Construction Of GA-HP-CpG ODN Nanoparticles And Its Synergistic Effect On Antitumor Immune Activation

Objective:In this study,the natural antitumor drug Gambogic Acid(GA),vascular inhibitor Heparin(HP)and synthetic immunostimulant CpG Oligonucleotide(CpG ODN)were used to prepare the PLGA core-shell nanosystem(GA-HP-CpG ODN)by the simple double emulsion solvent evaporation.The optimal preparation process of PLGA core-shell nanosystem was determined.GA-HP-CpG ODN nanosystem was characterized and its release behavior under acidic conditions in vitro was investigated.The antitumor effect of GA-HP-CpG ODN on human liver cancer cell line Hep G2 was studied in vitro.A model of H22 orthotopic liver cancer in mice was established to investigate the anti-tumor effect of GA-HP-CpG ODN in vivo.Methods:(1)Preparation and characterization of GA-HP-CpG ODN:A blank PLGA nanoparticle was prepared by a double emulsion solvent evaporation method.The particle size was used as the detection index,and the optimal preparation process was established through single-factor investigation.Agarose gel electrophoresis experiments and differential scanning calorimetry(DSC)were used to detect the CpG ODN,GA,and HP encapsulation;Malvern laser scattering particle size analyzer was used to detect the particle size and potential of GA-HP-CpG ODN;The transmission electron microscopy was used to observe the internal structure of GA-HP-CpG ODN;establish the ultraviolet(UV)or fluorescent content detection and analysis methods of GA,HP,and CpG ODN,and determine the encapsulation efficiency(EE%)and drug loading(DL%)of the three drugs in GA-HP-CpG ODN;The in vitro release behavior of GA-HP-CpG ODN was detected by sampling and separation method.(2)Anti-tumor activity of GA-HP-CpG ODN in vitro:Hep G2 cells were used as the research object,and the cytotoxicity of GA-HP-CpG ODN was observed by MTT method.Fluorescence microscopy was used to observe the uptake behavior of Flu-GA-HP-CpG ODN by Hep G2 cells.Laser confocal microscopy was used to observe the TLR-9 targeting effect of AFM-GA-HP-CpG ODN in RAW264.7 cells.(3)Anti-tumor activity of GA-HP-CpG ODN in vivo:H22 orthotopic liver cancer model were established and administered in groups.Histomorphological observation,body weight change,survival time and survival rate analysis,tissue section immunohistochemical staining,immunofluorescence staining,and ELISA kit tests were performed to evaluate the tumor suppressive activity and immune activation of each administration group.Results:(1)Preparation and characterization of GA-HP-CpG ODN:Optimal prescription was obtained through single-factor investigation,and the internal phase water-oil ratio was determined to be 1:3,the colostrum/re-emulsion volume ratio was1:2.5,the PLGA concentration was 3%,and the PVA concentration was 0.5%.Agarose gel electrophoresis and DSC spectra showed that CpG ODN,GA,and HP were successfully loaded.Transmission electron microscopy results showed that GA-HP-CpG ODN has a double-layered core-shell structure.The Malvern particle size analyzer showed that the average particle size of GA-HP-CpG ODN was(208±15.9)nm and the potential was(-2.00±1.21)mV.The EE%of GA,HP,and CpG ODN in GA-HP-CpG ODN were(92.33±2.22)%,(90.83±3.49)%,and(33.19±3.05)%,respectively.The DL%of GA,HP,and CpG ODN were(10.57±5.15)%,(12.37±5.44)%and(1.02±0.48)%,respectively.Meanwhile,GA-HP-CpG ODN have slow in vitro release behavior.(2)Anti-tumor activity of GA-HP-CpG ODN in vitro:The results of MTT experiments showed that both free GA and GA-HP-CpG ODN had cytotoxic effects on Hep G2 cells.In the first 24 h,the cytotoxicity of free GA was stronger than that of GA-HP-CpG ODN(P<0.01).As time went on,GA was gradually released from GA-HP-CpG ODN,and the cytotoxic effect of GA-HP-CpG ODN gradually tended to free GA at 48 h and72 h(P>0.05).In addition,GA-HP-CpG ODN can be efficiently taken in Hep G2 cells,and has a targeting TLR-9 in RAW264.7 cells.(3)Anti-tumor activity of GA-HP-CpG ODN in vivo:Frozen section images of H22liver cancer model mice showed that GA-HP-CpG ODN is mainly targeted to the liver tissues.The results of antitumor studies showed that the GA-HP-CpG ODN group significantly inhibited tumor growth and prolonged the survival time of liver cancer mice.Compared with the other three groups,the GA-HP-CpG ODN group significantly inhibited neovascularization at the tumor site,inhibited tumor cell proliferation,and promoted tumor cell apoptosis.In addition,the GA-HP-CpG ODN group also significantly up-regulated CD4~+and CD8~+T cells,promoted the secretion of IFN-γ,IL-12 and TNF-α,and had an immune activating effect.In terms of safety,GA-HP-CpG ODN shows good biocompatibility.Conclusion:This project successfully constructed a novel multifunctional“core-shell”nano platform,which realized the common encapsulation of GA,HP and CpG ODN.Where GA is used as the outer layer of the carrier,it is first released at the target site to directly kill tumor cells.HP and CpG ODN are inside the carrier and subsequently released to provide anti-angiogenic and immunomodulatory effects.The released CpG ODN can effectively up-regulate CD4~+and CD8~+T cells,induce CD4 cells to differentiate into Th1 cells,and up-regulate Th1 anti-tumor immune responses.This strategy effectively combines anti-angiogenic therapy,chemotherapy and immunotherapy to significantly extend the survival time of hepatocellular carcinoma mice.