Study On The Selective Targeting Ability Of Macrophage Targeted Delivery System And Influence To Diseases

BackgroundBased on the recruitment and distribution of monocyte-macrophages in inflammation,tumor,atherosclerosis and other diseases,a large number of drug delivery systems using macrophages as transport cells or carriers have been developed,which are called macrophage targeted delivery systems（mtdss）.This kind of system has great advantages in many aspects,such as targeting efficiency,breakthrough of physiological barrier,disease applicability and so on.However,on the basis of previous studies,our team found that macrophage targeted delivery system not only has a wide range of disease applicability,but also shows difficulty in distinguishing different types of macrophage related diseases.Because clinical patients are different from animal models of single disease model,they often suffer from a variety of diseases.The lack of selective targeting ability for specific diseases may lead to the drug loaded by them being delivered to multiple diseased tissues and organs at the same time,which may cause serious safety risks.This will seriously limit the clinical transformation and further development of macrophage targeted delivery system in the future.Monocyte-macrophages can differentiate or activate into M1 macrophages and M2macrophages through different activation pathways.These two cell subtypes have great differences in function.M1 macrophages are mostly involved in inflammation related diseases,such as colitis,viral myocarditis,arthritis,acute peritonitis,etc.,and release a large number of pro-inflammatory cytokines in these lesions,resulting in severe inflammatory damage.M2 macrophages are more involved in tissue healing,angiogenesis and tumorigenesis.Taking tumor as an example,M2 macrophages create an immunosuppressive environment in tumor by releasing anti-inflammatory cytokines,and activate regulatory T cells to inhibit the anti-tumor effect of killer T cells.Based on the difference of macrophages in tumor and inflammatory diseases,it is theoretically possible to achieve selective targeting of tumor or inflammatory diseases by targeting specific receptors or antigens on the surface of macrophages with different phenotypes,which has been regarded as a disease-specific targeting strategy by many studies.However,previous studies lack systematic evaluation of this specific targeting strategy in vivo,and the performance of delivery system when it confronts a variety of different macrophage related diseases in vivo is still unclear,which needs further research.At the same time,the impact of specific macrophage subtype delivery system on the disease progression of other subtype related macrophage diseases is rarely reported.In addition,as a group of highly plastic cells,macrophages are easily affected by various external factors.Whether the macrophage delivery system itself will affect the biological activity of macrophages and further affect the disease process is also an issue that can not be ignored.In order to answer the above questions,dextran,which is commonly used in the design and construction of macrophage targeted delivery system,was selected as the targeting unit,and bioactive polystyrene nanoparticles were selected as the model nanoparticles.Dextran modified polystyrene（DEX-PS）nanoparticles were used to objectively evaluate the targeting characteristics and differences on macrophages in vivo and in vitro,as well as the mechanism of these differences and their effects on the progression of inflammatory diseases and tumor diseases.This study is an in-depth comparative evaluation and exploration of macrophage targeted delivery system.The research results will have important guiding significance and reference value for comprehensive and reasonable evaluation of macrophage targeted delivery system and future transformation application.Method1.Synthesis and characterization of DEX-PS nanoparticles.Amine dextran and Nile Red labeled cooh-ps nanoparticles were used as materials to form stable amide linked dextran modified PS nanoparticles through the condensation reaction of amino group and carboxyl group.The physicochemical properties of nanoparticles were evaluated by infrared spectroscopy,transmission electron microscopy,particle size distribution,potential and fluorescence properties.The dextran loading rate of DEX-PS nanoparticles was determined by sulfuric acid phenol method.2.Different subtypes of macrophages derived from RAW 264.7 cells and different subtypes of macrophages derived from bone marrow macrophages were established in vitro.Using LPS and IFN-γOr IL-4 polarized RAW 264.7 monocyte macrophages to obtain M1 or M2 macrophages.The results of cell activation were confirmed by measuring and observing the levels of cytokines,macrophage surface markers and morphological characteristics.Bone marrow cells of BALB/c mice were extracted and activated into bone marrow-derived macrophages（bmdm）by M-CSF.LPS and IFN were used-γBmdm-m1 or bmdm-m2 macrophages were obtained by polarization of bmdm with IL-4.The results of cell activation were confirmed by measurement and observation of cytokine levels and morphological characteristics.3.Establish the best use conditions of nanoparticles and investigate the uptake rate of nanoparticles by different subtypes of macrophages.RAW 264.7 cells were cultured with different concentrations of nanoparticles.CCK-8method was used to determine the survival rate of RAW 264.7 cells.RAW 264.7 cells were incubated with nanoparticles for 1,2,4,8 or 24 hours respectively,and the uptake rate of nanoparticles was calculated to determine the optimal time range of interaction between nanoparticles and cells.The nanoparticles were co cultured with M1 and M2 macrophages according to the optimal concentration and time.After the culture,the enrichment of nanoparticles in cells was measured by flow cytometry.The nanoparticles were incubated with different subtypes of macrophages,and the distribution of nanoparticles in the cells was observed by laser confocal microscopy.Nanoparticles were incubated with M1 and M2macrophages for 0.5,1,2 or 4 hours respectively.The uptake efficiency of nanoparticles was measured and calculated by flow cytometry.The nanoparticles were incubated with bmdm-m1 cells and bmdm-m2 cells respectively for 4 hours.The phagocytic efficiency of the cells to the nanoparticles was measured by flow cytometry.4.Biological distribution of nanoparticles in diseases related to M1 and M2macrophage subtypes.4T1 tumor bearing mice were established by subcutaneous injection of 4T1 tumor cells,and acute peritonitis mice were established by intraperitoneal injection of 4%yeast polysaccharide.Three kinds of nanoparticles were injected into the tail vein of mice with two kinds of disease models respectively,and the distribution of nanoparticles in the two kinds of disease models was investigated by in vivo imaging technology.The tumor tissues of tumor bearing mice after the injection of nanoparticles were stained with fluorescence to confirm the targeting ability of nanoparticles to M2 macrophages in tumor tissues.5.Metabolism and distribution of nanoparticles in peripheral blood.Peripheral blood samples were collected at 2,4,6 and 8 hours after the injection of nanoparticles.The clearance rate of nanoparticles in peripheral blood was evaluated by in vivo imaging.Peripheral blood cells were labeled with CD11b,Ly6C and B220 antibodies,and then the distribution of nanoparticles in the three antibody labeled cell populations was investigated by flow cytometry.6.Study on the uptake mechanism of nanoparticles by monocytes-macrophages.The phagocytic function of RAW 264.7,M1 and M2 macrophages was inhibited by a variety of phagocytic pathway inhibitors,and then the inhibited cells were incubated with different nanoparticles.The change of phagocytic efficiency of cells to nanoparticles was investigated by flow cytometry,so as to infer the phagocytic pathway of different nanoparticles into cells.7.Effects of nanoparticles on phenotype and function of macrophages.After the nanoparticles were incubated with RAW 264.7,M1 and M2 macrophages respectively,the cell culture medium was collected and the cytokine TNF-α,IL-1β,TGF-βAnd IL-10 was detected by ELISA.Flow cytometry was used to detect the expression of CD86.Transwell system was established.Macrophages activated by nanoparticles were planted in the upper chamber,and 4T1 tumor cells were planted in the lower chamber.After culture,TUNEL staining method was used to count the apoptosis rate of tumor cells in the lower chamber,and CCK-8 method was used to count the survival rate of tumor cells in the lower chamber.8.The effect of nanoparticles on the disease process of tumor.The mice bearing 4T1 tumor were injected intravenously with a dose of 2 mg/kg every4 days,a total of 4 times.The tumor size and weight of mice were recorded before each treatment.At the end of the treatment,TNF-α,IL-1β,TGF-βAnd IL-10 was detected by ELISA.After treatment,CD163 antibody and i NOS antibody were used for fluorescent staining of tumor tissue sections to investigate the regulatory effect of nanoparticles on the phenotype of tumor macrophages.9.The effect of nanoparticles on the disease progression of peritonitis.The mice with acute peritonitis induced by 4%glacial acetic acid or 4%yeast polysaccharide were established.The mice with acute peritonitis were injected by tail vein at the dose of 2 mg/kg.After a certain time of injection of nanoparticles,the ascites were collected,and the TNF-αand IL-1βexpression level in the abdominal water was detected by ELISA.The survival time of mice treated with different nanoparticles was counted and the survival rate was calculated.Results1.The DEX-PS nanoparticles synthesized from Amine-Dextran and COOH-PS nanoparticles were observed by transmission electron microscope.The shape of DEX-PS nanoparticles was regular spherical.The average particle size and potential were 586±3nm and-48.6±0.8 m V.Compared with PS nanoparticles and COOH-PS nanoparticles,the average particle sizes of 574±3nm and 517±5nm,and the surface potentials of-35.4±1.4 m V and-42.4±4.1 m V,the three nanoparticles are relatively uniform in size and surface potential.Through the investigation of the nanoparticles by confocal microscope and fluorescence spectrophotometer,the fluorescence properties of the three kinds of nanoparticles are also relatively consistent.Overall,the DEX-PS nanoparticles synthesized in this topic are ideal materials for investigating the fate of nanoparticles modified by different functional groups in vivo.2.After activation of RAW 264.7 cells using LPS and IFN-γ,the pro-inflammatory cytokine IL-1βand TNF-αwere decreased.The expression levels of i NOS and CD86 were significantly increased.After RAW 264.7 cells were activated with IL-10,anti-inflammatory cytokine IL-4 and TGF-βwere decreased.The expression levels of CD206 and CD163 were significantly increased.M1 and M2 macrophages were successfully established.3.After the nanoparticles were incubated with different subtypes of macrophages,DEX-PS nanoparticles were more enriched in M2 macrophages.At the same time,DEX-PS nanoparticles were also enriched in M2 macrophages derived from BMDM.4.The results of in vivo imaging showed that DEX-PS nanoparticles could successfully target to the tumor（M2 macrophage related disease）.After staining different subtypes of macrophages in tumor tissue sections,it can be seen that the nanoparticles were mainly distributed in M2 macrophages.5.In vivo imaging showed that DEX-PS nanoparticles also had a high enrichment rate in peritonitis（M1 macrophage related disease）.6.To investigate the dynamic behavior of nanoparticles in peripheral blood,DEX-PS nanoparticles had a slower clearance rate in peripheral blood.In addition,DEX-PS nanoparticles were mainly absorbed by Ly6C^hi monocytes in peripheral blood,which could be non differentially distributed to different subtypes of macrophage related diseases.7.The mechanism of DEX-PS nanoparticles into cells was studied.It was found that DEX-PS nanoparticles mainly rely on mannose receptor（MMR）mediated phagocytosis to enter cells.At the same time,after the nanoparticles entered cells,the expression level of MMR increased,so as to phagocytize more nanoparticles,which gradually increased the rate of DEX-PS nanoparticles into cells.However,PS and COOH-PS nanoparticles did not rely on MMR pathway to enter cells,and the phagocytosis rate of these two kinds of nanoparticles gradually became flat.8.After DEX-PS nanoparticles entered macrophages,the pro-inflammatory cytokine IL-1βand TNF-αof M2 and M1 macrophages were decreased.The expression level of TGF-βand IL-10 were significantly inhibited.At the same time,the level of pro-inflammatory surface marker CD86 on the cell surface was also up-regulated.On the other hand,the cytotoxic effect of macrophages was also up-regulated.9.DEX-PS nanoparticles have a certain anti-tumor effect.The expression of pro-inflammatory cytokine IL-1βAnd TNF-αin tumor tissue were up-regulated the expression levels of TGF-βand L-10 were significantly inhibited.At the same time,the number of M1 macrophages in tumor tissue of mice treated with DEX-PS nanoparticles increased significantly.10.The survival period of mice with acute peritonitis was significantly shortened after DEX-PS nanoparticles injection.DEX-PS nanoparticles up-regulated the expression levels of proinflammatory cytokine IL-1βand TNF-αin abdominal cavity.And aggravates the symptoms of acute peritonitis in mice.Conclusion:1.In this project,DEX-PS nanoparticles were successfully prepared by using amine-dextran and COOH-PS nanoparticles,which have good M2 macrophage targeting ability in vitro.2.After investigating the biodistribution of nanoparticles in tumor（M2 macrophage related disease）and acute peritonitis（M1 macrophage related disease）,it was found that DEX-PS nanoparticles could not only target the tumor site,but also had a high enrichment rate in acute peritonitis.By investigating the dynamic fate of nanoparticles in peripheral blood,it was found that DEX-PS was easily intercepted and ingested by Ly6C^hi monocytes,which would distribute to different subtypes of macrophage related diseases with no difference,and there was a positive feedback regulation phenomenon in this uptake,that is,after the phagocytosis of nanoparticles,the phagocytic ability was activated by nanoparticles,thus phagocytizing more DEX-PS nanoparticles.Combined with these two reasons,we can explain the reason why DEX-PS nanoparticles with M2 macrophage specific recognition are enriched in M1 predominant peritonitis lesions.This kind of peripheral blood interception makes nanoparticles unable to distinguish two subtypes of macrophage related diseases in vivo.3.In addition,DEX-PS nanoparticles have immune activation effect on macrophages,which can achieve certain anti-tumor effect by improving the tumor immunosuppressive environment in tumor therapy.After treatment with DEX-PS nanoparticles,the tumor growth of tumor bearing mice is inhibited,and the tumor immune microenvironment changes to pro-inflammatory type,which has a positive effect on tumor therapy.At the same time,after DEX-PS nanoparticles treatment,the condition of mice with acute peritonitis was aggravated,the levels of inflammatory cytokines were increased,the inflammatory injury was aggravated,the survival time was shortened,and the survival rate was greatly reduced.4.These results suggest that researchers should consider the complex internal environment faced by drug delivery system when designing and evaluating MTDSs,pay attention to the ability of mtdss to recognize different subtypes of macrophages in vivo and the dynamic process of mtdss in vivo,so as to avoid the wrong distribution of nanoparticles aggravating the condition of non target diseases.