Construction And Evaluation Of Pab Mediated Targeted Paclitaxel Liposomes For Drug Resistant Lung Cancer

Objective:Paclitaxel（PTX）,an insoluble natural diterpenoid broad-spectrum anticancer compound,has shown significant anti-tumor activity against breast cancer,ovarian cancer and non-small cell lung cancer.Paclitaxel mainly disrupts the dynamic balance between microtubules and tubulin that lead to cell death.Paclitaxel injection（Taxol）and paclitaxel liposome（Lipusu）on the market lack active targeting of tumors,which not only kills tumor cells,but also has a great killing effect on normal cells.P-glycoprotein（Pgp）,as a transport protein on cell membrane,can mediate the excretion of drugs from cells and reduce the toxicity of drugs as well.It is one of the self-protection mechanisms of tumor cells.Overexpression of Pgp is the main cause of multidrug resistance of cancer cells.Pgp antibodies（Pab）can specifically bind to Pgp on the cell membrane,and transport drugs specifically to the tumor cells with high expression of Pgp by linking drug carrier to Pab.Therefore,this project was intended to link Pab with long circulating liposome and load with paclitaxel.,The preparation,quality evaluation and in vitro pharmacodynamic were evaluated together with studing the specific killing effect of Pab paclitaxel long circulating immune liposome on multidrug resistant lung cancer cells.Method:1)Long cycle paclitaxel liposomes were prepared with thin film dispersion method.Pab long cycle immunoliposomes（Pab-PTX-L）were prepared by binding succinimide succinate at the PEG terminal of the liposome to Pab antibody.The encapsulation efficiency of Pab-PTX-L was determined with sephadex G-50 microcolumn centrifugation and high performance liquid chromatography.The effects of single factors such as phospholipid concentration,the cholesterol to phospholipid ratio and ultrasonic time on the encapsulation efficiency of Pab-PTX-L were investigated.Then response surface methodology was used to optimize the optimal formulation and process of Pab-PTX-L.2)A series of quality evaluations were carried out on the prepared Pab-PTX-L:the morphological characterization,particle size and Zeta potential of the prepared Pab-PTX-L were determined by transmission electron microscopy and laser nano analyzer,respectively.At the same time,dynamic dialysis method was used to evaluate the release of Pab-PTX-L,Taxol and Lipusu in vitro at 37℃and 100rpm·min^-1.The stability of Pab-PTX-L and Lipusu was studied by supernatant of rat liver homogenate.3)Human lung adenocarcinoma A549 cells and paclitaxel-resistant human lung adenocarcinoma A549/T cells were selected for in vitro evaluation of Pab-PTX-L.The expression of Pgp on A549 cells and A549/T membranes was detected by flow cytometry.The effects of Pab-PTX-L and positive drug Lipusu on the activity of A549 cells and A549/T cells was detected by CCK8kit,and their IC₅₀were calculated.The cell apoptosis was detected by Annexin V-FITC/7-AAD double staining to further confirm the inhibitory effect of Pab-PTX-L.FITC Pab blank long cycle immunoliposome（Pab-FITC-L）and Pab-FITC blank long cycle immunoliposome（FITC-L）2h cell uptake were observed under inverted fluorescence microscope.The tumor suppressive effect of Pab-PTX-L and positive drug Lipusu was investigated by establishing three dimension（3D）cell model of A549/T cell.Result:1)Pab long circulating immune liposome（Pab-PTX-L）was successfully prepared.The results showed that sephadex G-50 microcolumn centrifugation method could separate Paclitaxel from free paclitaxel and the paclitaxel content and encapsulation rate in Pab-PTX-L could be quickly and accurately detected with HPLC.The optimal prescription of response surface screening was 3.1%phospholipid concentration,1:16 the cholesterol to phospholipid ratio,96s ultrasonic time of the probe,and the predicted average encapsulation rate was 89.69%.The encapsulation rate verified by the actual process was87.50%.2)Pab-PTX-L showed spherical shape under transmission electron microscope,and the particle size was less than 200 nm.The average particle size was 172.2nm,the polydispersion coefficient was 0.269,and the Zeta potential was-3.11m V.The results of in vitro release under dynamic dialysis showed that both commercially available paclitaxel injection（Taxol）and paclitaxel liposome（Lipusu）and self prepared Pab-PTX-L had sustained release effects,and the order of sustained release was Pab-PTX-L>Lipusu>Taxol.The stability of Lipusu and Pab-PTX-L by active enzymes in rat liver homogenate supernatant showed that the release rate of Pab-PTX-L and Lipusu was greatly increased while Pab-PTX-L was more stable than the former.3)Pgp expression on cell membrane showed that the average fluorescence intensity of PE in A549 cells and A549/T cells was 77.7±3.2 and 314.7±17.1,respectively.The average fluorescence intensity of PE in A549/T cells was about 4.1 times that of A549 cells,the comparision of which was statistically significant（P<0.05）.Cytoactive assay results showed that there was no significant influence in A549 cell activity between Pab-PTX-L group and Lipusu group 24h after administration,the comparision of which was no significant difference（P>0.05）.48h after A549/T cells administration,the survival rate of A549/T cells in Pab-PTX-L group was significantly decreased compared with that in Lipusu group（P<0.05）.The IC50 of Pab-PTX-L group was 10.20±0.79μg·m L^-1,which was significantly different（P<0.01）from that of Lipusu group(25.34±1.11μg·m L^-1).The result of apoptosis showed that the apoptosis rate of A549 cells in blank group,Lipusu group and Pab-PTX-L group were 1.0±0.4%,15.8±0.7%and 14.8±1.7%,respectively.Compared with Lipusu group,there was no significant difference in apoptosis rate of Pab-PTX-L group（P>0.05）.The apoptosis rate of A549/T cells in blank group,Pab-PTX-L group and Lipusu group were 1.1±0.1%,12.8±2.3%and26.7±1.6%,respectively.The apoptosis rate of A549/T cells in Pab-PTX-L group was significantly higher than that in Lipusu group（P<0.05）,comparison of which were significant difference（P<0.01）.Using FITC-DSPE-PEG₅₀₀₀(300μg·m L^-1)as fluorescence indicator,the fluorescence intensity of A549/T was stronger than that of A549 in Pab-FITC-L group.However,in FITC-L group,there was no significant difference in fluorescence intensity between the two kinds of cells.3D cell model activity of A549/T showed that the IC₅₀of Pab-PTX-L and Lipusu groups were 16.35±0.99μg·m L^-1and83.33±3.93μg·m L^-1,respectively.Compared with the activity results of 2D cell model A549/T,Pab-PTX-L inhibited the growth of A549/T cells more strongly than Lipusu.In conclusion,Pab-PTX-L has a better therapeutic effect on positive drug Lipusu in paclitaxel-resistant lung cancer A549/T cells.Conclusion:In this study,we successfully constructed a Pgp-targeted Pab paclitaxel long circulating immune liposome,which can specifically and actively target the highly expressed Pgp on paclitaxel-resistant lung cancer cells to promote paclitaxel uptake of tumor cells.The drug concentration in tumor cells was increased with greatly improved the anti-tumor effect.Therefore,this study provides a new idea for the treatment of drug-resistant lung cancer.