In this work, we are proposing new delivery systems of microRNA (miRNA) as a means to suppress tumorigenesis in cancer patients. By carefully designing a nanoparticulate miRNA delivery system with incorporated stealth agents and cationic and lipophilic character to bypass the blood-brain barrier, we can protect the miRNA mimics from degradation and rapid clearance and, in turn, improve the likelihood of clinical applications of this treatment strategy. pH- and redox-responsive cationic nanogels were prepared for miRNA delivery for the treatment of glioblastoma multiforme. With this dual stimuli-responsiveness, the unique characteristics of the tumor microenvironment are employed to trigger miRNA release. The tumor microenvironment differs markedly from the normal cell environment due to hypoxic regions around tumor cells. These regions of lowered oxygen and increased glycolysis result in a region with lowered extracellular pH compared to normal cells. By using the pH-responsive monomer, diethylaminoethyl methacrylate (DEAEMA), we designed carriers that remain collapsed in basic and neutral conditions but is triggered by a change in local pH to swell and release the incorporated payload. By incorporating disulfide bonds throughout the polymer network, we impart redox-responsive degradation characteristics to the carrier that responds to the increased level of glutathione present in cancer cells. Stimuli-responsive release is associated with increased levels of cellular uptake and a more selective release near tumor sites.
Cationic nanogels were synthesized via the ARGET ATRP controlled polymerization technique previously reported by Forbes and Peppas[1]. Nanogels were characterized and analyzed using FTIR spectroscopy and NMR to characterize their chemical structure, dynamic light scattering to establish the carrier particles size distribution, and potentiometric titration to identify pH-responsiveness. Certain formulations with favorable characteristics were assessed for toxicity to healthy astrocytes via an MTS assay. Non-cytotoxic formulations were then incubated with miRNA mimics to allow for complexation and miRNA loading efficiency was determined. Confocal microscopy and Incucyte proliferation assays were used to visualize miRNA mimic release and cellular uptake by GSC 3565 cells.
Work supported by National Institutes of Health Grant R01-EB022025
[1] D.C. Forbes, N.A. Peppas. Macromol. Biosci. 2014, 14, 1096-1105
