Application Of Hyaluronic Acid Nanovesicle Delivery System In Tumor Immunotherapy

Immune escape is one of the ten characteristic phenotypes of tumors.Therefore,tumor immunotherapy,which takes reawakening the active recognition and killing effect of the body’s immune system on tumor cells as the main mode of action,shows great clinical therapeutic prospects and has become the most studied and applied tumor therapy method with the most success at present.However,in clinical application,the problems of short efficacy and low response rate of patients should not be ignored,which may be related to the incomplete activation of the immune system and the heterogeneity of tumors.Based on this,two nano drug delivery systems based on hyaluronic acid(HA)were designed and constructed in this study:1)A noval cell-like biomimetic nano drug delivery system with“core-shell”stuctrure using to tumor combining treatment targeting immune system;2)A noval nano cell-imitated vaccine for personalized immunization of tumor.The following are specific research contents:1)A noval cell-like biomimetic nano drug delivery system with“core-shell”stuctrure using to tumor combining treatment targeting immune systemMore and more studies have shown that chemotherapy and light therapy can activate the adaptive immune response system of the body through the mechanism of immunogenic cell death(ICD).Therefore,the combined application of ICD-related therapy and immunotherapy will help to enhance the effect of tumor immunotherapy.However,how to achieve the synergistic spatiotemporal delivery of different drugs is the main barrier to limit the effectiveness of combination therapy.Based on this,HA was used as drug delivery carrier skeleton,programmed cell death ligand 1(PD-L1)targeting gene silencing plasmid(sh PD-L1),doxorubicin(DOX)and Au nanoparticles(Au NPs)were used as model drugs for combination therapy to build a noval nano drug delivery system with“core-shell”stuctrure(HTCP-Au/sh PD-L1/DOX)for achieving immune system-targeting tumor combination therapy.In this study,we grafted HA with 12-aminododecanoic acid and L-cysteine as bridge chains,and grafted small molecular weight polyethyleneimine to build caccrier HTCP.When loading drugs,Au NPs were loaded by combining Au-S bond with Au and sulfhydryl group of cysteine.PEI could arrest sh PD-L1 by to form the PEI/sh PD-L1 kernel through electrostatic interaction.HA was simultaneously driven by dodecyl chains and PEI and automatically wrapped around PEI/sh PD-L1 to form a shell.The hydrophobic intermediate layer formed by the long alkyl chain between the PEI/DNA core and the HA shell provided the space necessary to accommodate chemotherapy drug DOX.Finally,a noval cell-like biomimetic nano drug delivery system with“core-shell”stuctrure HTCP-Au/sh PD-L1/DOX was prepared.Experimental results showed that the particle size and potential of HTCP-Au/sh PD-L1/DOX was 243.2±32.8 nm and-19.9±0.8 m V,respectively.At808 nm laser radiation,HTCP-Au/sh PD-L1/DOX effectively inhibited 4T1 cells proliferation with an inhibition rate of(89.37±3.64)%,and reduced the expression rate of PD-L1 in cells to(23.18±12.74)%.Subcutaneous 4T1 cells transplanted BALB/c mice were used as animal model,the tumor inhibition rate of combination therapy reached(89.29±2.80)%,PD-L1 gene and protein inhibitory rate in tumor tissues were(62.61±8.02)%and(77.05±0.33)%,respectively.CD8~+T cells infiltration and secretion of immune factor increase.Metastatic tumor nodes in lung tissues decreased.The results showed that the nano drug delivery system can not only realize the efficient loading of the three drugs and the tumor-targeted synergistic space-time delivery,but also have obvious advantages in activating the immune system of the body and effectively inhibiting the proliferation and metastasis of tumors.2)A noval nano cell-imitated vaccine for personalized immunization of tumorThe key problems limiting the development of cancer vaccines are the inability to simultaneously cover a wide range of T cell epitopes,encapsulate sufficient amounts of effective antigens,and achieve efficient delivery of antigens.Moreover,recent studies tend to focus on stimulating parenteral immunity while ignoring the role of mucosal system in peripheral immunity.Based on this,nanosized“imitated tumor cells(ITCs)”contained whole tumor antigen were prepared by membrane filtering method in this study,then nanovesicles were formed by interaction of HTCP and ITCs.Mucosal immune adjuvant all-trans-retinoicacid(RA)was loaded in hydrophobic layer to prepare nano cell-imitated vaccine(HTCP/ITCs/RA),which can reach deep penetration in lymphoid tissue and antigen delivery to dendritic cells by nanoscale size and HA-mediated targeting to dendritic cells.RA can induce dendritic cells to express gut homing receptors CCR9,and migrate to mesenteric lymph nodes and Peyer’s patch.As a result,systemic and mucosal responses were activated.Experimental results showed that the particle size of HTCP/ITCs/RA was 312.4±19.7 nm and the zeta potential was-18.7±1.3 m V.The uptake rate of HTCP/ITCs in mouse bone marrow-derived dendritic cells was 59.3%,much higher than the 26.8%of ITCs alone,and the expression of CD86 and the secretion of interleukin-10 and interleukin-12 were increased.In subcutaneously vaccinated BALB/c mice,4T1 cells were transplanted into other side.Results showed that the tumor inhibition rate reached(80.11±3.85)%,and the immune organ weight rised.Lymph node mobility and maturity of dendritic cells,number of CD8~+T cells in tumor tissues and dendritic cells in Peyer’s patch increased.Anti-tumor activity of spleen lymphocytes rised.Results showed that the nano vaccine could effectively induce the activation of anti-tumor immunity in the whole body by delivering whole tumor antigen to dendritic cells and combining with the application of mucosal adjuvant RA,and inhibit tumor growth.