Study On Thrombosis And Mechanism Of Thrombolysis Reagent Based On Magnetic Nanoparticles And Microfludic Device

Thromboembolic diseases is the major cause of threatening human health, it's of great urgency to study it as the morbidity is increasing every year. In order to strengthen the function and drug targeting of thrombolytic reagents, we conjugated two thrombolytic emzyms (nattokinase and lumbrukinase) to magnetic nanopartiles, optimized the conjugation conditions, characteristiced the products properties and tested the thrombolytic activity. In addition, a microfluidic chip was used to mimic the in vivo conditions of thrombosis in vessel, also the thrombolysis process was analyzed at the function of NK and LK, thus realized the foundation of micro and efficient detection system in vitro and provided groundwork and method for studying thromboembolic diseases and drugs.Two important thrombolytic enzymes, nattokinase (NK) and lumbrukinase (LK), were immobilized onto fine magnetic Fe3O4 nanoparticles using 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide (EDC) as the coupling reagent, and their thrombolytic activities were studied. The Fe3O4 nanoparticles and NK- and LK-conjugated magnetic nanoparticles were characterized by transmission electron microscopy, Fourier transform infrared spectrophotometry, vibrating sample magnetometry, X-ray diffraction, and UV–vis absorption spectroscopy. Dual kinetic absorbance measurements at 405nm and 630 nm were employed to measure their thrombolytic activity. Analysis of protein amount showed that the optimum conditions for NK and LK binding to nanoparticles were respectively at a mass ratio of 2 : 1 : 1, 2 : 1 : 2 (magnetic nanoparticles : protein : EDC), and pH6.00. Thrombolytic activity assay showed that the best thrombolytic activity could reach 91.89% for NK–nanoparticle conjugates and 207.74% for LK–nanoparticle conjugates, which are much higher than the pure enzymes (NK, 82.86%; LK, 106.57%).PDMS was used as the material of making this microfluidic chip with different size, pletelet rich plasma and thrombin were injected into the channel by syringe pump under a certain flow rate, then observed the thrombosis with converted fluorescence microscope. With smaller channel the thrombus formed was big and easy to block, also needed longer time to lysis, whereas the thrombus was small and thin in the bigger channel and the thrombolysis time was shorter. Different concentrations of NK and LK were infused into the channel with formed thrombi, watched the thrombolytic process by microscope and got the pictures in real time by CCD camera. Analysis of thrombolysis time and thrombus area showed that the clot lysised faster in high concentration of the thrombolytic agents, the thrombolysis time were 21min and 12min respectively when the concentration was 2.5 mg/mL of NK and LK, the anverage thrombolysis rate of LK was higher than NK (23409.5 a.u./min and 14208.4 a.u./min respectively) at the same conditions.