A Novel Reductive Cationic Liposomes For Delivery Of Oligonucleotide Drugs

At present,malignant tumors have become one of the serious threats to human health.The mortality rate of cancer patients in China is increasing year by year.Oligonucleotide drugs have good prospects in the field of tumor therapy.Nowadays,a large number of oligonucleotide drugs have entered the clinical trial stage.However,oligonucleotide drugs have the problem of being easily degraded and having a weak transmembrane capability,which limits their development.To overcome these obstacles,oligonucleotide drugs require efficient delivery systems to increase stability and transmembrane capabilities.Therefore,it is of great significance to study efficient and low-toxic delivery systems that can improve the stability and transmembrane capabilities of oligonucleotides.Polyethyleneimine(PEI)has a unique "proton sponge effect" compared to other cationic polymers.When PEI is used as a nucleic acid drug carrier,high molecular weight PEI has higher transfection efficiency but greater toxicity.In this paper,PEI(25 kDa)was modified to reduce cytotoxicity.Polyethylenimine was hydrophobically modified by disulfide linkage(S-S)to linkage oleylamine(OA)to synthesize PEI-SS-OA.PEI-SS-OA has a strong cationic charge and can bind to negatively charged oligonucleotides and then enter cells.After adding S-S,it gives liposomes a certain reduction response force,and can quickly release the loaded drug under reducing conditions.The cells contain a large amount of reduced glutathione.Therefore,the liposomes can increase the stability of oligonucleotides and transmembrane capacity,and release the loaded drug rapidly in cells.In this paper,cationic liposome carriers were prepared on the basis of PEI-SS-OA.The experimental results showed that the prepared cationic liposome drug delivery system can improve the efficiency of oligonucleotide drug transfection and effectively down-regulate the expression of target protein.The content of this paper mainly includes the following aspects: 1.Synthesis of materials and establishment of cationic liposome drug delivery systemIn this paper,PEI is used as the raw material,and the polyethylenimine is hydrophobically modified through the S-S linkage of oleylamine.The final synthesized polyethyleneimine modified polymer(PEI-SS-OA)has a strong positive charge.On this basis,cationic liposomes were prepared by ethanol injection and the preparation of cationic liposomes was optimized.The finally prepared cationic liposome system(PEI-SS-OA Liposomes,POLP)has a better buffer capacity,and can effectively release its own drugs after entering the cells.The average particle size of POLP was 140 nm,and the average zeta potential was +31.7 mV.Compared with PEI,the cytotoxicity of POLP was significantly reduced.2.Establishment and Evaluation of siRNA Delivery SystemFor the evaluation of POLP,the siRNA was selected for loading and siRNA delivery system was established.Firstly,the nitrogen-phosphorus ratio(N/P)of siRNA-loaded PEISS-OA liposome(PEI-SS-OA Liposomes loading siRNA,siRNA-POLP)was optimized using the particle size and zeta potential as indicators.The N/P is 8:1.Agarose gel electrophoresis results showed that siRNA-POLP was fully bound at a N/P ratio of 8:1.In this paper,siRNA-POLP was evaluated in vitro in cell lines Hep G2 and A549.Compared with free siRNA and siRNA-loaded PEI(PEI loading siRNA,siRNA-PEI),siRNA-POLP transfection efficiency was significantly improved.Western-blot experiments showed that siRNA-POLP had a significant down-regulation effect on Survivin protein.3.Establishment and Evaluation of LOR-2501 Delivery SystemAt the same time,POLP was loaded with an antisense oligonucleotide LOR-2501 to establish a LOR-2501 delivery system.Using liposome particle size and zeta potential as indices,the results of the agarose gel electrophoresis test showed that PEI-SS-OA Liposomes loading LOR-2501(PEI-SS-OA Liposomes loading LOR-2501,LOR-POLP)is fully bound at a N/P ratio of 6:1.In vitro cell experiments were performed on HepG2 cell line.The results of flow cytometry and confocal laser scanning microscopy showed that LOR-POLP can successfully enter into cells,with free LOR-2501 and PEI loaded with LOR-2501(PEI loading the LOR-2501,LOR-PEI)group had better transfection efficiency than the LOR-PEI group.The Western-blot assay was used to determine the R1 protein.The results showed that LOR-POLP had a significant down-regulation effect on R1 protein.In summary,the cationic liposome drug delivery system prepared in this article can be effectively combined with oligonucleotide drugs to prevent it from being degraded.It has high transfection efficiency in a variety of tumor cell lines.High-efficiency oligonucleotide drug carriers have a good application prospect.