Novel Modified Polyethylenimine Based Liposomes For Oligonucleotide Delivery

Malignant tumor has become a serious threat to human health and cancermortality is increasing yearly in China. Among the drugs for treatment of cancer, thecurrent oligonucleotide drugs have obtained one of the biggest concerns.Oligonucleotide material is an important kind of specific gene expression, includingsiRNA and antisense oligonucleotides nucleotides, etc. Although oligonucleotideseffect is better, but because of less stable, strong negative charge, it is hard to span thecell membrane, thus its development is restricted. Therefore, developing low toxicity,high efficiency and low cost oligonucleotide carrier is important, a lot of researchfocused on the carrier of oligonucleotide development at present.This dissertation mainly includes the following parts:1. The synthesis of novel, high efficient delivery system materials and theestablishment of drug delivery systems.We developed a novel oligonucleotide drug carrier, fatty acid modifiedpolyethylene imine (PEI-OA and PEI-LA) were synthesized. The modified polymerhad strong positive charge. On this basis, the two kinds of cationic liposomes wereprepared by modified polyethyleneimine. The process of the preparation of liposomeswas optimized, the best preparation process is: the preparation of liposomes was usedethanol injection method. PEI-LA or PEI-OA with10μg/μl ethanol solution, thephospholipids and cholesterol were dissolved in ethanol, and were mixed withPEI-LA or PEI-OA ethanol solution, respectively. Then,10μl mixed solution wasinjected to90μl PBS solution quickly, vortex mixed10s, further ultrasound1to2minutes. The PEI-LA liposome was prepared with average particle size140.5nm andthe average potential+36.7mV; and average particle size of the PEI-LA liposomewas132.6nm, the average potential was+32.4mV. The liposomes showed lowertoxicity compared with PEI, and with good stability. 2. The establishment and evaluation of the LOR-2501delivery systemFor the evaluation of the cationic liposomes carrier prepared by hydrophobicmodification of polyethyleneimine. Firstly, LOR-2501, which was a kind ofsingle-stranded oligonucleotides with20base-pairs was used. LOR-2501was wellkown as a kind of oligonucleotides with good antitumor activities, because it cancombine with ribonucleotide reductase (RNR) large subunit R1mRNA coding region.PEI-LA or PEI-OA liposomes were combined with LOR-2501as a delivery system inthis paper. The results showed that two kinds of liposomes, PEI-LA and PEI-OAliposomes/LOR-2501complexes formed after when the N/P reaches6:1with stableparticle sizes. And the zeta potential ranged positive from negative, which illustratedthe cationic liposomes combined with LOR-2501together. Agarose gelelectrophoresis test results showed that the PEI-LA liposome and PEI-OA liposomecan combine LOR-2501with strong positive charge. In vitro experiment, MTTexperiment showed that the complexes have stronger antitumor activity, inhibitionrate reached30%. At the same time, flow cytometry and laser confocal microscopeshowed PEI-LA liposome and PEI-OA liposome loaded LOR-2501into the cells. Interms of active verification, the R1mRNA was evaluated by RT-PCR and the R1protein was tested by Western blot, respectively. PEI-LA liposome/LOR-2501andPEI-OA liposome/LOR-2501complexes reduced R1mRNA and R1proteinobservably. At the same time, three kinds of proteins closely connected with tumorcell apoptosis, Bax, Bad and Bcl-2were determined by Western blot method. It wasfound that the amount of apoptosis proteins increased after transfection, and promotecells may appear the phenomenon of apoptosis.3. The establishment and evaluation of the siRNA delivery systemIn this paper, a survivin siRNA was also used for the evaluation of the cationicliposomes carrier prepared by hydrophobic modification of polyethyleneimine. Thehydrophobic modified polyethyleneimine cationic liposomes were prepared and as acarrier to load a survivin siRNA system. First, the N/P ratios of PEI-LAliposome/siRNA were optimized, results showed that when the N/P≥8, stable particleswere formed. In agarose gel electrophoresis tests, PEI-LA liposomes combined with siRNA completely when the N/P≥6. We also evaluate PEI-LA liposome/siRNAcomplexes of in vitro. According to the results, PEI-LA liposome/siRNA showedhigher transfection efficiency than that of free siRNA and PEI25K by HeLa andA549cells. The survivin mRNA was evaluated by RT-PCR and the survivin proteinwas tested by Western blot in HeLa and A549cell lines, respectively. The group ofPEI-LA liposome/siRNA complex reduced survivin mRNA and survivin proteinsignificantly, which showed the PEI-LA liposome can transfectt siRNA into cells, andinterfere with the survivin protein synthesis process. At the same time, three kinds ofproteins closely connected with tumor cell apoptosis, Bax, Bad and Bcl-2weredetermined by Western-blot method. It was found that the amount of apoptosisproteins increased when siRNA was transfected by PEI-LA liposome, cells showedthe phenomenon of apoptosis.Above all, fatty acid modified polyethyleneimine liposomes (PEI-LA liposomeand PEI-OA liposome) can be used as a new type of transfection reagents, whichcould overcome the high cytotoxicity of high molecular weight PEI. The liposomescan form relatively stable system with oligonucleotide. The cationic liposomescould protect the oligonucleotide degradation, transfect LOR-2501and survivinsiRNA into cells, identify the cell active site, lead to tumor cell apoptosis and death.In this paper, the preparation of fatty acid modified polyethyleneimine cationicliposomes, and the formation of complexes with oligonucleotide can further bestudied in vivo, the new type of fatty acid modified polyethyleneimine cationicliposome will have good application prospects.