Experimental Study On Anti-tumor Effect Of PLGA Nanoparticle Loaded Human Heparinase Vaccine In Vitro

Objective: To prepare and explore the killing effect of heparinase(Hpa)nano vaccine on tumor.Method: 1.In vitro synthesis of heparinase monopeptide(Hpa),4-branched peptide(MAP)and polylactic acid-glycolic acid copolymer(PLGA)nanoparticles,embedding these two heparinase antigen peptides with PLGA,constructing PLGA-embedded heparinase nano vaccine.2.Human mononuclear cells(PBMCs)were extracted from healthy human peripheral blood by density gradient centrifugation and divided into monocytes and lymphocytes.Mononuclear cells were induced into dendritic cells(DCs)by cytokines GM-CSF(100 ng/ml),IL-4(20 ng/ml)and TNF-?(20 ng/ml)for phagocytosis,The antigen is presented;the lymphocytes are stimulated with nano-vaccine and IL-2 to become effector cells,ie,cytotoxic lymphocytes(CTL),which are used to kill target cells.3.In vitro culture of gastric cancer cell lines SGC-7901(Hpa+,HLA-A2-),KATO-III(Hpa+,HLA-A2+),breast cancer cell lines MCF-7(Hpa-,HLA-A2+),BT-549(Hpa+,HLA-A2+),autologous lymphocytes and DCs were used as target cells,and the content of heparanase in the four tumor cells was detected by Western blot.4.To study whether the nano-vaccine can successfully stimulate lymphocytes to become CTL,co-culture the nano-vaccine with lymphocytes,add IL-2 to promote T cell activation,and detect ? by enzyme-linked immunosorbent assay(ELISA)after co-stimulation-Interferon-?(IFN-?)release.5.To investigate whether the CTL induced by the nano vaccine has a wide range of heparinase and HLA-A2 double positive tumor cells,and co-culture with CTL as an effector cell,KATO-III and BT-549 as target cells,and lactic acid Dehydrogenase(LDH)release assay detects the killing effect of CTL on the above two target cells.In order to study whether the CTL induced by the nano-vaccine has Hpa specificity,CTL is used as the effector cell and MCF-7 is used as the target cell for co-culture,and the killing effect of CTL on the target cell is detected by LDH release method.7.To investigate whether the CTL induced by the nano-vaccine has HLAA2 specificity,CTL was used as the effector cell and SGC-7901 as the target cell for co-culture,and the killing effect of CTL on the target cell was detected by LDH release method.In order to study the safety of the nano-vaccine,CTL was used as the target cell,autologous DC and lymphocyte as the target cells for co-culture,and the killing effect of CTL on the two target cells was detected by LDH release method.9.The experimental data were expressed as ?xes,and the results were analyzed by SPSS 19.P<0.05 was statistically significant.Results: 1.The heparinase monopeptide Hpa(KMLKSFLKA)and the 4-branched peptide synthesized by the single peptide were successfully synthesized,and their purity was 97.91% and 84.37%,respectively.2.Successfully synthesized PLGA nanoparticles with a diameter of 105.5 nm,and used them to embed Hpa and MAP.The particle sizes after embedding were 111.3 nm and 121.2 nm,respectively.3.Western blot was used to detect heparinase levels in four tumor cells.The results showed that heparinase was positive in SGC-7901,KATO-III and BT-549 cells,while heparinase was negative in MCF-7 cells..4.Successfully isolated monocytes from peripheral blood and induced them into mature DCs.Flow cytometry analysis of DC phenotypic results showed that CD1?,CD80,CD83 and HLA-A2 all met the mature DC phenotype,and the positive rate They were 85.4%,88.7%,90.1%,and 93.2%,respectively.5.We used ELISA to detect the amount of FN-? secreted by different nano-vaccine-induced effector cells.The results showed that the amount of FN-? produced by PLGA nano-vaccineembedded MAP vaccine(PLGA-MAP)and PLGA-Hpa nano-vaccine was significantly higher.In the MAP group,Hpa group,PLGA group and blank control group.6.Effector cells have significant killing effect on hepatinase and HLA-A2 double positive KATO-III and BT-549 tumor cells;positive for heparinase and HLA-A2 positive MCF-7 and heparanase positive HLA-A2-negative SGC-7901 cells had no killing effect.7.The effector cells have no killing effect on the lymphocytes and DCs of the daughter itself.Conclusion: 1.Successful preparation of PLGA-MAP heparinase nano vaccine of about 100 nm.2.PLGA-MAP nano vaccine can stimulate effector cells to produce IFN-? non-specific killing of tumor cells.3.PLGA-MAP nano vaccine-induced effector cells have a good killing effect on malignant tumors,which are heparinase specific and restricted by HLAA2.4.PLGA-MAP nano vaccine-induced effector cells have no killing effect on autologous lymphocytes and dendritic cells,and their safety is good.5.PLGA-MAP nano-vaccine has a killing effect on both HLA-A2 and Hpa double positive KATO-III gastric cancer cells and BT-549 breast cancer cells,and it has a wide range of anti-tumor characteristics.