Study On The Anti-tumor Immune Response Induced By Melittin-lipid Nanoparticle And Its Associated Mechanisms

As a kind of natural host defense peptides(HDP),melittin possesses multiple immunomodulatory effects.However,the applications of immunomodulatory effects of melittin in tumor immunotherapy are limited because of the main hemolysis side effect during vein injection.Meanwhile,subcutaneous injection of the small molecule also leads to rapidly enter blood circulation and be eliminated by kidney,preventing it from accumulating in immune organs(lymph node,liver,etc)and giving full play to its immune adjuvant effect in target organs.Previously,our research group had successfully loaded melittin on high-density lipoprotein-mimicking peptide-phospholipid nanocarrier,and obtained melittin lipid nanoparticle(?-melittin-NP)with good biocompatibility and extended half-life.Thus,we effectively solved the hemolysis side effect of melittin during the transportation process and established the foundation for the study of immunoregulatory effects of melittin in vivo and the application in tumor immunotherapy.In this study,we used two modes of administration to observe the stimulation effect of melittin on the immune system in vivo.We found that after subcutaneous injection,melittin,with the assistance of high-density lipoprotein-mimicking peptide-phospholipid nanocarrier,could be successfully drained to lymph nodes and engulfed by professional APCs in lymph node to activate and reshape the microenvironment.In addition,after intravenous injection,?-melittin-NP rapidly and efficiently targeted the specific nonprofessional APC(LSEC)in the liver,activated the LSEC,and promoted the secretion of cytokines/chemokines related to lymphocyte infiltration and activation,thereby improving the inherent immune tolerance microenvironment in liver and successfully inhibiting liver metastasis of a variety of tumors.The main results are as follows:(1)?-melittin-NP could significantly shield the toxicity of melittin to APCs with the help of high-density lipoprotein-mimicking peptide-phospholipid nanocarrier.By isolating bone marrow-derived dendritic cell(BMDC),bone marrow-derived macrophage(BMDM)and LSEC,we detected the cytotoxicity of ?-melittin-NP to APCs using dynamic imaging and MTS assays.It was shown that incubation with ?-melittin-NP had no obvious effect on the cell viability of the three APCs even the concentration of melittin in nanoparticle increase to 20 ?M,and conversely,treatment with melittin(5 ?M)resulted in more than 70% cell death of BMDC,and 90% of BMDM and LSEC.(2)?-melittin-NP could stimulate the activation of professional and nonprofessional APCs in vitro.Flow cytometry data showed that ?-melittin-NP(5 ?M)up-regulated the expression of co-stimulatory molecules(CD80,CD86)of BMDC and BMDM,and promoted the secretion of IL-12p70.After incubation with LSEC,it also induced the upregulation of CD80,and promoted the secretion of cytokines/chemokines related to lymphocyte infiltration and activation.(3)?-melittin-NP could target to lymph nodes and reshape the lymph node microenvironment for the induction of significant antitumor vaccine effect.?-melittin-NP(~20 nm)reached the inguinal and axillary lymph node within 3 h after subcutaneous injection,and were phagocyted by macrophage and DC.After 24 h,co-stimulatory molecules(CD80,CD86)were highly expressed on macrophage and DC.Fluorescent imaging of tumor tissue sections showed that ?-melittin-NP could induce the release of tumor antigens(t RFP).By establishing a bilateral tumor model,we found that unilateral intratumor injection of ?-melittin-NP could induce 92% distal tumor growth inhibition and 50% tumor regression.(4)?-melittin-NP could efficiently and rapidly target LSECs,stimulate its activation,reverse the immune tolerance in liver,and successfully inhibit liver metastasis of various tumors.Intravital imaging showed that LSECs fluoresced within 1 min after intravenous injection of fluorescent labeled ?-melittin-NPs,and the high-efficiency targeted labeling effect was maintained for up to 12 h.LSEC transcriptome sequencing results showed that the m RNA level of immunoactivation-related molecules significantly increased.Combined with flow cytometry and liquid chip assay,we found that the co-stimulatory molecules(CD80,CD86,MHC-?)were highly expressed on LSEC,and the levels of cytokines(IL-1?)and chemokines(CXCL9,CXCL10,etc)increased in liver along with increased immune cell infiltration and NK cell activation,resulting to effective suppression of the developments of experimental liver metastasis in various tumors(melanoma,breast cancer,colon cancer)and spontaneous liver metastasis in breast cancer.In summary,this study made full use of the excellent characteristics of HDLmimicking peptide phospholipid nanocarrier,and researched the targeting ability and immunomodulatory effects of ?-melittin-NPs on APCs as well as the application in tumor immunotherapy.In case of no load of adjuvant and tumor antigen,subcutaneous injection of ?-melittin-NPs could be rapidly drained to lymph nodes to activate professional APCs in lymph nodes,and induce whole-cell tumor antigen-specific cellular and humoral immune response,leading to the generation of tumor vaccine effect.After intravenous injection,?-melittin-NPs could specifically target the specific nonprofessional APC(LSEC),promote its activation,improve the immune tolerance in liver,and successfully inhibit liver metastasis of a variety of tumors.Therefore,this study provided a new targeting strategy and treatment method for APC-based tumor immunotherapy.