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RAFT-Derived Polymers in Nanomedicine

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Title:
RAFT-Derived Polymers in Nanomedicine New Synthetic Methods and Applications
Creator:
Tucker, Bryan S
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[Gainesville, Fla.]
Florida
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University of Florida
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english
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Thesis/Dissertation Information

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Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
SUMERLIN,BRENT S
Committee Co-Chair:
WAGENER,KENNETH B
Committee Members:
MILLER,STEPHEN ALBERT
STEWART,JON DALE
KESELOWSKY,BENJAMIN G

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nanomedicine -- raft
Chemistry -- Dissertations, Academic -- UF
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
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Chemistry thesis, Ph.D.

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Abstract:
Reversible addition-fragmentation chain transfer (RAFT) polymerization is a valuable tool in polymer synthesis to generate polymers with control over molecular weights, molecular weight distributions, and polymer topology. Because of the functional group tolerance, amenability toward aqueous conditions, and simple reaction setups, RAFT has been used to make materials with biological applications. Here, we used RAFT polymerization to synthesize novel drug delivery vehicles using a simple method of chain extending homopolymers in the presence of a divinyl monomer to form well-defined, star-shaped nanoparticles. By employing heterogeneous reaction conditions, reaction yields were improved and nanoparticle molecular weights were increased relative to nanoparticles made under homogeneous conditions. RAFT also finds use in the production of polymer-protein conjugates. Often, preformed polymers are attached to a protein after the polymerization (grafting-to). We used RAFT to form brush polymers with various branching densities, and attached these polymers to a therapeutically viable protein. The results showed unique protein activity for the conjugates containing polymers with intermediate branching densities. Finally, RAFT polymerization can be used to generate polymer-protein conjugates in situ (grafting-from) using a protein modified with a chain transfer agent. Here, we utilized visible light-mediated RAFT polymerization to synthesize polymer-protein conjugates, reaching nearly full monomer conversion in extremely fast reaction times. A range of monomers were grafted-from a protein to show the broad applicability of this method. Furthermore, the end group retention of the initial polymer-protein conjugates was demonstrated by a chain extension reaction to form block copolymer-protein conjugates. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2017.
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Adviser: SUMERLIN,BRENT S.
Local:
Co-adviser: WAGENER,KENNETH B.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2017-11-30
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by Bryan S Tucker.

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11/30/2017
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LD1780 2017 ( lcc )

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RAFT DERIVED POLYMERS IN NANOMEDICINE: NEW SYNTHETIC METHODS AND APPLICATIONS By BRYAN SCOTT TUCKER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017

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2017 Bryan Scott Tucker

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To my wife, Caitlin

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4 ACKNOWLEDGMENTS I am grateful to my advisor, Prof. Brent Sumerlin, for the opportunity to be a member of his research group, a nd for his guidance and support. His scientific creativity and ingenuity have altered my viewpoints, and I am a much better scientist because of his influence I also thank my committee members, Prof. Ken Wagener, Prof. Stephen Miller, Prof. Jon Stewart, and Prof. Ben Keselowsky, for their instruction and time. I am grateful for the Butler Polymer Research Laborat ory, which promotes a collaborative environment that greatly aided in my research. I thank the Department of Chemistry at the University of Florida for the opportunity to earn my Ph.D. and for financial support. I likely would have never attended graduate school had it not been for the mentorship of Prof. Greg Gabriel at Kennesaw State University and Dr. Phil Imbesi at Texas A&M University. I thank both of them for their kind and encouraging mentorship, and for convincing me I could succeed in a doctoral pr ogram. I also thank Phil for his guidance in transitioning from graduate school to a position in industry I could not have completed my doctoral studies without the constant help and support of the Sumerlin research group members In particular, I would like to thank Dr. William Brooks, who helped in my transition both into and out of graduate school. He never hesitated to stop what he was doing to offer help or a word of advice, whether it was personal or professional in nature and this was instrumental to my progress I would like to thank Megan Hill for her friendship and for many fruitful discussions about science that aided in my projects We have had countless conversations about science, politics, religion, and world events, which h ave greatly broa dened my perspectives, and for that I thank her.

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5 I would like to thank all those who were directly involved in my research projects. I am appreciative of all of my collaborators: Prof. Jon Stewart, Prof. Shannon Holliday, Prof. Ignacio Aguirre, and Prof. C oray Colina. I thank Adrian Figg for his contributions to two of my manuscripts. I also thank undergraduates, Stephen Getchell and McKenzie Coughlin who worked with me during my doctoral research. My parents Mike and Nancy, always provided for me, supported me, and encouraged me. I was always encouraged to set optimistic goals for myself and I was always told I could achieve such goals with hard work The work ethic and values they instilled in me have been critical in my d octoral research, and I thank them for all they have done and continue to do for me I am grateful for the love and support of my in law s, Randy and Sheila Pennington. I know they are proud of me and welcome me in their family, and I am thankful to have su ch encouraging in laws I am also appr eciative of my extended family brothers, sisters in law, aunts, uncles, and grandparents who have all played important roles in shaping me into who I am today And finally, I am forever indebted to my wife, Caitli n Few people are so willing to uproot themselves to support someone in graduate school, but she never hesitated. Caitlin was not just waiting for me to complete my studies, she served as an integral partner during these five years. She has celebrated with me in my successes, learned with me through the struggles, and cried with me during the most difficult times both personally and professionally reminded me of why I started and encouraged me to c ontinue Her kindness and compassion are truly humbling I admire her and could not be more grateful to have her as my partner.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURE S ................................ ................................ ................................ ........ 10 LIST OF SCHEMES ................................ ................................ ................................ ...... 13 LIST OF ABBREVIATIONS ................................ ................................ ........................... 14 ABSTRACT ................................ ................................ ................................ ................... 18 CHAPTER INTRODUCTION ................................ ................................ ................................ ........... 20 1.1 Overview ................................ ................................ ................................ ........... 20 1.2 Reversible Addition Fragmentation Chain Transfer Polymerization .................. 20 1.3 Polymer Delivery Vehicles in Nanomedicine ................................ ..................... 22 1.4 PHPMA Materials in Nanomedicine ................................ ................................ .. 24 1.4.1 Polymer drug conjugates ................................ ................................ ......... 24 1.4.2 Self assembled nanoparticles ................................ ................................ 29 1.4.3 Other PHPMA structures in nanomedicine ................................ .............. 33 1.5 Polymer Protein Conjugates ................................ ................................ ............. 36 1.5.1 Synthetic Methods to Obtain Polymer Protein Conjugates ...................... 37 1.5.2 In Vivo Applications of Polymer Protein Conjugates ................................ 41 1.5.3 In Vitro Applications of Polymer Protein Conjugates ............................... 45 1.6 Summary and Future Outlook ................................ ................................ ........... 47 RESEARCH OBJECTIVE ................................ ................................ ............................. 48 FACILE SYNTHESIS OF DRUG CONJUGATED PHPMA CORE CROSSLINKED STAR POLYMERS ................................ ................................ ................................ 50 3.1 Overview ................................ ................................ ................................ ........... 50 3.2 Results and Discussion ................................ ................................ ..................... 53 3.2.1 CCS Polymers From Varying Crosslinker Concentration ........................ 55 3.2.2 CCS Polymers From Varying Unimer M n ................................ ................. 57 3.3.3 Heterogeneous CCS Polymer Synthesis ................................ ................. 57 3.3.4 Drug Loaded PHPMA CCS Polymer Synthesis ................................ ....... 61 3.3 Summary ................................ ................................ ................................ .......... 65 3.4 Experimental ................................ ................................ ................................ ..... 66 3.4.1 Materials ................................ ................................ ................................ .. 66 3.4.2 Charac terization ................................ ................................ ...................... 67

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7 3.4.3 Synthesis ................................ ................................ ................................ 68 3.4.4 Synthesis of PHPMA CCS Polymers ................................ ....................... 72 3.4.4.1 Investigation of the effect of [crosslinker]:[unim er] ratio on star formation ................................ ................................ ................................ 72 3.4.4.2 Investigation of the effect of unimer M n on CCS formation ............. 73 3.4.4.3 Synthesis of PHPMA CCS using heterogeneous polymerizations ................................ ................................ ....................... 74 3.4.4.4 Purification of CCS polymers ................................ ......................... 75 3.4.5 In vitro drug release experiments ................................ ............................ 76 ROLE OF POLYMER ARCHITECTURE ON THE ACTIVITY OF POLYMER PROTEIN CONJUGATES ................................ ................................ ...................... 78 4.1 Introduction ................................ ................................ ................................ ....... 78 4.2 Results and Discussion ................................ ................................ ..................... 81 4.2.1 Polymer Synthesis ................................ ................................ ................... 81 4.2. 2 Protein Conjugations ................................ ................................ ............... 85 4.2.3 In Vitro Osteoclast Inhibition Assay ................................ ......................... 86 4.2.4 In Vivo Skeletal Effect Study ................................ ................................ ... 89 4.3 Summary ................................ ................................ ................................ .......... 91 4.4 Experimental ................................ ................................ ................................ ..... 92 4.4.1 Materials ................................ ................................ ................................ .. 92 4.4.2 Instrumentation ................................ ................................ ........................ 93 4.4.3 Synthesis ................................ ................................ ................................ 93 4.4.4 Mouse Marrow Culture ................................ ................................ ............ 99 4. 4.5 Cell Cytotoxicity Assay ................................ ................................ .......... 100 4.4.6 Animals and Experimental Groups ................................ ........................ 100 4.4.7 Peripheral Quantitative Computed Tomography (pQCT) ....................... 101 GRAFTING FROM PROTEINS USING METAL FREE PET RAFT POLYMERIZATIONS UNDER MILD VISIBLE LIGHT IRRADIATION .................. 102 5.1 Overview ................................ ................................ ................................ ......... 102 5.2 Results and Discussion ................................ ................................ ................... 104 5.3 Summary ................................ ................................ ................................ ........ 117 5.4 Experimental ................................ ................................ ................................ ... 118 5.4.1 Materials ................................ ................................ ................................ 118 5.4.2 Instru mentation ................................ ................................ ...................... 119 5.4.3 Methods ................................ ................................ ................................ 124 5.4.3.1 Synthesis of LYS chain transfer agent (LYS CTA) ...................... 124 5.4.3.2 Photoinduced electron/energy transfer reversible addition fragmentation chain transfer (PET RAFT) polymerization. ................... 127 5.4.3.3 PET RAFT grafting from polymeriz ation using LYS CTA ............. 130 CONCLUSIONS AND FUTURE DIRECTIONS ................................ ........................... 135 APPENDIX: REACTIVITY RATIOS FO R PEGMA AND HPMA ................................ .. 137

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8 LIST OF REFERENCES ................................ ................................ ............................. 138 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 152

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9 LIS T OF TABLES Table page 3 1 Results for the synthesis of poly( N (2 hydro xypropyl)methacrylamide (PHPMA) ................................ ................................ ................................ ............ 54 3 2 Reaction conditions and molecular weight and size results during preparation of PHPMA core crosslinked star polymers ................................ ......................... 56 3 3 Reaction conditions and molecular weight and size results during preparation of CCS5 ................................ ................................ ................................ .............. 65 5 1 PET RAFT polymerizations of DMA. ................................ ................................ 106 5 2 PET RAFT polymerizations using low MW CTA ................................ ............... 108 5 3 Area under the peak of deconvoluted capillary liquid chromatography mass spectrometry chromatogram ................................ ................................ ............. 110 A 1 Monomer feed ratios and copolymer composition data for the RAFT copolymerization of PEGMA and HPMA ................................ .......................... 137 A 2 Monomer reactivity rat ios for PEGMA and HPMA ................................ ............ 137

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10 LIST OF FIGURES Figure page 1 1 Proposed mechanism for reversible addition fragmentation chain transfer polymerization. ................................ ................................ ................................ ... 21 1 2 Summary of nanostructures based on poly( N (2 hydroxypropyl )methacrylamide. ................................ ................................ .......... 24 1 3 Structure of coiled coil polymer conjugates and a proposed pathway for c ellular uptake and fate ................................ ................................ ...................... 26 1 4 Backbone degradable PHPMA via copper catalyzed azide alkyne cycloaddition of telechelic PHPMA ................................ ................................ ..... 27 1 5 Self assembly and siRNA loading of neutral polymeric micelles with a PHPMA corona and a pH responsive core ................................ ......................... 30 1 6 Synthesis and thermoresponsive behavior of PHPMA b PHPMA DL and hydrolysis of dilactate to leave bi ocompatabile PHPMA homopolymers ............. 33 1 7 Synthesis of hollow polymer nanoparticles using gold nanoparticle templates. .. 36 1 8 Schematic overview of the grafting to method to form polymer protein conjugates. ................................ ................................ ................................ ......... 38 1 9 Schematic overview of the grafting from method to form polymer protein conjugates ................................ ................................ ................................ .......... 39 1 10 Schematic depicting the proposed benefits of proteins immobilized with linear po lymers versus branched polymers ................................ ................................ 42 1 11 Representation demonstrating the relationship between chymotrypsin (CT) and substrate without polymer.. ................................ ................................ .......... 44 1 12 Small angle X ray scattering patterns of PNIPAM antibody conjugates ............. 46 3 1 GPC traces of PHPMA macro chain transfer agents of varying molecular weights using RAFT polymerization. ................................ ................................ .. 54 3 2 CCS polymers prepared using constant unimer M n and varying [crossliner]:[unimer] ratios ................................ ................................ .................. 56 3 3 GPC chromatograms of crude CCS polymer reactions in DMAc with constant crosslinker concentration and varying unimer M n ................................ .............. 58

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11 3 4 GPC chromatograms as a function of reaction time during the synthesis of CCS2 in EtOH/H 2 O. ................................ ................................ ........................... 59 3 5 GPC chromatograms as a function of reaction time during CCS polymer synthesis in DMAc ................................ ................................ .............................. 60 3 6 GPC chromatograms as a function of reaction time during the synthesis of CCS3 in pure H 2 O ................................ ................................ .............................. 61 3 7 Percent of MTX released from HPMA MTX with pig liver esterase .................... 62 3 8 GPC chromatogram of CCS4 before and after purification by ultrafiltration. ...... 63 3 9 Crude and purified GPC chromatograms ................................ ........................... 65 3 10 1 H NMR spectrum and peak assignments for HPMA MTX. (DMSO d 6 500 MHz). ................................ ................................ ................................ .................. 70 3 11 ESI MS spectrum of HPMA MTX. ................................ ................................ ...... 70 3 12 Analytical HPLC chromatogram of HPMA MTX. R t = 4.4 min, 96%. .................. 71 3 13 Calibration curve of MTX using the area under the curve at R t = 2.1 min in the HPLC chromatogram. ................................ ................................ ................... 77 4 1 GPC chromatogram of P2 ................................ ................................ ................. 82 4 2 UV vis spectra of P2 before RAFT group removal, P2 SH after RAFT group removal, and P2 fluorescein after Michael addition. ................................ ......... 83 4 3 GPC chromatogram of P3 after trithiocarbonate removal. ................................ .. 84 4 4 UV vis spectra of P3 before RAFT group removal, P3 SH after RAFT group removal, and P3 fluorescein after Michael addition. ................................ ......... 85 4 5 SDS PAGE analysis of OPG bioconjugates. ................................ ...................... 86 4 6 Osteoclast inhibition assay using 1,25 dihydro xyvitamin D 3 stimulated mouse marrow cells. ................................ ................................ ........................... 88 4 7 Cell cytotoxicity assay using RAW 264.7 cells shown as the percentage of cell survival relative to a control, which was untreated cells. .............................. 88 4 8 Bone mineral density (BMD) of OPG bioconjugates. ................................ .......... 90 4 9 Bone mineral density, bone mineral content, and bone mineral area of OPG bioconjugates. ................................ ................................ ................................ .... 90

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12 4 10 pQCT images of unmodified OPG and OPG3 in 7 days post treated rats at a distance of 4 mm from the growth plate. ................................ ............................. 91 4 11 1 H NMR spectrum of mPEG COOH (CDCl 3 500 MHz). ................................ ..... 94 4 12 1 H NMR spectrum of mPEG NHS (CDCl 3 500 MHz). ................................ ........ 95 5 1 Pseudo first order kinetics plots of DM A polymerizations ................................ 107 5 2 Pseudo first order kinetics plot of PET RAFT polymerizations with various monomers ................................ ................................ ................................ ......... 109 5 3 Deconvoluted chromatogram from capillary liquid chromatography mass spectrometry experiment of intac t lysozyme chain transfer agent .................... 110 5 4 Capillary liquid chromatography mass spectrometry spectrum of intact lysozyme chain transfer agent. ................................ ................................ ......... 110 5 5 Polymerization of DMA by grafting from CTA modified lysozyme in the presence of 2 m ol% eosin Y relative to the CTA ................................ ............. 111 5 6 Polymerization of DMA by grafting from CTA modified lysozyme in the presence of 1 mol% eosin Y ................................ ................................ ............. 113 5 7 Control reaction where DMA was polymerized in a mixture of unmodified lysozyme (LYS) and compound 1a (CTA). ................................ ....................... 114 5 8 Periodic light irradiation (i.e., on and off cycles) during the polymerization of DMA while grafting from lysozyme ................................ ................................ ... 114 5 9 Polymerization of 2 hydroxyethyl acrylate (HEA) by grafting from CTA modified lysozyme (LYS) in the presence of 1 mol% eosin Y ........................... 116 5 10 Polymerization of sodium styrene sulfonate (NaSS) by grafting from CTA modified lysozyme (LYS) in the presence of 1 mol% eosin Y ........................... 116 5 11 GPC chromatograms showing the chain extension of lysozyme poly(dimethylacrylamide) (LYS PDMA) with N isopropylacrylamide. ................ 117 5 12 Sodium dodecyl sulfate polyacrylamide gel electrophoresis results of polymer cleavage from lysozyme (LYS). ................................ ................................ ........ 134 A 1 Fineman Ross plot for the cop olymerization of PEGMA and HPMA ................ 137

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13 LIST OF SCHEMES Scheme page 3 1 Synthesis of poly( N (2 hydroxypropyl)methacrylamide (PHPMA) macro chain transfer agents. ................................ ................................ ................................ ... 53 3 2 Synthesis of HPMA MTX. ................................ ................................ ................... 62 3 3 Synthesis of CCS5 ................................ ................................ ............................ 64 4 1 Ring opening of succinic anhydride with mPEG and formation of an NHS ester to give P1 ................................ ................................ ................................ .. 81 4 2 RAFT polymerization of PEGMA to affo rd a densely branched polymer ............ 82 4 3 RAFT copolymerization of PEGMA and HPMA ................................ .................. 84 4 4 OPG bioconjugation reactions for OPG1 from P1 OPG2 from P2 and OPG3 from P3 ................................ ................................ ................................ ............... 86 5 1 Synthetic pathway to obtain the lysozyme chain transfer agent ....................... 104 5 2 PET RAFT polymerizations of various monomers in aqueous solution under blue light irradiation. ................................ ................................ ......................... 108

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14 LIST OF ABBREVIATIONS ACVA 4,4 Azobis(cyanovaleric acid) AIBN 2,2 Azobisisobutryonitrile AMPS 2 Acrylamido 2 methyl 1 propane sulfonic acid ARGET Activators regenerated by electron transfer ATRP Atom transfer radical polymerization BCA Bicinichoninic acid BMC Bone mineral content BMD Bone mineral density BSA Bovine serum albumin CCS Core crosslinked star CDP 4 (Cyanopentanoic acid)dithiobenzoate CDTPA 4 Cyano 4 [(dodecylsulfanyl thiocarbonyl)sulfanyl]pentanoic acid CT Computed tomography CTA Chain transfer agent DCC N,N' Dicyclo hexylcarbodiimide DCM Dichloromethane DMAP 4 (Dimethylamino)pyridine DMSO Dimethylsulfoxide D h Hydrodynamic diameter DL Dilactate DLS Dynamic light scattering DMA N,N Dimethylacrylamide DMAc N,N Dimethylacetamide DMF N,N Dimethylformamide

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15 DOX Doxorubicin DSDMA B i s(2 methacryloyl)oxyethyl disulfi de DTT Dithiothreitol EDC HCl 1 E thyl 3 (3 dimethylaminopropyl)carbodiimide HCl EGDMA Ethylene glycol dimethacrylate EPR Enhanced permeation and retention EtOH Ethanol EY Eosin Y Fab Antibody fragment GC Gas chromatography GPC Gel permeation chromatography HEA 2 Hydroxyethyl acrylate HPLC High performance liquid chromatography HPMA N (2 Hydroxypropyl)methacrylamide LC MS Liquid chromatography mass spectrometry LYS Lysozyme macroCTA Macro chain transfer agent MALS Multi angle light scattering ML Monolactate mPEG Monomethoxy polyethylene glycol MRI Magnetic resonance imaging MTX Methotrexate MW Molecular weight MWCO Molecular weight cutoff MWD Molecular weight distribution

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16 NaSS Sodium styrene sulfonate NHS N Hydroxysuccinimide NIPAM N Isopropylacrylamide NMP Nitroxide mediated polymerization NMR Nuclear magnetic resonance OI Optical imaging OPG Osteoprotegerin PAMAM Poly(amido amine) PBS Phosphate buffered saline PCB Poly(carboxybetaine) PDMA Poly( N,N dimethylacrylamide) PDSMA Pyridyl disulfide methacrylamide PEG Poly ( ethylene glycol ) PEGA Poly(ethylene glycol) acrylate PEGMA Poly (ethylene glycol) methyl ether methacrylate PET Positron emission tomography PET RAFT Photinduced elect r on/energy transfer reversible addition fragmentation chain transfer PHEA Poly(2 hydroxyethyl acrylate) PHPMA Poly( N (2 hydroxypropyl)methacrylamide) PLE Pig liver esterase PLMA Poly(lauryl methacrylate) PMDETA N N N N N P entamethyldiethylenetriamine PNaSS Poly(sodium styrene sulfonate) PNIPAM Poly( N isopropylacrylamide)

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17 PolyPEGMA Poly((polyethylene glycol) methyl ether methacrylate) PPEGA Poly((polyethylene glycol) acrylate) PTX P aclitaxel pQCT Peripheral quantitative computed tomography RAFT Reversible addition fragmentation chain transfer RANK Receptor activator of nuclear factor kappa B RANKL Receptor activator of nuclear factor kappa B ligand RDRP Reversible deactivation radical polymerization SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SiRNA Small interfering RNA TCEP T ris(2 carboxyethyl)phosphine HCl TEA Triethylamine TEM Transmission electron microscopy TFA Trifluoroacetic acid TFF Tangential flow filtration

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18 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RAFT DERIVED POLYMER S IN NANOMEDICINE: N EW SYNTHETIC METHODS AND APPLICATIONS By Bryan Scott Tucker May 2017 Chair: Brent S. Sumerlin Major: Chemistry Reversible addition fragmentation chain tran sfer (RAFT) polymerization is a valuable tool in polymer synthesis to generate polymers with control over molecular weights, molecular weight distributions, and polymer topology. Because of the functional group tolerance, amenability toward aqueous conditions, and simple reaction setups RAFT has been used to make materials with biological applications. Here, we used RAFT polymerization to synthesize novel drug delivery vehicles using a simple method of chain extending homopolymers in the presence of a divinyl monomer to form well define d, star shaped nanoparticles. By employing heterogeneous reaction conditions, reaction yields were improved and nanoparticle molecular weights were increased relative to nanoparticles made under homogeneous conditions. RAFT also finds use in the productio n of polymer protein conjugates. Often, preformed polymers are attached to a protein after the polymerization (grafting to). We used RAFT to form brush polymers with various branching densities, and attached these polymers to a therapeutically viable prote in. The results showed unique protein activity for the conjugates containing polymers with intermediate branching densities.

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19 Finally, RAFT polymerization can be used to generate polymer protein conjugates in situ (grafting from) using a protein modified wi th a chain transfer agent Here, we utilized visible light mediated RAFT polymerization to synthesize polymer protein conjugates, reaching nearly full monomer conversion in extremely fast reaction times. A range of monomers were grafted from a protein to s how the broad applicability of this method Furthermore, the end group retention of the initial polymer protein conjugates was demonstrated by a chain extension reaction to form block copolymer protein conjugates.

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20 CHA PTER 1 INTRODUCTION 1.1 Overview This dissertation focuses on i) the formation of delivery vehicles for small molecule drugs, and ii) the conjugation of polymers to proteins including new synthetic strategies, in order to improve their therapeutic efficacy Since much of the general area of nanomedicine uses reversible deactivation radical polymerization (RDRP) techniques and because this dissertation research utilized reversible addition fragmentation chain transfer (RAFT) polymerization, a brief overview of RAFT is presented in this chapter, followed by a discussion of delivery vehicles and polymer protein conjugates. 1.2 Reversible Addition Fragmentation Chain Transfer Polymerization RAFT polymerization is a valuable tool to generate polymers wit h predetermined molecular weights (MW), narrow molecular weight distribution s chain ends capable of undergoing subsequent polymerizations to form block copolymers. 1 4 Additionally complex polymer topologies (hyperbranched, star, comb, etc.) are attainable by RAFT polymerization 5 Importantly, a tolerance for diverse functional groups and aqueous solvents, a lack of transition metal catalysts, the ability to use a variety of initiation methods (e.g., thermal redox, and photoirradiation ), mild reaction conditions, and facile reaction setup mak e RAFT polymerization a n especially useful tool for the production of biologically relevant p olymers and advanced materials 6 *A portion of this chapter was adapted with permission from Polym. Chem. 2014 5 1566 1572. Copyright 2014 Royal Society of Chemist ry.

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21 Figure 1 1. Proposed mechanism for reversible addition fragmentation chain transfer polymerization. In RAFT polymerization p olymer MW control is achieved by a series of dege nerative chain transfer steps as opposed to a reversible termination mechanism found in atom transfer radical polymerization (ATRP) 7 and nitroxide mediated polymerization (NMP) 8 The proposed RAFT polymerization mechanism is provided in Figure 1 1. Briefly, an initiator, often azo compounds such as 2,2' azobisisobutyronitrile (AIBN), decomposes to generate radicals, which add to monomer as in a conv entional free radical polymerization. However, in an ideal scenario these oligomers quickly add to a thiocarbonylthio chain transfer agent (CTA) to form intermediate 2 This intermediate can either fragment toward starti ng materials to reform the initiator derived radical, or it can fragment constructively to form a new radical species from the 4 of the CTA. This new radical then initiates a new cha in which

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22 again adds to the thiocarbonyl thio group of the CTA. Upon the completion of this 9 t he main equilibrium is governed by numerous degenerative chain transfer steps through intermediate 5 where monomer propagation occurs followed by fast deactivation to a dormant chain ( 3 or 6 ) affording all chains appro ximately equal opportunity for chain growth and, hence, a narrow MWD Finally, as in any exogenously initiated radical polymerization, termination events lead to the quenching of radical sp ecies resulting in dead chains. H owever, because most chains are in itiated by the CTA R group and the targeted MW s are often much lower than would be achieved in the absence of a CTA the percentage of living chains in RAFT polymerization can be quite high and have the ability to efficiently undergo subsequent polymerizations 1.3 Polymer Delivery Vehicles in Nanomedicine Recent advances in nanomedicine have demonstrated exceptional promise for enhancing the delivery of therapeutics and diagnostics to their respective site of acti on. In particular, polymer based nanomaterials have been formulated into delivery systems that reduce toxicity, increase biodistribution, and increase drug efficacy. 10 12 In 1975, Ringsdorf suggested that the components necessary for an effective polymer drug delivery system are i) a hydrophilic, water soluble polymer scaffold capable of increasing the s olubility of small, hydrophobic drugs, ii) a drug conjugated via a biodegradable linker, and iii) a targeting moiety for specific delivery. 13 In addition to these components, the polymeric scaffold should be of sufficiently high molecular weig ht to avoid clearance by the renal system. This high molecular weight not only prolongs the biodistribution of the conjugates but can also serve to localize therapeutics in cancer tissue due to the enhanced permeation and retention (EPR) effect, whereby

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23 th e loosely formed vasculature and poor lymphatic drainage in many cancerous tissues allows nanostructures to selectively penetrate and reside in diseased tissue while being largely excluded from healthy tissue. 14,15 Poly(ethylene glycol) (PEG) has long been the most common hydrophilic, water soluble polymer employed for delivery of drugs and proteins. Des pite clearly demonstrated potential, the use of PEG can be problematic in some instances, with recent results indicating PEG containing therapeutics can elicit complement activation, rapid clearance after repeated injections, and may suffer from peroxidati on. 16 Moreover, the inability to effectively functionalize the polyether backbone mitigates the utility of colleagues, poly( N (2 hydroxypropyl) methacrylamide) (PHPMA) is an alternative hydrophilic polymer that incorporates functionality via a se condary hydroxyl group and which has been shown to be a viable alternative to PEG in many nanomedicine applications. 17 Like other N substituted methacrylamides, PHPMA is hydrolytically stable, an important attribute for applications in vivo. PHPMA can be synthesized by a variety of polymerization techniques including conventional radical polymerization, 18 atom transfer radical polymerization (ATRP), 19 and reversible addition fragmentation chain transfer (RAFT) polymerization of N (2 hydroxypropyl) methacrylamide (HPMA). 20 Alternatively, HPMA based (co)polymers can be prepared by postpolymerization modification via aminolysis of a polymer with activated ester sid e chains with 1 amino 2 propanol, the precursor of HPMA. 21,22 While conventional radical polymerization of HPMA has been carried out over many decades, advances in controlled radical polymerization techniques have provided exceptional control over m olecular weight and

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24 Figure 1 2. Summary of nanostructures based on poly( N (2 hydroxypropyl)methacrylamide. molecular weight distribution of PHPMA and have given rise to a vast amount of new macromolecular architectures and HPMA based copolymer self assemblies (nanoparticles, micelles, polymersomes, etc.) (Figure 1 2 ). The following section will focus on recent advances in the use of PHPMA in nanomedicine. More exhaustive reviews can be found elsewhere, 17, 23,24 but here scope will be limited primarily to recent examples of polymer drug conjugates, self assembled nanoparticles, and other materials that may have a significant imp act on the future of the field. 1.4 PHPMA Materials in Nanomedicine 1.4.1 Polymer drug c onjugates Many of the initial studies concerning the biological applicability of PHPMA involved the polymer being used as part of anticancer therapeutics. As compared to the poor site specificity of low molecular weight drugs, polymer based a nticancer

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25 compounds can passively target tumor tissues by the EPR effect. By conjugating anticancer drugs via stimuli responsive linkages to the polymer backbone, PHPMA can both target and treat tumor microenvironments. 25,26 In a recent example, DNA intercalators were conjugated to PHPMA copolymers via pH sensitive hydrazone bonds. 25 It was shown that the acridine based intercalators retained th eir anticancer activity in vivo by penetrating the cell membrane and successfully inserting into DNA. Enzyme degradable amino acid linkers have also been used to conjugate anticancer drugs to PHPMA copolymers. 26 For instance, HPMA was copolymerized with a methacrylamide modified peptide by RAFT polymerization, and cyclopamine was conjugated by a postpolymerization reaction with a thiothiazolidine 2 thione group at the t erminus of the peptide sequence. These polymer drug conjugates demonstrated promising anticancer activity against prostate cancer stem cells. To either enhance targeting in cancer tissue or treat diseases at sites that do not benefit from the EPR effect, t argeting ligands have been incorporated into many PHPMA drug conjugates. For example, many cancer cells over express certain surface receptors that can be selectively targeted using folic acid, 27 31 sugars, 32 34 or peptides. 35 For example, Stayton and coworkers synthesized a copolymer of HPMA and allyl methacrylate by RAFT polymerization and subsequently conjugated trimannoside ligands via photo initiated, thiol ene chemistry. 32 The modified polymer successfully demonstrated binding activity against the mannose specific lectin concanavalin A, suggesting their potential utility as glycan targeted drug delivery systems. Therapeutics can also be conjugated to PHPMA copolymers via non covalent, stimuli responsive coiled coil binding motifs. 36 37 Klok et al. employed RAFT

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26 Figure 1 3. Structure of coiled coil polymer conjugates (left) and a proposed pathway for cellular uptake and fate (right). Reprinted with permission from ref. 38. Copyright 2010 American Chemical S ociety. polymerization to create a library of PHPMA copolymers containing either an E3 or K3 peptide methacrylate comonomer. 37 The complementary peptide drug conjugate was then mixed with the polymer to form a coiled coil E3/K3 heterodimer that was taken up by endocytosis and disassembled at acidic pH to effect intracellular drug release (Figu re 1 3 ). 38 The novel strategy of using a supramolec ular coiled coil motif for drug conjugation leads to a fa cile and modular system that can incorporate a range of therapeutics, potentially allowing rapid screening for pharmaceutical studies. Additionally, the heterodimer peptides can facilitate intracellular delivery and serve as cytosolic drug delivery structu res. These benefits have been exploited in the design of drug free therapeutics based on CCE/CCK coiled coil heterodimers. 39,40 CCE was tethered to an antibody fragment (Fab) that binds to the CD20 receptor, which is over expressed in non Hodgkin lymphoma. After Fab CCE binding to the surface of C D20+ Raji B cells, PHPMA CCK copolymers were introduced, and a coiled coil heterodimer

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27 formed. The cross linking of CD20 receptors induced apoptosis of the B cells. This elegant strategy shows great promise for cancer therapy because of the absence of toxi c, low molecular weight drugs prevalent in traditional cancer therapy. While polymer drug conjugates should initially be large enough to allow prolonged blood circulation, ideally the polymer should be capable of degrading or disassembling after delivery of its therapeutic cargo so that its eventual hydrodynamic size is below the renal excretion limits of about 3.8 nm. In this respect, the lack of degradability of the carbon carbon backbone of PHPMA poses a challenge. However, et al. have recently formulated a novel strategy to synthesize backbone degradable PHPMA through the use of a difunctional chain transfer agent in RAFT polymerization and subsequent chain extension by step growth polymerization via copper catalyzed azide alkyne cycloaddition or thiol ene chemistry (Figure 1 4 ). 4 1 45 By Figure 1 4. Backbone degradable PHPMA via copper catalyzed azide alkyne cycloaddition of telechelic PHPMA and enzyme degradable peptide sequences. Reprinted with permission from ref. 44. Copyright 2011 American Chemical Society.

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28 employing an enzymatically degradable peptide sequence in the backbone, high molecular weight polymers capable of increasing pharmacokinetics were readily cleared after drug release and subsequent backbone degradation. High molecular weight PHPMA paclitaxel (PTX) conj ugates demonstrated improved anti tumor efficacy versus low molecular weight PHPMA PTX and commercially available PTX vesicle formulations. In addition, the high molecular weight PHPMA conjugates eventually showed improved clearance in vivo and lower systemic toxicity. 43 Imaging is another rapidly growing area of na nomedicine, as it provides the ability to monitor the biodistribution of a therapeutic de livery agent and/or observe the specific location of targeted disease tissue. Using appropriately labeled macromolecules, in vivo monitoring can be achieved by magneti c resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), or optical imaging (OI). 46 Long term monitoring of biodistribution is necessary to ascertain the in vivo fate of polymer delive ry systems. For example, this type of information was recently obtained by using the long term positron emitter, As(III), conjugated to the thiol end group of a RAFT derived PHPMA. 47 PHPMA copolymers were also used in OI studies using an IRDye contrast ag ent that targeted inflamed tissue. 48 By attaching dexamethasone to the PHPMA copolymer, a combined therapeutic and diagnostic system, known as a theranostic was synthesized. Vari ous imaging techniques can also be combined, providing more precise and accurate insight into the biodistribution of drug delivery systems. Lammers et al. utilized fluorescence molecular tomography with microcomputed tomography to make a hybrid imaging pla tform to non invasively monitor the biodistribution of PHPMA copolymers. 49 MR imaging was used to monitor

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29 the delivery and treatment of prostate tumors using plasmonic photothermal therapy facilitated by gold nanorods and PHPMA copolymers. 50 Using these techniques, or a combination of these techniques, the in vivo fate of dr ug delivery systems can be monitored, and upon the incorporation of therapeutics, theranostic systems can be synthesized. 1.4.2 Self assembled n anoparticles Advances in controlled radical polymerizations have allowed access to complex self assembled struc tures of PHPMA (co)polymers, including polymeric micelles, stars, and vesicles ( i.e. polymersomes). These nanoassemblies can exploit the aforementioned EPR effect and, when other responsive comonomers are employed, can be engineered to respond to specific stimuli to deliver a payload. Small interfering RNA (siRNA) and other nucleic acid therapeutics have great potential in the treatment of a number of diseases. However, delivery of this genetic material has proven challenging due to degradation, ineffecti ve cellular uptake, and inefficient endosomal escape. 51 To make a more effective delivery system, negatively charge d nucleic acid sequences can be complexed with positively charged nanoparticles to form polyplexes. In a recent example, methacrylamide oligolysine macromonomers were copolymerized with HPMA to yield comb copolymers capable of complexing to nucleic acids. 52 ,53 Because the peptide side chain of the macromonomer was attached to the methacrylamide group via a disulfide linkage, endosomal uptake led to the positively charged oligolysine gene units being released due to the elevated concentrations of glutathio ne in the cytosol as compared to the extracellular environment. 53 Other reports of increasing transfection efficiency have relied on the

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30 Figure 1 5. Self assembly and siRNA loading of neutral polymeric micelles with a PHPMA corona and a pH responsive core. Adapted with permission from ref. 51. Copyright 2013 American Chemical Society. functionalization of PHPMA based gene carriers by conjug ation to either melittin, a membrane penetrating peptide, 52 or RGD, an integrin binding peptide. 54 To avoid the cytotoxicity common to positively charged peptides, Stayton et al. synthes ized neutral siRNA carriers from PHPMA block copolymers. 51 HPMA was copolymerized with pyridyl disulfide methacryla mide (PDSMA) to form a hydrophilic block that was extended to yield a pH responsive block during a terpolymerization of propylacrylic acid, butyl methacrylate, and dimethylaminoethyl methacrylate. Thiolated siRNA was then conjugated via thiol exchange with the PDSMA units. The resulting amphiphilic block copolymers formed micelles that were successfully conjugated with siRNA, internalized via endocytosis, and escaped from the lysosome to deliver a model knock out gene (Figure 1 5 ). This strategy demonstrate d great potential in creating an efficient, n on toxic gene delivery system. In another approach to synthesize neutral gene delivery systems, Hennink and coworkers prepared a block copolymer of PEG b lock (PHPMA co PDSMA), where the HPMA repeat unit containe d a dimethylaminoethanol cationic group conjugated to its hydroxyl via a pH sensitive carbonate linkage. 55 Plasmid DNA was conden sed via electrostatic intera ctions, the micelles were cross linked using a dithiol compound, and

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31 HPMA repeat units. Upon entering into a reducing environment such as the cyto plasm of a cell, the disulfide cross links were reduced, and the plasmid DNA was released to leave only the biocompatible PEG b lock PHPMA. While this system showed good cytotoxicity results, efficient cellular uptake and endosomal escape ligands have yet to be incorporated. Numerous examples of drug delivery systems based on polymeric micelles with PHPMA coronas and various hydrophobic cores have been reported. 24 The examples below highlight some recent advances in this field. Block copolymers of PHPMA b lock poly(L or D lactide) have been synthesized using RAFT polymerization and were capable of forming micelles that encapsulated PTX. 56 57 Interestingly, it was shown that the stereochemistry of the lactide block affected the micelle size and cell uptake. Micelles were also formed from statistical copolymers of HPMA and a hydrazide containing comonomer that was capable of forming a pH sensitive hydrazone bond with doxorubicin (DOX) and cholesterol. 58 At pH 5.0, the linkage to DOX was h ydrolyzed rapidly as compared to the very slow hydrolysis of the linkage to the cholesterol moieties, which resulted in micelle dissociation and subsequent clearing of the resulting unimer chains. As a few of the examples above have demonstrated, an added advantage of PHPMA based systems over PEG based materials lies in the ability to functionalize the incorporation of a variety of biologically useful groups ( e.g., drugs, imaging a gents, ligands, solubility modifiers). For example, reductively responsive, core crosslinked micelles were formed using PEG b lock (PHPMA lipoic acid) copolymers in which the

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32 lipoic acid was conjugated via an ester bond to the hydroxyl of HPMA to yield a su fficiently hydrophobic block for micellization and DOX encapsulation. 59 After crosslinking with catalytic DTT, the resulting nanoparticles were observed to breakdown to release DOX in the reductive microenvironment of tumor cells. In a similar approach of functionalizing the hydroxyl of HPMA, Hennink and coworkers used HPMA modified with mono or dilactate (ML/DL) to synthesize thermoresponsive micelles based on PEG b lock PHPMA DL. 60 It was found that copolymers composed of PHPMA ML co PHPMA DL also formed thermoresponsive micelles. 61 These early examples used conventional radical polymerization, which suffers f rom high molecular weight dispersity; however, RAFT polymerization has recently been utilized to form well defined block copolymers composed of PHPMA b lock PHPMA DL. Biocompatible homopolymers of PHPMA resulted after hydrolysis of lact ate groups at basic p H (Figure 1 6 ). 62 The homopolymers were small enough to be cleared via the renal system; however, a possible drawback in this strategy is the elevated pH of 10.0 required to achieve rapid cleavage of lactate side groups and disassembly of the micelles. In addition to micelles, polymeric vesicles (i.e., polymersomes) self assembled from amphiphilic PHPMA based blo ck copolymers have been extensively studied in recent years. For many systems, a hydrophobic content of approximately 60% is required for polymersome assembly, but in a recent report, Davis and Lowe et al. showed that polymersomes could be formed from amph iphilic homopolymers with a hydrophobic end group comprising as little as 6 wt% of the polymer. 63 A RAFT agent containing a bis cholesterol or bis pyrenyl group was used to polymerize HPMA, and

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33 Figure 1 6. Synthesis and thermorespons ive behavior of PHPMA b PHPMA DL and hydrolysis of dilactate to leave biocompatabile PHPMA homopolymers. Adapted with permission from ref. 62. Copyright 2013 American Chemical Society. the resulting polymers formed polymersomes on the order of 150 1000 nm. Rhodamine B was encapsulated as a model hydrophilic drug, and the possibility of stimuli responsive delivery was suggested by incorporating a disulfide linker that allowed cleavage of the hydrophobic end group upon exposure to DTT. In this case, polymersome disassembly should lead to payload release. 1.4.3 Other PHPMA structures in n anomedicine In addition to polyplexes, micelles, and polymersomes, there still exist a number of other P HPMA structures that self assemble into therapeutically effective nanoaggregates. Many of these are intended to address currently unsolved problems. For example, the co administration of multiple chemotherapeutic drugs can provide synergistic benefits that enhance tumor suppression. Ulbrich et al. devised such a combination therapy with the synthesis of sub 200 nm nanoparticles containing a biodegradable copolyester core to encapsulate docetaxel, and a DOX conjugated PHPMA polymer coating. 64 These core shell nanoparticles were effective against mice

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34 bearing EL 4 T cell lymphoma and had the capability of incorporating targeting ligands in the PHPMA shell. Nanoaggregates have also been formed from copolymers of PHPMA and poly(lauryl methacrylate) (PLMA) via the activated ester postpolymerization functionalization route. 65,66 While both random and block copolymers formed nanoaggregates in solution, random copolymers consisting of 10% LMA were the most efficie nt in delivering the model drug Rhodamine 123 across a model blood brain barrier. This same polymer system was also radiolabeled with 18 monitoring of biodistribution. 67 Similar nanoaggregates based on random copolymers of PHPMA and folate bearing comonomers led to cellular uptake enhanced via receptor mediated endocytosis for targeted therapy. 30 Macromolecular stars based on HPMA have also been considered as nanotherapeutic agents. For example, various molecular weights of HPMA star polymers were investigated for their ability to passively deliver DOX. 68 Semi telechelic PHPMA DOX was synthesized using radical polymerization and functionalized azo initiators. The linear polymer drug conjugates were then grafted to poly(amido amine) (PAMAM) dendrimers, and the resulting stars demonstra ted prolonged blood circulation time and higher anti tumor activity than lower molecular weight linear polymers. While giving positive results in vivo a possible drawback of this method is the requirement of coupling two high molecular weight molecules us ing small molecule conjugation strategies that require a number of purification steps. Additionally, synthetic steps involving drugs should generally be limited since their high cost amplifies inefficiencies.

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35 An alternative way to form PHPMA star nanopar ticles was recently reported by Liu and coworkers. 29 bromopropionate ATRP initiator moieties, PHPMA stars were synthesized via the g rafting from method to give 14 arm stars with seven azide groups. The hydroxyl groups of the PHPMA side chains were then modified with both folic acid for targeting and DOX to give ester and carbamate linkages, followed by a copper catalyzed azide alkyne c ycloaddition to conjugate alkynyl DOTA Gd complexes for MRI contrast. The hydrophobicity of DOX allowed self assembly into micelle like nanoparticles that were pH responsive and shown to degrade at pH 4.0 5.0. Hollow nanoparticles with an aqueous core have also been synthesized for protein encapsulation. 69,70 An amphiphilic macroRAFT agent composed of PHPMA b lock poly(methyl methacrylate) was used as a colloidal stabilizer for inverse miniemulsion periphery RAFT polymerization, eliminating the need for additional surfactants. The shell of the nanoparticle was crosslinked by polymerization of ethylene glycol dimeth acrylate, and the shell thickness could be tuned by altering reaction stoichiometry. Additionally, the use of RAFT polymerization provided the possibility of further modifying the nanoparticles after their formation. Another route to the formation of PHP MA hollow nanoparticles was recently suggested by Davis and coworkers using sacrificial gold nanoparticle templates. 71 P[HPMA block (styrene stat maleic anhydride)] was synthesized and grafted onto gold nanoparticles exploiting the affinity of RAFT end groups to gold. The anhydride units in the shell of the nanoparticle were then crosslinked using a small molecule diamine. Finally, the gold core of the nanoparticles was removed using aqua regia (Figure 1 7 ).

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36 This method was a modular pathway to hollow nanoparticles with control over particle size, crosslinking density, and core si ze by altering polymer molecular weight, stoichiometry, and gold nanoparticle size. Figure 1 7. Synthesis of hollow polymer nanoparticles using gold nanoparticle templates. (1) Nanoparticle assembly (2) crosslinking and (3) gold core removal. Reprinted with permission from ref. 71. Copyright 2010 American Chemical Society. 1.5 Polymer Protein Conjugates The covalent attachment of a synthetic or natural polymer to a protei n has been studied to alter both the in vivo and in vitro properties of the protein. The first polymer protein conjugate was reported by Abuchowski and coworkers, who showed the attachment of PEG to bovine serum albumin (BSA) could alter the immunological properties and blood circulation times of the conjugate compared to the native prote in. 72,73 Since these initial reports, a wide variety of methods and applications have been investigated, and this section will provide a brief overview on the current state of the field with pertinent examples.

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37 1.5.1 Synthetic Methods to Obtain Polymer Protein C onjugates Polymer protein conjugate s are most often synthesized using a method, where pre formed polymers with functional end groups are reacted with a protein of interest. 73,74 The diversity of functional groups on the surface of proteins provides a variety of synthetic strategies to immobilize the polymer. Amine residues are abundant in a wide range of pr oteins, and these are often exploited using polymers with electrophilic end groups such as activated esters for acylation reactions although product distributions are often observed due to the high reactivity and low specificity of the activated ester Al dehyde bearing polymers have also been used to functionalize amine residues by reductive amination reactions using sodium cyanoborohydride. 74,75 Cysteine residues are less plentiful on the protein surface and these can be targeted to provide improved site selectivity during the conjugation reaction. Thiols are conveniently exploited using disulfide exchanges, Michael addition reactions or substitution of dibromomalei mide s 76 Figure 1 8 provides representative examples of conjugation strategies. Recent examples from our research group ha ve used acylation reactions with polymers containing N hydroxysuccinimidyl activated esters 77 The simple access to thiol terminated polymers through the end group removal of RAFT derived polymers was exploited to generate polymer protein conjugates in two consecutive Michael addition reactions first between a bismaleimide linker to afford a maleimide terminated polymer followed by a subsequent reaction with the available thiol in BSA 78 Our group also used c opper catalyzed azide alkyne cycloaddition reactions between alkyne modified BSA and azide terminated polymers to afford site specific polymer protein conjugates. 79

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38 Figure 1 8. (a) Schematic overview of the grafting to method to form polymer protein conjugates, where preformed polymers are reacte d with the protein. (b) Representative examples of the conjugation chemistry used for grafting from reactions targeting various amino acid residues on the protein surface. The grafting to method is especially beneficial because of the ability to synthesize polymers under conditions that would disrupt the structure and functionality of the protein (e.g., in organic solvents at high temper atures). Additionally, the grafting to method allows the polymer to be fully characterized prior to conjugation reactions.

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39 However, b ecause of the high MW of both the protein and the polymer in these bimolecular processes the concentrations of the reactive functional groups are quite low, which detrimentally reduces t he rate of the reaction. This leads to the requirement of large excess es of polymer starting material, making purification tedi ous and difficult. More often, it is beneficial to use reactions containing a large bimolecular rate constant to afford very rapi d and efficient reactions under dilute solutions ; 80 82 however, purification remains difficult. In a related challen ge, the multiplicity of the targeted functional groups on the protein surface result s in low chemoselectivity forming inseparable product distributions where either the number of polymers or the site of attachment varies Finally, polymer protein conjuga tes with high polymer grafting densities (i.e., the number of polymers per protein) or MW are often unattainable by this strategy because of the steric difficulty of coupling macromolecules. An alternative approach to the grafting g where a protein is rendered an initiator or CTA to modulate a polymerization directly from the surface of the protein (Figure 1 9) 83 86 Because each propagation step during the polymerization occurs with a low MW monomer, the steric encumbrance present in the grafting to method is mitigated and conjugate s bearing polymers with significantly higher MW s and high er grafting densities are easily attainable. Of particular benefit in Figure 1 9. Schematic overview of the grafting from method to form polymer protein conjugates, where a protein is modified with an initiator or chain transfer agent for reversible deact ivation radical polymerization. Adapted with permission from ref. 83. Copyright 2012 American Chemical Society.

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40 the grafting from method is the ability to completely remove unreacted monomers and low MW byproducts by simple purification methods (e.g., dial ysis). The advent of RDRP techniques 1,2,4,7,8,87 89 has led to a plethora of polymer protein conjugates via the g rafting from method, with the first reports using ATRP 90 93 Copper mediated RDRP methods have been most often utilized due to advances that allow for facile aqueous polymerizations 94 97 For example, Matyjas z ewski et al. first demonstrated activators regenerated by electron transfer (ARGET) ATRP to polymerize a PEG macro monomer from the surface of a protein This route employ ed a slow feed of a biologically friendly reducing agent (ascorbic acid) to allow controlled poly merization with copper catalyst concentrations between 100 300 ppm. 95 RAFT polymerization is more seldom utilized in grafting from reactions for the generation of polymer protein conjugates, despite the applicability of RAFT in aqueous solutions, 98 the excellent functional group tolerance, and the lack of transition metal catalysts Initial reports using RAFT polymerization to form polymer protein conjugates immobilized a CTA to a protein via the CTA Z group, which resulted in chain propagation in solution followed by a transfer step to the protein. 99,100 Soon after, our group first reported grafting from polymerizations using RAFT polymerization to synthesize poly( N isopropylacrylamide) ( PNIPAM ) protein conjugates where the CTA R group was immobilized to the protein and chain propagation occurred directly from the protein surface. 101 103 A critical feature of RDRP methods is an ability to chain extend living polymers to make block copolymers, and we demonstrated consecutive grafting from polymerizations by chain extending the initial polymer protein conjugate to form block copolymer protein conjugates. 102,103 Recently, Konkolewicz and coworkers

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41 repor ted an interesting strategy combining the grafting to and grafting from methods to first form a protein CTA with oligomers of acrylamide using grafting to, followed by a grafting from polymerization to produce high MW conjugates. 104 Others have recently used similar strategies to form transient ly responsive conjugates 105 and to investigate the effect of polymer chemistry on protein activity. 104,106 1.5.2 In Vivo Applications of Polymer Protein C onjug ates Among the most important effects of polymer protein conjugates is their ability to prolong the blood circulation lifetime of the protein in vivo This characteristic is most often attributed to a decrease in the protein clearance rate by kidney filtra tion which has a basement membrane pore size generally considered to be 3 5 nm While a number of factors contribute to this slowed filtration rate including altered protein surface charge s and polymer composition s the predomina nt cause is ascribed to t he dramatic change in the hydrodynamic diameter of the protein after polymer conjugation. Because the polymer, most often PEG, adopts a random coil in aqueous solution with a much larger hydrodynamic diameter than globular proteins of similar MW it has be en shown that PEG conjugates with polymer MW ca. 10 kDa is sufficie nt to prevent kidney filtration Furthermore, the MW of each polymer chain, rather than the total number of polymer modification sites, was determined to be the dominant factor to reduce clearance by kidney filtration. 107 Polymers h ave also been shown to mask protein epitope sites, resulting in a deceased immunological response compared to unmodified proteins This critical feature mitigates the deleterious side effects often observed with biologic al therapeutics, and it aids in prol onged circulation by slowing protein clearance by components of the immune system. Interestingly, the polymer topology plays an important role in protecting

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42 the protein from both immune recognition and proteolytic degradation. 107 Branched PEG where two polymers were joined by a lysine amino acid spac er and subsequently conjugated to the protein at this midpoint, were shown to have lower immune activation and proteolytic digestion compared to a conjugate containing linear PEG The author s suggest ed like effect with branched polymers whic h better m ask ed the protein su rface compared to linear polymers (Figure 1 10) 107 109 A number of polymer protein conjugates are approved for clinical use, the first of which was Adagen (pegade mase bovine ) in 1990 to treat severe combined immunodeficiency Other examples include PEGASYS (PEGylated interferon alpha 2a) to treat chronic hepatitis C, which contains br anched PEG as descr ibed abov e, and Oncaspar ( PEGylated L aspariginase ) to treat acute lymphoblastic leukemia which has an in vivo half life of 350 h compared to 4 h for the native enzyme. 110 All of the conjugates approved for clinical use contain PEG; however, there is intensive research Figure 1 10. Schematic depicting the proposed benefits of proteins immobilized with linear polymers versus branched the branched polymers provide enhanced protection against proteolytic enzymes and immune components as compared to the linear polymers. Reprinted with permission from ref. 107. Copyright 2003 Elsevier.

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43 efforts fo cusing on alternatives to PEG that are perhaps even more biocompatible, offer improve d ultimate degradability, or provide improvements in retaining the protein activity (i.e., pharmacodynamics) while maintaining strong pharmacokinetics. 111 For example, Davis and coworkers have recently observed slight improvements in the retention of protein activity of a PHPMA protein conjugate compared to an analogous PEGylated protein. 112 The same research group used a difunctional RAFT CTA to generate midfunctional PHPMA, which was coupled to BSA to form a branched PHPMA protein conjugate, potentially improving protein stability in a similar manne r as the branched PEG conjugates previously studied 113 Among the potential drawbacks of PEGylated proteins, likely the most detrimental is the observed loss of conjugate activity compared to the native protein. For example, PEGASYS, which has greatly improved pharmacokinetics compared to the native protein only maintains ca. 7% in vitro activity compared to the unmodified protein. 107,114,115 An interesting alternative to PEGylated proteins was recently demonstrated by Keefe and Jiang, where they examined conjugates containing zwitte rionic polymers based on poly(carboxybetaines) (PCB) 116 The zwitterionic polymer conjugates provided similar thermal and chemical stability as an analogous PEGylated protein. However, remarkably, the conjugate containing PCB actually d isplayed increased substrate binding affinity compared to the PEGylated enzyme and the activity even surpassed the native enzyme when high MW PCB w as attached. The authors reasoned the improvement in activity compared to PEGylated proteins derived from th e superhydrophilic nature of PCB compared to the amphiphilic PEG which

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44 served to promote the hydrophobic hydrophobic interaction between the enzyme active site and substrate ( Figure 1 11 ). Another important consideration for biological therapeutics is not only their stability toward biological factors, but also their stability during storage and transportation, as many proteins must be refrigerated to maintain activity. Toward this end, Maynard and coworkers have studied heparin mimicking polyanionic prote in conjugates, which provided enhanced stability toward heat, acidic conditions, and proteolytic degradation while protein activity was maintained 117 The same research group also used trehalose polymers to stabilize a protein toward multip le lyophilization cycles in addition to thermal stress. 118 Figure 1 11. (a) Representation demonstrating the relationship between chymotrypsin (CT) and substrate without polymer. (b) The amphiphilic nature of PEG lowers enzy me substrate affinity by inhibiting enzyme substrate hydrophobic hydrophobic interactions. (c) The super hydrophilic nature of PCB strongly effects the alignment of water around the protein active site, which increases enzyme substrate affinity. (d) The st ructure of PCB can be likened to protein stabilizing ions found in the Hofmeister series including ammonium and acetate (R = methacrylate backbone). Reprinted with permission from ref. 116. Copyright 2012 Nature Publishing Group.

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45 1.5.3 In Vitro Application s of Polymer Protein C onjugates Although not explicitly nanomedicine in the context described previously in this chapter, the attachment of stimuli responsive polymers to proteins endows them with unique in vitro activity relevant to biological application s 119 Therefore, this section will briefly outline interes ting examples of such materials. Thermally responsive conjugates incorporating PNIPAM are some of the most studied materials in this area, with a demonstrated ability to control the catalytic activity of enzymes, aid in prote in recovery and isolation, and form nanoscopically ordered surfaces and films. 119 Hoffman, Stayton and coworkers first showed that the thermally triggered phase transition of PNIPAM when attached to a protein sterically block ed the active site, modulating the protein activity on and off with altered temperature. 120,121 Since their initial report, others have used RDRP methods to generate PNIPAM conjugates with tunable activity. 79,91,101,103 In addition to controlling the activity, temperature responsive p olymers are also useful to afford the ability to recover or purify a protein of interest. 91,122 Enzymes immobilized on surfaces can serve as us eful biosensors for glucose detection or medical diagnostic assays; however, a difficult but necessary requirement is the three dimensional spatial orientation of the enzyme in high densities on the surface To this end, Olsen and coworkers have extensivel y studied the bulk self assembly properties of polymer protein conjugates. 123 130 The group demonstrated unique self assembly behavior of PNIPAM protein conjugates to achieve complex nanodomains When films were cast using solvent conditions below the phase transition temperature of PNIPAM, a hexagonal ly packed lamellar morphology with long range order was formed. Interestingly, a less ordered lamellar morphology with larger domain

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46 spacing was observed when films were cast above the PNIPAM pha se transition temperature 129 Furthermore, they showed extremely diverse phase behaviors ranging from disordered micellar, lamellar, to hexagonally packed cylinders bas ed on the degree of polymerization of PNIPAM and both the concentration and temperature used during casting and annealing. 124 The same group used b ulk self asse mbled polymer protein conjugates for catalytically active thin films with enzyme activi ty 5 10 that of films cast using other methods of enzyme immobilization 128 Finally, they reported the Figure 1 12. Small angle X ray scattering patterns of PNIPAM antibody conjugates with PNIPAM MW = 22 kDa in (a) solution and (d) bulk; or with PNIPAM MW = 68 kDa in (b) solution and (e) bulk. Domain spacings are inlaid within each scattering pattern. Transmission electron microscopy images (c and f) reveal th e lamellar morphology of each conjugate. (h) Schematic representing the lamellar domain of bulk self assembled polymer antibody conjugates. Reprinted with permission from ref. 131. Copyright 2017 Wiley.

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47 spatially controlled deposition of PNIPAM antibody c onjugates to make arrays containing long range order with tunable nanochannels for substrate transport (Figu r e 1 12) 131 1.6 Summary and Future Outlook With increased understanding and devel opment of new synthetic techniques, drug delivery systems can be further tailored to the specific needs of particular diseases. There is still much to be explored with respect to creating a delivery system that is cost effective and efficiently translated into clinical practice. Many of the materials included in this chapter and others throughout the literature rely on complex multi step syntheses; however, when it comes to macromolecular design, there is a certain elegance in simplicity, 45 and the versatility of PHPMA provided by its side chain functionality can allow for nanoparticles composed solely of HPMA and its derivatives. In the area of polymer protein conjugates, the ability of polymers to improve the in vivo characteristic s of the protein to which they are attached is well demonstrated. While a majority of the literature reports and all of the clinically approved materials utilize PEG to a ffect protein pharmacokinetics, advancements in synthetic polymer chemistry have opene d pathways toward alternatives to PEG. An important consideration moving forwar d in this field is the negative effects on protein activity posed by the immobilized polymer. Lower activity can often be offset by greatly increased pharmacokinetics in vivo; h owever, reduction in protein activit y could be detrimental to in vitr o applications. With continued advancements over site selectivity, protein engineering, and bioconjugation methods provided by chemical biology, coupled with expanded polymerization proto cols, we expect greater control over these advanced materials.

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48 CHAPTER 2 RESEARCH OBJECTIVE The purpose of t his research was to investigate new synthetic methods using reversible addition fragmentation chain transfer (RAFT) polymerization to generate biologically relevant polymers and study the efficacy of these materials in nanomedicine. RAFT polymerization is a vital tool in macromolecular design and engineering and o ften the resultant polymers are self assembled into advanced materials with complex architectures which can be time c onsuming and tedious My research goals were to provide new convenient synthetic approaches in the development of materials in the areas of polymer delivery vehicles and polymer protein conjugates. I have investigated a facile strategy to obtain nanoscopically discreet and well defined star shaped particles composed of poly( N (2 hydroxypropyl)methacrylamide) using RAFT polymerization under dispersion reaction conditions. These nanoparticles were investigated as drug delivery vehicles by covalently immobilizing a can c er therapeutic into the core of the nanoparticles (Chapter 3). RAFT polymerization is also useful to append polymers to the surfaces of proteins (polymer protein conjugates) manner, where polymers are grown directly from the protein surfaces. Using a grafting to strate gy, the effect of polymer architecture on the ac tivity of a therapeutic protein was investigated. Brush shaped polymers with various branching densities were synthesized by RAFT polymerization and a series of postpolymerization modifications were utilized to fluorescently label the polymers and endow them with protein reactive functionality.

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49 Each polymer bearing a unique architecture was immobilized to the protein, and t he results showed distinct in vitro activity based on the polymer architecture (Chapter 4). Finally, a new grafting from strategy using photomediated RAFT polymerization was inve stigated where polymer protein conjugates were formed under very mild reaction conditions in an extremely rapid grafting from polymerization The conjugates contai ned polymers with controlled molecular weights and narrow molecular weight distributions This technique also proved useful to polymerize monomer classes that are less utilized with RAFT grafting from. wa s demonstrated by chain extending the conjugate to form bloc k copolymer protein conjugates. This strategy should allow efficient access to new materials useful to control both the in vitro and in vivo properties of a protein (Chapter 5)

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50 CHAPTER 3 FACILE SYNTHESIS OF DRUG CONJUGATED PHPMA CORE CROSSLINKED STAR POLYMERS 3 .1 Overview Advances in synthetic polymerization techniques, including atom transfer radical polymerization (ATRP), 7 nitroxide mediated polymerization (NMP), 89 and reversible addition fragmentation chain transfer (RAFT) polym erization 2 have led to the development of complex macromolecular architectures 5,7,132 including micelles, 89,133,134 polymeric vesicles, 2,135 and star nanoparticles, 136 139 with highly diverse functions including drug delivery vehicles, 6,11,140,141 imaging agents, 7,46 emulsifiers, 89,142 and nanoreactors. 2,143,144 In particul ar, core crosslinked star (CCS) polymers, in which linear arms emanate from a highly crosslinked core, have received increased research interest due to their core shell structure and possibility for drug delivery applic ations 5,7,132,145,146 method 89,133,134,147 2,135,148 in which a multifunctional monomer. The arm first method is most often utilized due to an ability to characterize arm precursor polymers and the facil ity with which a large number of arms can be incorporated into the final CCS polymer. The work of Gao and Matyjaszewski has demonstrated the utility of ATRP in forming CCS polymers via an a rm first approach *Reprinted with permission from Polym. Chem. 2015 6 4258 4263 Copyright 2015 Royal Society of Chemistry.

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51 with the production of homo arm 136 139,145,149 and miktoarm 6,11,140,141,150 star s with a variety of compositions. Potentially d ue to its degenerative chain transfer mechanism RAFT polymerization has been more seldom used to form well defined CCS polymers. However, Boyer, Davis, and coworkers ha ve recently demonstrated the abi lity to tune the solubility of a divinyl crosslinker compound to generate well defined CCS polymers in homogeneous RAFT polymerizations 151 153 The An group has used heterogeneous RAFT polymerization s in ethanol/water mixtures to form homoarm and miktoarm CCS polymers for use as emulsifiers, 142,154 156 and Whittaker and coworkers have recently used heterogeneous RAFT polymerization to form CCS polymers for use as 19 F imaging agents for magnetic resonance imaging applications. 157 We are interested in studying the applicability of star polymers in drug delivery. Polymeric systems have long been studied to deliver therapeutics, and early work by Ringsdorf proposed the characteristics of an effective delivery system, including (i) hydrophilicity to solubilize small, hydrophobic drugs, (ii) a biodegradable linker for drug attachment, and (iii) a targeting moiety that directs deliver y to a specific site of a ction. 13 In addition, the delivery system should be of sufficie nt size (~10 200 nm) to increase biodistribution by preventing renal filtration but small enough to avoid clearance by the reticuloendothelial system. 11 This size range also allows the nanoparticles to benefit from the enhanced permeation and retention (EPR) effect, which describes the loosely formed vasculature and poor lymphatic drainage often found in cancerous tissues that lead to accumulation of nanoparticles within diseased tissue while largely excluding it from healthy tissue. 14,15

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52 While the majority of materials used in nanomedicine are composed of poly(ethylene glycol) (PEG) based materials because of their wate r solubility and biocompatibility, poly( N (2 hydroxypropyl)methacrylamide) (PHPMA) based materials 17,24,158 have shown considerable promise as well. PHPMA can potentially overcome some of the suggested shortcomings of PEG, including dose dependent immunoresponses rapid clearance after repeated injections, and potential peroxi dation 16,23,159 Additionally, HPMA is readily synthesized and can be polymerized via a variety of methods including conventional radical polymerization, 1 8 ATRP, 19 and RAFT polymerization. 20 Furthermore the available hydroxyl group on HPMA can be exploited as a versatile handle for the incorporation of drugs, imag ing agents, and targeting ligands. A number of PHPMA based therapeutics have been synthesized; however, the use of star shaped PHPMA derivatives has only been investigated using a dendritic poly(amido amine) core with linear PHPMA attached via coupling. 68,160 164 While the se reports demonstrated useful properties for drug delivery systems, such as an extended blood circulation time compared to linear PHPMA and drug release at acidic conditions, tedious and labor intensive purifications including preparative gel permeation chromatography, were often required to isolate the macromolecular coupling product s. Here we demonstrate, for the first time, the synthesis of PHP MA CCS polymers using the arm first method to prepare well defined, crosslinked star nanoparticles in a facile and efficient method. For potential drug delivery applications we exploited the hydroxyl group of HPMA for attachment of methotrexate, a folic a cid antagonist used in the treatment of a number of cancers. 165 167 This novel drug conjugated monomer was

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53 then directly polymerized during CCS formation to provide drug loaded PHPMA nanoparticle s. Given that the drug was covalently bound this method should provide enhanced stability toward premature drug release (Scheme 3 1) Finally, we show that the drug can be released from the monomer via enzymatic hydrolysis. Scheme 3 1. Synthesis of poly( N (2 hydroxypropyl)methacrylamide (PHPMA) macro chain transfer agents (unimers), which were then chain extended in the presence of a methotrexate modified HPMA monomer and a divinyl crosslinker to prepare drug loaded PHPMA core crosslinked star polymers. 3 .2 Results and Discussion Polymeric nanoparticles for drug delivery should ideally be narrowly dispersed in both their size and composition to efficiently and consistently deliver their payload. The uniformity of CCS polymers is affected by the molecular w eight of the unimers the amount of crosslinker used, and the efficiency with which the unimers are incorporated into the nanostructure. Our goal was to develop a strategy to well defined PHPMA based star polymers that have potential utility in drug delive ry. To demonstrate the versatility of PHPMA CCS formation by our RAFT based strategy, a number of synthetic variables were investigated to tune the formation of well defined stars including [crosslinker]:[unimer] ratios, unimer molecular weight, and solve nt selection. Linear unimers of three distinct molecular weights (MW) were prepared by RAFT polymerization of N (2 hydroxypropyl)methacrylamide (HPMA) to control the MW and molecular weight distributions of the resulting polymers (Table 3 1 and Figure 3 1 )

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54 Table 3 1. Results for the synthesis of poly( N (2 h ydroxypropyl)methacrylamide (PHPMA). Absolute molecular weights were determined using gel permeation chromatography equipped with a multi angle light scattering detector Entry M n (g/mol) M w / M n P1 6260 1.08 P2 9470 1.08 P3 17300 1.24 Figure 3 1. GPC traces of PHPMA macro chain trans fer agents of varying molecular weights using RAFT polymerization. We reasoned the narrow molecular weight distribution in the unimers should aid in preparing stars that also had narrow size distributions. These unimers were employed in the arm first synthesis of CCS polymers using ethylene glycol dimethacrylate (EGDMA) as the divinyl crosslinker. The efficiency of each reaction ( i.e, star yield) was calculated by deconvolution of the gel permeation chromatography (GPC) refract ive index (RI) chromatogram and E quation ( 3 1) star yield = A star /( A star + A unimer ) (3 1) where A star and A unimer are the areas of the star and unimer peaks, respectively. The weight average MW ( M w ) of each CCS polymer was obtained via GPC equipped with a multi angle light scattering detector (MALS) using the d n/ dc value for the unimer. While

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55 this assumption that the scattering of the star is only due to the PHPMA arms is not id eal, we believe that this can be used to understand the general trends in star formation under varied reaction conditions. Also from the GPC MALS data, the arm number, f was calculated to give the average number of arms per star using E quation 3 2. (3 2) where WF arms is the weight fraction of the arms in the star and is given by Equation 3 3. (3 3) where m unimer is the mass of the unimer, p unimer is the conversion of the unimer from the deconvoluted GPC chromatogram m CL is the mass of the crosslinker, and p CL is the conversion of the crosslinker 3.2.1 CCS Polymers From Varying Crosslinker C oncentration PHPMA CCS polymers were synthesized using P1 (Table 3 1 ) and varying [crosslinker ]:[unimer] ratios (15:1, 10:1, and 5:1) in N N dimethylacetamide ( DMAc ) for 24 h (Table 3 2) The GPC chromatograms of each crude reaction showed a decrease in elution time, i ndicating the formation of higher MW CCS polymers (Fig ure 3 2 ). I ncreasing crosslinker concentration led to an increased star yield, as well as higher arm number, f and higher star M w which is likely due to a larger core M w and higher incorporation of un imers in each CCS polymer. T he highest concentration of crosslinker ([crosslinker ]:[unimer] = 15:1) led to the highest star yield but the star peak was broad and multimodal due to a broad molecular weight distribution. This observation may be attributed t o star star coupling which could be due to a high number of crosslinking

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56 Figure 3 2. CCS polymers prepared using constant unimer M n ( P1 ) and varying [crossliner]:[unimer] ratios (5:1, 10:1, 15:1, denoted as P1 5, P1 10, and P1 15, respectively, in the legend). The star yield of each reaction is given to show that increasing concentration of crosslinker resulted in an increase in the star yield. moieties that can potentially lead to cross propagation during star synthesis On the oth er hand, u sing the intermediate crosslinker concentration ([crosslinker ]:[unimer] = 10:1) resulted in a narrow, monomodal star peak in the GPC chromatogram and moderate star yield. H owever, this star polymer had limited water solubility, possibly due Table 3 2. Reaction conditions and molecular weight and size results during preparation of PHPMA core crosslinked star polymers Entry a Unimer [crosslinker]:[unimer] Solvent b CCS M w (kg/mol) c Star yield (%) d f e D h (nm) P1 5 P1 5:1 DMAc 73.3 30 10 P1 10 P1 10:1 DMAc 256 60 20 P1 15 P1 15:1 DMAc 1250 70 100 P2 10 (CCS1) P2 10:1 DMAc 211 50 14 43 P3 10 P3 10:1 DMAc 287 10 10 CCS2 P2 10:1 EtOH/H 2 O 553 70 40 20 CCS3 P2 10:1 H 2 O 1280 70 100 20 CCS4 P2 f 10:10:1 EtOH/H 2 O 124 60 10 20 a Refer to Table 3 1 for the molecular weights of unimers P1 P3. b Weight average molecular weight of core crosslinked star polymers determined by GPC MALS. c Star yield calculated using the deconvoluted GPC RI chromatograms and Equation 3 1. d Arm number calculated using Equation 3 2. e Hydrodynamic diameter from dynamic light scattering in water. Diameters are provided only for the samples that were purified to avoid convultion by unreacted unimers in solution. f [crosslinker]:[HPMA MTX]:[unimer].

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57 to th e relatively short hydrophilic arms that were unable to solvate a large hydrophobic core. As expected, t he lowest crosslinker concentration ([crosslinker ]:[unimer] = 5:1) re sulted in the lowest star yield. Considering the well defined star peak obtained us ing the intermediate crosslinker concentration this ratio was chosen for further studies to investigate conditions that provide well defined stars that have sufficient water solubility. 3.2.2 CCS Polymers From Varying U nimer M n We hypothesized that increasing the MW of the arms would result in higher water solubility and provide a more efficient steric shield to prevent star star coupling. PHPMA stars were synthesized with varying unimer M n and a constan t [crosslinker]:[unimer] ratio (10:1). The resu lts indicated that increasing the unimer molecular weight resulted in a decrease in star yield (Table 3 2, Fig ure 3 3 ), which is po ssibly due to the difficulty in incorporating a large number of higher MW unimers during star growth as a result of the incre ased steric congestion around the core Even though the star yield was higher with P1 the lower water solubility and concern of aggregation led us to choose P2 as the unimer for continued studies. The CCS polymers obtained in this system denoted CCS1 we re purif ied by fractional precipitation and were characterized by GPC MALS, dynamic light scattering (DLS), and transmission electron microscopy (TEM) ( Figure 3 3 ). 3.3.3 Heterogeneous CCS Polymer S ynthesis Despite the ability to synthesize and isolate CC S polymers from homogeneous polymerization conditions, the long reaction times of homogeneous systems and tedious polymer recovery limited the utility of this method. We employed similar conditions to those previously reported for the synthesis of poly(polyethylene glycol)methacrylate

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58 CCS polymers by dispersion polymerization, using an EtOH/H 2 O solvent mixture and a water soluble initiato r. 154 Thus, PHPMA CCS polymers wer e prepared by dispersion Figure 3 3 (a) GPC chromatograms of crude CCS polymer reactions in DMAc with constant crosslinker concentration and varying unimer M n Star yields of each reaction are provided in the corresponding color of the trace. (b) GPC c hromatograms before and after purification of CCS1 by fractional precipitation. (c) DLS histogram of CCS1 in pure water. (d) TEM image of CCS1 (0.5% uranyl acetate stain; scale bar = 100 nm). polymerization using P2 and a [ crosslinker ]:[unimer] ratio of 1 0:1. W ell defined stars ( CCS2 ) w ere formed in only 4 h, as compared to the 24 h required under homogeneous conditions (Table 3 2, Figure 3 4 a Figure 3 5 ). We believe the increased rate of star formation is due to the limited solubility of both the EGDMA crosslinker and the growing

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59 Figure 3 4. (a) GPC chromatograms as a function of reaction time during the synthesis of CCS2 in EtOH/H 2 O. (b) GPC chromatograms of CCS2 before and after purification by ultrafiltration. (c) DLS histogram of CCS2 in pure wat er. (d) TEM image of CCS2 (0.5% uranyl acetate stain; scale bar = 100 nm). core of the stars in the reaction medium, which creates a dispersion polymerization scenario. CCS2 was readily purified by ultrafiltration which provided a facile and rapid method to remove unreacted unimers and isolate our stars compared to preparative GPC me thods used in previous reports. T he purified stars were then analyzed by GPC MALS, DLS and TEM ( Figure 3 4 ) DLS analysis indicated stars with a hydrodynamic diameter ( D h ) of 20 nm, and TEM revealed the nanoparticles adopted a spherical morphology with sizes consistent with DLS data, when considering the dehydration of

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60 Figure 3 5. GPC chromatograms as a function of reaction time during CCS polymer synthesis in DMAc. ([cross linker]:[unimer] = 10:1; unimer = P2 ). the stars after deposition onto the TEM grid. TEM also revealed the presence of very small aggregates in addition to stars B ased on their size and the MW data from GPC M ALS, it is believed these polymers are very low MW stars ( e.g., two or three arm stars). The stars could also be efficiently prepared in p ure water in the absence of an organic cosolvent. Interestingly, when P2 was used as the unimer with a [ crosslinker ]:[unimer] ratio of 10:1 t he resul ting CCS polymers ( CCS3 Table 3 2, Fig ure 3 6 ) had approximately twice the MW and number of arm s as compared to CCS2 even though the Z average D h was approximately equal for both samples. This suggests the size of the CCS polymers is equal, despite a lar ge difference in the MW We believe this is due to a higher packing efficiency for CCS3 possibly due to pre assembly of the crosslinker in a poor solvent, increasing the efficiency with which arms are incorporated into the star. Since the MW of the arms i s equal and a spherical morphology is observed

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61 in each scenario, the arms of CCS3 are presumably more closely packed than that of CCS2 and the resultant stars have approximately equal size. Figure 3 6. (a) GPC chromatograms as a function of reaction time during the synthesis of CCS3 in pure H 2 O. (b) GPC chromatograms of CCS3 before and after purification by ultrafiltration. (c) DLS histogram of CCS3 in pure water. (d) TEM image of CCS3 (0.5% uranyl acetate stain; scale bar = 100 nm). 3.3.4 Drug L oaded PHPMA CCS Polymer Synthesis With efficient synthetic conditions for star formation having been determined, we next investigated the incorporat ion of an anticancer drug within the star cores Met hotrexate (MTX) is a therapeutic used to treat a diverse set of cancers and contains a carboxylic acid functional group that provid es a straightforward method for conjugation to the hydroxyl group of HPMA (Scheme 3 2 ) Enzyme catalyzed release of the drug

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62 Scheme 3 2. Synthesis of HPMA MTX. Figure 3 7. Percent of MTX released from HPMA MTX with pig liver esterase (blue, 150 U) and without (orange). from the HPMA MTX conjugate was investigated using porcine liver esterase an enzyme that readily cleave s ester bonds 168 172 After incubation with PLE (150 U/mg) for 96 h, 30% of MTX was cleaved to yield the free drug and HPMA (Figure 3 7 ). We believe that the relatively low amount of drug release could be due to the electrophilic methacrylamide group, which is susceptible to Michael addition by the nucleophilic active site of the enzyme. However, since no release was observed over the same period in the absence of the enzyme, we reasoned that PLE might selectively release the drug from our PHPMA based star polymers. HPMA MTX was used in the formation of a star polymer ( CCS4 ) u sing RAFT dispersion conditions, and t he star was purified by ultrafiltration (Table 3 2) GPC MALS and DLS were used to determine the molecular weight and size of the stars. UV Vis

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63 spectroscopy was used to confirm the incorporation of the drug in CCS4 as 20 wt% using a standard curve to determine the concentration of MTX in the star relative to the total star concentration Because the drug was directly polymerized, the amount of drug in the star could be tuned by controlling the degree of polymerizatio n of the drug monomer. Here, we achieved 55% conversion of the drug monomer, based on 1 H NMR spectroscopy, which corresponded to 40 MTX units per star. Finally TEM analysis revealed a spherical morphology for the drug containing stars (Fig ure 3 8 ). Altogether, these results demonstrated the drug could be directly polymerized without altering the integrity of the CCS polymer. Finally, enzymatic drug release for CCS4 was Figure 3 8. (a) GPC chromatogram of CCS4 before and after purification by ul trafiltration. (b) DLS histogram of CCS4 in pure water. (c) UV vis spectrum of CCS4 showing the successful incorporation of MTX into the CCS polymer. (d) TEM image of CCS4 (0.5% uranyl acetate; scale bar = 100 nm).

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64 investigated. In contrast to the monomer the drug loaded stars had significantly enhanced stability toward enzymatic hydrolysis, with no release observed after 48 h. We reasoned the stability was due to the highly crosslinked nature of the core, limiting access of the enzyme to the ester linkag es tethering the drug to the stars. A convenient way to study how the core sterics affect drug release was to synthesize a drug loaded PHPMA star using a degradable crosslinker, where cleavage of the crosslinker would result in unimers in solution, provid ing more facile enzymatic access. It is possible this strategy also improves the ultimate utility of the polymer, as the unimers that result from dissociation should be below the size limit for clearance via renal filtration. Toward this goal, a star polym er ( CCS5 ) was synthesized using a disulfide bearing crosslinker, which can be cleaved upon the additi on of a reducing agent (Scheme 3 3 Table 3 3 Figure S9). To investigate drug release, the star was first reduced using tributylphosphine and purified by dialysis. PLE (150 U/mg) was then added, and drug release was monitored by HPLC. However, no drug release was observed in this system after 48 h. It is possible that even the sterics of a linear polymer slow the hydrolysis of the ester. Therefore, we are c urrently investigating alternative methods of drug conjugation that are more susceptible to release under specific conditions found in the tumor microenvironment. Scheme 3 3. Synthesis of CCS5

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65 Table 3 3. Reaction conditions and molecular weight and size results during preparation of CCS5 Entry [crosslinker]:[HPMA MTX]:[unimer] CCS M w (kg/mol) Star yield (%) f D h (nm) MTX wt% CCS5 10:10:1 728 70 30 10 15 Figure 3 9. (a) Crude and purified GPC chromatograms, (b) DLS histogram in pure water (1 mg/mL), and (c) TEM image (0.5% uranyl acetate; scale bar = 100 nm) of CCS5 3.3 Summary In summary, well defined PHPMA macroCTAs were synthesized by RAFT polymerization and were subsequently used to produce star polymers via both homogeneous and heterogeneous reaction conditions. We found that high concentrations of crosslinker during the polymerization led to only partial ly water soluble CCS polymers, and the use of high MW unimers resulted in limited star yields in homogeneous reaction conditions due to the steric hindrance encountered when adding large unimers to a growing CCS polymer. High star yields could be obtained in short reaction times by dispersion polymerization in EtOH/H 2 O with unimers of intermediate MW and intermediate [crosslinker]:[unimer] ratios. To study these materials for drug delivery, a chemotherapeutic agent was conjugated to HPMA, and the resultin g monomer drug was used during CCS polymer synthesis under the optimized RAFT dispersion conditions to form drug loaded PHPMA based star s. Well defined, spherical aggregates with a high drug loading capacity were

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66 confirmed. Because the drug was covalently bound within the star cores it is expected that higher drug stability may be observed during in vivo circulation The HPMA MTX conjugate was observed to undergo enzyme triggered drug release, though the sterically hindered environment of the drug conjuga ted to the PHPMA backbone limited release under the conditions employed. Although studies are underway to optimize the rate of drug release from these star polymers, we believe this report provides a strategy toward PHPMA based drug delivery systems and va luable insight into the slow release of covalently conjugated drugs from PHPMA. 3.4 Experimental 3.4.1 Materials 1 Amino 2 propanol (94%), methacryloyl chloride (97%), 4,4 azobis(cyanovaleric acid) (ACVA) (98%), anhydrous methotrexate (98%), and Spectrum/ Por Float A Lyzer G2 dialysis devices with 3500 Da MWCO membranes were purchased from VWR and used as received. EMD Millipore Amicon Ultra 0.5 centri fugal filter units with 50 kDa MWCO membranes were purchased from Fisher Scientific. N,N dicylcohexylca rbodiimide (DCC) (99%), 4 (dimethylamino)pyridine (DMAP) (99%), 2,2 azobisisbutyronitrile (AIBN), ethylene glycol dimethacrylate (EGDMA) (98%), esterase protein/mL), tri n butylphosphine (99%), anhydrous N N dimethylformamide (DMF), N N purchased from Sigma Aldrich. D euterium oxide (D 2 O 99.9%), and dimethylsulfoxide d 6 (DMSO d 6 99.8%) were purchased from Cambridge Isotopes. AIBN was recrystallized from methanol, EGDMA was passed through a column of basic alumina to remove inhibitors, and all other chemicals were purchased with the highest available purity and

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67 used as received. 4 C yano 4 [(dodecylsulfanyl thiocarbonyl)sulfanyl]pentanoic acid (CDTPA) 2 and bi s(2 methacryloyl)oxye thyl disulfi de (DSDMA) 173 were synthesized according to a literature procedure. 3.4.2 Characterization 1 H N MR spectra were recorded on a Varian Inova2 500 MHz or a Varian Mercury 300 MHz NMR spectrometer using the residual solvent signal as reference. UV Vis spectra we re obtained on a Molecular Devices SpectraMax M2 multimode microplate reader. Analytical HPLC was performed using a gradient from 3:1 to 1:3 aq. trifluoroacetic acid (TFA) (0.1%):CH 3 CN at 35 C at a flow rate of 1 mL/min (Hitachi Elite LaChrom pump, column oven, and UV Vis detector operating at 303 nm; column: 150 18 ). Preparative HPLC was performed using 3:1 to 1:1 aq. TFA (0.1%):CH 3 CN at 35 C and a flow rate of 8 mL/min (Hitachi Elite LaChrom pump, column oven, and UV Vis detector operating at 303 nm; column: 250 21.2 mm 18 ). High resolution mass spectrometry to obtain accurate mass was obtained with an Agilent 6220 electrospray ionization time of flight mass spectrometer (ESI TOF MS). Polymer m olecular weight and molecular weight distributions were determined by gel permeation chromatography (GPC) in N N DMAc with 50 mM LiCl at 50 C and a flow rate of 1.0 mL/min (Agilent isocratic pump, ViscoGel I series G3078 10 3 10 4 g mol 1 ). Detection consisted of a Wyatt Optilab T rEX refractive index detector operating at 658 nm and a Wyatt miniDAWN Treos light scattering detector operatin g at 659 nm. Absolute molecular weights and molecular weight distributions were calculated using

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68 the Wyatt ASTRA software (PHPMA dn/dc = 0.0751 mL/g) D ynamic light scattering (DLS) measurements were recorded on a Zetasizer Nano ZS, ( Malvern Instrument Ltd ., laser beam operating at 633 nm at 25 C Samples were prepared in pure water and each measurement was repeated six times to obtain the average value. Transmissi on electron microscopy (TEM) was conducted on a Hitachi H7000 microscope operating at 100 kV. A formvar coated 200 me s h Cu grid that was freshly glow discharged (Pelco a drop of sample solution for 30 sec and wic ked off with filter paper. Uranyl acetate (0.5 % aqueous solution) was used as a negative stain. 3.4.3 Synthesis N (2 Hydroxypropyl)methacrylamide (HPMA) To a three neck, 2 L round bottom flask equipped with a mechanical stirring device, thermometer, and addition funnel was added 1 amino 2 propanol (75 mL, 0.96 mol) The reagent was dissolved in dichloromethane (1 L) and cooled to 5 C in a salt ice bath. Met hacryloyl chloride (46 mL, 0.47 mol ) was added drop wise via addition funnel. The reaction was stirred for 30 min at 0 C then slowly warmed to room temperature and left to stir overnight. The reaction was filtered to remove 1 amino 2 propanol hydrochlor ide, and the filtrate was concentrated to 500 mL and placed in a 20 C freezer overnight to crystallize the product The resultant HPMA was isolated by filtration and recrystallized from acetone at 20 C (5 2 g, 76% yield ). 1 H NMR (300 MHz, D 2 O, ppm ) : 5 .72 (1H, s, C H 2 =C), 5.47 (1H, t, C H 2 =C), 3.96 (1H, m, CH 2 C H (CH 3 )OH), 3.3 0 (2H, m, C H 2 CH(CH 3 )OH), 1.95 (3H,

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69 s, CH 2 CH(C H 3 )OH), 1. 1 9 (3H, d, CH 2 =CC H 3 ). 13 C NMR (500 MHz, D 2 O, ppm) : 172.05 139. 01, 120.98, 66.20, 46.19, 19.38, 17.63 HPMA Methotrexate (HPMA MTX) A 10 mL round bottom flask with magnetic stir bar was flame dried and cooled to room temperature under N 2 flow. Metho t rexate (202 mg, 0.444 mmol ) was dissolved in anhydrou s DMF (3 mL). DCC (110 mg, 0.54 mmol) was added with stirring, followed b y DMAP (10.9 mg, 0.0892 mmol ), and HPMA (317 mg, 2.22 mmol). The reaction was allowed to stir for 48 h at room temperature, at which time the white dicylohexylurea precipitate was removed by filtration. The resulting clear solution was precipitated into et her and the solids were isolated by filtration. The crude product was purified by preparative reverse phase HPLC. The yellow fractions were collected, CH 3 CN was removed by rotary evaporation, and the solution was lyophilized to isolate a yellow powder (73 mg, 23 % yield). 1 H NMR (500 MHz, DMSO d 6 ppm) : 9.28 (1H, s), 9.08 (1H, s), 8.72 (1H, s), 8.25 (1H, d), 8.01 (1H, t), 7.75 (2H, d), 6.82 (2H, d), 5.60 (1H, s), 5.29 (1H, t), 4.90 (3H, m), 4.36 (1H, m), 3.26 (3H, m), 3.14 (1H, m), 2.36 (2H, m), 2.08 (1H, m), 1.92 (1H, m), 3.81 (3H, s), 1.10 (3H, d). ESI MS m/z ; 602.2466 [M + Na ] + calculated for C 27 H 33 N 9 O 6 602.2467. Analytical HPLC R t = 4.3 min (96%) Poly(HPMA) (PHPMA) (P1) HPMA (3.00 g, 20.9 mmol), CDTPA (126 mg, 0.312 mmol), and AIBN (5 mg, 0.03 mmol ) were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar ([M]:[CTA]:[I] = 67:1:0.1) The reagents were dissolved in DMAc (5 mL), purged with N 2 for 30 min, and added to a preheated heating block at 70 C. The reactio n was monitored by GPC MALS, quenched after 3.5 h by exposing the contents to oxygen, and the polymer was purified by precipitation into cold

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70 Figure 3 10. 1 H NMR spectrum and peak assignments for HPMA MTX. (DMSO d 6 500 MHz). Figure 3 11. ESI MS spectrum of HPMA MTX.

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71 Figure 3 12. Analytical HPLC chromatogram of HPMA MTX. R t = 4.4 min, 96%. diethyl ether ( 3) and vacuum dried to yield a light yellow powder (43% conversion, M n, GPC = 6,260 g/mol, M w / M n = 1.05). Synthesis of P2 HPMA (15. 0 g, 105 mmol ), CDTPA ( 387 mg, 0.960 mmol), and AIBN (16.8 mg, 0. 102 mmo l) were added to a 5 0 mL Schlenk tube with a glass stopper and magnetic stir bar ([M]:[CTA]:[I] = 110:1:0.1) The re agents were dissolved in DMAc (30 mL), degassed with three freeze pump thaw c ycles, backfilled with N 2 and added to a preheated oil bath at 70 C. The reaction was monitored by GPC MALS, quenched after 4 h by exposing the contents to oxygen, and the polymer was purified by precipit ation into cold diethyl ether ( 3) and vacuum dri ed to yield a light yellow powder ( 4.5 g, M n, GPC = 9,470 g/mol, M w / M n = 1.05). Synthesis of P3 HPMA (2.20 g, 15.4 mmol ), CDTPA ( 26.8 mg, 0.0664 mmol), and AIBN (1 mg, 6 10 3 mmo l) were added to a 20 mL scintillation vial equipped with a

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72 septum cap and magnetic stir bar ([M]:[CTA]:[I] = 230:1:0.1) The reagents were dissolved in DMAc ( 3 mL), purged with N 2 for 30 min, and added to a preheated heating block at 70 C. The reaction was monitored by GPC MALS, quenched after 4 h by exposing the contents to ox ygen, and the polymer was purified by precipitation into cold diethyl ether ( 3) and vacuum dried to yield a light yellow powder ( 1.24 g, 51 % conversion, M n, GPC = 17,300 g/mol, M w / M n = 1.24 ). 3.4.4 Synthesis of PHPMA CCS Polymers 3.4.4.1 Investigation o f the effect of [crosslinker]:[unimer] ratio on star formation using EGDMA and a constant unimer MW P1 was used as the unimer while varying the [crosslinke r]:[unimer] ratio (15 :1 ; 10:1; and 5:1). The crude GPC chromatograms were deconvoluted using a Gaussian function in MagicPlot Pro software and the star yield was calculated using Equation 3 1. The arm number, f was calculated using Equation 3 2 and Equation 3 3. Synthesis of CCS with [EGDMA]:[unimer] = 15: 1 P1 (100 mg, 0.016 0 mmol), EGDMA (47.7 mg, 0.241 m mol), and AIBN (0.3 mg, 2 10 3 mmol ) were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar. The reagents were dissolved in DMAc (1 mL, [ P1 ] = 100 mg/mL), purged with N 2 for 30 min, and added to a preheated h eating block at 70 C. The reaction was quenched after 24 h by exposing the contents to oxygen and analyzed by GPC MALS (star yield = 70%, crude M w = 1250 kg/mol, f = 100). Synthesis of CCS with [EGDMA]:[unimer] = 10:1 P1 (100 mg, 0.016 0 mmol ) EGDMA ( 31.7 mg, 0. 160 m mol), and AIBN (0.3 mg, 2 10 3 mmol) were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar. The reagents were dissolved in DMAc (1 mL, [ P1 ] = 100 mg/mL), purged with N 2 for 30 min, and

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73 added to a prehea ted heating block at 70 C. The reaction was quenched after 24 h by exposing the contents to oxygen and analyzed by GPC MALS (star yield = 60%, crude M w = 256 kg/mol, f = 20). Synthesis of CCS with [EGDMA]:[unimer] = 5:1 P1 (100 mg, 0.016 0 mmol), EGDMA ( 15.5 mg, 0. 0783 m mol), and AIBN (0.3 mg, 2 10 3 mmol) were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar. The reagents were dissolved in DMAc (1 mL, [ P1 ] = 100 mg/mL), purged with N 2 for 30 min, and added to a preheated heating block at 70 C. The reaction was quenched after 24 h by exposing the contents to oxygen and analyzed by GPC MALS (star yield = 30%, crude M w = 73.3 kg/mol, f = 10). 3.4.4.2 Investigation o f the effect of unimer M n on CCS formation The M n of the PHPMA macroCTA was altered using P1 P2 or P3 while holding the [crosslinker]:[unimer] ratio constant at 10:1. Synthesis of CCS with P1 The star formed using P1 and a [crosslinker]:[unimer] ratio of 10:1 was described above, and that sample was used here in the comparison of varying unimer M n Synthesis of CCS with P2 (CCS1 ) P2 (100. mg, 0.0106 mmol), EGDMA (21.2 mg, 0.106 mmol ), and AIBN (0.2 mg, 1 10 3 mmol ) were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar. The reagents were dissolved in DMAc (1 mL, [ P2 ] = 100 mg/mL), purged with N 2 for 30 min, and added to a preheated heating block at 70 C. The reaction was quenched after 24 h by exposing the contents to oxygen and analyzed by GPC MALS (star yield = 50%, crude M w = 211 kg/mol f = 10).

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74 Synthesis of CCS with P3 P3 (100. mg, 5.78 10 3 mmol), EGDMA (11.8 mg, 5.95 10 2 mmol), and AIBN (0.1 mg, 6 10 4 mmol) were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar. The reagents were dissolved in DMAc (1 mL, [ P 3 ] = 100 mg/mL), purged with N 2 for 30 min, and added to a preheated heating block at 70 C. The reaction was que nched after 24 h by exposing the contents to oxygen and analyzed by GPC (star yield = 10%, crude M w = 287 kg/mol f = 10). 3.4.4.3 Synthesis of PHPMA CCS using heterogeneous polymerizations Synthesis of CCS2 P2 (100. mg, 0.0106 mmol), EGDMA (20.5 mg, 0.10 6 mmol), and A CVA (0.3 mg, 1 10 3 mmol) were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar. The reagents were dissolved in EtOH/water (1/1 v/v) (1 mL [ P2 ] = 100 mg/mL), purged with N 2 for 30 min, and added to a pre heated heating block at 70 C. T he reaction was quenched after 4 h by exposing the contents to oxygen and analyzed by GPC MALS (star yield = 70%, crude M w = 553 kg/mol f = 40). Synthesis of CCS3 P2 (100. mg, 0.0106 mmol), EGDMA (20.4 mg, 0.103 mmol), and A CVA (0.3 mg, 1 10 3 mmol) were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar. The reagents were dissolved in water (1 mL [ P2 ] = 100 mg/mL), purged with N 2 for 30 min, and added to a preheated heating b lock at 70 C. T he reaction was quenched after 4 h by exposing the contents to oxygen and analyzed by GPC (star yield = 70%, crude M w = 1280 kg/mol f = 100). Synthesis of CCS4 P2 (100. mg, 0.0106 mmol), EGDMA (20.8 mg, 0.105 mmol), HPMA MTX (61.4 mg, 0. 106 mmol), and A CVA (0.3 mg, 1 10 3 mmol) were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar.

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75 The reagents were dissolved in EtOH/water (1/1 v/v) (1 mL [ P2 ] = 100 mg/mL), purged with N 2 for 30 min, and added to a preheated heating block at 70 C. T he reaction was quenched after 3 h by exposing the contents to oxygen and analyzed by GPC (55% conversion HPMA MTX, star yield = 60%, M w = 151 kg/mol f = 10). Synthesis of CCS5 P2 (25.4 mg, 0.00268 mmol), DSDMA (8.1 mg, 0.028 mmol), HPMA MTX (15.3 mg, 0.0264 mmol), and A CVA (0.074 mg, 2.6 10 4 mmol) were added to a NMR tube equipped with a septum cap and magnetic stir bar. The reagents were dissolved in [ P2 ] = 100 mg/mL), purged with N 2 fo r 15 min, and added to a preheated oil bath at 70 C. T he reaction was quenched after 8 h by exposing the contents to oxygen and the products were analyzed by GPC (star yield = 70%, M w = 728 kg/mol f = 30). 3.4.4.4 Purification of CCS p olymers Fractional precipitation CCS1 was purified using fractional precipitation to remove unimers and low molecular weight star polymers. The crude reaction mixture (0.5 mL) was transferred to a 1.5 mL microcentrifuge tube. Cold diethyl ether was added drop wis e with intermittent mixing until a turbid solution persisted. The tube was then centrifuged 5 min at 4,000 rpm to collect precipitated stars. The ether was removed, and the pellet was dissolved in DMAc (0.5 mL). The fractional precipitation was repeated tw ice more, and the purification was confirmed by GPC MALS Ultrafiltration. The PHPMA CCS polymers synthesized in aqueous media were purified via ultrafiltration using centri fugal filter units with a 50 kDa MWCO membrane The crude reaction was washed with water (5 0.5 mL ), and the purified stars were

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76 dried by lyophilization to yield a faint yellow powder for CCS2 and CCS3 and a bright yellow orange powder for CCS4 3.4.5 In vitro drug release experiments Drug cleavage from monomer HPMA MTX (1.3 mg, 2.7 10 3 mmol) was MTX stock solution (60 n the absence of PLE. The reactions were left at room with 3:1 aq. TFA (0.1%):CH 3 determined by integrating the area of the peak at R t = 2.1 min and using a standard curve of MTX to determine the concentration. Drug cleavage from CCS4 CCS4 (1.3 mg) was added to a microcentrifuge tub experiment was used in which CCS4 (1.3 mg) was added to a microcentrifuge tube and temperature, and with 3:1 aq. TFA (0.1%):CH 3 temperature for 24 h. However, no drug release was observed after 24 h. Drug cleavage from CCS5 CCS5 (5.4 mg) was added to a 4 mL vial and dissolved in DMF (0.5 mL), and the solution was purged with N 2 for 10 min. Tri n butylphosphine (0.1 mL, 0.4 mmol) was then added, and th e reaction was left to stir at

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77 room temperature overnight. The reaction was dialyzed against water using a 3,500 Da MWCO membrane, and the polymer was isolated by lyophiliz ation. The recovered material (1.2 mg) was dissolve 150 U/mg) was added. The reaction was left at room temperature and monitored by HPLC by removing 3 CN). No apparent drug release was observed after 24 h. Figure 3 13. Calibr ation curve of MTX using the area under the curve at R t = 2.1 min in the HPLC chromatogram.

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78 CHAPTER 4 ROLE OF PO L YMER ARCHITECTURE ON THE ACTIVITY OF POLYMER PROTEIN CONJUGATES FOR THE TREATMENT OF ACCELERATED BONE LOSS DISORDERS 4 .1 Int r oduction The conjugation of synthetic polymers to proteins has provided a viable route to alter the solubility, activity, and blood circulation times of proteins. 77,78,83,90,112,174 176 In proven effective to improve the efficacy of a number of clinically approved therapeutic protein s. 72,107,114,177,178 This increase in therapeutic effectiveness is most often attributed to an in creased hydrodynamic diameter of the protein after bioconjugation, thereby decreasing renal filtration and prolonging blood circulation time. 107 Recent advances in reversible deactivation radical polymerization (RDRP) methods have provided a useful toolbox to control the molecular weights, molecular weight distributions, and architectures of polymers used in bioconjugations. Atom transfer radical polymerization (ATRP), 90,94 nitroxide mediated polymerization (NMP), 89,179,180 and reversible addition fragmentation chain transfer (RAFT) polymerization 78,79,99,100,103,104,119 have all been successfully utilized to immobilize well defined polymers to a variety of proteins. Traditionally, polymer protein bioconjugates a method, which uses a reactive end group on the polymer to react with a specific functional group on the protein, most often primary amines or thiols found in lysine or cysteine residues, respectively. 75,78,180 182 This method provides the ability to completely *Reprinted with permission from Biomacromolecules 2015 16 2374 2381. Copyright 2015 American Chemical Society.

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79 characterize pre formed polymers prior to reaction with a protein. The well defined polymer s derived from RDRP techniques ha ve allowed en d group control, either by post polymerization modifications or through the use of a functionalized initiator, leading to well defined bioconjugates with control over the site specificity and multiplicity of the polymers. 75,78,117,181 184 The versatility of RDRP techniques has also been further strategy, which uses a protein as a macro initiator or macro chain transfer agent to grow polymers directly from the protein. 83,84,90,94,182,185 The grafting from method provides some advantages compared to the grafting to method, such as purification (e.g separation of unreacted monomers versus separation of unreacted polymers) and control over the number of polymers per protein. There have been a number of studies that use polymer peptide bioconjugates to treat degenerative bone diseases and elicit bone growth. 186 188 In the present research, we investigated the effect of conjugating a variety of polymer architectures to osteoprotegerin (OPG), a therapeutically viable protein used in the treatment of osteoporosis and other degenerative bone diseases caused by increased osteocl astic bone resorption. 189 192 OPG is a naturally occurring soluble decoy receptor involved in the regulation of bone resorption by binding to the receptor activator of nuclear factor kappa B (RANK) ligand (L), preventing RANKL from binding to its target receptor, RANK on the surfa ce of premature osteoclasts. 193 196 By inhibiting the RANK/RAN KL interaction, OPG prevents osteoclast differentiation and activation, which reduces bone resorption. 197 202 Treatment of degenerative bone disorders with OPG has been promising, and a recent study indicates that OPG may also be useful in the treatment of

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80 muscular dystrophy. 203 However, OPG has a very short in vivo half life, and multiple doses are required to achieve a therapeutic benefit. Previous studies have shown that conjugating OPG with PEG can impro ve the blood circulation time of OPG (e.g., the pharmacokinetics), but binding with RANKL (e.g., the pharmacodynamics) was reduced. 204,205 Further studies suggested that changing the architecture of the polymer from linear to branched could provide the increased pharmacokinetics without complete elimination of the pharmacodynamics of the protein. 206 We sought to further investigate the effect of polymer branching density on the activit y of a serie s of OPG polymer bioconjugates. Three polymers of varying branching density were synthesized, including linear PEG, loosely branched PEG, and densely branched PEG, while holding the molecular weight of each architecture relatively constant. Eac h of the unique architectures was then conjugated to OPG via a grafting to approach using the activated ester, N hydroxysuccinimide (NHS), in the polymer end group. Control over the multiplicity of polymers per protein was elicited by adjusting the pH of t he solution to preferentially target the N terminus of the protein. 78 Using a grafting to method allowed complete polymer charac terization and end group modifications prior to coupling with a valuable therapeutic protein. The OPG PEG bioconjugates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS PAGE ) to ensu re successful conjugation, and t he retentio n of protein activity was demonstrated using an in vitro osteoclast inhibition assay, which showed that each bioconjugate retained high activity against osteoclast formation. Finally, preliminary in vivo studies using peripheral quantitative computed tomog raphy

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81 indicated improved bone mineral density of the loosely branched bioconjugate relative to other OPG polymer architectures. 4 .2 Results and Discussion 4.2.1 Polymer Synthesis Linear PEG, P1 was synthesized by modifying the end group of commercially available monomethoxy poly ( ethylene glycol ) (mPEG, M n = 5,000 g/mol) to contain an NHS activated ester (Scheme 1), rendering the linear polymer reactive toward amine residues in the protein. Scheme 4 1. Ring opening of succinic anhydride with mPEG and f ormation of an NHS ester to give P1 The densely branched PEG architecture, P2 (PolyPEGMA 11 ) was synthesized using reversible addition fragmentation chain transfer (RAFT) polymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) (Scheme 4 2, Figure 4 1 ). Because we aimed to study the effect of varying branching density on conjugate activity, the MW of the polymers was held constant, targeting an M n of 5,000 g/mol. Reports have suggested in vivo cytotoxicity of dithiobenzoate end groups; 207 therefore, the RAFT group was removed by aminolysis. The resultant thiol, a useful handle for further functionalization, 208 was exploited using M ichael addition to incorporate fluorescein as a convenient marker for in vitro and in vivo monitoring. The end group removal and fluores cein con jugations were confirmed by UV v is spectroscopy (Figure 4 2 ), and the end group was then converted to an NHS ester for bioconjugations.

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82 Scheme 4 2. (a) RAFT polymerization of PEGMA to afford a densely branched polymer, polyPEGM A 11 followed by aminolysis to remove the RAFT group. (b) Michael addition using acryloyl(fluorescein). (c) Formation of NHS activated ester to give P2 Figure 4 1. GPC chromatogram of P2

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83 Figure 4 2. UV vis spectra of P2 before RAFT group removal, P2 SH after RAFT group removal, and P2 fluorescein after Michael addition with acryloyl(fluorescein). T he loosely branched PEG architecture, P3 (Poly(HPMA 9 co PEGMA 6 ) was prepared by copolymerizing PEGMA with N (2 hydroxypropyl)methacrylamide (HPMA) via R AFT polymerization (Scheme 4 3, Figure 4 3 ). We first studied the copolymerization kinetics of PEGMA and HPMA by determining the monomer reactivity ratios. The Finemann Ross method was used to calculate r 1 (monomer reactivity ratio for PEGMA) = 0.98 and r 2 (monomer reactivity ratio for HPMA) = 0.52 ( Appendix 1 ). These values indicate the copolymerization of the monomers is not random, with PEGMA being preferentially consumed during the reaction. Therefore, we altered the monomer feed ratio to target approxi mately 50% of the number of PEGMA repeat units as that of the densely branched polymer described above. While the polymer likely contains a gradient micro structure rather than a random copolymer, we believe that the polymer contains the lower branching den sity needed to study the effect of branching density on protein activity. The RAFT group was removed by aminolysis, flu orescein was conjugated through Michael addition (Figure 4 4 ) and the carboxylic acid end group was converted to an NHS ester.

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84 Scheme 4 3 (a) RAFT copolymerization of PEGMA and HPMA. (b) RAFT group removal and Michael addition with acryloyl(fluorescein). (c) Formation of NHS ester to afford P3 Figure 4 3. GPC chromatogram of P3 after trithiocarbonate removal.

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85 Figure 4 4. UV vis spectra of P3 before RAFT group removal, P3 SH after RAFT group removal, and P3 fluorescein after Michael addition with acryloyl(fluorescein). 4.2.2 Protein Conjugations OPG was modified with each of the polymer architectures using a gra fting to approach, in which the pH value of the reaction was controlled to increase the probability of selectively deprotonating the amine terminus of the protein, while the primary amines of lysine residues were expected to be protonated and therefore ren dered less nucleophilic (Scheme 4 4 ). 209,210 Since coupling reactions of macromolecular reagents can be less efficient than those of small molecules; five molar equivalents of polymer were used for each bioconjugation. A control experiment was also performed, in which pure OPG in the absence of poly mer was subjected to the same reaction and purification conditions employed during the bioconjugation reaction and work up. The purified OPG PEG conjugates ( OPG1 OPG2 and OPG3 for the linear PEG, densely branched PEG, and loosely branched PEG architectur es, respectively) were analyzed by SDS PAGE which demonstrate d successful polymer conjugation to OPG in addition to some remaining unmodified protein (Figure 4 5 ).

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86 Scheme 4 4. OPG bioconjugation reactions for OPG1 from P1 OPG2 from P2 and OPG3 from P 3 Structure from the PDB (3URF). Figure 4 5. SDS PAGE analysis of OPG bioconjugates. Lane: (1) molecular weight marker, (2) unmodified OPG, (3) OPG1 (4) OPG3 and (5) OPG2 4.2 .3 In Vitro Osteoclast Inhibition A ssay The in vitro activity of each bioconjugate was measured using an osteoclast inhibition assay with 1,25 dihydroxyvitamin D 3 stimulated mouse marrow cells and bioconjugate concentrations of 2, 20, and 200 ng/mL administered on days 1 and 4. Cells were fixed at day 6 and stained for tartr ate resistance phosphatase (TRAcP), a marker for osteoclast activity. 211 Cells were divided into mononuclear, multinuclear (2 10 nuclei), and giant (>10 nuclei), with each cell population representing increasingly

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87 mature osteoclast cells. The osteocla st levels were counted, and the results indicated that while 2 ng/mL was too dilute to inhibit osteoclast growth for the unmodified OPG or OPG1 and OPG2 the osteoclast count was reduced for OPG3 Furthermore, a concentration of 20 ng/mL resulted in decrea sed osteoclast counts for all samples receiving OPG relative to the control (no OPG). At a concentration of 200 ng/mL, osteoclasts were completely inhibited for all of the conjugates (Figure 4 6 ). Importantly, each bioconjugate of OPG retained anti osteocl ast activity at concentrations of 20 and 200 ng/mL. To provide evidence that the decrease in osteoclast activity is due to the inhibition of osteoclast maturation by the OPG RANKL interaction and not due to general cytotoxicity of the bioconjugates, a cell cytotoxicity study was performed using cells that serve as a model for macrophages (Figure 4 7 ). The cells were shown to have near quantitative viability at 2 and 4 days at the concentrations used in the osteoclast inhibition assay described above, sugges ting that each bioconjugate was nontoxic and supporting the observation that the decrease in osteoclast maturation was indeed a result of OPG RANKL binding. Conjugation of even a single polymer can greatly reduce the activity of many proteins. 86,112,212 214 Thus, these results were promising, because the OPG bioconjugates retained their ability to prevent osteoclast formation, suggesting they may also decrease bone resorption in vivo. Furt hermore, the loosely branched bioconjugate, OPG3 actually had higher in vitro activity than either OPG1 or OPG2 as indicated in a lower osteoclast count (Figure 4 6 ).

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88 Figure 4 6. Osteoclast inhibition assay using 1,25 dihydroxyvitamin D 3 stimulated mo use marrow cells. The control is the osteoclast count of untreated cells. Background shading indicates different OPG samples with doses on days 1 and 4, each with three concentrations of 2, 20, and 200 ng/mL. Three cell types were counte d mononuclear, mu ltinuclear, and gian t indicated by the different bar colors blue, orange, and green, respectively. Figure 4 7. Cell cytotoxicity assay using RAW 264.7 cells shown as the percentage of cell survival relative to a control, which was untreated cells. Each bioconjugate, indicated by background shading, was administered at concentrations of 20 and 200 ng/mL, and live cells were counted at 2 d (blue) and fixed with formaldehyde and counted at 4 d (orange). Each sample is the average of four replicates.

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89 4 .2 .4 In Vivo Skeletal Effect S tudy We aimed to show that the effects of osteoclast inhibition observed in vitro could also be translated to in vivo effectiveness. Since OPG ultimately prevents differentiation and activation of osteoclasts, we reasoned that we could monitor the efficacy of our bioconjugates in vivo by monitoring the bone mass and bone volume in rats. A facile and reliable way to determine bone mass is by determining bone mineral density (BMD), a surrogate of bone mass, using peripheral quant itative computed tomography (pQCT). We tested the antiresorptive activity of each bioconjugate in vivo by measuring trabecular (trab) BMD, trabecular bone mineral content (trab BMC), and trabecular bone area (trab BA) at the distal femoral metaphyses of Sp rague Dawley rats at different distances from the growth plate. For this purpose, 20 cannulated rats were divided into five groups. One group was sacrificed at the beginning of the experiment and received no treatment to serve as a baseline control. The ot her four groups received single bolus intravenous injections of unmodified OPG, OPG1 OPG2 or OPG3 and were euthanized seven days post treatment. Femurs were collected postmortem and BMD was analyzed by pQCT at distances of 2, 4, and 6 mm proximal to the distal epiphyseal growth plate (Figure 4 8 ). The pQCT analyses showed increased femur trab BMD, trab BMC, and trab BA at the distal metaphysis of rats treated with OPG3 compared to rats from the baseline, unmodified OPG, OPG1 and OPG2 groups (Figure 4 9 Figure 4 10 ). While the linear and densely bra nched protein bioconjugates showed no increase in BMD from either unmodified OPG or the baseline control, we believe that the loosely branched bioconjugate has enhanced antiresorptive activity, consistent wit h the in vitro data

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90 Figure 4 8. Bone mineral density (BMD) of OPG bioconjugates at distances of 2 (blue), 4 (orange), and 6 (green) mm from the growth plate in the distal femur of 7 days post treated rats. Figure 4 9. (a) Bone mineral density, (b) bo ne mineral content, and (c) bone mineral area of OPG bioconjugates as the average from 2 6 mm from the growth plate. Statistical analysis was accomplished with one way analysis of variance (ANOVA) followed by post ANOVA: multiple comparison Tukey Test or nonparametric Kruskal Wallis test; a = different from baseline, b = different from unmodif ied OPG, and c = different from OPG1

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91 described above. Furthermore, since even unmodified OPG had no significant bone effect as compared to the control, it is possible that higher concentrations or multiple doses of the bioconjugate would further enhance the bone growth profile. When combined with the nontoxic characteristics shown in the in vitro studies, these in vivo results indicate promise for these materials as an effective therapy in bone degenerative disorders. Further pharmacokinetic studies are n ecessary to show the enhanced blood circulation half life of the bioconjugates, but the increase in BMD is an exciting preliminary result that gives hope to the translation from in vitro to in vivo efficacy. Figure 4 10. pQCT images of unmodified OPG an d OPG3 in 7 days post treated rats at a distance of 4 mm from the growth plate. Increasing BMD is shown with more red to yellow shading. 4.3 Summary I n summary, three polymers with linear, loosely branched, and densely branched architectures were conjugat ed to OPG using a grafting to strategy. Control over the polymer branching density was elicited using RAFT copolymerization of a PEGMA

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92 macromonomer with HPMA, a water soluble monomer that leads to vinyl polymers with good biocompatibility. 158 End group control allowed further fun ctionalization to render the polymers reactive toward primary amines on proteins. Importantly, each bioconjugate was shown to be nontoxic and retained high activity toward the inhibition of osteoclasts. Preliminary in vivo studies further supported the non toxic character of the OPG bioconjugates, and initial results suggested an increase in the bone mineral density of the loosely branched OPG bioconjugate. A more robust pharmacokinetics study is needed to unequivocally show the therapeutic benefit of OPG po lymer bioconjugates, but this report demonstrates the feasibility of using such a system to treat bone degenerative diseases. 4.4 Experimental 4.4.1 Materials 1 Amino 2 propanol (Alfa Aesar, 94%), methacryloyl chloride (Alfa Aesar, 97%), succinic anhydride (TCI America, >95%), triethylamine (TEA, Alfa Aesar, 99%), hydrazine (Alfa Aesar, 98+%), tris(2 carboxyethyl)phosphine HCl (TCEP, Alfa Aesar, 98%), methoxypoly ( ethylene glycol ) (mPEG, Fluka, M n = 5,000 g/mol), 1 ethyl 3 (3 dimethylaminopropyl)car bodiimide HCl (EDC HCl, Sigma Aldrich, 98%), N hydroxysuccinimide (NHS, Sigma Aldrich, 99%), trioxane (Acros Organics, 99.5%), dichloromethane (BDH, 99.5%), diethyl ether (Fisher Chemicals), 1,4 dioxane (Fisher Chemicals, 99%), N,N dimethylformamide (DMF, EMD, 99.8%), N,N dimethylacetamide (DMAc, Sigma Aldrich, 99%), deuterium oxide (D 2 O, Cambridge Isotope, 99.9%), and chloroform d (CDCl 3 Cambridge Isotope, 99.8%) were used as received. 2,2 Azobisisobutronitrile (AIBN, Sigma Aldrich, 98%) was recrystalli zed from ethanol. Poly ( ethylene glycol ) methyl ether methacrylate (PEGMA, Sigma Aldrich, M n = 500

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93 g/mol) was passed through a column of basic alumina prior to use. 4 (Cyanopentanoic acid) dithiobenzoate (CDB) and 4 cyano 4 [(dodecylsulfanyl thiocarbonyl)sulf anyl]pentanoic acid (CDTPA) were synthesized according to a previously published procedure. 2 4.4.2 Instrumentation 1 H NMR spectra were recorded on a Varian Innova2 500 MHz or a Varian Mercury 300 MHz NMR spectrometer using the residual solvent signal as a reference. Molecular weight and molecular weight distributions were determined by gel perme ation chromatography (G PC) in dimethylacetamide (DMAc) with 50 mM LiCl at 50 C and a flow rate of 1.0 mL/ min (Agilent isocratic pump, degasser, and autosampler, columns: (i) series G3078 mixed bed columns: molecular weigh t 10 3 10 4 g/mol Mixed D columns: molecular weight range 200 400,000 g/ mol). Detection consisted of (i) Wyatt Optilab T rEX refractive index detector operating at 658 nm and a Wyatt miniDAWN Treos light scattering detector operating at 659 nm or (ii) Wyatt OptilabDSP interferometric refractometer operating at 690 nm and a Wyatt DAWN EOS light scattering detector operating at 685 nm. Absolute molecular weights and molecular weight dist ributions were calculated using the Wyatt ASTRA software. UV Vis measurements were obtained using a Varian Carey 500 Scan UV Vis NIR spectrophotometer. 4.4.3 Synthesis N (2 hydroxypropyl)methacrylamide (HPMA) To a three neck, 2 L round bottom flask equipp ed with a mechanical stirring device, thermometer, and addition funnel was added 1 amino 2 propanol (75 mL, 0.96 mol). The reagent was dissolved in

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94 dichloromethane (1 L) and cooled to 5 C in a salt ice bath. Methacryloyl chloride (46 mL 0.47 mol) was th en added drop wise via addition funnel. The reaction was stirred for 30 min at 0 C then slowly warmed to room temperature and left to stir overnight. The reaction was filtered to remove 1 amino 2 propanol hydrochloride, and the filtrate was concentrated to 500 mL and placed in a 20 C freezer overnight to crystallize the product. The resultant HPMA was isolated by filtration and recrystallized from acetone at 20 C (52 g, 76%). 1 H NMR (300 MHz, D 2 O) : 5.72 (1H, s, C H 2 =C), 5.47 (1H, t, C H 2 =C), 3.96 (1H, m CH 2 C H (CH 3 )OH), 3.30 (2H, m, C H 2 CH(CH 3 )OH), 1.95 (3H, s, CH 2 =CC H 3 ), 1.18 (3H, d, CH 2 CH(C H 3 )OH). mPEG succinic acid (mPEG COOH) To a 40 mL scintillation vial with septum cap and stir bar were added mPEG (1.93 g, 0.386 mmol), succinic anhydride (201 mg, 2.01 mmol), and TEA (218 mg, 2.13 mmol). The reagents were dissolved in dry dichloromethane (10 mL) and allowed to stir at room tem perature. The reaction was monitored by 1 H NMR spectroscopy to observe the appearance of methylene protons adjacent to the forming ester at 4.2 ppm. The reaction was quenched at 4 h and the polymer was purified by prec ipitation into diethyl ether (3 200 mL) (Figure 4 11) Figure 4 11. 1 H NMR spectrum of mPEG COOH (CDCl 3 500 MHz).

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95 Figure 4 12. 1 H NMR spectrum of mPEG NHS (CDCl 3 500 MHz). mPEG NHS ester (P1) To a 20 mL scintillation vial with septum cap and stir bar were added mPEG COOH (500 mg, 0.1 mmol) and NHS (13 mg, 0.12 mmol). The reagents were dissolved in dry dichloromethane (5 mL) and purged with N 2 EDC HCl (24 mg, 0.13 mmol) was dissolved in dichloromethane (2 mL) and added to the reaction with stirring. The reaction was allowed to stir at room temperature overnight, followed by precipitation into diethyl ether (500 mL). The polymer was filtered and dried under vacuum. The activated ester polymer was used without further purification (Figure 4 12) PolyPEGMA 11 PEGMA (7.86 g, 15.7 mmol), CDB (223 mg, 0.798 mmol) and AIBN (25.6 mg, 0.156 mmol) were added to a 25 mL Schlenk flask with magnetic stir bar. Trioxane (142 mg) was added as an internal reference, and the reagents were dissolved in 1,4 dioxane (9 mL). The flask was sealed with a ru bber septum, and the bright red solution was degassed via three freeze pump thaw cycles. The mixture was placed in an oil bath at 70 C with stirring, and the reaction was monitored by GPC and quenched at 2 h. The polymer was purified by dialysis against w ater using a 3,500 Da MWCO dialysis membrane, and a dark red oil was isolated after lyophilization. The

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96 sample was then dissolved in DMF, and hydrazine was used to remov e the dithiobenzoate end group ( M n, GPC = 6,040 g/mol, M w / M n = 1.08). Fluorescein conjugation to PolyPEGMA 11 P oly PEGMA 11 (163 mg, 2.69 10 2 mmol) was dissolved in DMF (3 mL) in a 20 mL scintillation vial with septum cap and purged with N 2 TCEP (9.5 mg, 3.3 10 2 mmol) dissolved in DMF/H 2 O (9/1) was added to the reaction, followed b y the addition of TEA (4.9 mg, 3.5 10 2 mmol) and acryloyl(fluorescein) (0.12 mg, 3.1 10 4 mmol). The reaction was left to stir at room temperature for 16 h, and 1 (2 hydroxyethyl) 1H pyrrole 2,5 dione (4.9 mg, 3.5 10 2 mmol) was added as a solution in DMF. The reaction was allowed to stir for 2 h and then dialyzed against water using a 3,500 Da MWCO dialysis membrane. The product was isolated by lyophilization (158 mg). NHS activa tion of PPEGMA fluorescein (P2) P oly PEGMA 11 fluorescein (147 mg, 2.43 10 2 mmol) and NHS (4.1 mg, 3.6 10 2 mmol) were dissolved in dry dichloromethane (2 mL). The reaction was purged with N 2 and EDC HCl (5.5 mg, 2.9 10 2 mmol) was added as a solution in dichloromethane (0.5 mL). The reaction was left to stir overnight at room temperature. Dichloromethane was removed in vacuo and the polymer was used without further purification. Copolymerizations of PEGMA and HPMA RAFT copolymerizations were performed using CDTPA and AIBN as the CTA and initiator, respectively with [CTA]:[I] = 5:1. The monomer feed ratios were varied according to Table S1, and the total or, and trioxane were added to a 20 mL scintillation vial equipped with a magnetic stir bar and septum cap, dissolved in DMAc (3 M), purged with N 2 for 30 min, and added to a

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97 preheated reaction block at 60 C The monomer conversions were determined from 1 H NMR spectroscopy by integrating the vinyl protons from each monomer relative to the trioxane standard. The reactivity ratios were calculated graphically using the Finemann Ross method, where r 1 is given by the slope and r 2 is given by the negative interc ept of a plot of G vs. H ( Appendix ). Poly(HPMA 9 co PEGMA 6 ) HPMA (1.00 g, 6.98 mmol), PEGMA (1.18 g, 2.36 mmol), CDTPA (92.4 mg, 0.229 mmol), AIBN (8.2 mg, 0.050 mmol), and trioxane (100 mg) as an internal standard were added to a 20 mL scintillation vial equipped with a septum cap and magnetic stir bar. The reagents were dissolved in DMAc (3 mL), purged with N 2 for 30 min, and added to a preheated heating block at 60 C. The reaction was quenched after 4 h by exposing the contents to oxygen, and the polymer was purified by dialysis against water using a 3,500 MWCO membrane and lyophilized to yield an amorphous s olid ( M n, GPC = 7,020 g/mol, M w / M n = 1.07). Poly(HPMA 9 co PEGMA 6 ) fluorescein P oly (HPMA 9 co PEGMA 6 ) (150 mg, 2.0 10 2 mmol) was dissolved in DMF (3 mL) with stirring in a 20 mL scintillation vial with septum cap. Hydrazine (0.05 mL, 2 mmol) was added, a nd the reaction turned colorless immediately. After 30 min the mixture was placed in a 3,500 Da MWCO dialysis membrane and dialyzed against water followed by lyophilization to isolate the polymer. The end group removed P(HPMA 9 co PEGMA 6 ) (118 mg, 1.68 10 2 mmol) was dissolved in DMF (3 mL) in a 20 mL scintillation vial with septum cap and stir bar and purged with N 2 for 30 min. TCEP (5.6 mg, 2.0 10 2 mmol) was then added as a solution in water (0.5 mL), followed by TEA (2.64 mg, 2.61 10 2 mmol). Acryloyl(fluorescein) (0.1 mg, 2 10 4 mmol) was added, and the reaction was allowed

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98 to stir at r oom temperature for 20 h. 1 (2 H ydroxyethyl) 1H pyrrole 2,5 dione (3.2 mg, 2.3 10 2 mmol) in DMF (0.5 mL) was then added and stirred 2 h, and the mi xture was dialyzed against water using a 3,500 Da MWCO dialysis membrane. The product was isolated by lyophilization to give a yellow solid (100 mg). NHS activation of Poly(HPMA 9 co PEGMA 6 ) fluorescein (P3) P oly (HPMA 9 co PEGMA 6 ) fluorescein (89.1 mg, 1.27 10 2 mmol) and NHS (1.8 mg, 1.6 10 2 mmol) were dissolved in dry dichloromethane (2 mL). The reaction was briefly purged with N 2 and EDC HCl (3.0 mg, 1.6 10 2 mmol) was added as a solution in dichloromethane (0.5 mL). The reaction was left to stir overnight at room temperature. Dichloromethane was removed in vacuo and the polymer was used without further purification. Conjugation to OPG Two vials of recombinant human TNFRSF 11B 487 (OPG, 1 mg protein, Creative Biomart, His tagged, lot #265155) were warmed to room Both solutions were combined and placed in a Millipore Amicon Ultra 0.5 10,000 Da MWCO ultrafiltration unit and centrifuged at 14,000 rpm at room temperat ure for 10 min to remove interfering excipients. The protein solution was diluted with phosphate buffer more times to de salt the OPG prior to conjugation. The final OPG so lution ( ca. Activated polymers were dissolved in phosphate buffer (100 mM, pH 7.5) at concentrations of 2.5 mM immediately prior to conjugation. OPG conjugations were carried out i

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99 h at room temperature, each reaction mixture was placed in a separate Millipore Amicon Ultra 0.5 10,000 Da MWCO ultrafiltration unit, diluted with phosphate buffer temperature. This washing was re peated three additional times. The final concentrated protein samples were stor ed at 4 C prior to biological assays. 4.4.4 Mouse Marrow C ulture 1,25 dihydroxyvitamin D 3 (1,25D 3 ) stimulated mouse marrow, in which osteoblasts and osteoclasts differentiate coordinately over a period of 6 days was produced as described previously. 215 Femora and tibia from Swiss g) that had been ki lled by cervical dislocation, were dissected from adherent tissue, and marrow was removed by clipping both bone ends, inserting a syringe with a 25 gauge D10). The marrow was wash 10 6 cells/cm 2 8 M 1,25D 3 Cultures were fed on day 4 by replacing half the media per plate and adding fresh 1,25D 3 OPG and OPG conjugates we re added on Day 1 and refreshed on Day 4. After 6 days in culture, osteoclasts were abundant in control cultures. Cells were fixed with 2% paraformaldehyde in citrate buffer for 20 min, permeabilized by treatment with 1% Triton X 100 for 10 min, washed in citrate buffer, and osteoclasts were detected by staining for tartrate resistant acid phosphatase (TRAcP) activity, which is a specific marker for mouse osteoclasts, using the Leukocyte Acid Phosphatase (TRAP) kit from Sigma (St Louis, MO). Cells expressin g TRAcP activity were documented as described

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100 previously. 215 The University of Florida Institutional Animal Care and Usage Committee approved this protocol. 4.4.5 Cell Cytotoxicity A ssay RAW 264.7 cells were grown as described previously. 216 Raw 264.7 cells (ATCC) were plated at a density of 1.25 10 4 cells per well in 24 well plates and treated with OPG or derivatives of OPG as indicated. Adherent cells from 3 random fields per well 2 ) were counted and then averaged for each well. Live cells we re counted on Day 2. On Day 4 the cells were fixed with 2% formaldehyde prior to counting. No overt signs of toxicity, excess non adherent cells or cell debris, were noted on either Day 2 or 4. 4.4.6 A nimals and Experimental G roups A total of 20 jugular ve in cannulated male Sprague Dawley rats aged 8 10 weeks arrived to the Animal Care Services, University of Florida from a commercial vendor (Charles River, Ltd). After arrival, rats were housed individually in ventilated cages. The housing room was maintain ed at 68 79 F with an average humidity of 30 70% and a 12:12 h light:dark cycle. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida (Gainesville, FL). After a week of acclimation, rats were randomized into the following five experimental groups (n = 4): Group of rats euthanized at the beginning of the experiment and received no treatment ( Baseline control ). Group of rats that received a single bolus intravenous (IV) injection of OPG alone ( 0.4 mg/kg) diluted in phosphate buffer (pH 7.2) ( Unmodified OPG ). Group of rats that received a single bolus IV injection of the linear PEG OPG bioconjugate (0.4 mg/kg) diluted in phosphate buffer (pH 7.2) ( OPG1 ).

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101 Group of rats that received a single bolu s IV injection of the densely branched PEG OPG bioconjugate (0.4 mg/kg) diluted in phosphate buffer (pH 7.2) ( OPG2 ). Group of rats that received a single bolus IV injection of the loosely branched PEG OPG bioconjugate (0.4 mg/kg) diluted in phosphate buffe r (pH 7.2) ( OPG3 ). Only one of the four assigned rats was injected in this group due to loss of the bioconjugate sample during filtration. Rats from the baseline group and those rats after completion of the 7 day period were euthanized by CO 2 inhalation fo llowed by thoracotomy. Left femurs were excised and stripped of musculature, placed in 10% buffered formalin for 48 hours, and transferred to 70% ethanol. 4.4.7 Peripheral Q uantit ative Computed T omography (pQCT) For the pQCT analysis, left femurs from all rats were scanned using a Stratec XCT Research M instrument (Norland Medical Systems; Fort Atkinson, WI) with software version 5.40. Scans were performed at distances of 2, 4 and 6 mm proximal to the distal femur epiphyseal growth plate. At this locatio n, corresponding to the primary and secondary spongiosa, is where endochondral ossification takes place and bone grows in length and undergoes remodeling. Trabecular (trab) bone mineral content (BMC), trab bone mineral density (trab BMD) and trab bone are a (trab BA) were determined as previously describe d 217

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102 CHAPTER 5 GRAFTING FROM PROTEINS USING METAL FREE PET RAFT POL YMERIZATIONS UNDER MILD VISIBLE LIGHT IRRADIATION 5.1 Overview The covalent attachment of a polymer to a protein (i.e., polymer protein conjugation) strongly affects the in vivo properties of the protein by improving its solubility, stability, and biodistribution 107,110,212,213 Solution properties of polymer protein conjugates are manipulated in vitro by attaching stimuli responsive polymers to control enzymatic activity, protein recyclability and purification, and to form self assembled, catalytically active surfaces 119 121,128,185,218 These materials are most often made This approach is typically limited to polymers with l ow molecular weights because of the difficulty in coupling macromolecular products and purifying the resultant materials. containing high molecular weight polymers with high g rafting densities and facile purification methods by directly growing the polymer from the protein surface. 83 86 Reversibl e deactivation radical polymerization (RDRP) techniques 2,4,7,8,89,179,219 have afforded c onvenient pathways for the generat ion of polymer protein conjugates by the grafting from approach under mild conditions that preserve the structure, functionality, and utility of the protein The first examples used atom transfer radical polymerization (ATRP) 90,92,220 Moreover copper mediated methods for grafting from proteins have been greatly expanded upon over the last few years due to advances in rapid aqueous polymerizations with very low ca talyst loadings and moderate temperatures to achieve rapid polymerizations with go od control over molecular weights (MW ). 94 97

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103 Although less utilized than ATRP, we have pioneered gra fting from proteins using reversible addition fragmentation chain transfer (RAFT) polymerization to create thermally responsive poly( N is o propylacrylamide) (PNIPAM) protein conjugates, which demonstrated temperature regulated protein activity. 101 103 Importantly, these materials coul d be efficiently chain extended with a second mo n omer, which demonstrated high chain end fidelity of the initial conjugate. 102,103 In a similar strategy, Davis, Bulmus, and protein conjugates in situ, where the Z group of the chain transfer agent ( CTA) was immobilized to the protein and monomer propagation occurred on free polymer chains in solution rather than from chains that were directly bound to the protein surface. 99,100 More recently, Chen and coworkers used a photoinitiated RAFT grafting from method to form thermally responsive polymer protein conjugates. 221 Rec ently, photomediated transformations have been heavily pursued, and many of these techniques have been incorporated into polymer science 222 224 These methods typically rely on either i) direct pho ton absorption by an initiator followed by an intramolecular process to produce radicals capable of initiating polymerization, or ii) the use of a photocatalyst to generate ra dicals in a photoredox process. 222 Thiocarbonylthio compounds (e.g., dithiocarbamates, trithiocarbonates) have been used to mediate RDRP under UV irradiation using a photo ini tiator trans fer ter mi nator (photoiniferter) process, 225 and recent work has demonstrated an ability to use this method to prepare ultra high MW polymers 226 and to mediate polymerizations using a compact fluorescent bulb with phenothiazine catalysts 227 Additionally, blue light irradiation with either tertiary amine catalysts 228,229 or in the absence of catalysts 230 has been used to generate well

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104 defined polymers. Alternatively, Boyer and coworkers have performed extensive investigations of photoredox RAFT polymerizatio ns, deemed photoinduced electron/energy transfer RAFT (PET RAFT) polymerizations. The range of photocatalysts examined includes iridium 231 and ruthenium 232 complexes, po rphyrins (e.g., chlorophyll a ), 233,234 and organodye s 235 and these systems haven proven to effectively control polymerizations in organic or aq ueous solution, often in the presence of oxygen. 236 These methods have been utilized in a variety of applications, including polymerizatio n induced self assembly (PISA) 237,238 and responsive gels ; 239 however, their use in biological settings h as been more seldom considered. 232 5.2 Results and Discussion Herein, we report a new method for r apidly grafting from proteins via met al free PET RAFT polymerization under mild visible light irradiation using an organo photocatalyst to generate well defined polymer protein conjugates (Scheme 5 1). The resultant conjugates contained polymers with high degrees of livingness, demonstrated by efficient chain extensions in a consecutive grafting from manner. Importantly, we show this method is applicable for a variety of mono mer classes and functionalities. The diversity in the types of monomers that can be polymerized, combined with the mild conditions used during polymerization, makes this a promising technique in the field of protein engineering with synthetic polymers. Scheme 5 1. Synthetic pathway to obtain the lysozyme chain transfer agent and subseq uent grafting from polymerization using photoinduced electron/energy transfer reversible addition fragmentation chain transfer to generate polymer protein conjugates

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105 In traditional RAFT polymerization, an external initiator is used to generate radical spe cies, which leads to unavoidable and rapid termination events between propagating chains and low MW initiator derived radicals lowering the livingness of the resultant polymers. In the case of grafting from polymerizations ext ernal initiators also lead t o a small number of initiator derived chains that are free in solution rather than covalently bound to the protein, potentially contaminating the conjugate. Since many photomediated RAFT methods rely on initiation solely from photolysis of the C S bond in the CTA, we reasoned we could exploit PET RAFT to eliminate the presence of unbound polymers in solution, while simultaneously improving the end group fidelity of the protein immobilized chains Additionally, proteins often have low solubility in aqueous s olutions due to their high MW and the presence of hydrophobic amino acid residues, which makes it difficult to dissolve proteins at concentrations useful for most radical polymerizations. Because of the se unavoidably low concentrations, polymerization cond itions must be robust enough to allow significant monomer conversions in very dilute solutions to achieve high MW conjugates. Previous studies of grafting from with RAFT have circumvented this challenge by increasing the initiator concentrations or using l arge monomer to CTA feed ratios to give high MW polymers at low conversions. We chose to exploit the rapid rates of polymerization of many photomediated RAFT methods to achieve rapid grafting from at high monomer dilutions. Our initial screening reactions relied on the photochemical properties of trithiocarbonates serving as photoiniferters to mediate polymerization under mild visible light 230 However, we only observed high monomer conversions when using elevated monomer

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106 Table 5 1. PET RAFT polymerizations of DMA. Entry a DMA (equiv.) EY (mol%) PMDETA (equiv.) Conv. (%) b M n,theory (kg/mol) c M n,GPC MALS (kg/mol) d M w / M n d 1 500 10 1 95 46.2 75.3 1.19 2 500 5 1 98 48.9 75.8 1.09 3 500 2 1 93 47.9 73.8 1.01 4 500 1 1 79 37.2 45.8 1.01 5 500 0.1 1 5 6 100 1 1 92 9.32 11.8 1.01 7 100 1 0 29 3.11 4.93 1.02 a Reagents were calculated relative to the CTA; [DMA] = 0.6 M. b Monomer conversions were calculated using gas chromatography by integrating DMA ( R t = 5.9 min) relative to DMF ( R t = 4.5 min). c M n,theory (theoretical molecular weight) = ([DMA]/[CTA] conv. MW DMA ) + MW CTA d M n,GPC MALS (Number average MW determined by gel permeation chromatography (GPC) equipped with multi angle light scattering detection (MALS)) and M w / M n were determined using GPC MALS with 0.05 M LiCl in N,N dimethylacetamide as the eluent. The refractive index increment ( dn/dc ) for PDMA was determined to be 0.0699 mL/g using offline experiments and Astra software (Wyatt). concentrations (5 M) and a mor e energetic UV source ( max = 365 nm, 7.0 mW/cm 2 ) PET RAFT conditions using organo photocatalysts, including eosin Y (EY ), 240 have been recently shown to mediate the polymeriz ation of methacrylates in dimethylsulfoxide 235 Using a low MW trithiocarbonate CTA, we applied aqueous PET RAFT using EY to polymerize N,N dimethylacrylamide (DMA) which led to very fast polymerization rates and polymers with narrow molecular weight distributions (MWD). Furthermore, we showed t hat varying the catalyst concentration, monomer feed ratio, and add ition of a tertiary amine ( N N N N N p entamethyldiethylenetriamine PMDETA) could provide polymers with good agreement between theoretical and experimental MWs, even under very high mo nomer dilutions (0.1 M) (Table 5 1, Figure 5 1).

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107 Figure 5 1. Pseudo first order kinetics plot s of DMA polymerizations : (a) w ith and without 1 equiv. of N N N N N pentamethyldiethylenetriamine ( PMDETA). Conditions: [DMA] = 0.6 M; DMA:CTA:EY:PMDETA = 100:1:0.01:X with X = 1 (blue circles) or X = 0 (orange squares) ; (b) with varied feed ratios of DMA relative to the CTA. Conditions: [DMA] = 0.6 M; DMA:CTA:EY:PM DETA = X:1:0.01:1 with X = 100 (blue circles) or X = 500 (orange squares) ; and (c) with varied DMA concentrations. Conditions: DMA:CTA:EY:PMDETA = 100:1:0.01:1 with [DMA] = 0.6 M (blue circles) or 0.1 M (orange squares). (d) Number average molecular weight ( M n ) versus monomer conversion of DMA polymer izations. Conditions: [DMA] = 0.6 M; DMA:CTA:EY:PMDETA = X:1:0.01:1 with X = 1 00 (blue) or X = 500 (orange). The theoretical molecular weights ( M n,theory ) were calculated using the following equation: M n,theory = ([DMA]/[CTA] conv. MW DMA ) + MW CTA M n,G PC MALS (Number average MW determined by gel permeation chromatography (GPC) with multi angle light scattering detection (MALS)) were determined using GPC MALS with 0.05 M LiCl in N,N dimethylacetamide as the eluent.

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108 These conditions were also applied t o polymerize other acrylamides, including the acid functional 2 acrylamido 2 methyl 1 propane sulfonic acid (AMPS) and neutral N isopropylacrylamide (NIPAM), both of which displayed similar polymerization rates as DMA (Table 5 2 entries 2 3). Although mor e sluggish, acrylates (e.g., poly(ethylene glycol) acrylate (PEGA) and 2 hydroxyethyl acrylate (HEA)) and a water soluble styrenic (sodium styrene sulfonate (NaSS)) could also be polymerized in a controlled fashion using PET RAFT with EY (Table 5 2 entrie s 4 6, Scheme 5 2 Figure 5 2 ). Scheme 5 2. PET RAFT polymerizations of various monomers in aqueous solution under blue light irradiation. Table 5 2. PET RAFT polymerizations using low MW CTA Entry a Monomer Conv. (%) b M n,theory (kg/mol) c M n,GPC MALS (kg/mol) d M w / M n d 1 DMA 92 9.32 11.8 1.01 2 AMPS 89 18.6 15.8 1.08 3 NIPAM 85 9.36 12.5 1.01 4 PEGA 53 26.7 33.4 1.20 5 HEA 69 7.78 9.09 1.02 6 NaSS 95 17.7 21.0 1.74 a Conditions: [M]:[CTA]:[EY]:[PMDETA] = 100:1:0.01:1; [M] = 0.1 M. b Monomer conversions were calculated using either gas chromatography or 1 H NMR spectroscopy. c M n,theory (theoretical molecular weight) = ([M]/[CTA] conv. MW M ) + MW CTA d M n,GPC MALS (Number average MW determined by gel permeation chromatography (GPC) with multi angle light scattering detection (MALS)) and M w / M n were determined using GPC MALS with either 0.05 M LiCl in N,N dimethylacetamide or 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) as the eluent. The refractive index increment ( dn/dc ) of each polymer used for absolute MW calculations was determined using 100% mass recovery methods using Astra software (Wyatt).

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109 Figure 5 2. Pseudo first order kinetics plot of PET RAFT polymerizations with various monomers. Conditions: M:CTA:EY:PMDETA = 100:1:0.01:1; [M] = 0.1 M. Monomers studied were N,N dimethylacrylamide (DMA), 2 acrylamido 2 methyl 1 propane sulfonic acid (AMPS), N isopropylacrylamide (NIPAM), poly(ethylene glycol) acrylate (PEGA), 2 hydroxyethy l acrylate (HEA), and sodium styrene sulfonate (NaSS). For grafting from reactions, we first synthesized a protein CTA by the modification of lysozyme (LYS) with a novel CTA bearing an N hydroxysuccinimidyl ester separated from the trithiocarbonate moiety by a diethylene glycol unit (Scheme 5 3 ). The purified LYS CTA was subjected to intact liquid chromatography mass spectrometry (LC MS), which revealed a weighted average of 3.14 CTA moieties per LYS (Table 5 3 Figure 5 3, Figure 5 4 ). Trypsin digestion of the LYS CTA followed by LC MS /MS showed the CTA predominately modified lysine residues 33 and 97. Although not explicitly detected in this experimen t due to an abundance lower than the threshold value for peak selection, we expect the N terminus to be the next most likely site of attachment based on our reaction conditions and literature precedent 77,106,241,242 An efficient grafting from polymerization with LYS CTA was achieved using 2 mol% EY and 500 equiv. DMA relative to the CTA to reach 96% monomer conversion with linear pseudo first order kinetics in only 1 h of irradiation with blue LEDs (Figure 5

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110 Table 5 3. Area under the peak of deconvoluted capillary liquid chromatography mass spectrometry chromatogram to determine the relative percentage s of chain transfer agent (CTA) moieties per lysozyme. Number of CTA M oieties Area Under the Peak Percentage (%) 0 97927 1.3 1 22077 0.3 2 1705100 23 .0 3 2633976 35.5 4 2960543 39.9 Figure 5 3. Deconvoluted chromatogram from capillary liquid chromatography mass spectrometry experiment of intact lysozyme chain transfer agent. The number of chain transfer agent moieties on each lysozyme are indicated by a number over the peak at a given retention time. Figure 5 4. Capillary liquid chromatography mass spectrometry spectrum of intact lysozym e chain transfer agent showing the addition of 3 chain transfer agent moieties. Retention time = 19.4 min. Calculated [M + 10 + ] 1549.6.

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111 Figure 5 5. Polymerization of DMA by grafting from CTA modified lysozyme in the presence of 2 mol% eosin Y relative to the CTA. Conditions: DMA:CTA:EY = 500:1:0.02; [DMA] = 0.6 M. (a) Pseudo first order kinetics plot, (b) Number average molecular weight ( M n ) versus monomer conversion for the intact conjugate ; M n,theory (theoretical molecular weight) was calculated using an average of 3 CTA moieties per protein and the following equation: M n,theory = ([DMA]/[CTA] conv. MW DMA 3) + MW LYS where MW LYS = 14.3 kg/mol. M n,GPC MALS (Number average MW determined by gel permeation chromatography (GPC) with multi angle light scattering detection (MALS)) were determined using GPC MALS with 0.05 M LiCl in N,N dimethylacetamide as the eluent, (c) GPC analyses of the intact conjugate at inc reasing reaction time points (GPC MALS was conducted in 0.05 M LiCl in N,N dimethylacetamide as the eluent and the normalized refractive index (RI) signals were plotted versus elution time), and (d) sodium dodecyl sulfate polyacrylamide gel electrophoresis results for lysozyme (LYS) and conjugates at increasing reaction time points during the polymerization.

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112 5 ). The MWs of the intact conjugate increased linearly with conversion and agreed well with theoretical value s, and s odium dodecyl sulfate polyacrylami de gel electrophoresis (SDS PAGE) indicated the polymer was covalently bound to the protein with a very low amount of unmodified protein present in solution ( Figure 5 5 ). Reaction optimization using a lower DMA:CTA feed ratio (100:1), 1 mol% EY, and the ad dition of 1 equiv PMDETA relative to the CTA led to improved MW control with linear pseudo first order kinetics. SDS PAGE effectively resolved the conjugates, which revealed each protein 5 6 ). While t he GPC chromatogram of the intact conjugate was bimodal, which we ascribe to the distribution of CTA moieties attached to the protein, the GPC chromatogram of the polymer after cleavage from the protein indicated a monomodal and symmetrical peak, suggestin g that each polymer was well defined (Figure 5 6c). Importantly, polymerization of DMA in a mixture of unmodified LYS and low MW CTA proceeded at a similar rate and resulted in no conjugates based on SDS PAGE, which indicated the absence of non specific ch ain transfer events to protein during polymerization and suggested that each polymer was immobilized via the CTA moiety on lysine residues during the grafting from polymerization as described above (Figure 5 7 ). Additionally, by cycling the light source on and off during the polymerization, monomer conversion and MW growth w ere observed only in the presence of light ( Figure 5 8 ). Many reports of RAFT grafting from proteins via RAFT utilize acrylamide monomers, possibly due to their high propagation rate con stant ( k p ) in water, which allows high monomer conversion using thermal initiators at low temperatures. To demonstrate the feasibility of grafting from using PET RAFT for other monomer clas ses,

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113 Figure 5 6. Polymerization of DMA by grafting from CTA modified lysozyme in the presence of 1 mol% eosin Y and 1 equiv. of N N N N N pentamethyldiethylenetriamine relative to the CTA. (a) Pseudo first order kinetics plot, (b) SDS PAGE results of unmodified lysozy me (LYS) and the conjuga tes at increasing reaction time points and (c) GPC analyses of the intact conjugate and the polymers after cleavage from the protein in pH 12 buffer (GPC MALS was conducted in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) as the eluen t and the normalized refractive index (RI) signals were plotted versus elution time ). (d ) GPC analyses of the intact conjugate at increasing reaction time points ( GPC MALS was conducted in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) as the eluent and the n ormalized refractive index (RI) signals were plotted versus elution time ) (e ) M n,theory (theoretical molecular weight) was calculated using an average of 3 CTA moieties per protein and the following equation: M n,theory = ([DMA]/[CTA] conv. MW DMA 3) + MW LYS where MW LYS = 14.3 kg/mol. M n,GPC MALS (Number average MW determined by GPC MALS) were determined using GPC MALS conducted in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) as the eluent

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114 Figure 5 7. Control reaction where DMA was polymerized in a mixture of unmodified lysozyme (LYS) and compound 1a (CTA) to demonstrate that conjugates are only formed in the presence of protein bound CTA. Conditions: DMA:CTA:LYS:EY:PMDETA = 100:1:0.3:0.01:1; [DMA] = 0. 1 M (a) Pseudo first order kinetics plot showing the same apparent rate of polymerization for polymerizations conducted with only compound 1a (blue circles, conditions: DMA:CTA:EY:PMDETA = 100:1:0.01:1, [DMA] = 0.1 M) and with both compound 1a and unmodifi ed LYS (orange squares). (b) Sodium dodecyl sulfate polyacrylamide gel electrophoresis results of increasing reaction time points during the polymerization confirmed that no conjugates formed. Figure 5 8. Periodic light irradiation (i.e., on and off cycles) during the polymerization of DMA while grafting from lysozyme. (a) Pseudo first order kinetics plot showing each on and off cycle versus total reaction time, (b) pseudo first order kinetics plot versus total light exposure time, and (c) SDS PAGE re sults showing MW growth only in the presence of light for each on and off cycle

PAGE 115

115 we generated LYS PHEA and LYS PNaSS, reaching 74% and 63% monomer conversion, respectively, in 20 h (Figure 5 9 and Figure 5 10 ). Additionally, a critical characteristic of RDRP methods is the ability to form block copolymers due to the retention of end groups afforded by the mediating agents. Here, the presence of the trithiocarbonate end group was demonstrated by a consecutive grafting from polymerization, where LYS PDMA wa s chain extended with NIPAM (55% monomer conversion in 5 h) The GPC chromatogram of the LYS PDMA starting material was bimodal due to the CTA distribution with in the LYS CTA; however, the chain extension showed good blocking efficiency, although a low MW shoulder was present, which could be attributed to resi dual unmodified LYS (Figure 5 11 ). The narrow and symmetrical chromatogram of the chain extended conjugate suggest ed that the majority of the polymer chains contained a trithiocarbonate end group at th e conclusion of the initial grafti ng from polymerization with DMA

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116 Figure 5 9. Polymerization of 2 hydroxyethyl acrylate (HEA) by grafting from CTA modified lysozyme (LYS) in the presence of 1 mol% eosin Y and 1 equiv. of N N N N N pentamethyldiet hylenetriamine relative to the CTA. Conditions: HEA :CTA:EY :PMDETA = 100:1:0.01:1; [HEA] = 0.1 M (a) G el permeation chromatography (GPC) analysis of the quenched reaction mixture (GPC was conducted in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) as the eluent and the normaliz ed refractive index (RI) signal w as plotted versus elution time (b) Sodium dodecyl sulfate polyacrylamide gel electrophoresis results for LYS and the initial and final reaction time points Figure 5 10. Polymerization of sodium styrene sulfonate (NaSS) by grafting from CTA modified lysozyme (LYS) in the presence of 1 mol% eosin Y and 1 equiv. of N N N N N pentamethyldiethylenetriamine relative to the CTA. Conditions: NaSS :CTA:EY :PMDETA = 100:1:0.01:1; [NaSS] = 0.1 M (a) G el permeati on chromatography (GPC) analysis of the quenched reaction mixture (GPC was conducted in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) as the eluent and the normaliz ed refractive index (RI) signal w as plotted versus elution time (b) Sodium dodecyl sulfate pol yacrylamide gel electrophoresis results for LYS and the initial and final reaction time points

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117 Figure 5 11 GPC chromatograms showing the chain extension of lysozyme poly(dimethylacrylamide) (LYS PDMA) with N isopropylacrylamide (NIPAM) to generate LYS PDMA block poly(NIPAM) (LYS PDMA b PNIPAM) (GPC MALS was conducted in 0.05 M LiCl in N,N dimethylacetamide as the eluent, and the normalized refractive index (RI) signals were plotted versus elution time ) 5.3 Summary We have demonstrated aqueous PET R AFT polymerizations of a range of monomers in water to achieve polymers with predetermined MWs and well defined MWDs at high monomer conversions. This method proved effective to rapidly generate polymer protein conjugates over a range of targeted MWs and m onomers under mild visible light irradiation. We continue to investigate the polymerization mechanism and effect of amines on EY catalyzed PET RAFT polymerizations, and these results will be disclosed elsewhere. Importantly, the conjugates produced using t his polymerization method potentially possess greater purity than those previously reported, since no exogenous initiators are employed. We believe this technique will allow access to an expanded library of functional materials, such as stimuli res ponsive block co polymer protein conjugates for in vitro use or anti fouling polymer protein conjugates for in vivo applications.

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118 5.4 Experimental 5.4.1 Materials All reagents were used as received unless otherwise noted. Sodium ethanethiolate (80%), 2 bromoisobuty ric acid, and eosin Y (EY) were purchased from Santa Cruz Biotech. Carbon disulfide (99+%), succinic anhydride (99%), and triethylamine (TEA, 99%) were purchased from Alfa Aesar. Tribasic potassium phosphate was purchased from VWR. 1 E thyl 3 (3 dimethylami nopropyl)carbodiimide HCl (EDC HCl 98%) was purchased from Combi Blocks. Hen egg white lysozyme (LYS, 20,000 U/mg) was purchased from MP Biomedicals as a lyophilized powder. N Hydroxysuccinimide (99%), 2 (2 aminoethoxy)ethanol (98%), N N N N N p entamethyldiethylenetriamine (PMDETA, 98+%), bicinichoninic acid (BCA) kit, N,N dimethylacrylamide (DMA, 99%), sodium hydroxyethylacrylate (HEA, 96%), poly(ethylene glycol) methyl ether acrylate (PEGA, M n 480 g/mol), 2 acrylamido 2 methyl 1 propane sulfonic acid (AMPS), and N isopropylacrylamide (NIPAM, 97%) were purchased from Sigma Aldrich. DMA and PEGA were passed through basic alumina to remove inhibitors prior to polymerization AMPS was recrystallized from 90 wt% acetic acid. HEA was purified according to a literature procedure. 243 NIPAM was recrystallized from hexanes ( 3). Deuterium oxide (D 2 O, 99.9 %) and chloroform d (CDCl 3 99.9%) were purchased from Cambridge Isotopes. GelCode Blu e protein stain and Pierce lane marker sample buffer containing dithiothreitol (DTT) reducing agent (5 concentrated) were purchased from Thermo Fisher. Polymerizations were conducted

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119 using a commercially available blue LED light strip (SuperbrightLEDs.co m, 50 cm, 0.9 W/ft, 16459 mcd/ft) wound inside a 400 mL beaker wrapped in aluminum foil. 5.4.2 Instrumentation NMR spectroscopy 1 H NMR spectra were recorded on a Varian Inova2 500 MHz NMR spectrometer using the residual s olvent signal as a reference. UV visible spectroscopy UV v is ible spectra were obtained on a Molecular Devices SpectraMax M2 multimode microplate reader Measurements were conducted with 100 L of sample on clear 96 well polypropylene microplates (Greiner Bio One). Gel permeation chr omatography (GPC) Polymer molecular weight and molecular weight distributions were determined by GPC in N N dimethylacetamide (DMAc) with 50 mM LiCl at 50 C a t a flow rate of 1.0 mL/min (Agilent isocratic pump, degasser, and autosampler, columns: ViscoGe l guard column + two ViscoGel I series 10 3 10 4 g/ mol). Detection consisted of a Wyatt Optilab T rEX refractive index detector operating at 658 nm and a Wyatt miniDAWN Treos light scattering detector operating at 659 nm. Absolute molecular weights and molecular weight distribu tions were calculated using ASTRA software (Wyatt). Aqueous GPC was conducted in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) at a flow rate of 0.5 mL/min ( Viscotek GPCMax VE 2001 module; columns: TSKgel PW XL guard column (Tosoh) + TSKgel G4000PW XL analytical column (Tosoh; 7.8 mm 30 cm, 10 m particle size)). Detection consisted of a Wyatt DSP refractive index detector operating at 6 90 nm and a Wyatt DAWN EOS light scat tering detector operating at 690 nm. Absolute molecular weights and molecular weight distr ibutions were calculated

PAGE 120

120 using ASTRA software (Wyatt). The refractive index increment ( dn/dc ) of each polymer was calculated using 100% mass recovery methods in Astra software (Wyatt). Columns were calibrated with narrow molecular weight distribution poly(sodium styrene sulfonate) standards with a range of 10 3 10 5 g/mol. Gas chromatography (GC) Monomer conversions were determined by GC recorded on an Agilent 6 550 Series II equipped with a 30 m 0.32 mm ID Agen Tubular column with a 0.25 m HP 1 (polydimethylsiloxane) stationary phase film. Detection consisted of a flame ionization detector (FID) operating at 250 C. Gas flows were maintained at the following f low rate: hydrogen at 40 mL/min, air at 450 mL/min, and helium at 45 mL/min. The inlet te mperature was maintained at 250 C, and the column was heated according to the following profile: 70 C initial temperature, 10 C/min ramp for 5 mi n, 120 C isotherma l for 2 min. Chromatogram peak integrations were calculated using Agilent ChemStation software. Tangential flow filtration (TFF) TFF was used to purify the protein chain transfer agent using a KrosFlo Research II i TFF system (Spectrum Labs) with a modified polyether sulfone MidiKros filter module (10 kDa molecular weight cutoff (MWCO), surface area: 115 cm 2 ) operating at a feed flow rate of 35 mL/min and feed pressure of 15 20 PSI, collecting a permeate volume ca. 3 10 the starting volume. Protein samples were then concentrated to ca. 10 15 mL, and the protein concentration was determined using a BCA assay. Capillary liquid chromatography mass spectrometry (Cap LC MS) Cap LC MS was performed on a Bruker Impact I I Quadrupole Time of Flight (QTOF) mass spectrometer equipped with an Apollo II ion funnel ESI source (Bruker) operated in

PAGE 121

121 ( Thermo Scientific ) The mobile phase A was water containing 0 .1% formic acid and the mobile phase B was acetonitrile with 0.1% formic acid. Each sample (5 L) was first injected onto the Precolumn Cartridge (Thermo Scientific), and washed with mobile phase A. The injector port was switched to inject, and the sampl e was eluted from the trap onto the column. An Acclaim m 150 m, 2 m particle size 100 pore size ) was used for chromatographic separations. Proteins were eluted directly from the column into the LTQ system using a gradient of 2 80%B over 30 min, with a flow rate of 5 L /min. The tota l run time was 60 min. The MS were acquired according to standard conditions in the lab. Briefly, the instrument was calibrated using Tu ne mix purchased from Agilent. The Apollo ESI s ource was operated with a spray voltage of 4.5 kV, a capillary temperature of 200 C, and dry gas at 4.0 L/min. A full scan was recorded between 150 3000 Da at a scan rate of 1 Hz. In Gel Digestion. Gels were digested with sequencing grade trypsin or sequencing grade chymotrypsin from Promega (Madison WI) using manufacturer recommended protocols. Briefly, bands were trimmed as close as possible to minimize background polyacrylamide material. Gel pieces were washed in nanopure water for 5 min ( 2 ). Ge l pieces were washed and/or destained with 1:1 v/v methanol:50 mM ammonium bicarbonate for 10 min ( 2 ). The gel pieces were dehydrated with 1:1 v/v acetonitril e: 50 mM ammonium bicarbonate. The gel bands were rehydrated and incubated with DTT solution (25 mM DTT in 100 mM ammonium bicarbonate) for 30 min prior to the addition of iodoacetamide solution ( 55 mM iodoacetamide in 100 mM ammonium bicarbonate ) Iodoacetamide was incubated with the gel bands in the dark

PAGE 122

122 for 30 min before being removed. The gel band s were washed again with two cycles of water and dehydrated with 1:1 v/v acetonitrile:50 mM ammonium bicarobonate. The protease was driven into the gel pieces by rehydrating them in 12 ng/ml trypsin in 0.01% was then overlaid with 40 L of a shaker for 1 h. The digestion wa s stopped with addition of 0.5% trifluoroacetic acid. The MS analysis was immediately performed to ensure high q uality tryptic peptides with minimal non specific cleavage or frozen at 80 o C until samples could be analyzed. Nano liquid chromatography ta ndem mass spectrometry (Nano LC MS/MS) Nano LC MS/MS was performed on a Thermo Scientific LTQ XL mass spectrometer equipped with an EASY operated in positive ion mode. The LC system was an EASY Nano Scientific). The mobile phase A was water containing 0.1% formic acid and the mobile phase B was acetonitrile wi th 0.1% formic acid. Each sample (5 L) was first injected onto a Thermo Scientific Acclaim m ID, 2 cm length, 3 m particle size, 100 pore size) and washed with mobile phase A to desalt and concentrate the pe ptides. The injector port was switched to inject and the peptides were eluted from the trap onto the column. An EASY PepMAP column ( Thermo Scientific ) was used for chromatographic separations (C18, 75 m ID, 15 cm length, 3 m particle size, 100 pore size). The column temperature was maintained at 35 C, and peptides were eluted directly from the column into the LTQ system using a gradient of 2 80%B over 45 min, with a flow rate of 300 nL/min. The total run time was 60 min. The MS/MS spectra were acquired according to standard conditions established

PAGE 123

123 in the lab. The EASY was operated with a spray voltage of 1.5 kV and a capillary temperature of 200 C. The scan sequence of the mass spectrometer was based on the T between 350 2000 Da, and a MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive instrument scans of the ten most abundant peaks in the spectrum. The AGC Target ion number was set at 30000 ions for full scan and 10000 ions for MSn mode. Maximum ion injection time was set at 20 ms for full scan and 300 ms for MSn mode. Micro scan number was set at 1 for both full scan and MSn scan. The CID fragmentation energy was set to 35%. Dynamic exclusion was enabled with a repeat count of 1 within 10 seconds, a mass list size of 200, and an exclusion duration of 350 seconds. T he low mass width was 0.5 and the high mass width was 1.5. Sequence analysis. Sequence in formation from the MS/MS data was processed by converting the .raw files into a merged file (.mgf) using msConvert (ProteoWizard) or Mascot Distiller (Matrix Science). The resulting mgf files were searched using Mascot Daemon by Matrix Science version 2.4. 0 (Boston, MA) and the database searched against the full SwissProt database version version 2015_10 (549,646 sequences; 195,983,064 residues). The mass accuracy of the precursor ions was set to 1.5 Da and the fragment mass accuracy was set to 0.5 Da. Con sidered variable modifications were methionine oxidation and asparagine or glutamine deamidation. Fixed modification for carbamidomethyl cysteine was considered. Two missed cleavages for the enzyme were permitted. A decoy database was searched to determine the false discovery rate (FDR) and peptides were filtered according to the to the FDR and proteins identified required

PAGE 124

124 bold red peptides. Protein identifications were checked manually and proteins with a Mascot significance threshold p < 0.05 with a mini mum of two unique peptides from one protein having a b or y ion sequence tag of five residues or better were accepted. Gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) gels were cast and run using Mini PROTEAN sy stems (Bio Rad), and buffers were prepared according to Laemmli. 244 Samples were prepared in sample buffer (Pierce) and heated 10 min to denature proteins prior to running at 180 V for 45 min to separate the proteins, which were visualized with GelCode Blue staining. 5.4.3 Methods 5.4.3.1 Synthesis of LY S chain transfer agent (LYS CTA) 2 (((Ethylthio)carbonothioyl)thio) 2 methylpropanoic acid 1a 245 Carbon disulfide (4 mL, 70 mmol) was added dropwise to a stirred suspension of sodium ethanethiolate (1.94 g, 23.1 mmol) in acetone (150 mL) and allowed to react for 15 m in. Tribasic potassium phosphate (4.92 g, 23.2 mmol) and 2 bromoisobutyric acid (3.51 g, 21.0 mmol) were added, and the heterogeneous yellow reaction was allowed to stir at room temperature overnight. White precipitate was removed by filtration, and the ye llow filtrate was concentrated in vacuo dissolved in dichloromethane (DCM, 150 mL), and washed with HCl (1 M, 1 125 mL), water (3 125 mL), and saturated NaHCO 3 (1 125 mL). The basic aqueous layer was acidified to pH ~1 2 with 1 M HCl and extracted with dichloromethane (125 mL). The organic layer was washed with brine (1 125 mL), dried over anhydrous MgSO 4 and filtered. The product was isolated as a yellow solid after concentration in vacuo (3.46 g, 73% yield). 1 H NMR (5 00 MHz, CDCl 3 ) : 3.30 (2H, q, CH 3 C H 2 S), 1.73 (6H, s, SC(C H 3 )), 1.33 (3H, t, C H 3 C H 2 S).

PAGE 125

125 Ethyl (1 ((2 (2 hydroxyethoxy)ethyl)amino) 2 methyl 1 oxopropan 2 yl) carbonotrithioate 1b Compound 1a (0.500 g, 2.23 mmol) was dissolved in DCM (40 mL). NHS (284 mg, 2.47 mmol) and EDC HCl (471 mg, 2.45 mmol) were added, and the reaction was stirred at room temperature and monitored by TLC. After 2 h, 2 (2 aminoethoxy)ethanol (2.58 mg, 2.46 mmol) was added and allowed to react at room temperature for 16 h. The crude product was washed with deionized water (3 50 mL) and brine (1 50 mL), dried over anhydrous MgSO 4 and filtered. The filtrate was concentrated, and the crude product was further purified by column chromatography using ethyl acetate:hexanes (9:1) as the eluent to afford the pr oduct as a yellow oil after concentration in vacuo (435 mg, 63% yield). 1 H NMR (5 00 MHz, CDCl 3 ) : 6.86 (1H, br s, N H ), 3.70 (2H, t, OCH 2 C H 2 OH), 3.51 (4H, m, CH 2 C H 2 OC H 2 CH 2 OH), 3.44 (2H, m, NHC H 2 CH 2 O), 3.30 (2H, q, CH 3 C H 2 S), 1.73 (6H, s, SC(C H 3 )), 1.33 (3H, t, C H 3 C H 2 S). 6,6 Dimethyl 7,15 dioxo 4 thioxo 11,14 dioxa 3,5 dithia 8 azaoctadecan 18 oic acid 1c Compound 1b (430 mg, 1.38 mmol) was dissolved in DCM (10 mL). Succinic anhydride (153 mg, 1.53 mmol) and triethylamine (200. L, 1.44 mmol) were added, and the reaction was stirred at room temperature for 16 h. The crude reaction mixture was washed with dilute HCl (pH ~2, 2 20 mL ), neutral deionized water (2 20 mL), and brine (1 20 mL). The organic fraction was dried over a nhydrous MgSO 4 filtered, and concentrated in vacuo to afford the product as a yellow oil (413 mg, 73% yield). 1 H NMR (5 00 MHz, CDCl 3 ) : 6.88 (1H, br s, N H ), 4.21 (2H, t, CH 2 C H 2 O(CO)CH 2 ), 3.60 (2H, CH 2 C H 2 OC H 2 CH 2 O(CO)), 3.50 (2H, CH 2 C H 2 OC H 2 CH 2 O(CO)), 3.43 (2H, t, NHC H 2 CH 2 O), 3.30 (2H, q, CH 3 C H 2 S), 2.69 (4H, m, (CO) C H 2 C H 2 COOH ), 1 .73 (6H, s, SC(C H 3 )), 1.33 (3H, t, C H 3 C H 2 S).

PAGE 126

126 6,6 Dimethyl 7 oxo 4 thioxo 11 oxa 3,5 dithia 8 azatridecan 13 yl (2,5 dioxopyrrolidin 1 yl) succinate 1 Compound 1c (0.400 g, 0.972 mm ol) was dissolved in DCM (15 mL). NHS (124 mg, 1.08 mmol) and EDC HCl (205 mg, 1.07) were added, and the reaction was stirred at room temperature for 16 h. The crude product was washed with deionized water (3 20 mL) and brine (1 20 mL), dried over anhydrous MgSO 4 and filtered. The filtrate was concentrated in vacuo to afford the product as a yellow oil. 1 H NMR (5 00 MHz, CDCl 3 ) : 6.84 (1H, br s, N H ), 4.24 (2H, t, CH 2 C H 2 O(CO)CH 2 ), 3.61 (2H, CH 2 C H 2 OC H 2 CH 2 O(CO)), 3.50 (2H, CH 2 C H 2 OC H 2 CH 2 O(CO)), 3.42 (2H, t, NHC H 2 CH 2 O), 3.28 (2H, q, CH 3 C H 2 S), 2.97 (2H, t, CH 2 CO 2 CH 2 C H 2 CO 2 N), 2.84 (4H, s, C H 2 C H 2 ( succinimide)), 2.79 (2H, t, CH 2 CO 2 C H 2 C H 2 CO 2 N), 1 .69 (6H, s, SC(C H 3 )), 1.32 (3H, t, C H 3 C H 2 S). 1 3 C NMR (125 MHz, CDCl 3 ) : 220.03, 172.43, 170.88, 168.83, 167.63, 69.57, 68.69, 64.05, 57.10, 39.83, 31.26, 28.67, 26.28, 25.81, 25.56, 12.82. ESI MS calculated for C 19 H 28 N 2 O 8 S 3 [M + Na + ] 531.09 (2.8 ppm); found 531.0915 LYS CTA Lysozyme (201 mg, 14.1 mol) was dissolved in phosphate buffer (265 mL, 0.1 M phosphate, pH 7.5) in a 500 mL round bottom flask with gentle stirring. A solution of 1 (74.1 mg, 156 mol) in DMSO (14 mL) was added dropwise via addition funnel over 10 min, and the reaction was allowed to stir at room temperature for 3 h. Glyc ine (20 mg 0.3 mmol) was added to quench the reaction. The crude reaction mixture was centrifuged at 5,000 rpm for 30 min to remove excess CTA, and the pale yellow supernatant was further purified by TFF. Protein concentration was determined using a BCA as say with a bovine serum albumin standard curve, and the trithiocarbonate concentration was determined from a standard curve at 310 nm by UV

PAGE 127

127 vis. The number of CTA molecules per LYS was determined by deconvolution of the LC MS spectrum to give 3.14 CTA/LYS, which corroborated well with the CTA/LYS calculated from the concentrations of LYS and CTA (2.62). 5.4.3.2 Photoinduced electron/energy transfer reversible addition fragmentation chain transfer (PET RAFT) polymerization using compound 1a as the CTA. PDMA DMA was polymerized by PET RAFT using EY as a photocatalyst under the conditions outlined in Table S1. A general procedure is as follows (Table S1, entry 7): DMA (87.8 mg, 0.886 mmol) and compound 1a (2.0 mg, 0.0089 mmol) were dissolved in water (1.264 mL ) in a 10 mL Schlenk tube. PMDETA (1.7 mg, 0.0098 mmol) as a solution in DMF (74.4 L) and EY (0.0579 mg as 57.9 L of a 1 mg/mL solution in water, 8.94 10 5 mmol) were added to the reaction, which was covered with foil and purged with argon for 30 min. The reaction was irradiated with blue LED, and the DMA conversion was monitored by GC using DMF as an internal standard. The polymerization was quenched after 1.5 h (92% DMA conversion) by turning off the light source and exposing the contents to air. Reac tion aliquots were freeze dried and dissolved in 0.05 M LiCl in DMAc for GPC analysis ( M n,theory = 9.32 kg/mol, M n,GPC MALS = 11.8 kg/mol, M w / M n = 1.01). PDMA under high dilution ([DMA] = 0.1 M) DMA (89 mg, 0.90 mmol) and compound 1a (2.0 mg, 0.0089 mmol) were dissolved in water (8.333 mL) in a 10 mL Schlenk tube. PMDETA (1.5 mg, 0.0087 mmol), EY (0.0579 mg as 57.9 L of a 1 mg/mL solution in water, 8.94 10 5 mmol), and DMF (447 L) as an internal standard were added to the reaction which was covered with foil and purged with argon for 30 min. The reaction was irradiated with blue LED, and the DMA conversion was monitored by GC using DMF as an internal standard. The polymerization was quenched after 5 h

PAGE 128

128 (92% DMA conversion) by turni ng off the light source and exposing the contents to air. Reaction aliquots were freeze dried and dissolved in 0.05 M LiCl in DMAc for GPC analysis ( M n,theory = 9.37 kg/mol, M n,GPC MALS = 14.3 kg/mol, M w / M n = 1.01). PPEGA PEGA (424 mg, 0.884 mmol) and com pound 1a (1.9 mg, 0.0089 mmol) were dissolved in water (8.0 mL) in a 10 mL Schlenk tube. PMDETA (1.9 mg, 0.011 mmol) dissolved in DMF (447 L) and EY (0.0579 mg as 57.9 L of a 1 mg/mL solution in water, 8.94 10 5 mmol) were added to the reaction, which was covered with foil and purged with argon for 30 min. The reaction was irradiated with blue LED, and the PEGA conversion was monitored by GC using DMF as an internal standard. The polymerization was quenched after 7 h (53% PEGA conversion) by turning off the light source and exposing the contents to air. Reaction aliquots were freeze dried and dissolved in 0.05 M LiCl in DMAc for GPC analysis ( M n,theory = 26.7 kg/mol, M n,GPC MALS = 33.4 kg/mol, M w / M n = 1.20). PAMPS Compound 1a (1.8 mg, 0.0080 mmol) was d issolved in DMF (447 L) containing PMDETA (1.3 mg, 0.0075 mmol) in a 10 mL Schlenk tube. The solution was diluted with phosphate buffer (8.43 mL, 0.5 M, pH 7.4), and AMPS (166 mg, 0.802 mmol) and EY (0.0579 mg as 57.9 L of a 1 mg/mL solution in water, 8. 94 10 5 mmol) were added. The final pH of the reaction solution was ca. 7. The tube was covered with foil, purged with argon for 30 min, and irradiated with blue LED. AMPS conversion was monitored by 1 H NMR spectroscopy using DMF as an internal standard. The polymerization was quenched after 5 h (89% AMPS conversion) by turning off the light source and exposing the contents to air. Reaction aliquots were freeze dried and

PAGE 129

129 dissolved in 0.05 M aqueous Na 2 S O 4 /acetonitrile (80:20) for aqueous GPC analysis ( M n,theory = 18.6 kg/mol, M n,GPC MALS = 15.8 kg/mol, M w / M n = 1.08). PHEA HEA (103 mg, 0.887 mmol) and compound 1a (2.1 mg, 0.0094 mmol) were dissolved in water (8.322 mL) in a 10 mL Schlenk tube. PMDETA (1. 6 mg, 0.0093 mmol) dissolved in DMF (447 L) and EY (0.0579 mg as 57.9 L of a 1 mg/mL solution in water, 8.94 10 5 mmol) were added to the reaction, which was covered with foil and purged with argon for 30 min. The reaction was irradiated with blue LED, and the HEA conversion was monitored by GC using DMF as an internal standard. The polymerization was quenched after 22 h (69% HEA conversion) by turning off the light source and exposing the contents to air. Reaction aliquots were freeze dried and dissolv ed in 0.05 M LiCl in DMAc for GPC analysis ( M n,theory = 7.78 kg/mol, M n,GPC MALS = 9.09 kg/mol, M w / M n = 1.02). PNaSS NaSS (184 mg, 0.894 mmol) and compound 1a (2.0 mg, 0.0089 mmol) were dissolved in water (8.425 mL) in a 10 mL Schlenk tube. PMDETA (1.8 mg, 0.010 mmol) dissolved in DMF (447 L) and EY (0.0579 mg as 57.9 L of a 1 mg/mL solution in water, 8.94 10 5 mmol) were added to the reaction, which was covered with foil and purged w ith argon for 30 min. The reaction was irradiated with blue LED, and the NaSS conversion was monitored by 1 H NMR spectroscopy using DMF as an internal standard. The polymerization was quenched after 22 h (95% NaSS conversion) by turning off the light sourc e and exposing the contents to air. Reaction aliquots were freeze dried and dissolved in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) for aqueous GPC analysis ( M n,theory = 17.7 kg/mol, M n,GPC MALS = 21.0 kg/mol, M w / M n = 1.74).

PAGE 130

130 PNIPAM NIPAM (101 mg, 0.893 mm ol) and compound 1a (2.1 mg, 0.0094 mmol) were dissolved in water (8.425 mL) in a 10 mL Schlenk tube. PMDETA (1.7 mg, 0.0098 mmol) dissolved in DMF (447 L) and EY (0.0579 mg as 57.9 L of a 1 mg/mL solution in water, 8.94 10 5 mmol) were added to the re action, which was covered with foil and purged with argon for 30 min. The reaction was irradiated with blue LED, and the NIPAM conversion was monitored by GC using DMF as an internal standard. The polymerization was quenched after 6.5 h (85% NIPAM conversi on) by turning off the light source and exposing the contents to air. Reaction aliquots were freeze dried and dissolved in 0.05 M LiCl in DMAc for GPC analysis ( M n,theory = 9.36 kg/mol, M n,GPC MALS = 12.5 kg/mol, M w / M n = 1.01). 5.4.3.3 PET RAFT grafting from polymerization using LYS CTA LYS PDMA with 2 mol% EY in the absence of PMDETA DMA (54.0 mg, 0.545 mmol) was added to a 10 mL Schlenk tube. A solution of LYS CTA (850 L, [CTA] = 1.28 mM determined by UV vis spectroscopy, 0.00109 mmo l CTA functionality), EY (0.0141 mg as 14.1 L of a 1 mg/mL solution in water, 2.18 10 5 mmol), and DMF (45 L) as an internal standard were added, and the reaction was purged with argon for 30 min and irradiated with blue LED. DMA conversion was determi ned by GC, and the reaction was quenched after 1 h (96% DMA conversion) by turning off the light source and exposing the contents to air. Conjugate MWs were determined by DMAc GPC after lyophilizing 50 L of each reaction aliquot and dissolving in 0.05 M L iCl in DMAc Intact conjugates were analyzed by SDS PAGE. ( M n,theory = 110 kg/mol, M n,GPC MALS = 173 kg/mol, M w / M n = 1.39 )

PAGE 131

131 LYS PDMA with 1 mol% EY and 1 equiv. PMDETA DMA (19.6 mg, 0.198 mmol) was added to a 10 mL Schlenk tube. Solutions of LYS CTA (2.0 m L, [CTA] = 1 mM determined by UV vis spectroscopy, 0.002 mmol CTA functionality), EY (0.013 mg as 13 L of a 1 mg/mL solution in water, 2.01 10 5 mmol), and PMDETA (0.346 mg as 100 L of a 3.46 mg/mL solution in DMF, 0.00200 mmol) were added, and the reaction was purged with argon for 30 min and irradiated with blue LED. DMA conversion was determined by GC, and the reaction was quenched after 1.5 h (91% DMA conversion) by turning off the light source and exposing the contents to air. Conjugate MWs were determined by aqueous GPC after lyophilizing ca. 150 L of each reaction aliquot and dissolving in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) Conjugates were analyzed by SDS PAGE. The c rude reaction mixture was purified by dialysis using 3.5 kDa MWCO membranes. ( M n,theory = 40.8 kg/mol, M n,GPC MALS = 57.4 kg/mol, M w / M n = 1.24 ) LYS PHEA HEA (13.8 mg, 0.119 mmol) was added to a 10 mL Schlenk tube. Solutions of LYS CTA (1.0 mL, [CTA] = 1 m M determined by UV vis spectroscopy, 0.001 mmol CTA functionality), EY (0.00648 mg as 6.48 L of a 1 mg/mL solution in water, 1.00 10 5 mmol), and PMDETA (0.173 mg as 100 L of a 1.73 mg/mL solution in DMF, 0.00100 mmol) were added, and the reaction was purged with argon for 30 min and irradiated with blue LED. HEA conversion was determined by GC, and the reaction was quenched after 18 h (74% HEA conversion) by turning off the light source and exposing the contents to air. Conjugate MWs were determined by aqueous GPC after lyophilizing ca. 150 L of each reaction aliquot and dissolving in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) Intact conjugates were analyzed by SDS PAGE. ( M n,theory = 44.9 kg/mol, M n,GPC MALS = 79.7 kg/mol, M w / M n = 1.31 )

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132 LYS PNaSS NaSS (21.4 mg, 0.104 mmol) was added to a 10 mL Schlenk tube. Solutions of LYS CTA (1.0 mL, [CTA] = 1 mM determined by UV vis spectroscopy, 0.001 mmol CTA functionality), EY (0.00648 mg as 6.48 L of a 1 mg/mL solution in water, 1.00 10 5 mmol), and PMDE TA (0.173 mg as 100 L of a 1.73 mg/mL solution in DMF, 0.00100 mmol) were added, and the reaction was purged with argon for 30 min and irradiated with blue LED. NaSS conversion was determined by 1 H NMR spectroscopy, and the reaction was quenched after 20 h (63% NaSS conversion) by turning off the light source and exposing the contents to air. Conjugate MWs were determined by aqueous GPC after lyophilizing ca. 150 L of each reaction aliquot and dissolving in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) Inta ct conjugates were analyzed by SDS PAGE. ( M n,theory = 55.5 kg/mol, M n,GPC MALS = 52.2 kg/mol, M w / M n = 1.59) Light on/off cycle for LYS PDMA DMA (19.3 mg, 0.195 mmol) was added to a 10 mL Schlenk tube. Solutions of LYS CTA (2.0 mL, [CTA] = 1 mM determined by UV vis spectroscopy, 0.002 mmol CTA functionality), EY (0.013 mg as 13 L of a 1 mg/mL solution in water, 2.01 10 5 mmol), and PMDETA (0.346 mg as 100 L of a 3.46 mg/mL solution in DMF, 0.00200 mmol) were added, and the reaction was purged with argon for 30 min and irradiated with blue LED. The light source was turned on and off in 15 min increments for a total exposu re time of 90 min. DMA conversion was determined by GC, and conjugate MWs were determined by SDS PAGE. LYS PDMA block PNIPAM NIPAM (151 mg, 1.33 mmol) was added to a solution of LYS PDMA (1.5 mL containing 0.002 CTA functionality) in a 10 mL Schlenk tube EY (17.4 g as 17.4 L of a 1 mg/mL solution in water, 2.69 10 5 mmol) and

PAGE 133

133 PMDETA (0.46 mg as 75 L of a 6.2 mg/mL solution in DMF, 0.0030 mmol) were added, and the reaction was purged with argon for 30 min and irradiated with blue LED. NIPAM conversi on was determined by GC, and the reaction was quenched after 5 h (55% NIPAM conversion) by turning off the light source and exposing the contents to air. Conjugate MWs were determined by aqueous GPC after lyophilizing ca. 200 L of a reaction aliquot and d issolving in 0.05 M LiCl in DMAc ( M n,theory = 150 kg/mol, M n,GPC MALS = 230 kg/mol, M w / M n = 1.50 ) Polymerization of DMA in the presence of unmodified LYS DMA (23.8 mg, 0.240 mmol) and LYS (11 mg, 0.77 mol) were added to a 10 mL Schlenk tube and dissolve d in water (1.86 mL). Solutions of compound 1a (0.538 mg as 50 L of a 10.75 mg/mL stock solution in DMF, 0.00240 mmol), EY (0.0156 mg as 15.6 L of a 1 mg/mL solution in water, 2.41 10 5 mmol), and PMDETA (0.415 mg as 50 L of an 8.30 mg/mL solution in DMF, 0.00240 mmol) were added, and the reaction was purged with argon for 30 min and irradiated with blue LED. DMA conversion was determined by GC, and the reaction was quenched after 3 h (85% DMA conversion) by turning off the light source and exposing t he contents to air. Reaction aliquots were analyzed by SDS PAGE. Polymer cleavage from the conjugate A lyophilized sample of LYS PDMA from the crude reaction mixture was dissolved in pH 12 phosphate buffer (150 L) and reacted at room temperature for 24 h to hydrolyze the ester linkage in the spacer CTA. The reaction was analyzed by SDS PAGE, and the remaining sample was lyophilized, dissolved in 0.05 M aqueous Na 2 SO 4 /acetonitrile (80:20) and analyzed by aqueous GPC. ( M n,theory (PDMA) = 9.12 kg/mol, M n,G PC MALS = 23.0 kg/mol, M w / M n = 1.01)

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134 Figure 5 12 Sodium dodecyl sulfate polyacrylamide gel electrophoresis results of polymer cleavage from lysozyme (LYS) Conditions: pH 12 phosphate buffer, room temperature, 24 h.

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135 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS The research described in this dissertation utilized RAFT polymerization to afford well defined polymer ic materials relevant to nanomedicine applications. Polymer synthesis has long been an important factor in materials for biology, and the adve nt of polymerization methods that allow control over molecular weight, molecular weight distribution, and polymer topology have led to a plethora of new materials. Often, the final material is highly complex, and i t i s frequently the result of tedious self assembly events. The goal of this dissertation research was to utilize RAFT polymerization as a convenient and facile route toward materials with interesting and tunable properties based on the reaction strategies employed, and to demonstrate their proper ties in biological applications. Polymer drug delivery vehicles have been used to preferentially locate a small molecule therapeutic to a specific site within the body. Here, star shaped nanoparticles composed of poly( N (2 hydroxypropyl)methacrylamide) (PH PMA) were synthesized in a resulted in efficient formation of nanoparticles of sufficient size for nanomedicine applications, and their potential as delivery vehicles was inves tigated by incor porating a chemotherapeutic within the core. While release of the drug from the nanoparticle was limited, it demonstrated the importance of considering the chemical functionality and steric environment when releasing a small molecule from a macromolecular architecture. Covalent immobilization of a polymer to a protein greatly affects the in vivo and in vitro properties of the protein. Here, the effect of polymer architecture on the activity of a

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136 protein that regulates bone homeostasis was in vestigated. The results showed unique in vitro activity against cells for a polymer protein conjugate containing a branched polymer where the branching units were intermittently spaced along the backbone. This conjugate improved the activity of the protein as compared to conjugates containing e ither linear polymers or a more densely branched polymer. Ongoing work in this area includes a fundamental understanding of how the attached polymers might alter the tertiary structure and dynamics of the protein, the reby leading to broad changes in activity. This is currently being probed using computational methods to model protein conjugates. Finally, access to new materials was afforded using a grafting from strategy to grow a polymer directly from th e surface of a protein. Visible light mediated polymerization s offer exquisite spati o temporal control over the polymerization, and the mild irradiation sources might be useful for biological materials. Here, PET RAFT polymerization methods were used to grow acrylamide acrylate, and even styrenic monomer classes from a protein, greatly expanding the previously a ttainable compositions attainable by RAFT grafting from polymerizations. The ability to polymerize functional monomers could endow the protein with beneficial pro perties, including the ability to manipulate their solution properties with exposure to a particular stimulus (e.g., temperature, light).

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137 APPENDIX REACTIVITY RATIOS FO R PEGMA AND HPMA Table A 1. Monomer feed ratios and copolymer composition data for the RAFT copolymerization of PEGMA and HPMA using 4 cyano 4 [(dodecylsulfanyl thiocarbonyl)sulfanyl]pentanoic acid as the CTA. M 1 (PEMGA) M 2 (HPMA) m 1 (PEGMA) m 2 (HPMA) M = M 1 /M 2 m 1 /m 2 a P b G c H 0.64 0.11 0.58 0.10 5.8 5.8 6 4.85 5.64 0.53 0.17 0.49 0.16 3.1 3.1 4 2.34 2.43 0.90 0.83 0.80 0.74 1.1 1.1 1.1 0.108 1.06 0.90 2.65 0.71 2.29 0.34 0.31 0.53 0.304 0.219 0.27 1.48 0.25 1.42 0.18 0.18 0.33 0.365 0.0998 a P = (M 1 m 1 )/(M 2 m 2 ); b G = M M/P; c H = M 2 /P Figure A 1. Finema n Ross plot for the copolymerization of PEGMA and HPMA. The slope provides r 1 and the intercept provides r 2 The following equations are used for the above plot. (A 1) (A 2) Table A 2. Monomer reactivity ratios for PEGMA and HPMA r 1 (PEGMA) r 2 (HPMA) r 1 r 2 0.98 0.52 0.50

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138 LIST OF REFERENCES (1) Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Rizzardo, E.; Thang, S. H. Macromolecules 1998 31 5559 5562. (2) Moad, G.; Chong, Y. K.; Postma, A.; Rizzardo, E.; Thang, S. H. Polymer 200 5 46 8458 8468. (3) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2012 65 985 1076. (4) Hill, M. R.; Carmean, R. N.; Sumerlin, B. S. Macromolecules 2015 48 5459 5469. (5) Boyer, C.; Stenzel, M. H.; Davis, T. P. J Polym. Sc i., Part A: Polym Ch em. 2010 49 551 595. (6) Boyer, C.; Bulmus, V.; Davis, T. P.; Ladmiral, V.; Liu, J. Chem Rev 2009 109 5402 5436. (7) Matyjaszewski, K.; Tsarevsky, N. V. J Am Chem. Soc 2014 136 6513 6533. (8) Hawker, C. J.; Bosman, A. W.; Harth, E. Chem Rev 2001 101 3661 3688. (9) McLeary, J. B.; Calitz, F. M.; McKenzie, J. M.; Tonge, M. P.; Sanderson, R. D.; Klumperman, B. Macromolecules 2005 38 3151 3161. (10) Larson, N.; Ghandehari, H. Chem Mate r. 2012 24 840 853. (11) Elsabahy, M.; Wooley, K. L. Ch em Soc Rev 2012 41 2545 2561. (12) Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F. Acc Chem Res 2011 44 1029 1038. (13) Ringsdorf, H. J Polym Sci ., Symp. 1975 51 135 153. (14) Matsumura, Y.; Maeda, H. Cancer Res 1986 46 638 7 6392. (15) Fang, J.; Nakamura, H.; Maeda, H. Adv Drug Delivery Rev 2011 63 136 151. (16) Barz, M.; Luxenhofer, R.; Zentel, R.; Vicent, M. J. Polym Chem 2011 2 1900 1918. (17) Adv Drug Delivery Rev 2010 62 122 149. (18) ilov, H. Eur Polym J 1973 9 7 14. (19) Teodorescu, M.; Matyjaszewski, K. Macromolecules 1999 32 4826 4831.

PAGE 139

139 (20) Scales, C. W.; Vasilieva, Y. A.; Convertine, A. J.; Lowe, A. B.; McCormick, C. L. Biomacromolecules 2005 6 1846 1 850. (21) Gibson, M. I.; Frhlich, E.; Klok, H. A. J Polym Sci ., Part A: Polym Chem 2009 47 4332 4345. (22) Eberhardt, M.; Theato, P. Macromol Rapid Commun. 2005 26 1488 1493. (23) Duncan, R.; Vicent, M. J. Adv Drug Delivery Rev 2010 62 272 28 2. (24) Talelli, M.; Rijcken, C. J. F.; van Nostrum, C. F.; Storm, G.; Hennink, W. E. Adv Drug Delivery Rev 2010 62 231 239. (25) Bioorg Med C hem 2012 20 4056 4063. (26) Biomaterials 2012 33 1863 1872. (27) York, A. W.; Huang, F.; McCormick, C. L. Biomacromolecules 2010 11 505 514. (28) Bareford, L. M.; Avaritt, B. R.; Ghandehari, H.; Nan, A.; Swaan, P. W. Pharm Res 2013 30 1799 1812. (29) Liu, T.; Li, X.; Qian, Y.; Hu, X.; Liu, S. Biomaterials 2012 33 2521 2531. (30) Barz, M.; Canal, F.; Koynov, K.; Zentel, R.; Vicent, M. J. Biomacromolecules 2010 11 2274 2282. (31) Wei, C.; Wu, K.; Li, J.; Ma, W.; Guo, J.; Hu, J.; Wang, C. Macromol. Chem. Phys. 2012 213 557 565. (32) Roy, D.; Ghosn, B.; Song, E. H.; Ratner, D. M.; Stayton, P. S. Polym. Chem. 2013 4 1153 1160. (33) Journo Pharm Res 201 2 29 1121 1133. (34) Yang, Y.; Zhou, Z.; He, S.; Fan, T.; Jin, Y.; Zhu, X.; Chen, C.; Zhang, Z. R.; Huang, Y. Biomaterials 2012 33 2260 2271. (35) Studenovsk, M.; Pola, R.; Pechar, M.; Etrych, T.; Ulbrich, K.; Kovar, L.; Macro mol. Biosci. 2012 12 1714 1720. (36) Griffiths, P. C.; Paul, A.; Apostolovic, B.; Klok, H. A.; de Luca, E.; King, S. M.; Heenan, R. K. J Controlled Release 2011 153 173 179. (37) Apostolovic, B.; Deacon, S. P. E.; Duncan, R.; Klok, H. A. Macromol Rap id Commun 2011 32 11 18.

PAGE 140

140 (38) Apostolovic, B.; Deacon, S. P. E.; Duncan, R.; Klok, H. A. Biomacromolecules 2010 11 1187 1195. (39) J Controlled Release 2012 157 126 131. (40) Angew. Chem. Int. Ed. 2010 49 1451 1455. (41) React Funct Polym 2011 71 294 302. (42) Biomacr omolecules 2011 12 247 252. (43) Zhang, R.; Luo, K.; Yang, J.; Sima, M.; Sun, Y.; Jant J. J Controlled Release 2013 166 66 74. (44) Macromolecules 2011 44 2481 2488. (45) Sumerl in, B. S.; Vogt, A. P. Macromolecules 2010 43 1 13. (46) Boase, N. R. B.; Blakey, I.; Thurecht, K. J. Polym Chem 2012 3 1384 1389. (47) Herth, M. M.; Barz, M.; Jahn, M.; Zentel, R.; Rsch, F. Bioorg Med Chem Lett 2010 20 5454 5458. (48) Ren, K. ; Purdue, P. E.; Burton, L.; Quan, L. D.; Fehringer, E. V.; Thiele, G. M.; Goldring, S. R.; Wang, D. Mol. Pharmaceutics 2011 8 1043 1051. (49) Kunjachan, S.; Gremse, F.; Theek, B.; Koczera, P.; Pola, R.; Pechar, M.; Etrych, T.; Ulbrich, K.; Storm, G.; Ki essling, F.; Lammers, T. ACS Nano 2013 7 252 262. (50) Gormley, A. J.; Larson, N.; Banisadr, A.; Robinson, R.; Frazier, N.; Ray, A.; Ghandehari, H. J Controlled Release 2013 166 130 138. (51) Lundy, B. B.; Convertine, A.; Miteva, M.; Stayton, P. S. Bioconjugate Chem 2013 24 398 407. (52) Schellinger, J. G.; Pahang, J. A.; Johnson, R. N.; Chu, D. S. H.; Sellers, D. L.; Maris, D. O.; Convertine, A. J.; Stayton, P. S.; Horner, P. J.; Pun, S. H. Biomaterials 2013 34 2318 2326. (53) Shi, J.; Johnson, R. N.; Schellinger, J. G.; Carlson, P. M.; Pun, S. H. Int J Pharm 2012 427 113 122. (54) Shi, J.; Schellinger, J. G.; Johnson, R. N.; Choi, J. L.; Chou, B.; Anghel, E. L.; Pun, S. H. Biomacromolecules 2013 14 1961 1970.

PAGE 141

1 41 (55) Novo, L.; van Gaal, E. V. B.; Mastrobattista, E.; van Nostrum, C. F.; Hennink, W. E. J Controlled Release 2013 169 246 256. (56) Barz, M.; Armin, A.; Canal, F.; Wolf, F.; Koynov, K.; Frey, H.; Zentel, R.; Vicent, M. J. J Controlled Release 2012 163 63 74. (57) Barz, M.; Wolf, F. K.; Canal, F.; Koynov, K.; Vicent, M. J.; Frey, H.; Zentel, R. Macromol Rapid Commun 2010 31 1492 1500. (58) Chytil, P.; Etrych, T.; Kostka, L.; Ulbrich, K. Macromol. Chem. Phys. 2012 213 858 867. (59) Wei, R.; Cheng, L.; Zheng, M.; Cheng, R.; Meng, F.; Deng, C.; Zhong, Z. Biomacromolecules 2012 13 2429 2438. (60) Soga, O.; van Nostrum, C. F.; Ramzi, A.; Visser, T.; Soulimani, F.; Frederik, P. M.; Bomans, P. H. H.; Hennink, W. E. Langmuir 2004 20 9388 9395. (61) Sog a, O.; van Nostrum, C. F.; Hennink, W. E. Biomacromolecules 2004 5 818 821. (62) Shi, Y.; van den Dungen, E. T. A.; Klumperman, B.; van Nostrum, C. F.; Hennink, W. E. ACS Macro Lett. 2013 2 403 408. (63) Xu, J.; Tao, L.; Boyer, C.; Lowe, A. B.; Davis, T. P. Macromolecules 2011 44 299 312. (64) J Controlled Release 2013 165 153 161. (65) Hemmelmann, M.; Metz, V. V.; Koynov, K.; Blank, K.; Postina, R.; Zentel, R. J Controlled Release 2012 163 170 177. (66) Hemmelmann, M.; Knoth, C.; Schmitt, U.; Allmeroth, M.; Moderegger, D.; Barz, M.; Koynov, K.; Hiemke, C.; Rsch, F.; Zentel, R. Macrom ol Rapid Commun 2011 32 712 717. (67) Allmeroth, M.; Moderegger, D.; Biesalski, B.; Koynov, K.; Rsch, F.; Thews, O.; Zentel, R. Biomacromolecules 2011 12 2841 2849. (68) J Cont rolled Release 2012 164 346 354. (69) Utama, R. H.; Stenzel, M. H.; Zetterlund, P. B. Macromolecules 2013 46 2118 2127. (70) Utama, R. H.; Guo, Y.; Zetterlund, P. B.; Stenzel, M. H. Chem. Commun. 2012 48 11103 11105

PAGE 142

142 (71) Boyer, C.; Whittaker, M. R.; Nouvel, C.; Davis, T. P. Macromolecules 2010 43 1792 1799. (72) Abuchowski, A.; McCoy, J. R.; Palczuk, N. C.; van Es, T.; Davis, F. F. J Biol Chem 1977 252 3582 3586. (73) Hermanson, G. T. Bioconjugation Techniques ; Academic Press, 1996. (74) Rober ts, M. J.; Bentley, M. D.; Harris, J. M. Adv Drug Delivery Rev 2002 54 459 476. (75) Sayers, C. T.; Mantovani, G.; Ryan, S. M.; Randev, R. K.; Keiper, O.; Leszczyszyn, O. I.; Blindauer, C.; Brayden, D. J.; Haddleton, D. M. Soft Matter 2009 5 3038 3046 (76) Smith, M. E. B.; Schumacher, F. F.; Ryan, C. P.; Tedaldi, L. M.; Papaioannou, D.; Waksman, G.; Caddick, S.; Baker, J. R. J Am Chem Soc 2010 132 1960 1965. (77) Li, H.; Bapat, A. P.; Li, M.; Sumerlin, B. S. Polym Chem 2011 2 323 327. ( 78) Li, M.; De, P.; Li, H.; Sumerlin, B. S. Polym Chem 2010 1 854 859. (79) Li, M.; De, P.; Gondi, S. R.; Sumerlin, B. S. Macromol Rapid Commun 2008 29 1172 1176. (80) Sletten, E. M.; Bertozzi, C. R. Angew Chem Int E d. 2009 48 6974 6998. (81) Stephanopoulos, N.; Francis, M. B. Nat. Chem. Biol. 2011 7 876 884. (82) ElSohly, A. M.; Francis, M. B. Acc Chem Res 2015 48 1971 1978. (83) Sumerlin, B. S. ACS Macro Lett 2012 1 141 145. (84) Wallat, J. D.; Rose, K. A.; Pokorski, J. K. Polym Chem 2014 5 1545 1558. (85) Obermeyer, A. C.; Olsen, B. D. ACS Macro Lett 2015 4 101 110. (86) Qi, Y.; Chilkoti, A. Polym Chem 2014 5 266 276. (87) Wang, J. S.; Matyjaszewski, K. J Am Chem Soc 1995 117 5614 5615. (88) Kato, M.; Kamigaito, M.; Sawamoto, M.; Higashimura, T. Macromolecules 1995 28 1721 1723. (89) Nicolas, J.; Guillaneuf, Y.; Lefay, C.; Bertin, D.; Gigmes, D.; Charleux, B. Prog Polym Sci 2013 38 63 235.

PAGE 143

143 (90) Lele, B. S.; Murata, H.; Matyjaszewski, K.; Russell, A. J. Biom acromolecules 2005 6 3380 3387. (91) Heredia, K. L.; Bontempo, D.; Ly, T.; Byers, J. T.; Halstenberg, S.; Maynard, H. D. J Am Chem Soc 2005 127 16955 16960. (92) Bontempo, D.; Maynard, H. D. J Am Chem Soc 2005 127 6508 6509. (93) Nicolas, J.; Miguel, V. S.; Mantovani, G.; Haddleton, D. M. Chem. Commun. 2006 4697 4699 (94) Averick, S.; Simakova, A.; Park, S.; Konkolewicz, D.; Magenau, A. J. D.; Mehl, R. A.; Matyjaszewski, K. ACS Macro Lett 2012 1 6 10. (95) Simakova, A.; Averick, S. E.; Konkolewicz, D.; Matyjaszewski, K. Macromolecules 2012 45 6371 6379. (96) Zhang, Q.; Li, M.; Zhu, C.; Nurumbetov, G.; Li, Z.; Wilson, P.; Kempe, K.; Haddleton, D. M. J Amer Chem Soc 2015 137 9344 9353. (97) Boyer, C.; Corrigan, N. A.; Jung, K.; Nguyen, D.; Nguyen, T. K.; Adnan, N. N. M.; Oliver, S.; Shanmugam, S.; Yeow, J. Chem Rev 2015 116 1803 1949. (98) Lowe, A. B.; McCormick, C. L. Prog Polym Sci 2007 32 283 351. (99) Liu, J.; Bulmus, V.; Herlambang, D. L.; Barner Kowollik, C.; Stenzel, M. H.; Davis, T. P. Angew. Chem. Int. Ed. 2007 46 3099 3103. (100) Boyer, C.; Bulmus, V.; Liu, J.; Davis, T. P.; Stenzel, M. H.; Barner Kowollik, C. J Am Chem Soc 2007 129 7145 7154. (101) De, P.; Li, M.; Gondi, S. R. ; Sumerlin, B. S. J Am Chem Soc 2008 130 11288 11289. (102) Li, M.; Li, H.; De, P.; Sumerlin, B. S. Macromol Rapid Commun 2011 32 354 359. (103) Li, H.; Li, M.; Yu, X.; Bapat, A. P.; Sumerlin, B. S. Polym Chem 2011 2 1531 1535. (104) Falatach, R.; McGlone, C.; Al Abdul Wahid, M. S.; Averick, S.; Page, R. C.; Berberich, J. A.; Konkolewicz, D. Chem. Commun. 2015 51 5343 5346. (105) Vanparijs, N.; De Coen, R.; Laplace, D.; Louage, B.; Maji, S.; Lybaert, L.; Hoogenboom, R.; De Geest, B. G. Chem. Commun. 2015 51 13972 13975. (106) Lucius, M.; Falatach, R.; McGlone, C.; Makaroff, K.; Danielson, A.; Williams, C.; Nix, J. C.; Konkolewicz, D.; Page, R. C.; Berberich, J. A. Biomacromolecules 2016 17 1123 1134.

PAGE 144

144 (107) Caliceti, P.; Veronese, F. M. Adv Drug Delivery Rev 2003 55 1261 1277. (108) Monfardini, C.; Schiavon, O.; Caliceti, P.; Morpurgo, M.; Harris, J. M.; Veronese, F. M. Bioconjugate Chem 1995 6 62 69. (109) Veronese, F. M. Biomaterials 2001 22 405 417. (110) Alconcel, S. N. S.; Baas, A. S.; Maynard, H. D. Polym Chem 2011 2 1442 1448. (111) Pelegri W.; Maynard, H. D. J Am Chem Soc. 2014 136 14323 14332. (112) Tao, L.; Liu, J.; Xu, J.; Davis, T. P. Org. Biomol. Chem. 2009 7 3481 3485. (113) Tao, L.; Liu, J.; Davis, T. P. Biomacromolecules 2009 10 2847 2851. (114) Rajender Reddy, K.; Modi, M. W.; Pedder, S. Adv. Drug Delivery Rev. 2002 54 571 586. (115) Parrott, M. C.; DeSimone, J. M. Nat Chem 2012 4 13 14. (116) Keefe, A. J.; Jiang, S. Nat Chem 2012 4 59 63. (117) Nguyen, T. H.; Kim, S. H.; Decker, C. G.; Wong, D. Y.; Loo, J. A.; Maynard, H. D. Nat Chem 2013 5 221 227. (118) Mancini, R. J.; Lee, J.; Maynard, H. D. J Am Chem Soc 2012 134 8474 8479. (1 19) Cobo, I.; Li, M.; Sumerlin, B. S.; Perrier, S. Nat Mater 2015 14 143 159. (120) Stayton, P. S.; Shimoboji, T.; Long, C.; Chilkoti, A.; Chen, G.; Harris, J. M.; Hoffman, A. S. Nature 1995 378 472 474. (121) Hoffman, A. S.; Stayton, P. S. Prog Pol ym Sc i. 2007 32 922 932. (122) Chang, C. W.; Nguyen, T. H.; Maynard, H. D. Macromol Rapid Commun 2010 31 1691 1695. (123) Thomas, C. S.; Olsen, B. D. Soft Matter 2014 10 3093 3102. (124) Lam, C. N.; Olsen, B. D. Soft Matter 2013 9 2393 2402. (12 5) Huang, A.; Olsen, B. D. Macromol Rapid Commun 2016 37 1268 1274. (126) Chang, D.; Olsen, B. D. Polym Chem. 2016 7 2410 2418.

PAGE 145

145 (127) Chang, D.; Lam, C. N.; Tang, S.; Olsen, B. D. Polym Chem 2014 5 4884 4895. (128) Huang, A.; Qin, G.; Olsen, B. D. ACS Appl. Mater. Interfaces 2015 7 14660 14669. (129) Thomas, C. S.; Glassman, M. J.; Olsen, B. D. ACS Nano 2011 5 5697 5707. (130) Lam, C. N.; Kim, M.; Thomas, C. S.; Chang, D.; Sanoja, G. E.; Okwara, C. U.; Olsen, B. D. Biomacromolecules 2014 15 1248 1258. (131) Dong, X. H.; Obermeyer, A. C.; Olsen, B. D. Angew. Chem. Int. Ed. 2017 56 1273 1277. (132) Mai, Y.; Eisenberg, A. Chem Soc Rev 2012 41 5969 5985. (133) Stenzel, M. H. Chem. Commun. 2008 3486 3503. (13 4) Roy, D.; Sumerlin, B. S. ACS Macro Lett 2012 1 529 532. (135) Blanazs, A.; Armes, S. P.; Ryan, A. J. Macromol Rapid Commun 2009 30 267 277. (136) Bapat, A. P.; Ray, J. G.; Savin, D. A.; Hoff, E. A.; Patton, D. L.; Sumerlin, B. S. Polym Chem 201 2 3 3112 3120. (137) Mukherjee, S.; Bapat, A. P.; Hill, M. R.; Sumerlin, B. S. Polym Chem 2014 5 6923 6931. (138) Bapat, A. P.; Ray, J. G.; Savin, D. A.; Sumerlin, B. S. Macromolecules 2013 46 2188 2198. (139) Ghosh Roy, S.; De, P. Polym Chem 2014 5 6365 6378. (140) Bapat, A. P.; Roy, D.; Ray, J. G.; Savin, D. A.; Sumerlin, B. S. J Am Chem Soc 2011 133 19832 19838. (141) Roy, D.; Sumerlin, B. S. Macromol Rapid Commun 2013 35 174 179. (142) Qiu, Q.; Liu, G.; An, Z. Chem. Commun. 2011 47 12685 12687. (143) Gao, H. Macromol Rapid Commun 2012 33 722 734. (144) Macromolecules 2011 44 7233 7241. (145) Blencowe, A.; Tan, J. F.; Goh, T. K.; Qiao, G. G. Polymer 2009 50 5 32. (146) Gao, H.; Matyjaszewski, K. Prog Polym Sc i. 2009 34 317 350.

PAGE 146

146 (147) Gao, H.; Matyjaszewski, K. J Am Chem Soc 2007 129 11828 11834. (148) Gao, H.; Matyjaszewski, K. Macromolecules 2008 41 1118 1125. (149) Gao, H.; Matyjaszewski, K. Macromolecules 2006 39 3154 3160. (150) Gao, H.; Matyjaszewski, K. Macromol. Symp. 2010 291 292 12 16. (151) Syrett, J. A.; Haddleton, D. M.; Whittaker, M. R.; Davis, T. P.; Boyer, C. Chem. Commun. 2011 47 1449 1451. (152) Ferreira, J.; Syrett, J.; Whittaker, M.; Haddleton, D.; Davis, T. P.; Boyer, C. Polym Chem 2011 2 1671 1677. (153) Liu, J.; Duong, H.; Whittaker, M. R.; Davis, T. P.; Boyer, C. Macromol Rapid Commun 2012 33 760 766. (154) Shi, X.; Miao, M.; An, Z. Polym Chem 2013 4 1950 1959. (155) Shi, X.; Zhou, W.; Qiu, Q.; An, Z. Chem. Commun. 2012 48 7389 7391. (156) Ma, K.; Xu, Y.; An, Z. Macromol Rapid Commun 2015 36 566 570. (157) Duong, H. T. T.; Marquis, C. P.; Whittaker, M.; Davis, T. P.; Boyer, C. Macromolecules 2011 44 8008 8019. (158) Tucker, B. S.; Sumerlin, B. S. Polym Chem 2014 5 1566 1572. (159) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Angew Chem Int E d. 2010 49 6288 6308. (160) Biomacromolecules 2000 1 313 319. (161) J Controlled Release 2011 154 241 248. (162) Etrych, T.; Strohalm, J.; Ch ytil, P.; Cernoch, P.; Starovoytova, L.; Pechar, M.; Ulbrich, K. Eur J Pharm Sci 2011 42 527 539. (163) Polym Chem 2015 6 160 170. (164) Chytil, Macromol. Biosci. 2015 15 839 850. (165) Farber, S.; Diamond, L. K. N. Engl. J. Med. 1948 238 787 793. (166) Bertino, J. R. J. Clin. Oncol. 1993 11 5 14.

PAGE 147

147 (167) Goldman, I. D.; Matherly, L. H. Pharmacol. Ther. 1985 28 77 102. (168) Tian, L.; Yang, Y.; Wysocki, L. M.; Arnold, A. C.; Hu, A.; Ravichandran, B.; Sternson, S. M.; Looger, L. L.; Lavis, L. D. Proc. Natl. Acad. Sci. U.S.A. 2012 109 4756 4761. (169) Lau W.; Heard, C.; White, A. Pharmaceutics 2013 5 232 245. (170) Amir, R. J.; Albertazzi, L.; Willis, J.; Khan, A.; Kang, T.; Hawker, C. J. Angew. Chem. Int. Ed. 2011 50 3425 3429. (171) Oh, S. S.; Lee, B. F.; Leibfarth, F. A.; Eisenstein, M.; Robb, M. J .; Lynd, N. A.; Hawker, C. J.; Soh, H. T. J Am Chem Soc 2014 136 15010 15015. (172) Azagarsamy, M. A.; Sokkalingam, P.; Thayumanavan, S. J Am Chem Soc 2009 131 14184 14185. (173) Wang, K.; Peng, H.; Thurecht, K. J.; Puttick, S.; Whittaker, A. Polym Chem 2014 5 1760 1771. (174) Broyer, R. M.; Grover, G. N.; Maynard, H. D. Chem. Commun. 2011 47 2212 2226. (175) Ryan, S. M.; Fras, J. M.; Wang, X.; Sayers, C. T.; Haddleton, D. M.; Brayden, D. J. J Controlled Release 2011 149 126 132. (176 ) Duncan, R. Nat Rev Cancer 2006 6 688 701. (177) Harris, J. M.; Chess, R. B. Nat Rev Drug Discov ery 2003 2 214 221. (178) Veronese, F. M.; Harris, J. M. Adv Drug Delivery Rev. 2002 54 453 456. (179) Becker, M. L.; Liu, J.; Wooley, K. L. Chem. C ommun. 2003 180 181. (180) Chenal, M.; Boursier, C.; Guillaneuf, Y.; Taverna, M.; Couvreur, P.; Nicolas, J. Polym Chem 2011 2 1523 1530. (181) Jones, M. W.; Strickland, R. A.; Schumacher, F. F.; Caddick, S.; Baker, J. R.; Gibson, M. I.; Haddleton, D. M. J Am Chem Soc 2012 134 1847 1852. (182) Pokorski, J. K.; Breitenkamp, K.; Liepold, L. O.; Qazi, S.; Finn, M. G. J Am Chem Soc 2011 133 9242 9245. (183) Grover, G. N.; Lee, J.; Matsumoto, N. M.; Maynard, H. D. Macromolecules 2012 45 4958 4965. (184) Bays, E.; Tao, L.; Chang, C. W.; Maynard, H. D. Biomacromolecules 2009 10 1777 1781.

PAGE 148

148 (185) Murata, H.; Cummings, C. S.; Koepsel, R. R.; Russell, A. J. Biomacromolecules 2013 14 1919 1926. (186) Policastro, G. M.; Lin, F.; Smith Callaha n, L. A.; Esterle, A.; Graham, M.; Sloan Stakleff, K.; Becker, M. L. Biomacromolecules 2015 16 1358 1371. (187) Tang, W.; Ma, Y.; Xie, S.; Guo, K.; Katzenmeyer, B.; Wesdemiotis, C.; Becker, M. L. Biomacromolecules 2013 14 3304 3313. (188) Moore, N. M.; Lin, N. J.; Gallant, N. D.; Becker, M. L. Biomaterials 2010 31 1604 1611. (189) Yamaguchi, K.; Kinosaki, M.; Goto, M.; Kobayashi, F.; Tsuda, E.; Morinaga, T.; Higashio, K. J Biol Chem 1998 273 5117 5123. (190) Boyle, W. J.; Simonet, W. S.; Lacey, D L. Nature 2003 423 337 342. (191) Simonet, W. S.; Lacey, D. L.; Dunstan, C. R.; Kelley, M.; Chang, M. S.; Lthy, R.; Nguyen, H. Q.; Wooden, S.; Bennett, L.; Boone, T.; Shimamoto, G.; DeRose, M.; Elliott, R.; Colombero, A.; Tan, H. L.; Trail, G.; Sulliv an, J.; Davy, E.; Bucay, N.; Renshaw Gegg, L.; Hughes, T. M.; Hill, D.; Pattison, W.; Campbell, P.; Sander, S.; Van, G.; Tarpley, J.; Derby, P.; Lee, R.; Boyle, W. J. Cell 1997 89 309 319. (192) Lacey, D. L.; Timms, E.; Tan, H. L.; Kelley, M. J.; Dunstan C. R.; Burgess, T.; Elliot, R.; Colombero, A.; Elliot, G.; Scully, S.; Hsu, H.; Sullivan, J.; Hawkins, N.; Davy, E.; Capparelli, C.; Elie, A.; Qian, Y. X.; Kaufman, S.; Sarosi, I.; Shalhoub, V.; Senaldi, G.; Guo, J.; Delaney, J.; Boyle, W. J. Cell 1998 93 165 176. (193) Luan, X.; Lu, Q.; Jiang, Y.; Zhang, S.; Wang, Q.; Yuan, H.; Zhao, W.; Wang, J.; Wang, X. J Immunol 2012 189 245 252. (194) Yasuda, H.; Shima, N.; Nakagawa, N.; Yamaguchi, K.; Kinosaki, M.; Mochizuki, S. I.; Tomoyasu, A.; Yano, K.; Go to, M.; Murakami, A.; Tsuda, E.; Morinaga, T.; Higashio, K.; Udagawa, N.; Takahashi, N.; Suda, T. Proc Natl Acad Sci U.S.A. 1998 95 3597 3602. (195) Hofbauer, L. C. Eur. J. Endocrinol. 1999 141 195 210. (196) Hsu, H.; Lacey, D. L.; Dunstan, C. R.; Solovyev, I.; Colombero, A.; Timms, E.; Tan, H. L.; Elliott, G.; Kelley, M. J.; Sarosi, I.; Wang, L.; Xia, X. Z.; Elliott, R.; Chiu, L.; Black, T.; Scully, S.; Capparelli, C.; Morony, S.; Shimamoto, G.; Bass, M. B.; B oyle, W. J. Proc Natl Acad Sci U.S.A. 1999 96 3540 3545. (197) Ominsky, M. S.; Li, X.; Asuncion, F. J.; Barrero, M.; Warmington, K. S.; Dwyer, D.; Stolina, M.; Geng, Z.; Grisanti, M.; Tan, H. L.; Corbin, T.; McCabe, J.; Simonet, W. S.; Ke, H. Z.; Kos tenuik, P. J. J Bone Miner Res 2008 23 672 682.

PAGE 149

149 (198) Ominsky, M. S.; Stolina, M.; Li, X.; Corbin, T. J.; Asuncion, F. J.; Barrero, M.; Niu, Q. T.; Dwyer, D.; Adamu, S.; Warmington, K. S.; Grisanti, M.; Tan, H. L.; Ke, H. Z.; Simonet, W. S.; Kostenuik P. J. J Bone Miner Res 2009 24 1234 1246. (199) Morony, S.; Capparelli, C.; Sarosi, I.; Lacey, D. L.; Dunstan, C. R.; Kostenuik, P. J. Cancer Res 2001 61 4432 4436. (200) Bekker, P. J.; Holloway, D.; Nakanishi, A.; Arrighi, M.; Leese, P. T.; Duns tan, C. R. J Bone Miner Res 2001 16 348 360. (201) Body, J. J. Clin Cancer Res 2006 12 1221 1228. (202) Bucay, N.; Sarosi, I.; Dunstan, C. R.; Morony, S.; Tarpley, J.; Capparelli, C.; Scully, S.; Tan, H. L.; Xu, W.; Lacey, D. L.; Boyle, W. J.; Sim onet, W. S. Genes Dev. 1998 12 1260 1268. (203) Dufresne, S. S.; Dumont, N. A.; Bouchard, P.; Lavergne, E.; Penninger, J. M.; Frenette, J. Am. J. Pathol. 2015 185 920 926. (204) Saito Yabe, M.; Kasuya, Y.; Yoshigae, Y.; Yamamura, N.; Suzuki, Y.; Fukuda, N.; Honma, M.; Yano, K.; Mochizuki, S. I.; Okada, F.; Okada, A.; Nagayama, Y.; Tsuda, E.; Fischer, T.; Hpner, U.; Zaja, S.; Mueller, J.; Okada, J.; Kurihara, A.; Ikeda, T.; Okazak i, O. J Pharm Pharmacol 2010 62 985 994. (205) Miyaji, Y.; Kurihara, A.; Kamiyama, E.; Shiiki, T.; Kawai, K.; Okazaki, O. Xenobiotica 2009 39 113 124. (206) Miyaji, Y.; Kasuya, Y.; Furuta, Y.; Kurihara, A.; Takahashi, M.; Ogawara, K. I.; Izumi, T.; Okazaki, O.; Higaki, K. Pharm Res 2012 29 3143 3155. (207) Pissuwan, D.; Boyer, C.; Gunasekaran, K.; Davis, T. P.; Bulmus, V. Biomacromolecules 2010 11 412 420. (208) Roth, P. J.; Boyer, C.; Lowe, A. B.; Davis, T. P. Macromol Rapid Commun 2011 32 1123 1143. (209) Klok, H. A. J Polym Sci ., Part A: Polym Chem. 2005 43 1 17. (210) Slo, I.; Ngroni, L.; Crminon, C.; Grassi, J.; Wal, J. M. J. Immunol. Methods 1996 199 127 138. (211) Minkin, C. Calcif. Tissue Int. 1982 34 285 290. (212) Hered ia, K. L.; Maynard, H. D. Org. Biomol. Chem. 2007 5 45 53. (213) Gauthier, M. A.; Klok, H. A. Chem. Commun. 2008 2591 2611. (214) Lee, S.; Greenwald, R. B.; McGuire, J.; Yang, K.; Shi, C. Bioconjugate Chem 2001 12 163 169.

PAGE 150

150 (215) Ostrov, D. A.; Magis, A. T.; Wronski, T. J.; Chan, E. K. L.; Toro, E. J.; Donatelli, R. E.; Sajek, K.; Haroun, I. N.; Nagib, M. I.; Piedrahita, A.; Harris, A.; Holliday, L. S. J Med Chem 2009 52 5144 5151. (216) Toro, E. J.; Zuo, J.; Ostrov, D. A.; Catalfamo, D.; Bradaschia Correa, V.; Arana Chavez, V.; Caridad, A. R.; Neubert, J. K.; Wronski, T. J.; Wallet, S. M.; Holliday, L. S. J Biol Chem 2012 287 17894 17904. (217) Ke, H. Z.; Qi, H.; Chidsey Frink, K. L. ; Crawford, D. T.; Thompson, D. D. J Bone Miner Res 2001 16 765 773. (218) Gauthier, M. A.; Klok, H. A. Polym Chem 2010 1 1352 1373. (219) Wang, J. S.; Matyjaszewski, K. J Am Chem Soc. 1995 117 5614 5615. (220) Nicolas, J.; Miguel, V. S.; Man tovani, G.; Haddleton, D. M. Chem. Commun. 2006 4697 4699. (221) Li, X.; Wang, L.; Chen, G.; Haddleton, D. M.; Chen, H. Chem. Commun. 2014 50 6506 6508. (222) Chen, M.; Zhong, M.; Johnson, J. A. Chem Rev 2016 116 10167 10211. (223) Dadashi Silab, S.; Doran, S.; Yagci, Y. Chem Rev 2016 116 10212 10275. (224) McKenzie, T. G.; Fu, Q.; Uchiyama, M.; Satoh, K.; Xu, J.; Boyer, C.; Kamigaito, M.; Qiao, G. G. Adv. Sci. 2016 3 1500394. (225) Otsu, T.; Yoshida, M.; Tazaki, T. Makromol. C hem., Rapid Commun. 1982 3 133 140. (226) Carmean, R. N.; Becker, T. E.; Sims, M. B.; Sumerlin, B. S. C hem 2017 2 93 101. (227) Chen, M.; MacLeod, M. J.; Johnson, J. A. ACS Macro Lett 2015 4 566 569. (228) Fu, Q.; McKenzie, T. G.; Tan, S.; Nam, E.; Qiao, G. G. Polym Chem 2015 6 5362 5368. (229) Allegrezza, M. L.; DeMartini, Z. M.; Kloster, A. J.; Digby, Z. A.; Konkolewicz, D. Polym Chem 2016 7 6626 6636. (230) McKenzie, T. G.; Fu, Q.; Wong, E. H. H.; Dunstan, D. E.; Qiao, G. G. Macromolecules 2015 48 3864 3872. (231) Xu, J.; Jung, K.; Atme, A.; Shanmugam, S.; Boyer, C. J Am Chem Soc 2014 136 5508 5519. (232) Xu, J.; Jung, K.; Corrigan, N. A.; Boyer, C. Chem Sci 2014 5 3568 3575.

PAGE 151

151 (233) Shanm ugam, S.; Xu, J.; Boyer, C. Chem Sci 2015 6 1341 1349. (234) Shanmugam, S.; Xu, J.; Boyer, C. J Am Chem Soc 2015 137 9174 9185. (235) Xu, J.; Shanmugam, S.; Duong, H. T.; Boyer, C. Polym Chem 2015 6 5615 5624. (236) Corrigan, N.; Shanmugam, S.; Xu, J.; Boyer, C. Chem Soc Rev 2016 45 6165 6212. (237) Yeow, J.; Xu, J.; Boyer, C. ACS Macro Lett 2015 4 984 990. (238) Yeow, J.; Shanmugam, S.; Corrigan, N.; Kuchel, R. P.; Xu, J.; Boyer, C. Macromolecules 2016 49 7 277 7285. (239) Zhou, H.; Johnson, J. A. Angew Chem Int E d. 2013 52 2235 2238. (240) Avens, H. J.; Bowman, C. N. J Polym Sci ., Part A: Polym Chem 2009 47 6083 6094. (241) Baker, D. P.; Lin, E. Y.; Lin, K.; Pellegrini, M.; Petter, R. C.; Chen, L. L.; Arduini, R. M.; Brickelmaier, M.; Wen, D.; Hess, D. M.; Chen, L.; Grant, D.; Whitty, A.; Gill, A.; Lindner, D. J.; Pepinsky, R. B. Bioconjugate Chem 2006 17 179 188. (242) MacDonald, J. I.; Munch, H. K.; Moore, T.; Francis, M. B. Nat. Chem. Biol. 2 015 11 326 331. (243) Coca, S.; Jasieczek, C. B.; Beers, K. L.; Matyjaszewski, K. J Polym Sci ., Part A: Polym Chem 1998 36 1417 1424. (244) Laemmli, U. K. Nature 1970 227 680 685. (245) Chem. Commun. 2008 4183 4185.

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152 BIOGRAPHICAL SKETCH Bryan Tucker was born in Chickamauga, Georgia, a small town approximately 100 miles northwest of Atlanta. He grew up in Chickamauga, graduating from Gordon Lee High School in 2008 before attending Kennesaw State University in Kennesaw, Georgia. There he ea rned a Bachelor of Science degree in biochemistry in 2012. During his undergraduate studies, he conducted research in the laboratory of Prof. Greg Gabriel, where he studied water soluble polymers for antifouling surfaces. In the summer of 2011, he complete d a Research Experience for Undergraduates program at Texas A&M University, where he conducted research in the laboratory of Prof. Karen Wooley under the mentorship of Dr. Phil Imbesi. It was here where he first decided doctoral studies might be right for him. In 2012 he began the doctoral program in the Department of Chemistry at the University of Florida under the direction of Prof. Brent Sumerlin. His research focused on using controlled polymerization methods to generate polymer materials for biomedical applications. He completed his Ph.D. in chemistry in spring 2017. While studying at Kennesaw State, Bryan met his future wife, Caitlin, and they married in 2012. Together they have an energetic Australian Shepherd, and enjoy outdoor activities, including hiking and beach trips. Bryan and Caitlin love to travel, have a passion for culture, and enjoy good food and wine.