Construction Of Nanomedicine For Tumor Immune Synergy Therapy Based On Host-Guest Interactions

Background and purposeTraditional cancer treatment methods such as surgical treatment,chemotherapy,and radiotherapy,often have shortcomings including single treatment,low selectivity,and large toxicity and side effects.Therefore,in order to solve the most difficult cancer treatment problem in medicine,a more efficient,longer-lasting and more complete cancer treatment method is required to be developed.Tumor immunotherapy controls and kills tumor cells by stimulating and enhancing the immune function of the body.On the basis of tumor immunotherapy,it is promising to achieve a more powerful tumor killing effect through different tumor killing mechanisms combined with other treatment methods,such as photothermal therapy,chemotherapy,gene therapy,etc.Based on the above ideas,this paper designed two different drug-delivery systems based on the host-guest chemistry:(1)Construction of a mannose-targeted amino-modified cyclodextrin and double-chain alkylated adamantane polymer type.In the delivery system CHTMD,the photothermal therapy reagent indocyanine green(ICG)is packaged on this system combined with the model antigen ovalbumin,and the antigen adjuvant CPG is loaded at the same time to achieve the high-efficiency therapeutic effect of the tumor vaccine.By stimulating the immunity response of the tumor site,this system can achieve the purpose of synergistic treatment of tumors with light,heat and immunity.(2)Employing a kind of immunoagonist,imiquimod(R848),as a guest molecule and cyclodextrin as host to construct a CD47 siRNA nanosystem CHTR through host-guest interaction,and this system use R848 and CD47 siRNA to promote macrophages repolarization in vivo.The ‘re-education’function inhibits the growth of tumors and achieves immune synergistic therapy.Research methodsIn this paper,experiments were carried out according to the following two parts.With amino-functionalized β-cyclodextrin as the main part,two host-guest polymer nanocarriers,CHTMD and CHTR were constructed respectively,which were used as nanocarriers to carry photothermal reagents and immunotherapy reagents in anti-tumor experiments in vivo and in vitro.Prat Ⅰ: A nano-assembly system was constructed through the host-guest interaction of amino-functionalized β-cyclodextrin and phospholipid-modified adamantane.In order to solve the targeting problem of nanovaccine,we introduced small molecule mannose into the nanoparticles,which can efficiently target DC 2.4cells and be internalized.Furthermore,the photothermal therapy reagent indocyanine green(ICG)is contained in the particles,and it is excited by 808 nm near infrared light(NIR)to convert light energy into heat energy at the tumor site,thereby causing tumor cells damage.Finally,CpG is loaded by electrostatic interaction and the phospholipid group of the assembly is combined with the model antigen ovalbumin(OVA)to construct an efficient targeted nanovaccine to realize the synergistic treatment of tumor immunity and photothermal therapy.Part Ⅱ: A nano-assembly system was constructed through the host-guest interaction of amino-functionalized β-cyclodextrin and immunoagonist R848,and CD47 siRNA is loaded through electrostatic interaction.R848 can "re-educate" macrophages from the M2 subtype,that promotes tumor growth,to inhibitive M1subtype;CD47 siRNA can down-regulate the "don’t eat me" signal on the surface of cancer cells and enhance the phagocytosis of cancer cells by macrophages.Through the dual regulation of macrophages,the supressive tumor microenvironment is relieved,and the nano-host-guest drug-carrying system has a synergistic immunotherapy effect on tumors and inhibits tumor growth.Research resultsPart Ⅰ: In this study,an assembly CHTMD modified with mannose was successfully prepared and confirmed by NMR.The MTT experiment showed that CHTMD has low toxicity in COS 7,DC 2.4 and other cells.The photothermal experiment proved that when the ICG drug loading efficiency is 16%,CHTMD has a more significant lethality to 4T-1 cells than ICG alone.The agarose gel electrophoresis experiment explored the optimal nitrogen-phosphorus compound ratio when CHTMD is combined with CpG.The particle size and distribution of untargeted and targeted nanocarriers were measured after compounding with CpG and OVA by dynamic light scattering(DLS).Through in vitro experiments such as confocal laser scanning microscopy and flow cytometry,it is proved that the nanocarrier modified by mannose is more easily taken up by DC 2.4 cells.Mouse melanoma and breast cancer are used as animal models to confirm that the targeted nanovaccine is effective.The therapeutic effects of the co-loaded photothermal reagent and immunotherapeutic reagent on breast cancer in mice were performed.Part Ⅱ: In this study,we successfully prepared an amino-modifiedβ-cyclodextrin and R848-loaded nano-medicine carrier CHTR after host-guest self assembly,confirmed by NMR,and successfully loaded CD47 siRNA.Agarose gel electrophoresis experiment and dynamic light scattering experiments explored the particle size of CHT-R loaded with CD47 siRNA at different mass compound ratios,and selected the optimal mass ratio as the basis for subsequent experiments.MTT experiments showed that the different mass compound ratios of CHTR and CD47 siRNA has a higher toxicity in CT26 cells than COS 7,RAW 264.7 and other cells,and they have better anti-tumor effects in vitro.In vitro experiments such as confocal laser scanning microscopy and flow cytometry have shown that R848 in CHTR/siRNA can make macrophages "re-educated" to the M1 type that inhibits tumor growth.Finally,the mouse CT26 cancer was used as animal model,and the tumor size monitoring during treatment in tumor-bearing mice and the in vivo immunotherapeutic effect evaluation proved the immune-synergistic therapeutic effect of CHTR/CD47 siRNA.Research conclusionPart Ⅰ: In this study,mannose-targeted amino-modified cyclodextrin and phospholipid-modified adamantane were designed,and the host-guest assembly of the two was used to construct a polymer carrier,which can encapsulate the photothermal therapy reagent ICG.And together with the model antigen OVA and immune adjuvant CpG,the nanocarrier can form a highly efficient nanovaccine to achieve the purpose of photothermal and immune synergistic therapy against tumors.Part Ⅱ: In this study,polymeric nanocarriers were constructed by the host-guest interaction between amino-modified β-cyclodextrin and immunoagonist R848 with CD47 siRNA loaded.R848 and CD47 siRNA in the drug-loading system could regulate the macrophages from M2 type to M1 type.Reversing the tumor microenvironment and "re-educate" M2 subtype macrophages into M1 subtype macrophages to encourage them to engulf more tumor cells and realizes the purpose of immune synergistic therapy of tumors.