316L不鏽鋼表面活化改質及固定抗凝血藥物肝素於冠心病支架上之應用

Abstract

冠心病是國人常見的疾病，此疾病是因提供心臟所需的冠狀動脈發生狹窄阻塞所致。目前常見的治療方式是支架的置入，但支架不理想的血液相容性，常使得撐開後的血管在半年後再度的狹窄。為了避免血管的再狹窄，本研究固定抗凝血藥物肝素於支架表面以改善其血液相容性。由於金屬支架表面並不具有具反應性之官能基，本研究先以表面偶合劑二異氰酸己烷將之活化，使支架得以進行後續的藥物固定反應。支架表面改質後，肝素分子也必須進行活化，方可固定於支架表面。乙基二甲基氨丙基碳二亞胺是目前常用來固定肝素至材料表面的交聯劑。此試劑可將肝素的羧酸基轉換成高反應性的中間產物，使肝素固定於支架表面。但有研究指出，經此試劑處理後的肝素，將喪失其部分的抗凝血功效。為了避免羧酸基的耗損，本研究以氧化劑過碘酸鈉處理肝素。此分子可使肝素分子中特定且無關肝素活性的氫氧基轉成極具反應性的醛基，使肝素能在不損失羧酸基的情況下，固定於材料表面。本研究我們分別以乙基二甲基氨丙基碳二亞胺及過碘酸鈉將肝素固定於支架表面，並進行各種肝素活性及血液相容性測試。研究結果顯示，以乙基二甲基氨丙基碳二亞胺法固定肝素後之支架較未經處理之支架有相對較好的血液相容性，但結果仍未盡理想。而經過碘酸鈉法固定肝素之支架，則有明顯較好的血液相容性。因此我們相信，本研究所研發的肝素固定技術，能改善過去支架不理想的血液相容性，解決血管再狹窄的問題。

Poor compatibility between blood and metallic coronary artery stents is one reason for arterial restenosis. Immobilization of anticoagulant heparin on the stent’s surface is feasible for improving compatibility. Prior to heparin immobilization, we examined possible surface-coupling agents for heparin immobilization. Hexamethylene diisocyanate (HMDI) and 3-aminopropyl-triethoxysilane (APTS) were examined as surface-coupling agents to activate 316L stainless steel (e.g., stent material). The activated surface was characterized by Fourier transformation infrared spectroscopy (FTIR), atomic force microscope (AFM), surface plasmon resonance (SPR), and trinitrobenzene sulfonic acid (TNBS) assay. In the FTIR analysis, HMDI and APTS were both covalently linked to 316L stainless steel. In the AFM analysis, it was found that the HMDI-activated surface was smoother than the APTS-activated one. In the SPR test, the shift of the SPR angle for the APTS-activated surface was much higher than that for the HMDI-activated surface after being challenged with acidic solution. The TNBS assay was utilized to determine the amount of immobilized primary amine groups. The HMDI-activated surface was found to consist of about 1.32 μmole/cm2 amine group, whereas the APTS-activated surface consisted of only 0.89μmole/cm2 amine group. We conclude that the HMDI-activated surface has more desirable surface characteristics than the APTS-activated surface, such as surface roughness, chemical stability, and the amount of active amine groups. The HMDI-activated 316L stainless steel (SS) was then utilized for heparin linking. The compound 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDAC) has often been utilized for the immobilization of heparin, but the critical carboxyl groups of heparin (with regards to heparin’s anticoagulant activity) will be reduced by this method. We were trying to examined possible methods of heparin immobilization without consuming these carboxyl groups. Sodium periodate (NaIO4; SP) was then used to oxidize heparin to form aldehyde groups and then coupled with bis-amine-terminated poly(ethylene glycol) (Bis-amine PEG) so as to form heparin-PEG complexes. The complexe could then be grafted onto the activated surface of the test material without losing its carboxyl groups. The heparin-PEG complex formed by EDAC method was used as comparison group. Effective surface modification of the HMDI-activated and heparin-PEG grafted 316L SS surface was confirmed using Fourier Transform Infrared Spectroscopy (FTIR), Electron Spectroscopy for Chemical Analysis (ESCA) and a water contact angle test. After the heparin grafted by SP, the surface showed an improvement in antithrombrin (AT) binding ability, its anticoagulant property, and hemocompatibility in comparison to heparin grafted by EDAC.