Cancer cells can be intrinsically drug resistant or can become drug resistant when exposed to chemotherapeutics. Specifically, cancer cells can develop resistance through up-regulation of efflux pumps or anti-apoptotic pathways. In addition, chemotherapeutic regimens can essentially select for a cancer cell subpopulation resistant to the chosen therapy. Currently, the preferred method of treatment for the majority of solid tumors is surgical resection followed by chemotherapy and radiation. As such, methods to improve chemotherapeutic regimens to combat drug resistant cancer are needed. Small interfering RNAs (siRNAs) are 21-23 nucleotide double stranded RNAs that have the ability to specifically silence the expression of a target gene. However, siRNAs are rapidly degraded by nucleases in the body. As such, delivery vehicles including cationic polymers and liposomes have been studied to enhance chemotherapeutic efficacy because of their ability to load and release chemotherapeutic agents and siRNA. Here, cationic nanogels were synthesized with either randomly incorporated or localized pH responsive and hydrophobic functional groups. The presence of localized functional groups are hypothesized to enhance pH responsive properties as well as drug and siRNA loading to reduce off target affects associated with premature release of therapeutic agents.
In this work, activators regenerated by electron transfer (ARGET) atom transfer radical polymerization (ATRP) was utilized to synthesize cationic nanogels with either localized or randomly distributed functional groups. For random polymerizations, tertiary amino ethyl methacrylate monomers, poly ethylene glycol methyl ether methacrylate, surfactants, crosslinker and ATRP reagents were combined in water and sonicated into an emulsion. The emulsion was nitrogen purged and polymerized for 24 hours upon addition of a reducing agent. Cationic nanogels with localized functional groups were similarly synthesized through sequential addition of monomers to a PEG macroinitiator. Cationic nanogels were purified though five cycles of swelling and ionmer collapse in hydrochloric acid and acetone followed by dialysis against water for one week and lyophilized. Incorporation of functional groups for nanogels with localized functional groups was tracked with proton nuclear magnetic resonance and gel permeation chromatography on linear analogs of nanogels.
The swelling properties of cationic nanogels were observed with dynamic light scattering from pH 4-9. The onset of the pH dependent swelling was modulated by varying monomer feed ratios. Typical nanogels exhibited hydrodynamic diameters of 80-100nm in collapsed state to 120-140nm swollen in 1x PBS at 25C. Similarly, zeta potential was used to track apparent surface charge on the nanogels through the same pH range. Positive zeta potential on the order of 15mV was observed for swollen gels with almost neutral zeta potential observed at pH 7.5.
Initial screening of the randomly polymerized nanogels exhibited minimal cytotoxicity when incubated with RAW 264.7 murine macrophages at concentrations up to 0.1mg/mL for 24 hours. Results were calculated using a lactate dehydrogenase membrane integrity assay. Further, randomly polymerized cationic nanogels exhibited the ability to electrostatically bind siRNA with high efficiency post synthesis. In a typical experiment, 95% of siRNA incubated with the nanogels was electrostatically bound as determined by Quant-iT™ RiboGreen® RNA Assay.
Cationic nanogels with randomly incorporated and localized functional group were synthesized. Swelling and surface potential tracked as a function of pH demonstrated desirable pH responsive behavior, concentration dependent cytotoxicity demonstrated limited toxicity in the anticipated dosing range, and high siRNA loading demonstrated the promise of these cationic nanogels for the co-delivery of siRNA and chemotherapeutics. Further studies will compare the co-delivery potential of nanogels with localized functional group to those with randomly distributed functional groups with respect to swelling properties, drug and siRNA loading and release, and in vitro toxicity and transfection.
This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program.
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