Target Delivery CSF-1R Inhibitor To Tumor-associated Macrophages For Cancer Immunotherapy

Background:The recurrence rate of tumors after conventional treatment is extremely high,and it is difficult to eradicate from patients.Regarding the treatment of malignant tumors,the classic method is still radical resection of the lesion combined with radiotherapy and chemotherapy,but these methods cannot completely remove the residual or metastatic tumor cells to achieve the desired radical cure effect.Immunotherapy is gradually becoming a radical cure for malignant tumors with broad prospects.In recent years,tumor microenvironment(TME)has gradually become a hotspot of tumor immunity research.TME is quite different from the normal tissues in cell and cytokine composition,nutrient metabolism,tissue oxygen supply and p H value.Tumor cells and tumor microenvironment are mutually causal,coexist and symbiotic.The differentiation and function of immune cells in TME have different degrees of defects,which also leads to the complexity and variability of TME.A large number of studies have shown that the TME has an important influence on promoting tumor angiogenesis,inducing drug resistance and tumor metastasis.Therefore,in addition to adjuvant conventional treatment,tumor immunotherapy should also have a long-lasting immune monitoring function to avoid tumor recurrence by reshaping the tumor microenvironment.Tumor-associated macrophages(TAM)are one of the most abundant tumor-infiltrating leukocytes.TAM is mainly presented as anti-tumor M1 subtype and pro-tumor M2 subtype in the TME.M1 macrophages have strong antigen presentation ability and can secrete pro-inflammatory cytokines such as TNF-?,IL-12 and IL-23.On the contrary,M2 macrophages have strong anti-inflammatory activity,which can release IL-4,IL-10,IL-13,and inhibit T cell activity.Therefore,the ideal immunotherapy strategy is to increase the M1 subtype and reduce the M2 subtype in TEM,which can reshape the TME and enhance anti-tumor immunity.Colony stimulating factor 1 receptor(CSF-1R)belongs to the type III receptor tyrosine kinase family.The overexpression of CSF-1R and its ligand CSF-1 is widely present in solid tumors,which plays an important role in tumor proliferation,metastasis and microenvironment regulation.CSF-1R signal transduction is essential for the differentiation and survival of macrophages,which plays a critical role in the promotion and immunosuppressive function in tumor.BLZ-945 is a highly selective CSF-1R inhibitor.It can reduce the number of TAMs or change their phenotype,promote CD8~+T cell infiltration,and ultimately inhibit tumor growth.However,BLZ-945 has shortcomings such as low tumor targeting efficiency,poor bioavailability,and many adverse reactions.How to increase its effective accumulation at the tumor site,reduce adverse reactions and increase treatment benefits remains to be further studied.Nano drug-delivery system(NDDS)has been proven to have good advantages in cancer treatment.The sensitivity of the tumor microenvironment is very important for the effectiveness and safety of the formulation.Dextran can specifically bind to CD206receptor,but CD206 receptor is widely distributed in the body,especially in liver tissue.This makes the dextran targeting nano formulations easy to be swallowed by the liver and spleen,resulting in poor distribution and rapid clearance in the body.The biomimetic drug-delivery system has many advantages,such as good biocompatibility,low immunogenicity,long circulation time in the body.Hybrid cell membranes can balance immune escape and homology targeting,which lead to broader application prospects.Drug release directly affects anti-tumor effect.After the carrier reaches the tumor site,how to break through the cell membrane and fully release the drug is another problem that needs to be solved.Based on the above,the weak acid characteristics of the TME aroused our attention.Due to the rapid growth of tumor cells,the abnormal vasculature cannot meet its high metabolic oxygen demand.Long-term hypoxia increases the anaerobic glycolysis of tumor cells to produce a large amount of lactic acid,but abnormal blood vessels cannot clear the accumulated lactic acid in time.The proton pump accelerates the transport of acidic substances outside the cell,which eventually leads to acidosis in tumor tissues.The p H-responsive nano-drug delivery system designed based on the characteristics of the tumor microenvironment can maintain a stable structure in normal tissues.Responsively releasing drugs under low p H conditions in the tumor microenvironment can enhance the drug delivery efficiency at the tumor site.In summary,this project designed and constructed a p H-responsive erythrocyte-cancer cell hybrid membrane coated polymer micelle for the TME.Through the combination of active targeting mediated by cell membrane and passive targeting mediated by EPR effect,with the p H change of tumor tissue enable the drug BLZ-945to be released accurately,quickly and efficiently at the tumor site.This formulation can improve the bioavailability of the drug effectively,and reshapes the tumor immune microenvironment to achieve anti-tumor efficacy.Objectives:This project integrates immunology,materials science,pharmacy,molecular biology with drug immunotherapy and nanotechnology,which complements each other.It plans to construct a erythrocyte-cancer cell hybrid membrane camouflaged bionic nano drug-delivery system for TAM.Taking in vitro cytology and in vivo zoology models as the research methods,comprehensively evaluate the cytotoxicity and uptake,tissue distribution,biological safety of the micelle complex,as well as the anti-tumor effect.It provides new strategies and more choices for the development of intelligent drug delivery systems with TAM,meets the urgent clinical need for high-efficiency and low-toxicity pharmaceutical formulations,opens up new directions for immunotherapy to reshape the TME.Part I Preparation and characterization of hybrid membrane coatedmicellar complexMethods:The p H-sensitive copolymer micelles coated with a erythrocyte-cancer hybrid membrane were prepared by co-extrusion.First synthesize dextran-grafted-poly(histidine)copolymer(DH),then dope BLZ-945 into micelles through hydrophobic force,finally coat erythrocyte-cancer cell hybrid membrane(ECm),prepared as drug-loaded DH@ECm.The structure of DH was identified by ~1H-nuclear magnetic resonance(~1H-NMR).The cell membrane coating efficiency was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).High performance liquid chromatography(HPLC)was used to determine the encapsulation efficiency of DH and DH@ECm.The transmission electron microscopy(TEM)was used to observe and compare the morphology of drug-loaded DH and DH@ECm.Dynamic light scattering(DLS)was used to characterize the particle size and Zeta potential of different formulations under different p H conditions.And conventional dialysis method was used to investigate the release behavior of each group under different p H conditions in vitro.Results:~1H-NMR characterization shows the successful synthesis of DH.SDS-PAGE showed that the hybrid membrane was coated on the surface of drug-loaded DH successfully.The entrapment efficiency of DH and DH@ECm are 82.1%and 81.5%respectively.TEM showed that DH and DH@ECm are spherical particles with uniform size and regular shapes,and the“core-shell”structure can be clearly observed on the surface of DH.The blank DH,DH loaded with BLZ-945 and the DH@ECm loaded with BLZ-945 have particle sizes of 163.4 nm,172.1 nm and 194.3 nm respectively.The Zeta potential of different formulations is around-20 m V.The cumulative release of drug-loaded DH and DH@ECm at p H 6.5 is above 65%.Also their release behavior in vitro is similar,showing a certain p H dependence.Summary:1.In this chapter,the mediator copolymer DH was successfully synthesized.Erythrocyte and cancer cell membranes were successfully extracted.DH@ECm was successfully prepared and the pharmaceutical properties was systematically characterized.2.DH@ECm can release drugs quickly in the acidic microenvironment of tumors.The reason may be due to its potential“membrane escape effect”,which needs to be further verified in subsequent experiments.3.The results of this experiment lay the foundation for subsequent cytology and animal experiments.Part II In vitro study of hybrid membrane coated micellar complexMethods:This chapter investigates the cytotoxicity,cellular uptake of micellar complexes and other effects on target cells through cytological experiments in vitro.Taking 4T1cells and M2 TAMs as research objects,the thiazolyl blue tetrazolium bromide(MTT)was used to investigate the cytotoxicity of different formulations to the two kinds of cells under different p H conditions.Confocal laser scanning microscopy(CLSM)and flow cytometry(FCM)were used to investigate M2 TAMs'uptake behavior of different formulations under different p H conditions qualitatively and quantitatively.In addition,the active targeting effect mediated by dextran was also verified by FCM.Construct M1 and M2 subtypes of TAMs,co-culture method was used to determine the targeting ability of the formulations under different p H conditions.The phosphorylation level of CSF-1R(p CSF-1R)of different formulations was measured by western blot(WB)under different p H conditions,so that the CSF-1R inhibitory ability can be evaluated.The effect of the formulations on 4T1 and M2 TAMs was observed through co-culture.Results:The MTT results showed that the cell survival rates of blank carriers acting on 4T1and M2 TAMs were all above 80%,indicating low cytotoxicity.The WB method was used to determine the protein content of p CSF-1R in M2 TAMs.The results suggest that BLZ-945 has a significant inhibitory effect on CSF-1R under the condition of p H7.4.The inhibitory effect of DH group on CSF-1R is higher than that of BLZ-945 group in p H 7.4.The inhibitory effect of DH group on CSF-1R is higher than that of DH@ECm group in p H 7.4.Under the condition of PH 6.5,there was no significant difference in the inhibitory effect of DH group and DH@ECm group on CSF-1R.In the DH@ECm group,the inhibitory effect on CSF-1R under the condition of p H 6.5was significantly higher than that under the condition of p H 7.4.It is consistent with the results of cell uptake and cytotoxicity experiments in vitro.The cell uptake results also indicate that DH has the ability to target M2 macrophages,and DH@ECm has the ability to target M2 macrophages in acidic environment.The 4T1 and M2 TAMs co-culture results showed that the formulations had a significant inhibitory effect on M2TAMs,but there was no significant apoptosis induction for 4T1 cells.Summary:1.DH@ECm is safe and biocompatible,without obvious cytotoxicity.2.In acidic microenvironment,DH@ECm has p H response characteristics and the unique“membrane escape”effect.3.Dextran residues can promote the recognition and internalization of the formulation by TAMs in order to deplete them with M2 type targeting.4.DH@ECm doped with BLZ-945 only has depletion effect on TAMs,but has no effect on tumor cells.5.DH@ECm has certain application prospects in tumor immunotherapy.Part?Anti-tumor effect and immunological evaluation of hybridmembrane coated micellar complexMethods:Taking tumor-bearing mice as the research object,lipophilic cell membrane green fluorescent probes(Di O)and in vivo image system(IVIS)were used for this investigation.The circulation and the biodistribution of Di O-labeled micellar complexes were evaluated for their tumor tissue targeting ability in vivo.Immunofluorescence technique(Immunofluorescence technique,IFT)was used to stain the in vitro sections,focusing on the anti-tumor pharmacodynamics of micellar complexes.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression level of cytokines in tumor tissues.The safety of micellar complexes was evaluated in C57BL/6 mice.The body weight of mice and the histological change of the main tissue paraffin sections stained by Hematoxylin-Eosin staining(HE)were used as indicators to investigate the safety of the formulations in vivo.Results:IVIS observed the biodistribution in tumor-bearing mice.Along with the circulation in the body,the Di O-labeled micellar complexes show good tumor tissue targeting ability compared with Di O solution.The DH@ECm group had the strongest tumor tissue fluorescence intensity,showing significant long-circulation effects and tumor targeting ability.Pharmacodynamics in tumor-bearing mice showed that compared with free BLZ-945 drug group,the DH@ECm group could significantly inhibit tumor growth.As DH@ECm can quickly play a“membrane escape”effect in the acidic TME to increase the targeting of TAMs,release BLZ-945 at the same time,both contribute to high-efficiency TAMs depletion.The results of immunofluorescence experiments showed that the DH@ECm group could increase CD8~+T cell infiltration and activate anti-tumor immunity.ELISA test showed that DH@ECm significantly increased the expression of IL-12p70,but decreased the expression of IL-10,matrix metalloproteinase 9(MMP-9),and tumor tissue vascular endothelial growth factor(VEGF).The above results indicate that it can reshape the tumor microenvironment.The safety study of the formulations showed that the micellar complexes of the two groups had no obvious organ toxicity,and the mice were well tolerated.Summary:1.DH@ECm can target TAMs in tumor tissue,and reduce the distribution in normal tissues effectively.2.DH@ECm can assist BLZ-945 to exert its anti-tumor activity and efficacy.3.DH@ECm doped with BLZ-945 can reshape the tumor immunosuppressive microenvironment,activate the anti-tumor immune response,and inhibit tumor growth effectively.4.DH@ECm doped with BLZ-945 is a safe and effective drug delivery system which has no significant non-specific damage to the body.Conclusion:1.In this project,we successfully prepared the erythrocyte-cancer cell hybrid membrane camouflaged p H-responsive copolymer micelle doped with selective CSF-1R inhibitor(BLZ-945)abbreviated as DH@ECm.It can target and deplete TAMs to reshape the tumor immune microenvironment to achieve immunotherapy for malignant solid tumors.2.This biomimetic nano drug-delivery system is safe with good biocompatibility,low cytotoxicity,and stable in circulation.3.In the tumor immune microenvironment,DH@ECm doped with BLZ-945 can deplete TAMs by targeting M2 type macrophages,cause CD8~+T cell infiltration,activate anti-tumor immune response,and achieve immunotherapy.4.This study provides new ideas for targeted immunotherapy of solid tumors.