Sequential Targeting Cancer Immunotherapy By Macrophage Membranes Coated Glycolipid-like Drug Delivery System

The action sites of PD-L1 antibody and immune adjuvant CpG are located on the surface of tumor cells and in dendritic cells,respectively.Existing combination therapy of tumor immunity lacks clear selectivity for the site of drug action,affecting the efficacy of combined applications.It is an urgent problem to realize sequential targeting and drug release based on different targets sites of cancer immunotherapy.In this study,biodegradable chitosan was chosen as the basic skeleton,and hydrophobic stearic acid was grafted to construct a cationic oligosaccharide stearic acid polymer(CSO-SA).CSO-SA was further modified with mannose(M-CSO-SA),M-CSO-SA could self-assemble into micelle in aqueous water.As a chitosan-based cationic polymer,M-CSO-SA could condense negatively charged immunoadjuvant CpG-ODN by ionic interactions to construct M-CSO-SA/CpG drug delivery system.The macrophage membrane was modified with anti-PD-L1 peptides(PPA)to form Mem-PPA.Mem-PPA coated onto the drug delivery system to construct M-CSO-SA/CpG@Mem-PPA.It is mediated by the binding of CCR2 on the surface of macrophages membrane to the CCL2 receptor ligand at the tumor site,and could be actively distributed to the breast cancer site of model animals.Upon fulfilling its mission of tumor homing,the macrophage-membrane coating could be shed via morphological changes driven by extracellular microenvironment stimuli and the buffering capacity of M-CSO-SA,and the core M-CSO-SA/CpG continued to capture antigen in the tumor microenvironment,and then CpG and tumor-associated antigen could be delivered to dendritic cells,significantly increasing the expression of CD86 and CD80 in dendritic cells.The M-CSO-SA/CpG@Mem-PPA particle size was 100.4±6.22 nm in PBS(p H7.4),the Zeta potential was 6.85±0.72 mV in aqueous.When the p H was lowered to 6.5,the particle size of M-CSO-SA/CpG@Mem-PPA was increased to 865.6±97.3 nm,and a double peak appeared.TEM showed the outer bilayer structure of M-CSO-SA/CpG@Mem-PPA was disappeared.Gel retardation analysises showed when the N/P ratio is 8:1,the micelles can effectively encapsulate CpG.The drug loading of the M-CSO-SA/CpG was 7.38±2.3% and the encapsulation efficiency was 95.74±3.9% measured by UV spectrophotometer.DC 2.4 cell-based competitive uptake experiments showed that M-CSO-SA was taken up by DC2.4 cells via the mannose receptor pathway,the uptake of M-CSO-SA was significant increased due to mannose expressing DC2.4 cells.CLSM observed that M-CSO-SA/CpG after detachment from the cell membrane could capture free antigen and deliver it to dendritic cells.The antigen capture of M-CSO-SA has a synergistic effect with CpG-induced maturation of DCs,which can significantly increase the expression of CD86 and CD80 on the surface of DCs.Establishing 4T1 tumor-bearing BALB/c mice were used as animals models,macrophage membrane coating was used to improve the distribution of grafted micelles in situ breast cancer,and to achieve targeting of breast cancer in situ.Western Blot confirmed that the targeting of macrophages to tumor sites is mediated by CCR2 on the surface of macrophage membranes.The tumor inhibition rate of M-CSO-SA/CpG@Mem-PPA was raised to 82.3%,compared to PPA+CpG group(48.9%).Compared with the PPA+CpG group,the CD80CD86 positive ratio of DCs in the tumor site increased from 34.0% to 49.9%,The expression of MHC-I increased from 10.9% to 12.1%,and the CD8~+/IFN-γand CD4~+/IL-4 double positive ratios at the tumor site increased from 0.75% and 0.66% to 1.33% and 1.05%,respectively.The results showed that M-CSO-SA/CpG@Mem-PPA can strongly activate anti-tumor immune responses in vivo,and this study can provide new strategies for further anti-tumor immunotherapy.