Study On Cytotoxicity And Function Influence Of F-MWNTs On Primary Macrophages

Carbon nanotubes(CNTs) are cylindrical molecules with a length of up to micrometers and a diameter of 0.4-2 nm for single-walled carbon nanotubes(SWNTs) and 2-100 nm for coaxial multi-walled carbon nanotubes(MWNTs).CNTs have long been speculated and tested as new materials for biological and biomedical applications.Because of their abilities to bind cells and across the cell membrane.The solubility and biocompatibility of CNT will be improved by modification,so functionalized CNTs can be used as nanovectors for drug delivery and cancer phototherapy.As important cells in immune systerm, macrophages function as 'pathogen sensors' and are 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 and to rapidly discover nanotubes with the effect of macrophages and reveal structure-activity relationships.OBJECTIVES:1.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.Four kinds of f-MWNTs were synthesized from the functionalization of pristine MWNT. The effect of f-MWNTs on cytotoxicity and immunocyte function of primary mouse peritoneal macrophages are studied.2.The difference of macrophages derived from Human Peripheral Blood Monocyte incubating with or without f-MWNTs were investigated. METHODS:1.The effect of f-MWNTs on cytotoxicity and immunocyte function of primary mouse peritoneal macrophages1.1 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.1.2 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.1.3 Four kinds of f-MWNTs with deferent cytotoxicity were seclected based on the results of stage 1.2,which were 2,43,49 and 67.1.4 Primary mouse peritoneal macrophages were obtained.1.5 Mouse peritoneal macrophages were incubated with f-MWNTs for 3～24 hours. The uptake of f-MWNTs by macrophages was observed under microscope.The cell viability and ROS concentration were detected.2.The difference of macrophages derived from Human Peripheral Blood Monocyte incubating with or without f-MWNTs2.1 PBMCs were obtained from blood buffy coats of healthy donors through Ficoll gradient centrifugation and from cytapheresis products of nonpathological peripheral blood Monocytes were then prepared by a 2-h adhesion step.2.2.These monocytic cells were next cultured in RPMI 1640 medium supplemented with GM-CSF to get macrophages.2.3 During the derivation the cells were incubated with or without f-MWNTs.RESULTS:1.The effects of f-MWNTs on cytotoxicity and immunocyte function of primary mouse peritoneal macrophages1.1 83 kinds of f-MWNTs were synthesized by combinatorial modifications.1.2 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.1.3 Primary mouse peritoneal macrophages were obtained.The cell morphology was typical.1.4 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.Mouse peritoneal macrophages were incubated with f-MWNTs for 3 and 24 hours in various concentrations.The uptake of f-MWNTs by macrophages was observed under microscope at 24h.f-MWNTs could affect the cell viability and ROS increased obviously at the same time.2.The difference of macrophages derived from Human Peripheral Blood Monocyte incubating with or without f-MWNTs2.1 The macophages derived from Human Peripheral Blood Monocyte were obtained2.2 The macophages derived from Human Peripheral Blood Monocyte incubating with f-MWNTs were large and had phagosome.CONCLUSIONS:1.An f-MWNT library with maximal surface structural diversity was designed and synthesized using combinatorial chemistry.2.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.3.The effect of f-MWNTs on monocytes and macrophages is due to the diversity of modifications too.After further research,f-MWNT 49 and 67 was considered as the materials with the most weakly immune toxicity.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.4.As foreign substances,f-MWNTs can induce respiratory burst of monocytes and macrophages as soon as f-MWNTs bind cells and across the cell membrane.