Research On The Application Of Synthetic High-Density Lipoprotein Nano Drug Delivery System In The Immunotherapy Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC),the third leading cause of cancer-associated deaths in the worldwide.So,how to prolong the progression-free survival(PFS)of HCC patients has become a important problem needed to be solved.Especially for the patients that are diagnosed in the middle and advanced stages which is cannot be removed by surgery.For middle-stage patients,transcatheter arterial chemoembolization(TACE)is a common treatment,but this method can only make the patient's median overall survival extended to about 26 months.For patients with advanced liver cancer,chemotherapy with sorafenib can only make the median overall survival to about 5 months.For sorafenib progressors,anti-PD-1(programmed cell death protein-1)antibodies could prolong PFS,but the improvement is not statistically significant in the Phase III clinical trials(Keynote-240),this may be related to the abundant M2 macrophages in the liver tumor immune microenvironment.Moreover,in order to achieve the maximal efficacy therapeutic effect,repeated administration are often needed during the treatment process,it may also increase the toxicity of the drug to non-targeted normal organs,resulting in side effects of the drug on the body during the treatment process.Recently,some studies have shown that treated with PD-1 antibodies can prolong the PFS of HCC patients,which also provide a new strategy of HCC treatment by activating HCC patients'innate immunity against tumor cells.However,for patients in advanced-stage,the response rate of PD-1 antibody immunotherapy is very low(<20%)because of the intratumoral heterogeneity of HCC tumors.Which also leads to the rarely detection of tumor antigen-specific CD8~+T cells in the peripheral blood of patients.For HCC early-stage patients whose tumor can be surgically removed to obtain tumor-associated antigens,vaccination can achieve a good therapeutic effect on HCC.Therefore,in the process of clinical immunotherapy for unresectable intermediate and advanced HCC,how to overcome the heterogeneity and change the immunosuppressive microenvironment of the tumor therefore to achieve long-term disease control has become a critical problem in the immunotherapy of hepatocellular carcinoma.At present,for unresectable advanced-stage HCC tumors,an alternative immunotherapy is to induce immunogenic cell death(ICD)of tumor cells at the tumor site by local injection of vaccines,thereby achieving the release of tumor-associated antigens,then priming the body's specific immunity.ICD induction can be achieved through traditional radiotherapy and chemotherapy.However,due to the lack of liver function,traditional radiotherapy and highly toxic chemotherapy are not suitable for the treatment of HCC patient due to their side effects.Therefore,the clinical treatment of hepatocellular carcinoma usually uses thermal ablation.This also suggests the possibility to use photothermal therapy(PTT)that has recently been proven can induce ICD to promote HCC immunotherapy.Simultaneously.PTT can kill heat-sensitive tumor cells,weaken the tumor barrier,and promote drug penetration,which have become its beneficial properties for stimulating tumor-associated immunity.In addition,the enrichment of M2 macrophages in HCC has also become an important reason for the cause of poor prognosis and low survival rate.Therefore,reversing the immunosuppressive microenvironment within the tumor has also become a compulsory course for HCC immunotherapy.It can also become another solution for the poor effect of immunotherapy for HCC.In this paper,a biomimetic synthetic high-density lipoprotein(sHDL)was synthesized by hydration and membrane formation method,sHDL can specifically transport the photosensitizer DiR and other hydrophobic drugs to the cytoplasm of HCC cells through the SR-B1 receptor on the cell surface.At the same time,sHDL has a similar structure and long circulation characteristics with natural HDL,which can make sHDL achieve a good therapeutic effect with only one single injection.Besides,by properly choose the drugs encapsulated in sHDL,tumor immunotherapy and tumor immune microenvironment(TIME)regulation can be fully realized.The research in this thesis mainly includes the following research contents:1.Preparation and characterization of sHDLs.In order to encapsulate hydrophilic drug in sHDL,the hydrophobic prodrug of vadimezan(DMXAA),vadimezan-cholesterol ester(VC),was first synthesized by esterification reaction,and identified by mass spectrometry(ESI-LR).The molecular weight of the prodrug was determined,and the purity of the prodrug(>90%)was determined by high performance liquid chromatography(HPLC).Then the empty and drug-loaded sHDL was prepared by the method of hydration and film formation,and the morphology and particle size of the sHDL were measured by transmission electron microscope,and the zeta potential was measured.In addition,the cellular uptake of sHDL in vitro and the biodistribution in vivo were both investgated.The results showed that on the basis of the successful synthesis of VC,sHDL was successfully synthesized by the method of hydration and film formation.Its particle size,morphology,zeta potential,and properties of long circulation in the body are similar with natural HDL.Therefore,sHDL can be used as an ideal,tunable nanocarrier for tumor immunotherapy.2.PTT-based cancer Immunotherapy induces ICD and activate tumor-specific immunity.The photosensitizer DiR and the microtubule stabilizer mertansine(DM1)are co-loaded to prepare mertansine-DiR-sHDL(MR-sHDL),mouse stimulator of interferon genes(STING))vadimezan derived VC was co-loading with DiR to prepared VC-DiR-sHDL(VR-sHDL).The biological effects and mechanism of MR-sHDL and VR-sHDL were studied in vivo and in vitro.The results showed that both sHDL can induce ICD and activate the body's immune system in Hepa1-6 tumor bearing C57BL/6 mice.However,the effect of MR-sHDL is weaker than VR-sHDL.And this has been proved that probably because of the high toxicity of chemotherapeutic drug DM1 to immune cells,which then suppress the immune reaction.This means that compared with mertansine,the lower cytotoxic vadimezan is more suitable in the use of combination with the photosensitizer DiR,and it also provides ideas for our follow-up prescription drug selection.3.sHDL promotes the formation of immune memory and extends PFS of tumor-bearing mice when combined with PD-1 antibodies.Compared the effects of sHDLs on PFS in tumor-bearing mice,and introduce PD-1 antibody to enhance the immune effect.The effects of two sHDLs on the aspect of immune memory formation were evaluated.The results showd that both prescriptions can appropriately prolong PFS in mice.After combined use with PD-1antibody,the effect of prolonging PFS is more obvious,and the two kinds of sHDL have different degrees of positive effects on promoting immune memory.4.Research on the interaction between sHDLs and cells in the tumor microenvironment.DiD-encapsulated sHDL(D-sHDL)and DiD-encapsulated PEG-PLGA nanoparticles(D-PEG-PLGA)were used as two model nanocarriers to explore the effect of sHDL on Hepa1-6 through the SR-B1 receptor-mediated pathway.The difference in the affinity of each cell in the tumor microenvironment.The results showd that sHDL has the strongest affinity for M2 and Hepa1-6 tumors in the tumor microenvironment,and the order of the affinity between sHDL and tumor cells is consistent with the strong and weak trend of SR-B1 receptor expression on the cell surface.At the same time,for the non-specific model particle D-PEG-PLGA,there is no significant difference in the uptake of various cells.In order to verify this conclusion,the method of in vitro induction of macrophages was adopted,and corresponding cytokines were added to induce bone marrow-derived mouse macrophages.It was verified that the expression of bone marrow-derived macrophages induced in vitro was similar to that of mouse intratumoral macrophages.Bone marrow derived macrophages have the same surface antigens and ability to secrete specific cytokines and chemokines.At the same time,the expression level of SR-B1 receptor on the surface of bone marrow derived macrophages and the uptake ability of D-sHDL were investigated,and the results of in vitro and in vivo was consistent.5.VG-sHDL preparation,characterization and regulation of monocytes in tumor microenvironment.Bone marrow derived immune cells,including macrophages,dendritic cells(DC),bone marrow-derived suppressive cells(MDSC),are obtained in vitro by extracting monocytes from mouse bone marrow and cytokine induction.Among them,macrophages include two different compression types,M1 and M2.MDSC contains two different types of compaction,namely polymorphic nuclei(PMN-MDSC)and mononuclear(M-MDSC).The effects of free drugs gemcitabine(Gem),Gem E,Gem E-sHDL(G-sHDL),VC-sHDL(V-sHDL)and Gem E-VC-sHDL(VG-sHDL)on monocyte differentiation were determined in vitro.The results showed that G-sHDL and VG-sHDL can increase the ratio of M1/M2.In order to explore the mechanisms for the increase of the M1/M2 ratio,the toxicity of the prescription to macrophages and the influence of the differentiation process of M2 to M1 were studied.The results showed that the toxicity of the prescription containing Gem to M1 was significantly lower than that of M2,and the prescription containing V was less toxic to M1 and M2.At the same time,it was proved that Gem does not significantly promote the differentiation of M2to M1.Therefore,it is concluded that Gem can specifically control the tumor immunosuppressive microenvironment by specifically killing M2.Based on the conclusions of the above in vitro experiments,we have proved that prescriptions containing Gem can also specifically reduce the amount of M2 in the tumor microenvironment to achieve the effect of promoting immunity.At the same time,we have proved that Vadimezan is also indispensable in the process of obtaining the desired therapeutic effect.An important factor that is missing is that it can enhance the effect of immunotherapy by promoting the body's immune activation and increasing the infiltration of immune cells in the tumor.Finally,some related cytokines and chemokines were also tested to further support the above conclusion.6.Research on the effect of VG-sHDL on anti-tumor and specific immune memory formation.The efficacy of the above six prescriptions,the survival curve and the formation of tumor-specific immune memory were evaluated on Hepa1-6 tumor bearing C57BL/6mouse.It was proved that VG-sHDL has a strong inhibitory effect on tumors,promotes the formation of memory cells and forms specific immune memory.In this project,based on the high-density lipoprotein system,PTT-based photothermal-immune combination therapy and Gem-based immunotherapy that can specifically regulate the M2-related immunosuppressive microenvironment in TIME are designed respectively.A program that has the effect of promoting HCC immunotherapy.Among them,the constructed VR-sHDL can generate a large number of ICDs through the thermal effect of photothermal therapy,and induce strong specific antitumor immunity.At the same time,through the comparison of MR-sHDL and VR-sHDL,the relationship and the impact of the drug combination were both clarified.From another perspective,the constructed VG-sHDL achieves the enhancement of HCC immunotherapy effect by specifically regulating the M2 pathway in TIME.Both strategies have a good effect on HCC,and induce tumor-specific immune memory,prevent tumor recurrence,and prolong PFS.This topic provides new ideas for the clinical immunotherapy of inoperable HCC,emphasizes the importance of attaching importance to the interaction between drug combination and immune cells in the body,and clarifies the interaction between sHDL and immune cells in TIME and the effect of tumor immunosuppression.The mechanism of environmental regulation highlights the importance of understanding the interaction between the carrier and the body before the formulation is designed.Provides a sHDL-based biomimetic high-density lipoprotein nanocarrier platform that can promote the effect of HCC immunotherapy.It is of great significance For the clinical immunotherapy of HCC.