Construction And Study Of Multifunctional Co-loaded Liposome For Targeting Of Tumor Organelles In Cancer Cells

Objective:This study construted hierarchical targeting liposomes with characteristics of simultaneous transport drugs to nucleus and mitochondria in cancer cells,then synergistically induce cell apoptosis in different pathway.Meanwhile,in vitro and in vivo anti-tumor effects and mechanisms of the liposomes were studied and evaluated.Methods:The multifunctional liposomes were prepared by film dispersion method and ammonium sulfate gradient method.The particle size,Zeta potential,the encapsulation efficiency and in vitro drug release was characterized.We choose MDA-MB-231 cells as model cells.The cell viability of different liposomes on MDA-MB-231 cells was evaluated by MTT method.Flow cytometry method was used to investigate the cellular uptake.And the confocal microscope was used to observe the transport process of multifunctional liposomes in cells.Besides,in order to investigate the apoptosis mechanism of the liposomes,some experiments such as Annexin V-FITC/PI staining,JC-1 assays and detection of the activity of the apoptotic protease were conducted.In vivo antitumor experiments were performed on MDA-MB-231 tumor-bearing nude mice after tail vein injection of the liposomes formulations.And the pathological changes of tumor tissue and major organs tissue were observed by HE staining.Results:¹H NMR spectrum shows that the DSPE-PEG₂₀₀₀-RGD and DSPE-PEG₂₀₀₀-TAT were successfully synthesized.The particle size of the prepared multifunctional liposomes was 100¹50 nm and the Zeta potential was-20 to-10 mV,PDI<0.3.The optimal ratio of DOX:TR was less than 2:1.The in vitro release assay showed that the release rate of the two drugs in the co-loaded liposomes was faster than that of the single drug-loaded liposomes and the cumulative release rate increased significantly after 72 hours.MTT assay showed that the multifunctional liposomes had significantly inhibited proliferation of MDA-MB-231 cells.The results of flow cytometry showed that the uptake of dual target liposomes was 1.19 times of RGD liposomes and 1.24 times of TAT liposomes,indicating that dual target liposomes promote cell uptake synergistically.By cofocal experiments,it was found that the multifunctional liposomes could release DOX to the nucleus and release TR to the mitochondria.Study on apoptosis mechanism confirmed that TR liposomes could lead to mitochondrial depolarization,activate apoptotic protease cascade to induce apoptosis of tumor cells.In vivo imaging results showed that dual-target liposomes can target tumor tissue and reduce drug accumulation in normal tissues,and the target efficiency is superior to single target liposomes.In vivo anticancer study indicated that multifunctional liposomes can effectively inhibit tumor growth,significantly reduce the side effects of free DOX at the same time.HE staining showed that the multifunctional liposomes caused tumor necrosis in large area but were safe to normal tissues.Conclusions:The multifunctional liposomes T/R-LP-（DOX+TR）was successfully prepared.The prepared liposomes had uniform size,high encapsulation efficiency of two drugs and performed good release characteristics.In vivo and in vitro antitumor activity experiments showed the multifunctional liposomes could enhance the therapeutic effect and alleviate the toxic effects of free DOX by targeting the drugs to tumor tissue,tumor cells and tumor organelles step by step.All of this showed that the multifunctional liposomes T/R-LP-（DOX+TR）have a broad application prospect in the field of tumor target drug delivery system.