Spatio-temporal delivery of both intra-and extracellular toll-like receptor agonists for enhancing antigen-specific immune responses

For cancer immunotherapy,triggering toll-like receptors(TLRs)in dendritic cells(DCs)can potentiate antigen-based immune responses.Nevertheless,to generate robust and long-lived immune responses,a well-designed nanovaccine should consider different locations of TLRs on DCs and co-deliver both antigens and TLR agonist combinations to synergistically induce optimal antitumor immunity.Herein,we fabricated lipid-polymer hybrid nanoparticles(LPNPs)to spatio-temporally deliver model antigen ovalbumin(OVA)on the surface of the lipid layer,TLR4 agonist monophosphoryl lipid A(MPLA)within the lipid layer,and TLR7 agonist imiquimod(IMQ)in the polymer core to synergistically activate DCs by both extra-and intra-cellular TLRs for enhancing adaptive immune responses.LPNPs-based nanovaccines exhibited a narrow size distribution at the mean diameter of 133.23 nm and zeta potential of−2.36 mV,showed a high OVA loading(around 70.83μg/mg)and IMQ encapsulation efficiency(88.04%).Our data revealed that LPNPs-based nanovaccines showed great biocompatibility to immune cells and an excellent ability to enhance antigen internalization,thereby promoting DCs maturation and cytokines production.Compared to Free OVA,OVA-LPNPs promoted antigen uptake,lysosome escape,depot effect and migration to secondary lymphatic organs.In vivo immunization showed that IMQ-MPLA-OVA-LPNPs with dual agonists induced more powerful cellular and humoral immune responses.Moreover,prophylactic vaccination by IMQ-MPLA-OVA-LPNPs effectively suppressed tumor growth and increased survival efficacy.Hence,the nanovaccines we fabricated can effectively co-deliver antigens and different TLR agonists and realize coordinated stimulation of DCs in a spatio-temporal manner for enhanced immune responses,which provides a promising strategy for cancer immunotherapy.