Laboratory of Nanotechnology, Biotechnology and Cell Culture, Academic Center of Vitória, Federal University of Pernambuco, 55608-680, Vitória de Santo Antão, Pernambuco, Brazil.
Laboratory of Bioactive Technology, Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, 52171-900, Recife, Pernambuco, Brazil.
Protein Expr Purif. 2022 Apr;192:106044. doi: 10.1016/j.pep.2022.106044. Epub 2022 Jan 6.
This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on FeO magnetic nanoparticles (MNPs) produced by precipitation of FeCl·6HO and FeCl·4HO, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.
这项工作报道了将纤维蛋白溶解酶（FP）固定在通过沉淀 FeCl·6HO 和 FeCl·4HO 制备的 FeO 磁性纳米粒子（MNPs）上，这些 MNPs 用聚苯胺涂层并用戊二醛活化。FP 通过固态发酵获得，用 40-60%的硫酸铵沉淀，并通过 DEAE-Sephadex A50 离子交换层析进行纯化。FP 的固定化程序允许酶保留率为 52.13%。与非固定化酶相比，固定在磁性纳米粒子上的纤维蛋白溶解酶（MNPs/FP）在 40 至 60°C 和 pH 7 至 10 的温度下保持超过 60%的活性。MNPs 和 MNPs/FP 对 HEK-293 和 J774A.1 细胞均无细胞毒性。MNPs/FP 不具有溶血作用，并将 MNPs 诱导的溶血率从 2.07%降低至 1.37%。MNPs/FP 对血栓的降解表明，固定化过程保证了酶的溶栓活性。MNPs/FP 在 90 分钟内完全降解了人纤维蛋白原的 γ 链。这些结果表明，MNPs/FP 可以作为一种替代策略，通过外部磁场实现靶向释放，用于治疗心血管疾病。
