Construction Of A Dual-Drug Sequential Release Platform For Heparin And Urokinase Based On Double Auxiliary Of NIR-Ⅱ And Bubbles And Its Evaluation In Vivo And In Vitro

Background and objective:Venous thromboembolism（VTE）is a common and fatal disease including deep venous thrombosis（DVT）and pulmonary embolism（PE）.DVT is one of the diseases that seriously endanger human health.Its morbidity,mortality and disability are high,and acute thrombosis can easily lead to disability and even death.At present,the traditional thrombolytic drugs based on urokinase（UK）have some disadvantages,such as large dosage,short half-life,easy bleeding side effects and so on.Therefore,there is an urgent need for a more efficient method of drug thrombolysis.The nano-drug delivery platform has the characteristics of diversified size,large specific surface area,easy surface modification,large drug loading and biodegradability,which can change the drug release rate and increase the biocompatibility and safety of the drug delivery system,and improve the bioavailability of drugs and reduce their side effects.At present,the combination of nanotechnology and drug therapy has become a research hotspot and direction in the field of medicine and biomaterials.Hollow mesoporous silicon（HMSNs）has the advantages of high permeability,low density,stable thermodynamics,excellent properties and characteristics of mesoporous silicon（MSNs）,large specific surface area and pore volume,adjustable pore size,highly ordered structure,surface rich in active hydroxyl（-OH）groups,easy modification,good biocompatibility,biodegradability and so on.It is mainly used in nano-scale sensors,catalytic reactions,drug carriers,biomedical and other fields,especially as carriers are widely used in drug delivery systems.Unfractionated heparin（UH）and UK are commonly used antithrombotic drugs in clinic,and the mechanism of thrombolysis is different.Theoretically,the combination of UH and UK is a promising combination of drugs.But in fact,due to the opposite charge of UH and UK,the thrombolytic effect of direct combination is not good.In order to change the disadvantage of charge repulsion of these two drugs,we intend to develop a multi-functional thrombolysis platform（UK-UH@PDA@HMSNs）with double auxiliary（NIR-Ⅱ,bubbles）dual-drug sequential drug release.This sequence release can successfully solve the problem that UK and UH are difficult to be directly mixed and combined because of charge repulsion.The purpose of this study was to investigate the characterization,photothermal properties,drug loading,drug release and biocompatibility of PDA@HMSNs,and to evaluate the thrombolytic efficacy and biosafety of UK-UH@PDA@HMSNs nano-thrombolytic composites in vivo and in vitro.Methods:1.Based on the comprehensive comparison of various reported methods for preparing HMSNs,we chose a cheap,simple and practical method to prepare biodegradable HMSNs by combining oil-water double reaction with template.After loading UK,polydopamine（PDA）film was formed on the surface of HMSNs by interfacial polymerization.Finally,UH was loaded on the outside of PDA film to form UK-UH@PDA@HMSNs nano-thrombolytic complex.A series of morphologies and structures of HMSNs and PDA@HMSNs were characterized by scanning electron microscope（SEM）,transmission electron microscope（TEM）,atomic force microscope（AFM）,nano-particle size potentiometer,specific surface area and porosity tester（BET）,Fourier transform infrared spectrometer（FTIR）,X-ray diffractometer（XRD）,X-ray photoelectron spectrometer（XPS）,thermogravimetric analyzer（TGA）.2.The performance and biosafety of PDA@HMSNs were further studied by photothermal heating test,UV-vis spectrophotometer,drug loading and release test,hemolysis test,cytotoxicity test,living-dead cell staining and other experiments.3.The thrombolytic performance and biological safety of UK-UH@PDA@HMSNs were evaluated comprehensively by thrombolysis test in vitro,thrombolysis test in vivo,blood biochemical analysis and Hype staining of important tissues and organs.Results:1.SEM results showed that HMSNs had a porous structure,and the corresponding TEM results showed that HMSNs was a hollow sphere with a huge cavity.AFM further studied the morphology and three-dimensional structure of PDA@HMSNs and HMSNs.The diameter of PDA@HMSNs is about 20 nm than that of HMSNs.After PDA was coated on the surface of HMSNs,the zeta potential of HMSNs changed from-6.86 mV to 2.68 mV.The average hydrodynamic diameters of HMSNs and PDA@HMSNs were 251.37±8.30 nm and 601.77±85.88 nm,respectively.Compared with CTAC/m Si O₂@s Si O₂,the specific surface area and pore volume of HMSNs were significantly increased.The results of FTIR,XRD,XPS and TGA further illustrated that PDA had been successfully modified on the surface of HMSNs.2.In vitro studies had found that PDA@HMSNs had a good photothermal heating effect.According to the UH standard curve and the drug loading rate formula,the drug loading rate of UH in UK-UH@PDA@HMSNs was 16.15±1.39%.According to the urokinase kit and the drug loading rate formula,the drug loading rate of UK was 28.68±3.96%.Within 40 min of NIR-II irradiation,the release rate of UK increased significantly,about 80.67%.However,without NIR-II irradiation,the cumulative release rate of UK-UH@PDA@HMSNs dropped to 17.33%.Under the excitation of NIR-II,UH released about 81%in 25 min,but without NIR-II irradiation,UH only released about 20%in 30 min.The results of hemolysis experiments,cytotoxicity experiments,and live-dead cell staining experiments showed that PDA@HMSNs had good cell compatibility and low toxicity.3.The results of in vitro thrombolysis experiments showed that the thrombolytic effect of the UK after UH group was better than that of the UK group,with a significantdifference（***p<0.01）.The thrombolysisrateof UK-UH@PDA@HMSNs+NIR-II group was significantly higher than that of UK+NIR-II group（***p<0.001）.The results of in vivo thrombolysis experiments showed that UK-UH@PDA@HMSNs+NIR-II had a better thrombolytic effect than UK+NIR-II（**p<0.01）.Finally,we conducted an in vivo biosafety assessment on SD rats.The results showed that after SD rats were injected with UK-UH@PDA@HMSNs,the H&E stained images of major organs did not show any histopathological abnormalities such as inflammation or tissue damage.phenomenon.The liver and kidney function and blood coagulation function of rats were within the normal range,and there were no side effects such as bleeding during the treatment,indicating that the thrombolytic system had certain biological safety in the body.Conclusion:In summary,for the treatment of vascular embolic diseases,especially for lower limb DVT,the efficiency and biosecurity of drug based thrombolysis system are important factors directly related to the rehabilitation quality of patients.In this paper,a multi-functional thrombolysis platform（UK-UH@PDA@HMSNs）with double physical assistance（NIR-II and bubbles）and dual-drug sequential release was developed.Both in vivo and in vitro studies had confirmed that UK-UH@PDA@HMSNs had a safe and efficient thrombolytic effect under the excitation of NIR-Ⅱ.