The Application Of Thrombus Targeted Liposomes In Thrombolysis Therapy

The ischemic stroke,myocardial infarction and venous thromboembolism caused by thrombosis are the three killers of cardiovascular and cerebrovascular diseases.Thrombolytic drugs are usually applied in the clinical treatment of thromboembolism.As a kind of cheap and good plasminogen activator,urokinase has been widely used in the clinical practice in China.However,urokinase has short half-life(about 15 min)and no thrombus targeting ability.Thus,a higher dosage of urokinase is needed in thrombolytic therapy and therefore prone to produce bleeding side effects.As a drug carrier,liposome has been widely applied in clinic for the delivery of antitumor and antifungal drugs,which can significantly improve drug efficacy while reducing toxic and side effects.The liposomes also show the advantage of good biocompatibility and facile surface modification by target ligands and are endowed with long circulating half-life in blood by PEGylation,thus serving as an appropriate drug delivery system.Platelet activation plays a key role in the formation and development of thrombus.When platelets are activated,GPIIb/IIIa forms an active dimeric form on the surface,which binds to fibrinogen in plasma,causing platelet aggregation and thrombosis.The molecules targeting the active GPIIb/IIIa can be used for the targeted delivery of drugs in vivo.The cyclic RGD peptide can target the GPIIb/IIIa on the activated platelet surface in thrombus site while does not bind to the unactivated platelets.In this study,cRGD peptide was coupled to the surface of PEGylated liposomes and urokinase was encapsulated to achieve targeted thrombolysis,aiming to increase thrombolytic efficiency and reduce bleeding side effects,thus providing a new type of urokinase formulation for the clinical treatment of thrombotic diseases.In this research,the cRGD peptide was first ligated to DSPE-PEG2000-NHS through the amide formation,resulting in the preparation of DSPE-PEG2000-cRGD.Urokinase loaded liposomes(DPPC,Cholesterol,DSPE-mPEG2000,and DSPE-PEG2000-cRGD)were prepared by membrane hydration method and then homogenized by probe sonication.The encapsulation efficiency was about 29%,and the drug loading capacity was about 15%.The hydrodynamic size of prepared liposomes was approximately 150 nm.The dynamic light scattering results showed that the particle size remained stable for 21 days at 4?.In platelet-targeted experiments,flow cytometric and fluorescence microscopy results showed that cRGD liposomes specifically targeted activated platelets but not unactivated platelets.In vitro release study revealed that the release percentage reached stationary phase in about 5 hours with 60%urokinase being released from liposomes.The pharmacokinetics of FITC-cRGD liposomes was also performed on BALB/c mice.By analyzing the fluorescence intensity of plasma and fitting the data using a two-compartment model,the blood half-life of cRGD liposomes was approximately 2.5 h,which was about 10 times that of free urokinase.The capillary simulating thrombolysis experiments in vitro showed that the thrombolysis of urokinase loaded cRGD liposomes within 4 h was inferior to free urokinase,which was consistent with the release profile of urokinase in vitro.We further prepared a ferric chloride-injured mouse mesenteric vascular thrombosis model to evaluate the thrombolytic effect of urokinase loaded cRGD liposomes.By injecting rhodamine 6G to label platelets and leukocytes through the tail vein,the formation and dissolution of mesenteric vessel thrombi can be observed in real-time by fluorescence microscopy.The mouse mesenteric vascular thrombosis model results showed that cRGD liposomes specifically targeted thrombi in vivo and achieved the same thrombolytic effect with a 75%reduction in the dosage of urokinase.The mouse tail bleeding assay also showed that compared with free urokinase,urokinase-encapsulated cRGD liposomes can significantly reduce bleeding side effects.In summary,this study exploited cRGD modified and PEGylated liposome to deliver urokinase to achieve targeted thrombolysis,which can significantly reduce the dosage of urokinase and achieve the same thrombolytic effect as the higher dosage of free urokinase,while reducing the bleeding side effects.The results of this study are expected to provide a new economic,effective and safe drug formulation for the clinical thrombolytic therapy.