【作者】 赵铮；

【导师】 蹇锡高； 王锦艳；

【作者基本信息】 大连理工大学 ， 高分子材料， 2023， 博士

【摘要】 近年来,镁合金支架作为新一代治疗冠脉狭窄的可降解植入器械展现出巨大的应用优势。然而,镁合金支架植入后过快的降解造成其支撑功能丧失过早,使得未修复的血管发生弹性回缩现象;过快的降解也造成支架表面药物洗脱功能可控性下降,使得支架抑制血管早期再狭窄的效果不佳。改善镁合金支架的上述不足并实现其安全应用已经成为该领域的研究热点。复合涂层表面修饰可以降低镁合金支架的降解速率并调控其生物功能性,被认为是改善镁合金支架不足的有效策略。其中,聚三亚甲基碳酸酯（PTMC）为主体的复合涂层具有可降解、腐蚀防护性能强和生物相容性好的特点,展现出巨大优势。基于此,为了降低镁合金支架的降解速率,并实现可控的药物洗脱功能,本文在镁合金支架表面设计了以PTMC为主体的三层复合涂层。具体研究内容如下:为了增强PTMC涂层在镁合金表面的附着力并初步改善镁合金的耐蚀性,选用腐蚀防护性能优异的氟化镁无机涂层作为AZ31镁合金的打底层,并进一步对其表面进行Na OH溶液改性处理,制备新型羟基化氟化镁涂层。重点研究了Na OH溶液处理时间对样品表面PTMC涂层附着、镁合金的耐蚀性及生物相容性的影响。结果表明,Na OH溶液处理使镁合金氟化镁涂层表面的微纳结构从原有的Mg F_x（OH）_2-x纳米球逐渐转变为Mg（OH）₂纳米片,并形成新型Mg（OH）₂/Mg F_x（OH）_2-x涂层。此外,Na OH溶液处理可以改善PTMC涂层在样品表面的附着力,处理20 min以上可使附着力等级提升至1级。同时,Na OH溶液处理使氟化镁涂层的孔隙缺陷被形成的片状Mg（OH）₂封堵,镁合金的耐蚀性进一步提升,处理40 min时样品展现出最强的耐蚀性,在0.1 Hz下的阻抗模量达到3.0×10⁵Ω·cm²。体外生物学实验表明,改性镁合金的人脐静脉血管内皮细胞（ECs）和人脐动脉血管平滑肌细胞（SMCs）相容性得到显著改善。其中,Na OH溶液处理40 min得到的Mg（OH）₂/Mg F_x（OH）_2-x涂层修饰的镁合金具有最优的综合性能,并用于后续研究。为了有效降低镁合金的降解速率,在最优Mg（OH）₂/Mg F_x（OH）_2-x底层上制备氧化石墨烯（GO）/PTMC腐蚀防护增强层。重点考察了GO添加量对镁合金耐蚀性的影响,并对改性样品的体外生物相容性进行了评价。结果表明,GO/PTMC涂层的修饰可以显著增强镁合金的耐蚀性。在GO不超过6 wt.%的情况下,随着PTMC涂层中GO含量的增加,涂层的腐蚀防护性能改善,镁合金的耐蚀性逐渐增强。GO添加量达到6 wt.%时,镁合金耐蚀性最强,0.1 Hz下的阻抗模量达到9.8×10⁷Ω·cm²。不过,GO添加过量时,会发生大范围团聚并在GO/PTMC涂层中形成新的溶液通道,造成涂层腐蚀防护性能下降。体外生物学实验表明,GO/PTMC涂层的修饰进一步改善了镁合金的细胞相容性,使ECs和SMCs可在其表面进行完全正常的贴附和增殖。其中,6 wt.%GO含量的改性样品展现出最优的综合性能,并用于进一步的生物功能化修饰。为了赋予镁合金抑制早期再狭窄的功能,在腐蚀防护层表面制备雷帕霉素（RAPA）/PTMC药物释放面层。探究了样品RAPA释放的可控性,考察了RAPA添加量对样品耐蚀性和药物释放行为的影响,并测试了RAPA/PTMC涂层修饰后样品的体外生物学功能。结果表明,RAPA/PTMC涂层的修饰可以进一步提升镁合金的耐蚀性,且耐蚀性随着RAPA添加量的增加而增强,但由于RAPA溶出在RAPA/PTMC涂层中留下了缺陷,使RAPA/PTMC涂层在浸泡后期的腐蚀防护性能下降。样品的RAPA释放速率随着药物载量的增加而加快,并具有良好的可控性,尽管碱性环境会加速RAPA释放,但由于复合涂层对镁合金基底具有较强的保护,浸泡28天内降解产生的碱性较弱,不会对RAPA的释放造成显著影响。体外生物学实验表明,RAPA/PTMC涂层的修饰使样品具备了抑制ECs和SMCs贴附和增殖的功能,改性样品组的细胞活力下降到对照组的50%左右。为了改善内皮化前聚合物涂层在血液中暴露造成的血栓风险,对RAPA/PTMC药物释放层中的PTMC进行了抗凝血功能化结构改性,合成了可催化抗凝因子一氧化氮（NO）产生且生物相容性良好的双硒-PTMC共聚物（PUSP）。在腐蚀防护增强层表面制备RAPA/PUSP药物释放层,并考察了PUSP主链中双硒单体含量对样品NO释放、RAPA释放和生物功能性的影响。结果表明,使用RAPA/PUSP作为药物释放层的样品不仅可满足28天的RAPA可控释放,实现对ECs和SMCs的贴附和增殖抑制,同时可在此期间催化NO的产生,实现对血小板激活的抑制并延长活化部分凝血活酶时间,降低血栓发生的风险。此外,PUSP中硒元素含量越高,NO的释放量越大,活化部分凝血活酶时间越长,抗凝性能越强。其中,双硒单体含量为30%的PUSP修饰的镁合金具有最优的抗凝性能,并进一步用于动物实验研究。最后,将优化后的RAPA/PUSP-GO/PTMC-Mg（OH）₂/Mg F_x（OH）_2-x复合涂层制备到镁合金支架表面,并进行14天家兔皮下植入实验和28天白猪冠脉植入实验。家兔皮下植入实验表明,裸镁合金支架在植入14天后已经基本降解消失,而改性镁合金支架由于复合涂层的保护,体内降解速率较慢,14天植入后的支架结构依旧完整。此外,改性镁合金支架的植入对组织、血液及肝肾功能没有显著负面影响,具有良好的体内生物相容性。猪冠脉植入实验表明,改性镁合金支架的早期支撑功能良好,支架在植入28天内被修复的血管内皮完全覆盖,没有出现血管的弹性回缩现象。此外,血管没有出现明显的支架内再狭窄,且没有支架内血栓的产生,展现出不错的早期再狭窄及血栓抑制效果。更多还原

【Abstract】 Magnesium alloy stents as biodegradable implant devices for treating coronary artery stenosis have demonstrated significant advantages in recent years.However,the rapid degradation of magnesium alloy stents leads to premature loss of their support function after implantation,causing vascular elastic retraction.In addition,the rapid degradation causes instability of their drug release,leading to an inadequate inhibition of early restenosis.Improving magnesium alloy stents and realizing their safety application has become a hotspot in this field.Composite coating is considered as an effective strategy to solve these problems,which can reduce the degradation rate of magnesium alloy stents and regulate their biological functionality.Among all kinds of composite coatings,composite coatings based on polytrimethylene carbonate（PTMC）have the characteristics of biodegradability,excellent corrosion protection performance,and good biocompatibility,which have shown huge advantages.Herein,in order to control the degradation rate of magnesium alloy stents and realize stable drug release function,a three-layer composite coating based on PTMC was designed for AZ31 magnesium alloy stent.The specific research contents are as follows:To enhance the adhesion of the PTMC coating to the surface of magnesium alloy,and meanwhile improve the corrosion resistance of the magnesium alloy,a magnesium fluoride inorganic coating was selected as the basis layer of the magnesium alloy,and further modified by using Na OH solution.The impacts of Na OH treatment time on the adhesion of the PTMC coating and the corrosion resistance and biocompatibility of the modified magnesium alloy were studied.The results showed that the Na OH treatments transformed the micro-nano structure of the magnesium fluoride surface from Mg F_x（OH）_2-x nanospheres to Mg（OH）₂nanosheets,forming new Mg（OH）₂/Mg F_x（OH）_2-x coatings.The formed Mg（OH）₂ sheets improved the adhesion of the PTMC coating（The adhesion level raised to level 1 after more than 20 minutes Na OH treatment）.At the same time,the defects of magnesium fluoride coating were gradually blocked by formed Mg（OH）₂ sheets,and thus improved the corrosion resistance of the sample.The sample with 40 minutes Na OH treatment exhibited the strongest corrosion resistance,and the impedance modulus at 0.1 Hz achieved 3.0×10⁵Ω·cm².In vitro biological experiments showed that the compatibility of the magnesium alloy on human umbilical vein endothelial cells（ECs）and human umbilical artery smooth muscle cells（SMCs）was significantly improved after modified with the Mg（OH）₂/Mg F_x（OH）_2-x coatings.Overall,the Mg（OH）₂/Mg F_x（OH）_2-x coating modified magnesium alloy with 40 minutes Na OH treatment showed the best comprehensive performance and was used for subsequent study.To effectively inhibit the corrosion rate of magnesium alloy,the graphene oxide（GO）/PTMC corrosion protection layer was prepared on the optimal inorganic coating.The effect of GO content on the corrosion resistance of the sample was investigated.In addition,the in vitro biocompatibility of the modified samples was evaluated.The results showed that the modification of GO/PTMC coating could significantly enhance the corrosion resistance of the magnesium alloy.With the increase of GO content（less than 6 wt.%）,the corrosion protection performance of the coating and the corrosion resistance of the magnesium alloy gradually enhanced.When the content of GO was 6 wt.%,the corrosion resistance of the magnesium alloy reached the maximum,and the impedance modulus at 0.1Hz achieved 9.8×10⁷Ω·cm².However,when the addition of GO exceeded 10 wt%,GO aggregated on the coating surface,resulting in the formation of new defects and a rapid decrease in corrosion resistance.In vitro biological experiments showed that the modification of the GO/PTMC coatings further improved the cell compatibility of the magnesium alloy,and ECs and SMCs can adhere and proliferate normally on its surface.Overall,the modified sample with 6 wt.%GO content in the GO/PTMC coating exhibited the best comprehensive performance and was used for further biological functionalization modifications.To give magnesium alloy the function of inhibiting early restenosis,the rapamycin（RAPA）/PTMC drug release coating was prepared on the optimal corrosion protection layer.The effect of RAPA content on the corrosion resistance and drug release behavior of the samples was investigated.Besides,the stability of RAPA release and the biological function of the modified samples was tested.The results showed that the RAPA/PTMC modification further enhanced the corrosion resistance of the magnesium alloy.However,the corrosion protection performance of the RAPA/PTMC coating decreased at the later stage of the immersion due to the RAPA release.The drug release experiments showed that the release rate of RAPA was stable,and accelerated with the addition of the RAPA content.Because of the strong corrosion protection of the composite coating,the p H value of the environment within 28days changed little,which could not affect the release stability of RAPA.In vitro biological experiments showed that the modification of the RAPA/PTMC coating effectively inhibited the proliferation of ECs and SMCs,and the cell activity decreased about 50%compared to the control group.To reduce the risk of thrombosis before endothelialization,the PTMC of the RAPA/PTMC drug release coating was structurally modified.A series of biocompatible diselenium-PTMC copolymer（PUSP）,which can catalyze the production of anticoagulant nitric oxide（NO）,were synthesized.The RAPA/PUSP drug release layer was prepared on the surface of the corrosion protection layer,and the effects of structural modification on NO release,RAPA release and biological functions of the samples were studied.The results showed that the modified samples could achieve stable release of RAPA for 28 days,which inhibited the adhesion and proliferation of ECs and SMCs.Besides,the modified samples could catalyze the generation of NO during this period,which inhibited platelet activation,prolonged partial prothrombin time and could reduce the risk of thrombosis.In addition,the higher the selenium content,the greater the release of NO and the longer the partial prothrombin time,indicating a better anticoagulant function.Overall,the RAPA/PUSP modified sample with the selenide monomer content of 30%exhibited the best anticoagulant performance,which was further studied in animal experiments.Finally,the optimal RAPA/PUSP-GO/PTMC-Mg（OH）₂/Mg F_x（OH）_2-x composite coating was prepared on the magnesium alloy stent.The 14-day rabbit subcutaneous implantation experiment and the 28-day white pig coronary artery implantation experiment were performed.The rabbit subcutaneous implantation experiment showed that the uncoated magnesium alloy stent basically disappeared after 14 days of implantation,while the modified magnesium alloy stent showed a slower degradation rate and the structure of the stent remained intact.Besides,the modified magnesium alloy stent had no obviously negative effect on tissue,blood,liver and kidney,showing good biocompatibility in vivo.Pig coronary artery implantation experiments showed the early support function of the modified magnesium alloy stent was good,the stent was completely endothelialized within 28 days,and no vessel elastic retraction occurred.In addition,no in-stent restenosis and thrombus occurred,exhibiting good early restenosis and thrombus inhibition functions.更多还原