Tunable, responsive nanoscale hydrogels for intracellular delivery of small interfering RNA
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Responsive, polybasic nanoscale hydrogels were synthesized using photoemulsion polymerization. The nanoscale hydrogels (nanogels) are approximately 50 nm in diameter and consist of a pH-responsive poly(2-(diethylaminoethyl methacrylate)) core with a poly(ethylene glycol) corona. The nanogels undergo a volume phase transition in response to environmental pH, and possess tunable physicochemical properties based on the copolymer composition. Aqueous solution properties of the nanogels were investigated using dynamic light scattering, pyrene fluorescence spectroscopy, and zeta potential measurements. Nanogel-mediated disruption of biological membranes was investigated with erythrocytes, giant unilammelar vesicles, and live cells to evaluate the potential of these nanogels to act as endosomolytic carriers for intracellular delivery of small interfering RNA. Selective membrane disruption was achieved by increasing nanogel hydrophobicity via copolymerization with tert-butyl methacrylate (TBMA). Nanogels with TBMA possessed an additional advantage of increased cytocompatibility owing to their reduced cationic charge density. These nanogels are able to efficiently entrap siRNA in the nanogel core and enhance cellular internalization of siRNA in model cell lines. The cell-line dependent mechanism of nanogel internalization and uptake and intracellular distribution of fluorescent nanogel/siRNA complexes was investigated with imaging flow cytometry. Functional activity of delivered siRNA was assessed using gene silencing assays. Modifications to the polymer chemistry were also explored to enhance the biological activity. A disulfide-containing crosslinker was synthesized, characterized and incorporated into nanogels. This crosslinker enabled the rapid nanogel degradation in response to reductive environments. A functional phenylalanine-derivative monomer (MAPA) was synthesized and characterized to serve as a bio-inspired substitute for TBMA. Nanogels copolymerized with MAPA demonstrate comparable siRNA delivery efficiency to its TMBA analogue. The ability to exert control over physicochemical properties and biological interactions of these nanogels by tuning polymer composition is a facile strategy to tailor material properties for specific applications. The hydrophobically-modified nanogels possess great potential as delivery vehicles to enhance the cellular internalization and endosomal escape of siRNA cargoes.