Preparation Of Responsive EGCG/Cystamine Crosslinked Film And Its Properties

Coronary artery disease is a common disease that threatens human health,and its morbidity and mortality have gone beyond cancer and become the first killer to human health.The one of the main treatment for coronary artery disease is interventional therapy with the drug-eluting stent(DES).The drug in widely used in clinic DES inhibits smooth muscle hyperplasia while also inhibits the endothelium healing.Moreover the long-term presence of the polymer carrier could initiate inflammation response,which would cause neonatal atherosclerosis,late restenosis and late thrombosis.Coronary artery disease is mainly caused by coronary atherosclerosis,and the trigger step of atherosclerosis is the low-density lipoprotein oxidized to oxidized low-density lipoprotein in the oxidative stress.For oxidative stress caused by reactive oxygen species associated with free radicals,such as superoxide anion,hydroxyl radical,H2O2,a modified layer with better biocompatibility and oxidative response is beneficial to vascular stents application property.In this work,two kinds of functional molecules,epigallocatechin gallate(EGCG)and cystamine were introduced into the surface of 316L stainless steel.Utilizing the biological functions of EGCG such as anti-oxidation and inhibiting the proliferation of smooth muscle cells,and the disulfide bond of cystamine break property under oxidative or reductive condition,to obtain a multi-functional coating with treatment effect and redox response ability.This coating was expected to adapt to the microenvironment of the lesion,and improve the biocompatibility of the vascular stents.316L stainless steel plates(SS)were immersed into EGCG and cystamine mixed solution to construct crosslinking film on the surface of stainless steel by Michael addition and Schiff base reaction between EGCG and cystamine.Attenuated total reflection fourier transform infrared spectroscopy(ATR-FTIR)and X-ray photoelectron spectroscopy(XPS)demonstrated that the EGCG/cystamine crosslinked film was successfully constructed on the SS surface.The phenolic hydroxyl content detection showed the amount of phenolic hydroxyl groups of the film was significant higher than that of the stainless steel,thus improved the hydrophilicity.In vitro culture of endothelial cells(ECs)and smooth muscle cells(SMCs)showed that the EGCG and cystamin film could promote the growth and proliferation of ECs and inhibit the growth and proliferation of SMCs.In vitro culture of macrophages exhibited that the EGCG and cystamin film could inhibit the growth of macrophages.In vitro platelet adhesion and whole blood test indicated the number of platelet activation and adhesion decreased.The result of subcutaneous implantation samples into SD rats showed that the EGCG/cystamine crosslinked film had good histocompatibility.The redox response behavior of the EGCG/cystamine film was evaluated.The results of FTIR and XPS showed that after the EGCG/cystamine film was immersed into the H2O2 solution,the surface S chemical state was S-S and-SO3.This may due to the further oxidization of-S-formed from the S-S breaking down to-SO3.After the EGCG/cystamine film was immersing into the GSH solution,with the prolongation of soaking time,the percentage of S element on the surface decreased.The morphology of the sample was observed by light microscope,scanning electron microscope(SEM)and atomic force microscope(AFM).The morphology of the sample was changed after redox treatment.After immersing into the GSH solution,the EGCG/cystamine film is severely damaged.Morphology change can be observed through AFM image after immersing into the H2O2 solution,which indicated that the EGCG/cystamine film had a response under the redox condition.QCM test also demonstrated that the film was destroyed under oxidation and reduction conditions.The experiment results of the soaking solution from EGCG/cystamine samples in redox solution indicated the soaking solution under the conditions of GSH could promote the growth and proliferation of ECs,while which of H2O2 solution could kill the cells.However,since the GSH and H2O2 in the solution were difficult to remove completely,thesel results have been interfered.In the later investigation,it is need to explore new methods in order to truly evaluate the effect of soaking solution on cells.EGCG and cystamine in EGCG/cystine solution can react each other to form circular EGCG/cystamine particles with regular shape and particle size of about 300 nm.The observation of transmission electron microscopy(TEM)showed that after treated with GSH solution,the surface of EGCG/cystamine was destroyed,and the profile became unclear and the pits appeared.The particles changed to irregular shape and the surface morphology was obviously damaged after the treatment with oxidized H2O2 solution,indicating that the EGCG/cystamine particles also had a redox response property.The EGCG/cystamine particles loaded with Rhodamine were broken under the redox condition,and it can be found that Rhodamine has released.The results of macrophages culture proved that the certain concentrations of EGCG/cystamine nanoparticles could inhibit the growth of macrophages.In summary,EGCG/cystamine particles and cross-linked film both can respond to the redox conditions,occur structural damage and morphology change,also they can inhibit the growth of macrophages.The EGCG/cystamine cross-linked film could promote endothelial cell growth,inhibit smooth muscle cell proliferation,and has good blood compatibility.The EGCG/cystamine coating studied in this paper has good biocompatibility and has the responsiveness property to the microenvironment of the lesion,which provides a new idea for the development of functionalized modified coating for vascular stent.