| In the field of anticancer research, antisense oligonucleotide(AS-ODN) drugs and the related treatment technologies are taken more attention by us. Currently, although the researches of AS-ODN drugs are in clinical stage, but there are still some defects with AS-ODN drugs, such as poor stability, transmembrane ability, easy to be degraded by nuclease and so on. LOR-2501 is a 20-mer phosphorothioate AS-ODN drug. LOR-2501 has antitumor activity because it is complementary to a coding region of the mRNA of R1, a subunit of ribonucleotide reductase(RNR). Lots of researches focused on the development and application of the AS-ODN drugs carriers. Polyethyleneimine(PEI) has the unique advantage, which called “proton sponge effect” compared with other cationic polymers, PEI can absorb protons by its large numbers of amines after loading drugs, which causes lysosomal osmotic swelling and burst, then the lysosome will release the drugs into the cytoplasm. As a kind of AS-ODN carriers, high molecular weight of PEI(25 kDa) has high transfection efficiency but higher toxicity at the same time. Low molecular weight PEI(800 Da)has much lower toxicity but the transfection efficiency is lower. So how to improve the transfection efficiency while reducing the toxicity by the hydrophobic modification and targeting ligand modification of PEI has become a research hotspot.In this paper the research content mainly includes the following sections: 1. The synthesis and establishment of drug delivery system of PEI-SS-OAIn this paper, first of all, on the basis of the PEI, a polymer containing the disulfide bond(S-S) was synthesized, which was hydrophobic modified by oleylamine(named PEI-SS-OA). At the same time, the corresponding drug delivery system was prepared by ethanol injection method. The polymer of PEI-SS-OA had the buffer capacity. It was suitable for the carriers of AS-ODN. The particle size of the delivery system was 201.0±5.9 nm, the zeta potential value was 43.5±0.9 mV under the optimal preparation conditions. There was no obvious toxicity of PEI-SS-OA in HeLa and A549 cell lines, the cell vitalities were both above 90%. The results of the stability showed that the particle size of the PEI-SS-OA system showed a slight increase and the zeta potential values had no obvious change in 30 days. 2. The establishment and evaluation of PEI-SS-OA/LOR-2501 delivery systemTo evaluate the efficiency of PEI-SS-OA, a oligonucleotide drugs LOR-2501 was chosen in this paper. PEI-SS-OA/LOR-2501 delivery system was prepared. The best nitrogen and phosphorus ratio(N/P) was 8:1, which was chosen by the size and zeta potential as an index. At this ratio, PEI-SS-OA combined with LOR-2501 completely, the addition of reductant could lead to the release of LOR-2501. In vitro studies, MTT tests and flow cytometry illustrates showed that the PEI-SS-OA could transfect LOR-2501 into HeLa and A549 cells and inhibit the growth of the tumor cells. Large quantities of the complexes were uptaken into the tumor cells, which was observed by laser confocal microscop. The results of real-time PCR showed that the down-regulation rates of R1 mRNA were 40.8% and 43.6% in He La and A549 cells, which were improved 17.8% and 23.3% compared with PEI. The results of Western-blot also showd that the R1 protein levels were reduced by PEI-SS-OA/LOR-2501 more obviously than PEI. 3. The synthesis and establishment of drug delivery system of PEI-NH-OAIn this paper, on the basis of the PEI, a polymer containing the amido bond(N-H) was synthesized at the same time, which was hydrophobic modified by oleoacyl(named PEI-NH-OA). The corresponding drug delivery system was prepared by ethanol injection method at the same time. The polymer of PEI-NH-OA also had the buffer capacity. The particle size of the delivery system was 102.0±3.9 nm, the zeta potential value was 54.5±3.1 mV under the optimal preparation conditions. There was no obvious toxicity of PEI-NH-OA in HeLa and A549 cell lines, the cell vitalities were both above 90%. Results of the stability showed that the particle size and zeta potential of the PEI-NH-OA system showed no obvious change in 30 days. PEI-NH-OA system has much higher cations, smaller size and better stability than PEI-SS-OA system. 4. The establishment and evaluation of PEI-NH-OA/LOR-2501 delivery systemTo evaluate the efficiency of PEI-NH-OA, PEI-NH-OA/LOR-2501 delivery system was preared in this paper. The best nitrogen and phosphorus ratio(N/P) was 6:1, which was chosen by the size and zeta potential as an index. At this ratio, PEI-NH-OA combined with LOR-2501 completely. In vitro studies, MTT tests showed that the inhibition rates were 46.2% and 41.5% in HeLa and A549 cells by PEI-NH-OA/LOR-2501 complex. It was observed that PEI-SS-OA could transfect LOR-2501 into HeLa and A549 cells by flow cytometry illustrates and laser confocal microscope. The results of real-time PCR showed that the down-regulation levels of R1 mRNA were 37.7% and 34.5% in HeLa and A549 cells, which was only about 20% of PEI. The results of Western-blot showd that the R1 protein levels were reduced 50.1% and 46.3% by PEI-NH-OA/LOR-2501 in HeLa and A549 cells. Above all, PEI-NH-OA could transfect LOR-2501 into cells efficiently. At the same time, PEI-NH-OA showed smaller particle size, higher stability compared to PEI-SS-OA system. PEI-NH-OA was chosen to be modified in the later experiment. 5. The establishment and evaluation of FA/PEI-NH-OA/LOR-2501 delivery systemIn this paper, PEI-NH-OA was further modified by folic acid(FA), the method of the combination was by electrostatic adsorption, FA/PEI-NH-OA/LOR-2501 delivery system was prepared. Results showed that the particle sizes increaseed gradually with the increase of amount of FA, at the best proportion, the particle size was 159.0±1.1 nm and the zeta potential value was-12.2±1.3 m V. PEI-NH-OA could still combine with LOR-2501 after adding FA. FA/PEI-NH-OA had no obvious toxicity, the cell vitality were all above 80% with different concentrations of FA. MTT tests showed that the inhibition rates were 55.6% and 53.7% in HeLa and A549 cells by FA/PEI-NH-OA/LOR-2501 complex. The results of flow cytometry experiment showed that the fluorescence intensity of the group of FA/PEI-NH-OA/LOR-2501 was 2 times more than PEI-NH-OA/LOR-2501 in HeLa cells(FR+) and A549 cells(FR-). In another two kinds of tumor cell lines, KB cells(FR+) and SK-HEP-1 cells(FR-), this phenomenon was also appeared. Large quantities of FA/PEI-NH-OA/LOR-2501 complexes were uptaked into the tumor cells, which was observed by laser confocal microscope. The results of real-time PCR showed that the down-regulation levels of R1 mRNA were 51.7% and 45.7% in HeLa and A549 cells, respectively. The results of Western-blot showd that the R1 protein levels were reduced 72.0% and 68.9% by FA/PEI-NH-OA/LOR-2501 in HeLa and A549 cells. Above all, PEI-NH-OA could transfect LOR-2501 into cells efficiently, which was improved obviously than PEI-NH-OA/LOR-2501 complexes. 6. The preliminary study of the internalization mechanism ofFA/PEI-NH-OA/LOR-2501 delivery systemFlow cytometry was firstly used to investigate the influence of free FA on the uptake of LOR-2501 by FA/PEI-NH-OA system in cancer cells. Results showed that the free FA with the concentration of 0.01, 0.1and 1 mmol/L had no influence to the uptake of FA/PEI-NH-OA/LOR-2501 complexes in HeLa and KB cells(FR+), A549 and SK-HEP-1 cells(FR-). The addition of free FA did not inhibit the uptake progress. Three kinds of internalization inhibitors, sugar, cytochalasin D and nystatin were also used to study the endocytosis pathway. Results showed that the complexes might be uptaken into cells mainly by clathrin-mediated endocytosis pathway. Cellular uptake of FA/PEI-NH-OA/LOR-2501 complex was also studied by confocal microscopy. Cellular markers of the internalization process were used. The results further verified the mainly internalization pathway of the complex was clathrin-mediated endocytosis pathway.In summary, we prepared the hydrophobic modified PEI and its FA modified delivery system with low toxicity and high transfection efficiency. The FA/PEI-NH-OA/LOR-2501 complex was high-efficiency, regardless of the expression levels of the FR on the surface of tumor cells. There did not appear to be any limitation of the delivery system based on the type of tumor cell lines. The system is expected to be safe and efficient as AS-ODN drugs carrier. |
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