Study On Succinate Dehydrogenase-like Activity Of Fe₃O₄ Nanoparticles

Magnetic iron oxide nanoparticles are widely used in in biomedical fields such as magnetic resonance imaging,magnetothermal therapy,magnetic targeting drug delivery,due to their magnetic responsiveness,excellent biocompatibility,diversified modification as well as size and morphology controllability.In the past 15 years the study of nano-enzyme based on iron oxide nanoparticles,in particular,peroxidase and catalase have also greatly advanced its application in in vitro detection.Recently,Fe₃O₄ nanoparticles cytotoxicity was studied by thiazolyl blue tetrazolium bromide（MTT）assay in our group,it was found that MTT were reduced to formazan in the present of Fe₃O₄ nanoparticles,suggesting that Fe₃O₄nanoparticles may act as succinate dehydrogenate enzyme.Succinate dehydrogenase（SDH）is a function enzyme oxidizing succinate into fumarate in a tricarboxylic acid cycle.Because of succinate dehydrogenase activity in vivo related to the cancer occurrence,it is of importance to explorer whether Fe₃O₄ nanoparticles can mimic the succinate dehydrogenase activity as a nanozymes.Herein,18 nm Fe₃O₄ nanoparticles in oil phase were prepared by size controllable thermal decomposition,and water-soluble Fe₃O₄ nanoparticles were obtained by ligand exchange method;Then,the succinate dehydrogenase in the mitochondrial were extracted and some detection methods were developed to evaluate its dehydrogenase activity.Finally,the developed detection methods were used to evaluate the Fe₃O₄ nanoparticles whether can mimic succinate dehydrogenase activity;the specific results are as follows:（1）Preparation,characterization,and surface modification of 18 nm Fe₃O₄ nanoparticles:thermal decomposition with precursor hydroxy iron oxide were used to prepared the oil phase nanoparticles.After exchanged by the ligand of 3,4-dihydroxyphenylpropionic acid（DHCA）,Fe₃O₄ nanoparticles were water soluble.TEM,XRD,FTIR and DLS are used to characterize the nanoparticles.The experimental data show that the preparation of 18 nm of Fe₃O₄ nanoparticles size is uniform,good crystallization,and the Fe₃O₄ nanoparticles in water have a hydrated particle size of 33 nm with good stability.（2）Extraction of SDH and Validation SDH activity:Since SDH is unstable after being isolated SDH has no commercial product available.It is necessary to be used or detected immediately after extraction for SDH.After extracted from non-small cell lung cancer cell A549,the activity of SDH was evaluate by the commercial kit（PMS assay with phenazine methyl sulfate and 2,6-dichloroindoxyl）.Beside the PMS assay,the extracted SDH activity was also determined by potassium ferricyanide assay and tetrazolium salt MTT method.the activity of SDH evaluated by PMS assay was 0.0385 U/10⁴ cell,while that of potassium ferricyanide assay was 0.050 U/10⁴ cell.The optical density of formazan produced by SDH in MTT assay in 3×10⁴ live cells was 1.025±0.062.Finally,succinate and fumarate were quantitatively detected by HPLC to evaluate the activity of SDH.The retention times of succinate and the reduced product fumarate in HPLC were 5.36 min and 6.08 min,respectively.Quantitative detection showed that the conversion rates of succinate in the reaction systems of succinate dehydrogenase by PMS assay and potassium ferricyanide assay were 92.2%and 99.8%,respectively,but fumarate could not be detected.（3）Investigation of SDH-like activity of Fe₃O₄ nanoparticles:PMS assay,potassium ferricyanide assay and MTT assay were used to evaluate whether Fe₃O₄ had SDH-like activity by detecting absorption of DCPIP at 600 nm,Pusher Blue at 685 nm and formazan at 550 nm and HPLC detection.The PMS assay showed that no obvious catalytic reaction of Fe₃O₄ nanoparticles was observed,In the potassium ferricyanide assay,it was found that the potassium ferricyanide can react Fe₃O₄ nanoparticles to produce Turnbull’s blue in the control group,indicating that the potassium ferricyanide assay is not suitable for Fe₃O₄nanoparticles.It was found the absorbance of formazan at 550 nm reduced from MTT in the presence of Fe₃O₄ nanoparticles is higher than that without Fe₃O₄ nanoparticles,but no fumarate was detected in HPLC.