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Biomimetic Drug-loaded Bacterial Ghosts Formulation For The Treatment Of Metastatic Lung Cancer

Posted on:2024-04-05Degree:MasterType:Thesis
Country:ChinaCandidate:D D LingFull Text:PDF
GTID:2531307082966659Subject:Pharmacy
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Objective:In view of the problems of poor drug targeting and difficult control of immune response in chemotherapy and immunotherapy of metastatic lung cancer,the liposomal paclitaxel-loaded bacterial ghosts formulation encapsulated by cancer cell membrane was prepared to obtain efficient and safe treatment of metastatic lung cancer through tumor targeting,immune stimulation and drug treatment.Methods:Liposomal paclitaxel(LP)was prepared by film dispersion method.Bacterial ghosts(BGs)were prepared by genetic engineering.Paclitaxel liposome was introduced into the lumen of bacterial ghosts in the form of electroporation to prepare paclitaxel liposome-loaded bacterial ghosts(LP@BG),and the conditions of electroporation voltage were investigated by single factor to optimize the loading prescription.The 4T1 mouse breast cancer tumor cells were cultured in vitro,and the cancer cell membranes(CCMs)were extracted and purified by differential centrifugation.The cancer cell membranes were mixed with the LP-loaded bacterial ghosts formulation and repeatedly extruded through the polycarbonate porous membrane(1μm)for 15 times to prepare the CCM-coated LP@BG(LP@BG@CCM).The formulation was evaluated by particle size,potential,scanning electron microscopy and transmission electron microscopy.The antitumor and cell targeting of each formulation were evaluated by in vitro cell experiments.The safety of the formulation was evaluated by hemolysis test in vitro.A mouse model of metastatic lung cancer was established by tail vein injection of4T1 cells,and each formulation group was treated by tail vein injection.The appearance of lung tumor,tumor weight,pathological section of tumor tissue and apoptosis of tumor cells were investigated.The distribution of drug-loaded bacterial ghosts coated by cancer cell membrane or lipid membrane in vivo was observed by small animal in vivo imaging,and the in vivo targeting effect of cancer cell membrane was evaluated.The relative percentage of CD4~+T cells and CD8a~+T cells in spleen cells of mouse in each group was detected by flow cytometry,and the stimulating effect of LP@BG@CCM and other formulation on immune cells was evaluated.Results:According to the ratio of soybean phospholipids:cholesterol=10:1(w/w),paclitaxel liposomes with small and uniform particle size were successfully prepared.Under scanning electron microscope,obvious holes were formed at both ends or in the middle of Escherichia coli.Transmission electron microscopy showed that the lumen of the bacterial ghosts were transparent and there were basically no content,indicating that the bacterial content had flowed out of the cell through the lysis pores.Transmission and confocal experiments show that the electroporated conditions are:voltage 1000 V,the number of electric shocks is 10,can achieve the best drug loading.The results of fluorescence microscopy showed that the cancer cell membrane was successfully purified.Transmission electron microscopy showed that the cancer cell membrane had a complete nanoscale vesicle structure.Under transmission electron microscopy,a transparent cell membrane layer was coated around the paclitaxel-loaded liposome BGs wrapped by the cancer cell membrane,and the bacterial lumen was filled with content.Confocal observation further showed that the erythrocyte membrane stained by Di I was successfully wrapped around the drug-loaded coumarin liposome BGs.In vitro cell experiments showed that when the concentration of paclitaxel was 2μmol/L,the inhibition rate of cancer cell membrane reached more than 50%.The results of cellular uptake of Coumarin-6(C6)-labeled formulation bacterial ghosts that each formulation group had different degrees of promotion of cell uptake,among which the CCM-coated group had the most obvious promotion.The results of in vivo imaging of small animals showed that the fluorescence of lung tumor in LP@BG@CCM group was the strongest.On the third day after the end of administration,the mice were dissected and found that the fluorescence intensity of the lung tumor area was 2.5 times that of the lipid membrane-coated drug-loaded bacterial shadow group.The results of in vivo safety evaluation and in vitro hemolysis test showed that each formulation group had good biosafety.Through the analysis of lung appearance,pathological section and apoptosis detection results,LP@BG and LP@BG@CCM have a certain inhibitory effect on tumors in vivo,and the LP@BG@CCM group has the strongest inhibitory effect on tumors.The percentage of CD4~+T cells and CD8a~+T cells in spleen was detected by flow cytometry.The results showed that LP@BG and LP@BG@CCM could effectively activate immune cells and exert antitumor effects.Conclusion:The multifunctional tumor-targeting LP@BG@CCM formulation can effectively treat metastatic lung cancer through tumor targeting,immune stimulation,and drug therapy.It is an antitumor targeted formulation with potential clinical application value.
Keywords/Search Tags:Metastatic lung cancer, Bacterial ghosts, Cancer cell membrane, Paclitaxel, Liposome
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