Ferritin H-chain Nanoparticles For Targeting Therapeutics Of Thrombolysis

Thrombotic diseases have seriously damaged human health.In recent years,the development goal of thrombolytic drugs is to obtain new thrombolytic agents with good thrombolytic effect and high drug safety.At present,the thrombolytic effect is improved mainly through the targeting strategy,and the insoluble fibrin is one of the main targets of thrombus;the reduction of side effects of systemic hemorrhage is mainly through the modification of thrombolytic agents,which can selectively dissolve the forming blood clot(pathological thrombus)rather than the established clot.As excellent carriers,human ferritin H-chain protein(FTH1)existing widely in organisms,have high biosafety,and can carry multiple targets and drug molecules simultaneously by its modification.Therefore,FTH1-based nanoparticles can be targeted at the tumor site for imaging and treatment,but there are few reports about its application in thrombus therapy.In this study,we selected FTH1 as basic carrier for carrying a variety of targeted and thrombolytic molecules at the same time by means of genetic engineering;their characterization,targeting,activity,thrombolytic effect and safety were then carefully investigated.Firstly,the fibrin-binding peptide(FibPep)as targeted molecular was modified at the N terminus of FTH1,the low molecular weight single chain urokinase-type plasminogen activator activated by thrombin(T-uPA)as thrombolytic molecule was modified at the C terminus of FTH1.To obtain these two proteins,the two recombinant plasmids were successfully constructed and induced to express corresponding target proteins.Then The resulted fusion proteins of FibPep-FTH1 and FTH1-T-uPA were purified,denatured,and refolded together to obtain the FibPep-FTH1/FTH1-T-uPA protein nanoparticles.In the same way,FTH1/FTH1-T-uPA as a control group was generated by refolding the denatured FTH1 and FTH1-T-uPA proteins together.The native-PAGE,protein composition analysis,TEM and DLS experiments showed that denatured FTH1 and FTH1-T-uPA,or FibPep-FTH1 and FTH1-T-uPA subunits were successfully folded and assembled into the characteristic cages of ferritin.The FibPep-FTH1/FTH1-T-uPA and FTH1/FTH1-T-uPA were obtained in the shape of spherical particles with a diameter of 14.45 nm and 13.41 nm respectively.DLS also showed that they got very narrow size distribution in solution with the hydrodynamic diameter of 29.8 nm and 24.2 nm respectively.Thus,the study first shows that Fibpep and T-uPA can be simultaneously modified on the surface of ferritin and provides new ferritin nanoparticles for the clinical application of thrombus targeting or treatment.Secondly,the fibrin-binding affinity and enzymatic properties of FTH1/FTH1-T-uPA and FibPep-FTH1/FTH1-T-uPA were analyzed.In vitro fibrin binding studies showed that the FibPep-FTH1/FTH1-T-uPA exhibited significantly higher binding to fibrin compared to non-targeted FTH1/FTH1-T-uPA.The percentage of binding to fibrin was even greater than eighty percent due to the FibPep peptides on the suface of the cages,which could significantly enhance the fibrin-binding affinity by multivalent binding.The thrombin activation assay further showed that the T-uPA molecules were on the both surfaces of the two kinds of nanoparticles and could be successfully activated by thrombin;and the fibrinolytic activity of FTH1/FTH1-T-uPA and FibPep-FTH1/FTH1-T-uPA were 1200 and 1400 IU/mg,and their amidolytic activity were 860 and 1100 IU/mg,respectively.All the results showed that the targeting ability of FibPep-FTH1/FTH1-T-uPA was enhanced by multivalent binding approach,and thrombin was required to activate T-uPA for a higher safety.In this way,it showed that those nanoparticles acquired the key characteristics of thrombolytic drugs with an excellent targeting ability and low bleeding side effects.Thirdly,the thrombolytic efficacy and safety issues of FTH1/FTH1-T-uPA and FibPep-FTH1/FTH1-T-uPA are evaluated.It was found that the fibrin and blood clot lysis of the targeted FibPep-FTH1/FTH1-T-uPA was statistically significant higher than that of the non-targeted FTH1/FTH1-T-uPA.Futhermore,in the FeCl3-induced carotid artery thrombosis rat model the thrombolytic effect of the targeted FibPep-FTH1/FTH1-T-uPA was also significantly better than that of the non-targeted FTH 1/FTH 1-T-uPA for the nascent thrombi;compared with the commercialized urokinase,the dosage of FibPep-FTH1/FTH1-T-uPA was lower(5000 IU/kg)when achieving similar thrombolysis effect in vivo.As comes to the safety assessments,in vitro established clot lysis assay showed that both FTH1/FTH-uPA and FibPep-FTH1/FTH1-T-uPA,need to be activated by thrombin,could hardly dissolve the established blood clots.However,the urokinase could still significantly dissolve the blood clots.It means that thrombolytic activity of T-uPA could be largely turned off in the circulation and thus gave a safer treatment.At the same time,the in vivo activated partial thromboplastin time(APTT)remained at nearly normal level even after the mice were administrated with double dosage of the FTH1/FTH-uPA or FibPep-FTH1/FTH1-T-uPA(10000 IU/kg).With this,we here provide the method for constructing new thrombolytic drugs by targeting delievery and modifing uPA,which may improve thrombolytic effect and reduce side effects.All together,FibPep-FTH1/FTH1-T-uPA has been developed with a better targeting towords fribin and a thrombin-required activation.It can improve the nascent thrombi lysis in vivo and thus obtain a better safety by reducing the therapeutic dose and its side effects.All these studies provide the methods for designing the thrombolytic drugs by combining the ability of targeting thrombi with the uPA modification.It is very promising for the construction of an ideal thrombolytic agent,and also allows us to explore the applications of protein-based nanoparticles in medicine.