Studies On Targeting, Anti-tumor Effect And Acute Toxicity Of Paclitaxel And Epirubicin Co-loaded Sterically Stabilized And Targeted Liposome

Objectives:Studies have shown that estrogen receptors are over-expressed on the surface of breast cancer cells.Based on this feature,the drug can be targeted delivered to tumor sites.In our lab,the targeting fragment ES-DSPE-PEG₂₀₀₀,which can specifically bind to estrogen receptors,was synthesized.The ES-DSPE-PEG₂₀₀₀ was incorporated into lipid materials,at the same time,paclitaxel and epirubicin were encapsulated to prepare a long-acting targeted liposome ES-SSL-PTX/EPI.On the basis of the previous studies,we continued to study the in vivo and in vitro targeting effects and in vivo antitumor effects.In terms of safety,acute toxicity study was carried out.Methods:Based on the fluorescence characteristic of epirubicin,ES-SSL-EPI and SSL-EPI were prepared.The cellular uptake of free EPI,ES-SSL-EPI and SSL-EPI by MCF-7 cells and MDA-MB-231 cells were observed by inverted fluorescence microscope,respectively.The targeting effect of ES-SSL-EPI was verified by adding excess estrone,and the endocytosis mechanism of ES-SSL-EPI by MCF-7 cells was further studied using three kinds of endocytosis inhibitors.MCF-7tumor-bearing animal model was established,and ES-SSL-DiR and SSL-DiR were prepared to investigate the in vivo targeting effect.We used the small animal imager to observe their distribution in various organs and tumors.We established MCF-7tumor-bearing animal models,and the anti-tumor effect of ES-SSL-PTX/EPI in vivo was studied by weighing and comparing the body weight,tumor weight and volume of each group.ICR mice were selected to investigate the acute toxicity of L-PTX/EPI,ES-SSL-PTX,ES-SSL-EPI,and ES-SSL-PTX/EPI.The status and death of the mice were recorded,and the LD50 of each preparation was calculated.Results:As for MCF-7 cells,in vitro targeting experiments showed that,compared with the SSL-EPI group,the EPI fluorescence intensity of the ES-SSL-EPI group was significantly enhanced.After adding excess estrone,the fluorescence intensity of the ES-SSL-EPI group decreased significantly.After the addition of three endocytosis inhibitors,the fluorescence intensity of the amiloride group was the strongest,genistein group was the second,and the sucrose group was the weakest,indicating that clathrin-dependent endocytosis played a dominant role,followed by caveolin-induced endocytosis,although macrocytoyin also participated in cellular uptake,but had less effect.For MDA-MB-231 cells,the fluorescence intensity of the SSL-EPI group was comparable to that of the ES-SSL-EPI group.In vivo targeting experiments showed that both SSL-DiR and ES-SSL-DiR were accumulated in the tumor site,and the cumulative amount increased with time,but the ES-SSL-DiR group accumulated more,while no fluorescence was found at the tumor site in free DiR group.The in vivo anti-tumor experiments showed that for MCF-7 tumor-bearing animal models,the tumor volumes of ES-SSL-PTX/EPI group were smallest and the mice had the best status,indicating that ES-SSL-PTX/EPI has the strongest anti-tumor effect.The results of the acute toxicity study showed that the LD₅₀ of the PTX/EPI,L-PTX/EPI,ES-SSL-EPI,ES-SSL-PTX,and ES-SSL-PTX/EPI groups were 17.039,83.009,91.592,96.188,and 85.983,respectively.The LD₅₀ of the ES-SSL-PTX/EPI was significantly higher than that of the free PTX/EPI group.Conclusions:The ES-SSL-PTX/EPI preparation had a good targeting effect both in vitro and in vivo.It could target into tumor sites,and enter tumor cells through estrogen receptor-ligand specific binding,so that increased the uptake of cancer cells,and achieve excellent anti-tumor effects.At the same time,it showed that the ES-SSL preparation could significantly increase the LD₅₀ value,reduce the toxicity of chemotherapeutics,and have good safety.