Chitosan and PEGylated Chitosan Nanoparticles for Functional Gene Silencing in Colorectal Cancer Cells
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RNA interference (RNAi) technology is a useful mechanism for inhibiting gene expression. This technology involves the use of small interfering RNA (siRNA) for gene therapy purposes. We have made an effort to use this technology to reduce the expression of beta-catenin in colon cancer cells. The pharmaceutical application of siRNA is dependent on an efficient delivery system. The positively charged nature of chitosan provides interesting properties with respect to interaction with oppositely charged materials, especially those of high molecular weight. Interaction between positively charged chitosan and negatively charged siRNA can be exploited; making chitosan an ideal candidate to serve as a delivery vehicle for siRNA. The most important problem associated with the use of chitosan is its limited solubility at pH higher than its pKa (5.5-6.5). Being a weak base chitosan is only completely soluble under acidic conditions. To overcome the problem of solubility we have grafted poly (ethylene glycol) to chitosan. PEGylated chitosan/siRNA and chitosan/siRNA nanoparticles have been synthesized and characterized. Size characterization of nanoparticles was carried out using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dynamic light scattering (DLS) techniques. The encapsulation efficiency was evaluated using a nanodrop (UV absorption detector). HCT 116 colorectal cancer cell line was used to evaluate the transfection of siRNA across the cellular membrane. The cell lines were treated with 0.1 and 0.5 nmol of siRNA encapsulated in PEGylated chitosan and chitosan for a period of 24 and 48 hours. The percent reduction in beta-catenin protein level was evaluated using the western blot technique. The chitosan/siRNA and PEGylated chitosan/siRNA nanoparticle approach was compared to the classical approach used for siRNA delivery; namely siRNA in Lipofectamine 2000 to identify the difference in the levels of beta-catenin protein in HCT 116 cells. We were successful in reducing the cellular beta-catenin protein levels and both the chitosan/siRNA and PEGylated chitosan/siRNA approaches were successful in reducing beta catenin protein levels after 48 h. Another significant result was that the chitosan/siRNA had a similar ability as Lipofectamine/siRNA to reduce beta catenin protein levels when administered at a sufficient concentration of siRNA.