# Conjugation of Urokinase to Water-Soluble Magnetic Nanoparticles for Enhanced Thromolysis
## Materials and Methods
### Characterization of Magnetic Nanoparticles (NPs)  
The morphology of magnetic NPs was observed by transmission electron microscope (TEM) (JOEL JEM-2010, JOEL, Tokyo, Japan). Figure 2a,b are the TEM micrographs of PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs and urokinase-coated PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs, respectively. Neither micrograph shows much aggregation. The magnetization of the NPs was evaluated by a vibrating sample magnetometer (VSM) (JDM-13, Jilin University, Jilin, China) by changing the applied magnetic field from \(-\)150 kA/m to 150 kA/m at room temperature. The magnetization curve shown in Figure 2c indicates that the coercivity and remanence of OA-Fe\({}_{3}\)O\({}_{4}\) NPs, PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs, and urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) are all zero. The saturation magnetization of OA-Fe\({}_{3}\)O\({}_{4}\) NPs is 47.6 Am\({}^{2}\)/kg, and that of PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs and urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs are 40 Am\({}^{2}\)/kg and 34.5 Am\({}^{2}\)/kg, respectively. The structure of these NPs were studied by X-ray diffraction (XRD) (X'Pert PRO, PANalytical B.V., Almelo, The Netherlands)  
Figure 1: Schematic of synthesis process of urokinase@poly(maleic anhydride-_alt_-1-octadecylene) (PMAO)-oleic acid (OA)-Fe\({}_{3}\)O\({}_{4}\) Nanoparticles (NPs) conjugate. at diffraction angles from 20\({}^{\circ}\) to 80\({}^{\circ}\). As shown in Figure 2d, the peaks at (220), (311), (400), (422), (511), and (440) with respective crystal planes at 2\(\theta\) = 30.1\({}^{\circ}\), 35.4\({}^{\circ}\), 43.1\({}^{\circ}\), 53.4\({}^{\circ}\), 57\({}^{\circ}\), and 62.6\({}^{\circ}\) on Fe\({}_{3}\)O\({}_{4}\) (JCPDS card no. 85-1436) [30] are clearly displayed in the various stages of synthesis process, indicating that Fe\({}_{3}\)O\({}_{4}\) are embedded in these synthesized products. The spectra of urokinase-conjunted materials and NPs were also recorded with Fourier transform infrared spectroscopy (FTIR) (VERTEX 70, Bruker, Munich, Germany), as shown in Figure 2e. The 1710 cm\({}^{-1}\) peak assignable to the C=O vibration in OA is shifted to two new bands at 1540 and 1638 cm\({}^{-1}\) in OA-Fe\({}_{3}\)O\({}_{4}\) NPs, which are the asymmetric and symmetric features of OA-Fe\({}_{3}\)O\({}_{4}\) NPs. The 563 cm\({}^{-1}\) peak corresponds to the vibration of Fe-O in the crystalline lattice of Fe\({}_{3}\)O\({}_{4}\). The result reveals that the surface of NPs is covered with a shell of OA. After further conjunction, all characteristic vibrations in PMAO can be seen in PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs, and 563 cm\({}^{-1}\) peak indicating the Fe-O vibration is also found. The existence of 1710 cm\({}^{-1}\) peak both in PMAO and PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs is due to the decomposition of anhydride and the release of the -COOH. These results reflect that the PMAO is immobilized on the outermost layer of OA-Fe\({}_{3}\)O\({}_{4}\) NPs. The 2927 cm\({}^{-1}\) and 2854 cm\({}^{-1}\) assigned to the asymmetric stretch and symmetric stretch of -CH\({}_{2}\) respectively are found in all materials except pure urokinase. In addition, 563 cm\({}^{-1}\) (Fe-O) and 1778 cm\({}^{-1}\) (anhydride) in PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) are also observed. In the FTIR spectrum of urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\), three characteristic peaks of urokinase are present at 1656 cm\({}^{-1}\) (C=O), 1542 cm\({}^{-1}\) (N-H), and 1450 cm\({}^{-1}\) (amide band). They show that urokinase is immobilized on the surface of PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs. To ensure that the protein-coated magnetic NPs have a good response to magnetic field in aqueous phase, the magnetic performance of PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs in physiological saline under external magnetic field was observed by an UV-vis spectrophotometer (Nanodrop 2000, Thermo Fisher, Waltham, MA, USA) at 460 nm wavelength. The magnetic separation property of NPs solution can be confirmed by detecting its transmittance after being subjected to external magnetic field for a certain time.