Structure-function relationships of biodegradable cationic nanogels for intracellular drug delivery




Spencer, David Stephen

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Biodegradable, cationic nanogels were synthesized via a one-pot controlled radical heterogeneous emulsion polymerization using activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP). Nanogels were synthesized with the tertiary amine methacrylate monomers 2-(diethylamino)ethyl methacrylate or 2-(diisopropylamino)ethyl methacrylate to impart pH responsiveness. Nanogels were copolymerized with poly(ethylene glycol) to impart colloidal stability and biocompatibility. Hydrophobic comonomers were used to modulate responsive properties, and a disulfide crosslinking agent, bis(2-methacryloyl)oxyethyl disulfide was used to impart biodegradability. The reaction scheme was modified to synthesize up to 24 formulations in parallel to enable optimization of reaction conditions and to determine structure-function relationships. Multiple sets of nanogels were synthesized including formulations that varied with respect to PEG mole fraction (0 – 5 mol %), comonomer fraction (0 - 50 mol %), crosslinking density (0.31 to 5 mol %), cationic monomer (substitution of DEAEMA for DPAEMA from 0 to 100%), and choice of hydrophobic comonomer (or hydrophilic monomer with a hydrophobic protecting groups). In each case, nanogel composition was determined by Fourier Transform Infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR) to correlate nanogel composition with feed ratios. Monomer incorporation followed feed ratios except for the PEG macromonomer which demonstrated poor conversion (< 10%) under the initially tested conditions. As such, specific attention was placed on the tuning the degree of PEG incorporation into nanogels. PEG macromonomer feed concentration, surfactant concentration, and surfactant hydrophilic-lipophilic balance were determined to be key factors and were subsequently utilized to synthesize nanogels with predictable degrees of PEGylation. The pH-responsive swelling profiles of nanogels formulations were determined by dynamic light scattering. Nanogels were approximately 130 nm in the swollen state and 70 nm in the collapsed state with critical swelling pH that ranged from pH 6.3 – 7.8 and volume swelling ratios from 2.5 – 8.0. Nanogel mediated cytotoxicity and erythrocyte hemolysis were evaluated as a function of concentration. Nanogels pK [subscript a] was the strongest predictor of both cytocompatibility and hemolysis at pH 7.4. Formulations with pKa < 7.0 demonstrated limited toxicity in both assays. Hemolytic activity at pH 6.8 and below was a function of pKa, hydrophobicity, and volume swelling. Knockdown of eGFP was utilized to assess transfection efficiency of nanogels formulations. Nanogels electrostatically complexed siRNA with high efficiency. Knockdown of eGFP was enhanced by the incorporation of comonomers with increasing hydrophobicity. The cationic nanogels discussed in this work are excellent candidates for intracellular drug delivery. The described structure-function relationships will allow for rapid optimization of formulations, and the ability to synthesize cationic nanogels with targeted material properties.


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