Synthesis of functional copolymers via aqueous RAFT polymerization for bioconjugation and targeted delivery of small interfering RNA

Adam Wesley York


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. 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 ω-chain ends were developed. First, a facile method for the amine functionalization of the thiocarbonylthio ω-chain end was developed. The key to labeling the ω-chain end of HPMA-b-DMAPMA is to first reduce the dithioester chain end with the reducing agent NaBH 4 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. 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). 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 1 H 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. (Abstract shortened by UMI.)