Conjugates of Gold Nanoparticles and Cross-linked Polynorbornenes: Enhancement of Nanoparticle Stability and Templated Synthesis of Polymer Nanocapsules By Xiang Liu B.S., University of Science and Technology of China, 2004 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Chemistry at Brown University Providence, Rhode Island May 2010 © Copyright 2009 by Xiang Liu     This dissertation by Xiang Liu is accepted in its present form by the Department of Chemistry as satisfying the dissertation requirement for the degree of Doctor of Philosophy. Date_____________________ ____________________________________ Professor Amit Basu, Advisor Recommended to the Graduate Council Date_____________________ ____________________________________ Professor Matthew Ben Zimmt, Reader Date_____________________ ____________________________________ Professor Paul Gregory Williard, Reader Approved by the Graduate Council Date_____________________ ____________________________________ Sheila Bonde, Dean of the Graduate School iii    CURRICULUM VITAE Born in Lasa, Xizang, China on 25 July 1981, Xiang Liu grew up and was educated in Chengdu, Sichuan, China, graduating from Shude High School in 1996. His study in chemistry began at the University of Science and Technology of China (USTC) in 1999, culminating in a Bachelor of Science degree. He was awarded Outstanding Student Award (Gold Prize) for 4 consecutive years from 2000 to 2003. In 2004, he graduated with the highest glory of USTC, Guo Moruo Scholarship which was named after the first president of the USTC. He spent two years in the Laboratory of Nanoscience and Nanotechnology under the guidance of Professor Yi Xie. The work focused on the synthesis of cadmium sulfide and copper sulfide nanoparticles. Following the completion of that work, Xiang started doctoral studies in 2004 in the Department of Chemistry at Brown University under the supervision of Professor Amit Basu, which leads to this dissertation. At Brown University, Xiang has been awarded Wernig Fellowship in 2007, Dissertation Fellowship in 2008 and Sigma Xi Award of Excellence in Graduate Research in 2009. He has also been recognized for his work in chemistry at the American Chemical Society with Excellence in Graduate Polymer Research in 2009. He was a teaching assistant from 2004 to 2006, teaching Inorganic Chemistry (CHEM 33), Organic Chemistry I (CHEM 35), Organic Chemistry II (CHEM 36) and Advanced Organic Chemistry (CHEM 145). He has been a member of the American Chemical Society since 2006. iv Publications and Presentations (1) Liu, X.; Basu, A., Core Functionalization of Hollow Polymer Nanocapsules. J. Am. Chem. Soc., 2009, 131, (16), 5718-5719 (2) Liu, X.; Basu, A., Pyrene Functionalized Hollow Polymer Capsules. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.), 2009, 50 (1), 55. (3) Liu, X.; Basu, A., Pyrene Functionalized Hollow Polymer Capsules 2009, 237th ACS National Meeting, Salt Lake City, UT. (4) Liu, X.; Basu, A., Cross-Linked Polynorbornene-Coated Gold Nanoparticles: Dependence of Particle Stability on Cross-Linking Position and Cross-Linker Structure. Langmuir, 2008, 24, (19), 11169-11174. (5) Liu, X.; Basu, A., Impact of Polymer Cross-linking on the Stability of Nanoparticles. 2008, 235th ACS National Meeting, New Orleans, LA. (6) Liu, X.; Basu, A., Olefin Metathesis on Nanostructures. J. Organomet. Chem. 2006, 691 (24-25), 5148-5154. v PREFACE AND ACKNOWLEDGEMENTS The completion of my Ph.D. study has been a reflection of how scientific research is done. Research is never a creation of one person. At Brown University, I was always surrounded by knowledgeable professors, intellectual collaborators and enthusiastic friends. Without their generous support, this dissertation would never come into being. First of all, I must thank my advisor, Professor Amit Basu. His has provided the opportunity and support (moral and financial) to pursue my own path in becoming a competent chemist. From my first contact with him in 2004 in the class of Organic Reactions, he has shown broad knowledge in chemistry and biochemistry. I have enjoyed working with him and learning from him. Secondly, I must thank the group members, especially Dr. Anand Rai and Dr. Nicole Seah for sharing their valuable experience on organic synthesis. They taught me how to set up organic reactions, purify the products by column chromatography, and analyze the structures using various techniques. In terms of technical support, I must thank Dr. Russell Hopson for his help on NMR spectroscopy, Dr. Tun-Li Shen on mass spectroscopy, and Anthony McCormick on electronic microscopy. Their analytical help is gratefully acknowledged. My friends at Brown University have been particularly kind and helpful. Dr. Jin Xie and Dr. Sheng Peng spent a lot time helping me take TEM images, Dr. Zhenbo Ma and Dr. Shuangbin Han helped me on thermogravimetric analysis, and Xinyuan Liu is helpful on obtaining DLS data. Assistance from Dr. Fengtian Xue, Dr. Deyu Li, Dr. vi  Yanhu Wei, Sa Wang, and Wenjun Tong is recognized. I wish them great success in the future. I am grateful to Professor Matthew Zimmt and Professor Paul Williard for being my committee members. I also appreciate their support when I looked for post-doctoral positions. Particularly importantly, I must thank the Wernig Fellowship and Dissertation Fellowship for the financial support. Lastly, many thanks to my mother and father! vii  TABLE OF CONTENTS Preliminary Pages Curriculum Vitae…………………………………………………………………………iv Preface and Acknowledgements……………………………………………….…………vi Table of Contents………………………………………………………………………..viii List of Tables……………………………………………………………………………..xi List of Figures………………………………………………...………………………….xii List of Abbreviations……………………………………………………………………xix Chapter 1 Cross-linked polymer shells at the surface of nanoparticles and the enhancement of nanoparticle stability 1.1 Introduction………………………………………………………………………..1 1.1.1 Structure and stability of nanoparticles……………………………………1 1.1.2 Assembly of polymers to the surface of nanoparticles……………………3 1.1.3 Effect of cross-linking on nanoparticle stabilities……………………….10 1.2 Preparation of cross-linked polymers coated gold nanoparticles………………..14 1.2.1 Strategy on the synthesis of cross-linked polymer coated nanoparticles..14 1.2.2 Synthesis of the norbornene-derivatized thiol and gold nanoparticles…..15 1.2.3 Synthesis of monomers…………………………………………………..21 1.2.4 Immobilization of catalysts to the surface of gold nanoparticles………..24 1.2.5 Surface-initiated ROMP of block copolymers at gold nanoparticles……26 viii  1.2.6 Cross-linking……………………………………………………………..32 1.3 Study of nanoparticle stability affected by ligand structures…………………….36 1.3.1 Effect of cross-linking positions…………………………………………37 1.3.2 Effect of cross-linker strucutures………………………………………...41 1.3.3 Effect of polymer chain length…………………………………………..42 1.4 Conclusion……………………………………………………………………….43 1.5 Experimental……………………………………………………………………..45 Chapter 2 Functionalized polymer nanocapsules as a potential drug delivery vehicle 2.1 Introduction………………………………………………………………………57 2.1.1 Preparation of polymer nanocapsules via self-assembly approach………57 2.1.2 Preparation of polymer nanocapsules via template approach……………62 2.2 Synthesis of hollow polymer nanocapsules using gold nanoparticle templates…68 2.3 Core functionalization through disulfide exchange reactions……………………75 2.3.1 Core functionalization with pyrene………………………………………76 2.3.2 Core functionalization with ferrocene…………………………………...79 2.4 Encapsulation and release of small molecules based on charge interactions……83 2.5 Encapsulation of inorganic cations and gold nanoparticles……………………...92 2.6 Functionalization in the cross-linking domain via SN2 substitution……………..95 2.7 Kinetic study on encapsulation and release processes………………………….101 2.7.1 Effect of cross-linker structures………………………………………...102 2.7.2 Effect of cross-linking positions………………………………………..114 ix  2.7.3 Effect of environmental pH…………………………………………….115 2.8 Conclusion…………………………………………………………………….117 2.9 Experimental……………………………………………………………………117 Appendix 1 Structures of other cationic dyes for encapsulation study …………133 1 Appendix 2 H-NMR and 13C-NMR spectra of synthetic compounds…………...134 x  LIST OF TABLES Table 1.2.1 Different norbornene derivatives for ROMP Table 1.3.1a T1 relaxation time (ms) of olefinic peaks at 5.23 ppm Table 1.3.1b T1 relaxation time (ms) of olefinic peaks at 5.36 ppm Table 1.3.2 Hydrodynamic diameter (nm) of different cross-linked polymer- nanoparticle conjugates by DLS Table 1.3.3 tlag [s] (k [×10-3 s-1]) Table 2.2.1 Water contact angles of hollow polymer capsules and analogues Table 2.3.1 Excimer to monomer emission ratio Ie/Im for pyrene functionalized nanocapsules and polymers in different solutions Table 2.3.2 Concentrations of Fe and S standard solutions for ICP measurement Table 2.7.1 T1 values of hollow polymer capsules with different cross-linking positions xi  LIST OF FIGURES Figure 1.1.1 Schematic structure of butanethiol coated gold nanoparticles Figure 1.1.2 Cyanide-induced decomposition of alkanethiol coated gold nanoparticles Figure 1.1.3 Cyanide-induced decomposition rates of gold nanoparticles coated by different alkanethiol ligands Figure 1.1.4 Cyanide-induced decomposition of PEG-AuNP and C12-AuNP Figure 1.1.5 Surface functionalization of nanoparticles with polymers Figure 1.1.6 Olefin metathesis reactions Figure 1.1.7 Mechanism of ROMP Figure 1.1.8 ROMP of ferrocene functionalized polynorbornene block copolymer at nanoparticle surface Figure 1.1.9 ROMP using cyclooctene at nanoparticle surface Figure 1.1.10 Shell cross-linked gold nanoparticles by amidation Figure 1.1.11 Photo-cross-linked gold nanoparticles showing enhanced stability towards cyanide etching Figure 1.1.12 Dendritic nanoparticles cross-linked by cross-metathesis Figure 1.1.13 Absorbance of CdSe quantum dots upon the treatment of H2O2 Figure 1.1.14 Location change of the cross-linking domain at nanoparticle surfaces Figure 1.2.1 Preparation of cross-linked polymer coated gold nanoparticles Figure 1.2.2 Synthesis of the norbornene-derivatized thiol ligand Figure 1.2.3 Synthesis of gold nanoparticles with norbornene at surfaces xii  Figure 1.2.4 Hydrodynamic size of Au-13/14 by DLS Figure 1.2.5 TEM of Au-13/14 (top) and size distribution (bottom) 1 Figure 1.2.6 H-NMR of the mixture of 13 and 14 cleaved from Au-13/14 Figure 1.2.7 TGA result of Au-13/14 Figure 1.2.8 Calculation of the amount of norbornene in Au-13/14 Figure 1.2.9 Synthesis of monomer 20 Figure 1.2.10 Steric effects on endo and exo isomer of norbornene derivatives Figure 1.2.11 Synthesis of exo isomers of norbornene derivatives Figure 1.2.12 Immobilization of Grubbs’ catalyst to the surface of nanoparticles Figure 1.2.13 1H-NMR of Au-13/14 with Grubbs’ catalyst immobilized at surface Figure 1.2.14 Synthesis of block copolymers by ROMP Figure 1.2.15 Structure of surface grafted block copolymers Figure 1.2.16 Preparation of linear polymers for structural characterization Figure 1.2.17 1H-NMR of p-methoxybenzylamine quenched HS-20102530 Figure 1.2.18 1H-NMR of p-methoxybenzylamine quenched HS-252020102510 Figure 1.2.19 MALDI-ToF MS of p-methoxybenzylamine quenched HS-20102530 Figure 1.2.20 MALDI-ToF MS of p-methoxybenzylamine quenched HS-252020102510 Figure 1.2.21 Side reactions (backbiting and cross-metathesis) during ROMP Figure 1.2.22 Structures of diamine cross-linkers Figure 1.2.23 Capping unreacted ester and amine Figure 1.2.24 1H-NMR of Au-20102530 (top) and Au-252020102510 (bottom) after TEG cross-linking and p-methoxybenzylamine capping xiii  Figure 1.2.25 1H-NMR of polymer-nanoparticle conjugates after cross-linking and allyl isocyanate capping. Top: Au-20102530 cross-linked by 0.5 eq. of TEG; middle: Au-252020102510 cross-linked by 0.5 eq. of TEG; bottom: Au- 20302530 treated by 1 eq. of C8 Figure 1.2.26 UV-vis of nanoparticles in dichloromethane Figure 1.2.27 TEM of Au-20102530 crosslinked by C8 diamine Figure 1.3.1 Cyanide mediated decomposing rate of polymer-nanoparticle conjugates cross-linked with given diamines at different positions Figure 1.3.2 Plots of dA/dt vs time for cross-linked polymer-nanoparticle conjugates: a) TEG; b) C8; c) C4; d) Cy Figure 1.3.3 Cyanide mediated decomposing rate of polymer-nanoparticle conjugates cross-linked at given positions with different diamines Figure 1.3.4 Cyanide mediated decomposing rate of nanoparticle with different length of polymers Figure 2.1.1 Cross-linking of PMOXA-PDMS-PMOXA triblock vesicles by radical polymerization of methacrylate end groups Figure 2.1.2 Preparation of polymer nanocapsules by polymerization of monomers incorporated in the lipid bilayer Figure 2.1.3 Hollow polymer capsules by PI-b-PAA micelles Figure 2.1.4 Hollow polymer nanocapsules by PI-b-PCEMA-b-PTBA micelles Figure 2.1.5 Polymer capsules by layer-by-layer adsorption of polyelectrolyte Figure 2.1.6 AuNP templated synthesis of pNIPAm nanogels Figure 2.1.7 Synthesis of pH-sensitive polymer capsules using a AuNP template xiv  Figure 2.1.8 Selective functionalization of polymer capsules within the capsule or throughout the shell Figure 2.2.1 Strategy for the synthesis of hollow polymer capsules Figure 2.2.2 Nominal structure of p-methoxybenzylamine quenched HS-2030-block- 2530 Figure 2.2.3 MALDI spectrum of p-methoxybenzylamine quenched HS-2030-block- 2530 1 Figure 2.2.4 H-NMR spectrum of p-methoxybenzylamine quenched HS-2030-block- 2530 1 Figure 2.2.5 H NMR spectrum of cross-linked Au-20302530 after capping with p- methoxybenzylamine and allyl isocyanate Figure 2.2.6 TEM image of Au-25200 Figure 2.2.7 Size of HS-capsules by DLS Figure 2.2.8 TEM image of HS-capsules and size distribution Figure 2.3.1 Functionalization of the capsule hollow core Figure 2.3.2 Synthesis of pyrene derivatized disulfide Figure 2.3.3 Fluorescence spectra of pyrene functionalized capsules (λexcitation = 338 nm) (a) compound 35; (b) pyrene functionalized capsules using 35; (c) pyrene functionalized capsules after treatment with tributylphosphine; (d) pyrene functionalized capsules after treatment with tributylphosphine followed by dialysis. Inset: UV-vis absorption of pyrene functionalized capsules Figure 2.3.4 Fluorescence of pyrene functionalized capsules in different solvents Figure 2.3.5 Synthesis of ferrocene derivatized disulfides xv  Figure 2.3.6 TEM images of capsules functionalized ferrocene in the core Figure 2.4.1 Synthesis of allyl, carboxylate and amine derivatized disulfides Figure 2.4.2 Functionalization of the capsule core with COOH Figure 2.4.3 Size (diameter) of COOH and NH2 functionalized capsules by DLS Figure 2.4.4 Structure of alizarin red s (ARS) and methylene blue (MB) Figure 2.4.5 Uptake and release of ARS. a) ARS mixed with NH2-capsules; b) ARS mixed with COOH-capsules; c) ARS incorporated NH2-capsules mixed with water; d) ARS incorporated NH2-capsules mixed with 0.1 M NaOH. Figure 2.4.6 Structure of bromophenol blue Figure 2.4.7 Uptake and release of MB. a) MB mixed with NH2-capsules; b) MB mixed with COOH-capsules; c) MB incorporated COOH-capsules mixed with water; d) MB incorporated COOH-capsules mixed with 0.1 M HCl. Figure 2.4.8 Uptake of ARS and MB in different polymer capsules Figure 2.4.9 Nominal molecular weight of polymer chain in NH2-capsules Figure 2.4.10 Correlation of ARS concentration and absorbance at 424 nm Figure 2.4.11 Release of ARS from NH2-capsule to different aqueous media Figure 2.4.12 Release of MB from COOH-capsule to different aqueous media Figure 2.5.1 Absorption of gold nanoparticles taken up by hollow polymer capsules Figure 2.5.2 TEM of gold nanoparticles taken up by hollow polymer capsules Figure 2.6.1 Functionalization of the cross-linking domain via amide substitution Figure 2.6.2 Synthesis of pyrene derivatized alkyl bromide Figure 2.6.3 Emission of pyrene functionalized capsules (capsules made from C8 cross-linked Au-20302530 and functionalized with 50 on amide nitrogens) xvi  1 Figure 2.6.4 H-NMR of pyrene functionalized capsules (capsules made from C8 cross- linked Au-20302530 and functionalized with 50 on amide nitrogens) 1 Figure 2.6.5 H-NMR of ferrocene functionalized capsules (capsules made from C8 cross-linked Au-20302530 and functionalized with 51 on amide nitrogens) Figure 2.6.6 Hollow polymer capsules with cross-linking domain in the exterior Figure 2.6.7 TEM of ferrocene functionalized capsules (capsules made from C8 cross- linked Au-25252030255 and functionalized with 51 on amide nitrogens) 1 Figure 2.6.8 H-NMR of ferrocene functionalized capsules (capsules made from C8 cross-linked Au-25252030255 and functionalized with 51 on amide nitrogens) Figure 2.7.1 Use of ARS as a model to study the encapsulation and release kinetics Figure 2.7.2a Amount of unreacted ester and amine in capsules cross-linked by C8 Figure 2.7.2b Amount of unreacted ester and amine in capsules cross-linked by TEG Figure 2.7.2c Amount of unreacted ester and amine in capsules cross-linked by C4 Figure 2.7.2d Amount of unreacted ester and amine in capsules cross-linked by Cy Figure 2.7.3 Uptake of ARS with different concentrations Figure 2.7.4 Equipment setup for the kinetics study Figure 2.7.5 ARS uptake rate into capsules with different cross-linkers Figure 2.7.6 Repeated measurements of ARS uptake rate Figure 2.7.7 UV-vis spectra showing increased baseline Figure 2.7.8 UV-vis spectra of ARS release from C8 cross-linked capsules. Left: kinetics. Right: Full UV-vis absorbance before and after release xvii  Figure 2.7.9 UV-vis spectra of ARS release from TEG cross-linked capsules. Left: kinetics. Right: Full UV-vis absorbance before and after release Figure 2.7.10 UV-vis spectra of ARS release from C4 cross-linked capsules. Left: kinetics. Right: Full UV-vis absorbance before and after release Figure 2.7.11 Equipment setup for monitoring the aqueous layer Figure 2.7.12 Increase of ARS in the aqueous layer during release. (Capsule: C8 cross- linked 20302530) Figure 2.7.13 Release from capsules with different cross-linkers Figure 2.7.14 Effect of cross-linking positions on the uptake kinetics Figure 2.7.15 Release kinetics of capsules with different cross-linking positions Figure 2.7.16 Effect of pH on ARS uptake rate xviii  LIST OF ABBREVIATIONS Ac acetyl Abs absorbance ADMET acyclic diene metathesis polymerization AFM atomic force microscopy ARS alizarin red s ATRP atom transferradical polymerization AuNP gold nanoparticles b block C4 diaminobutane C8 diaminooctane Cy trans-1,4-diaminocyclohexane CM cross metathesis d doublet DCM dichloromethane DLS dynamic light scattering DMF N,N-dimethylformamide DMAP 4-(dimethylamino)pyridine DTAB dodecyltrimethylammonium bromide EDC 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide Et ethyl xix  EtOAc ethyl acetate FAB fast-atom bombardment G1 generation 1 G2 generation 2 GPC gel permeation chromatography HRMS high resolution mass spectroscopy ICP inductively coupled plasma IR infra-red spectroscopy J coupling constant LCST lower critical solution temperature m multiplet MALDI-ToF matrix assisted laser desorption/ionization-time of flight MB methylene blue Me methyl MeOH methanol MF melamine formaldehyde MS mass spectroscopy NMR nuclear magnetic resonance PAA poly(acrylate acid) PAH poly(allylamine hydrochloride) PCEMA poly(2-cinnamoylethyl methacrylate) PDMS poly(dimethylsiloxane) PEG polyethylene glycol xx  ph phenyl PI polyisoprene PMOXA poly(2-methyloxazoline) pNIPAm poly(N-isopropylacrylamide) PS polystyrene PSS poly(sodium styrenesulfonate) PTBA poly(tert-butyl acrylate) PVBP poly(4-vinyl benzophenone) pyr pyridine q quartet RAFT reversible addition-fragmentation polymerization RCM ring closing metathesis ROM ring opening metathesis ROMP ring opening metathesis polymerization s singlet s second SCK shell cross-linked knedel-like SDBS sodium dodecylbenzenesulfonate SEM scanning electron microscopy SI-ROMP surface-initiated ring metathesis polymerization t triplet TEA triethylamine TEG triethylene glycol diamine xxi  TEM transmission electron microscopy TFA trifluoroacetic acid TGA thermogravimetric analysis THF tetrahydrofuran TOPO tri-noctylphosphineoxide Tr triphenylmethyl Ts p-toluenesulfonyl UV qultraviolet vis visible xxii  CHAPTER 1 Cross-linked Polymer Shells at the Surface of Nanoparticles and the Enhancement of Nanoparticle Stability 1.1 Introduction 1.1.1 Structure and stability of nanoparticles Nanoparticles are defined as particles within the size range between 1 and 100 nanometers in diameter.1 Nanoparticles are of great scientific interest as they are effectively a bridge between bulk materials and atomic or molecular structures, and they have found wide application as sensors, catalysts and biomedical diagnostics.2, 3 Due to the nature of the small size, nanoparticles have high surface energy and tend to aggregate. Therefore nanoparticles are frequently coated by a layer of organic ligands at the surface to segregate particles from each other. For example, gold nanoparticles (AuNP) can be stabilized by alkanethiol ligands. The structure of butanethiol coated gold nanoparticles is shown in Figure 1.1.1. Figure 1.1.1 Schematic structure of butanethiol coated gold nanoparticles. (Adapted with permission from ref 1. Copyright 2003 American Chemical Society) 1 2 To characterize the structure of nanoparticles—the conjugates of organic ligands and inorganic core, a variety of techniques are available. Nuclear magnetic resonance spectroscopy (NMR) and infra-red spectroscopy (IR) provide the chemical and structural information of the organic ligands. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) measure the size of the core. DLS (dynamic light scattering) measures the hydrodynamic radius of the conjugates. Ultraviolet-visible spectroscopy (UV-Vis) also provides information about the dimension of nanoparticles, as the absorption wavelength is correlated to the particle size. The stability of nanoparticles is of great interest. Cyanide-induced decomposing of alkanethiol coated gold nanoparticles is widely used as a model to study the stability of nanoparticles, due to the ease of synthesis, characterization and surface functionalization of gold nanoparticles.4-7 Alkanethiol ligands are covalently immobilized at gold surfaces through Au-S bond. After the treatment of cyanide, the Au-S bond breaks, producing alkyl disulfide and gold(I) dicyanide as products (Figure 1.1.2). The decomposition rate of nanoparticles can be monitored by UV-vis spectroscopy and correlates to the protective barrier that the alkanethiol monolayer provides to the gold surface. Figure 1.1.2 Cyanide-induced decomposition of alkanethiol coated gold nanoparticles 3 The structure of the peripheral ligand at nanoparticle surfaces has a considerable effect on nanoparticle stability.4 Cyanide-induced decomposition rates of gold nanoparticles coated by different alkanethiol ligands are shown in Figure 1.1.3. Nanoparticle concentrations were correlated to UV-vis absorption. When gold nanoparticles were coated by different butanethiol isomers (n-C4, iso-C4 and sec-C4), n- C4 coated gold nanoparticles were etched by cyanide most rapidly. In other words, sterically bulkier iso-C4 and sec-C4 provided better protection to the nanoparticles. When gold nanoparticles were coated by alkanethiols with different chain length (C4, C8 and C12), the nanoparticle stability increased along with the ligand chain length. Figure 1.1.3 Cyanide-induced decomposition rates of gold nanoparticles coated by different alkanethiol ligands (Adapted with permission from ref 4. Copyright 1998 American Chemical Society) 1.1.2 Assembly of polymers to the surface of nanoparticles Since larger ligands provide better protection to the nanoparticles, polymers are frequently used as stabilizing agents.8 Polyethylene glycol (PEG) coated gold 4 nanoparticles were synthesized using thiol terminated PEG polymers (PEG-SH, MW 5000). UV-vis absorbance at 340 nm was recorded to represent the concentration of 2.8 nm AuNP. Compared to dodecanethiol coated gold nanoparticles (C12-AuNP), PEG-SH coated gold nanoparticles (PEG-AuNP) showed enhanced stability towards cyanide etching (Figure 1.1.4), as the absorbance dropped slowly. Figure 1.1.4 Cyanide-induced decomposition of PEG-AuNP and C12-AuNP. (Adapted with permission from ref 8. Copyright 1998 American Chemical Society) Approaches for modifying nanoparticles with polymers include ‘grafting onto’ and ‘grafting from’.9, 10 The difference between ‘grafting into’ and ‘grafting from’ is illustrated in Figure 1.1.5.11 The ‘grafting onto’ method involves the addition of an already synthesized polymer to a nanoparticle which is not stabilized or is stabilized with a labile ligand. The ‘grafting from’ method is also called surface-initiated polymerization. It involves immobilization of a monomer or initiator at the nanoparticle surface followed by an outward, or radial, polymerization. Several approaches for surface-initiated 5 polymerization at gold nanoparticles have been reported, including atom transfer radical polymerization (ATRP)12, 13, reversible addition-fragmentation transfer (RAFT)14 or ring opening metathesis polymerization (ROMP).15, 16 m gr f ro af tin ng g ti af on gr to Figure 1.1.5 surface functionalization of nanoparticles with polymers (Adapted with permission from ref 16. Copyright 2006 Springer) Olefin metathesis is an important branch of organic synthesis. The transformation can be generally considered as rearrangement of double bonds. As shown in Figure 2.5, olefin metathesis reaction can be categorized to ring-closing metathesis (RCM), acyclic diene metathesis (ADMET), ring-opening metathesis polymerization (ROMP), ring- opening metathesis (ROM) and cross-metathesis (CM)17. The Grubbs’ catalysts are widely used in olefin metathesis reactions. The structure of the first generation of the Grubbs’ catalysts is shown in Figure 1.1.8 (compound 1). 6 Figure 1.1.6 Olefin metathesis reactions (Adapted with permission from ref 17. Copyright 2001 American Chemical Society) Among the subcategories of olefin metathesis, ROMP is widely used to functionalize surfaces at micro- or nano-scale. The mechanism of ROMP is summarized in Figure 1.1.7.18 Initiation begins with the coordination of a metal catalyst to a cyclic alkene. A metallacyclobutane intermediate is generated through [2+2] cycloaddition. This intermediate undergoes a cycloreversion reaction to generate a new metal stabilized carbene (alkylidene). As the new metal complex has similar reactivity to the original initiator, analogous steps are repeated in the propagation stage. When all monomers are consumed or the reaction is quenched by the addition of specialized reagents, the polymerization is terminated. For most Ru mediated ROMP, ethyl vinyl ether, which provides a [Ru]=CHOEt type complex and a methylidene end-functionalized polymer, is used to quench the reaction. 7 Figure 1.1.7 Mechanism of ROMP (Adapted with permission from ref 18. Copyright 2001 American Chemical Society) Mirkin and Nguyen showed that gold nanoparticles coated with a norbornene functionalized alkane thiol 2 could be used to initiate polymer growth outward from the particle surface (Figure 1.1.8).15 The norbornene particles were treated with 1 equivalent of 1 to immobilize an active ruthenium alkylidene on the particle, followed by addition of a ferrocene linked norbornene and subsequent termination with ethyl vinyl ether. Block copolymers were prepared by the sequential addition of two different norbornenes, 3 and 4. Purification of the polymer nanoparticle hybrids was facilitated by the differential solubility of the monolayer coated nanoparticle and the polymer functionalized particle. The progress of the reaction was monitored by 1H-NMR, since alkene protons in 3 and 4 showed different chemical shift from alkene protons in poly3 and poly4. 8 Figure 1.1.8 ROMP of ferrocene functionalized polynorbornene block copolymer at nanoparticle surface (Adapted with permission from ref 15. Copyright 1999 American Chemical Society) A similar strategy was reported by Coughlin and Emrick, using cadmium selenide nanoparticles (Figure 1.1.9).19 A styrene modified trialkyl phosphine oxide, 5, was assembled to tri-noctylphosphineoxide (TOPO) coated CdSe nanoparticles by ligand exchange. Treatment of the styrene coated particles with catalyst 1 followed by cyclooctene generates a polymer coated quantum dot composite. Loading of the catalyst on the nanoparticle surface was monitored by 1H-NMR, which clearly resolved the alkylidene protons of the free catalyst and the various particle bound species. The benzylidene proton in free catalyst 1 (highlighted in red in Figure 1.1.9) had a resonance at 19.97 ppm. After the addition of 1 to a solution of 5 coated nanoparticles, the chemical shift of the particle bound benzylidene protons (highlighted in blue) moved to 19.91 ppm. 9 Dicyclopentadiene and oxanorbornene derivatives were also successfully polymerized. The absorption and emission spectra of the quantum dots were not affected when the particles were embedded in the polymer matrix. However, the product must be dialyzed extensively to completely remove the ruthenium from the composite, as residual metal can quench particle-derived fluorescence. The particles remain well dispersed within the polymer, and aggregation was not observed. Figure 1.1.9 ROMP using cyclooctene at nanoparticle surface (Adapted with permission from ref 19. Copyright 2002 American Chemical Society) 10 1.1.3 Effect of cross-linking on nanoparticle stabilities Besides ligand length, ligand cross-linking also impacts nanoparticle stability.20-23 Figure 1.1.10 shows the self-assembly of polystyrene-block-poly(acrylate acid) (PS-b- PAA) copolymers around gold nanoparticles. In the core-shell architecture, hydrophobic PS block is in the interior and hydrophilic PAA block is in the exterior. The PAA moiety was cross-linked by 2, 2’-(ethylenedioxy)bis(ethylamine) via the coupling reaction using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). The shell cross-linked gold nanoparticles exhibit enhanced stability towards cyanide etching.21 Figure 1.1.10 Shell cross-linked gold nanoparticles by amidation (Adapted with permission from ref 21. Copyright 2001 Wiley) Taton also demonstrated the enhancement of nanoparticle stability by cross- linking (Figure 1.1.11).22 Photo-cross-linkable copolymer [polystyrene-co-poly(4-vinyl benzophenone)]-block-poly(acrylic acid) [(PS-co-PVBP)-b-PAA] was self-assembled to gold nanoparticle surfaces, and the PVBP moiety was cross-linked by UV irradiation. When the degree of cross-linking increased with longer time of UV irradiation, the continuously enhanced nanoparticle stability was observed towards cyanide etching. 11 Figure 1.1.11 Photo-cross-linked gold nanoparticles showing enhanced stability towards cyanide etching (Adapted with permission from ref 22. Copyright 2007 American Chemical Society) Another example of the enhancement of nanoparticle stability by cross-linking is by Chechik.23 Figure 1.1.12 shows the immobilization of alkene terminated dendrimers (6 or 7) to gold nanoparticle surfaces. The surface alkenes were cross-linked by cross- metathesis, and the cross-linked gold nanoparticles exhibited enhanced stabilities towards heat and cyanide etching. The enhanced stability induced by cross-linking was observed on both generations (G1 and G2) of the dendritic ligands coated gold nanoparticles. 12 Figure 1.1.12 Dendritic nanoparticles cross-linked by cross-metathesis (Adapted with permission from ref 23. Copyright 2007 American Chemical Society) Peng used a similar strategy to cross-link dendritic ligands at the surface of CdSe quantum dots.24 The octa-alkene derivatized thiol ligand was assembled on nanoparticles, and after cross-metathesis using 1, the encapsulated nanostructures showed improved stability towards chemical, photochemical, and thermal degradation. Figure 1.1.13 shows that the cross-linking by cross-metathesis enhanced the stability towards strong oxidant. The cross-linked quantum dots were also more stable towards strong acid HCl, demonstrated by UV-vis spectroscopy. 13 Figure 1.1.13 Absorbance of CdSe quantum dots upon the treatment of H2O2 (Adapted with permission from ref 24. Copyright 2003 American Chemical Society) While previous studies established a correlation between the degree of cross- linking and particle stability, we were interested in how the location of cross-linking influences nanoparticle stabilities, as depicted in Figure 1.1.14. Cross-linked polymer shells with controlled cross-linking locations were coated at the surface of gold nanoparticles. The stability of gold nanoparticles was then evaluated by cyanide- mediated etching. The results provide new insight into how fine-tuning the polymer cross-linking architecture can modulate nanoparticle stability. Figure 1.1.14 Location change of the cross-linking domain at nanoparticle surfaces 14 1.2 Preparation of cross-linked polymers coated gold nanoparticles 1.2.1 Strategy on the synthesis of cross-linked polymer coated nanoparticles The general strategy on the synthesis of cross-linked polymer coated gold nanoparticles and the control over cross-linking locations are shown in Figure 1.2.1. Surface-initiated ROMP, a well developed method to assemble functional block copolymers to nanoparticles, was chosen as the method to synthesize the polymer backbones. A norbornene derivatized thiol ligand was immobilized at gold nanoparticles during the synthesis of gold nanoparticles. After the treatment of the Grubbs’ catalyst (generation II), the ruthenium stabilized benzylidene was bound to nanoparticles. The addition of appropriate norbonene derivatives resulted cross-linkable linear block copolymers. The location of the cross-linking was controlled by the addition sequence of the monomers. In Figure 1.2.1, the block for cross-linking is highlighted in red, and the inert block is in blue. Lastly, the stability of gold nanoparticles after ligand cross-linking was evaluated by cyanide-mediated etching. Figure 1.2.1 Preparation of cross-linked polymer coated gold nanoparticles 15 1.2.2 Synthesis of the norbornene-derivatized thiol and gold nanoparticles The synthesis of gold nanoparticles has been well developed. Aqueous soluble gold nanoparticles capped by citrate were first reported by Turkevich in 195125 and modified by Frens in 1973.26 Typically, this method is used to synthesize spherical gold nanoparticles around 10-20 nm in diameter. To produce larger particles, less citrate should be used, but the nanoparticles may loss the spherical shape. Another method by Brust and Schiffrin in 1994 produces alkanethiol coated gold nanoparticles that could be dispersed in organic solutions.27 In this method, AuCl4¯ is transferred to toluene using tetraoctylammonium bromide as the phase-transfer reagent and reduced by NaBH4 in the presence of alkanethiol. The organic phase changes color from orange to deep brown within a few seconds upon addition of NaBH4, indicating the formation of gold nanoparticles. Control over the particle size can be easily achieved by tuning the relative amount of alkanethiol to AuCl4¯. Occasionally, other stabilizing ligands are used for gold nanoparticle synthesis.28 To prepare the norbornene-functionalized gold nanoparticles, a norbornene- derivatized thiol 13 was synthesized (Figure 1.2.2). The bromide in 8 was substituted by triphenylmethyl mercaptan (TrSH) to give 9 as a protected thiol. The hydroxyl group in 8 was activated by p-toluenesulfonyl chloride (TsCl), and the resulted 10 was attacked by 11 in the presence of sodium hydride. 12 was formed as a norbornene derivative. 12 was then deprotected by TFA in the presence of triethylsilane (Et3SiH) to give 13, a norbornene-derivatized thiol. The products at each step were purified by flash chromatography, and the overall yield over 4 steps was about 36%. As 11 was used as a mixture of endo and exo isomers, the final product 13 is consequently a mixture. 16 Figure 1.2.2 Synthesis of the norbornene-derivatized thiol ligand With this norbornene-derivatized thiol 13 in hand, gold nanoparticles were prepared using a slightly modified Brust method (Figure 1.2.3).15 Gold nanoparticles were synthesized by the reduction of HAuCl4 using NaBH4 in the presence of 13 and 1- dodecanethiol 14. Gold nanoparticles coated by a mixture of 13 and 14 are named as Au- 13/14. Dodecanethiol was used as a co-ligand because gold nanoparticles coated by only 13 were not soluble in common organic solvents including chloroform, dichloromethane, tetrahydrofuran, hexanes and toluene. This low solubility was also noticed by Mirkin and Reinhoudt.15, 29 This could be attributed to the low packing density of alkanethiol chains due to the steric hindrance from the norbornene headgroups. After the formation, Au- 13/14 was precipitated by the addition of methanol, washed by methanol while in a bath sonicator, and stored as a solution in dichloromethane. 17 Figure 1.2.3 Synthesis of gold nanoparticles with norbornene at surfaces The particle size was measured by DLS and TEM. The hydrodynamic diameter of the Au-13/14 was 7.7 ± 0.8 nm by DLS, as shown in Figure 1.2.4. Samples for TEM analyses were prepared by drying dichloromethane solutions (absorbance@512 nm ~ 0.5) of the particles on amorphous carbon coated copper grids (400 mesh). As shown in Figure 1.2.5, the average diameter of Au-13/14 is 3.6 ± 0.6 nm. Figure 1.2.4 Hydrodynamic size of Au-13/14 by DLS 18 35 30 25 particle number 20 15 10 5 0 1 2 3 4 5 6 7 particle diameter (nm) Figure 1.2.5 TEM of Au-13/14 (top) and size distribution (bottom) 19 Since the two thiol ligands, norbornene-thiol 13 and dodecanethiol 14, had characteristic peaks with different chemical shift, the molar ratio of the ligands was determined by 1H-NMR integration. Since the particle bound ligands showed broad peaks in 1H-NMR spectra which could not be accurately integrated, the thiol ligands was cleaved off the particle surface by cyanide etching prior to NMR analysis. Typically, etching was carried out by mixing nanoparticles in dichloromethane with an aqueous solution of sodium cyanide. The mixture was stirred at room temperature until the organic layer turned from black to colorless or light yellow. The organic layer was separated and washed by saturated sodium bicarbonate and water. After drying over granular anhydrous Na2SO4, the organic solution containing the thiol mixture was concentrated and redissolved in CDCl3 for 1H-NMR measurement. As each 14 had 3 protons on methyl (0.9 ppm) and each 13 had 2 olefinic protons on norbornene (around 6 ppm), the molar ratio of 13 to 14 was 1:2.62 (Figure 1.2.6). The calculation is shown in the inset equation. Figure 1.2.6 1H-NMR of the mixture of 13 and 14 cleaved from Au-13/14 20 The weight fraction of organic components in Au-13/14 was measured by TGA. In Figure 1.2.7, X axis is the temperature, and the heating rate, the percentage of weight loss and the derivative weight loss is highlighted in red, green and blue, respectively. Control experiment on pure 13 coated AuNP and pure dodecanethiol 14 coated AuNP both showed significant weight loss at 214 oC, so the peak at 214 oC in Figure 1.2.7 was attributed to the hybrid thiols. The weight fraction of the thiol ligands was about 17.3%. Figure 1.2.7 TGA result of Au-13/14 Knowing the molar ratio of the two thiol ligands (1:2.6) and the collective weight fraction (17.3%) in the Au-13/14, the exact amount of norbornene groups could be calculated (Figure 1.2.8). As a result, every 1 gram Au-13/14 contained 0.214 mmol norbornene groups at the surface. Figure 1.2.8 Calculation of the amount of norbornene in Au-13/14 21 1.2.3 Synthesis of monomers Before ROMP at nanoparticle surfaces, polymerization of different monomers (norbornene derivatives) was carried out in solution to optimize the reaction conditions. A series of monomers is listed in Table 1.2.1. Compound 11 and 15~19 were used as commercially obtained, and 20 was synthesized (Figure 1.2.9). 2 mole% of the Grubbs catalyst (generation II) was added to the solution of monomers in dichloromethane (0.01 M). After the stirring at room temperature for 20 minutes, excess ethyl vinyl ether was added to quench the reaction. The products were then dried under nitrogen flow and redissolved in CDCl3 for 1H-NMR measurement. The polymerization of 15~17 didn’t proceed, as only monomer peaks (6.2 ppm) were detected instead of polymer peaks (5.2 ppm). During the polymerization of 11, the solution turned from clear to turbid, indicating an insoluble product.In the 1H-NMR spectrum of the crude product, the integration of monomer alkene protons was more than 10 folds of the polymer alkene protons, indicating a large amount of monomer remained unreacted. Polymerization of 18~20 worked well, as only polymer peaks were observed in 1H-NMR spectra. Since 20 easily react with primary amines, it was used as the monomer for the cross-linkable block. Figure 1.2.9 Synthesis of monomer 20 22 Table 1.2.1. Different norbornene derivatives for ROMP Norbornene derivatives Problems towards ROMP Failure towards ROMP Insoluble product (in DCM, THF, DMF or methanol) obtained after ROMP O O None H CH3 18 19 None It has been reported that endo and exo isomers of norbornene derivatives have different reactivity in ROMP.30 For example, exo-dicyclopentadiene was found to be more than an order of magnitude more reactive than endo-dicyclopentadiene.31 It is believed that the cause of the rate difference between exo and endo isomers is primarily sterics. It has been suggested that the formation of the metallacyclobutane ring may be the rate-determining step in metathesis with first generation Grubbs’ catalysts.32, 33 In this case, as the metallacyclobutane is formed, an unfavorable steric interaction may exist between the protons on the newly formed sp3 center and the substituent (Figure 1.2.10). However, because the carbon with star mark in the ester is sp2 hybridized, the steric interaction is not as disfavorable since the sp2 center is planar. Therefore, the polymerization rate of endo and exo isomer of 20 should be similar, and 20 was used as a mixture of endo and exo isomers even if the endo isomer was the major component. 23 Figure 1.2.10 Steric effects on endo and exo isomer of norbornene derivatives But still, it is worth mentioning the exploration of the synthetic methods to enrich the exo isomers from the mixture (Figure 1.2.11). For example, the endo and exo isomers of 5-norbornene-2-carboxaldehyde (18) could be separate by flash chromotography. The treatment of 18 with lithium hydroxide increased the exo isomer fraction in the endo/exo mixture.34 The carboxaldehyde 18 was converted to carboxylic acid using mild oxidant NaClO2 which was unreactive to alkenes. The final product was 15 in its pure exo form. Another method to prepare the pure exo isomer of norbornene derivatives involves norbornadiene 21 as the starting material and norbornenyl acetate 22 as an important intermediate. 35 Figure 1.2.11 Synthesis of exo isomers of norbornene derivatives 24 1.2.4 Immobilization of catalysts to the surface of gold nanoparticles Since the amount of norbornene groups at nanoparticle surface was known, stoichiometric amount of the second generation of Grubbs’ catalyst, 24, was added to a dichloromethane solution of Au-13/14 to activate the nanoparticle surface. The Grubbs’ catalyst opened the norbornene rings and was covalently immobilized to the nanoparticle surface (Figure 1.2.12). The product was dried under nitrogen flow, washed by methanol and redissolved in CDCl3 for 1H-NMR measurement. In 1H-NMR spectrum in Figure 1.2.13, the original peaks around 6.2 ppm for the norbornene olefinic protons were no longer observed due to the ring opening caused by the Grubbs’ catalyst. A new peak appeared at 19.1 ppm for the alkylidene proton as an evidence for the particle bound catalyst. Figure 1.2.12 Immobilization of Grubbs’ catalyst to the surface of nanoparticles 25 Figure 1.2.13 1H-NMR of Au-13/14 with Grubbs’ catalyst immobilized at surface When less than stoichiometric amount of 24 (5 mole% per surface norbornene) was mixed with the Au-13/14, the norbornene olefinic resonances at 6.2 ppm remained after reaction. Because every 1 molecule of 13 was surrounded by 2.6 molecules of 14 at the surface of Au-13/14, the sterics from 14 prevented 13 from laterally reacting with other peripheral norbornenes. In other words, the surface norbornene preferentially reacted with exogenous Grubbs’ catalysts other than the peripheral catalysts due to the sterics from dodecanethiol. Lateral ROMP could only be achieved by the simultaneous addition of exogenous norbornene and Grubbs’ catalyst to cross the steric barrier of dodecanethiol.36 Another control experiment was carried out using dodecanethiol coated gold nanoparticles (Au-14). When the Grubbs’ catalyst was mixed with Au-14 instead of Au- 13/14, negligible amount of Grubbs’ catalyst could by detected by 1H-NMR after the same reaction and purification processes as above. This result indicated the Grubbs’ catalyst could not be immobilized to gold nanoparticles without norbornene functionality 26 at surfaces. It also confirmed any unbound catalyst could be washed away by methanol wash. 1.2.5 Surface-initiated ROMP of block copolymers at gold nanoparticles ROMP is one type of living polymerization in which the chain termination and chain transfer are negligible. It is widely used to synthesize block copolymers, by the addition of the second monomer after the completion of the first monomer. Norbornene (25) and norbornene derivatives are frequently used as monomers. The reaction scheme for the synthesis of block copolymers is shown in Figure 1.2.14. Figure 1.2.14 Synthesis of block copolymers by ROMP In our case, diblock or triblock copolymers were grafted at nanoparticle surfaces using 20 and 25. 20 and 25 were sequentially added to a dichloromethane solution of 24 1 treated Au-13/14. Successful polymerization was confirmed by H-NMR, as the norbornene peaks at 6.2 ppm shifted upfield to 5.2-5.3 ppm for polynorbornene. The absence of norbornene peaks also confirmed the complete conversion of monomers to polymers. The polymer-nanoparticle conjugates were named as Au-20102530, Au- 251020102520 and Au-252020102510. In Au-20102530, 10 units of 20 were grafted to the nanoparticle surface, followed by a block of 30 units of 25 which was further away from 27 the surface. In Au-252020102510, the triblock copolymer was assembled to the nanoparticle surface in the sequence of 20 units of 25, followed by 10 units of 20 and then 10 units of 25. The structures of Au-20102530, Au-251020102520 and Au-252020102510 are shown in Figure 2.29. Each type of linear polymers contained 40 repeating units of the 1,3-divinylcyclopentane structure in the polymer backbones. The polyester blocks for diamine cross-linking always consisted of 10 units of the polynorbornene substructure, which afforded the same extent of cross-linking regardless of the cross-linking positions. The polynorbornene block, which was inert to amine, was used as a spacer to modulate the distance of polyester block from nanoparticle surface. For example, the polyester block was close to nanoparticle surface in Au-20102530, and it was further away from the surface in Au-252020102510. All of the particles were terminated with a short polynorbornene block to a) minimize interparticle cross-linking and b) to maximize solubility in organic solvents such as tetrahydrofuran and dichloromethane. AuNP S a 10 b O Au-20102530: a=0, b=30 O Au-251020102520: a=10, b=20 Au-252020102510: a=20, b=10 NO2 Figure 1.2.15 Structure of surface grafted block copolymers To characterize their structure, the linear polymers were cleaved off the nanoparticle surface by cyanide etching. During the aqueous etching, the active ester 20 was hydrolyzed to carboxylic acid 15, and insoluble polymers were obtained. To overcome the low solubility caused by poly(carboxylic acid), polymers were quenched by 28 p-methoxybenzylamine prior to etching, yielding stable and soluble polyamide (Figure 1.2.16). 5 equivalents of p-methoxybenzylamine per ester were added to the polymer coated nanoparticle solution in dichloromethane, and the mixture was stirred at room temperature for 24 hours. The nanoparticles were then etched by cyanide, releasing free polymers which were sequentially purified by dialysis. The dialyzed polymers were treated with tributylphosphine which reduced the disulfide to thiol. The polymer cleaved from Au-20102530 was named as HS-20102530, and HS-252020102510 was from Au- 252020102510. The molecular weight and structural composition of linear polymers were characterized by MALDI-ToF MS and 1H-NMR, respectively. S O 9 a 10 b O Ph O Au-20102530: a=0, b=30 Au-252020102510: a=20, b=10 NO2 1) p-methoxybenzylamine 2) NaCN 3) Bu3P, MeOH HS O 9 a 10 b O Ph HN HS-20102530: a=0, b=30 HS-252020102510: a=20, b=10 MeO Figure 1.2.16 Preparation of linear polymers for structural characterization 29 The 1H-NMR spectra of p-methoxybenzylamine quenched linear polymers are shown in Figure 1.2.17 and 1.2.18. The integration of the aromatic protons ortho to the methoxy group (6.81 ppm) to the polynorbornene alkene resonances (5.23 and 5.36 ppm) was 25:81 for HS-20102530 and 26:80 for HS-252020102510. This indicated the fraction of ester to the entire polymer backbone was 13:40. Although the value was larger than the target 10:40, the composition for these two polymers were very similar regardless of the position of polyester block. The molecular weight of the linear polymers was measured by MALDI-ToF mass spectrometry. Linear polymers HS-20102530 (Figure 1.2.19, Mw=5045, Mn=3710, PDI=1.36) and HS-252020102510 (Figure 1.2.20, Mw=4786, Mn=3682, PDI=1.30) had very close molecular weight, although it was smaller than the nominal molecular weight, 5798. The difference between every two peaks, 257, was consistent with the molecular weight of p-methoxybenzylamine quenched ester. Figure 1.2.17 1H-NMR of p-methoxybenzylamine quenched HS-20102530 30 Figure 1.2.18 1H-NMR of p-methoxybenzylamine quenched HS-252020102510 Figure 1.2.19 MALDI-ToF MS of p-methoxybenzylamine quenched HS-20102530 31 Figure 1.2.20 MALDI-ToF MS of p-methoxybenzylamine quenched HS-252020102510 ROMP at nanoparticle surfaces was carefully studied by Seery using silica nanoparticles.37 Polynorbornene was grafted at the surface of silica nanoparticles via surface-initiated ROMP. The resulting polymers were cleaved off the particles by HF treatment. Subsequent analysis by gel permeation chromatography (GPC) was used to determine molecular weight, polydispersities, and the number of polymer chains per particle. Increases in the number of grafted chains could be obtained, but often at the cost of greater polydispersity. The surface initiated polymerization can potentially give cyclic polymers or particle bound loops through undesirable backbiting (Figure 1.2.21). A careful study of the reaction conditions revealed that these side reactions could be minimized by using short polymerization times and high monomer concentrations. The side reactions (backbiting and cross-metathesis) could be the reason why our polymers 32 were smaller than designed. However, the chemical compositions and molecular weights of HS-20102530 and HS-252020102510 were very similar. Figure 1.2.21 Side reactions (backbiting and cross-metathesis) during ROMP 1.2.6 Cross-linking The surface-bound block copolymers were cross-linked by the addition of 0.5 equivalents of diamines per ester. Four different diamines were used – triethyleneglycol diamine (TEG), diaminooctane (C8), diaminobutane (C4) and trans-1,4- diaminocyclohexane (Cy). The structures of diamine cross-linkers are shown in Figure 1.2.22. The cross-linking locations were controlled in the interior (close to nanoparticle 33 surface), in the middle and in the exterior, corresponding to the locations of polyester blocks. Figure 1.2.22 Structures of diamine cross-linkers The extent of cross-linking was determined by detecting the unreacted amine and ester in the cross-linked polymer-nanoparticle conjugates. For example, Au-20102530 and Au-252020102510 that had been cross-linked with TEG diamine were treated with excess allyl isocyanate to cap any free amines and excess p-methoxybenzylamine to quench any unreacted esters. The chemical reactions of capping are shown in Figure 1.2.23. S O 9 O O O Ph HN O HN HN NH2 O NO2 1) p-methoxybenzylamine followed by dialysis 2) allyl isocyanate followed by dialysis S O 9 O O O Ph HN HN HN HN HN O O HN MeO Figure 1.2.23 Capping unreacted ester and amine 34 After removal of excess capping reagents by dialysis, the cross-linked polymers were examined using 1H-NMR. Both Au-20102530 and Au-252020102510 after TEG cross- linking and capping had a ratio of 7-8 to 80 for the integration of the protons ortho to the methoxy group (6.81 ppm) to the polynorbornene alkene resonances (5.23 and 5.36 ppm) (Figure 1.2.24). This indicated that about 27% of the esters didn’t react with the diamine. As for the amount of unreacted amine in either Au-20102530 or Au-252020102510, the olefinic region of the 1H-NMR spectrum after the capping reaction with allyl isocyanate did not show any evidence of allyl group incorporation, indicating that there was a negligible amount of free amine present after cross-linking (Figure 1.2.25). In contrast, the internal olefinic proton from the allyl group was clearly visible in the 1H-NMR spectrum at 5.92 ppm when 1 equivalent of C8 diamine was used for cross-linking of a related polymer-nanoparticle conjugate. Taken together, these experiments suggested that the amounts of cross-linking in Au-20102530 and Au-252020102510 were the same. Figure 1.2.24 1H-NMR of Au-20102530 (top) and Au-252020102510 (bottom) after TEG cross-linking and p-methoxybenzylamine capping 35 Figure 1.2.25 1H-NMR of polymer-nanoparticle conjugates after cross-linking and allyl isocyanate capping. Top: Au-20102530 cross-linked by 0.5 eq. of TEG; middle: Au- 252020102510 cross-linked by 0.5 eq. of TEG; bottom: Au-20302530 treated by 1 eq. of C8 The UV-vis spectra of the nanoparticles before polymerization and after cross- linking were the same, indicating that radial polymerization followed by lateral cross- linking had no effect on the properties of nanoparticles (Figure 1.2.26). This spectral result suggested that substantial particle aggregation or interparticle cross-linking did not take place. The same conclusion was supported by TEM. The TEM image of C8 cross- linked Au-20102530 in Figure 1.2.27 showed monodispersed nanoparticles with the same particle size as original Au-13/14 particles (Figure 1.2.4). 36 Figure 1.2.26 UV-vis of nanoparticles in dichloromethane Figure 1.2.27 TEM of Au-20102530 crosslinked by C8 diamine 1.3 Study of nanoparticle stability affected by ligand structures The stability of nanoparticles was determined by the decomposition rate of cyanide mediated etching. To a solution of nanoparticles (0.50 absorbance at 512 nm) in 1 mL tetrahydrofuran was added potassium cyanide in methanol (0.10 mL, 0.1M). UV- vis spectroscopy was used to monitor the absorbance change at 512 nm. 37 1.3.1 Effect of cross-linking positions Cyanide etching experiments were carried out on each of the cross-linked materials with variable cross-linking positions. For any given diamine (TEG, C8, C4 or Cy), cross-linking of Au-20102530 resulted in faster etching relative to its counterpart Au- 252020102510 (Figure 1.3.1, a-d). The particles with the ester block in the middle, Au- 251020102520, were crosslinked with the TEG and C8 diamines. These particles were etched at an intermediate rate (Figure 1.3.1, a and b). Figure 1.3.1 Cyanide mediated decomposing rate of polymer-nanoparticle conjugates cross-linked with given diamines at different positions 38 1 H-NMR and MALDI analyses on linear polymers from Au-20102530 and Au- 252020102510 indicated that the polymer precursors had similar molecular weight distributions and block ratios, so the differences in etching rate were not due to differences in the composition or length of the polymers. By quantitating the amount of unreacted ester and amine after cross-linking, the extents of cross-linking on interior or exterior cross-linked polymer-nanoparticle conjugates were demonstrated very close. Therefore the differences in etching rate were not due to differential amount of cross- linking in Au-20102530 and Au-252020102510. Further analysis of the cross-linked particles was carried out using 1H-NMR. Spin-lattice relaxation time (T1) is the decay constant for the recovery of the z component of the nuclear spin magnetization towards its thermal equilibrium value. As nuclear spins exchange energy with their surroundings (the lattice) during the process to reach the thermal equilibrium distribution, the values of T1 reveal information about the rigidity of chemical structures.38 T1 relaxation times for the olefinic peaks at 5.23 and 5.36 ppm are listed in Table 1.3.1. T1 values were smaller for the cross-linked Au-252020102510 relative to cross-linked Au-20102530, indicating that the mobility of polynorbornene became more restricted when it was cross-linked at the exterior. Table 1.3.1a T1 relaxation time (ms) of olefinic peaks at 5.23 ppm diamine cross-linker nonea TEG C8 C4 Cy Au-20102530 (interior cross-linking) 1161 1132 1150 1170 1158 Au-252020102510 (exterior cross- 1165 1117 1093 1167 1137 linking) 39 Table 1.3.1b T1 relaxation time (ms) of olefinic peaks at 5.36 ppm diamine cross-linker nonea TEG C8 C4 Cy Au-20102530 (interior cross-linking) 1402 1304 1321 1390 1372 Au-252020102510 (exterior cross- 1399 1301 1233 1378 1330 linking) a active polyester quenched with p-methoxybenzylamine DLS measurements of Au-20102530 and Au-252020102510 demonstrated that for any given diamine cross-linker, the diameters of the former particles were always smaller than the latter (Table 1.3.2). Based on the results of T1 relaxation by NMR and hydrodynamic radius by DLS, it could be concluded that when the polymer shell was cross-linked in the interior, the terminal polynorbornene block existed in a random coil or mushroom conformation. However, when the cross-linking was situated in the exterior, the internal polynorbornene block adopted a more extended conformation upon cross- linking, which reduced the mobility of the polymer strands.39 Table 1.3.2 Hydrodynamic diameter (nm) of different cross-linked polymer-nanoparticle conjugates by DLS diamine cross-linker TEG C8 C4 Cy Au-20102530 (interior cross-linking) 30.7 23.5 25.7 26.2 Au-252020102510 (exterior cross- 39.0 29.6 31.8 33.0 linking) An alternative possibility was that the diamines underwent more intrastrand reactions in Au-20102530 than in Au-252020102510, resulting in faster etching of the former. However, it was unlikely that intrastrand reactions would be favored in the interior of the 40 polymer shell. Rather, it was expected that the ratio of intrastrand to interstrand reaction would be greater at the exterior due to the radial nature of polymer extension off the highly curved nanoparticle surface. Thus, although this possibility couldn’t be unequivocally ruled out, the light scattering, NMR and MALDI data were best explained by a model involving conformational changes in the polymer backbone. Absorbance data for the dodecanethiol coated particles were fit to a rate law corresponding to pseudo first-order kinetics, consistent with previous reports.4 The etching of the polymer coated and cross-linked particles all exhibited an initial lag phase, followed by a subsequent pseudo first-order decay of the absorbance. The time of onset of this pseudo first-order decay (tlag) was determined by plotting the first derivative of the etching plot (Figure 1.3.2). The minimum of the first derivative plot of the cross-linked polymer-nanoparticle conjugates was fixed as tlag. As the cross-linking progressed from the interior to the exterior, tlag increased as well. Plots of ln A versus time starting from tlag were linear, indicating pseudo first-order etching from that point. Values of tlag and pseudo first-order rates are provided in Table 1.3.3. For any given diamine, tlag increased as the cross-linking positions moved from the interior to the exterior. Additionally, there did not appear to be a direct correlation between tlag and the rate of etching. For example, C8 cross-linked Au-252020102510 and Cy cross-linked Au-20102530 were etched at comparable rates, but had different tlag values. Tlag may be related to significant conformation change of the polymer shells. 41 Figure 1.3.2 Plots of dA/dt vs time for cross-linked polymer-nanoparticle conjugates: a) TEG; b) C8; c) C4; d) Cy Table 1.3.3 tlag [s] (k [×10-3 s-1]) TEG C8 C4 Cy 137 152 185 Au-20102530 (interior cross-linking) 92 (6.0) (3.8) (2.5) (2.1) Au-251020102520 (middle cross- 143 224 linking) (3.3) (2.9) Au-252020102510 (exterior cross- 206 311 338 389 linking) (2.6) (2.0) (1.2) (1.1) 1.3.2 Effect of cross-linker structures For a given cross-linking position, the rate of etching was dependent on the diamine used for cross-linking. Polymers cross-linked by TEG were etched the fastest, while its more hydrophobic analogue C8 offered increased protection (Figure 1.3.3). 42 Cross-linking by diamines with fewer degrees of conformation freedom increased particle stabilization (i.e., C8 vs C4 vs Cy). Figure 1.3.3 Cyanide mediated decomposing rate of polymer-nanoparticle conjugates cross-linked at given positions with different diamines 1.3.3 Effect of polymer chain length Figure 1.3.4 shows the results of cyanide etching of the nanoparticles Au-13/14, Au-2540, Au-25120, and Au-20102530 cross-linked with TEG or C8. As expected, Au- 13/14 was etched faster than those coated with polymers. Au-25120 was etched slower than Au-2540, because larger ligands provided better protection to the nanoparticles. Cross-linked Au-20102530 was more stable against cyanide etching than the noncross- linked Au-2540. In order for a noncross-linked polymer to provide a comparable degree of protection, the polymer length must be tripled. The decomposition rate of Au-25120 was 43 slightly slower than that of TEG cross-linked Au-20102530, but faster than that of C8 cross-linked Au-20102530. Figure 1.3.4 Cyanide mediated decomposing rate of nanoparticle with different length of polymers The time of onset of this pseudo first-order decay (tlag) was also observed for the noncross-linked nanoparticles. Values of tlag and pseudo first-order rates are summarized in Table 1.3.4. As the polymeric ligands grew larger, tlag increased and reaction rate decreased. Table 1.3.4 tlag [s] (k [×10-3 s-1]) Au-13/14 0 (17.6) Au-2540 32 (9.9) Au-25120 71 (4.4) 1.4 Conclusion It was demonstrated that gold nanoparticles can be coated with cross-linked block copolymers. The protection afforded by the polymer coating was sensitive to the structure 44 of the cross-linker, as well as the distance of the cross-links from the nanoparticle surface. Cross-links which were further away from the gold nanoparticle provided better protection against cyanide etching. Cross-linkers with fewer degrees of conformation freedom increased particle stabilization. The radial distance dependent correlation with cyanide etching was also observed for dendron coated nanoparticles.23 The surface alkenes were cross-linked by cross- metathesis, and the cross-linked gold nanoparticles exhibit enhanced stabilities towards cyanide etching (Figure 1.1.12). However, G1-AuNP was always more stable than G2- AuNP. This result was contrary to what was observed in this study—the particles were more stable when the exterior was cross-linked. The difference could be attributed to the ligand packing density at nanoparticle surfaces. Increases in generation number resulted in ligands of different sizes, complicating the packing density of ligands at nanoparticle surfaces which also dramatically affected the nanoparticle stability. Generally, it was believed that ligands with large heading groups (G2) were less densely packed at nanoparticle surfaces. Therefore G2-AuNP was less stable. The nanoparticle-polymer conjugates prepared in the current study were derived from the same precursor, so the ligand packing densities were always the same. In summary, subtle changes in the architecture of the polymer coating around a gold nanoparticle could influence the degree of stabilization provided by the coating. The results of this study suggested that properties such as nanoparticle stability and access of small molecules (reagents, analytes, etc.) to the particle surface could be tuned by careful selection of the polymer cross-linking architecture. 45 1.5 Experimental General Experimental Protocols: Dry solvents (DMF/THF/CH2Cl2/MeOH) were obtained using a commercially available solvent purification system from GlassContour based on purification principles reported by Grubbs.40 All other reagent chemicals were purchased from Aldrich. All reactions were carried out under an atmosphere of nitrogen. NMR spectra were recorded on a Bruker instrument at 300 or 400 MHz for 1H and 75 or 100 MHz for 13 C. 1H chemical shifts were referenced to TMS at 0 ppm or residual solvent peaks. 13C chemical shifts were referenced to the solvent peak, typically CDCl3 at 77 ppm. T1 values of olefinic protons at 5.23 and 5.36 ppm were measured by an inversion recovery series on a Bruker 400 spectrometer. The variable delay times were set to 2.40 s, 2.10 s, 1.80 s, 1.70 s, 1.60 s, 1.50 s, 1.40 s, 1.30 s, 1.20 s, 1.10 s, 1.00 s, 0.90 s, 0.80 s, 0.70 s, 0.60 s, and 0.40 s. UV-Vis spectra were recorded on an Agilent 8453 UV-Visible diode-array spectrophotometer. Samples for TEM analyses were prepared by drying dichloromethane solutions (A512 ~ 0.5) of the particles on amorphous carbon coated copper grids (400 mesh). Particles were imaged using a Philips EM 420 (120 kV). Particles sizes were obtained from the TEM images using ImageJ software. The size of the polymer- nanoparticle hybrid structures in dispersion was evaluated using a Malvern Zeta Sizer Nano S-90 dynamic light scattering (DLS) instrument in dichloromethane at room temperature. Light scattering analyses were carried out at nanoparticle concentrations corresponding to an absorbance of 0.5 at 512 nm. Thermogravimetric analysis was performed under nitrogen on TA instruments TGA Q500 Hi-Res from room temperature 46 to 650 °C. The molecular weight of linear polymers was measured by MALDI-ToF using a Voyager-DE Pro BioSpectrometry Workstation. Compound 9: A mixture of 11-bromoundecanol (5.53 g, 20.0 mmol) and triphenylmethyl mercaptan (5.02 g, 20.0 mmol) and potassium carbonate (5.53 g, 40.0 mmol) in 200 mL EtOH/H2O (1:1 v/v) was heated at reflux for 16 hours. The solution was cooled to room temperature and neutralized using 0.1 M HCl aqueous solution. The crude product was extracted with dichloromethane, concentrated and purified by flash column chromatography (SiO2, ethyl acetate/hexanes, 1/4) to give 8.42 g (94%, 18.8 mmol) of 9 as a yellow oil. 1 H NMR (400 MHz, CDCl3): δ 1.10-1.46 (m, 16H, alkyl H), 1.53-1.63 (m, 2H, - CH2CH2OH), 2.13 (t, J=7.3 Hz, 2H, TrSCH2-), 3.63 (t, J=6.6 Hz, 2H, -CH2OH), 7.19- 7.44 (m, 15H, aromatic H). 13 C NMR (75.5 MHz, CDCl3): δ 25.7, 28.5, 28.6, 29.0, 29.2, 29.4, 29.5, 29.6, 32.1, 32.8, 63.1 (–CH2OH), 66.4 (TrSCH2-), 126.5, 127.8, 129.6, 145.1 (Ph3-C-). HRMS (FAB): calculated for C30H38OS (M+Na)+ 469.2541, found 469.2535 Compound 10: 47 To a solution of 9 (8.42 g, 18.8 mmol), pyridine (5 mL, 61.8 mmol) and 4- (dimethylamino)pyridine (0.05 g, 0.4 mmol) in distilled dichloromethane (100 mL) was added p-toluenesulfonyl chloride (5.72 g, 30.0 mmol). The mixture was stirred at room temperature for 24 hours. The crude product was concentrated and purified by flash column chromatography (SiO2, dichloromethane/hexanes, 2/3) to give 9.95 g (88%, 16.6 mmol) of 10 as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 1.10-1.46 (m, 16H, alkyl H), 1.60-1.70 (m, 2H, CH2CH2OH), 2.13 (t, J=7.3 Hz, 2H, TrSCH2-), 2.44 (s, 3H, CH3 on OTs), 4.01 (t, J=6.5 Hz, 2H, -CH2OTs), 7.19-7.47 (m, 17H, aromatic H), 7.79 (d, J=8.3, 2H, aromatic H). 13 C NMR (75.5 MHz, CDCl3): δ 21.7, 25.3, 32.1, 28.6, 28.8, 28.9, 29.0, 29.2, 29.3, 29.4, 32.1, 66.4 (TrSCH2-), 70.7 (–CH2OTs), 126.5, 127.8, 127.9, 129.6, 129.8, 133.3, 144.6, 145.1. HRMS (FAB): calculated for C37H44O3S2 (M+Na)+ 623.2630, found 623.2640. Compound 11: To a solution of 10 (1.98 g, 3.3 mmol) and 5-norbornene-2-methanol (0.31 g, 2.5 mmol) in 30 mL distilled DMF was added NaH (0.07 g, 2.9 mmol). The mixture was stirred at RT for 3 days, and worked up with saturated NH4Cl aqueous solution. The crude product was extracted by dichloromethane and purified by flash column chromatography (SiO2, dichloromethane/hexanes, 2/3) to give 1.22 g (66%, 2.2 mmol) of 12 as a colorless oil. 48 1 H NMR (300 MHz, CDCl3): δ 0.45-0.55 (m, minor diasteromer), 1.01-1.85 (m, 23H, alkyl H), 2.13 (t, J=7.3 Hz, 2H, TrSCH2-), 2.28-2.43 (m, minor diasteromer), 2.73-2.96 (m, 2H, -CH-CH=CH-CH-), 2.97-3.53 (m, 4H, -CH2OCH2-), 5.91-6.17 (m, 2H, - CH=CH-), 7.18-7.47 (m, 15H, aromatic H). 13 C NMR (75.5 MHz, CDCl3): δ 26.2, 28.6, 29.05, 29.2, 29.4, 29.5, 29.6, 29.75, 32.15, 38.8, 38.9, 41.6, 42.2, 43.7, 44.0, 45.0, 49.4, 66.4 (TrSCH2-), 71.1, 71.2, 74.5, 77.25, 126.5, 127.8, 129.6, 132.5, 136.6, 137.1, 145.1 MS (FAB): calculated for C38H48OS (M+Na)+ 575.8, found 575.8. Compound 13: To a solution of 12 (0.92 g, 1.66 mmol) and triethylsilane (0.40 mL, 2.5 mmol) in 20 mL distilled dichloromethane was added dropwise trifluoroacetic acid (5 v% in dichloromethane). The reaction was monitored by TLC. The reaction mixture was worked up by saturated aqueous NaHCO3. The crude product was concentrated and purified by flash column chromatography (SiO2, dichloromethane/hexanes, 2/3) to give 0.34 g (66%, 1.1 mmol) of 13 as a colorless oil. 1 H NMR (300 MHz, CDCl3): δ 0.45-0.55 (m, minor diasteromer), 1.01-1.85 (m, 23H, alkyl H), 2.28-2.43 (m, minor diasteromer), 2.52 (dt, J= 7.4, 14.7 Hz, 2H, HSCH2-), 2.73- 2.96 (m, 2H, -CH-CH=CH-CH-), 2.97-3.53 (m, 4H, -CH2OCH2-), 5.91-6.17 (m, 2H, - CH=CH-), 7.18-7.47 (m, 15H, aromatic H). 49 13 C NMR (75.5 MHz, CDCl3): δ 24.70, 26.19, 28.41, 29.09, 29.19, 29.50, 29.52, 29.54, 29.60, 29.72, 29.74, 29.77, 34.08, 38.81, 38.88, 41.55, 42.21, 43.71, 44.00, 45.01, 49.43, 71.07, 71.17, 74.54, 75.53, 132.52, 136.62, 136.67, 137.08. HRMS(FAB): calculated for C19H34OS (M+H)+ 311.2409, found 311.2416. Compound 20: To a solution of 5-norbornene-2-carboxylic acid (0.22 g, 1.6 mmol) and 4- (dimethylamino)pyridine (20 mg, 0.16 mmol) in 10 mL dichloromethane was added N,N′-diisopropylcarbodiimide (0.5 mL, 3.2 mmol). The mixture was stirred at room temperature for 30 minutes. p-Nitrophenol (0.22 g, 1.6 mmol) was added and stirred at room temperature overnight. The crude product was concentrated and purified by flash column chromatography (SiO2, ethyl acetate/hexanes, 1/6) to give 0.36 g (87%, 1.4 mmol) of 20 as a yellow solid. 1 H NMR (300 MHz, CDCl3): δ 1.35-1.62 (m, 3H, alkyl H), 2.00-2.12 (m, 1H), 2.49-2.57 (m, minor diasteromer), 3.00-3.06 (m, 1H), 3.23-3.31 (m, 1H), 3.37-3.44 (m, 1H), 6.04- 6.35 (m, 2H, -CH=CH-), 7.20-7.34 (m, 2H, aromatic H meta to NO2), 8.21-8.32 (m, 2H, aromatic H ortho to NO2). 13 C NMR (75.5 MHz, CDCl3): δ 29.40, 30.63, 41.78, 42.70, 43.41, 43.74, 46.01, 46.40, 46.86, 49.84, 122.40, 125.17, 125.21, 131.84, 135.46, 138.51, 138.64, 155.73, 172.40. HRMS (FAB): calculated for C14H13NO4 (M+Na)+ 282.0742, found 282.0750. 50 Synthesis of gold nanoparticles (Au-13/14) To a solution of HAuCl4·3H2O (2.55 g, 6.64 mmol) in 180 mL H2O was added tetraoctylammonium bromide (6.00 g, 11.0 mmol) and 500 mL of toluene. The two phase mixture was stirred at room temperature for 0.5 h, until the aqueous layer turned colorless and the organic layer turned dark red. The aqueous layer was removed. The thiol ligand 13 (0.34 g, 1.1 mmol) in 100 mL of toluene was added, and the solution was stirred for 15 min. NaBH4 (2.70 g, 71.4 mmol) in 180 mL of water was added rapidly. After stirring at room temperature for 3 h, dodecanethiol 14 (0.26 mL, 1.1 mmol) was added. The reaction mixture was stirred at room temperature for another 2 h. The aqueous layer was removed and the nanoparticles were precipitated by adding methanol (3 L). The particles were collected by centrifugation and washed by methanol (5×10 mL) in a bath sonicator (approximately 5 minutes each wash) to provide Au-13/14 (1.56 g) as a black solid. Etching Typically, etching was carried out by mixing nanoparticles in dichloromethane (5 mg/mL) with an aqueous solution of sodium cyanide (1.0 M) in a 1:1 volume ratio. Molecular oxygen in air was used as the oxidant. The mixture was stirred at room temperature until the organic layer turned from black to colorless or light yellow. The organic layer was separated and washed by an equal volume of saturated NaHCO3 (aq.) and water (3×). ROMP 51 To prepare Au-20102530, 18 mg of second generation Grubbs’ catalyst (0.021 mmol, 1 equivalent relative to particle-bound norbornene) was added to a solution of nanoparticle Au-13/14 (0.10 g, 0.021 mmol) in 10 mL dichloromethane. The mixture was stirred at room temperature for 20 min. Compound 20 (55 mg, 0.21 mmol) was added and stirred for 20 min. Norbornene (25) (59 mg, 0.63 mmol) was added and stirred for 20 min. Ethyl vinyl ether (1 mL) was added to terminate the polymerization. To prepare Au-251020102520, 18 mg of second generation Grubbs’ catalyst (0.021 mmol, 1 equivalent relative to particle-bound norbornene) was added to a solution of nanoparticle Au-13/14 (0.10 g, 0.021 mmol) in 10 mL dichloromethane. The mixture was stirred at room temperature for 20 min. 25 (20 mg, 0.21 mmol) was added and stirred for 20 min. Compound 20 (55 mg, 0.21 mmol) was added and stirred for 20 min. Finally additional 25 (40 mg, 0.43 mmol) was added and stirred for 20 min. Ethyl vinyl ether (1 mL) was added to terminate the polymerization. To prepare Au-252020102510, 18 mg of second generation Grubbs’ catalyst (0.021 mmol, 1 equivalent relative to particle-bound norbornene) was added to a solution of nanoparticle Au-13/14 (0.10 g, 0.021 mmol) in 10 mL dichloromethane. The mixture was stirred at room temperature for 20 min. 25 (40 mg, 0.43 mmol) was added and stirred for 20 min. Compound 20 (55 mg, 0.21 mmol) was added and stirred for 20 min. Finally additional 25 (20 mg, 0.21 mmol) was added and stirred for 20 min. Ethyl vinyl ether (1 mL) was added to terminate the polymerization. p-methoxybenzylamine quenching and allyl isocyanate capping 52 To quench the active ester for linear polymer analysis, excess p- methoxybenzylamine was used at 7 equivalents per ester. To a solution of Au-20102530 or Au-252020102510, prepared as described above, was added p-methoxybenzylamine (0.20 mL, 1.5 mmol) to convert the p-nitrophenyl esters to stable amides. The mixture was stirred at room temperature for 3 days. To quench the unreacted ester after cross-linking, excess amount of p- methoxybenzylamine to the ester prior to cross-linking was used. To a dichloromethane solution of nanoparticles after diamine cross-linking (0.21 mmol ester cross-linked by 0.105 mmol diamine), was added 0.42 mmol p-methoxybenzylamine (56 μL). The mixture was stirred at room temperature for 3 days. Products were purified by dialysis. To cap the unreacted amine after cross-linking, excess amount of allyl isocyanate was used. To a dichloromethane solution of nanoparticles after diamine cross-linking (0.21 mmol ester cross-linked by 0.105 mmol diamine), was added 0.42 mmol allyl isocyanate (37 μL). The mixture was stirred at room temperature for 3 days. Products were purified by dialysis. Cross-linking Diamine (0.5 equiv. relative to 9) was added to a solution of the nanoparticle- polymer conjugates, prepared as described above, after polymerization was terminated. The reaction was stirred at room temperature for 3 days. The polymer coated nanoparticles were purified by dialysis. Dialysis 53 Polymers, either bound to or cleaved from particle surface, were purified by dialysis. In general, approximately 5 mg polymer was dissolved in 10 mL dichloromethane, transferred into regenerated cellulose dialysis tubing (SpectrumLabs, MW cutoff 1000), and dialyzed at room temperature in 500 mL solvent. A mixture of THF and DMF (1:1) was used for 3 rounds, and pure THF was used for another three rounds. The solvent reservoir was changed every 12 h. MALDI MALDI samples were prepared by combining the analyte and matrix. The condition was modified based on reported procedures.41-45 Polymers were dissolved in THF to prepare stock solutions with concentrations of approximately 2x10-3 M (based on nominal mass for calculation). The 2,5-dihydroxybenzoic acid (DHB) matrix was prepared as a 0.4 M solution in THF. A 100 μL sample solution was prepared by mixing 5 μL of the polymer solution in THF, 50 μL of the matrix solution in THF, and 45 μL of toluene. 1 μL of the mixture was added to the MALDI probe tip and allowed to air dry. The experimental condition was set to laser intensity 2500, accelerating 25000 V, grid 95%, guide wire 0.035%, delay time 800 ns, and 100 shots per spectrum. Etching kinetics The stability of nanoparticles is determined by the rate of cyanide mediated etching. To a solution of nanoparticles (0.50 absorbance at 512 nm) in 1mL tetrahydrofuran was added potassium cyanide in methanol (0.10 mL, 0.1M). UV-vis 54 spectroscopy was used to monitor the absorbance change at 512 nm. The absorbance was recorded every 5 seconds for 1000 seconds. Reference 1. Shenhar, R.; Rotello, V. M., Nanoparticles: Scaffolds and building blocks. Acc. Chem. Res. 2003, 36, (7), 549-561. 2. Daniel, M. 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CHAPTER 2 Functionalized Polymer Nanocapsules as a Potential Drug Delivery Vehicle 2.1 Introduction In recent years materials with defined structures are of great research interest. Controls over the composition, structure and function of materials at nano-scale may lead to new properties and hence new applications. Polymer nanocapsules have attracted particular interest due to their potential for encapsulation of guest molecules in their empty core domain. These materials are useful as confined reaction vessels, drug carriers and protective shells for reactive species. However, although various applications of polymer nanocapsules have been discussed, they are yet not realized. The major challenge is the preparation of these materials and control over the composition, structure and function. Two general approaches will be discussed here: the self-assembly approach and the template approach. 2.1.1 Preparation of polymer nanocapsules via self-assembly approach Amphiphilic block copolymers may self-assemble to vesicular structures under particular conditions.1, 2 The use of polymer vesicles as potential drug carriers has been widely discussed.3-6 For example, polyisoprene-block-poly(2-cinnamoylethyl methacrylate) (PI-b-PCEMA) formed vesicles in THF/hexanes.3 The PI-b-PCEMA vesicles took up a large amount of rhodamine B in methanol and released the compound into water at a tunable rate, depending on the amount of ethanol added to the aqueous 57  58  medium. However, a major disadvantage of polymer vesicles is their dynamic nature which leads to disassembly at high temperature, at low concentration or under certain changes in solvent conditions. As a result, cross-linking is used to stabilize the vesicle structure, producing cross-linked polymer capsules.7-16 Meier reported the synthesis and characterization of a poly(2-methyloxazoline)- block-poly(dimethylsiloxane)-blockpoly(2-methyloxazoline) (PMOXA-PDMS-PMOXA) triblock copolymer carrying polymerizable groups at both chain ends.17 This copolymer formed vesicular structures in dilute aqueous solution, the size of which could be controlled in the range from 50 nm up to 500 nm. The methacrylate end groups of the triblock copolymer were laterally polymerized in the vesicular aggregates using an UV- induced free radical polymerization. This strategy of synthesizing polymer nanocapsules is also called polymerization of the vesicles. Static and dynamic light scattering, scanning electron microscopy, and transmission electron microscopy on both the resulting nanocapsules and their nonpolymerized precursors showed that the cross-linking polymerization did not lead to any change in size, size distribution or even molecular weight of the vesicles. Moreover, due to their cross-linked structure, the nanocapsules maintained their shape under the conditions in which the noncross-linked vesicles disassembled.   59  Figure 2.1.1 Cross-linking of PMOXA-PDMS-PMOXA triblock vesicles by radical polymerization of methacrylate end groups (Adapted with permission from ref 17. Copyright 2000 American Chemical Society) Another strategy of synthesizing polymer nanocapsules relies on vesicle templates. Vesicles or liposomes are able to trap hydrophobic molecules within the lipid bilayer. If such hydrophobic molecules carry polymerizable groups, their subsequent polymerization should lead to a polymer chain or network entrapped in the hydrophobic domain of the vesicles. The removal of the vesicle template yields a polymer network which is considered as a nanocapsule (Figure 2.1.2).18 This strategy is also called polymerization in the vesicles. Several examples are available. German reported the polymerization of styrene in the vesicle of dioctadecyldimethylammonium bromide, which was 160 nm in diameter.19 Kaler reported the formation of polystyrene network by polymerization of divinylbenzene incorporated in the vesicle of dodecyltrimethylammonium bromide (DTAB) / sodium dodecylbenzenesulfonate (SDBS).20 Meier reported a poly(methyl methacrylate) type of quasi-two-dimensional 60  polymer network using a dimethyldioctadecylammonium chloride vesicle template.21 Compared to the polymerization of vesicles strategy, this polymerization in vesicles strategy allows better control over the structure and composition of polymer nanocapsules through the amount and identity of monomers incorporated, as well as the degree of cross-linking. Depending on the dimensions of the vesicle templates, the polymer nanocapsules can be produced with diameter ranging from tens of nanometers to hundreds of micrometers. But the practical applications are limited by the low economical efficiency. In spite of the energy consumed on the preparation and removal of vesicle templates, the synthesis of polymer nanocapsules always requires approximately 1.5-2 times of their weight of the vesicles and a large amount of solvents. Figure 2.1.2 Preparation of polymer nanocapsules by polymerization of monomers incorporated in the lipid bilayer (Adapted with permission from ref 18. Copyright 1998 Wiley) Not only vesicular aggregates but also micelles can be used for the preparation of polymer nanocapsules. The method relies on the self-assembly of amphiphilic block copolymers to micelles as a core-shell structure, followed by selectively cross-linking in the shell layer and then degradation and extraction of the core material. For example, amphiphilic diblock copolymer PI-PAA (polyisoprene-poly(acrylic acid)) self-assembles 61  to form micelles in aqueous solution (Figure 2.1.1).8 Condensation between diamine cross-linkers and pendant carboxylic acid groups along the PAA segments in the micelle exterior layer yielded the shell cross-linked knedel-like (SCK) nanostructures. These SCKs, containing a polyisoprene core surrounded by a cross-linked polyacrylamide shell, served as precursors for the hollow polymer capsules. The polyisoprene segment in the SCK cores degraded upon the treatment with ozone, leaving the cross-linked polyacrylamide shell as a hollow polymer capsules. The formation of hollow polymer capsules was confirmed by DLS, TEM and AFM. For triethyleneglycol diamine cross- linked SCKs, the average of hydrodynamic diameter (Dh) was 27 ± 9 nm, and the Dh increased to 133 ± 1 nm after ozonolysis. Figure 2.1.3 Hollow polymer capsules by PI-b-PAA micelles (Adapted with permission from ref 8. Copyright 1999 American Chemical Society) 62  Another early example of micelle-templated synthesis of polymer nanocapsules was reported by Liu15, based on a similar strategy. A poly(isoprene)-block-poly(2- cinnamoylethyl methacrylate)-block-poly(tert-butyl acrylate) (PI-PCEMA-PTBA) triblock copolymer forms micelles with a PTBA corona, PCEMA shell and a PI core in THF–methanol mixtures. After the micelle structure was “locked” by UV induced cross- linking of PCEMA, the PI core of the cross-linked aggregate was degraded by ozonolysis to yield a hollow polymer nanocapsule with a central cavity (Figure 2.1.4). The size of the micelles before and after cross-linking was the same, and the size of cross-linked aggregates before and after PI degradation was also the same. The potential application for encapsulation of small molecules was demonstrated by loading the cross-linked PCEMA-PTBA nanocapsules with rhodamine B. The incorporation of the dye into the central cavity of the capsule was directly visualized by TEM.   Figure 2.1.4 Hollow polymer nanocapsules by PI-b-PCEMA-b-PTBA micelles (Adapted with permission from ref 15. Copyright 1999 American Chemical Society) 2.1.2 Preparation of polymer nanocapsules via template approach Another method for synthesizing polymer capsules involves nanoparticles as a sacrificial template. Nanoparticles of desired size can be used to grow cross-linkable 63  polymers which are subsequently cross-linked to form a polymer shell. After dissolution of the nanoparticle template, hollow polymer capsules can be formed.22-30 Mӧhwald reported the formation of a polymer capsule by layer-by-layer assembly of polyelectrolyte to melamine formaldehyde (MF) colloidal particles (Figure 2.1.5).31 The polyelectrolyte multilayer film was formed the alternate adsorption of the negatively charged poly(sodium styrenesulfonate) (PSS) and the positively charged poly(allylamine hydrochloride) (PAH). The adsorption began with PSS, as MF colloidal particles were positively charged. After each adsorption step, the nonadsorbed polyelectrolyte was removed by repeated centrifugation and washing. The desired number of polyelectrolyte layers could be deposited, allowing the control of the multilayer film thickness. After then formation of 9-layer of polyelectrolyte film, MF template was removed by the treatment of acid which decomposed MF. Characterized by SEM, TEM and AFM, the resulted polyelectrolyte capsules was 4.0±0.5 μm in diameter which was larger than the MF template (3.3 μm), due to polymer swelling after the removal of the template. Figure 2.1.5 Polymer capsules by layer-by-layer adsorption of polyelectrolyte (Adapted with permission from ref 31. Copyright 1998 Wiley) 64  Feldheim reported the synthesis of a polymer capsule of polypyrrole and poly(N- methylpyrrole) using gold nanoparticles as a template.32 The polymer bilayer was immobilized at the nanoparticle surface through a membrane-supported polymerization method. Polymer-nanoparticle conjugates with core diameter from 5 to 200 nm, shell thickness from 5 to 200 nm and multilayers of chemically distinct polymers (polypyrrole/poly(N-methylpyrrole)) were synthesized. Consequently, the resulted polymer capsules had controlled core size from 5 to 200 nm and thickness from 5 to 200 nm. In addition, the use of polymer capsules to encapsulate small molecules (rhodamine B as a model compound) was demonstrated by UV-vis spectroscopy. Lyon reported the synthesis of a poly(N-isopropylacrylamide) (pNIPAm) nanogel using gold nanoparticles as a template (Figure 2.1.6).28 Polymer-nanoparticle conjugates were formed by the adsorption of NH2-terminated pNIPAm polymer on citrate stabilized gold nanoparticles. When heated above LCST, the adsorbed pNIPAm layer collapsed to the nanoparticle surface, and served as a hydrophobic nucleus for the polymerization and formation of a cross-linked pNIPAm shell. N, N’-methylenebis(acrylamide) was used as the cross-linker. Dissolution of gold nanoparticle by cyanide yielded the hollow nanogel. The architecture, shape, and morphology of the nanogels before and after dissolution of the template were characterized by TEM and AFM. 65  Figure 2.1.6 AuNP templated synthesis of pNIPAm nanogels (Adapted with permission from ref 28. Copyright 2007 American Chemical Society) Mohwald also reported the use of gold nanoparticle for the synthesis of hollow polymer nanocapsules (Figure 2.1.7).24 The work relies on the immobilization of an ATRP initiator to the surface of citrate stabilized gold nanoparticles, followed by surface- initiated ATRP to grow cross-linkable block copolymers (pDMA-block-pDEA). After cross-linking the polymer brushes at pDMA moieties using 1,2-bis(2-iodoethoxy)ethane, followed by etching out the Au cores, hydrophilic polymer capsules were formed. Due to the presence of positive charges in the cross-linking domain, the capsules swell in acidic condition and shrink in basic condition. The encapsulation and release of a dye, Rhodamine 6G (R6G), have been carried out using this hollow polymer capsule. Capsules were at first incubated with R6G in aqueous solutions at pH 6. Then pH was gradually adjusted to pH 12 by the addition of 1 M NaOH aqueous solution. R6G precipitated at pH 12 and was removed by centrifugation, leading R6G-loaded capsules in aqueous solution. The encapsulation of R6G was confirmed by photoluminescence 66  spectroscopy, as a blue shift of 10 nm was observed after encapsulation. When the R6G- loaded capsules were dialyzed against water of pH 6, the dialyzing water immediately turned fluorescent, indicating the release of R6G molecules. Figure 2.1.7 Synthesis of pH-sensitive polymer capsules using a AuNP template (Adapted with permission from ref 24. Copyright 2005 American Chemical Society) Besides gold nanoparticles, the use of other inorganic templates including silica nanoparticles has also been reported for synthesizing hollow polymer nanocapsules.30 The preparation of polymer nanocapsules with control over the composition, size and properties leads to various applications of these materials, including encapsulation and release of small molecules14, 15, 24, 33 , protection of nanoparticles25 or enzymes27 and nanoreactors.34 However, different applications may require different properties and functions of the polymer nanocapsules. Despite the diversity of methods for nanocapsule preparation, methods for efficiently functionalizing the core of hollow nanocapsules are scarce. One example of capsule functionalization is shown in Figure 2.1.8 by Wooley.16 Polymer hollow capsules, possessing carbonyl groups on their internal surfaces and acrylic acid 67  residues throughout their structure were prepared via self-assembly of amphiphilic block copolymers. Phosphatidylethanolamine-based lipids were covalently attached within the polymer capsule through Schiff-base chemistry, or attached throughout the shell via carbodiimide-mediated coupling. The use of phosphatidylethanolamine lipids labeled with 7-nitrobenz-2-oxa-1,3-diazole allowed for determination of the environmental polarities of the lipid domains within the lipid-capsule constructs. Figure 2.1.8 Selective functionalization of polymer capsules within the capsule or throughout the shell (Adapted with permission from ref 16. Copyright 2005 Elsevier) The functionalization of the core domains of the polymer hollow capsules is an exciting area of research, as the application of capsules requires control over cargo entry and exit and installation of diverse chemical functionality in the capsule interior. Functionalized groups attached in the capsule cores can be segregated from the environment and protected from degradation. In this Chapter, the development of a method on the preparation and functionalization of hollow polymer capsules will be discussed. 68  2.2 Synthesis of hollow polymer nanocapsules using gold nanoparticle templates The synthesis of hollow polymer nanocapsules involves gold nanoparticle as a sacrificial template and block copolymer 20302530. The schematic strategy is shown in Figure 2.1.1. Norbornene functionalized gold nanoparticles (Au-13/14) were synthesized using the norbornene derivatized thiol (13) and dodecanethiol (14) as a coligand. Cross- linkable block copolymer 20302530 was covalently attached to the nanoparticle surface via surface-initiated ROMP, and was laterally cross-linked by diaminooctane (27) on the polyester moiety. Lastly, the gold nanoparticles were removed by cyanide etching, yielding hollow polymer nanocapsules. Figure 2.2.1 Strategy for the synthesis of hollow polymer capsules 69  The structure of HS-2030-block-2530 was analyzed by 1H-NMR and MALDI-ToF MS, after the sequential treatment with Au-20302530 by p-methoxybenzylamine, potassium cyanide and tributylphosphine. The nominal molecular weight of the resulting block copolymer was 10959, calculated using the structure shown in Figure 2.2.2. Figure 2.2.3 shows the molecular weight distribution (Mn=7704, Mw=9399, PDI=1.22) by MALDI-ToF mass spectroscopy. The experimental molecular weight was smaller than the target value, as chain transfer or backbiting during polymerization might occur35 (Figure 1.2.21). In the 1H-NMR spectrum between 4 and 8 ppm (Figure 2.2.4), the broad peak from 5.1 to 5.3 ppm was representative for polynorbornene. The integration of the aromatic protons ortho to the methoxy group (6.81 ppm) to the overall polynorbornene alkene resonances (5.23 and 5.36 ppm) was about 54.5:120. This indicated the fraction of the original ester to the entire polymer backbone was 27:60. Figure 2.2.2 Nominal structure of p-methoxybenzylamine quenched HS-2030-block-2530 70  Figure 2.2.3 MALDI spectrum of p-methoxybenzylamine quenched HS-2030-block-2530 Figure 2.2.4 1H-NMR spectrum of p-methoxybenzylamine quenched HS-2030-block-2530 The cross-linking was achieved by the addition of 0.5 equivalent diaminooctane per ester. To evaluate the extent of cross-linking, particles after cross-linking were treated with allyl isocyanate to cap any free amines and with p-methoxybenzylamine to quench any unreacted esters. After removal of excess capping reagents by dialysis, the cross- linked polymers were examined using 1H-NMR (Figure 2.2.5). The ratio of 1H-NMR 71  integration of the aromatic protons ortho to the methoxy group of p-methoxybenzylamine (6.81 ppm) to the overall polynorbornene alkene resonances (5.23 and 5.36 ppm) indicated that 6% of the esters remain unreacted after cross-linking. The amount of unreacted amine was negligible, as no resonances from the allyl group were observed. The sharp peak at 6.98 ppm was an impurity from the dialysis step. Overall, the degree of cross-link was about 94%. Figure 2.2.5 1H NMR spectrum of cross-linked Au-20302530 after capping with p- methoxybenzylamine and allyl isocyanate TEM images of polymer coated gold nanoparticles were taken. However, only gold nanoparticles were observed instead of the core-shell structures for the nanoparticle- polymer conjugates. The difficulty of the TEM measurement could be attributed to the amorphous nature of the polymer shell and its low contrast under TEM. In fact, gold nanoparticles coated with a 200-mer of polynorbornene were prepared, and even for the nanocomposites with large portion of organic components, the organic shell could not be easily observed (Figure 3.9). 72  Figure 2.2.6 TEM image of Au-25200 After the removal of gold templates by cyanide etching followed by tributylphophine treatment, the hollow polymer capsules (HS-capsules) were characterized by TEM and DLS. The average hydrodynamic size of hollow capsules in diameter was 107 ± 29 nm in THF (Figure 2.2.7). TEM image in Figure 2.2.8 indicates larger diameters (143 nm), presumably arising from capsule flattening on the TEM grid.28 Figure 2.2.7 Size of HS-capsules by DLS 73  20 18 16 14 12 Frequency 10 8 6 4 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 60 70 80 90 e 11 10 12 13 14 15 16 17 18 19 20 21 22 23 24 25 or M Diameter [nm] Figure 2.2.8 TEM image of HS-capsules and size distribution 74  After the removal of the gold nanoparticle, the cross-linked polymers may maintain the capsular structure with polynorbornene as the exterior or collapse to random polymer aggregations. To confirm the formation of hollow capsules, capsules and two analogues were analyzed with surface contact angles, since chemical patterning at substrate surface would change the surface contact angles.36 Normally, contact angles are measured by dropping a small droplet of liquid on a flat horizontal surface, and the angles are determined by the interactions across the interface. The maximum contact angle is called advancing contact angle. The advancing contact angle is measured just before the wetting line starts to advance. The minimum contact angle is called receding contact angle. The receding contact angle is measured while the volume of the drop in decreasing – practically this is done just before the wetting line starts to recede. Presumably, capsules exhibit similar surface properties to polynorbornene if the capsule structure maintains after etching, as polynorbornene is the exterior block. But if capsules collapse to random aggregations, the surface properties are similar to the linear block copolymers, butylamine quenched 20302530. Thin films of different polymers were spin-coated onto glass slides at 50 rounds per second, and the water contact angles of these surfaces were measured and listed in Table 2.2.1. The advancing water contact angle of the capsule coated slides (80.5°) lied between the values for polynorbornene 2560 (87.6°) and butylamine quenched 20302530 (73.3°). This result suggested that the cross- linked polyamide block is partly shielded from water on the spin-coated surface. In the other word, the exterior of the hollow polymer capsules was preferentially composed by polynorbornene, and the polyamide domain in the interior was accessible to water. The porous characteristic provided the capsules as a potential drug delivery vehicle. 75  Table 2.2.1 Water contact angles of hollow polymer capsules and analogues advancing contact angle receding contact angle sample (degree) (degree) bare glass 68.4 ± 0.6 33.2 ± 1.5 capsule 80.5 ± 0.7 69.5 ± 3.7 polynorbornene 2560 87.6 ± 0.3 61.3 ± 0.6 butylamine quenched 20302530 73.3 ± 0.5 52.6 ± 0.5 2.3. Core functionalization through disulfide exchange reaction With the capsules in hand, the research interest was extended to the functionalization of the capsules. Figure 2.3.1 shows the strategy of functionalizing the capsule core through disulfide exchange reactions. When the Au-S bond breaks during cyanide etching, sulfur stays at the polymer termini preferentially in the form of disulfide. The treatment of tributylphosphine reduces disulfide to thiol as a better nucleophile. Lastly, thiols are functionalized through the disulfide exchange reaction using either symmetric disulfides or mixed disulfides. 76  Figure 2.3.1 Functionalization of the capsule hollow core 2.3.1 Core functionalization with pyrene It is known than pyrene and pyrene derivatives exhibit excimeric emissions when they are at close proximity.37-42 Pyrene derivatized disulfide 35 was used as a probe to study the structure of the core domain of the hollow capsules. The synthesis of 35 is shown in Figure 2.3.2. Capsules were functionalized with pyrene via disulfide exchange, purified by dialysis and dissolved in dichloromethane for fluorescence spectroscopy analysis. The rough concentration of the sample solution was about 1 x 10-7 M. The 77  absorption spectrum is shown as inset in Figure 2.3.3. The pyrene functionalized capsule exhibited a strong excimer emission at 478 nm (Figure 2.3.3, trace b), in addition to a monomer emission at 378 nm, indicating close proximity betweeen adjacent disulfide linked pyrene moieties. Only monomer emission was observed (Figure 2.3.3, trace c) after the capsules were treated with tributylphosphine, which cleaves the disulfide linkages to liberate pyrene. Dialysis of the phosphine treated capsules provided capsules with no fluorscence emission (Figure 2.3.3, trace d). These results indicated that the pyrene moieties in nanocapsules functionalized with 35 were covalently attached to the capsules and not physically adsorbed or trapped within polymer shell. Figure 2.3.2 Synthesis of pyrene derivatized disulfide 78  Figure 2.3.3 Fluorescence spectra of pyrene functionalized capsules (λexcitation = 338 nm) (a) compound 35; (b) pyrene functionalized capsules using 35; (c) pyrene functionalized capsules after treatment with tributylphosphine; (d) pyrene functionalized capsules after treatment with tributylphosphine followed by dialysis. Inset: UV-vis absorption of pyrene functionalized capsules It is well known that the polymer morphology is dependent on the solvent environment.43-46 For example, hydrogel swells in aqueous condition and shrinks in organic solvents. It was noticed that the excimeric emission of pyrene functionalized capsules was related to the solvent polarity (Figure 2.3.4). The values of Ie/Im are listed in Table 2.3.1. The Ie/Im ratio increased from 1.29 to 1.93 as the water content of the solution increased from 0% to 50%. In contrast, pyrene functionalized linear polymer didn’t show the same relationship between Ie/Im ratio and solvent polarity. Butylamine quenched linear polymer 20302530 was functionalized with pyrene via the same disulfide exchange reaction using 35. Pyrene functionalized linear polymers had an excimer/monomer ratio of 0.75 in THF, and this ratio did not increase when water was 79  added. These data suggested that increasing water content induced capsule constriction, resulting in a closer packing of pyrene groups within the core. Figure 2.3.4 Fluorescence of pyrene functionalized capsules in different solvents Table 2.3.1 Excimer to monomer emission ratio Ie/Im for pyrene functionalized nanocapsules and polymers in different solutions THF 3:1 THF:H2O 1:1 THF:H2O pyrene functionalized capsules 1.29 1.33 1.93 pyrene functionalized linear polymer 0.75 0.70 0.58 35 1.59 2.3.2 Core functionalization with ferrocene After the core functionalization was confirmed by fluorescence spectroscopy using the pyrene probe 35, the degree of functionalization was studied using ferrocene derivatized disulfides 37 and 39, the syntheses of which are shown in Figure 2.3.5. Ferrocene was functionalized in the capsule core using either the symmetric disulfide 37 80  or the mixed disulfide 39. After the disulfide exchange reaction, the content of Fe and S elements was measured by inductively coupled plasma (ICP) to study the degree of functionalization. The ferrocene functionalized capsule sample was treated with concentrated HNO3 at 100 oC. Solvent was dried under nitrogen flow and heat, and then the residue was redissolved in 2% HNO3 aqueous solution for ICP measurement. The content of Fe and S elements was measured at the same time. The concentrations of Fe and S standard solutions are listed in Table 2.3.2. Figure 2.3.5 Synthesis of ferrocene derivatized disulfides Table 2.3.2 Concentrations of Fe and S standard solutions for ICP measurement Fe content (ppm) S content (ppm) Correlation 0.99928 Correlation 0.99979 prepared measured Prepared measured 0 0.305 0 0.025 2.17 2.21 2.17 2.13 5.48 5.57 5.41 5.57 10.20 9.71 10.13 10.18 15.83 15.69 15.79 15.49 20.55 20.89 20.57 20.73 81  The results of ICP analysis on ferrocene functionalized capsules are listed in Table 2.3.3. The experimental values of Fe:S (molar ratio) in 37 and 39 were 0.9715 and 0.4693, respectively, which were very close to the ideal numbers. If the capsule core was completely functionalized, the ideal number of Fe:S should be 0.5. When the capsules were functionalized using 37, the Fe:S molar ratio was 0.232, indicating 46% functionalization. When the capsules were functionalized using 39, the Fe:S molar ratio was 0.3989, indicating 80% functionalization. Higher degree of functionalization was achieved using the mixed disulfide which carried a better leaving group. 47-51 Table 2.3.3 ICP analysis of ferrocene functionalized nanocapsules sample Fe (ppm) S (ppm) Fe:S (molar ratio) Compound 37 23.32±0.788 13.78±0.927 0.9715 (ideally 1) Capsule functionalized using 37 5.39±0.251 13.33±0.485 0.232 Compound 39 9.63±0.295 11.78±0.594 0.4693 (ideally 0.5) Capsule functionalized using 39 17.21±0.264 24.77±1.854 0.3989 Capsules functionalized with ferrocene in the core using 39 were also characterized by TEM. Compared to the non-functionalized capsules with thiols in the core (Figure 2.2.8), regions with increased contrast towards the capsule cores were observed (Figure 2.3.6), due to the presence of ferrocene in the core region. 82  83  Figure 2.3.6 TEM images of capsules functionalized ferrocene in the core 2.4 Encapsulation and release of small molecules based on charge interactions The core functionalization via disulfide exchange reactions was demonstrated using pyrene functionalized capsules by fluorescence spectroscopy. The degree of functionalization was studied using ferrocene functionalized capsules by ICP analysis. To explore the applications of the capsules, disulfides 41, 43 and 45 which would correspondingly functionalize the capsule core with allyl, COOH and NH2 groups were synthesized (Figure 2.4.1). 84  Figure 2.4.1 Synthesis of allyl, carboxylate and amine derivatized disulfides Figure 2.4.2 shows an example of functionalization of the capsule core with COOH. The functionalization with NH2 or allyl is very similar. Capsules functionalized with COOH and NH2 contain negative and positive charges in the core, respectively, and allyl functionalized capsules are neutral. The size of COOH and NH2 functionalized capsules was measured by DLS (Figure 2.4.3). Both COOH and NH2 functionalized capsules were larger than the bare capsules with thiols in the core (107 nm, Figure 2.2.7), probably because capsules swelled more due to the electrostatic repulsions from charges in the capsule core. Figure 2.4.2 Functionalization of the capsule core with COOH 85  Figure 2.4.3 Size (diameter) of COOH and NH2 functionalized capsules by DLS Both COOH and NH2 functionalized capsules contained charges in the hollow core, and interacted with charged molecules through electrostatic interaction. Alizarin red s (ARS, Figure 2.4.4), an anionic dye molecule, interacted with NH2-capsule through electrostatic attractions. 2 mL of capsule (NH2-capsule or COOH-capsule) solution in dichloromethane (10 mg/mL) and 1 mL of ARS aqueous solution (14.6 mM) were shaken vigorously at room temperature for 1 day. ARS, which was poorly soluble in dichloromethane, was efficiently extracted into the organic layer by NH2-capsule, as the dichloromethane solution turned yellow (Figure 2.4.5, a). The dichloromethane solution of COOH-capsule remained colorless, indicating little ARS transferred into organic layer (Figure 2.4.5, b). The dichloromethane solution of NH2-capsule with ARS incorporated was separated and washed extensively with water, and the color of organic solution stayed yellow, implying ARS was firmly trapped in the capsule core by strong electrostatic attraction (Figure 2.4.5, c). However, when the organic solution was washed with 0.1 M NaOH aqueous solution, the color of organic layer turned to almost colorless, 86  and the color of aqueous layer turned to violet (Figure 2.4.5, d). The color change indicated that most ARS released to aqueous solution, because NaOH neutralized the positive charges in the core and eliminated the electrostatic attraction. After extensive wash by NaOH (aq., 0.1 M), the organic layer turned colorless, and capsules stayed in the organic layer and could be recycled by solvent evaporation. Figure 2.4.4 Structure of alizarin red s (ARS) and methylene blue (MB) Figure 2.4.5 Uptake and release of ARS a) ARS mixed with NH2-capsules; b) ARS mixed with COOH-capsules; c) ARS incorporated NH2-capsules mixed with water; d) ARS incorporated NH2-capsules mixed with 0.1 M NaOH. 87  Besides ARS, bromophenol blue (Figure 2.4.6) was also tried as the anionic model compound to study the encapsulation. Bromophenol blue was also taken up by NH2-capsules based on UV-vis absorption. But compared to ARS which is barely soluble in dichloromethane, bromophenol blue has higher solubility, providing strong background for the encapsulation. Figure 2.4.6 Structure of bromophenol blue Similarly, methylene blue (MB, Figure 2.4.4), a positively charged molecule, interacted with COOH-capsule. MB could be extracted into the organic solution of COOH-capsule, and released back to aqueous solution by the addition of hydrochloric acid (Figure 2.4.7). Unlike ARS, MB has relatively high solubility in organic solutions, yielding high background signal for the extraction. Beside MB, other cationic dye were tried, including alpha-naphthyl red, fat brown rr, basic red 29, basic blue 41, basic fuchsin, methyl violet 2B and safranine O, but these dyes are as soluble as MB. Alcian yellow and bismarck brown y may work better, as they are polycationic (structures shown in appendix I). 88  Figure 2.4.7 Uptake and release of MB a) MB mixed with NH2-capsules; b) MB mixed with COOH-capsules; c) MB incorporated COOH-capsules mixed with water; d) MB incorporated COOH-capsules mixed with 0.1 M HCl. The uptake process was quantitatively studied, using allyl-capsule (neutral core), COOH-capsule (polyanionic core), NH2-capsule (polycationic core), ARS (anionic small molecule) and MB (cationic small molecule). Polymer capsules (1 mg/mL) were dissolved in dichloromethane. The amount of dye taken up in dichloromethane was determined by UV-vis spectroscopy, monitoring at 424 nm for ARS and at 656 nm for MB. Dye solutions were 14.6 mM (ARS) and 2.7 mM (MB) in water. 2 mL polymer solution or dichloromethane was mixed with 1 mL dye solution in a 4 mL cuvette and agitated gently at room temperature for 1 hour. After the incubation with aqueous dye solutions (ARS or MB), dichloromethane solutions of capsules were separated and dried with Na2SO4. After the incubation with ARS, the absorbance at 424 nm was 0.63 for the dichloromethane solution of incorporated NH2-capsule, 0.06 for dichloromethane, 0.08 for the dichloromethane solution of allyl-capsule, and 0.01 for the dichloromethane solution of COOH-capsule. Conversely, MB selectively interacted with COOH-capsules. 89  The absorbance of solution with MB incorporated was 0.61 for COOH-capsule, 0.18 for dichloromethane, 0.22 for allyl-capsule, and 0.14 for NH2-capsule (Figure 2.4.8). Figure 2.4.8 Uptake of ARS and MB in different polymer capsules The loading capacity of a single capsule was evaluated. First of all, the number of amine groups functionalized in a single was calculated. Murray prepared different size of alkanethiol coated gold nanoparticles and systematically studied the number of thiol ligands at the surface of nanoparticles. For 3.6 nm diameter gold nanoparticles, the number of thiol ligands can be estimated to be 261.52 In this case, each polymer shell around a nanoparticle contained 261 thiol groups. After the removal of dodecanethiol following etching, PBu3 reduction and dialysis, the number of thiols remaining in each capsule would be 73, as the ratio of dodecanethiol to norbornene-thiol was 2.6 to 1. The number of amine groups was assumed the same as thiol groups. Second, the concentration of amine groups and ARS was calculated. As each amine group was associated with a molecular weight of 9006 (Figure 2.4.9), the concentration of amine groups was 1.10x10-4 mol/L when the concentration of the amino functionalized capsule was 1.0 mg/mL in DCM. The concentration of ARS was calculated from the calibration 90  curve (Figure 2.4.10). When the amino functionalized capsule (1.0 mg/mL in DCM, 10.0 mL) was incubated with ARS (5.0 mM aq., 10.0 mL), the equilibrium absorbance at 424 nm was 0.48. So the concentration of ARS in the DCM layer was 1.16x10-4 M. Lastly, since [NH2]/[ARS] equals 0.98, each capsule encapsulated 74 ARS molecules. Figure 2.4.9 Nominal molecular weight of polymer chain in NH2-capsules Figure 2.4.10 Correlation of ARS concentration and absorbance at 424 nm To study the release process, the NH2-capsule (1.0 mg/mL in dichloromethane, 20 mL) was incubated with ARS solution (14.6 mM in water, 10 mL) for 24 hours to ensure the maximum loading of ARS. Then the organic layer (dichloromethane) was separated, washed with 10 mL water for 3 times, and dried with Na2SO4. The final absorbance of 91  the dichloromethane solution was 1.10 at 424 nm. The solution was diluted with dichloromethane to give 1.00 absorbance at 424 nm, and treated with different aqueous solution, including 3.4 M NaCl, 0.0010 M KOH, 0.10 M KOH, and 0.10 M KOH mixed with 3.4 M NaCl. 2 mL of the ARS incorporated NH2-capsule solution (dichloromethane) was stirred with 1 mL aqueous at room temperature for 1 hour, and the dichloromethane layer was separated and dried with Na2SO4 for UV-vis measurement. The absorbance at 424 nm indicating the ARS amount is listed in Figure 2.4.11. The release to water was less than 10% (abs@424 nm = 0.910). The amount of release increased along with the increase of solution pH, as positive charges in the capsule core were neutralized at high pH. 45% incorporated ARS released to 0.0010 M KOH aq. (abs@424 nm = 0.555), and 64% released to 0.10 M KOH aq. (abs@424 nm = 0.364). Also, the release could be controlled by the ionic strength of the aqueous solution. To the mixture of KOH solution (0.20 M in water, 0.5 mL) and NaCl solution (6.8 M in water, 0.5 mL), 56% release was observed (abs@424 nm = 0.438). Figure 2.4.11 Release of ARS from NH2-capsule to different aqueous media 92  Similarly, the release of MB from COOH capsule was controlled by the addition of acid. The COOH-capsule (1 mg/mL in dichloromethane, 20 mL) was incubated with MB solution (2.7 mM in water, 10 mL) for 24 hours. After wash and drying, dichloromethane layer exhibited blue color due to the incorporated MB (abs@656 nm = 0.847). 2 mL of the COOH-capsules in dichloromethane with MB incorporated was mixed with 1 mL of 0.1 M or 1.0 M HCl aqueous solution at room temperature for 1 hour. The final absorbance at 656 nm in dichloromethane was 0.489 or 0.413, after the treatment of 0.1 M and 1.0 M HCl solution, respectively (Figure 2.4.12). Figure 2.4.12 Release of MB from COOH-capsule to different aqueous media 2.5 Encapsulation of inorganic cations and gold nanoparticles The encapsulation of charged organic molecules into core-functionalized hollow polymer capsules has been demonstrated using ARS and MB as model compounds. The encapsulation is based on electrostatic interaction and can be modulated by solution pH. In addition, the encapsulation of inorganic cations and nanoparticles has been investigated. 93  1 mL of capsules solution (1 mg/mL, dichloromethane) with SH, NH2 or COOH functionality in the core was mixed with 1 mL of saturated CoSO4 or CuSO4 aqueous solution. After the mixture was stirred at room temperature for 12 hours, the organic layer was separated and measured by UV-vis. The organic layer remained colorless, and no cations were taken up into any types of polymer capsules. In addition to stirring the heterogeneous solution at room temperature, the mixture was treated with bath sonication for 1 hour, but still no uptake was observed by UV-vis. Moreover, saturated solutions of the salts in methanol were prepared and mixed polymer capsule solution (1 mg/mL, dichloromethane or tetrahydrofuran) at 1:10 volume ratio. The salts precipitated and solution was still colorless after filtration. It seems small molecules must be slightly soluble in organic solutions before they diffuse into the capsule cores. Besides Co2+ and Cu2+ cations, the encapsulation of gold nanoparticles has also been studied. The uptake experiment of nanoparticles may reveal the pore size of the polymer capsules. Gold nanoparticles (15 nm in diameter) stabilized by citrate were obtained from Xiaoliang Wei from Zimmt lab. Capsules (1 mL, 1 mg/mL in THF) with SH functionality in the core was mixed with gold nanoparticles (1 mL, aqueous). After the mixture was stirred at room temperature for 1 day, 5 mL of dichloromethane was added. The organic layer was washed with water, separated, dried with Na2SO4 and measured by UV-vis spectrometer and TEM. A plasmon band was clearly observed at 520 nm (Figure 2.5.1), indicating the presence of gold nanoparticles. However, TEM showed aggregation of nanoparticles (Figure 2.5.2). As the capsules couldn’t be easily observed when gold nanoparticles were present, it was difficult to tell whether the 94  nanoparticles were taken up in the hollow core or physically trapped within the polymer brushes. Figure 2.5.1 Absorption of gold nanoparticles taken up by hollow polymer capsules Figure 2.5.2 TEM of gold nanoparticles taken up by hollow polymer capsules 95  2.6 Functionalization in the cross-linking domain via SN2 substitution A versatile and modular approach for the synthesis of hollow polymeric capsules that can be readily and diversely functionalized in the core through disulfide exchange reactions has been developed. These core functionalized capsules can selectively extract hydrophilic cationic or anionic dyes into organic solutions based on the charge complementarity of the core functionality. Besides the core functionalization, the cross- linking domains can be functionalized via SN2 substitution. As shown in Figure 2.6.1, the amide nitrogens can be deprotonated by sodium hydride, and the deprotonated amides react with good electrophiles (e.g. alkyl bromide) by SN2 reactions. The capsules can be functionalized with different functional groups in the cross-linking domain using different alkyl bromide. O R-Br, NaH O R HN N THF, reflux NH N R O O O Br Br R-Br: or Fe 50 51 Figure 2.6.1 Functionalization of the cross-linking domain via amide substitution Pyrene derivatized bromide was synthesized using 1-pyrenemethanol and 1,5- dibromopentane (Figure 2.6.2). 96  Figure 2.6.2 Synthesis of pyrene derivatized alkyl bromide Pyrene functionalized capsules in dichloromethane (roughly 10-7 M) was analyzed by fluorescence spectroscopy, and showed strong excimeric emission at 480 nm, while 52 showed only monomeric emission (Figure 2.6.3). The fluorescence spectra confirmed the presence of pyrene groups, and also indicated that pyrenes were closely packed at the proximity of each other. 1H-NMR spectrum showed a broad aromatic peak around 8 ppm (Figure 2.6.4). By comparing the integration of aromatic protons from pyrene and olefinic protons from polynorbornene, the degree of functionalization was 35.6%, as the ideal integration ratio was 270:120 if the alkylation was 100% complete. Figure 2.6.3 Emission of pyrene functionalized capsules (capsules made from C8 cross- linked Au-20302530 and functionalized with 50 on amide nitrogens) 97  Figure 2.6.4 1H-NMR of pyrene functionalized capsules (capsules made from C8 cross- linked Au-20302530 and functionalized with 50 on amide nitrogens) Capsules were also functionalized with ferrocene in the cross-linking domain, 1 using (6-bromohexyl)ferrocene. H-NMR of the feccorene functionalized capsules showed characteristic peaks for mono-substituted ferrocene (Figure 2.6.5). The degree of functionalization was 9.7%, based on the comparison between the integration of ferrocene protons and alkene resonance. 98  Figure 2.6.5 1H-NMR of ferrocene functionalized capsules (capsules made from C8 cross-linked Au-20302530 and functionalized with 51 on amide nitrogens) As demonstrated previously, the location of cross-linking domain could be modulated by controlling the polymerization sequence of the linear polymer precursor.53 When the capsules were synthesized from 20302530 block copolymer precursor, the cross- linking domain was consequently in the interior. If the structure of the linear polymer precursor changed to 25252030255 (2525 was close to NP surface), the position of the cross- linking domain moved to the capsule exterior (Figure 2.6.6). Consequently, the functional groups introduced by alkylation of the amide would be preferentially located on the capsule exterior. 99  Figure 2.6.6 Hollow polymer capsules with cross-linking domain in the exterior The capsules prepared by cross-linking 25252030255 using diaminooctane was functionalized by (6-bromohexyl)ferrocene. Thanks to the enrichment of ferrocene, TEM analysis of the ferrocene functionalized capsules showed ring structures, as an evidence of hollow architectures. The overall size was about 150 nm, and the core size was difficult to measure (Figure 2.6.7). Calculated from 1H-NMR integrations (Figure 2.6.8), the degree of functionalization was 26.4%. 100  Figure 2.6.7 TEM of ferrocene functionalized capsules (capsules made from C8 cross- linked Au-25252030255 and functionalized with 51 on amide nitrogens) Figure 2.6.8 1H-NMR of ferrocene functionalized capsules (capsules made from C8 cross-linked Au-25252030255 and functionalized with 51 on amide nitrogens) 101  Lastly, the whole capsules can be functionalized on the alkenes throughout the polymer backbones. The ability of the capsules being functionalized with different functional groups at different positions makes this polymer hollow capsule a versatile platform for various applications. 2.7 Kinetic study on encapsulation and release processes Cross-linked polymers are widely used as drug delivery vehicles,54, 55 sensors56 and catalysts.57 The use of cross-linked polymers for controlled uptake and release has been a subject of great interest because of their properties that allow them to respond to external stimuli. The use of pH-responsive polymers in drug delivery has been widely studied.58 In addition to pH, functionalized polymers also respond to other environmental stimuli, such as temperature,59, 60 light,61 ionic strength62 and solvent.63 A versatile and modular approach for the synthesis of hollow polymeric capsules that can be readily and diversely functionalized in the core through disulfide exchange reactions was discussed previously. The potential application of the functionalized capsules in drug delivery was demonstrated by selective extraction of hydrophilic cationic or anionic dyes into organic solutions based on the charge complementarity of the core functionality. Since the diffusion rate of small molecules through the polymer shell could be modulated by tuning the structure of the cross-linked polymers (i.e. cross- linking positions and cross-linker structures), the research attention was extended to the impact of capsule structure on the uptake/release rate. The result would be significant for designing drug delivery vehicles. 102  2.7.1 Effect of cross-linker structures Organic soluble polymer capsules with NH2 groups functionalized in the polymer hollow core via disulfide bonds exhibited high selectivity on the extraction of ARS from aqueous solution to organic solution with little background adsorption. The use of ARS would be a good model to study how the capsule structure change affects the encapsulation and release rate.   Figure 2.7.1 Use of ARS as a model to study the encapsulation and release kinetics The preparation of functionalized capsules followed by kinetic study of encapsulation and release is shown in Figure 2.7.1. Block copolymer 20302530 was grafted at the surface of 3.6 nm gold nanoparticles by surface-initiated ROMP. The 103  polyester moiety was cross-linked by different diamines, including TEG, C8, C4 and Cy diamines. After removal of the gold template and regeneration of the free thiol at polymer termini, the capsule core was functionalized with NH2 groups using 45 through disulfide exchange reactions. The extent of cross-linking on C8 cross-linked capsules has been reported in Chapter 2.2. The extents of cross-linking by other diamines were determined using the same method: the amount of unreacted ester and amine were analyzed by 1H-NMR, after the capping by p-methoxybenzylamine and allyl isocyanate, respectively. The 1H-NMR spectra are shown in Figure 2.7.2 (a-d). When the capsules were cross-linked by TEG, C8 or C4 diamines, the amount of unreacted ester was less than 5%, and the amount of unreacted amine was negligible. Therefore the overall extent of cross-linking for TEG, C8 or C4 diamines cross-linked capsules was higher than 95%. When the capsule was cross-linked by Cy diamine, the amount of unreacted ester was about 40%, although the amount of unreacted amine was still negligible. Therefore the overall extent of cross- linking for Cy cross-linked capsules was about 60%. Capsules cross-linked by TEG, C8 and C4 were used to study the effect of cross-linker structures, as they had the same extent of cross-linking. The capsule cross-linked by Cy was not studied, because the system would be too complicated if multiple variables are changed at the same time (i.e. cross-linker structure and degree of cross-linking). 104  Figure 2.7.2a Amount of unreacted ester and amine in capsules cross-linked by C8 Figure 2.7.2b Amount of unreacted ester and amine in capsules cross-linked by TEG 105  Figure 2.7.2c Amount of unreacted ester and amine in capsules cross-linked by C4 Figure 2.7.2d Amount of unreacted ester and amine in capsules cross-linked by Cy 106  Before the kinetics study on encapsulation, the experimental condition of the interaction between NH2 functionalized capsules and ARS was optimized. Different concentrations of ARS aqueous solutions were prepared: 1.00 mM, 5.00 mM, 10.0 mM, 15.0 mM, and 20.0 mM. C8 diamine cross-linked and NH2 functionalized capsule was dissolved in dichloromethane at 1mg/mL. 2 mL capsule solution was stirred with 1 mL ARS solution at room temperature overnight. The organic layer was separated, dried over Na2SO4, and analyzed by UV-Vis spectroscopy. As shown in Figure 2.7.3, the absorbance increased along with the ARS concentration and gradually reached the saturation. Moreover, there was a critical ARS concentration between 1.00 mM and 5.00 mM, as the absorption spectra of capsule solution incubated with 1.00 mM or 5.00 mM ARS were significantly different. The capsule solution incubated with 1.00 mM ARS showed negligible uptake. Therefore, 5.00 mM ARS solution was used in the kinetic study. Figure 2.7.3 Uptake of ARS with different concentrations The uptake rate of ARS by different capsules was measured by monitoring the UV-vis absorption in the organic solution (bottom layer). The equipment setup is shown in Figure 2.7.4. The organic layer was tall enough so that the beam passed through only 107  the organic layer and absorbance was exclusively attributed by ARS in the organic solution. Figure 2.7.4 Equipment setup for the kinetics study The kinetics of the encapsulation of ARS by NH2 functionalized capsules are shown in Figure 2.7.5. The absorbance at 424 nm was recorded every 30 seconds for 1 hour. Each sample was measured twice with good reproducibility. The amount of ARS taken up by any capsules was more than that by dichloromethane, due to the presence of electrostatic attraction between the positively charged capsules and negatively charged ARS molecules. The uptake rates of capsules cross-linked by different diamines were compared. The uptake rate of TEG cross-linked capsules was faster than that of C8 cross- linked capsules, and C4 cross-linked capsules was the fastest. The UV-vis absorbance indicating this trend is shown in Figure 2.7.5. Each measurement was done twice, and the results were reproducible (Figure 2.7.6). 108  Figure 2.7.5 ARS uptake rate into capsules with different cross-linkers Figure 2.7.6 Repeated measurements of ARS uptake rate 109  Noticeably but unexpectedly, a second stage of absorbance increase was sometimes observed during the uptake study, mostly after 2000 seconds. For example, in the second measurement of C8 cross-linked capsules, the absorbance increased much faster after 2500 s (Figure 2.7.6, trace C8 2). Full UV-vis spectra were recorded at 3600 s and 5200 s (Figure 2.7.7). The baseline in the spectrum at 5200 s was very high, and thus the absorbance used to represent ARS concentration was not accurate. After the dichloromethane layer was separated and dried with Na2SO4, the baseline dropped down. Figure 2.7.7 UV-vis spectra showing increased baseline As discussed in chapter 2.4, ARS encapsulated in the capsules could be released upon the treatment of base. The kinetics of pH triggered ARS release was studied to investigate the effect of cross-linker structures on the release rate.Capsules which were made from TEG, C8 or C4 cross-linked 20302530 and functionalized with NH2 in the core 110  were dissolved in dichloromethane. The capsules solution was stirred vigorously with ARS aqueous solution at room temperature for 1 day to achieve the maximum loading. The organic solutions were separated, washed with water, dried by Na2SO4 and diluted with dichloromethane to the same concentration of ARS (Abs@424nm=0.40). First of all, the release was studied by monitoring the organic layer during the biphasic release. 2.0 mL of ARS incorporated capsule solution in dichloromethane, 1.0 mL of NaOH and a mini stir bar were placed in a 4 mL cuvette (Figure 2.7.4). The absorbance at 424 nm of the organic layer (bottom) was monitored by UV-vis spectroscopy. The release kinetics is shown in Figure 4.8-4.10. The absorbance dropped at first, but started to increase after a while. The final absorbances were even greater than that before release. According to the full UV-vis spectra before and after release, the increase of the absorbance was caused by the baseline, which was also observed during encapsulation. Figure 2.7.8 UV-vis spectra of ARS release from C8 cross-linked capsules Left: kinetics. Right: Full UV-vis absorbance before and after release 111  Figure 2.7.9 UV-vis spectra of ARS release from TEG cross-linked capsules Left: kinetics. Right: Full UV-vis absorbance before and after release Figure 2.7.10 UV-vis spectra of ARS release from C4 cross-linked capsules Left: kinetics. Right: Full UV-vis absorbance before and after release Since the baseline problem was always encountered, the absorbance for ARS in the organic layer could not be accurately acquired. The second method of studying the release process would be monitoring the increase of ARS concentration in the aqueous layer. The UV-vis equipment setup was modified by decreasing the height of organic layer, so that the beam only passed through the aqueous layer. A mechanical stirrer was introduced to stir the aqueous layer (Figure 2.7.11). 1.0 mL of ARS incorporated capsule solution in dichloromethane, 2.0 mL of NaOH (aqueous, 1.0 mM) and a stir bar were placed in a 4 mL cuvette. The mechanical stirrer was positioned in the middle of the 112  aqueous layer. As ARS changed to violet in basic solution, the absorbance at 555 nm was monitored to represent the concentration of ARS in aqueous layer. Theoretically, the ARS concentration in aqueous layer should increase consecutively, but the actual kinetics showed spikes (Figure 2.7.12) because ARS molecules in aqueous layer were not evenly distributed. The mechanical stirrer was lowered down, but the beam was blocked. Figure 2.7.11 Equipment setup for monitoring the aqueous layer Figure 2.7.12 Increase of ARS in the aqueous layer during release (Capsule: C8 cross-linked 20302530) Lastly, the release was studied by static measurement at different time points. ARS incorporated capsule solution (dichloromethane, 8.0 mL) and NaOH aqueous solution (1.0 mM, 4.0 mL) were mixed and stirred in a scintillation vial. 300 μL of the 113  aqueous solution (top) was taken out and measured by Tecan plate reader every 10 minutes (10, 20, 30, 40, 50, 60 min). After each measurement, the aqueous solution was immediately transferred back to the vial. Figure 2.7.13 shows the release kinetics from capsules cross-linked by different diamines in 20302530. The fastest release was from C4 cross-linked capsules, and the slowest release was from C8 cross-linked capsules. Figure 2.7.13 Release from capsules with different cross-linkers In conclusion, how the cross-linker structures affected the encapsulation and release kinetics of a hydrophilic small molecule by hydrophobic polymer capsules were studied, using UV-vis spectroscopy. The fastest encapsulation was from C4 cross-linked capsules, the slowest encapsulation was from C8 cross-linked capsules, and the encapsulation rate was medium in TEG cross-linked capsules. The release kinetics followed the same trend: fastest from C4 cross-linked capsules and slowest from C8 cross-linked capsules. 114  2.7.2 Effect of cross-linking positions Besides the cross-linkers, how the cross-linking positions of polymer capsules affected the encapsulation rate was also studied. The general strategy was similar to the one used in the project of nanoparticle stability (Chapter 1). The capsules were derived from 20302530 or 25252030255 block copolymers and cross-linked by C8 diamine at the polyester moiety. Capsules were functionalized with NH2 groups in the core. The uptake kinetics of ARS by exterior cross-linked capsules is shown in Figure 2.7.14 (green triangles). The uptake rate was faster than that of the interior cross-linked capsules. To rationalize this observation, T1 values of the alkene protons in different capsules were measured by 1H-NMR and are listed in Table 2.7.1. As larger T1 value indicated more flexible conformation, the exterior cross-linked capsules were less rigid than the interior cross-linked capsules. The result was the same as in chapter 1: the diffusion of small molecules is faster in a more flexible polymer. Figure 2.7.14 Effect of cross-linking positions on the uptake kinetics 115  Table 2.7.1 T1 values of hollow polymer capsules with different cross-linking positions T1 @ 5.23 ppm [s] T1 @ 5.36 ppm [s] (standard deviation) (standard deviation) Exterior cross-linking 1.139 (1.471x10-3) 1.337 (2.194x10-3) Interior cross-linking 1.107 (9.638x10-4) 1.278 (7.813x10-4) In addition to encapsulation, the effect of cross-linking positions on the rate of release was also studied. C8 cross-linked capsules from 20302530 or 25252030255 block copolymers were incubated with ARS aqueous solution. The release by pH stimulus was studied by the static measurement of ARS concentration in aqueous layer (method c in Experimental). Figure 2.7.15 shows the release kinetics: the release from exterior cross- linked capsules was faster than from interior cross-linked capsules. Figure 2.7.15 Release kinetics of capsules with different cross-linking positions 2.7.3 Effect of environmental pH The effect of environment pH on the uptake rate was studied, using the capsules cross-linked by C8 in the interior. The uptake was faster in acidic condition (Figure 2.7.16), as the protonation of amine groups in the capsule core was favored. The effect of 116  environment pH on the release was also studied. The result had been discussed in Chapter 2.4. The release was faster when a more concentrated NaOH (aq.) solution was used. Figure 2.7.16 Effect of pH on ARS uptake rate Overall, hollow polymer capsules have been prepared from cross-linked block copolymers, using gold nanoparticles as a template. The core functionalized capsules can be used as a potential drug delivery vehicle, as charged small molecules can be taken up or released, based on charge complementarity. How the cross-linker structures of polymer capsules, the cross-linking positions and the environment pH influence the uptake and release rate has been studied by UV-vis spectroscopy. The result is useful for designing of drug delivery vehicles. 117  2.8 Conclusion A versatile and modular approach has been developed for the synthesis of hollow polymer nanocapsules that can be readily and diversely functionalized. using gold nanoparticles as a template. Block copolymers were grafted at the surface of gold nanoparticles via surface-initiated ring-opening metathesis polymerization. The polyester moiety in the block copolymer was cross-linked by diamine to afford cross-linked polymer shells around gold nanoparticles. After the removal of the gold nanoparticle template, hollow polymer nanocapsules were obtained. The core of polymer nanocapsules could be functionalized through disulfide exchange reactions. Capsules functionalized with pyrene, ferrocene, amines, and carboxylic acids were prepared and characterized using dynamic light scattering, fluorescence spectroscopy, and transmission electron microscopy. These core functionalized capsules selectively extracted hydrophilic cationic or anionic dyes into organic solutions based on the charge complementarity of the core functionality. The encapsulation and release rates associated with different capsules were measured by UV-vis spectroscopy. 2.9 Experimental Compound 35: To a solution of 1-pyrenebutyric acid (0.29 g, 1.0 mmol) in 2 mL dichloromethane was added oxalyl chloride (1.0 mL of 2.0 M solution in dichloromethane, 2.0 mmol). The 118  mixture was stirred under nitrogen at room temperature for 2 hours. The solvent and excess oxalyl chloride were removed under reduced pressure. The resulted solid was redissolved in 5 mL dichloromethane. Cystamine dihydrochloride (0.090g, 0.40 mmol) and triethylamine (0.42 mL, 3.0 mmol) were added. The mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the product was purified by flash column chromatography (SiO2, ethyl acetate) to give 35 (0.17 g, 0.25 mmol, 62%) as a white solid. 1 H NMR (400 MHz, CDCl3): δ 2.15 (quint, J=7.1 Hz, 4H, -CH2CH2CH2-), 2.26 (t, J=7.1 Hz, 4H), 2.75 (t, J=6.4 Hz, 4H), 3.31 (t, J=7.5 Hz, 4H), 3.50 (q, J=6.3 Hz, 4H, - CONHCH2CH2-), 6.21 (t, J=5.7 Hz, 2H, aromatic), 7.77 (d, J=7.8 Hz, 2H, aromatic), 7.92-8.05 (m, 10H, aromatic), 8.09-8.15 (m, 4H, aromatic), 8.21 (d, J=9.3 Hz, 2H, aromatic), 13 C NMR (100 MHz, CDCl3): δ 27.4 (alkyl), 32.9 (alkyl), 35.9 (alkyl), 37.9 (alkyl), 38.6 (alkyl), 123.5 (aromatic), 124.9 (aromatic), 125.0 (aromatic), 125.1 (aromatic), 125.2 (aromatic), 126.0 (aromatic), 126.8 (aromatic), 127.4 (aromatic), 127.5 (aromatic), 127.7 (aromatic), 128.9 (aromatic), 130.1 (aromatic), 131.0 (aromatic), 131.5 (aromatic), 135.9 (aromatic), 173.4 (-CONH-). HRMS(FAB): calculated for C44H40N2O2S2 (M+Na)+ 715.2429, found 715.2446. Compound 37: 119  To a solution of ferrocenecarboxylic acid (0.23 g, 1.0 mmol) in 2 mL dichloromethane was added oxalyl chloride (1.0 mL of 2.0 M solution in dichloromethane, 2.0 mmol). The mixture was stirred under nitrogen at room temperature for 2 hours. Solvent and excess oxalyl chloride were removed under vacuum. The resulted solid was redissolved in 5 mL dichloromethane. Cystamine dihydrochloride (0.090g, 0.40 mmol) and triethylamine (0.42 mL, 3.0 mmol) were added. The mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the product was purified by flash column chromatography (SiO2, ethyl acetate/hexanes, 4/1), to provide 37 (0.13 g , 0.23 mmol 58%) as an orange solid. 1 H NMR (400 MHz, CDCl3): δ 3.00 (t, J=6.4 Hz, 4H, -CH2CH2S-), 3.74 (q, J=6.5, 6.4 Hz, 4H, -CONHCH2CH2-), 4.25 (s, 10H, unsubstituted Cp), 4.37 (t, J=1.9 Hz, 4H, substituted Cp), 4.80 (t, J= 1.9 Hz, 4H, substituted Cp), 6.62 (t, J=5.9 Hz, 2H, -CONH-). 13 C NMR (75.5 MHz, CDCl3): δ 38.3 (alkyl), 39.1 (alkyl), 68.5 (ortho or meta to the substitute in Cp), 70.0 (unsubstituted Cp), 70.8 (ortho or meta to the substitute in Cp), 76.0 (substituted C in Cp), 171.1 (-CONH-). HRMS(FAB): calculated for C26H28N2O2S2Fe2 (M+Na)+ 599.0189, found 599.0171. Compound 38: To a solution of 37 (1.15 g, 2.00 mmol) in 10 mL THF were added tributylphosphine (1.0 mL, 4.0 mmol) and 1.0 mL MeOH. The mixture was stirred at room temperature under 120  nitrogen for 1 day. The solvent was removed under reduced pressure and the product was purified by flash column chromatography (SiO2, Hexanes/EtOAc 1/1) to provide 38 (0.53 g , 1.8 mmol, 92%) as an orange liquid. 1 H NMR (400 MHz, CDCl3): δ 1.40 (t, J=8.4 Hz, 1H, -SH), 2.78 (q, J=7.0 Hz, 2H, - CH2CH2SH), 3.56 (q, J=6.2 Hz, 2H, -CONHCH2CH2-), 4.22 (s, 5H, unsubstituted Cp), 4.36 (t, J= 1.9 Hz, 2H, substituted Cp), 4.68 (t, J= 1.9 Hz, 2H, substituted Cp), 6.11 (s, 1H, -CONH-). 13 C NMR (100 MHz, CDCl3): δ 25.2 (alkyl), 42.5 (alkyl), 68.3 (ortho or meta to the substitute in Cp), 70.66 (unsubstituted Cp), 70.73 (ortho or meta to the substitute in Cp), 76.0 (substituted C in Cp), 170.6 (-CONH-). HRMS(FAB): calculated for C13H15NOSFe (M+Na)+ 312.0121, found 312.0129. Compound 39: To a solution of 38 (0.29 g, 1.0 mmol) in 10 mL THF was added 4,4’-dithiodipyridine (0.33 g, 1.5 mmol). The mixture was stirred at room temperature for 1 day. The solvent was removed under reduced pressure and the product was purified by flash column chromatography (SiO2, EtOAc) to give 39 (0.31 g , 0.78 mmol, 78%) as an orange solid. 1 H NMR (400 MHz, CDCl3): δ 2.98 (t, J=6.2 Hz, 2H, -CH2CH2S-), 3.67 (q, J=6.2 Hz, 2H, -CONHCH2CH2S-), 4.21 (s, 5H, unsubstituted Cp), 4.36 (t, J= 1.9 Hz, 2H, 121  substituted Cp), 4.65 (t, J= 1.9 Hz, 2H, substituted Cp), 6.01 (t, J=5.4 Hz, 1H, -CONH-), 7.48 (d, J=6.2 Hz, 2H, meta to N in pyridine), 8.51 (d, J=6.2 Hz, 2H, ortho to N in pyridine). 13 C NMR (100 MHz, CDCl3): δ 38.2 (alkyl), 38.3 (alkyl), 68.3 (ortho or meta to the substitute in Cp), 70.0 (unsubstituted Cp), 70.8 (ortho or meta to the substitute in Cp), 75.7 (substituted C in Cp), 120.3 (pyridine, meta to N), 148.5 (pyridine, substituted C), 150.0 (pyridine, ortho to N), 170.8 (-CONH-). HRMS(FAB): calculated for C18H18N2OS2Fe (M+Na)+ 421.0108, found 421.0122. Compound 41: To a solution of allyl mercaptan (0.165 mL, 2.0 mmol) in 10 mL THF was added 4,4’- dithiodipyridine (0.44 g, 2.0 mmol). The mixture was stirred at room temperature for 1 day. The solvent was removed under reduced pressure and the product was purified by flash column chromatography (SiO2, ethyl acetate/hexanes 2/3) to provide 41 (1.5 mmol, 0.27 g, 73%) as a pale yellow oil. 1 H NMR (300 MHz, CDCl3): δ 3.39 (d, J=7.4 Hz, 2H, =CHCH2S-), 5.13 (d, J=9.6 Hz, 1H, terminal alkene trans to the alkyl), 5.16 (d, J=16.9 Hz, 1H, terminal alkene cis to the alkyl), 5.74-5.89 (m, 1H, internal alkene), 7.46 (d, J=5.8 Hz, 2H, meta to N in pyridine), 8.47 (d, J=5.5 Hz, 2H, ortho to N in pyridine). 122  13 C NMR (75.5 MHz, CDCl3): δ 41.9 (alkyl), 120.0 (terminal alkene), 120.3 (pyridine, meta to N), 132.1 (internal alkene), 149.1 (pyridine, substituted C), 149.7 (pyridine, ortho to N). HRMS(FAB): calculated for C8H9NS2 (M+H)+ 184.0255, found 184.0262 Compound 43: The synthesis of 7 followed a modified reported procedure.2 To a solution of 3- mercaptopropionic acid (0.21 g, 2.0 mmol) in 10 mL THF was added 4,4’- dithiodipyridine (0.44 g, 2 mmol). The mixture was stirred at room temperature for 1 day. The resulted precipitates were collected by filtration and purified by flash column chromatography (SiO2, methanol/dichloromethane 1/4) to provide 43 (0.20 g, 1.1 mmol, 53%) as a solid. 1 H NMR (400 MHz, DMSO-d6): δ 2.61 (t, J=6.8 Hz, 2H, alkyl), 2.99 (t, J=6.8 Hz, 2H, alkyl), 7.57 (d, J=6.2 Hz, 2H, meta to N in pyridine), 8.49 (d, J=6.2 Hz, 2H, ortho to N in pyridine), 12.50 (s, 1H, HOOC-). 13 C NMR (100 MHz, DMSO-d6): δ 33.3 (alkyl), 33.4 (alkyl), 119.8 (pyridine, meta to N), 147.7 (pyridine, substituted C), 149.7 (pyridine, ortho to N), 172.4 (HOOC-). Compound 45: 123  The synthesis of 45 followed a reported procedure.64 The yield is 60%, and the product is a yellow solid. 1 H NMR (400 MHz, D2O): δ 2.97 (t, J=6.6 Hz, 2H, alkyl), 3.24 (t, J=6.5 Hz, 2H, alkyl), 7.72-7.77 (m, 2H, aromatic), 7.96 (d, J=8.5 Hz, 1H, aromatic). 13 C NMR (100 MHz, D2O): δ 33.8 (alkyl), 37.4 (alkyl), 125.9 (aromatic), 125.4 (aromatic), 128.4 (aromatic), 128.7 (aromatic), 143.9 (aromatic), 144.9 (aromatic), 169.3 (-COOH). MS (FAB): Calculated for C9H11N2O4S2 (M+H)+ 275.0, found 275.1 Compound 50: The synthesis of 50 followed a reported procedure.37 The product is a light yellow solid. The yield is 62%. 1 H NMR (400 MHz, D2O): δ 1.50-1.58 (m, 2H, alkyl), 1.67-1.73 (m, 2H, alkyo), 3.37 (t, J=6.8 Hz, 2H, -OCH2CH2- or -CH2Br), 3.62 (t, J=6.4 Hz, 2H, -OCH2CH2- or -CH2Br), 5.22 (s, 2H, pyrene-CH2O-), 8.00-8.06 (m, 2H, aromatic), 8.14-8.18 (m, 2H, aromatic), 7.72-7.77 (m, 2H, aromatic), 8.40 (d, J=9.2 Hz, 1H, aromatic). 13 C NMR (100 MHz, D2O): δ 25.2, 29.2, 32.8, 33.9, 70.3, 71.8, 123.7, 135.0, 125.2, 125.41, 125.42, 126.1, 127.1, 127.59, 127.64, 127.9, 129.6, 131.1, 131.5, 131.9. 124  HRMS (FAB): calculated for C22H21OBr (M+Na)+ 403.0673, found 403.0680 ROMP To prepare Au-20302530, 18 mg of second generation Grubbs’ catalyst (0.021 mmol, 1 equivalent relative to particle-bound norbornene) was added to a solution of nanoparticle Au-13/14 (0.10 g, 0.021 mmol of 13 at surfaces) in 10 mL dichloromethane. The mixture was stirred at room temperature for 20 min. Compound 20 (165 mg, 0.63 mmol) was added and stirred for 20 min. Norbornene (25) (59 mg, 0.63 mmol) was added and stirred for 20 min. Ethyl vinyl ether (1 mL) was added to terminate the polymerization. To prepare Au-25252030255, 18 mg of second generation Grubbs’ catalyst (0.021 mmol, 1 equivalent relative to particle-bound norbornene) was added to a solution of nanoparticle Au-13/14 (0.10 g, 0.021 mmol) in 10 mL dichloromethane. The mixture was stirred at room temperature for 20 min. Norbornene (25) (49 mg, 0.53 mmol) was added and stirred for 20 min. Compound 20 (165 mg, 0.63 mmol) was added and stirred for 20 min. Finally, compound 25 (10 mg, 0.11 mmol) was added and stirred for 20 min. Ethyl vinyl ether (1 mL) was added to terminate the polymerization. Cross-linking Diaminooctane (0.5 equiv. relative to 20) was added to a dichloromethane solution of the nanoparticle-polymer conjugates, prepared as described above. The reaction was stirred at room temperature for 3 days. The polymer coated nanoparticles were purified by dialysis. 125  Disulfide exchange reaction When the symmetric disulfides were used for thiol functionalization, 10 mL capsule solution in THF at 1 mg/mL was mixed with 0.10 g 35 (0.14 mmol) or 0.10 g 37 and 1 mL diisopropylethylamine. The mixture was stirred at reflux for 2 days, and the product was purified by dialysis. When the mixed disulfides 39, 41, 43 and 45 were used for ferrocene, allyl, carboxylate and amino functionalization, 10 mL nanocapsule solution (1 mg/mL) in THF was mixed with 0.050 g 39 (0.13 mmol), 41 (0.27 mmol), 43 (0.23 mmol) or 45 (0.18 mmol). The mixture was stirred at room temperature for 1 day, and the products were purified by dialysis. Contact angle measurements Glass sheets were purchased from Fisher Scientific (0.25 mm thickness and 18 mm diameter). 20 mg/mL polymer solutions in 1:1 DCM/DMF were prepared. 200 μL of polymer solution was dropped at the center of glass sheets and spin-casted at 50 rounds per seconds. 10 μL of water was placed at the center of polymer coated glass sheets. Contact angles were measured using static method. DLS and TEM The experimental conditions were the same as described in Chapter 1. Solutions of polymers in THF (5.0 mg/mL) were used instead of solutions of gold nanoparticles. Amide substitution 126  Capsules (free thiol in the interior) in THF (5 mg/mL, 5 mL) was mixed with 53 (86 mg, 0.23 mmol) or 55 (80 mg, 0.23 mmol) at the presence of NaH (10 mg, 0.42 mmol). The amount 53 or 55 was about 3 times to the amount of amide, based on the nominal molecular weight of polymers. The mixture was stirred under reflux condition for 2 days. After the addition of 0.5 mL water to quench the reaction, N-functionalized capsules were extracted with dichloromethane, washed with water, and purified by dialysis. Biphasic uptake NH2 functionalized capsules were prepared as solutions in dichloromethane (5.0 mg/mL). 2.0 mL of the capsule solution and a mini magnetic stir bar were placed in a 4 mL cuvette. The mini stir bar inside the cuvette was agitated by an external mechanical stirring machine to which another magnetic stir bar was attached. The equipment setup is shown in Figure 2.7.4. After adding the aqueous solution of ARS (5.0 mM, 1.0 mL) to the cuvette, the absorbance at 424 nm was recorded by Agilent UV-vis spectrometer. The absorbance was continuously recorded every 30 seconds for 3600 seconds. When the effect of environment pH was studied, the mixture of ARS (10 mM, 0.50 mL) and buffer solution (0.50 mL) were added to start the measurement. Biphasic release The capsules solution (5.0 mg/mL in dichloromethane, 10 mL) was vigorously stirred with ARS aqueous solution (5.0 mM, 10 mL) at room temperature for 1 day to achieve the maximum loading. The organic solution was separated, washed with water, 127  and dried by Na2SO4. The solutions were diluted by dichloromethane to the same ARS concentration (Abs@424nm=0.40). The release was then studied using three methods. Method a: 2.0 mL of ARS incorporated capsule solution and a mini magnetic stir bar were placed in a 4 mL cuvette (Figure 3.2.4). After adding the aqueous solution of NaOH (1.0 mM, 1.0 mL) to the cuvette, the absorbance at 424 nm was recorded by Agilent UV-vis spectrometer. The absorbance of the bottom layer was continuously recorded every 30 seconds for 3600 seconds. Method b: 1.0 mL of ARS incorporated capsule solutions, a mini magnetic stir bar and a mechanical stir bar (on top) were placed in a 4 mL cuvette (Figure 3.2.11). After adding the aqueous solution of NaOH (1.0 mM, 2.0 mL) to the cuvette, the absorbance at 555 nm was recorded by Agilent UV-vis spectrometer. The absorbance of the top layer was continuously recorded every 30 seconds for 3600 seconds. Method c: ARS incorporated capsule solution (dichloromethane, 8.0 mL) and NaOH aqueous solution (1.0 mM, 4.0 mL) were mixed and stirred in a scintillation vial. A magnetic stir bar was placed at the bottom. A mechanical stir bar was placed right above the aqueous/organic interface. 300 μL of the aqueous solution (top) was taken out and measured by Tecan plate reader every 10 minutes. After each measurement, the aqueous solution was immediately transferred back to the vial. Buffer solutions Buffer solutions were prepared using the Aldrich protocol (http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning- center/buffer-reference-center.html). 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APPENDIX 1 Structures of other cationic dyes for encapsulation study Cl N O S O S N N N O H2N N N N NH2 O S N N N OH fat brown rr basic red 29 basic blue 41 N H2N NH Cl N Cl N NH2 Cl N N NH2 basic fuchsin methyl violet 2B safranine O N N N N H2N NH2 H2N NH2 S Cl Cl S N N N N N N N N S S . 2 HCl alcian yellow bismarck brown y   133  APPENDIX 2 1 H-NMR and 13C-NMR spectra of synthetic compounds   134  135  136  137  138  139  140  141  142  143  144  145  146  147  148  149  150  151  152  153  154  155  156  157  158  159  160