Study On The Application Of Detachable Core-shell Nano Delivery System In Enhancing Chemoimmunotherapy

Chemotherapy is still the main treatment for malignant tumors.In recent years,with the development of chemotherapy immunotherapy,the concept of tumor cell immunogenic death(ICD)has been well known.It can effectively promote tumor cells to release tumor associated antigen(TAAs)and a variety of cytokines,and promote the efficiency of antigen-presenting cells,so as to activate cytotoxic T cells(CTLs)to a greater extent,so as to produce immunotherapeutic effect.However,the immune response induced by chemotherapy alone is greatly limited by tumor immunosuppression microenvironment(itme),which can not achieve satisfactory therapeutic effect.Tumor associated macrophages(TAMs)are the main immunosuppressive cells in itme.Reversing TAMs from M2 phenotype to M1 phenotype is beneficial to eliminate the immunosuppression of TME and enhance the overall effect of immunotherapy.However,most of the current chemoimmunotherapies deliver chemotherapeutic drugs and immunomodulators without any difference.Chemotherapeutic drugs kill tumors,destroy immune microenvironment and damage beneficial immune cells.At the same time,when the normal immune cells in tumor microenvironment are damaged,more immunosuppressive cells will be collected to aggravate itme.Based on this,we constructed a kind of detachable double targeting core-shell nanoparticles to enhance the chemoimmunotherapy of tumor.Method:(1)The construction and characterization of nanosystemFirstly,we synthesized ha-dox macromolecular prodrug by using tumor targeting hyaluronic acid(HA)and chemotherapy drug DOX.The prodrug was then linked to the peptide GPLGLAGC,a matrix metalloproteinase-2(MMP-2)responsive broken peptide,to form CGALGLPG-HA-DOX.Finally,M2 TAMs targeting peptide(M2pep)and gplglagc-ha-dox were connected with albumin to form two blocks:HSA-M2pep(HM2)and HSA-CGALGLPG-HA-DOX(HMHD).Two blocks were loaded with chloroquine(CQ)during the coupling process to form the final preparation HMHD/HM2-CQ.The synthesis of nano drug delivery system was characterized by UV and 1H NMR.The particle size potential and morphology of the system were studied by laser particle size analyzer and transmission electron microscopy(TEM).(2)In vitro drug release characteristicsThe response of the drug delivery system to MMP-2 enzyme was studied by high performance liquid chromatography(HPLC)and TEM.The results showed that under the action of MMP-2 enzyme,the system could be divided into HA-DOX and HM2-CQ.In addition,the release of DOX and CQ in vitro was studied under the conditions of MMP-2 enzyme and different pH.(3)Study on immunogenicity and repolarization in vitroCell uptake experiments were carried out by fluorescence microscopy and flow cytometry to investigate the targeting ability of the core and shell of the removable core-shell nanosystem to macrophages and 4T1 cells.In addition,MTT method was used to study the biocompatibility of the blank carrier and the inhibition of the delivery system on 4T1 cells.Scratch test was used to study the anti metastasis ability of the nano system.(4)In vivo targeting and antitumor activityThe tissue distribution of IR783 in mice labeled with free IR783 and HMHD/HM2-IR783 NPs was studied by mouse in vivo fluorescence imager.BALB/C female mice bearing 4T1 breast cancer were established.The antitumor activity of nano delivery system was investigated by tumor volume,weight,change of survival curve and he staining pathological section.The lung metastasis animal model of BALB/C female mice bearing 4T1 breast cancer was established,and the anti metastasis effect of nano system in vivo was studied with the number of lung metastasis nodules as the index.(5)The study on immune response in vivoThe BALB/C female mice bearing 4T1 breast cancer were established.The immune response of mice was effectively activated by nano drug delivery system through the detection of cytokines in vivo,flow cytometry and immunofluorescence sections.Results:The results of various characterization experiments show that the HMHD/HM2-CQ NPs was successfully synthesized.The morphology of the HMHD/HM2-CQ NPs is core-shell spherical,the particle size is about 180 nm,and the potential is about-27 mV.The results of enzyme response showed that the detachable nano system could be separated into HA-DOX prodrug shell and HM2-CQ core in response to MMP-2 enzyme.The results of drug release in vitro showed that the drug was released more easily under the condition of MMP-2 enzyme and acid than under normal physiological conditions.In addition,in vitro cell experiments showed that HMHD/HM2-CQ NPs targeted their respective target cells through CD44 receptor and M2pep on the surface of cancer cells,making chemotherapy drugs enter tumor cells accurately,avoiding their damage to macrophages,while immunomodulators enter macrophages precisely,effectively regulating itme;in vitro studies on CRT and transformation showed that HMHD/HM2-CQ NPs can successfully induce ICD and transform M2 TAMs into M1 TAMs.In vivo imaging results show that the system is accumulating in tumor tissue,which indicates that it has strong tumor targeting.The results of pharmacodynamics and anti metastasis showed that HMHD/HM2-CQ NPs could effectively inhibit tumor growth and metastasis.On the other hand,the results of in vitro immune response study also showed that HMHD/HM2-CQ NPs can effectively activate the immune response n mice.Conclusion:In this study,we have developed a precise two-way targeted combined drug delivery strategy,which can deliver drugs to TAMs and tumor cells through CQ and DOX based chemotherapy drugs.The nano delivery system can improve the microenvironment of immunosuppressive tumor,promote the recruitment of cytotoxic T lymphocytes(CTLs)to tumor,inhibit tumor growth,inhibit tumor metastasis,and prolong the survival period of tumor bearing mice.Compared with the traditional simultaneous and indiscriminate co delivery system,using the different distribution characteristics of tumor microenvironment to deliver a variety of therapeutic drugs to tumor cells and target other cells respectively may be a new strategy to optimize the therapeutic effect of chemotherapy and immunotherapy.