Nanovaccine Incorporated With MPG^?NLSor Hydroxychloroquine Enhances Antigens Escape From Lysosome And Promotes Anti-tumor Immunity

Objectives:Immunotherapy that guides the immune system to recognize and eliminate malignant cells has emerged as a promising strategy for cancer treatment.With the development of nanotechnology,nanovaccines have become a promising approach in the cancer immunotherapy toolbox.Compared to traditional vaccines,the nanovaccine delivery systems have unique multifaceted advantages,for example constant release of antigens from nanoparticles at the injected site for a prolonged period,protection of antigens from enzymatic degradation,and facilitating the access of antigens to antigen-presenting cells by modification of nanovaccine carriers with ligands/molecules targeting the DCs or draining lymph nodes.Recently,numerous studies have demonstrated that the clutch of cancer nanovaccine in achieving cross-presentation is antigen spatiotemporal orchestration and antigen escape from lysosome of the antigen-presenting cells.In this study,we chose poly(lactide-co-glycolide)acid nanoparticles(PLGA NPs)as a scaffold and developed two nanovaccines,one is PLGA NPs encapsulated with OVA(ovalbumin,a model antigen)chemically linked with MPG?NLS which was one of cell-penetrating peptides(MPG?NLS-OVA-PLGA NPs),the other was made by physically mixing OVA(a model antigen)and hydroxychloroquine(HCQ)which was an endosomal membrane disrupting agent,and then coencapsulating the mixture into the PLGA NPs(HCQ-OVA-PLGA NPs).Our objectives were to investigate whether the use of MPG?NLS or HCQ facilitates the cytosolic delivery of antigen encapsulated in PLGA NPs and hence promotes MHC-I restricted CD8+ T cell responses and to evaluate these resultant antigen-specific immune responses.Methods:PLGA NPs encapsulated with conjugation MPG?NLS-OVA or mixture of OVA and HCQ were formulated using a double emulsion(W/OW)and solvent evaporation method.The size of nanoparticles was measured using dynamic light scattering(DLS).Zeta potential was detected using Zetasizer Nano ZP by laser Doppler electrophoresis.The particle morphology of nanoparticles was analyzed by transmission electron microscopy(TEM).The loading capacity of OVA in PLGA NPs and the in vitro release behavior of antigen were quantified by measuring OVA concentration using Enhanced BCA Protein Assay Kit.In order to observe whether MPG?NLS or HCQ encapsulated facilitates OVA lysosomal escape into cytoplasm of BMDCs,fluorescein isothiocyanate(FITC)labeled OVA was used to prepare nanoparticles and cells treated with nanoparticles were fixed and imaged with laser scanning confocal microscope.To test whether nano vaccines could activate and help BMDCs mature,membrane MHC-?co-stimulatory molecules on CD11c+ DCs were analyzed using a BD AccuriTM C6 flow cytometer,and concentrations of IL-12(p70)and tumor necrosis factor-a(TNF-a)in culture supernatants were quantified with ELISA kits.In vivo experiments,OVA-specific antibody response was detected using ELISA kits,the proportion of IFN-?-producing CD8+ T cells and T cells memory immune responses were analyzed with BD Accuri C6 flow cytometer.To further investigate the tumor inhibition effects,mice were vaccinated after tumor inoculation,and then tumor progression was monitored by measuring tumor growth over time and mouse survival percentage was calculated.Results:The TEM images show that the nano vaccine features were uniform as spherical particles.Antigen was released from OVA-loaded PLGA nanoparticle in a continuous manner for about 22 days.Images taken with confocal fluorescence miroscopy showed that both HCQ and MPG?NLS facilitate OVA escape from the lysosome into the cytoplasm in BMDCs and suggest that protein antigens delivered by MPG?NLS-OVA-PLGA NPs or HCQ-OVA-PLGA NPs could be processed and presented through both the MHC-? and MHC-? pathways.After immature BMDCs co-incubation with the two nanovaccines,class ? MHC molecules(MHC-?)and co-stimulatory molecules on CD11c+ DCs were upregulated signifncantly,and BMDCs treated with MPG?NLS-OVA-PLGA NPs or HCQ-OVA-PLGA NPs secreted more Thl-type cytokines.Taken together,all our results indicated that both HCQ and MPG?NLS improved antigen cross-presentation and assisted in triggering CTL/Thl-polarizing immune responses in vitro.In vivo studies showed that treatment with MPG?NLS-OVA-PLGA NPs or HCQ-OVA-PLGA NPs remarkably enhanced antigen-specific IgG antibody responses,and induced IgG antibodies featured a higher IgG2a/IgGl ratio when compared to that of other groups.The two nanovacciens significantly stimulated the proliferation of IFN-?+ CD4+ T cells and CD8+ T cells(CTL)in spleens from immunized mice,and be capable of significantly enhancing frequency of memory T cells in splenocytes.Results of in vivo experiments confirmed that both MPG?NLs and HCQ were capable of promoting Thl-type immune responses and CTL.Moreover,in E.G7-OVA tumor mouse models,treatment with MPG?NLS-OVA-PLGA NPs or HCQ-OVA-PLGA NPs did result in remarkable suppression of tumor growth and prolonged mice survival.Conclusion:The two nanovaccines not only significantly facilitated OVA lysosomal escape into the cytoplasm in BMDCs,enhanced expression levels of molecules MHC-I on DC cells and improved cross-presentation of antigen efficiently,but also elicited Thl-type responses and strong CD8+ T-cell responses.Moreover,both MPG?NLS-OVA-PLGA NPs and HCQ-OVA-PLGA NPs could generate effective antigen-specific antitumor memory immune response and have long-term tumor preventive effect.The method to produce nanovaccine reported here can be applied broadly to preparation of many types of nanovaccines using different nanocarriers that are loaded with tumor antigens as well as microbial and viral antigens to prevent and treat tumors and infectious diseases.