Study On Preparation, Characterization And Biologic Action Of Survivin SiRNA/Chitosan And Survivin SiRNA/Water-soluble Chitosan Nanoparticles

Survivin is newly discovered as inhibitor of apoptosis proteins which prominently expressed in all the most common human cancers but not expressed in the other surrounding normal tissue.Cancer cells will regain the ability of apoptosis when Survivin gene is silenced by siRNA using RNA interference technology, thus, cancer can be cured. Chitosan(CS) as a non-viral vector deliver small interference RNA(siRNA) into cells protecting it from enzymolysis,has good biocompatibility and biodegradability, low toxicity and immunogenicity, but its low solvability in water also arouses extensive attention.In order to explore the feasibility of water-soluble CS as survivin siRNA vector and effects of inhibiting gene expression, firstly we modify CS and discuss the influences on solubility by the different factor levels loaded with a single-factor method. Optimized modified-CS is detected by Infrared spectrum and X-ray diffraction. And then, The modified CS-Survivin siRNA nanoparticles are prepared by complex coacervation process binded with sodium sulfate or sodium tripolyphosphate and plasmid gene, the diameter is measured by TEM, the binding capability of DNA is evaluated by agrose gel electrophoresis, the encapsulation rate is determined by colorimetry, transfection in vitro studies and effects of inhibiting gene expression are performed with Hep-2 cells and use lipofectamine as active control.The experimental results show that the optimized modified-chitosan has good water-solubility and the deacetylation degree (DD) is 50.73%. Infrared spectroscopy analysis shows that CS has bonded with acetyl smoothly and X-ray diffraction pattern discovers the Crystal form of CS has greatly destroyed ,the corresponding regularity of structure and crystallinity are also declined which result in its solubility.CS and optimized water-soluble CS are used to binded with different cross linkers. The result of TEM shows the diameter of CS-TPP/siRNA nanoparticles is 50~100nm, water-soluble CS-TPP/siRNA nanoparticles is about 100nm, they are both sphere.The embedding rate is 88%~92%.SiRNA binds with CS and water-soluble CS both well.These findings have prepared for further applications of water-soluble chitosan as a non-viral vector of nucleic acid molecules.