 2.97 (br. s., 1.61H), 2.66 (br. s., 2.46H), 2.37 (br. s., 2H), 2.14 - 2.21 (m, 1.64H), 1.78

- 2.01 (m, 1H), 1.42 (br. s., 9H). Mn=79 kDa, Mw=106 kDa, PDI =1.34.

4.3.4 Conjugation of rhodamine B alkoxyamine derivative with p(mPhe-Keto-BocGlu)

1.06 g of p(mPhe-Keto-BocGlu) was dissolved in 6 mL anhydrous THF. 8 mg of

M4.5 was dissolved in 4 mL anhydrous THF and 1.5 µL triethylamine was added to M4.5 solution. Then M4.5 solution was added into polymer solution. 4 mg of PTSA was added and the reaction mixture was stirred at RT overnight. The polymer was precipitated three times from MeOH.

91 4.3.5 Electrospinning of p(mPhe-Keto-BocGlu), 7-(diethylamino)coumarin-3-carboxylic acid, and rhodamine B

300 mg p(mPhe-Keto-BocGlu) was dissolved in 500µL chloroform. 75 µL 7-

(diethylamino)coumarin-3-carboxylic acid (3 mg) in DMF and 75 µL rhodamine B (1.5 mg) in DMF were added into polymer solution. The mixture was vortexed to mix thoroughly. Then the solution was transferred in to a glass pipette with outlet of 0.5 mm. A thin wire was inserted into the pipette, the end was connected to a direct current. An aluminum foil collecting plate was connected to a grounding electrode. The distance between the tip of glass pipette and aluminum foil was 6 cm. To concentrate the fibers, a copper ring was inserted between the pipette and aluminum foil and the copper ring is 2 cm above pipet tip. 11 kV of direct current was applied to the wire inserted into the pipette and the resulting electrospun fibers were collected on the aluminum foil. The fiber was dried under vacuum overnight. The fiber mat was peeled off from aluminum foil by quick immersion into water and careful removal with tweezers.

4.3.6 Electrospinning of p(mPhe-Keto-BocGlu)-RB and 7-(diethylamino)coumarin-3- carboxylic acid

p(mPhe-Keto-BocGlu)-RB (300 mg) was dissolved in 500µL chloroform. 150 µL

7-(diethylamino)coumarin-3-carboxylic acid (3 mg) in DMF was added to the polymer solution. The solution was vortexed and transferred to a glass pipette with 0.5 mm outlet.

A thin wire was inserted into the pipette, the end was connected to a direct current. An aluminum foil collecting plate was connected to a grounding electrode. The distance between the tip of glass pipette and aluminum foil was 6 cm. To concentrate the fibers, a

92 copper ring was inserted between the pipette and aluminum foil and the copper ring is 2 cm above pipet tip. 12 kV of direct current was applied to the wire inserted into the pipette and the resulting electrospun fibers were collected on the aluminum foil. The fiber was dried under vacuum overnight. Fiber mat was peeled off from aluminum foil by quick immersion into water and careful removal with tweezers.

4.3.7 UV-Vis standard curve of rhodamine B in PBS

6.36 mg rhodamine B was dissolved in 20 mL PBS. 200 µL solution of this solution was taken and 1.8 mL PBS was added to achieve a 10x dilution. The solution was serially diluted from 100x to 1000x, and the corresponding absorbance of each was measured.

Rhodamine B concentration vs absorbance was plotted.

0.7

0.6

0.5

0.4

0.3

Absorbance y = -0.0166 + 0.0948x 0.2

0.1

0.0 0 1 2 3 4 5 6 7 rhodamine B concentration (10-6 mol/L)

Figure 4.1. UV-Vis standard curve of rhodamine B in PBS.

93 4.3.8 UV-Vis standard curve of 7-(diethylamino)coumarin-3-carboxylic acid in PBS

2.87 mg 7-(diethylamino)coumarin-3-carboxylic acid was dissolved in 15 mL PBS.

The above solution was diluted to 30, 40, 50, 60, 70, 80, 90, 100, 150, 200x concentrations.

UV absorbance was measured for each solution. 7-(diethylamino)coumarin-3-carboxylic acid concentration vs absorbance was plotted.

0.9

0.8

0.7

0.6

0.5

0.4

Absorbance

0.3 y = -0.000283 + 0.347x 0.2

0.1

0.0 0.5 1.0 1.5 2.0 2.5 Coumarine dye Concentration (10-5 mol/L)

Figure 4.2. UV-Vis standard curve of 7-(diethylamino)coumarin-3-carboxylic acid in PBS.

4.3.9 Release study of two dyes

In a 20 mL scintillation vial, 2 cm × 2 cm fiber mat of the polyester was immersed in 20 ml PBS. The vial was stored in 37 °C incubator. 2 mL PBS was removed at each time point and replaced with 2 mL fresh PBS, and UV absorbance was measured. These measurements were performed in triplicates. Dye release was calculated according to the

UV absorbance standard curve.

94 4.3.10 Degradation study of the fiber mat

An electrospun fiber mat (~8 mg) and 20 mL 1×PBS was added to a 20 mL scintillation vial and placed in a 37 °C incubator for a fixed time. Experiments were repeated twice per time point. At each point, PBS was decanted and the sample was rinsed with deionized water. Degradation was analyzed using GPC with DMF as solvent.

Figure 4.3. 1H NMR of p(mPhe-Keto-BocGlu).

4.4 Results and Discussion

The polymer p(mPhe-Keto-BocGlu) was synthesized from a mixture of diols which contains 80% mPhe monomer, 20% Keto monomer, and Boc protected glutamic acid and polymerized via DIC mediated room temperature polyesterification. Figure 4.3 shows the proton NMR of polymer p(mPhe-Keto-BocGlu) with peak designations. The peak at 2.89 corresponds to the methylene group next to the phenyl group with a peak

95 integration of 1.61. The peak at 3.46-3.53 are methylene groups adjacent to the tertiary amine with a peak integration of 4.00. A comparison of the two peaks demonstrates that the polymer contains 80% mPhe pendant group and 20% ketone groups, similar to the feed ratio. This polymer has a Mn of 79 kDa, Mw of 106 kDa, and a PDI of 1.34. Larger molecular weights are better for electrospinning so this polymer can be electrospun easily.

Thermogravimetric analysis shows a decomposition temperature of 219 °C, and DSC analysis shoews a glass transition temperature (Tg) of 50.1 °C. A Tg above body temperature is important for integrity of electospun fibers during application. The conjugation of rhodamine B derivative to the polymer was efficiently carried out by oxime bond from the reaction between alkoxyamine and ketone.

Fiber mats with uniform diameter were obtained by electrospinning mixture of p(mPhe-Keto-BocGlu) and rhodamine B and 7-(diethylamino)coumarin-3-carboxylic acid, or rhodamine B conjugate p(mPhe-Keto-BocGlu)-RB and 7-

(diethylamino)coumarin-3-carboxylic acid. Figure 4.4A shows the scanning electron microscope (SEM) image of obtained fiber mat from p(mPhe-Keto-BocGlu) mixed with rhodamine B and 7-(diethylamino)coumarin-3-carboxylic acid. The SEM describes continuous fibers with no bead formation. Figure 4.4B describes the fiber distribution, analyzed through Nano Measurer 1.2 software. Five SEM images were used for calculation and 50 measurement of diameter for each image was taken. The average nanofiber diameter is 810 nm with a standard deviation of 220 nm. Figure 4.4C shows an SEM image of fiber mats from p(mPhe-Keto-BocGlu)-RB mixed with 7-(diethylamino)coumarin-3- carboxylic acid. Figure 4.4D describes an average nanofiber diameter of 881 nm with a standard deviation of 180 nm.

Figure 4.4. (A) SEM image of p(mPhe-Keto-BocGlu)/1% Coumarin dye/ 0.5%

Rhodamine B electrospun mat, (B) Diameter distribution of image A, (C) SEM image of p(mPhe-Keto-BocGlu)-RB/1% Coumarin dye electrospum mat, (D) Diameter distribution of image C.

The nanofiber mats were characterized with fluorescence microscopy (Figure 4.5).

Excitation of the nanofiber mat at 345 nm showed a blue fluorescence on the entire mat, which results from coumarin dye fluorescence. Excitation of the nanofiber mat at 555 nm results in yellow fluorescence of the entire mat, describing conjugated rhodamine B dye

Figure 4.5. (A), (C) Fluorescence image of p(mPhe-Keto-BocGlu)-RB/1% Coumarin dye electrospun mat excited at 345 nm. (B), (D) Fluorescence image of p(mPhe-Keto-

BocGlu)-RB/1% Coumarin dye electrospun mat excited at 555 nm.

fluorescence. This means that rhodamine B and coumarin dyes are dispersed in the fiber.

Figure 4.6 describes cumulative release of coumarin dye and rhodamine B from the fiber mat, made by mixing the two dyes with p(phe-keto-BocGlu). The release was tested up to

90 days. As shown in this figure, the release of each dye has three phases. The release kinetics were characterized by an initial burst release of coumarin from days 0-7, followed by a slower release rate and then by an increase kinetics after day 55. Rhodamine B release demonstrated similar kinetics. Coumarin dye and rhodamine B were released 12.9±1.6%

98 and 7.4±1.6% respectively from days 0-7 and coumarin dye and rhodamine B were released

27.1±3.2% and 17.0±3.2% respectively after day 55. Coumarin dye and rhodamine B were released at 48.6±6.3% and 30.4±6.5% respectively after day 90. PLA or PLGA fiber mats has been reported to demonstrate encapsulated drug burst release and the released time usually within month.156-158 The release rate of rhodamine B was lower than coumarin dye, which may indicate stronger polymer-dye interactions between rodamine B compared to coumarin.

The rhodamine B conjugated polymer with encapsulated coumarin dye p(mPhe-

Keto-BocGlu)-RB was also elctrospun to create a fiber mat. Figure 4.7 describes cumulative release of coumarin dye and rhodamine B. Coumarin dye encapsulated by RB-

60 Coumarin Dye 55 Rhodamine B 50

Cumulative release Cumulative (%) 10

0 0 10 20 30 40 50 60 70 80 90 Time (Days)

Figure 4.6. Cumulative release of coumarin dye and rhodamine B from fiber mat of p(mPhe-Keto-BocGlu) mixed with rhodamine B and 7-(diethylamino)coumarin-3- carboxylic acid.

99 70 Coumarin dye 60 Rhodamine B

CumulativeResease (%)

0 20 40 60 80 100 Time (Days)

Figure 4.7. Cumulative release of coumarin dye and rhodamine B from fiber mat of p(mPhe-Keto-BocGLU)-RB mixed with 7-(diethylamino)coumarin-3-carboxylic acid.

conjugated polymer has a similar release profile coumarin dye encapsulated by p(mPhe-

Keto-BocGlu), described by Figure 4.6. The covalently bonded rhodamine B through oxime bond did not release after 90 days, due to the stable oxime bond in 1×PBS (pH =

7.4). Raines and coworkers4 studied the hydrolytic stability of hydrazones and oximes and found that oximes are more stable than simple hydrazones. Therefore, oxime bond can be used to tether a bioactive compound.

The hydrolytic degradation of p(mPhe-Keto-BocGlu)-RB fiber mat was studied by measuring molecular weights of the fibers at 15, 30, and 60 days. The original and degraded molecular weights are listed in Table 4.1. After 15 days, Mn decreased from 79 kDa to 40 kDa, while the Mw decreased from 106 kDa to 61 kDa and the PDI increased

100 from 1.34 to 1.53. After 60 days, Mn was 25 kDa and Mw was 41 kDa, PDI was 1.64. After

60 days, Mn was 8.3 kDa and Mw was 14.2 kDa. Figure 4.8 describes molecular weight vs time immersed in 1x PBS at 37 °C. Figure 4.9 shows GPC traces of degraded polymers.

Table 4.1. Molecular weights of p(mPhe-Keto-BocGlu)-RB fiber mats immersed in 1x

PBS at 37 °C for different time.

Time (Day) Mn (kDa) Mw (kDa) PDI

0 79 106 1.34

15 40 61 1.53

30 25 41 1.64

60 8.3 14.2 1.53

Figure 4.8. Plot of molecular weights vs days of immersing in 1x PBS.

101

Figure 4.9. GPC traces of degraded polymers.

4.5 Conclusion

A polyester with high molecular weight was synthesized from carbodiimide mediated step growth polycondensation of mPhe, ketone bearing monomer and Boc protected glutamicacid. The ketone pendant group was further modified with akoxylamine through oxime bond. The polyester can be electrospun into fiber mats with nanofiber diameter around 800 nm. Coumarin dye and rhodamine B were physically incorporated to fiber by mixing the two dyes with polymer solution. We demonstrated sustained release of both dyes over 90 days. Additionally, a rhodamine B conjugated polymer was synthesized and electrospun with coumarin dye. The release data demonstrates that while coumarin can be released from the fiber mat, the oxime bond between polymer and rhodamine B was stable in PBS and rhodamine B was not released. The fiber degraded over 60 days and showed a decrease in Mw from 106 kDa to 14.2 kDa.

102 CHAPTER V

FUNCTIONAL POLYESTERS FOR OSTEOBLAST DIFFERENTIATION

5.1 Abstract

Three polymers with about 40% of carboxylic, amine, and hydroxyl pendant group were synthesized to study the differentiation of mouse pre-osteoblast cell line(MC3T3) into osteoblasts. Alkaline Phosphatase (ALP) staining and ALP acitivity of MC3T3 differentiated for 14 days were performed. Alizarin Red staining and von Kossa staining of the MC3T3 differentiated for 21 days were performed to study the mineralization.

Qualitatively, polymer with COOH groups demonstrated the largest influence on differentiation to osteoblasts as determined by positive staining showing mineralization. In addition, two polymers with the same amount of COOH but with different hydrophilicity were synthesized. Both polymers were electrospun with the mixing of osteoactivin (OA) peptide. The release study showed different OA peptide release profile from the two polymer fibers.

5.2 INTRODUCTION

Stem cells can differentiate to several cell lineages and hence the ability to control the differentiation to a particular cell type can potentially lead to tissue regeneration.

Several growth factors have been shown to influence stem cell differentiation to particular

103 lineages. In addition, synthetic small molecules and polymeric functional groups have demonstrated the ability to modulate differentiation. For example, Garcia et al159 have shown that differentiation of human mesenchymal stem cells plated on self-assembled monolayers (SAM) is influenced strongly by the identity of the terminal functional group on the SAM. Anseth et al100 have shown that the presence of various functional groups of polyacrylamide copolymer hydrogels modulates the differentiation of MSCs.

In the current work, the influence of various pendant functional groups on the differentiation of MC3T3 cells were examined. The effect of carboxylic acid, hydroxyl and amine functional groups were examined through this study. The ratio of the above three functional groups were maintained the same for all the polymers. Three different polyesters were designed from four different functionalized diols. The ratio between a phenylalanine functionalized diol and the alanine functionalized diol were maintained for all the compositions. In addition to the chosen functional group, pheylalanine and alanine, butene diol was incorporated to enable the preparation of crosslinked hydrogels in future studies.

In the second part of this work, the above polymer with the strongest influence on osteoblast differentiation was chosen and the ability of two copolymers containing the

COOH functionality to modulate the release rate of osteoactivin peptide was examined.

Ostoeactivin has been shown to exhibit a strong influence on the differentiation and function of stem cells. In this study, two polymers with different ratios of the phenylalanine and alanine diols, and the same amount of COOH functionality were designed. In addition to the above three functional groups, a diol with a ketone functional group was incorporated to enable future covalent conjugation of osteoactivin. In this study only the release of non- covalently osteoactivin was examined.

104 5.2 Experimental Section

Experimental section describes the chemicals needed for synthesis of targeted product, the method of making monomers and polymers and the characterization and tests of materials.

5.2.1 Materials and instrumentation

Materials: All the reagents were purchased from Sigma Aldrich or Alfa Aesar and used without further purification unless otherwise noted. N,N'-diisopropylcarbodiimide

(DIC, 99%) was purchased from Oakwood Chemical and used as received. 4-

(dimethylamino) pyridinium 4-toluene sulfonate (DPTS) was prepared according to

Leu-Leu-Gln-Asp-Pro-Leu-Gln-Asp-Lys-OH was synthesized from solid-phase peptide synthesizer.

1 13 Instrumentation: All H and C NMR spectra in CDCl3 of the monomers and polyesters were recorded on either a Varian Mercury 300 MHz or 500 MHz spectrometer.

Chemical shifts were recorded in ppm (δ) relative to solvent signals. Polyester molecular weights were analyzed on a TOSOH EcoSec HLC-8320 GPC equipped with a refractive index detector (RI) and UV detector. Separation occurred over two PSS Gram Analytical

GPC Columns in series using 25 mM LiBr in DMF as eluent at a flow rate of 0.8 mL/min.

The column and detector temperatures were maintained at 50 °C. Molecular weights were

105 obtained relative to PS standards using the RI signal. Thermogravimetric analysis (TGA) measurements were performed with TA Q500 over an interval of ambient temperature to

600 °C at a heating rate of 10 °C/min under a N2 atmosphere. Differential scanning calorimetry (DSC) measurements were performed on TA Q2000 DSC with a liquid N2 cooling unit and a heating/cooling rate of 10 °C/min. ESI MS was performed on Bruker

HTC ultra QIT.

Scheme 5.1. Synthesis of polyester with carboxylic pendant group.

5.2.2 Synthesis of polyesters with carboxylic, amine, and hydroxyl group

Synthesis of p(mPhe-mAla-mAsptBu-But): mPhe (237.3 mg, 1 mmol), mAla

(241.8 mg, 1.5 mmol), cis-2-Butene-1,4-diol (But) (58.5 mg, 0.66 mmol), mAspt Bu

(529.3 mg, 2.03 mmol), succinic acid ( 612.8 mg, 5.19 mmol), and DPTS (606.8 mg, 2.08 mmol) were added to a 50 mL Schlenk flask. To the flask, vacuum was applied and back filled with N2 three times. After purging the flask of air, 5.0 mL anhydrous DCM was added.

The mixture was heated with warm water for one min. Then, the flask was cooled with ice and 2.5 mL DIC (15.6 mmol) was added dropwise through a syringe. After the addition of

DIC, the reaction was brought to room temperature and stirred under the protection of N2

106 for 68 h. The resulting polymer was precipitated twice from MeOH and dried in vacuum oven. 1.0 g of white solid polymer was obtained. Yield was 66.7%. 1H NMR (500 MHz,

CDCl3):  7.19 - 7.30 (m, 1.59 H), 5.74 (br. s., 0.35H) 4.70 (br. s., 0.68H), 4.15 - 4.23 (m,

4H), 3.54 - 3.66 (m, 4H), 2.98 (t, J = 7.46 Hz, 0.54H), 2.57 - 2.69 (m, 6.63H), 2.37-2.41

(m, 0.79H), 1.44 (s, 3.85H), 1.15 (t, J = 7.21 Hz, 1.08H).

Synthesis of p(mPhe-mAla-mAsp-But): 415 mg of p(mPhe-mAla-mAsptBu-But) was dissolved in 3.0 mL DCM. Then, 3.0 mL TFA and 50 µL TIPS was added. The solution was stirred at room temperature for 1 h. Following the reaction, the TFA and DCM were removed under reduced pressure by rotary evaporation. The polymer was precipitated in

1 diethyl ether and dried in vacuum oven at RT overnight. H NMR (500 MHz, CDCl3): 

7.22-7.27 (m, 2H), 6.73 (br. s., 1.68H), 5.74 (br. s., 0.85H), 4.70 (br. s., 0.97H), 4.17 - 4.24

(m, 4H), 3.71 - 3.95 (m, 8H), 2.97 (br. s., 0.96H), 2.64 (br. s., 6.71H), 2.28 - 2.47 (m, 1.12H),

1.11 - 1.19 (m, 1.51H).

Scheme 5.2. Synthesis of polyester with amine pendant group.

Synthesis of p(mPhe-mAla-mLysBoc-But): mPhe (281.3 mg, 1.19 mmol), mAla

(316.6 mg, 1.96 mmol), cis-2-Butene-1,4-diol (75 mg, 0.85 mmol), mLysBoc (760.8 mg,

107 2.39 mmol), succinic acid ( 754.6 mg, 6.39 mmol), and DPTS (747.1 mg, 2.56 mmol) were added to a 50 mL Schlenk flask. To the flask, vacuum was applied and back filled with N2 three times. After purging the flask of air, 5.0 mL anhydrous DCM was added. The mixture was heated with warm water for one min. Then, the flask was cooled with ice bath and 3.0 mL DIC (19.2 mmol) was added dropwise through a syringe. After the addition of DIC, the reaction was brought to room temperature and stirred under the protection of N2 for 70 h.

The resulting polymer was precipitated three times from cold 2-propanol and dried in vacuum oven at 45 °C. 1.15 g white solid polymer was obtained. Yield was 58.7%. 1H

NMR (500 MHz, CDCl3):  7.13 - 7.34 (m, 1.64H), 5.74 (br. s., 0.35H), 4.70 (br. s., 0.98H),

4.15 - 4.24 (m, 4H), 3.55 - 3.61 (m, 4H), 3.11 - 3.12 (m, 0.95H), 2.98 (t, J = 7.70 Hz,

0.54H), 2.51 - 2.72 (m, 5.1H), 2.31 - 2.43 (m, 1.68H), 1.60 - 1.72 (m, 1.36H), 1.35 - 1.56

(m, 5.67H), 1.15 (t, J = 7.34 Hz, 1.21H).

Scheme 5.3. Synthesis of polyester with hydroxyl pendant group.

Synthesis of p(mPhe-mAla-mLys-But): p(mPhe-mAla-mLysBoc-But) was dissolved in 3.0 mL DCM. Then, 3.0 mL TFA and 50 µL TIPS was added. The solution

108 was stirred at room temperature for 2 h. Following the reaction, the TFA and DCM were removed under reduced pressure by rotary evaporation. The resulting polymer was precipitated twice from cold diethyl ether and dried in vacuum oven at RT overnight. 1H

NMR (300 MHz, DMSO-d6):  7.72 (br. s., 1.2H), 7.04 - 7.37 (m, 1.06H), 5.67 (s, 0.27H),

4.64 (d, J = 4.10 Hz, 0.61H), 3.95 - 4.25 (m, 4.81H), 3.48 - 3.56(m, 4H), 2.74 - 2.85 (m,

1.22H), 2.48 - 2.65 (m, 6.94H), 2.29 - 2.33 (m, 1.64H), 1.39 - 1.63 (m, 1.65H), 1.27 - 1.33

(m, 0.77H), 0.96 (t, J = 7.32 Hz, 0.83H).

Synthesis of p(mPhe-mAla-mSerTBDMS-But): mPhe (282.2 mg, 1.19 mmol), mAla (293.8 mg, 1.82 mmol), cis-2-Butene-1,4-diol (69.4 mg, 0.79 mmol), mSerTBDMS

(662.4 mg, 2.27 mmol), succinic acid (717.1 mg, 6.07 mmol), and DPTS (710 mg, 2.43 mmol) were added to a 50 mL Schlenk flask. To the flask, vacuum was applied and back filled with N2 three times. After purging the flask of air, 5.0 mL anhydrous DCM was added.

The mixture was heated with warm water for one min. Then, the flask was cooled with ice and 3.0 mL DIC (19.2 mmol) was added dropwise through a syringe. After the addition of

DIC, the reaction was brought to room temperature and stirred under the protection of N2 for 68 h. The resulting polymer was precipitated twice from cold 2-propanol and dried in vacuum oven at 45 °C. 1.18 g white solid polymer was obtained. Yield was 61.6%. 1H

NMR (500 MHz, CDCl3):  7.16 - 7.34 (m, 1.64H), 5.74 (br. s., 0.34H), 4.70 (br. s., 0.61H),

4.15 - 4.24 (m, 4H), 3.95 (t, J = 6.36 Hz, 0.89H), 3.54 - 3.69 (m, 4H), 2.98 (m, t, J = 7.58

Hz, 0.55H), 2.51 - 2.69 (m, 5.85H), 2.34 -2.41(m, 0.96H), 1.15 (t, J = 7.34 Hz, 1.08H),

0.88 (s, 3.84H), 0.06 (s, 2.52H).

Synthesis of p(mPhe-mAla-mSer-But): 340 mg of p(mPhe-mAla-mSerTBS-But) was dissolved in 2.5 mL DCM. Then, 2.5 mL TFA and 50 µL TIPS was added. The solution

109 was stirred at room temperature for 2 h. Following the reaction, the TFA and DCM were removed under reduced pressure by rotary evaporation. The polymer was precipitated twice from cold diethyl ether and dried in vacuum oven overnight. 1H NMR (500 MHz,

CDCl3):  7.19 - 7.33 (m, 1.49H), 5.74 (br. s., 0.36H), 4.70 (br. s., 1.47H), 4.15 - 4.24 (m,

4H), 3.87 (s, 0.19H), 3.54 – 3.62 (m, 4H), 2.97 (t, J = 7.58 Hz, 0.52H), 2.80 - 2.92 (m,

0.87H), 2.50 - 2.71 (m, 4.98H), 2.33 - 2.44 (m, 0.83H), 1.14 (t, J = 7.34 Hz, 1.10H).

Scheme 5.4. Synthesis of polymer for carrier of OA peptide.

t Synthesis of p(mPhe0.1-mAla0.4-mAsp Bu0.3-Keto0.2): mPhe (374.7 mg, 1.58 mmol), mAla (1.0329 g, 6.41 mmol), mAsp tBu (1.2474 g, 4.77 mmol), Keto monomer

(666.7 mg, 3.28 mmol), and succinic acid ( 1.8942 g, 16.04 mmol), and DPTS (1.8754 g,

6.4 mmol) were added to a 100 mL Schlenk flask. To the flask, vacuum was applied and back filled with N2 three times. After purging the flask of air, 20 mL anhydrous DCM was added. The mixture was heated with warm water for one minute. Then, the flask was cooled with ice and 8.0 mL DIC (48.1 mmol) was added dropwise through a syringe. After addition

110 of DIC, the reaction was brought to room temperature and stirred under the protection of

N2 for 72 h. The resulting polymer was precipitated once from 2-propanol/MeOH (2:1).

Then, the polymer was redissolved in DCM was precipitated from 2-propanol/MeOH (1:1).

In the final precipitation, polymer was redissolved in DCM and precipitated in 2-

1 propanol/MeOH (1:2) and dried in vacuum oven at 45 °C. H NMR (500 MHz, CDCl3): 

7.13 - 7.34 (m, 0.68H), 4.09 - 4.33 (m, 4H), 3.54 - 3.67 (m, 4H), 2.97 (t, J = 7.58 Hz,

0.19H), 2.77 (t, J = 5.99 Hz, 0.44H), 2.57 - 2.68 (m, 5.77H), 2.38 (q, J = 7.34 Hz, 0.81H),

2.20 (s, 0.62H), 1.44 (s, 2.6H), 1.14 (t, J = 7.46 Hz, 1.26H).

Synthesis of p(mPhe0.1-mAla0.4-mAsp-Keto0.2): 437 mg of p(mPhe0.1-mAla0.4-

t mAspBu 0.3-Keto0.2) was dissolved in 5 mL DCM. Then, 5 mL TFA and 50 µL TIPS was added. The solution was stirred at room temperature for 2 h. Following the reaction, the

TFA and DCM were removed under reduced pressure by rotary evaporation. The polymer was precipitated twice from diethyl ether and dried in vacuum oven overnight. 1H NMR

(500 MHz, CDCl3):  7.17 -7.29 (m, 0.68H), 6.92 (br. s., 0.51H), 4.15 - 4.23 (m, 4H), 3.54

- 3.68 (m, 4H), 2.96 - 2.98 (m, 0.2H), 2.53 - 2.78 (m, 6.29H), 2.39 - 2.41 (m, 0.81H), 2.19

(s, 0.6H), 1.14 (t, J = 7.09 Hz, 1.19H).

t Synthesis of p(mPhe0.4-mAla0.1-mAsp Bu0.3-Keto0.2): mPhe (1.0183 g, 4.29 mmol), mAla (169.4 mg, 1.05 mmol), mAsptBu (0.8333 g, 3.19 mmol), Keto monomer

(446.3 mg, 2.2 mmol), and succinic acid ( 1.2667 g, 10.73 mmol), and DPTS (1.2542 g,

4.3 mmol) were added to a 100 mL Schlenk flask. To the flask, vacuum was applied and back filled with N2 three times. After purging the flask of air, 20 mL anhydrous DCM was added. The mixture was heated with warm water for one min. Then, the flask was cooled with ice, 5.5 mL DIC (32.2 mmol) was added dropwise through a syringe. After addition

111 of DIC, the reaction was brought to room temperature and stirred under the protection of

N2 for 72 h. The resulting polymer was precipitated once from 2-propanol/MeOH (2:1).

Then, the polymer was redissolved in DCM and precipitated from 2-propanol/MeOH (1:1).

In the final precipitation, the polymer was redissolved in DCM and precipitated in 2-

1 propanol/MeOH (1:2) and dried in vacuum oven at 45 °C. H NMR (500 MHz, CDCl3): 

7.11 - 7.34 (m, 2.24H), 4.14 - 4.22 (m, 4H), 3.54 - 3.66 (m, 4H), 2.97 (t, J = 7.46 Hz,

0.79H), 2.77 (br. s., 0.45H), 2.52 - 2.66 (m, 6.49H), 2.19 (br. s., 0.62H), 1.44 (s, 2.65H),

1.14 (t, J = 7.71 Hz, 0.32H).

Synthesis of p(mPhe0.4-mAla0.1-mAsp0.3-Keto0.2): 390 mg of p(mPhe0.4-mAla0.1-

t mAsp Bu0.3-Keto0.2) was dissolved in 5 mL DCM. Then, 5 mL TFA and 50 µL TIPS was added. The solution was stirred at room temperature for 2 h. Following the reaction, the

TFA and DCM were removed under reduced pressure by rotary evaporation. The polymer was precipitated twice from diethyl ether and dried in vacuum oven at RT overnight. 1H

NMR (500 MHz, CDCl3):  8.26 (br. s., 0.71H), 7.20 - 7.35 (m, 2.26H), 4.15 - 4.23 (m,

4H), 3.61 - 3.68 (m, 4H), 2.96 (t, J = 7.21 Hz, 0.82H), 2.53 - 2.79 (m, 6.96H), 2.43 (br. s.,

0.22H), 2.19 (s, 0.6H), 1.15 (t, J = 7.71 Hz, 0.31H).

Spin coating of p(mPhe-mAla-mAsp-But), p(mPhe-mAla-mLys-But), p(mPhe- mAla-mSer-But) onto glass coverslips: Coverslips (12 mm diameter) were cleaned by sonicating with acetone, ethanol, and deionized water for 30 min each. Then, they were dried by compressed air. The solutions of p(mPhe-mAla-mSer-But) were prepared to 2%

(w/v) in CHCl3. After allowing the polymer to dissolve, the solution was filtered with a

0.45 µm PTFE syringe filter. This solution was spin coated onto coverslips a spin coater machine (WS-400B-6NPP/LIFT) set to a speed of 2500 rpm and allowed to spin for 1 min.

112 The coated coverslips were dried in vacuum oven at RT overnight. The solutions of p(mPhe-mAla-mLys-But) were prepared to 2% (w/v) in hexafluoroisopropanol (HFIP).

After allowing the polymer to dissolve completely the solution was filtered with 0.45 µm

PTFE syringe filter. This solution was spin coated onto coverslips using the spin coater machine set to a speed of 2500 rpm and allowed to spin for 1 min. The coated coverslips were dried in vacuum at r.t. oven overnight. The solutions of of p(mPhe-mAla-mAsp-But) were prepared to 2% (w/v) in HFIP. After allowing the polymer to dissolve completely, the solution was filtered with 0.45 µm PTFE syringe filter. This solution was spin coated onto coverslips using a spin coater machine set to a speed of 5000 rpm and allowed to spin for

1 min. The coated coverslips were dried in a RT vacuum oven overnight.

Electrospinning of p(mPhe0.1-mAla0.4-mAsp-Keto0.2) and p(mPhe0.4-mAla0.1- mAsp-Keto0.2) with OA peptide: 2 mg of OA peptide was dissolved into 110 µL DMF and

60 µL DMSO. 107 mg of p(mPhe0.1-mAla0.4-mAsp-Keto0.2) was dissolved in 240 µL

DCM and 60 µL of the peptide solution was added to polymer solution to yield a working concentration of 3.3 mg/mL peptide and 357 mg/mL polymer. The mixture was then transferred to a glass pipette for electrospinning. An 11 kV electric field was applied between polymer solution and collector. The distance between the pipette tip and collector was 18 cm. The electrospun fibers were collected onto 22 mm diameter round glass cover slips placed on top of the steel collector to collect fiber for 10 min, 20 min, 30 min, and 40 min. The collected samples were dried in vacuum oven at RT. For p(mPhe0.4-mAla0.1- mAsp0.3-Keto0.2), 121 mg of the polymer was dissolved in 240 µL DCM, and 60 µl peptide solution was added to polymer solution to give a working concentration of 3.3 mg/mL peptide and 403 mg/mL polymer. The mixture was also transferred to a glass pipette for

113 electrospinning. For this polymer solution, a 10 kV electric field was applied between polymer solution and collector. The distance between pipette tip and collector was18 cm.

Just as with the other polymer, the electrospun fibers were collected onto 22 mm diameter round glass cover slips placed on top of the steel collector to collect fiber for 10 min, 20 min, 30 min, and 40 min. The collected samples were dried in vacuum oven at RT.

Water contact angle measurement: Water contact was measured using a contact angle goniometer (Ramé-Hart). Each spin coated coverslip was loaded onto the sample stage and one drop of 18.2 MΩ*cm water was deposited on the polymer film surface. An image was taken every 30 seconds for 7 min. Contact angle was analyzed via DROPimage

Advanced software. A total of 10 measurements for each sample were performed. The results were reported as average ± standard deviation.

5.3 Results and Discussion

A summary of the chemical characterization of the synthesized polymers is shown in Table 5.1. The obtained ratios were calculated from 1H NMR and were similar to the feed ratio. Each polymer contains about 36 mol% of repeat units which contain either

COOH, OH, or NH2 functional groups. The ratios of other repeat units are similar for all three polymers. Figure 5.1 shows the water contact angle of the three polymers over 480 seconds. For p(mPhe-mAla-mLys-But), the water contact angle was very low. It started at 15° and decreased to around 10° after 30 s. After that, the contact angle was not measurable.

For p(mPhe-mAla-mAsp-But) and p(mPhe-mAla-mSer-But), the water contact angle decreases fast for the first 150 s and then decreases more slowly after this point. This

114 could because of the rearrangement of polymer chain upon the contact with water. The contact angle of p(mPhe-mAla-mSer-But) is about 35° and p(mPhe-mAla-mAsp-But) is about 50° after 480 seconds.

Table 5.1. Characterization of the polymers.

Feed ratio Obtained ratio Mn Mw Td* Polymer PDI (x:y:z:w) (x:y:z:w) (kDa) (kDa) (°C)

p(mPhex-mAlay- 19:29:39:13 21:31:34:13 104 178 1.72 191.7 mAspButz-Butw)

p(mPhex-mAlay- 19:31:37:16 20:31:36:13 22 33.5 1.52 181.6 mLysBocz-Butw)

p(mPhex-mAlay- 20:30:37:13 22:29:36:14 54 91 1.68 150.1 mSerTBSz-Butw) * data for the deprotected polymer

Figure 5.1. Water contact angle of polymers with COOH, NH2, and OH functional groups.

115 In vitro study of differentiation of MC3T3-E1 cells on polymer coated coverslips were carried out by a collaborator at the Northeast Ohio Medical School (NEOMED).

Figure 5.2. Vinculin Immunofluorescent Staining of the MC3T3 cultured for 24 hours on

A) Blank coverslip, coverslips coated with COOH (B), OH (C), NH2 (D) functionalized polymers.

To test the cell adhesion on the different surfaces, MC3T3 cells were seeded at a density of 5,000 cells/well in 4-well chamber slides and incubated overnight in standard cell culture conditions. The next day, they were fixed with paraformaldehyde. Their cytoskeletons (actin, red) and focal adhesion points (vinculin, green) as well as their nuclei

(DAPI, blue) were stained. 116 Vinculin staining appears as green dots or “arrowheads” at the ends of actin fibers.

The TCPS group was excluded from immunofluorescent staining because it is difficult to image through 24-well plates.

For polymer with amine group, many of the cells in the center of the coverslip were wiped away due to a handling error. In order to image intact cells, images were taken near the periphery of the coverslip near the sealing agent.

Figure 5.3. Alkaline Phosphatase (ALP) Staining of the MC3T3 differentiated for 14 days on A) Tissue Culture treated Poly(styrene) (TCPS), B) Blank coverslip, coverslips coated with COOH(C), OH(D), NH2(E) functionalized polymers.

MC3T3 is an osteoblast precursor cell line derived from Mus musculus (mouse) calvaria. The cells were plated on tissue culture treated poly(styrene) (TCPS), blank coverslips, or coverslips spin coated with the polymers which have either COOH, OH or

117 NH2 functional groups. After 14 days, ALP staining was carried out (Figure 5.3). The color red indicates cells positive for the Alkaline Phosphatase enzyme, which is an early differentiation marker, and is important in bone matrix mineralization. Closely-grouped

ALP-positive cells are indicative of cells which are beginning to mineralize the extracellular matrix around them. Pictures were taken at 2X magnification so as to show the greatest possible area of the well. Qualitatively, the following trend of increased ALP activity is true based on the pictures, COOH> Blank>NH2>TCPS>OH.

Figure 5.4. Alkaline phosphatase activity.

Additionally, after 14 days of culture, an assay was performed to directly quantify

ALP activity of the MC3T3-E1 cells. This was achieved by lysing the cells, taking a small sample of the cell lysate and then adding it to a substrate which is chemically modified by the enzyme to produce a compound that absorbs at 405 nm. Since there may be slight 118 variation in the total amount of cells present in each well plate, the ALP activity was normalized to the total protein content.

Figure 5.4 displays the average ALP activity/protein values with error bars representing standard error. Asterisks indicate significance when compared to the group with the corresponding color of the asterisk where * is p<0.05, ** is p<0.01, and *** is p<0.001. Interestingly, TCPS and polymer with NH2 functionality have similar ALP activity while the blank coverslip and polymer with COOH groups show slightly less activity. The polymer with OH group shows even less expression of ALP activity

In this experiment, the cells were lysed in the well, meaning that a small percentage of the cells lysed were growing on the TCPS for all groups. In future studies, the coverslips

Figure 5.5. Alizarin Red staining of the MC3T3 differentiated for 21 days on A) TCPS, B)

Blank coverslip, coverslips coated with COOH(C), OH(D), NH2(E) functionalized polymers.

119 will be removed from the wells and then the cells will be lysed to make these measurements more sensitive.

After 21 days, cells were stained with Alizarin Red S, which is a dye that stains calcium dark red to brown in color. The results of the satining are shown in Figure 5.5.

Pictures were taken at 4X to enable the largest area to be visible while still conserving our ability to view cell morphology. Pictures were taken mainly of the most positively stained region; multiple images were taken in cases of good staining or to demonstrate greater variation in staining for a given polymer.

Qualitaitvely, the polymer with COOH group appeared to demonstrate the best results and the most positive staining showing prominent mineralization. The polymer with

Figure 5.6. von Kossa staining of the MC3T3 differentiated for 21 days on A) Tissue

Culture treated Poly(styrene) (TCPS), B) Blank coverslip, coverslips coated with

COOH(C), OH(D), NH2(E) functionalized polymers.

120 amine group also performed well and displayed confluent cells but did not show significant calcium deposition. Polymer with hydroxyl group did show confluence, but lacked consistency between different samples. The Blank cover slip performed the worst out of all groups, lacking consistency between trials, mineralization, and confluence, even being surpassed by the TCPS trials. A lack of confluence in the Blank group is indicative of cells lifting.

To complement the Alizarin Red S staining, von Kossa staining of the MC3T3 osteoblast-like cell line differentiated for 21 days was carried out as well (Figure 5.6). von

Kossa staining utilizes silver nitrate and its precipitation with phosphate ions, followed by photochemical degradation to visualize the phosphate content of mineralized bone matrix.

The precipitate is grey to black, depending on the extent of mineralization.

Pictures were taken at 40X to enable the largest area to be visible while still conserving our ability to view cell morphology. Pictures were taken of the most positively stained region of the wells. A picture of a Blank well has been included that shows variation in cell attachment between wells for a given treatment. In general, there is a trend of polymer with carboxylic acid or amine group performed the best of all the different growth conditions. Polymer with hydroxyl group also showed positive growth but was not consistent between its three wells nor between the von Kossa and Alizarin Red S stains.

From the previous MC3T3 cell differentiation study, it shows polymer with carboxylic group performs best for the mineralization. So we designed polymer with carboxylic group for electrospinning mixture with osteoactivin (OA) peptide. OA peptide is a peptide sequence that can promote osteoblast adhesion.160 For these experiments, p(mPhe0.1-mAla0.4-mAsp0.3-Keto0.2) and p(mPhe0.4-mAla0.1-mAsp0.3-Keto0.2) polymers

121 were synthesized. The purpose in synthesizing these polymers using these mole ratios of monomer was to vary the amount of mPhe and mAla to create polymers with different hydrophilicity. By changing the hydrophilicity of the polymer, the peptide release profile of the polymers will vary. The ketone group was used to conjugate OA peptide to polymer through oxime bond. In this work, OA peptide was mixed with the two polymers and electrospun into nanofiber mat and the release of OA peptide was studied. The further experiments will be carried out by our collaborator.

Table 5.2. Characterization of polymers.

Feed ratio Obtained ratio Mn Mw Polymer PDI (x:y:z:m) (x:y:z:m) (kDa) (kDa)

p(mPhe0.1-mAla0.4-mAsp0.3- 10:40:30:20 9:40:29:22 115 138 1.20 Keto0.2)

p(mPhe0.4-mAla0.1-mAsp0.3- 40:10:30:20 39:10:29:22 63 80 1.27 Keto0.2)

Table 5.2 shows the characterization of the two polymers, and the obtained ratio of each component is close to the feed ratio. Molecular weights for the two polymer are high, which allows for electrospinning. Water contact angle was also carried out in 300 s and the results are shown in Figure 5.7. As expected, the polymer with 40% mPhe has higher contact angle compared to the polymer with 10% mPhe. For both polymers, contact angle decreased with time which may be because the rearrangement of polymer chain upon the contact with water.

122

Figure 5.7. Water contact angle of polymers.

Figure 5.8. Cumulative release of OA peptide from electrospun fiber.

123 Figure 5.8 shows the release of OA peptide from electrospun fiber. The release behaviors are similar for the sample of electrospun time of 10 min and 40 min. For polymer with 10% mPhe which is the more hydrophilic, OA peptide releases more quickly while the polymer with 40% mPhe shows a slower release.

5.4 Conclusion

Three polymers with about 40% of carboxylic, amine, and hydroxyl pendant group respectively were synthesized. The polymers contain about 10% of alkene in the backbone, which may be used for crosslinking in future. The three polymers were used for study of the differentiation of MC3T3 mouse pre-osteoblast cell line into osteoblasts. ALP staining and ALP acitivity of MC3T3 differentiated for 14 days was performed. From the ALP staining picture, qualitatively, the following trend of increased ALP activity is observed,

COOH> Blank>NH2>TCPS>OH. Alizarin Red staining and von Kossa staining of the

MC3T3 differentiated for 21 days was performed to study the mineralization. For Alizarin

Red S staining, polymer with COOH group demonstrated best results and the most positive staining showing prominent mineralization. For von Kossa staining, there is a trend of polymer with carboxylic acid or amine group performed the best of all the different growth conditions.

Two polymer with the same amount of COOH but with different hydrophilicity were synthesized. Both polymers were electrospun with mixing of OA peptide. The release study showed different OA peptide release profile from the two polymer fiber.

124 CHAPTER VI

SUMMARY

In this dissertation we focused on designing and synthesizing multifunctional polyesters. We also studied their applications on the biomedical adhesives, extended drug release and stem cell differentiation.

6.1 The Platform of Functional Polyesters with Peptide-Like Pendant Groups

Biomaterials including polylactic acid have good mechanical and biodegradable properties.1 But they are devoid of functional groups that enable integration with the cellular environment. We have designed a platform of modular multifunctional polyesters with pendant functional groups that address the lack of functional cues in current biomaterials.2 The polyesters were synthesized at room temperature by carbodiimide- mediated polymerization of pendant functionalized diols and succinic acid (Scheme 6.1).

Scheme 6.1. Polyesterification of functional diol(s) with succinic acid.

125

Figure 6.1. Variation of contact angles with copolymer composition.

Figure 6.2. Variation of Tg with polymer composition and its comparison to calculated values from Fox equation.

The pendant groups were designed to mimic the side chains of peptides. It was shown that the physical properties of the polyesters such as water contact angle (Figure

126 6.1), glass transition temperature Tg can be modulated over a wide range by the selection of pendant groups (Figure 6.2).

One of the advantages of these functional polyesters is their ability to tag multiple ligands (dyes, drugs, growth factors etc.) via orthogonal pendant functional groups.

Tethering of various ligands is an effective method for presentation of imaging, therapeutic and signaling moieties.95-97 As a proof of concept, we were able to functionalize 10% of the propargyl groups of a p(mNHBoc-co-propargyl) polyester with azido anthracene

(AA). This AA conjugate was characterized by 1H NMR , IR and SEC. Subsequently we deprotected the amine groups of p(mNHBoc-co-propargyl-AA) and covalently attached fluorescein isothiocyanate (FITC) to 1% of amine groups. As seen in Figure 6.3, duplication of Figure 2.9, the polyester exhibits the absorbance and fluorescence of both dyes, proving the orthogonal conjugation.

Figure 6.3. a) Polyester tethered with AA and FITC b) UV absorbance spectrum of FITC-

AA conjugated polyester (in water) and fluorescence spectrum of FITC-AA conjugated polyester (at 370 nm excitation) in DMSO-water (90:10).

127 We also cojugated RGD and PEG to functional polyester and did the cell adhesion study. The polyester we used for the conjugation was with mAla, mAsp and propargyl pendant groups in a ratio of 2:2:1. A short PEG chain [CH3O-(CH2CH2O)2-CH2CH2NH2], was covalently tethered to 20% of the COOH groups and is denoted as BP. The resulting functionalized polymer was spin coated on glass coverslips. Then the propargyl groups on the polymer surface were functionalized with the cell attachment peptide, N3-

(CH2)5CONHGRGDSCO2H via Cu catalysed azide-alkyne cycloaddition. Similarly, coverslips were made with either PEG (BP) or RGD (BR). These functionalized coverslips were plated with smooth muscle cells and the attachment and spreading were analyzed on the base polyester and compared to the same polyester functionalized with RGD or PEG.

The results show that the base polymer induces high cell attachment and spreading relative to the glass control (Figure 2.10). The RGD functionalized polyester showed increased cell attachment and spreading relative to the base polymer. A decrease in cell attachment was observed with the PEG functionalized polymer.

6.2 Mussel Inspired Adhesives Based on ‘Peptide-Like’ Functional Polyester

In the ocean, mussels secrete protein holdfasts that are capable of securing themselves on a variety of wet surfaces, namely all kinds of inorganic and organic surfaces.

The characteristic presence of a unique amino acid, L-3, 4-dihydroxyphenylalanine

(DOPA), were found in at least five adhesive protein subtypes of the widely studied blue mussel, Mytilus edulis. Inspired by mussel adhesives, DOPA group were incorporated into the side chain of functional polyesters. The first generation adhesive polyester contains 20%

DOPA and 80% of alanine mimic monomer. The adhesion strength was measured by the

128 lap shear test on aluminum substrates. As shown in Figure 6.4, there was effect of DOPA groups for the increasing of adhesion strength, but the strength decreased in wet conditions.

The second generation adhesive polyester was a copolymer with soybean oil based monomer, coumarin and DOPA monomer (Scheme 6.2). The polymer was viscous and the glass transition temperature was about -50 °C. It showed good adhesion under both dry and wet conditions (Figure 6.5, duplication of Figure 3.6). Adhesion tests on porcine skin was also performed and the results demonstrated that our polymer has higher adhesion strength than the commercial available fibrin glue after 5 min UV irradiation (Figure 6.6).

Figure 6.4. Lap shear geometry to test the adhesive strength of the polyesters (left). Lap shear strength of the p(mAla0.8-co-mDOPA0.2) (catechol) and p(mAla0.8-co-mPhe0.2)

(control) polyesters under dry and wet conditions (right).

129 Scheme 6.2. Synthesis of viscoelastic polyester p(SCD) from pendant functionalized diols

of long alkyl chain (S), coumarin (C) and DOPA (D) units.

Figure 6.5. Lap shear measurements of p(SCD) and p(SCP) under dry conditions (A) and

wet conditions (B).

130

Figure 6.6. End to end test adhesion test geometry on porcine skin (A), adhesion strength for p(SCD), p(SCP), and fibrin glue (B).

6.3 Electrospun Fiber Mat for Sustainable Drug Release

Nanofibers can be made by electrospinning from a variety of materials. Nanofiber electrospun mats were fabricated from pendant functionalized polyesters. These electrospun mats contained dyes that were either conjugated or encapsulated within the polymer matrix. The fibers were characterized with SEM and fluorescence microscope

(Figure 4.4, 4.5), both images showed that the fibers were smooth and relatively uniform.

The release of the dyes was evaluated over 90 days. The cumulative release profile of mixed dyes showed that the release of each dye had three phases (Figure 6.7). The coumarin dye exhibited a bust release over days 0-7, followed by a decreased release rate and followed by accelerated rate after day 55. When Rhodamine B was encapsulated, it demonstrated similar kinetics as coumarin, but with lower cumulative release. However when the rhodamine B was conjugated to the polyester through oxime coupling to a pendant ketone functionality, the dye did not release even 90 days, as the oxime bond is stable in 1×PBS (pH = 7.4) (Figure 6.8). 131 60 Coumarin Dye 55 Rhodamine B 50

Cumulative release (%)Cumulative 15

0 0 10 20 30 40 50 60 70 80 90 Time (Days)

Figure 6.7. Cumulative release of coumarin dye and rhodamine B from fiber mat of p(mPhe-Keto-BocGlu) mixed with rhodamine B and 7-(Diethylamino)coumarin-3- carboxylic acid.

70 Coumarin dye 60 Rhodamine B

CumulativeResease (%)

0 20 40 60 80 100 Time (Days)

Figure 6.8. Cumulative release of coumarin dye and rhodamine B from fiber mat of p(mPhe-Keto-BocGLU)-RB mixed with 7-(Diethylamino)coumarin-3-carboxylic acid.

132 6.4 Functional Polyesters for Osteoblast Differentiation

Stem cells can differentiate to several cell lineages and there are many factors which can influence the differentiation to a particular cell type. Among these factors, the chemistry of surface have been demonstrated to have ability to modulate differentiation. In this context, three polymers with about 40% of carboxylic (COOH), amine (NH2), and hydroxyl (OH) pendant group were synthesized (Scheme 5.1-Scheme 5.3) to study the differentiation of MC3T3 mouse pre-osteoblast cell line into osteoblasts. Alkaline

Phosphatase (ALP) staining and ALP acitivity of MC3T3 differentiated for 14 days were performed. From the ALP staining picture( Figure 5.3, qualitatively, there is a increased

ALP activity based on the pictures, COOH> Blank>NH2>TCPS>OH. Alizarin Red staining and von Kossa staining of the MC3T3 differentiated for 21 days were performed to study the mineralization(Figure 5.5, 5.6). Qualitatively, polymer with COOH group

Figure 6.9. Cumulative release of OA peptide from electrospun fiber.

133 demonstrated the largest influence on differentiation to osteoblasts as determined by positive staining showing mineralization. In addition, two polymers with the same amount of COOH but with different hydrophilicity were synthesized (Scheme 5.4). Both polymers were electrospun with mixing of osteoactivin (OA) peptide. The release study showed different OA peptide release profile from the two polymer fiber (Figure 6.9).

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148 APPENDIX

Figure A.1. 1H NMR (DMSO-d6) of mTrp monomer.

1 Figure A.2. H NMR (CDCl3) of mTyrBn monomer.

149

1 Figure A.3. H NMR (CDCl3) of Keto monomer.

1 Figure A.4. H NMR (CDCl3) of Azide monomer. 150

1 Figure A.5. H NMR (CDCl3) of mPhe monomer.

1 Figure A.6. H NMR (CDCl3) of mAla monomer.

151

1 t Figure A.7. H NMR (CDCl3) of mAsp Bu monomer.

1 Figure A.8. H NMR (CDCl3) of mSerTBDMS monomer.

152

1 Figure A.9. H NMR (CDCl3) of mLysBoc monomer.

1 Figure A.10. H NMR (CDCl3) of mGluBn monomer.

153 7.27 6.66 6.63 6.62 6.62 6.60 3.90 3.89 3.87 3.73 3.71 3.59 3.58 3.56 3.46 3.44 3.43 2.97 2.92 2.89 2.86 2.76 2.68 2.66 2.63 1.66 1.57

g b b h HO OH N a a O c e d

O O f f f

e a b g d h c

3.02 4.00 4.20 1.00 2.16 1.00 2.11 6.34

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)

1 Figure A.11. H NMR (CDCl3) of Cat monomer.

1 Figure A.12. H NMR (CDCl3) of Cou monomer.

154 SOYBEANOILMONOMER-012814.ESP

7.27 5.40 5.39 5.37 5.36 5.35 5.34 5.33 5.32 3.85 3.84 3.83 3.79 3.78 3.77 3.56 3.55 3.51 3.50 2.78 2.77 2.40 2.39 2.37 2.06 2.04 2.03 2.02 1.63 1.36 1.35 1.32 1.28 1.27 1.26 0.91 0.89 0.88 0.88

2.88 5.50 4.00 1.29 2.09 3.38 2.11 17.940.21 2.76

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm)

1 Figure A.13. H NMR (CDCl3) of SBO monomer.

1 Figure A.14. H NMR (CDCl3) of p(mAla).

155

1 Figure A.15. H NMR (CDCl3) of p(mPhe).

1 Figure A.16. H NMR (CDCl3) of p(mLysBoc).

156

1 Figure A.17. H NMR (CDCl3) of p(mSerTBDMS).

Figure A.18. 1H NMR (DMSO-d6) of p(mSer).

157

1 Figure A.19. H NMR (CDCl3) of p(mGluBn).

1 Figure A.20. H NMR (CDCl3) of p(N3).

158 k

1 Figure A.21. H NMR (CDCl3) of p(mTyrBn).

Figure A.22. 1H NMR (DMSO-d6) of p(mTrp).

159

1 Figure A.23. H NMR (CDCl3) of p(mAla-co-mPhe)0.66:0.34.

1 Figure A.24. H NMR (CDCl3) of p(mAla-co-mPhe)0.5:0.5.

160

1 Figure A.25. H NMR (CDCl3) of p(mAla-co-mPhe)0.25:0.75.

1 Figure A.26. H NMR (CDCl3) of p(mPhe-co-mGluBn)0.87:0.13.

161

1 Figure A.27. H NMR (CDCl3) of p(mPhe-co-mGluBn)0.73:0.27.

1 Figure A.28. H NMR (CDCl3) of p(mPhe-co-mGluBn)0.5:0.5.

162

1 t Figure A.29. H NMR (CDCl3) of p(mPhe-co-mAsp Bu)0.9:0.1.

1 t Figure A.30. H NMR (CDCl3) of p(mPhe-co-mAsp Bu)0.75:0.25.

163

1 t Figure A.31. H NMR (CDCl3) of p(mPhe-co-mAsp Bu)0.6:0.4.

1 Figure A.32. H NMR (CDCl3) of p(mSerTBDMS-co-mPhe)0.75:0.25. 164

1 Figure A.33. H NMR (CDCl3) of p(mSer-co-mPhe)0.75:0.25.

1 Figure A.34. H NMR (CDCl3) of p(mSerTBDMS-co-mPhe)0.5:0.5.

165

1 Figure A.35. H NMR (CDCl3) of p(mSer-co-mPhe)0.5:0.5.

1 Figure A.36. H NMR (CDCl3) of p(mSerTBDMS-co-mPhe)0.25:0.75.

166

1 Figure A.37. H NMR (CDCl3) of p(mSer-co-mPhe)0.25:0.75.

1 Figure A.38. H NMR (CDCl3) of p(mLysBoc-co-Propargyl)0.5:0.5. 167

1 t Figure A.39. H NMR (CDCl3) of p(mAla-co-mAsp Bu-co-Propargyl)0.4:0.4:0.2.

Figure A.40. 1H NMR (DMSO-d6) of p(mAla-co-mAsp-co-Propargyl).

168 7.31 7.28 7.27 7.27 7.23 7.21 7.20 4.24 4.23 4.16 4.15 3.62 3.61 3.55 3.51 3.49 2.97 2.95 2.67 2.62 2.61 2.57 2.56 2.43 2.40 2.38 2.35 1.61 1.17 1.14 1.12

k j O O i O b b c d f O O d N N O O y a a c x n O e e f O O g h c, f, i h

a, d b, e j g k

1.68 4.00 4.05 0.40 4.43 1.68 2.29

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm)

1 Figure A.41. H NMR (CDCl3) of p(mAla-co-mPhe)0.8:0.2.

1 Figure A.42. H NMR (CD3OD) of Rhodamine B-Base.

169

1 Figure A.43. H NMR (CD3OD) of Rhodamine B Piperazine amide.

1 Figure A.44. H NMR (CD3OD) of Rhodamine B NHBoc.

170

1 Figure A.45. H NMR (CDCl3) of p(mPhe-keto-BocGLU)0.8:0.2.

1 t Figure A.46. H NMR (CDCl3) of p(mPhe-mAla-mAsp Bu-But). 171

1 Figure A.47. H NMR (CDCl3) of p(mPhe-mAla-mLysBoc-But).

1 Figure A.48. H NMR (CDCl3) of p(mPhe-mAla-mSerTBS-But).

172

1 t Figure A.49. H NMR (CDCl3) of p(mPhe0.1-mAla0.4-mAsp Bu0.3-Keto0.2).

1 Figure A.50. H NMR (CDCl3) of p(mPhe0.1-mAla0.4-mAsp0.3-Keto0.2).

173

1 t Figure A.51. H NMR (CDCl3) of p(mPhe0.4-mAla0.1-mAsp Bu0.3-Keto0.2).

1 Figure A.52. H NMR (CDCl3) of p(mPhe0.4-mAla0.1-mAsp-Keto0.2). 174