Study On Preparation Of Nattokinase Magnetic Nanoparticles And Its Antithrombotic Effect

Nattokinase（NK）is an alkaline protease with a strong fibrinolytic effect.It is produced by fermenting natto from Bacillus subtilis natto.Studies have shown that NK has a good lytic effect on thrombus,and has the advantages of being able to be taken orally and safety.The thrombolytic mechanism of NK is diversified,especially sensitive to fibrin in the thrombus state,which can be directly thrombolytic.And tissue-type plasminogen activator（t-PA）,urokinase and plasmin were produced to achieve indirect thrombolytic effect.In this paper,magnetic nanoparticles with sustained-release effect were prepared to load NK produced by a high-yield NK strain（Bacillus subtilis natto LNUB236）isolated in our laboratory.Characterization of NK magnetic nanoparticles and evaluation of their antithrombotic and thrombolytic effects provided a theoretical basis for the development of NK drugs.Magnetic nanoparticles were prepared by a co-precipitation method.Fe Cl₃·6H₂O and Fe Cl₂·4H₂O were added to the carboxymethyl chitosan solution and stirred for 10 minutes to obtain a mixed solution.25%ammonia was added.Under the protection of nitrogen,reactants were stirred in the oil bath（60°C for 1 hour）,then cooled to room temperature and deposited overnight.Then,dialysis was performed with gentle stirring in distilled water（dialysis bag:cut-off molecular weight 800-14000）.Sodium alginate was added to the solution.After stirring for 24 h,the solution passed through a 0.22μm filter.Magnetic nanoparticles can be obtained after freeze-drying.The diffraction of x-ray showed five characteristic peaks of 220,311,400,422,511,and 440,which were consistent with the standard Fe₃O₄.Infrared Spectroscopy showed that carboxymethyl chitosan and sodium alginate have successfully modified Fe₃O₄nanoparticles.The particle size and the zeta potential were measured by the particle size analyzer,and it was further confirmed that the magnetic material was synthesized.The magnetic material and NK were dissolved in distilled water.Added 1-hydroxybenzotriazole（HOBT）and 1-ethyl-（3-dimethylaminopropyl）carbodiimide hydrochloride（EDCI）to the solution,and wrapped with tin foil to avoid the effects of light.Then,stirred in a shaker for 24h.Finally,dialysis was performed to remove unattached NK.The main factors in the preparation process were investigated through a single factor,and the optimal prescription was determined by orthogonal experiments.Infrared spectroscopy and particle sizing systems showed that NK was successfully loaded.The antithrombotic and thrombolytic effects of NK magnetic nanoparticles were determined by in vitro and in vivo pharmacodynamics.In vitro pharmacodynamics,the cumulative release in a solution with a p H of 7.4 in 1 h is less than 40%,indicating there is no sudden release;the release in 72 h exceeds 70%,indicating that it can be completely released.In the magnetic property test,the light transmittance at 24 h was 87.84%,and the magnetic property was good.The experimental results on blood clot dissolution showed that the blood clot had no obvious change within 24 h in the saline group and the magnetic material group.The free NK group could completely dissolve the blood clot in about 10 h.The NK magnetic nanoparticle group can completely dissolve the blood clot in about 21 h,further indicating that it has a sustained-release effect.In the hemolysis test,the hemolysis rate of red blood cells increased with the concentration of the NK magnetic particles.When the enzyme activity was 50 to 150 IU/m L,the hemolysis rate of red blood cells was less than 5%,and there was no hemolysis.In the study of in vivo pharmacodynamics,SD rats were used as research objects.In the test of tail bleeding time,after 5 days of administration,the cross-section was cut at 6 mm from the tip of the rat tail.Compared with the blank control group,the bleeding time of the urokinase group,the free NK group,and the NK magnetic nanoparticle group was significantly increased,and the NK magnetic nanoparticle group were also significantly different from the urokinase group（P<0.001）.In the thrombus length of rat tail test,after administration for 5 days,4%carrageenan was injected into the back toes of SD rats to prepare rat tail thrombus.Then,the administration continued for 2 days and the thrombus length was recorded.Compared with the control group,the urokinase group can significantly reduce the length of tail thrombus in SD rats（P<0.001）,the free NK group（P<0.0001）and the NK magnetic nanoparticle group（P<0.0001）;Compared with the urokinase group,the length of tail thrombus of the NK magnetic nanoparticle group was significantly shorted（P<0.001）.It can be seen that NK magnetic nanoparticles have good antithrombotic and thrombolytic effects.