# Conjugation of Urokinase to Water-Soluble Magnetic Nanoparticles for Enhanced Thromolysis
## 3 Results and Discussions of Thrombysis Experiment In Vitro
### Thrombysis Experiment In Vitro  
Before the mimic blood vascular vessel experiment, thrombolysis induced by the prepared urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs outside the magnetic control system was first observed. Four test tubes were prepared and each tube was inserted with 3 mL of saline and 3 mg of thrombus. Then, three of the tubes were respectively added into pure PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) magnetic NPs, pure urokinase and urokinase coated magnetic NPs, at 3 mg each. A NdFeB magnet was placed under the tubes to attract the magnetic NPs to the surface of the thrombus at the bottom. Within 90 min, the color state  
Figure 7: Effect of alternating magnetic field on triggering urokinase release. of the solution was recorded with a camera (EOS 80D, Canon, Tokyo, Japan); the image was then converted to gray scale. The more thrombus dissolved in the solution, the darker the color and the lower the gray scale. Because the maximum gray level in the image channel is 255, the normalized gray level of the solution over time can be obtained by dividing the gray scale by 255, as shown in Figure 8. The red color in the test tubes gradually changed from light to deep within 90 min; accordingly, the gray level also changed from high to low. The results display that the thrombus in tube containing pure physiological saline or pure PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs solution did not change much, the thrombus in pure urokinase solution decreased partially, and the thrombus in the tube of urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs solution almost completely dissolved. Therefore, the thrombolysis speed in urokinase@PMAO-OA-Fe\({}_{3}\)O NPs solution is obviously the fastest. This may be because the static magnetic field helps to increase the concentration of urokinase on the thrombus surface and improve thrombol efficiency. The prepared urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs can directly dissolve thrombus regardless of whether urokinase is released from the NPs. Subsequently, thrombosis in vitro was performed in the mimic blood-vascular system shown in Figure 6. In each experiment, 30 mg of thrombus was placed in the working area of the magnetic control system inside the plastic tube. Then, the solutions including physiological saline, 1 mg/mL pure urokinase, 0.7 mg/mL urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs, and 1 mg/mL urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs were inserted into the tube respectively through the micro-injection pump. Saline experiment without urokinase or magnetic NPs was conducted as control. During the experiment, by inputting 2 ampere DC into the y-axis electromagnetic coil, a static magnetic field was introduced to accumulate the flowing nano-drug into the thrombus region. Meanwhile, a rotating magnetic field was applied by activating the coils of the x-axis and z-axis at an amplitude of 2 ampere and a frequency of 50 Hz, which helps trigger the release of urokinase. Thromolysis experiments in all solutions were conducted with and without the rotating magnetic field. Each experiment was repeated five times, each time lasting 90 min. Thrombus was removed from the tube every 30 min, and the wet weight of the thrombus was weighed by a precision micro-balance (FA1104J, Shanghai Sunny Henping Scientific Instrument Co., Shanghai, China). Figure 9 shows the variation of thrombus wet weight with time in different solutions. As shown, almost no mass change of saline treated thrombus was observed. This illustrates that, even if the flowing fluid exerts impact force, the thrombus cannot break down automatically. When thrombus was exposed in pure urokinase, the mass loss was 7 mg \(\pm\) 0.3 mg after 90 min. However, when 0.7 mg/mL urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs and 1 mg/mL urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs were injected, the thrombolysis amount was increased to 13 mg \(\pm\) 0.4 mg and 20 mg \(\pm\) 0.4 mg, respectively, after the same treatment time even without the action of AC magnetic field.