Biomembrane Hydrogel For Prophylactic Tumor Vaccine

Tumor vaccines are designed to prevent the occurrence and development of tumor or treat an existed tumor by activating immune system,which has the potential to reduce the incidence of tumors and improve prognosis.Therefore,tumor vaccines have great research value and broad clinical application prospect.Immunizing healthy individuals with prophylactic tumor vaccines is an efficient and cost-effective strategy to reduce tumor occurrence and thus alleviate the heavy economic burden of tumor treatment.However,the immune activation effect of prophylactic tumor vaccines needs to be improved.Three kinds of signals,including antigenic signal,co-stimulatory signal and cytokine signal,are required for all-sided activation of cytotoxic T cells(CTL).At the same time,adequate antigen exposure is significant for stimulating immune system and establishing immune memory.Vaccines are often quickly cleared after injection,and individuals usually need to receive multiple injections to ensure establishing immune memory.The resulting pain and poor compliance are also serious problems in vaccine use.Based on the above,a biomembrane-based hydrogel,E64@O-MHCM,was designed as tumor vaccine and expected to achieve effective immune activation and prolonged in vivo retention,so as to reduce the number of vaccine injections and improve the immune effect of tumor vaccine.The gel factor(O-MHCM)was prepared by linking mannose-modified hybrid biomembrane(MHCM)with sodium alginate oxide(OSA)via Schiff base bond.The hybrid biomembrane(HCM)was obtained by fusion of tumor cell membrane(TCM)with bacterial outer membrane vesicle(OMV),and subsequently modified by DSPE-PEG-mannose.The protease inhibitor E64 was loaded into hydrophilic chamber of the hydrogel.As the antigen source,TCM retained all surface antigens of tumor cells and could stimulate immune system to produce a variety of tumor-specific CTL after being presented by dendritic cells(DC).OMV contained a variety of pathogen-related molecular patterns(PAMP)derived from the parent bacteria,which could bind to the pattern recognition receptors(PRR)on DC surface.Therefore,OMV had strong immunogenicity and could promote the maturation of DC as well as the secretion of cytokines,and was an effective adjuvant.The protease inhibitor E64 acted on DC lysosomes to inhibit the degradation of tumor antigens,thus improving the efficiency of antigen cross-presentation when used in combination with tumor antigens.Mannose modification was designed to bind to the mannose receptor on DC surface for better targeting.The synergistic use of these components comprehensively enhanced the antigenic signal,costimulatory signal and cytokine signals transmitted to naive T cells.Preparing gel factors by reacting MHCM with OSA could achieve continuous release of tumor antigens and adjuvants with hydrogel degradation,which finally provided stable and sufficient antigen stimulation after a single subcutaneous immunization,avoided the rapid elimination of vaccine and reduced the number of vaccine administration.The research content of this project is as follows:(1)Firstly,TCM was extracted from mouse tumor tissue by probe ultrasonic crushing and multiple centrifugation,and OMV was extracted from the culture supernatant of E.coli DH5α by ultrafast centrifugation as previously reported.TCM and OMV were mixed with ultrasonic and co-extrusion to prepare HCM,and then MHCM was prepared by stirring DSPE-PEG-mannose with HCM;OSA and MHCM were stirred in dark to prepare O-MHCM.Finally,tumor vaccine E64@O-MHCM was obtained by mixing O-MHCM solution with E64 solution.Confocal experiments confirmed the successful fusion of the two membranes.The results of dynamic light scattering showed that the average particle sizes of HCM,MHCM and O-MHCM were 183.3 nm,210.6 nm and 310.6 nm,respectively.The gel formation test demonstrated that E64@O-MHCM solution could be changed into gel after adding CaCl2.The microstructure of E64C@O-MHCM hydrogel was observed by SEM and proved that it had a network porous structure.The needle-through test showed that E64@O-MHCM solution had certain fluidity and could be used for injection.In vitro release results supported that E64@O-MHCM hydrogel could achieve sustained release of antigens and adjuvants.The results of subcutaneous degradation demonstrated the hydrogel was formed after subcutaneous injection and degraded slowly in vivo within 16 days.(2)The cytotoxicity of E64@O-MHCM to mouse fibroblasts L929 was tested via CCK-8 method,and the results showed that this tumor vaccine was safe to normal cells.Bone marrow-derived dendritic cells(BMDC)were co-incubated with E64@O-MHCM and detected by flow cytometry to explore the immune stimulation effect of the preparation in vitro.The mature markers CD80 and CD86 on BMDC surface were determined and the results showed that E64@O-MHCM had the ability to promote DC maturation.The proportion of SIINFEKL-H-2Kb+CD11c+cells was determined,confirming that E64@OMHCM could effectively improve the cross-presentation efficiency of DC.ELISA assay showed that the preparation could promote the secretion of IL-6,IL-12 and TNF-α of DC.Moreover,T cell proliferation experiments demonstrated that E64@O-MHCM significantly enhanced the activation and proliferation of T cells by acting on DC.(3)Subsequently,the retention effect,immune effect,tumor prevention effect,tumor treatment effect and biological safety of E64@O-MHCM were determined in mice.In vivo retention test confirmed that E64@O-MHCM hydrogel could remain in vivo for a long time after injection.After a single subcutaneous injection of E64@O-MHCM in healthy mice,DC and T cells in lymph nodes and spleen were determined by flow cytometry.The results showed that the proportion of DC maturation and the efficiency of antigen cross-presentation in lymph nodes were both increased,and the number of activated T cells in lymph nodes and spleen were also increased after vaccination.Healthy mice were injected with E64@OMHCM for a single time,and were attacked with 4T1 tumor cells 21 days later to observe the tumor growth.The results showed that preimmunization with E64@O-MHCM could significantly slow the growth of tumors,confirming that the vaccine had an excellent preventive effect.After the prevention test,tumor infiltrating CTL and spleen CTL were detected by flow cytometry,and the levels of them in vaccine group were both increased compared with normal saline(NS)group,indicating that mice immunized with E64@OMHCM were able to rapidly produce large amounts of CTL when attacked by tumor cells.Serum TNF-α and IFN-y levels were also increased in E64@O-MHCM group.Subsequently,the tumor treatment experiment was carried out.After 4T1 breast cancer model was established in mice,treatment with E64@O-MHCM showed a reduction in tumor growth compared with NS group.The body weight of mice did not decrease significantly within three weeks after injection with E64@O-MHCM.After 3 weeks,serum creatinine(CREA)and aspartate aminotransferase(AST)levels were determined and there was no significant difference between the E64@O-MHCM group and the NS group.H&E(hematoxylin-eosin)staining showed no significant toxicity in major tissues and organs of mice.All above indicated that E64@O-MHCM hydrogel had good biosafety.In conclusion,this project designed a biomembrane hydrogel as a preventive tumor vaccine,which comprehensively enhanced the three signals required for naive T cells activation and realized the enhanced in vivo retention of vaccine,solving the problem of rapid elimination and multiple injection of vaccines and showing a favourable effect of activating cellular immunity and preventing tumor in vivo.This project provides an innovative way for tumor vaccine design.