Multi-walled Carbon Nanotubes Activate The Limulus Reagent Reaction And The Application Study Of Its Extinction Spectroscopy

Objective:Carbon nanotubes have multiple potentials in biomedical application,which can be used as delivery vehicle for cancer treatment and imaging diagnosis.Endotoxin contamination is inevitable during the preparation of carbon nanotubes,and endotoxin can interfere with the biological effects of carbon nanotubes.China Food and Drug Administration(CFDA)has very strict requirement for the endotoxin content in medical devices used clinically.Therefore,endotoxin testing is a key factor for evaluating carbon nanotubes biological effect and potential medical application.The LAL assay is recommended by the CFDA,the International Organization of Standardization(ISO)and the International Pharmacopoeia for specific detection of endotoxin in vitro.Whether LAL assay can be applied for the detection of endotoxin in carbon nanotubes has not been reported in the literatures.The interaction of carbon nanotube with protein molecules in LAL assay is not clear yet.Hence the aim of this work was to investigate the interactions of multi-walled carbon nanotubes with the LAL assay and the potential mechanism.Methods:Oxidized multi-walled carbon nanotubes(O-MWCNT)was treated at 250?for 45 min to eliminate any possible endotoxin prior to be dispersed in water.The O-MWCNT was dispersed in endotoxin-free water with the aid of ultrasonicaton.The supernatant of the dispersion was collected(Os-MWCNT).The Os-MWCNT was analyzed by UV-Vis-NIR absorption spectroscopy in wavelength range of 200-800 nm.The average hydrodynamic diameter and zeta potential of Os-MWCNT was mesured by Dynamic Light Scattering(DLS).The interactions of Os-MWCNT with LAL were studied with gel-clotting and end point chromogenic LAL assay.The mechanism of zymogen activation was analyzed by SDS-PAGE.In our previous work,the application of the UV-vis-NIR absorption spectroscopy for characterization of the aqueous dispersed MWCNT was established.Three commercialized MWCNT dispersions were obtained and their UV-vis-NIR spectra were measured.The characterized UV-vis-NIR absorption spectra for MWCNT were resolved into three peaks by Origin software.Results:The characteristic absorption of Os-MWCNT at 200-300 nm was displayed and its intensity of the absorbance was dependent on the concentration of Os-MWCNT.The position of the peak was related to the size and the oxidation degree of MWCNT.The UV-vis-NIR absorption spectroscopy of MWCNT provided a way to distinguish different MWCNTs.The average hydrodynamic diameter of Os-MWCNT was about 150 nm and the zeta potential was about-30 mV.The Os-MWCNT activated gel-clotting and end point chromogenic LAL assay in a concentration dependent manner.The Os-MWCNT interfered with the detection of known endotoxin by gel-clotting LAL assay.The Os-MWCNT induced the conversion of coagulogen to coagulin when its concentration was higher than 0.01?g/mL.The activation of LAL assay induced by Os-MWCNT was retarded by the BS adsorption.The Os-MWCNT relied on the cascade of proteases in LAL assay to cause gel formation.Conclusion:The endotoxin-free Os-MWCNT activates the cascade reaction of the enzyme in LAL assay through surface adsorption and induced the positive reaction of the gel-clotting and the end point chromogenic LAL assay resulting in a "false positive" result.The positive reaction is related with the concentration of Os-MWCNT.The Os-MWCNT also interferes with the detection of endotoxin by LAL assay,resulting in high endotoxin values.The position of the peak was related to the size distribution and the oxidation degree of MWCNT.The UV-vis-NIR spectroscopy of MWCNT can help distinguish MWCNT and provide characterization of physicochemical property.