| As new members of carbon material family,carbon nanotubes(CNTs)have become the subject of intense investigation because of their unique structural,electrical and mechanical properties.In recent years,increasing studies are focused on exploring CNTs' properties for biological applications both at molecular and cellular levels.As a kind of CNTs,multi-walled carbon nanotube used in medicine must exhibit several properties,including biocompatibility,non-toxicity, non-carcinogenicity,non-mutagenicity,non-antigenicity,and non-teratogenicity, The solubility and biocompatibility of CNTs will be improved by modification.As important cells in immune systerm,macrophages function are 'pathogen sensors' and essential for the elimination of pathogens as well as for the regulation of the adaptive immune response and the inflammatory response.Whether functionalized MWNT can affect the macrophages has not been known clearly.We took the lead in this filed that combinatorial modifications of the nanotube's surface would enable us to map unknown chemical space more effectively.We also rapidly discover the effect of nanotubes on macrophages and reveal the structure-activity relationships.OBJECTIVES:1.Three kinds of f-MWNTs were synthesized from the functionalization of pristine MWNT.The effects of f-MWNTs on cytotoxicity and immunocyte function of primary mouse peritoneal macrophages are studied.2.A series of f-MWNTs were synthesized from the functionalization of pristine MWNT using combinatorial chemistry.The effect of f-MWNTs on cytotoxicity and immunocyte function of PMA-stimulated THP-1 cells are studied.Then the mechanism and structure-activity relationships were also analysised.METHODS:1.The effects of f-MWNTs on cytotoxicity and immunocyte function of primary mouse peritoneal macrophages 1.1 Three f-MWNTs,MWNT-COOH.MWNT-iBA and MWNT-Tyr,were synthesized by the oxidation of pristine MWNT,the amidation and esterification of MWNT-COOH with iso-butyl amine and tyrosine.The products were well characterized by FTIR and Elemental analysis.1.2 Primary mouse peritoneal macrophages were obtained.1.3 Mouse peritoneal macrophages were incubated with p-MWNT and f-MWNTs for 6,24,and 48 hours.The uptake of f-MWNTs by macrophages was observed under microscope.The cell viability and NO concentration were detected.2.The effects of f-MWNTs on cytotoxicity and immunocyte function of PMA-stimulated THP-1 cells2.1 An f-MWNT library containing 83 members was synthesized using combinatorial chemistry method.All intermediates and the products were well characterized by FTIR,HPLC and Elemental analysis。2.2 The effect of 83 kinds of f-MWNTs on cytotoxicity and immunocyte function of PMA-stimulated THP-1 cells are studied.PMA-stimulated THP-1 cells were incubated with p-MWNT and f-MWNTs in various concentrations.The cell viability and NO concentration were detected.2.3 Four kinds of f-MWNTs with deferent cytotoxicity were seclected based on the results of stage 2.2,which were 2,43,49 and 67.PMA-stimulated THP-1 cells were incubated with f-MWNTs in various concentrations.The cell viabilities and the concentrations of NO,TNF-α,ROS were tested.RESULTS:1.The effects of f-MWNTs on cytotoxicity and immunocyte function of primary mouse peritoneal macrophages1.1 MWNT-COOH,MWNT-Tyr and MWNT-iBA were synthesized.1.2 Primary mouse peritoneal macrophages were obtained.The cell morphology was typical.1.3 Mouse peritoneal macrophages were incubated with p-MWNT and f-MWNTs for 6,24,and 48 hours.The uptake of f-MWNTs by macrophages was observed under microscope at 24h.p-MWNT and f-MWNTs could affect the cell viability and the immune function seriously at the dose of 400μg/mL.It is noticeable that the cytotoxicity induced by MWNT-iBA was least.The cell viabilities and the concentrations of NO induced by p-MWNT and MWNT-COOH were depended on the dose of materials.MWNT-COOH and p-MWNT could not injure the cell function below the dose of 100μg/mL,although the effect of the later was more remarkably.2.The effects of f-MWNTs on cytotoxicity and immunocyte function of PMA-stimulated THP-1 cells2.1 83 kinds of f-MWNTs were synthesized by combinatorial modifications.2.2 PMA-stimulated THP-1 cells were incubated with p-MWNT and f-MWNTs in various concentrations for 24h.The cell viability reduced from about 60%to 30% when the doses increased from 50μg/mL to 200μg/mL.The NO concentrations varied with the dose and functionalization.2.3 f-MWNTs 2,43,49 and 67,with deferent cytotoxicities,were seclected based on the results of stage 2.2.PMA-stimulated THP-1 cells were incubated with f-MWNTs in various concentrations.Immune toxicity was not investigated at the dose of 10μg/mL.But MWNT-COOH could inhibit the cell viability to below 40% at the dose of 400μg/mL,The concentrations of NO,TNF-αand ROS increased obviously at the same time,especially the concentration of TNF-α.Even f-MWNT 49 and 67 could affect the cell viability,the concentration of NO,TNF-αand ROS were smaller than f-MWNT 2.CONCLUSIONS:1.An f-MWNT library with maximal surface structural diversity was designed and synthesized using combinatorial chemistry.2.After carboxylation,the solubility of f-MWNT was improved while the immune toxicity became more severed than p-MWNT.The biocompatible of f-MWNT improved further after modified with amino acid and amide group.Due to the diversity of modifications,the effects of f-MWNT on PMA-stimulated THP-1 cells were diferent.The cytotoxicity induced by AC005-containing f-MWNTs in the library was unconspicuous generally.After further research,f-MWNT 49 and 67 was considered as the materials with the most weakly immune toxicity.3.The effect of f-MWNTs on monocytes and macrophages may be related to the activation of NF-κB,because of the release of TNF-α,NO and oxidative stress.
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