Studies On Survivin SiRNA And Its Nano Drug Delivery Systems Based On Modified Cell Penetrating Peptides

Cancer is a serious threat to human health. As the increase of the deaths from cancer, cancer therapy has aroused widespread attention all over the world. When RNA interference was induced by si RNA in the cytoplasm, the m RNA which was complementary with the si RNA, was silenced, then the expression of protein was reduced. The cancer therapy based on RNAi is a promising approach. However, since si RNA has poor stability, hydrophilic and negative charge, so it is not easy to penetrating the cell membrane and targeting the cytoplasm. Hence, it is not able to play a role in the process of RN Ai. So the lack of effective si RNA sequences and delivery systems limited their applications. We synthesized a newly designed survivin si RNA modified with 2′-O-methyl and we also used four fatty acids to modified the cell penetrating peptide octa-arginine. A series of experiments were carried out to obtain a high efficient nanoparticle that has better capacity of condensation, protection and delivery of si RNA. In order to overcome the poor cell specificity and low efficiency through the cell membranes from the modified cell penetrating peptides and the targeting ligands, the excellent modified cell penetrating peptide and transferrin were used to prepare the multi- function liposomes. The delivery efficiencies of liposomes were examined in vitro and vivo. The specific topics of this thesis are as follows:1. The evaluation of RNAi effect by si RNA in the tumor cel s.We synthesized a modified si RNA sequences targeting survivin gene. Real-time fluorescent quantitative PCR, Western blot and flow cytometry were used to detect the activity of si RNA. Results showed that after 48 h treatment with 20 n M si RNA, survivin transcription was significantly reduced by 90%. Moreover, survivin protein expression was reduced by more than 80%. According to the experimental result,40 n M si RNA could inhibit proliferation of tumor cell, and block the cell cycle in G2/M phase as well as promote apoptosis, the percentage of apoptotic cells was reached more than 40%. At the same time, si RNA could lead the abnormal expression of apoptosis protein and change the mitochondrial membrane potential.2. The synthesis and toxicity assessment of modified cell penetrating peptides4 kinds of fatty acid were covalent bonded to the free amino terminal of R8 using solid-phase peptide synthesis method. The purification and identification of the amphiphilic cell penetrating membrane peptides were detected by MALDI-TOF-MS and HPLC. The viability of the cells treated by cell penetrating membrane peptides was determined by MTT. The results demonstrated that the modified cell penetrating membrane peptides had low cytotoxicity and can be used as the delivery material of drugs.3. The preparation and delivery efficiency of modified CPP nanoparticlesThe modified cell penetrating peptides are amphiphilic and able to form the self-assembled systems. The nanoparticles loaded si RNA were prepared by electrostatic interaction, and the cellular uptake and silencing efficiency of target gene were studied. The results showed hydrophobic derivatives of R8 with oleic acid had strong si RNA binding, protection, delivery efficiency and low cytotoxicity. At the charge ratio of 1:1, si RNA was completely condensed by OA-R8. In addition, at the charge ratio of 4:1, OA-R8/si RN A nanoparticles had better serum and polyanion stability. They have well uniformity distribution of particle size and Zeta potential, the average particle size were 191.9±17.2 nm, Zeta potential were 13.2±5.4 m V. As a si RNA carrier, OA-R8 showed high delivery efficiency. The transcriptional level of survivin m RNA in Hep G2 and A549 cells were only 30.2% and 38.9% respectively, and the expression of survivin protein were only 42.7% and 54.6%, respectively. The results of endocytosis inhibitor test showed that OA-R8/si RNA nanoparticles were internalized through the clathrin and actin mediated endocytosis, and the si RNA could take effect in cytoplasm leading the inhibition of cell proliferation, cell cycle arrest and apoptosis.4. The preparation of multifunctional liposome and anti- tumor research in vitro and vivoMulti- function liposomes were modified by transferrin and OA-R8. The liposomes loading si RNA(s TOLP) were used to delivery si RNA in vitro and vivo. The results showed that s TO LP had positive charge, uniform particle size and spherical shape, Zeta potential were 13.2±5.4 m V and the average particle size were 191.9±17.2 nm. TOLP had less cytotoxicity on human normal cells and tumor cells. The cellular uptake of liposomes which possess OA-R8 were higher than other non OA-R8 liposomes. Confocal microscopy confirmed the results. These results illustrated that OA-R8 could improve the delivery capable of liposomes and did not exhibit cytotoxicity.In vivo experiment, we injected s TOLP to the caudal vein of mice and measured the tumor volume and weight. The results showed that s TOLP could significantly inhibit the growth of liver tumor, and tumor inhibitory rate was 61.7%. Si RNAs delivered by s TOLP were mainly accumulated in the liver and tumor tissues.There were more si RNAs in tumour tissues delivered by s TO LP than s TLP. The s TOLP do not have systemic toxicity and can be used for delivering si RNA in vivo.In conclusion, we synthesized a high efficiency survivin si RNA, which could depress the survivin expression. The synthesized modified CPP, OA-R8, had better capacity of condensation, protection and delivery of si RNA. The multi- function liposomes modified by OA-R8 and Tf could successfully deliver si RNA to tumor tissue and inhibit the growth of tumor cells.