Construction Of M2 Macrophages Targeted Drug Delivery System For Cancer Immunotherapy

ObjectiveCancer immunotherapy has become a novel method following surgery,chemotherapy and radiotherapy in cancer therapy.As the largest number of immune cells in tumor microenvironment,M2 macrophages play an important role in promoting tumor growth and metastasis.They can secrete some cytokines,such as IL-4 and IL-10,to inhibit T cell immune response and reduce the production of ROS and NO,thus promoting tumor angiogenesis,invasion and metastasis,as well as inhibiting the anti-tumor immune response.Previous studies have shown that tumor-associated macrophages were highly variability and M2 macrophages could be polarized to the anti-tumor M1 phenotype.Therefore,the polarization of M2 macrophage can reverse the immunosuppressive tumor microenvironment driven by M2 macrophages and achieve specific anti-tumor effects.In order to effectively polarize M2 macrophages,reduce unnecessary cellular uptake and elimination by other wide distributed macrophages in vivo,as well as retain the potentially beneficial of M1 macrophages,a specific drug delivery system for cancer immunotherapy was designed and constructed by using the over-expressed CD206?mannose receptors?on M2 macrophages as the target.When choosing zoledronic acid which could polarize M2macrophages to M1 macrophages as the model drug,we hope that the constructed drug delivery system could selectively target the M2 macrophages and polarize them to an anti-tumor M1 phenotype,thus reversing the immunosuppressive tumor microenvironment derived from M2 macrophages,activating the anti-tumor immune response,and finally inhibiting the tumor growth effectively.Methods1.In this study,DOPA,cholesterol,DOTAP and DSPE-PEG₂₀₀₀-Ma with mannose targeting groups were employed to prepare zoledronic acid loaded M2 macrophages targeted nanoparticles?M2-TNPs?via reverse microemulsion method.When DSPE-PEG₂₀₀₀-Ma was replaced by DSPE-PEG₂₀₀₀,the M2 macrophages non-targeted nanoparticles?Non-TNPs?were prepared in the similar way.The stability and zeta potential of M2-TNPs and Non-TNPs were measured by Delsa?Nano C particle analyzer.Transmission electron microscopy and scanning electron microscopy were used to assess the morphology of M2-TNPs.Ultraviolet-visible spectrophotometer was carried out to test the drug loading efficiency and entrapment efficiency of M2-TNPs and Non-TNPs.2.The cellular uptake of NBD-labeled M2-TNPs and Non-TNPs by BMDMs,M1and M2 macrophages was evaluated by fluorescence microscopy and luminometer in order to estimate their targeting ability.Mannan competitive inhibition experiment was employed to further investigate the M2 macrophages targeting ability of Non-TNPs and M2-TNPs.CCK-8 cell viability assay was utilized to measure the cytotoxicity of free zoledronic acid,Non-TNPs and M2-TNPs to macrophages.Flow cytometry,western blot and ELISA assay were carried out to evaluate the ability of polarizing M2 macrophages by M2-TNPs comparing with free zoledronic acid and Non-TNPs.The transwell co-culture model of B16-F10 and M2 macrophages was established to verify the anti-tumor effect of M2-TNPs through polarizing M2 macrophages to M1 macrophages.Mannan was also used in competitive experiment to further evaluate the M2 macrophages targeting ability of M2-TNPs.The transwell co-culture model of B16-F10 and T cells was established to investigated the anti-tumor activity of M2-TNPs through the activation of T cell mediated anti-tumor response.3.The anti-tumor activities of free zoledronic acid,Non-TNPs,M2-TNPs,as well as the M2-TNPs and dacarbazine combined therapy were tested by C57BL/6 tumor-bearing mice in vivo.The apoptosis of tumor cells was investigated by TUNEL staining.H&E staining was employed to further investigate the anti-tumor effect of free zoledronic acid,Non-TNPs,M2-TNPs and the combined therapy group.H&E staining was utilized to estimate the biocompatibility of M2-TNPs.The targeted delivery ability of M2-TNPs was evaluated in vivo by immunofluorescence staining.Flow cytometry,western blot,immunofluorescence staining and ELISA assay were carried out to assess the immunoregulatory mechanism of M2-TNPs.Results1.Zoledronic acid loaded M2-TNPs were prepared by reverse microemulsion method.Characterization of nanoparticles showed that the particle size of M2-TNPs was about 140nm.Besides,M2-TNPs had good stability since the particle size did not change obviously within 28 days.Results of scanning electron microscopy and transmission electron microscopy demonstrated that M2-TNPs were uniform and spherical.The spectroscopic results indicated that drug loading efficiency and entrapment efficiency of M2-TNPs were9.4±0.9%and 52.0±5.0%,respectively.2.The results of fluorescence microscopy and luminometer revealed that there was little cellular uptake of Non-TNPs and M2-TNPs when incubated with BMDMs and M1macrophages.However,M2 macrophages could capture a greater amount of M2-TNPs than Non-TNPs.Besides,the uptake of M2-TNPs by M2 macrophages was significantly reduced after incubated with mannan,whereas the uptake of Non-TNPs by all above macrophages did not change obviously.These data indicated that after modification,M2-TNPs could target mannose receptor and selectively uptake by M2 macrophages.Results of CCK-8 cell viability,flow cytometry,western blot and ELISA assay demonstrated that in the concentrations which had no cytotoxicity to macrophages,free zoledronic acid,Non-TNPs and M2-TNPs could polarize M2 macrophages to M1phenotype,thus increasing the expression of cytokines such as iNOS and TNF-?,decreasing the expression of immunosuppressive cytokines such as Arg-1 and IL-10,and finally,inhibiting tumor growth.Among them,M2-TNPs displayed the strongest M2macrophages polarization ability.The results of CCK-8 test indicated that free zoledronic acid,Non-TNPs and M2-TNPs did not inhibit cell viability of B16-F10 at therapeutic concentrations.However,when free zoledronic acid,Non-TNPs and M2-TNPs were added into M2 macrophages in co-culture model,the cell viability of B16-F10 was decreased,suggesting the anti-tumor effect originated from the polarization of M2macrophages to an anti-tumor M1 phenotype.Among them,M2-TNPs exhibited the highest anti-tumor efficiency.Besides,after pre-incubated with mannan,the cell viability of B16-F10 in M2-TNPs treatment group was greatly increased,while the cell viability in free zoledronic acid and Non-TNPs did not change obviously.The mannan competitive experiment further proved that M2-TNPs could specially target M2 macrophages and be internalized through mannose receptor medicated endocytosis.Besides,compared with the control group,free zoledronic acid,Non-TNPs and M2-TNPs treated M2 macrophages culture medium successfully inhibited the cell viability of B16-F10 in B16-F10 and T cells co-culture model.Besides,free zoledronic acid,Non-TNPs and M2-TNPs treated M2macrophages culture medium effectively promoted the activation and proliferation of T cells,suggesting that free zoledronic acid,Non-TNPs and M2-TNPs could activate T cell mediated anti-tumor immune response through polarizing M2 macrophages.Among them,M2-TNPs which could effectively polarize M2 macrophages to the anti-tumor M1phenotype,exhibited the highest anti-tumor efficiency.3.In vivo anti-tumor experiment using B16-F10 tumor-bearing mice showed that compared with the control group,M2-TNPs dramatically retarded the tumor growth and the tumor suppression efficiency was 45.2%,whereas the tumor suppression efficiency of free Zol and Non-TNPs was 11.7%and 6.9%,respectively.Results of TUNEL showed the highest apoptotic rate of tumor cells in M2-TNPs group was found when comparing with free zoledronic acid and Non-TNPs.And H&E staining indicated that comparing with free zoledronic acid and Non-TNPs,M2-TNPs group exhibited the greatest cytotoxicity to tumor cells.After combing with the chemotherapy drug dacarbazine,the tumor suppression efficiency was up to 93.2%,indicating an enhanced anti-tumor effect of chemotherapy combined with immunotherapy.Besides,H&E staining of organ samples showed that M2-TNPs had superior biocompatibility.Results of immunofluorescence staining showed that M2-TNPs revealed an enhanced tumor-targeting efficiency compared with Non-TNPs.Moreover,colocalization of M2-TNPs with mannose receptor was observed,while no obvious colocalization was found in Non-TNPs group.Results of flow cytometry showed that the number of M1 macrophages in M2-TNPs group was dramatically increased while the number of M2 macrophages was decreased,indicating that M2-TNPs successfully polarized M2 macrophages to M1 phenotype.Western blot and immunofluorescence staining showed that M2-TNPs significantly increased the expression of CD80 and decreased the expression of CD206,which was consistent with the results of flow cytometry.ELISA results showed that the expressions of M1-releated markers CD80,iNOS and TNF-?in M2-TNPs group were increased while the expressions of CD206,Arg-1 and IL-10 were decreased,indicating that M2-TNPs exhibited the strongest ability of polarizing M2 macrophages comparing with free zoledronic acid and Non-TNPs.Results of flow cytometry also showed that compared with the control group,free zoledronic acid and Non-TNPs,the number of CD8⁺T cells and secretion of IFN-?increased significantly after M2-TNPs treatment.Besides,results of Ki-67 proliferation experiment showed that M2-TNPs significantly promoted the proliferation of CD8⁺T cells.These data suggested that M2-TNPs could effectively activate T cell mediated anti-tumor immune response through polarizing M2 macrophages into anti-tumor M1 macrophages.ConclusionsIn this study,we designed and constructed a novel zoledronic acid loaded M2macrophages targeted nanoparticles?M2-TNPs?through reverse microemulsion method.M2-TNPs could specially targeted M2 macrophages and be internalized through mannose receptor medicated endocytosis.M2-TNPs could effectively polarize M2 macrophages into an anti-tumor M1 phenotype,thus revering the immunosuppressive tumor microenvironment driven by M2 macrophages,activating the anti-tumor immune response and finally,inhibiting melanoma growth in vitro and in vivo.After further research and evaluation,the above constructed M2 macrophages targeted delivery system can offer new opportunities to develop the novel and efficient drug delivery system for cancer immunotherapy.