Study On The Targeting Of Folate-chitosan-SiRNA Nanocomposites To The Heparanase In Tumor Cells

Objectives:The siRNA were designed and synthesized according to the cDNA of human heparanse gene, and the effective one should be screened. In order to sieve out the gene sites which will effectively reduce the expression of heparanase, we use the siRNA transfected with the breast cancer cell (MCF-7). The effect on tumor invasion and metastasis were determined by Tumour Invasion Assay, providing a theoretical basis for the development of drugs with tumor cell invasion and metastasis. Morever, we use chitosan as the nano-materials to package siRNA, and couple it with FA which is a specific targeting molecule of tumor cell. Then, FA identifies its receptors on the cell surface, which can increase the efficiency of the siRNA delivery. So we can achieve a safe and effective targetting gene therapy.Methods:The siRNA were syntheticed by GenePharma Co., Ltd.Shanghai.The experiment was divided into 6 groups (n=3). The groups as follows:①positive control group;②Negative control group;③siRNA fragmentⅠgroup;④siRNA fragmentⅡgroup;⑤siRNA fragmentⅢgroup;⑥control group. The siRNA was transiently transfected into MCF-7cells by Lipofectamine2000, and the one which can reduce the expression of the mRNA and protein effectively was selected by RT-PCR and Western blot.The proliferation of MCF-7 cells which were transfected by siRNA was determined by MTT. The invasion and metastasis of MCF-7cells were detected by Tumour Invasion Assay.The folate-conjugated (FA-CS) chitosan can be obtained by the reaction of the activated folate ester with the amine group on chitosan. The FA-CS was verified by the infrared spectroscopy. And the coupling rate was measured by UV spectrophotometry. The FA-CS and siRNA was used to prepare FA-CS-siRNA nanocomposites, and detected its particle size and eneapsulating rate. The Transfection Assay was divided into 5 groups:the Negative control group; FA-CS nanocomposites group; FA-CS-siRNA nanocomposites group; CS-siRNA nanocomposites group and Lipofectamine2000-siRNA group. After 48h the expression of mRNA was detected by RT-PCR, while the expression of heparanase was detected by western blot.Tumour Invasion Assay was divided into 3 groups:the Negative control group and the lipo2000-siRNA fragment I group and the FA-CS-siRNA fragment I group. After the transfection experiments and the cells should be cultured for 24h. And than cells were seeded in the Cell Culture Inserts which containing Matrigel matrix, and cultured for 24h, cells were observed under microscope and cell counts.Results:1. MCF-7 cells were transfected by different concentrations of FAM-siRNA, showing the different transfection efficiency. And the better transfection efficiency was achieved at the concentration of 80nM/L and 100nM/L2. The siRNA fragment I reduce the expression of heparanase specifically in MCF-7 cells.3. We can know from the infrared spectroscopy that the FA have been successfully coupled with CS.4. The FA-CS-siRNA nanoparticle is round-form, distribute evenly, and the particle size is 197.7nm.5. The FA-CS-siRNA nanoparticle reduced the expression of heparanase effectively in MCF-7 cells.6. Tumors Invasion Assay showed that the lipo2000-siRNA group and the FA-CS-siRNA nanocomposites group can inhibit the invasion and metastasis of MCF-7 cells.Conclusion:1. Different concentrations FAM-siRNA transfected MCF-7 cell showed that when the concentration of siRNA transfection were at 80nM/L and 100nM/L, the transfection efficiency is better. So we use the siRNA concentration of 80nM/L with transfection for the follow-up experiment.2. The siRNA of siRNA fragment I reduce the expression of heparanase specifically in MCF-7 cells, and inhibited the invasion and metastasis of MCF-7 cells. So we can use siRNA fragment 638 for the follow-up experiment.3. The FA-CS-siRNA can be prepared successfully. And it can be taken as a delivery vector for siRNA to transfected MCF-7 cells.4. FA-CS-siRNA nanocomposites and lipo2000-siRNA group can reduce the expression of heparanase effectively in MCF-7 cells. The efficiency of FA-CS-siRNA nanocomposites group is close to lipo2000-siRNA group.