Neutral Polymeric Micelles for RNA Delivery


RNA interference (RNAi) drugs have significant therapeutic potential, but delivery systems with appropriate efficacy and toxicity profiles are still needed. Here, we describe a neutral, ampholytic polymeric delivery system based on conjugatable diblock polymer micelles. The diblock copolymer contains a hydrophilic poly[N-(2-hydroxypropyl)methacrylamide-co-N-(2-(pyridin-2- yldisulfanyl)ethyl)methacrylamide) (poly[HPMA-co-PDSMA]) segment to promote aqueous stability and facilitate thiol-disulfide exchange reactions and a second ampholytic block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The poly[(HPMA-co-PDSMA)-b- (PAA-co-DMAEMA-co-BMA)] was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization with an overall molecular weight of 22 000 g/mol and a PDI of 1.88. Dynamic light scattering and fluorescence measurements indicated that the diblock copolymers self-assemble under aqueous conditions to form polymeric micelles with a hydrodynamic radius and critical micelle concentration of 25 nm and 25 μg/mL, respectively. Red blood cell hemolysis experiments show that the neutral hydrophilic micelles have potent membrane destabilizing activity at endosomal pH values. Thiolated siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was directly conjugated to the polymeric micelles via thiol exchange reactions with the pyridal disulfide groups present in the micelle corona. Maximum silencing activity in HeLa cells was observed at a 1:10 molar ratio of siRNA to polymer following a 48 h incubation period. Under these conditions 90% mRNA knockdown and 65% protein knockdown at 48 h was achieved with negligible toxicity. In contrast the polymeric micelles lacking a pH-responsive endosomalytic segment demonstrated negligible mRNA and protein knockdown under these conditions. The potent mRNA knockdown and excellent biocompatibility of the neutral siRNA conjugates demonstrate the potential utility of this carrier design for delivering therapeutic siRNA drugs.


Materials Science and Engineering

Keywords and Phrases

diblock copolymer; glyceraldehyde 3 phosphate dehydrogenase; polymer; small interfering RNA; unclassified drug, article; biocompatibility; cell pH; chemical composition; controlled study; critical micelle concentration; drug conjugation; female; gel electrophoresis; gel permeation chromatography; gene silencing; gene targeting; hemolysis; human; human cell; hydrophilicity; in vitro study; light scattering; micelle; molecular weight; nonviral gene delivery system; polymerization; proton nuclear magnetic resonance; reverse transcription polymerase chain reaction; RNA interference; synthesis; transmission electron microscopy, Erythrocytes; Gene Transfer Techniques; HeLa Cells; Humans; Micelles; Polymers; RNA, Small Interfering, Paa

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Article - Journal

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© 2013 American Chemical Society (ACS), All rights reserved.

Publication Date

01 Mar 2013

PubMed ID