Nanotechnology-mediated Immunochemotherapy Combined With Docetaxel And PD-L1 Antibody

This study designed a smart pH-sensitive mmunoliposome(PDL)combined with docetaxel(DTX)-mediated chemotherapy and PD-L1-mediated tumor immunotherapy to synergistically exert high anti-tumor effect.Exploring the physicochemical properties,release behavior,in vitro cytotoxicity,anti-tumor effect,safety and the primary mechanism of liposomes.Hence this study can provide theoretical and experimental basis for clinical transformation and further apply tin the clinic.The specific research contents are as follows:(1)Preparation,characterization and release behavior of immunoliposomes co-loaded docetaxel with PD-L1 antibodyThe DTX-LP and PDL were successfully prepared by thin-film hydration and post-insertion method,the antibody and PEG were modified on the surface of the liposome by amide binding.The TEM images showed that the liposomes were round and uniform;the average diameters of DTX-LP and PDL were 113.6±2.991 nm and 126.8±0.651 nm,respectively,and PDI was 0.252±0.005 and 0.232±0.010.After modification,the average diameter increased,while the zeta potential increased from-41.5± 0.306 to-31.5± 0.961,initially indicating that the antibody was successfully linked to the surface of the liposome.SDS-PAGE and co-localization results further confirmed the successful binding of antibodies on the surface of liposomes.The encapsulation efficiency of DTX-LP was 94.32±0.10%,which was determined by HPLC,and the drug loading was about 5.91±0.001%.The entrapment efficiency of PDL was 91.39±1.09%,and the drug loading was 4.49±0.042%.The large negative charge on the surface of the liposome can help itself to be more stable in the solution,and effectively reduce the occurrence of hemolysis,hence has better safety and stability.The in vitro release behavior of liposomes was determined by dynamic membrane dialysis.The results showed that under neutral conditions of pH=7.4,both the cumulative release rate of DTX-LP and PDL were slower than free drug after 72 h,which are approximately 57.99%and 62.41%.They significantly prolonged drug release behavior,ensured the stability of liposomes before reaching the target tissue.However,while in pH 5.0 medium,the drug could be released from liposomes more quickly,which can reach 86.80%and 90.03%after 72 h,respectively.The in vitro release of DTX-LP and PDL has obvious pH sensitivity,and the drug can be released quickly at the targeted site.(2)Evaluation of in vitro cytotoxicity and the targeting ability of immunoliposomesMelanoma B16-F10 cells were selected,the activity and function of each formulation were studied at the cellular level.Since the target was PD-L1 on the surface of tumor cells,the expression of PD-L1 on the surface of B16-F10 cells was first verified by flow cytometry.The blank carrier showed low cytotoxicity and good biocompatibility by CCK-8 method.In the drug group,cell survival rates were time-and concentration-dependent in different groups.The IC50 of free DTX,DTX-LP and PDL at 48 h were 12.46 ?g/mL,6.60?g/mL and 0.93 ?g/mL,respectively.The IC50 of DTX-LP and PDL were approximately 0.52-fold and 0.07-fold compared to free drug,which demonstrated that actively targeted liposomes have higher cytotoxicity Apoptosis experiments also showed that combined delivery has a stronger ability to induce apoptosis than free combination.The apoptosis induction rates of DTX-LP and PDL were 15.74±2.67%and 26.17±3.71%,respectively,which were significantly higher than the 9.26±0.90%of the free DTX.The toxicity of the formulation increased by nanotechnology and active targeting.Then we used immunofluorescence staining and flow cytometry to explore the mechanism and efficiency of liposome,the results showed that PDL uptake was higher than DTX-LP due to the presence of PD-L1 on the surface of B16-F10 cells,so the active targeting ability of PDL helped itself enriched more.Competitive experiments have further verified that their targeting ability is mediated by PD-L1.Therefore,it can be preliminarily concluded that active targeting of liposomes can significantly improve the efficacy of the drug and help the drug to concentrate more on the target site.(3)In vivo anti-tumor effect and active targeting ability of immunoliposomesB16-F10 melanoma cells were inoculated subcutaneously in mice to construct a mouse melanoma model,the anti-tumor effect and targeting ability of different drugs were examined at the animal level.The liposomes(DiR-LP and DiR-PDL)were prepared by using lipid small molecule fluorescent probe DiR instead of DTX,and the real-time biodistribution of free drug or carrier in vivo was monitored by using near-infrared living imaging system.DiR-PDL showed more enrichment in both in vivo and ex vivo imaging of tumor tissues,while tumor tissue distribution was almost undetectable in the free drug group.The accumulation of DiR-PDL and DiR-LP in tumor tissues was 11.98 times and 3.95 times higher than that of free drugs,respectively,indicating that immunoliposomes can effectively target tumor tissues,thereby reducing side effects.Anti-tumor results showed that PDL had the strongest effect,the survival time of mice was significantly prolonged,and no significant weight loss.Tumor tissue sections TUNEL immunofluorescence and Ki-67 immunohistochemical staining were consistent with anti-tumor experiment.The positive rates of PDL group were 58.81 ±14.16%and 65.95± 3.5%,respectively.Therefore,we deduced immunochemotherapy enhances anti-tumor immunity while effectively killing tumor cells to inhibit tumor growth and prolong survival in mice.(4)Safety evaluation and preliminary mechanism of immunoliposomesTo investigate the safety and anti-tumor mechanism of liposome,provide data support and theoretical basis for its clinical transformation,the safety of the drug was indirectly assessed by measuring the levels of liver function indicators ALT and AST and renal function index BUN.The results showed that all the parameters were within the normal range,which indicated that the formulations did not produce significant physical damage to the liver and kidney.Histopathological examination of major organs showed that,except for the free combination,the morphology of each tissue section showed no obvious pathological abnormalities.It is indicated that there is relatively serious systemic toxicity in the free combination,which further validated PDL can reduce side effects.At the same time,preliminary study on anti-tumor immune mechanism by immunofluorescence staining showed that the most abundant TILs and granzyme B in tumor tissues in PDL group,indicating that immunoliposomes can promote the recruitment and toxicity of TILs in vivo.Therefore,it can be inferred that PDL triggers CD8 + T cell-mediated potent tumor cell killing by turning off the PD-1/PD-L1 pathway,and synergistically produces a strong anti-tumor effect with DTX cytotoxicity,with has good safety and application prospects.