Construction Of A Novel Drug Loaded Nano System And Its Effect On Promoting Thrombolysis

Objective:Thrombus is one of the most common cardiovascular and cerebrovascular diseases in clinical field.Previous studies have shown that Fe₃S₄ nanoparticles have a good magnetocaloric effect and can be used in deep thrombosis hyperthermia.Therefore,in this study,we synthesized Fe₃S₄ nanoparticles,identified their characteristics,and investigated the potential roles of Fe₃S₄ for dissolving the venous thrombosis as well as the protective function of Fe₃S₄on vascular endothelial cells with oxidative damage condition.In addition,the potential mechanism of Fe₃S₄ nanoparticles was preliminarily discussed in combination with the advanced second-generation sequencing technology in the field of scientific research.Methods:1)The size as well as morphology of the Fe₃S₄nanoparticles have been determined by a transmission electron microscopy?TEM?;moreover,the crystal structure of the Fe₃S₄ nanoparticles have been evaluated by the XRD methods;furthermore,X-ray photoelectron spectroscopy was used to analyzed the oxidation state of the Fe₃S₄ nanoparticles;meanwhile,we used inductively coupled plasma atomic emission spectrometry?ICP-AES?to determine the concentration of iron ions that released by Fe₃S₄ nanoparticles in vitro.The magnetic thermal conversion properties of Fe₃S₄ nanoparticles were evaluated by AMF treatment.The photothermal properties of Fe₃S₄ nanoparticles were evaluated by 808nm laser treatment.After intravenous injection of Fe₃S₄ nanoparticles?100?L,12mg/kg?and stimulation with AMF,the thrombolytic effect of Fe₃S₄ nanoparticle dispersion in vivo was confirmed.The tissues and organs of the mice have collected and embedded with paraffin,and then stained with H&E.The histological changes of the samples were imaged by microscope.Moreover,the blood samples were collected,and the activities of alanine aminotransferase as well as aspartate aminotransferase?AST?in blood have also been examined;2)Human umbilical vein endothelial cells?HUVEC?as well as pulmonary vascular endothelial cells?PVECs?were cultured and treated with hydrogen peroxide to establish the in vitro oxidative damage model.Cells have been treated by 808nm laser as well as AMF?4.2×109amm-1s-1?.MTT,flow cytometry,ELISA and protein immunoassay were used to investigate the effect of Fe₃S₄ nanoparticles on HUVEC and PVECs stimulated by NIR and AMF;3)HUVEC in logarithmic growth phase were treated with hydrogen peroxide,and treated with 808nm?0.33wcm-2?laser and AMF?4.2×109amm-1s-1?.The cells were collected from HUVEC treated with hydrogen peroxide and Fe₃S₄+NIR+AMF groups.Gene expression differences between HUVEC treated with Fe₃S₄ nanoparticles and untreated HUVECs were compared by second generation sequencing.According to the sequencing results,HSP70 was selected as the target gene.HUVEC have been randomly divided into:blank control group,H₂O₂,H₂O₂+Fe₃S₄ nanoparticle group?H₂O₂+NIR+AMF?.m RNA and protein levels of HSP70 in HUVEC cells have been examined by RT-q PCR and Western blot;moreover,HUVEC cells were transfected with the sh RNA of HSP 70,MTT,flow cytometry,ELISA and protein immunoassay were used to investigate the effects of Fe₃S₄ nanoparticles on behaviors of HUVEC cells;4)Urokinase type plasminogen activator?u PA?-Fe₃S₄ nanocomposite?upa-Fe₃S₄?was synthesized by chemical method.Transmission electron microscopy?TEM?was used to measure the size and evaluate the morphology of upa-Fe₃S₄ nanoparticles.The photothermal properties of upa-fe3s4nanoparticles were evaluated by 808nm laser treatment.The release of u PA was detected by UV Vis NIR spectrophotometer in vitro.The animal thrombus model was established and the animal tissues were collected.The thrombolytic effect of upa-Fe₃S₄nanoparticles stimulated by NIR was verified by HE staining.Results:1)The size of Fe₃S₄nanoparticles is about 17.7nm,and the spacing between crystal planes is 0.298nm,which accords with the parameters of Fe₃S₄.X-ray diffraction,X-ray photoelectron spectroscopy and analysis results show that the sample is Fe₃S₄.The results of Fe valence state analysis show that there are mixed oxidation states of Fe2+and Fe3+in Fe3s4 nanoparticles,indicating that there are defects in Fe3s4 nanoparticles.Fe₃S₄ nanoparticles have good magnetothermal and photothermal conversion properties,and there is a synergistic thermal conversion effect when the light source and external magnetic field are treated at the same time.Fe₃S₄ nanoparticles have good thrombolytic effect in vitro and in vivo under the stimulation of NIR and AMF,which can effectively reduce the size of thrombus.Fe₃S₄nanoparticles have good histocompatibility and little toxicity to tissues.They are mainly degraded in liver and spleen in vivo;2)Fe₃S₄ nanoparticles could significantly enhance the activity of HUVEC and pvecs cells and inhibit the apoptosis of HUVEC and pvecs under the action of NIR and AMF.Fe₃S₄ nanoparticles could significantly increase the contents of no and SOD and decrease the contents of LDH and MDA in HUVEC and pvecs cells treated with hydrogen peroxide.Compared with the single NIR or AMF treatment group,the reduction of oxidative damage was higher after the combination of NIR and AMF.The mechanism of Fe₃S₄ nanoparticles protecting endothelial cells under the action of NIR and AMF may be through regulating the expression levels of Caspase-3,Bax,Bcl-2 and oxidative stress related proteins SOD and e NOS;3)Sequencing results showed that 28 genes were significantly up-regulated and 47 genes were significantly down regulated in H₂O₂ group.Heat shock protein 70 is one of the up-regulated genes.Hydrogen peroxide treatment decreased the expression of HSP70 in HUVEC cells,while Fe₃S₄ nanoparticles and NIR and AMF treatment significantly increased the expression of HSP70 in HUVEC cells.HSP70 sh RNA could reverse the effect of Fe₃S₄ nanoparticles on the activity and apoptosis of HUVEC treated with hydrogen peroxide.HSP70 sh RNA could reverse the increase of no and SOD contents and the decrease of LDH and MDA contents in HUVEC cells treated with hydrogen peroxide under the action of NIR and AMF.HSP70 sh RNA could affect the levels of Caspase-3,Bax,Bcl-2,SOD and e NOS in HUVEC cells in H202 group.Upa-Fe₃S₄ nanoparticles have good photothermal conversion properties;4)u PA-Fe₃S₄ nanoparticles can effectively release the loaded u PA drugs under the action of NIR photothermal response.u PA-Fe₃S₄ nanoparticles can effectively reduce the size of thrombus.Conclusion:1)the preparation of Fe₃S₄nanoparticles is feasible,and the nanoparticles have good magnetocaloric and photothermal effects.In animal experiments,Fe₃S₄ nanoparticles have good histocompatibility,no obvious toxicity,and can effectively metabolize and decompose;2)Fe₃S₄ nanoparticles can protect HUVECs from H₂O₂ injury induced by NIR and/or AMF;3)After bioinformatics analysis,we found that Hsp70 may be involved in the protection of Fe₃S₄ nanoparticles on human umbilical vein endothelial cells injured by hydrogen peroxide under the action of NIR and AMF;4)Through the construction of nanoparticles Fe₃S₄ mediated u PA drug delivery system,it was found that the system can effectively slow release the loaded drug under the action of NIR,and play a role in promoting thrombolysis.