Studies of the aqueous RAFT polymerization of methacrylamido-monomers and the application of their polymers in the protection and delivery of novel siRNA-based gene silencing agents

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Polymers and High Performance Materials

First Advisor

Charles L. McCormick

Advisor Department

Polymers and High Performance Materials


The extension of the reversible addition-fragmentation chain transfer (RAFT) process to the controlled polymerization of α-substituted, methacrylamido-monomers in aqueous media has been investigated and employed in the preparation of homo- and block-copolymer systems for applications in the effective protection and stabilization of a novel RNA-based gene silencing agent. Building on the previously reported controlled polymerization of a cationic, methacrylamido, monomer, namely N -[3-(dimethylamino)propyl]methacrylamide (DMAPMA) (65 ), the first study involves the controlled polymerization of the neutral, hydrophilic monomer, methacrylamide (MAM) (74 ), and its subsequent chain-extension polymerizations with MAM and acrylamide (AM) (70 ). Homopolymers of poly(MAM) (PMAM) were obtained using 4-cyanopentanoic acid dithiobenzoate (77 ) and sodium 4,4'azobis(4-cyanopentanoic acid) (V-501) as the RAFT chain transfer agent (CTA) and primary initiating species, respectively, under buffered aqueous conditions at 70°C. Under these conditions a PMAM macroCTA was prepared and employed in chain extension with additional MAM monomer as well as AM to form poly(MAM- b -MAM) and poly(MAM-b -AM) block copolymers, respectively. To elucidate the blocking order dependence of MAM and AM, a separate AM macroCTA was prepared and employed in a chain-extension experiment under said conditions with MAM. This resulted in the corresponding poly(AM-b -MAM) block copolymer, however, significant amounts of homopolymer impurity were formed, indicating that block copolymers of MAM and AM should be prepared by first synthesizing the MAM macroCTA and then blocking with AM in a second polymerization. The second study concerns the synthesis and characterization of homo- and diblock copolymers with a neutral, hydroxy-functional methacrylamido monomer, namely, N -(2-hydroxypropyl)methacrylamide (HPMA), and the previously reported cationic methacrylamido monomer, DMAPMA. HPMA was chosen as the first monomer because its homopolymer is permanently hydrophilic and is well known for its biocompatible properties in drug delivery applications. DMAPMA ( 65 ) was chosen as the second monomer because of its cationic nature at physiological pH. Initially, HPMA was polymerized via RAFT in buffered aqueous media using CTP and V-501 as the CTA and initiator, respectively. Following characterization via size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS), the resulting HPMA macroCTA was subjected to a second block-copolymerization with DMAPMA, yielding well-defined, near-monodisperse poly(HPMA-b -DMAPMA) copolymers. To test the blocking order dependence of HPMA and DMAPMA a DMAPMA macroCTA was prepared under identical conditions and employed in chain-extension experiments to yield well defined block copolymers with minimal formation of homopolymer impurity. The third study concerns the formation of block ionomer complexes with a series of the above mentioned HPMA/DMAPMA block copolymers and a 43-nucleotide single-stranded segment of ribonucleic acid (RNA) known as short interfering RNA (siRNA). This siRNA represents the new state-of-the-art in the areas of gene silencing for anticancer and gene therapies. The specific sequence employed is able to silence the gene in human cells that ultimately codes for the synthesis of Human RNA Polymerase II, an enzyme whose production is required for the cell to survive. The degree of soluble complex formation obtained with these block copolymer systems is determined by centrifugal filtration experiments and quantitated by scintillation counting of 32P ATP-labeled siRNA to determine complex solubility and estimate the degree of complexation as functions of cationic and neutral block-lengths. Dynamic and static light scattering methods are employed to determine the hydrodynamic radii, molecular weights, and second virial coefficients of the complexes and to demonstrate their unimodal size distributions. In vitro enzymatic degradation studies of selected siRNA/block copolymer complexes were conducted to demonstrate the enhanced stability of siRNA when bound to poly(HPMA-b -DMAPMA) copolymers.