Clot Structure-based Physical-matching Design Of Platelet Cloaking Nano-delivery System Facilitates Specific Arteriovenous Thrombolysis

Thrombus,a pathologic product of a solid mass(blood clot),can lead to myocardial infarction,ischemic cerebral infarction and other cardio-cerebrovascular diseases.From the 1990 s to the present,prevalent cases suffering cardiovascular diseases increase year by year,nearly doubled from 271 million in 1990 s to 523 million in 2019,and the death from 12.1 million to 18.6 million,which account for global mortality.Thrombus is a blood clot that forms in the circulation system and blocks the flow of blood.The blood clot usually consists of insoluble fibrin and blood cells(i.e.,red blood cells,white blood cells and platelets).According to the anatomical position of thrombosis,it can be divided into arterial thrombosis and venous thrombosis.Venous thrombosis mainly composes a large number of red blood cells,fibrin and a small number of white blood cells,while arterial thrombosis mainly composes a large number of platelets and fibrin.In addition to the differences in compositon,the abundant active platelets contact and drive the structure of fibrin network,which compress the whole thrombus and make arterial thrombus denser and lower porosity than venous thrombus.At the same time,the shear stress at the site of arterial thrombus is very high,compared with whichat the site of venous thrombus is low.Differences in the composition,structure and pathological environment of thrombus lead to different clinical strategies for thrombus treatment.The treatment of arterial thrombosis mainly uses anti-platelet drugs,such as Abciximab,while the treatment of venous thrombus usually adopts anti-coagulant strategies targeting the coagulation cascade,such as low molecular weight heparin,etc.The complexity of the thrombus treatment strategies and the risk of bleeding bring great difficulties to clinical application.The different physiology and fluid parameters(i.e.,shear stress,blood flow velocity)lead to varied accumulation of the nanoparticle in blood vessel.All above suggests that a specific delivery system for thrombus in arteries and veins respectively may achieve a better therapeutic effect.Nano-drug delivery systems have the advantages of improving the targeting of drugs for thrombus and reducing the risk of bleeding.However,most nano-drug delivery systems designed for single-structure thrombus and lacked of effectiveness in the treatment of other types or structures of thrombosis.In order to realize the specific treatment of arteriovenous thrombus by nano-drug delivery system,this study designed a set of physically matched thrombolytic drug-delivery system based on the differences of arteriovenous thrombus to achieve the specific treatment.The system consists of three monodisperse mesoporous silica nanoparticles of different sizes,platelet membranes and urokinase(termed UNPs).The main results are as follows:1.Construct a successful physically-matched platelet membrane cloaking nano-delivery system.In this study,monodispersed mesoporous silica nanoparticles(MSNs)were prepared by template method.The particles were spherical and uniform by scanning electron microscope(SEM),and their sizes were ～40nm(MSN-S),～70nm(MSN-M)and ～110nm(MSN-L),respectively.The platelet membrane was then coated on the surface of the nanoparticles using ultrasound.The hydrodynamic particle size of platelet membrane cloaking nanoparticles increased slightly and the surface potential is close to that of platelet membrane.At the same time,transmission electron microscopy(TEM)observed platelet membrane cloaking particles have obvious core-shell structure.These results jointly proved the success of platelet membrane encapsulation.SDS-PAGE Coomash Blue staining results showed that the location and number of platelet membrane protein bands on the source platelet membrane were basically the same as those on the nanoparticles,indicating good protein integrity on the platelet membrane cloaking particles.Next,the platelet membrane cloaking nanoparticles loaded with urokinase,UNPs,were prepared,and their drug loading efficiency and encapsulation efficiency were detected.The results showed that the drug loading efficiency of UNP-S,UNP-M and UNP-L were 34.66 ±0.99%,29.04 ± 0.92% and 14.78 ± 1.47%,respectively.The encapsulation efficiency of UK was 25.41 ± 1.12%(UNP-S),20.48 ± 0.92%(UNP-M),and 8.69 ±1.00%(UNP-L),respectively.The UK release profiles in PBS were detected.After 4hours,more than 50% UK was released from the all three formulations.2.Systematically evaluate the safety of platelet membrane cloaking nano-delivery system.This study verified the safety of platelet membrane cloaking nanoparticles from the aspects of cell survival rate,blood compatibility,liver function and coagulation system function.(1)The results of cell survival experiment showed that platelet membrane cloaking nanoparticles did not affect the cell survival of human umbilical vein endothelial cells(HUVECs),hepatoma cells(Hep G2)and normal liver cells(L-02).The results of apoptosis assay showed that platelet membrane cloaking nanoparticles could not induce hepatocellular carcinoma cell apoptosis.(2)Morphology of red blood cells and hemolysis test showed that red blood cells were uniform in size and good in shape,and no significant hemolysis occurred,which proved that platelet membrane cloaking nanoparticles had good blood compatibility.(3)The expression level of endothelin(ET-1)in HUVECs and the changes of activated partial thrombin time(APTT)and thrombin time(TT)in plasma were detected.The results showed that platelet membrane cloaking nanoparticles had no significant effect on the function of HUVECs and did not cause significant changes in coagulation function.(4)The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)did not change significantly,which proved that platelet membrane cloaking nanoparticles could not induce liver injury.Combined with these results,platelet membrane cloaking nanoparticles have good biosafety and can be used as a drug delivery system in the treatment of thrombus.3.Investigate the metabolism and biological distribution of platelet membrane cloaking nano-delivery system.Plasma was collected at different time points after i.v.injection of fluorescent-labeled platelet membrane cloaking nanoparticles.The fluorescence intensity of platelet membrane cloaking nanoparticles was detected and the blood half-life was calculated.The blood half-life of MSN-S,MSN-M and MSN-L were 4.91 hours,4.42 hours and 3.81 hours,respectively.The blood half-life of PNP-S,PNP-M and PNP-L were 9.19 h,7.08 h and 6.15 h,respectively.Subsequently,the main organs(heart,liver,spleen,lung and kidney)of the rats were collected at 90 min and 24 h respectively for fluorescence imaging ex vivo.The results showed that the platelet membrane cloaking nanoparticles were mainly distributed in liver,kidney and lung at 90 min.Compared with the fluorescence imaging results at90 min,the fluorescence intensity of liver,kidney and lung showed a downward trend at 24 h,indicating that platelet membrane cloaking nanoparticles can be metabolically cleared from the body.The reason is that mesoporous silica nanoparticles can be degraded into silicic acid in physiological environment and be excreted with urine.And platelet membrane encapsulation significantly reduced the uptake of nanoparticles by mouse macrophage(RAW264.7).In conclusion,platelet membrane cloaking nanoparticles could reduce the uptake of macrophages,delay the entry into major organs(such as liver and kidney)for metabolic clearance,thus prolonging blood circulation time of platelet membrane cloaking nanoparticles.4.Evaluate the enrichment effect of platelet cloaking nano-delivery system in red/white blood clots.In this study,the enrichment of platelet membrane cloaking nanoparticles in red/white blood clots was first verified under static conditions in vitro.The results showed that the permeability of platelet membrane cloaking nanoparticles decreased with the increase of particle size in red blood clots.PNP-S showed better permeability.In white blood clots,PNP-S and PNP-M could penetrate into the interior of blood clots to varying degrees.However,the fluorescence signal of PNP-S was stronger than that of PNP-M,and PNP-L was only distributed on the outer surface of the blood clot.Then,carotid arterial thrombus and deep vein thrombosis(DVT)models in rats were prepared for in vivo verification.The results showed that in the X DVT model,PNP-L showed stronger thrombotic permeability,which was inconsistent with the results under static conditions in vitro.In the carotid arterial thrombus model,PNP-S also showed stronger enrichment ability.In order to explore the reasons affecting the enrichment ability of platelet membrane cloaking nanoparticles in vivo and in vitro,this study conducted in vitro experiments on platelet membrane cloaking nanoparticles enrichment ability in red/white blood clots under different pressures.The results showed that under the condition of simulating the shear stress at the site of venous thrombus in vitro,the enrichment capacity of PNP-L in blood clots was stronger.Under the condition of simulating the shear stress at the site of arterial thrombus in vitro,the enrichment ability of PNP-S in blood clots was stronger,which was consistent with the results in vivo.In conclusion,the lower shear stress at the thrombus site makes the larger platelet membrane cloaking nanoparticles(PNP-L)easier to be captured in the larger occluded venous thrombus.In the denser,smaller occluded arterial thrombus,the large flow shear stress drives the enrichment of smaller platelet-membrane cloaking nanoparticles(PNP-S)in the arterial thrombus.5.Evaluate the thrombolytic effect of platelet membrane cloaking nano-delivery system.In this study,the rapid and long-term thrombolytic effects of platelet membrane cloaking nanoparticles were evaluated.(1)In the rapid thrombolytic experiment,the thrombolytic results of DVT model showed that the three groups of UNPs with different sizes had a certain degree of thrombolytic effect,and the thrombolytic effect of UNP-L was slightly better than that of the other two groups of UNPs.In the rat carotid arterial thrombus model,three groups of UNPs of different sizes showed similar thrombolytic effect to the low-dose urokinase group(100U/g),and the thrombolytic effect of PNP-S was slightly better than that of the other two groups of UNPs.(2)In the long-acting thrombolytic experiment,the thrombolytic results of DVT model showed that UNP-L could significantly reduce the weight of thrombus,reduced the degree of vascular closure,and showed better thrombolytic effect.In the rat carotid arterial thrombus model,UNP-S could significantly reduce the weight of thrombus,reduced the degree of vascular closure,and showed better thrombolytic effect.Based on the above experimental results,the platelet membrane-coated nano-drug delivery system could reduce the uptake of macrophages,avoid the clearance of reticuloendothelial system and prolong the blood half-life through platelet membrane coating.At the same time,this delivery system had good biocompatibility,did not affect the vascular endothelial function,liver function and coagulation system,and reduced the side effects of urokinase.We also found that different sizes of platelet membrane cloaking nanoparticles had different enrichment ability in vivo and in vitro in arteriovenous thrombosis.The effect of blood flow shear force on the enrichment ability of nanoparticles of different sizes in arteriovenous thrombosis was verified under different pressure conditions in vitro.The relatively loose structure of venous thrombus traps relatively larger nanoparticles(PNP-L)under the action of low flow shear stress.The dense structure of arterial thrombus makes it difficult for cloaking particles to penetrate,but driven by high blood flow shear force,relatively smaller nanoparticles(PNP-S)are enriched in arterial thrombus.Therefore,UNP-L and UNP-S have better long-termed thrombolytic effect in animal models of venous thrombus and arterial thrombus,respectively.In conclusion,nano-delivery systems coated with platelet membrane of different sizes were constructed in this study,which physically matched the characteristics of arteriovenous thrombus and realized the specific thrombolytic therapy of nano-drug delivery system.This provides a reference for the design of nano-delivery system in arterial and venous thrombolytic therapy.