Preparation Of Paclitaxel Loaded Sterically Stabilized And Targeted Liposomes, And Its Study On Antitumor Activity

Objective: Sterically stabilized and targeted liposomes can be specifically target to tumor cells, thus they are supposed to be able to improve the cellular uptake, extend the biologic half- life period and reduce the accumulation of drugs in normal tissues. This study aimed to develop a new type of paclitaxel(PTX) loaded sterically stabilized liposome modified with ES. This liposome was designed to enhance the targeting specificity and improve the therapeutic effect of PTX on Michigan Cancer Foundation- 7(MCF-7) cells, which are covered with over-expressed estrogen receptors(ER). We also focused on the characterization of the prepared liposomes, the in-vitro and in-vivo target effects and the anti-tumor efficiency of ES-SSL-PTX in MCF-7 cells.Method:(1) A new method was established by using HPLC to determine the concentration of PTX. Four types of PTX loaded liposomes(PTX loaded conventional liposomes L-PTX, PTX loaded targeted liposomes ES-L-PTX, PTX loaded sterically stabilized liposomes SSL-PTX and PTX loaded sterically stabilized and targeted liposomes ES-SSL-PTX) were prepared by using thin film hydration method. Furthermore, the physicochemical characteristics of the PTX loaded liposomes were investigated, including the morphological examination, particle size, zeta potential, encapsulation efficiency, drug-loading capacity and the stability study at 4℃ or room temperature.(2) To evaluate the in vitro target effect, the fluorescence intensity of MCF-7 cells, which were treated with various Rhodamine B loaded liposomes, was measured via fluorescence microscope. By adding different inhibitory agents of endocytosis the mechanism of cellular uptake was determined.(3) The DiR-loaded liposomes were applied to investigate the tumor targeting efficacy in female BALB/c nude mice models bearing MCF-7 cells.(4) The in vitro anti-tumor efficacies of PTX and the PTX-loaded liposomes were analyzed via MTT assay based on the cytotoxicity of MCF-7 cells.(5)The MCF-7 cell tumor bearing mice model was injeced different paclitaxel preparations for the in vivo anti-tumor effect.Result:(1) Four types of PTX loaded liposomes were successfully prepared. Their particle sizes were average in the range of 120nm~140nm, the PDI were in the range of 0.1~0.2 and the zeta potential of the liposomes were negative. These four types of PTX loaded liposomes obtained similarly high encapsulation efficiency in the range of 86.0%~88.0% and high drug-loading capacity in the range of 6.1%~7.5%. The stability study indicated that after storing for 48 h at 4°C ES-SSL-PTX and SSL-PTX showed an obviously advantage over other liposomes.(2) After cellular uptake of liposomes, the MCF-7 cells, treated with ES-L-RhB or ES-SSL-RhB showed higher fluorescence intensity and higher uptake efficiency than the cells treated with L?RhB or SSL-Rh B. This result confirmed our hypothesis, that ER?mediated endocytosis facilitated the cellular uptake of ES-L-RhB and ES-SSL-RhB into MCF-7 cells. By adding different inhibitory agents of endocytosis including sucrose, genistein, amiloride hydrochloride and ES, the cellular uptake of ES-SSL-RhB was decreased. This result hinted that, the cellular uptake depended mainly on macropinocy-tosis, caveolin-dependent endocytosis and estrogen receptor-mediated endocytosis.(3)The result of in vivo imaging experiment showed that the stronger fluorescence intensity was found in tumor tissue after administration of ES-SSL-DiR compared with that in the SSL-DiR group. ES-SSL-DiR can quickly and selectively targeted to the estrogen receptor on the tumor and significantly increased the drug accumulation amount in the tumor site.(4) From the in vitro anti-tumor experiment showed that, ES-SSL-PTX possessed the greatest inhibiting effect on MCF-7 cells.(5)From the in vivo anti-tumor experiment showed that, ES-SSL-PTX possessed the greatest inhibiting effect on tumor.Conclusion: A new type of liposome, PTX loaded sterically stabilized and estrogen receptor-targeted liposomes modified with ES(ES-SSL-PTX) was successfully prepared. ES-SSL-PTX is a kind of long-circulating liposome, which is able to avoid the rapid clearance by RES, thus it could enhance the accumulation of drugs in tumor cells. Benefit by the specific targeting to ER, which are over-expressed on the cytomembrane of MCF-7 cells, ES-SSL- PTX could markedly reduce the side effects of paclitaxel and significantly improve its anti-tumor effect. We expect that in the further ES-SSL-PTX could be a novel anti-tumor drug with full of huge potential for the clinical therapy of breast cancer.