Date of Award

Spring 5-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Polymers and High Performance Materials

Committee Chair

Charles L. McCormick

Committee Chair Department

Polymers and High Performance Materials

Committee Member 2

Sergei Nazarenko

Committee Member 2 Department

Polymers and High Performance Materials

Committee Member 3

Sarah Morgan

Committee Member 3 Department

Polymers and High Performance Materials

Committee Member 4

Faqing Huang

Committee Member 4 Department

Chemistry and Biochemistry

Committee Member 5

Robert Lochhead

Committee Member 5 Department

Polymers and High Performance Materials

Abstract

The versatility of reversible addition-fragmentation chain transfer (RAFT) polymerization has moved this controlled radical technique to the forefront of copolymer construction for bioapplications including polymeric drug/gene delivery vehicles. Strengths of the RAFT process include the capacity to control the polymerization of a wide variety of vinyl monomers under mild conditions, its tolerance to numerous chemical groups that allow the preparation of functional copolymers for facile modification, and the range of copolymer architectures with predetermined end group functionalities which may be synthesized. Given these strengths, RAFT polymerization was utilized in this research to synthesize functional/reactive copolymers for bioconjugation and targeted delivery of small interfering RNA (siRNA).

The first section describes the successful aqueous RAFT polymerization of water soluble, biocompatible N-(2-hydroxypropyl)methacrylamide-b-N-[3-(dimethylamino) propyl] methacrylamide (HPMA-b-DMAPMA) block copolymers and subsequent chain end conjugation. Well-defined, HPMA-b-DMAPMA copolymers were synthesized in the presence of the carboxylic acid containing chain transfer agent, 4-cyanopentanoic acid dithiobenzoate (CTP; C1), and the initiator 4,4’-azobis(4-cyanopentanoic acid) (V- 501; I3). Following copolymer characterization, bioconjugation methods to both the α- and -chain ends were developed. First, a facile method for the amine functionalization of the thiocarbonylthio -chain end was developed. The key to labeling the -chain end of HPMA-b-DMAPMA is to first reduce the dithioester chain end with the reducing agent NaBH4 and then functionalize the resulting polymeric thiol with a primary amine through a disulfide exchange reaction with cystamine. It was demonstrated that this disulfide exchange reaction is efficient and that the amine-functionalized HPMA-b- DMAPMA can be easily labeled with an amine-reactive fluorescein fluorophore. Primary amines were detected via a ninhydrin assay while fluorescein conjugation was analyzed via UV-vis spectroscopy. Building on the success of this end group conjugation, the focus was then turned to the conjugation of folate, a cancer cell targeting moiety, to the α-terminal chain end of HPMA-b-DMAPMA copolymers for targeted siRNA delivery. The carboxylic acid α-chain ends of the block copolymers were activated via carbodiimide chemistry to form an activated ester that was subsequently modified with an amine and folate containing PEG. However, poor conjugation yields, determined via UV-vis spectroscopy and MALDI-ToF mass spectrometry, to the α-terminal chain ends led to the development of an alternate synthetic pathway for folate conjugation.

The second section concerns the cell specific delivery of small interfering ribonucleic acid (siRNA) using well-defined multivalent folate-conjugated block copolymers. Primary amine functional, biocompatible, hydrophilic-b-cationic copolymers were synthesized via aqueous RAFT polymerization. HPMA, a permanently hydrophilic monomer, was copolymerized with a primary amine containing monomer, N-(3- aminopropyl)methacrylamide (APMA). Poly(HPMA) confers biocompatibility, while APMA provides amine functionality, allowing conjugation of folate derivatives. HPMA s-APMA was chain extended with a cationic monomer, DMAPMA, to promote electrostatic complexation between the copolymer and the negatively charged phosphate backbone of siRNA. Notably, the HPMA polymer block stabilizes the neutral complexes in aqueous solution, while APMA allows the conjugation of a targeting moiety, thus, dually circumventing problems associated with the delivery of genes via cationically charged complexes (universal transfection). As demonstrated through zeta potential, fluorescence microscopy and gene down-regulation studies, this tailored copolymer allows formation of neutral complexes that can be specifically delivered to cancer cells that over-express folate receptors.

In the third section, a well-defined HPMA-s-APMA copolymer, synthesized via RAFT polymerization, was utilized for the rational design of multiconjugates containing both a gene therapeutic, siRNA, and a cancer cell targeting moiety, folate. After isolating HPMA-s-APMA, a small fraction of the pendent primary amines were converted to activated thiols utilizing N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), providing a copolymer with two distinct reactive sites for both thiol containing compounds and activated esters. Characterization of the intermediates was performed by ASEC-MALLS and 1H NMR and UV-vis spectroscopy. Conditions for the bioconjugation of both 5’-thiolated siRNA and modified folic acid were developed and carried out in two separate steps. It was demonstrated that this pathway provides a facile and robust route for producing well-defined targeted siRNA delivery vehicles. In addition, siRNA release through disulfide cleavage was demonstrated under intracellular conditions, while the presence of attached folates allows for site-directed delivery to cancer cell lines that over-express folate receptors. Conjugation reactions and subsequent siRNA release were confirmed by polyacrylamide gel electrophoresis and UV-vis spectroscopy.

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