Study On The Modified Cell-penetrating Peptide And Its Nanoparticles System For Oligonucleotide Delivery

In recent years,with the accelerated pace of life,environmental pollution,and changes in diet structure,the incidence of cancer has increased year by year.Therefore,drug development for the treatment of tumors is of great significance.In the field of oncology treatment,Anti-Sensitive Oligonucleotide(AS-ODN)drugs and related therapeutic technologies have entered everyone's field of vision and received great attention.Oligonucleotides are an important class of specific gene expression substances,including small interfering RNAs and antisense oligonucleotides.Currently,they have poor stability,high negative charge,weak transmembrane capability,and are susceptible to degradation by nucleases.Problems limit the research and development.LOR is a 20-base AS-ODN that is complementary to the mRNA coding region of the large R1 subunit of Ribonucleotide Reductase(RNR)and has a certain anti-tumor activity.The 2 ?-methoxy modified siRNA targeted to survivin gene can effectively inhibit the expression of survivin protein,thus inhibiting the proliferation of tumor cells.In this paper,stearic acid-modified octa-arginine was synthesized on the basis of transmembrane peptide octa-arginine,and a liposome delivery system was prepared for antisense oligonucleotide LOR and a siRNA targeting survivin gene.The LOR and siRNA was used as an oligonucleotide drug to investigate the effect of modified transmembrane peptide liposomes on the delivery of oligonucleotide drugs.At the same time,the liposomes were modified with targeting groups to investigate their effect on AS-ODN delivery.The specific content of this paper's research is summarized as follows:1.StA-R8 synthesis and establishment of delivery systemIn this paper,firstly,on the basis of R8,modified transmembrane peptide StA-R8 was synthesized.The StA-R8 liposome delivery system(SLP)was prepared using ethanol injection method.The preparation process of SLP delivery system was optimized and its stability was investigated.The optimum conditions for preparation were HEPES(pH=7.4).The particle size of liposomes prepared at 20/35/45 molar ratio of StA-R8,cholesterol,and egg-yolk lecithin was 126.4±6.2 nm.The zeta potential value was obtained.It is 18.3±1.3 mV.The cytotoxicity of StA-R8 on HeLa and A549 cells was low,and the cytotoxicity of StA-R8 liposomes was lower.The cell viability was all above 80%.Within 25 days,the particle size of the SLP delivery system increased slightly but did not change significantly,while the zeta potential value did not change significantly.2.Establishment and evaluation of StA-R8 liposome/LOR delivery systemTo evaluate the transfection efficiency of the prepared SLP delivery system,LOR was selected for loading and delivery.LOR is an AS-ODN drug with antitumor activity.Firstly,the particle size and zeta potential were used as indicators.The results showed that when N/P reached 6,the particle size of the nanoparticles formed between StA-R8 cationic liposome and LOR did not change substantially.The N/P ratio changes from negative to positive,indicating that with the increase in the amount of cationic liposomes,it can combine with LOR.In vitro cell evaluation experiments,the tumor cell inhibitory effect of SLP/LOR was examined by MTT assay.The results showed that the inhibition rate exceeded 30%;cells were examined by flow cytometry and confocal laser scanning microscopy.The Cellular uptake assay results showed that SLPs can deliver large amounts of LOR into tumor cells and have a good transfection effect.R1 mRNA was measured by RT-PCR.The results showed that the efficiency of R1 mRNA down-regulation of the LOR liposome in Hep G2 cells was 39.1%.Compared with StA-R8,it increased by 19.2%.Western-blot was used to determine the expression of R1 protein.The results showed that the down-regulation efficiency of R1 protein in SLP group was significantly higher than that in StA-R8 group.3.Establishment and evaluation of StA-R8 liposome/siRNA delivery systemAt the same time,a cationic liposome prepared from modified polyarginine was loaded with a siRNA targeting the survivin gene,and its properties were evaluated.Firstly,the N/P ratio of StA-R8 liposome/siRNA was optimized.The particle size and potential were used as indicators.The results showed that when N/P?6,stable composite particles could be formed.Agarose gel electrophoresis experiments showed that SLP completely bind to siRNA when N/P ratio is bigger than 6.At the same time,the composite particles formed by SLP and siRNA were studied in vitro.The tumor cell inhibitory effect of SLP/siRNA was examined by MTT assay.The results showed that the inhibition rate exceeded 35%;the uptake of cells was examined by flow cytometry and laser confocal microscopy.The results showed that SLP can efficiently deliver siRNA into tumor cells and have a good transfection effect.Western-blot method was used to determine the expression of survivin protein.The results showed that the efficiency of survivin protein down-regulation in SLP group was significantly higher than StA-R8 group,indicating that SLP can transfect si RNA into cells and then interfere with the synthesis of survivin protein and inhibit the growth of tumor cells.4.Establishment and evaluation of Tf/StA-R8 cationic liposome delivery siRNA systemBased on the StA-R8 cationic liposome prepared in this paper,Tf-PEG-DSPE was inserted into the liposome using the post-insertion method to form the transferrin-modified liposome delivery system(TLSP).The prepared TLSP structure includes a tumor-targeting transferrin ligand in the outer layer,a PEG structure that prolongs blood circulation time,and a modified transmembrane peptide StA-R8 that can assist the delivery system to penetrate the cell membrane in the phospholipid layer,thereby increasing internalization of siRNA into tumor cells.The stability test showed that the transferrin-modified liposomes prepared by the post-insertion method had better stability.The particle size and potential of TSLP/siRNA were changed compared to liposomes containing no targeting ligand.The particle size increased to 148.7±11.95 nm with a uniform particle size distribution and a potential of 4.7±0.2 mV.TSLP as a carrier has the same low cytotoxicity as SLP.Observation of cell uptake by flow cytometry and laser confocal microscopy revealed that TSLP can deliver siRNA into tumor cells more efficiently than liposomes.Western-blot method was used to measure the expression of survivin protein.The results showed that the protein expression levels of SLP/siRNA and TSLP/siRNA groups were 44.1% and 20.4%,respectively.Transfection of survivn siRNA with TSLP as a vector is more efficient.In summary,the modified poly-arginine liposome and its Tf-modified drug delivery system prepared in this paper have the characteristics of low cytotoxicity and high transfection efficiency,and can combine with oligonucleotides to form a stable system.The efficient delivery of oligonucleotide drugs into tumor cells lays a solid foundation for future in vivo studies.The obtained transferrin-targeted PEGylated polyarginine cationic liposome will have a good application prospect.