Endolytic, pH-responsive HPMA-b-(L-Glu) copolymers synthesized via sequential aqueous RAFT and ring-opening polymerizations.

A facile synthetic pathway for preparing block copolymers with pH-responsive L-glutamic acid segments for membrane disruption is reported. Aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization was first used to prepare biocompatible, nonimmunogenic poly[N-(2-hydroxypropyl)methacrylamide]. This macro chain transfer agent (CTA) was then converted into a macroinitiator via simultaneous aminolysis and thiol-ene Michael addition using the primary amine substituted N-(3-aminopropyl)methacrylamide. This macroinitiator was subsequently utilized in the ring-opening polymerization of the N-carboxyanhydride monomer of γ-benzyl-L-glutamate. After deprotection, the pH-dependent coil-to-helix transformations of the resulting HPMA-b-(L-Glu) copolymers were monitored via circular dichroism spectroscopy. HPMA segments confer water solubility and biocompatibility while the L-glutamic acid repeats provide reversible coil-to-helix transitions at endosomal pH values (~5-6). The endolytic properties of these novel [HPMA-b-(L-Glu)] copolymers and their potential as modular components in drug carrier constructs was demonstrated utilizing red blood cell hemolysis and fluorescein release from POPC vesicles.

Facile synthesis of multivalent folate-block copolymer conjugates via aqueous RAFT polymerization: targeted delivery of siRNA and subsequent gene suppression.

Cell specific delivery of small interfering ribonucleic acid (siRNA) using well-defined multivalent folate-conjugated block copolymers is reported. Primary amine functional, biocompatible, hydrophilic-block-cationic copolymers were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. N-(2-hydroxypropyl)methacrylamide) (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-stat-APMA was chain extended with a cationic block, poly(N-[3-(dimethylamino)propyl]methacrylamide), to promote electrostatic complexation between the copolymer and the negatively charged phosphate backbone of siRNA. Notably, poly(HPMA) 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). Fluorescence microscopy and gene down-regulation studies indicate that these neutral complexes can be specifically delivered to cancer cells that overexpress folate receptors.

Block ionomer complexes consisting of siRNA and aRAFT-synthesized hydrophilic-block-cationic copolymers II: The influence of cationic block charge density on gene suppression.

Block ionomer complex (BIC)-siRNA interactions and effectiveness in cell transfection are reported. Aqueous RAFT polymerization was used to prepare a series of hydrophilic-block-cationic copolymers in which the cationic block statistically incorporates increasing amounts of neutral, hydrophilic monomer such that the number of cationic groups remains unchanged but the cationic charge density is diluted along the polymer backbone. Reduced charge density decreases the electrostatic binding strength between copolymers and siRNA with the goal of improving siRNA release after targeted cellular delivery. However, lower binding strength resulted in decreased transfection and RNA interference pathway activation, leading to reduced gene knockdown. Enzymatic siRNA degradation studies with BICs indicated lowered binding strength increases susceptibility to RNases, which is the likely cause for poor gene knockdown.