Preparation Of Hemoglobin-based Oxygen Carrier Modified By Tempol And Its Physicochemical Properties

Hemoglobin-based oxygen carriers (HBOCs) is a kind of blood substitutes and can replace part of function of red blood cells, which was used to treat hemorrhagic shock, surgery and so on. However, when the HBOCs was used to ischemic perfusion, it was induced oxidative stress in body, which must be produce large amounts of reactive oxygen species substances and reactive nitrogen species substances. Among this species substances, the superoxide radical is hurt for tissue and the organs of body. It should be a important method for suppressing or eliminating superoxide free radicals, which is the solvement of the damage from the processing of ischemia reperfusion of HBOC. Piperidine nitroxides (Tempol) is a kind of a small molecule compound which with the mimetics of the enzyme SOD. It can make oxygen radicals into hydrogen peroxide and oxygen through conversion of between form of Tempol’s nitrogen-oxygen radical and form of its nitro-state, which is eliminating damage of tissue and organ with superoxide free radical. This study used of Tempol modified HBOCs to eliminated damage of oxygen stress when process of ischemia reperfusion of HBOCs.We have prepared4-carbonyl-2,2,6,6-tetramethylpiperidine-l-nitrogen radical (carbo nyl-Tempol);4-hydroxy-2,2,6,6-tetramethylpiperidine-l-nitrogen radical (hydroxy-Tempol);4-nitroxide-amino-2,2,6,6-tetramethylpiperidine-l-nitrogen radical (amino-Tempol) by2,2,6,6-tetramethyl piperidone. When cultured of vascular endothelial cells with Tempols to test cell survivals and study cytotoxic of Tempols for vascular endothelial cell. W hen the concentration of Tempols was1.2mmol/L, the cell cytotoxic for vascular end othelial cell of three Tempols have no significant differencs compered with normal co ntrol cell group that was cultured with just have culture medium.Three kinds of Tempol cross-linked with hemoglobin respectively to prepared Tempol modified HBOCs (Tempol-polyHb). We found that amino-Tempol was higher levels in the same concentration of Tempol-PolyHb compared with other kinds of Tempol. The average molecular weiht of Tempol-polyHb is383kDa by size exclusion chromatography-eighteen angle laser light scattering instrument (SEC-MALLS). Amino-Tempol content of Tempol-polyHb is44μmol/g by electron spin resonance (ESR), which the rat of amino-Tempol to Hb is3:1.To srudy the protective effect of Tempol-polyHb in oxidative stree, we have established a model of oxidative stress by culturing vascular endothelial cells wiht xanthine/xanthine oxidase. The results show that vascular endothelial cells survival rate is60%when the concentration of Tempol-polyHb is0.08g/L. This is higher than control group in same concentration which survival rate of vascular endothelial cell is40%.The above results show that amino-Tempol is not only modify easily to HBOCs, more sample prepare process of Tempol-polyHb and low toxicity of vascular endothelial cell. When the rate amino-Tempol to Hb is3:1, Tempol-polyHb is very well porctect vascular endothelial cell form oxidative stress compared with control group that is instant of polyHb in the environment of oxygen free radicals in cells. Tempol-polyHb is a way of resolve oxidative stress induced with ischemia reperfusion of HBOCs.