PH-Responsive Polymeric Micelle Carriers for SiRNA Drugs


The ability of small interfering RNA (siRNA) to efficiently silence the expression of specific genes provides the basis for exciting new therapies based on RNA interference (RNAi). The efficient intracellular Delivery of siRNA from cell uptake through the endosomal trafficking pathways into the cytoplasm remains a significant challenge. Previously we described the synthesis of a new family of diblock copolymer siRNA carriers using controlled reversible addition-fragmentation chain transfer (RAFT) polymerization. The carriers were composed of a positively charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA binding and a second pHresponsive endosome releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios and butyl methacylate (BMA). Here we describe the Development of a new generation of siRNA Delivery polymers based on this design that exhibit enhanced transfection efficiency and low cytotoxicity. This design incorporates a longer endosomolytic block with increased hydrophobic content to induce micelle formation. These polymers spontaneously form spherical micelles in the size range of 40 nm with CMC (critical micelle concentration) values of approximately 2µg/mL based on dynamic light scattering (DLS), 1H NMR, electron microscopy, and selective partitioning of the small molecule pyrene into the hydrophobic micelle core. The siRNA binding to the cationic shell block did not perturb micelle stability or significantly increase particle size. The self-assembly of the diblock copolymers into particles was shown to provide a significant enhancement in mRNA knockdown at siRNA concentrations as low as 12.5 nM. Under these conditions, the micelle-based systems showed an 89% reduction in GAPDH mRNA levels as compared to only 23% (10 nM siRNA) for the nonmicelle system. The reduction in mRNA levels becomes nearly quantitative as the siRNA concentration is increased to 25 nM and higher. Flow cytometry analysis of fluorescent-labeled siRNA showed uptake in 90% of cells and a 3-fold increase in siRNA per cell compared to a standard lipid transfection agent. These results demonstrate the potential utility of this carrier design for siRNA drug Delivery.


Materials Science and Engineering

Keywords and Phrases

Block copolymers; Copolymerization; Critical micelle concentration; Cytology; Design; Drug delivery; Dynamic light scattering; Flow cytometry; Gene expression; Gene therapy; Hydrophobicity; Micelles; Polymers, Carrier design; Cell uptake; Diblock copolymer; Dimethylaminoethyl methacrylates; Endosomes; Equimolar ratio; Flow cytometry analysis; Hydrophobic content; Intracellular delivery; Micelle formation; Micelle stability; MRNA level; ON dynamics; PH-responsive; Polymeric micelle; Positively charged; Potential utility; Reversible addition-fragmentation chain transfer polymerization; RNA interference; siRNA delivery; Size ranges; Small interfering rna (siRNA); Small molecules; Spherical micelles; Transfection agents; Transfection efficiency, RNA, copolymer; drug carrier; fluorescent dye; lipid; messenger RNA; polymer; pyrene; small interfering RNA; drug carrier; micelle; small interfering RNA, article; controlled study; critical micelle concentration; cytotoxicity; DNA transfection; drug delivery system; electron microscopy; endosome; flow cytometry; gene silencing; gene targeting; human; human cell; hydrophobicity; light scattering; micelle; particle size; pH; priority journal; proton nuclear magnetic resonance; RNA binding; cell survival; chemistry; dose response; drug effects; HeLa cell line; micelle; structure activity relation; surface property; synthesis; toxicity, Cell Survival; Dose-Response Relationship, Drug; Drug Carriers; HeLa Cells; Humans; Hydrogen-Ion Concentration; Micelles; Particle Size; Polymers; RNA, Small Interfering; Structure-Activity Relationship; Surface Properties

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Article - Journal

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© 2010 American Chemical Society (ACS), All rights reserved.

Publication Date

01 Nov 2010

PubMed ID