# Conjugation of Urokinase to Water-Soluble Magnetic Nanoparticles for Enhanced Thromolysis
## 1 Introduction  
Thrombosis is a serious disease that threatens the lives of people all over the world [1]. Although research on rapid thrombolysis has progressed in recent decades [2, 3], the treatment of possible hemorrhage by using large dosage of thrombol drugs (e.g., urokinase and streptokinase) continues to greatly restrict the improvement of medical care [4, 5]. It remains an urgent task to develop new technologies for the treatment of such a complicated disease and relieve the discomfort of patients [6, 7]. Nanomaterials are considered an emerging trend with future prospects in biomedical applications because of their possible enhanced physical, chemical, and biological functionality owing to high surface-to-volume ratios and surface charges [8]. Recently, magnetic nanoparticles (NPs) have gained significant attentions for their applications in magnetic resonance image (MRI) [9, 10], cell separation [11, 12], and gene therapy [13]. Magnetic NPs have also been used as drug carriers for targeting therapies, such as cancer [14, 15], cardiovascular diseases [16], and infectious diseases [17].  
Magnetic carrier drugs can be guided to accumulate at specific sites under the action of external magnetic field to improve the drug concentration at the lesion locations and reduce the adverse side effects on the normal parts. However, two risk factors, hidden within these drug delivery methods for cardiovascular diseases, such as thrombotic diseases [18; 19; 20], deter expectation for clinical use. One is the possible toxicity introduced by chemicals that may damage endothelial tissue and cells [21; 22]; the other is the micron scale of particles that may form new blockages in certain capillaries [23; 24]. In this study, a new synthesis of a nanoscale magnetic drug that has good biocompatibility and low cytotoxicity is proposed for targeted therapy of thrombus. The magnetic carrier drug can be guided by magnetic field for rapid thrombolysis and controlled by oscillating magnetic field for drug release.  
Efforts have been made to improve the thrombol efficiency by binding thrombol drug to the surface of magnetite NPs [18; 25; 26; 27; 28]. In these studies, only a static magnetic field is introduced to guide and accumulate the urokinase-coated NPs for targeted thrombolysis. In our novel, magnetic carrier drug delivery system, Fe\({}_{3}\)O\({}_{4}\) NPs are first prepared with an oleic acid (OA) coating to form a hydrophobic surface. Amphipathic copolymer poly(maleic anhydride-_alt_-1-octadec) (PMAO) is then chemically bonded to OA-Fe\({}_{3}\)O\({}_{4}\) NPs to provide carboxyl groups, and the hydrophobic surface of OA-coated NPs is transformed into hydrophilic characterization. Next, urokinase is covalently bound to the magnetic NPs modified by PMAO under the action of \(N\)-Ethyl-\(N^{\prime}\)-(3-dimethylaminopropyl) carbodiimide (EDC) and \(N\)-hydroxysuccinimide (NHS). In consideration of the therapeutic function of magnetic carriers in treating thrombosis, the presence of PMAO is considered to be an advantage for providing a large number of carboxyl groups for drug loading. In addition, nano-drugs can effectively release urokinase under the action of alternating magnetic field, which is helpful to improve thrombol efficiency. The prepared urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs are very stable in maintaining the protein drug urokinase on carriers. In vitro thrombolysis experiments show that the thrombolysis efficiency of urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs can be increased to about four times of pure urokinase by using the coupling magnetic field composed of static magnetic field and alternating magnetic field. This indicates that effective thrombol effect can be achieved even if the proposed magnetic carrier is used at a lower dose. Therefore, the application of magnetically controlled urokinase@PMAO-OA-Fe\({}_{3}\)O\({}_{4}\) NPs is expected to be a solution for the treatment of thrombotic diseases in the future.