Investigation On Thrombin-Responsive Nanoparticles For Accurate Thrombolysis And Improvement Of Thrombus Microenvironment

Nowadays,cardiovascular diseases led by acute myocardial infarction and stroke have become the first killer endangering human health.Thrombosis is the direct cause of acute myocardial infarction,stroke and pulmonary embolism.So,it is particularly important to administer thrombolytic therapy immediately to realize vascular recanalization and restore blood supply.However,traditional fibrinolytic drugs show poor tissue specificity,short half-life,and they have systemic bleeding complications which could even be life-threatening in severe cases.Therefore,the targeted delivery and controlled release of fibrinolytic drugs is an urgent problem to be solved.In this study,a thrombosis microenvironment-responsive nano-drug delivery system（NDDS）was developed,which used flowerlike mesoporous manganese dioxide（MnO₂）nanoparticles as carriers and natural polysaccharide fucoidan（Fuco）as the encapsulating and targeting agent.Fuco was modified on the surface of MnO₂ by a thrombin-responsive peptide sequence of GGLVPRGFGG（peptide,pep）.Finally,the fibrinolytic drug urokinase（uPA）was loaded to obtain MnO₂/uPA@pep-Fuco NDDS which was used for accurate thrombolysis and improvement of the inflammatory thrombus microenvironment.In this study,flowery mesoporous MnO₂ nanoparticles were synthesized by the template method,and their surface was modified by carboxylation.In order to endow the MnO₂ nanoparticles with thrombus targeting ability,we chose Fuco as the targeting material.The thrombin-responsive peptide of pep was grafted onto the skeleton of Fuco by Yamaguchi reaction to get pep-Fuco.Then,pep-Fuco was modified to the surface of MnO₂-COOH nanoparticles via amide bonds formed by-COOH groups of MnO₂-COOH NPs and-NH2 groups of pep-Fuco.Finally,MnO₂@pep-Fuco was loaded with uPA under the condition of ultrasound at 4 °C to obtain the final MnO₂/uPA@pep-Fuco NDDS.Fuco could simulate P-selectin Glycoprotein Ligand 1（PSGL-1）and thus enabled MnO₂/uPA@pep-Fuco to specifically bind with P-selectin overexpressed by activated platelets at thrombus sites.Upon arrival at thrombus sites,pep could be cleaved by abundant thrombin,causing the decomposition of MnO₂/uPA@pep-Fuco and achieving the controlled release of uPA.In addition,based on the H₂O₂-like enzyme activity of MnO₂,the NDDS could effectively remove the high level of H₂O₂ at thrombus sites,which improved the inflammatory thrombus microenvironment and realized synergistic thrombolytic therapy.In vitro experiments showed that MnO₂/uPA@pep-Fuco was successfully prepared with encapsulation and drug loading rates of 14.60% and22.60%,respectively.In vitro drug release experiments showed that there was a significant difference in drug release behavior of MnO₂/uPA@pep-Fuco under different media.Compared with the PBS buffer（simulated normal physiological environment）,the release rate of nano-agents in the PBS buffer containing 10 U/m L thrombin（simulated thrombus microenvironment）was significantly faster,indicating that the pep-Fuco coating endowed MnO₂/uPA@pep-Fuco NDDS with a thrombin-responsive characteristic.In vitro live-dead cell double staining experiment showed that MnO₂@pep-Fuco had little effect on Human umbilical vein endothelial cells（HUVEC）during the concentration range of 1 ～ 30 μg/m L.The fluorescence co-localization experiments showed that,compared with MnO₂-COOH,the adhesion of MnO₂@pep-Fuco to activated platelets was obviously enhanced by the modification of Fuco,which resulted in more nanoparticles entering activated platelets.In vitro H₂O₂ detection results showed that MnO₂@pep-Fuco could remove H₂O₂ produced by damaged endothelial cells and activated platelets and effectively reduce the H₂O₂ level.The clearance of H₂O₂ improved the inflammatory microenvironment of thrombus and was expected to hinder thrombosis formation and dilation.In vitro thrombus adhesion experiments showed that MnO₂/uPA@pep-Fuco could be specifically enriched on platelet-rich thrombi due to ligand-receptor interactions between Fuco and P-selectin.In vitro thrombolysis experiments showed that MnO₂/uPA@pep-Fuco exhibited weak thrombolytic ability in the absence of thrombin.When exposed to 10 U/m L of thrombin,the thrombolytic rate of MnO₂/uPA@pep-Fuco reached 92.23% after 2 h and the thrombus was completely dissolved after 4 h.This indicated that MnO₂/uPA@pep-Fuco with thrombin responsive drug release characteristics could achieve efficient thrombolysis in vitro.The FeCl₃-induced carotid thrombosis of SD rats were used as the models for in vivo experiments.The optical imaging results showed that the fluorescence intensity of left carotid artery thrombus was 3.43 times that of right normal carotid artery after intravenous injection of MnO₂/IR783@pep-Fuco for 30 min.However,there was no significant difference in fluorescence intensity between the left and right carotid arteries of the MnO₂-COOH/IR783 and the free IR783 group,indicating that only MnO₂/IR783@pep-Fuco could actively recognize and bind to P-selectin overexpressed by activated platelets at thrombus sites.In vivo thrombolysis results showed that the thrombolytic efficiency of MnO₂/uPA@pep-Fuco was significantly increased compared with the free uPA.And the thrombus closure rate of MnO₂/uPA@pep-Fuco decreased to 5.99 ± 5.07% after 2 h of administration,demonstrating its superior thrombolytic ability.In addition,the tail bleeding analysis showed that the tail bleeding time of MnO₂/uPA@pep-Fuco group was effectively reduced to 33 ± 8 s,indicating a lower risk of bleeding complications.Finally,we investigated the serum biochemical indexes levels and tissue histopathological changes of rats after treatment of different nanoparticles.The results proved that MnO₂/uPA@pep-Fuco NDDS had good safety for application in vivo.