Dendritic Cell Targeted Glycolipid-like Micelles As Nanovaccines For Cancer Immunotherapy

Cancer immunotherapy has been regarded as an effective way to treat malignant tumors because of its unique advantages.Cancer nanovaccines,as one of the most promising treatments in cancer immunotherapy,can induce antigen-specific immune responses to inhibit and kill cancer cells by activating antigen presenting cells(APCs)with antigens.However,various nanovaccines fail to induce potent antitumor immune responses due to insufficient major histocompatibility complex I(MHC I)-restricted antigen presentation.In this study,we focus on developing novel glycolipid-like nanovaccines to target dendritic cells(DCs)for enhanced MHC I-restricted antigen presentation,so as to improve the efficacy of cancer immunotherapy.Chitosan(Ct)can graft with stearic acid(SA)by amide reaction,which resulted in glycolipid-like stearic acid-grafted chitosan(CtSA).DCs are the strongest antigen presenting cells in APCs and highly express mannose receptors.Thus,mannose was chosen to modify CtSA to obtain mannose modified stearic acid-grafted chitosan(MCtSA).The in vitro uptake of MCtSA by DCs was stronger than that of CtSA,which indicated that mannose modification could promote the cellular uptake of MCtSA by DCs.There are a lot of DCs in skin tissue.The low express of chemokine receptor type7(CCR7)results in deficient DC migration to lymph nodes,which restricted the antitumor immune responses of nanovaccines by leading to insufficient MHC I-restricted antigen presentation.MCtSA micelles were used as targeted co-delivery vector of antigen ovalbumin(OVA)and plasmid DNA encoding CCR7(CCR7 pDNA)to prepare MCtSA/OVA/pDNA glycolipid-like nanovaccines.MCtSA/OVA/pDNA targeted skin DCs,promoted DC migration to lymph nodes by improving the expression of CCR7,and achieved the lysosomal escape of OVA by protonation effect,thus boosting MHC I-restricted antigen presentation and enhancing antitumor efficiency.The size of MCtSA/OVA/pDNA was 184.3±3.21 nm and the zeta potential was9.14±0.35 mV.OVA and pDNA achieved lysosomal escape.MCtSA/OVA/pDNA improved CD40 and CD86 molecule expression on DCs,and stimulated the secretion of tumor necrosis factor-?(TNF-?)and interleukin-6(IL-6)cytokines,thus promoting the activation and maturation of DCs.Besides,MCtSA/OVA/pDNA showed no obvious cytotoxicity against DCs.In addition,MCtSA/OVA/pDNA obviously enhanced the expression of CCR7 and RAC1 so as to enhance the migration capacity of DCs.MCtSA/OVA/pDNA showed obvious antigen depot effect,could target skin DCs and promote skin DC migration to lymph nodes.MCtSA/OVA/pDNA dramatically inhibited tumor growth by inducing potent CD8~+T cell immune responses.Compared with OVA,MCtSA/OVA/pDNA apparently increased the inhibition rate of tumor growth.Lymph nodes contain a large number of DCs and have been proposed as an intriguing target in cancer targeted immunotherapy.Among which,tumor-draining lymph node(TDLN)has already bathed in tumor antigens.Targeted delivery of adjuvants to TDLN could induce personalized immunotherapy.MCtSA,as the immune adjuvants and antigen-capturing agents,targeted TDLN and captured endogenous antigens in TDLN,which enhaced the cellular uptake of antigens by DCs and promoted antigen lysosomal escape,thus improving the antitumor efficacy by promoting MHC I-restricted antigen presentation.The size of MCtSA was 94.2±0.60 nm and the zeta potential was 11.8±1.25 mV.After mixing with antigens,the size of MCtSA+OVA was 126.0±3.46 nm and the zeta potential was 6.75±0.40 mV.Compared with MCtSA,the size of MCtSA+OVA significantly increased and the zeta potential decreased,indicating the capture of OVA by MCtSA.Furthermore,the result of quantitative analysis showed that the amount of proteins captured by MCtSA was 308.6?g/mg,and MCtSA could enhance antigen uptake by DCs,which indicated the strong antigen-capturing ability of MCtSA.MCtSA significantly up-regulated CD40 and CD86 expression and increased the secretion of TNF-?and IL-6,which demonstrated the strong adjuvant activity of MCtSA.Besides,the result of cytotoxic evaluation indicated the safety of MCtSA.MCtSA showed strong TDLN targeting ability and could be taken up by DCs in TDLN,thus promoting the activation and maturation of DCs.In addition,MCtSA induced potent CD4~+T and CD8~+T cell immune responses,and stimulated the secretion of Interferon-?(IFN-?),TNF-?,Interleukin-12(IL-12)and IL-6.MCtSA obviously inhibited the tumor growth and the inhibition rate was 71.5%.Directly delivering antigens to lymph node DCs is being considered as an important strategy for immunotherapy with nanovaccines.Nanoparticles can target lymph nodes by tailoring the size and zeta potential so as to target lymph node DCs.Histidine modified stearic acid-grafted chitosan(HCtSA),as the antigen delivery vectors,was further enveloped with dendritic cell membrane(DCM)to prepare DCM/HCtSA/OVA glycolipid-like nanovaccines.The envelop of DCM can improve lymph node targeting of DCM/HCtSA/OVA nanovaccines so as to further enhace the antigen uptake by DCs in lymph nodes,and the modification of histidine can promote the lysosomal escape of antigens,which results in enhanced MHC I-restricted antigen presentation so as to induce potent antitumor immune responses.The size of DCM/HCtSA/OVA was 149.9±7.85 nm and the zeta potential was-5.63±0.31 mV.PBS solutions with pH 7.4 and pH 5.0 were used as the release medium,and the cumulative release efficiency of DCM/HCtSA/OVA within 24 h were 52.9%and 76.4%respectively,which indicated the pH responsive release of OVA.DCM/HCtSA/OVA promoted antigen uptake by DCs and antigen lysosomal escape.In addition,the expression of CD40 and CD86 and the secretion of TNF-?and IL-6 were enhanced by DCM/HCtSA/OVA.DCM/HCtSA/OVA targeted lymph nodes and promoted antigen uptake by DCs in lymph nodes,resulting in the activation and maturation of DCs.Besides,DCM/HCtSA/OVA induced potent CD4~+T and CD8~+T cell immune responses,and stimulated the secretion of IFN-?,TNF-?,IL-12 and IL-6.The tumor growth inhibition rate of DCM/HCtSA/OVA was 85.8%and was significantly higher than that of OVA which was only 39.5%,indicating that DCM/HCtSA/OVA could dramatically inhibit tumor growth.