可降解载药聚羟基丁酸—羟基乙酸酯涂层的生物性能研究
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摘要
近年来,冠状动脉内支架植入术在国内外被广泛应用于治疗心血管疾病领域中,但其术后血管再狭窄严重影响其治疗效果,亟待解决。为此,本文设计一种以可应用于血管支架表面上的以可生物降解的羟基丁酸-羟基乙酸酯的共聚物(PHBHHx)为药物载体、以中国传统中药当归的有效提取物阿魏酸为药物的可降解载药涂层。本文首先采用体外检测方法研究了不同分子量PHBHHx的血液相容性和体外生物降解性能。其次,采用NaOH表面处理对材料表面进行改性,并研究了表面改性及其时效性对PHBHHx生物相容性的影响。最后,本文还研究了载药PHBHHx的体外生物降解性能及释药性能,探讨了阿魏酸及NaOH处理对加速载药PHBHHx表面内皮化和抑制血管平滑肌细胞(VESCs)在其表面过度增殖所起到的作用。得到如下结果:
通过溶血实验、血浆复钙时间实验、凝血酶原时间实验、动态凝血时间实验以及体外血小板粘附实验评价了三种不同分子量PHBHHx的血液相容性,并与PLLA相比较。结果表明:PHBHHx的血液相容性随着材料分子量的增加而降低,三种不同分子量PHBHHx的血液相容性都比PLLA好。
研究PHBHHx的细胞粘附和细胞增殖性能可知:由于PHBHHx的表面较为疏水,其细胞粘附和增殖性能较差。采用NaOH处理可以有效地提高PHBHHx的亲水性进而提高PHBHHx的抗凝血性能、细胞粘附及细胞增殖性能。究其原因,主要是由于NaOH对PHBHHx表面的蚀刻作用以及大量的亲水性极性基团引入到PHBHHx表面,使得PHBHHx的亲水性有了较大提高。
进一步的研究发现:NaOH处理存在时效性的问题。产生时效性的根本原因是由于材料表面极性基团的部分减少。影响表面改性时效性的主要因素包括材料结晶度、材料的存储环境以及存储温度。通过适当提高材料的结晶度、在常规生物医用材料存储条件下将NaOH处理后的材料存储到4℃、亲水性环境中可有效降低时效性带来的不良影响,保持PHBHHx膜表面良好的亲水性进而保持NaOH处理后材料良好的抗凝血性和细胞相容性。
通过比较不同载药PHBHHx的宏观、微观形貌及体外静态降解速度,确定了阿魏酸与PHBHHx重量比为5%和10%为研究范围内载药涂层中较为合适的载药量。研究了PHBHHx (5%)及PHBHHx (10%)膜的体外药物释放性能,结果表明:载药膜的体外药物释放存在快速释放期和稳定释放期两个释药阶段。快速释放期的药物释放以药物扩散为主,稳定释放期的药物释放与载药膜的降解行为有着紧密的关联性。
正交实验结果表明:影响载药涂层与镁合金基体间结合强度最大的因素是镁合金表面预处理工艺,其次是载药量,再次是涂层制备温度。研究范围内载药涂层与镁合金基体之间达到最高结合强度的制备工艺是:使用磷酸对镁合金基体进行表面预处理,载药涂层中的阿魏酸与PHBHHx重量比为5%,制备温度为40℃。此工艺下镁合金与载药涂层之间的结合强度为4.08±0.45MPa。
体外生物相容性实验表明:载药PHBHHx中阿魏酸的释放对血小板聚集及血栓形成有明显的抑制作用,可以有效地提高载药PHBHHx的抗溶血性能以及抗内源、外源凝血性能。而且阿魏酸的释放可以显著地促进内皮细胞在载药膜表面的粘附和增殖,达到加快内皮化的目的,同时,可以有效地抑制平滑肌细胞在其表面的过度增殖。
对载药PHBHHx表面进行NaOH处理后发现:一方面,NaOH处理提高载药膜表面的亲水性进而提高了载药膜表面内皮细胞的粘附和增殖,加速了内皮化进程;另一方面,NaOH蚀刻作用使得表面阿魏酸含量大幅减少,减少了阿魏酸在随后的释放,减弱了其对平滑肌细胞过度增殖的抑制作用。但是,总体来说,NaOH处理仍然有望通过加快内皮化进程来抑制平滑肌细胞的过度增殖,达到防治血管再狭窄的目的。
Recently, coronary artery stent implantation techniques are widely applied to the treatment of cardiovascular disease at home and abroad. However, some problems such as cardiovascular restenosis are crying out for solution. For this reason, a bioabsorbable drug-loaded coating which can be applied on the surface of blood vessel stents was designed in this work. The coating includes biodegradable poly (hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) as drug-loaded material and extract (ferulic acid) from Chinese angelica as drug. First, blood compatibilities and degradation properties of PHBHHx with different molecular weights were studied by in vitro test methods. Second, PHBHHx was surface-modified by a NaOH treatment, and the influence of the surface modification and its aging effect on the biocompatibility of PHBHHx were studied. Furthermore, the in vitro degradation property and the drug release property of the drug-loaded PHBHHx films were studied in this work. The function of the ferulic acid and the NaOH treatment on the endothelialization and inhibition of the blood vessel smooth muscle cell (VMSCs) proliferation was also discussed. The results are summarized as follows:
The blood compatibility of PHBHHx with different molecular weights was evaluated in comparison with poly (L-lactic acid) (PLLA) by a haemolysis assay, plasma recalcification time (PRT), plasma prothrombin time (PT), kinetic clotting time and in vitro platelet adhesion test. The results showed that the blood compatibility of PHBHHx decreased with the increasing of molecular weight of PHBHHx. All PHBHHx films exhibited better blood compatibility than PLLA.
It was thought that the cell adhesion and proliferation of PHBHHx were both worse than those of PLLA, due to the bad surface hydrophilic property of PHBHHx. The hydrophilic property of PHBHHx was effectively improved by a NaOH treatment, and thus the anticoagulation property, the cell adhesion and proliferation of the PHBHHx after the NaOH treatment also had a significant improvement.
Further research found that the NaOH treatment exhibited an aging effect, which was caused by the partial reduction of polar groups on the sample surface. The main influence factors on the aging effect included the crystallinity of PHBHHx, the storage environment and the storage temperature. The aging effect speed could be effectively decreased by increasing the surface crystallinity of the samples, decreasing the storage temperature and laying the samples at 4℃in a hydrophilic environment under the normal biomaterials storage conditions. The good anticoagulation property and cell compatibility of PHBHHx after the NaOH treatment could be maintained by the above methods.
By comparing macro-morphologies, micro-morphologies and in vitro static degradation speeds of the different drug-loaded PHBHHx films, it was confirmed that the appropriate weight ratios of ferulic acid to PHBHHx in the drug-loaded coating beyond the scope of our study were 5% and 10%. In vitro drug release properties of PHHBHx (5%) and PHBHHx (10%) films were studied and the result showed that the drug release from the drug-loaded PHBHHx films exhibited two stages: burst release and stabilization release. Primary mechanism of the drug release in the burst release stage was drug diffusion while the drug release in the stabilization release stage had a close relevancy and regularity to the degradation behaviors of the drug-loaded films.
The results of the orthogonal test showed that the surface pretreatment of a magnesium alloy plays the most important role on the bonding strength between the drug-loaded coating and the magnesium alloy, closely followed by the drug charge and the preparation temperature. The preparation process which could lead to the highest bonding strength was that the magnesium alloy was surface pretreated by H3PO4 solution, the weight ratio of ferulic acid and PHBHHx was 5% and the preparation temperature was 40℃, corresponding to a bonding strength of 4.08±0.45MPa.
In vitro biocompatibility test results showed that the ferulic acid release of the drug-loaded films had an obviously inhibition on the platelet aggregation and thrombosis and an effective improvement on the antihemolytic property and the anti-intrinsic and extrinsic coagulation properties of the drug-loaded films. Moreover, the human umbilical vein endothelial cells (HUVECs) adhesion and proliferation on the drug-loaded films could be significantly promoted and the VMSCs excessive proliferation could be effectively inhibited by the ferulic acid release.
It was found that the NaOH treatment could improve the hydrophilic property of the drug-loaded films, further improve the HUVECs adhesion and proliferation properties, and accelerate the endothelialization process. On the other hand, NaOH treatment could sharply reduce the content of ferulic acid on the sample surface due to the etching action of NaOH, and then weakened the inhibition on VMSCs excessive proliferation on the drug-loaded films. In general, NaOH treatment was expected to control the cardiovascular restenosis by speeding up the endothelialization.
通过溶血实验、血浆复钙时间实验、凝血酶原时间实验、动态凝血时间实验以及体外血小板粘附实验评价了三种不同分子量PHBHHx的血液相容性,并与PLLA相比较。结果表明:PHBHHx的血液相容性随着材料分子量的增加而降低,三种不同分子量PHBHHx的血液相容性都比PLLA好。
研究PHBHHx的细胞粘附和细胞增殖性能可知:由于PHBHHx的表面较为疏水,其细胞粘附和增殖性能较差。采用NaOH处理可以有效地提高PHBHHx的亲水性进而提高PHBHHx的抗凝血性能、细胞粘附及细胞增殖性能。究其原因,主要是由于NaOH对PHBHHx表面的蚀刻作用以及大量的亲水性极性基团引入到PHBHHx表面,使得PHBHHx的亲水性有了较大提高。
进一步的研究发现:NaOH处理存在时效性的问题。产生时效性的根本原因是由于材料表面极性基团的部分减少。影响表面改性时效性的主要因素包括材料结晶度、材料的存储环境以及存储温度。通过适当提高材料的结晶度、在常规生物医用材料存储条件下将NaOH处理后的材料存储到4℃、亲水性环境中可有效降低时效性带来的不良影响,保持PHBHHx膜表面良好的亲水性进而保持NaOH处理后材料良好的抗凝血性和细胞相容性。
通过比较不同载药PHBHHx的宏观、微观形貌及体外静态降解速度,确定了阿魏酸与PHBHHx重量比为5%和10%为研究范围内载药涂层中较为合适的载药量。研究了PHBHHx (5%)及PHBHHx (10%)膜的体外药物释放性能,结果表明:载药膜的体外药物释放存在快速释放期和稳定释放期两个释药阶段。快速释放期的药物释放以药物扩散为主,稳定释放期的药物释放与载药膜的降解行为有着紧密的关联性。
正交实验结果表明:影响载药涂层与镁合金基体间结合强度最大的因素是镁合金表面预处理工艺,其次是载药量,再次是涂层制备温度。研究范围内载药涂层与镁合金基体之间达到最高结合强度的制备工艺是:使用磷酸对镁合金基体进行表面预处理,载药涂层中的阿魏酸与PHBHHx重量比为5%,制备温度为40℃。此工艺下镁合金与载药涂层之间的结合强度为4.08±0.45MPa。
体外生物相容性实验表明:载药PHBHHx中阿魏酸的释放对血小板聚集及血栓形成有明显的抑制作用,可以有效地提高载药PHBHHx的抗溶血性能以及抗内源、外源凝血性能。而且阿魏酸的释放可以显著地促进内皮细胞在载药膜表面的粘附和增殖,达到加快内皮化的目的,同时,可以有效地抑制平滑肌细胞在其表面的过度增殖。
对载药PHBHHx表面进行NaOH处理后发现:一方面,NaOH处理提高载药膜表面的亲水性进而提高了载药膜表面内皮细胞的粘附和增殖,加速了内皮化进程;另一方面,NaOH蚀刻作用使得表面阿魏酸含量大幅减少,减少了阿魏酸在随后的释放,减弱了其对平滑肌细胞过度增殖的抑制作用。但是,总体来说,NaOH处理仍然有望通过加快内皮化进程来抑制平滑肌细胞的过度增殖,达到防治血管再狭窄的目的。
Recently, coronary artery stent implantation techniques are widely applied to the treatment of cardiovascular disease at home and abroad. However, some problems such as cardiovascular restenosis are crying out for solution. For this reason, a bioabsorbable drug-loaded coating which can be applied on the surface of blood vessel stents was designed in this work. The coating includes biodegradable poly (hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) as drug-loaded material and extract (ferulic acid) from Chinese angelica as drug. First, blood compatibilities and degradation properties of PHBHHx with different molecular weights were studied by in vitro test methods. Second, PHBHHx was surface-modified by a NaOH treatment, and the influence of the surface modification and its aging effect on the biocompatibility of PHBHHx were studied. Furthermore, the in vitro degradation property and the drug release property of the drug-loaded PHBHHx films were studied in this work. The function of the ferulic acid and the NaOH treatment on the endothelialization and inhibition of the blood vessel smooth muscle cell (VMSCs) proliferation was also discussed. The results are summarized as follows:
The blood compatibility of PHBHHx with different molecular weights was evaluated in comparison with poly (L-lactic acid) (PLLA) by a haemolysis assay, plasma recalcification time (PRT), plasma prothrombin time (PT), kinetic clotting time and in vitro platelet adhesion test. The results showed that the blood compatibility of PHBHHx decreased with the increasing of molecular weight of PHBHHx. All PHBHHx films exhibited better blood compatibility than PLLA.
It was thought that the cell adhesion and proliferation of PHBHHx were both worse than those of PLLA, due to the bad surface hydrophilic property of PHBHHx. The hydrophilic property of PHBHHx was effectively improved by a NaOH treatment, and thus the anticoagulation property, the cell adhesion and proliferation of the PHBHHx after the NaOH treatment also had a significant improvement.
Further research found that the NaOH treatment exhibited an aging effect, which was caused by the partial reduction of polar groups on the sample surface. The main influence factors on the aging effect included the crystallinity of PHBHHx, the storage environment and the storage temperature. The aging effect speed could be effectively decreased by increasing the surface crystallinity of the samples, decreasing the storage temperature and laying the samples at 4℃in a hydrophilic environment under the normal biomaterials storage conditions. The good anticoagulation property and cell compatibility of PHBHHx after the NaOH treatment could be maintained by the above methods.
By comparing macro-morphologies, micro-morphologies and in vitro static degradation speeds of the different drug-loaded PHBHHx films, it was confirmed that the appropriate weight ratios of ferulic acid to PHBHHx in the drug-loaded coating beyond the scope of our study were 5% and 10%. In vitro drug release properties of PHHBHx (5%) and PHBHHx (10%) films were studied and the result showed that the drug release from the drug-loaded PHBHHx films exhibited two stages: burst release and stabilization release. Primary mechanism of the drug release in the burst release stage was drug diffusion while the drug release in the stabilization release stage had a close relevancy and regularity to the degradation behaviors of the drug-loaded films.
The results of the orthogonal test showed that the surface pretreatment of a magnesium alloy plays the most important role on the bonding strength between the drug-loaded coating and the magnesium alloy, closely followed by the drug charge and the preparation temperature. The preparation process which could lead to the highest bonding strength was that the magnesium alloy was surface pretreated by H3PO4 solution, the weight ratio of ferulic acid and PHBHHx was 5% and the preparation temperature was 40℃, corresponding to a bonding strength of 4.08±0.45MPa.
In vitro biocompatibility test results showed that the ferulic acid release of the drug-loaded films had an obviously inhibition on the platelet aggregation and thrombosis and an effective improvement on the antihemolytic property and the anti-intrinsic and extrinsic coagulation properties of the drug-loaded films. Moreover, the human umbilical vein endothelial cells (HUVECs) adhesion and proliferation on the drug-loaded films could be significantly promoted and the VMSCs excessive proliferation could be effectively inhibited by the ferulic acid release.
It was found that the NaOH treatment could improve the hydrophilic property of the drug-loaded films, further improve the HUVECs adhesion and proliferation properties, and accelerate the endothelialization process. On the other hand, NaOH treatment could sharply reduce the content of ferulic acid on the sample surface due to the etching action of NaOH, and then weakened the inhibition on VMSCs excessive proliferation on the drug-loaded films. In general, NaOH treatment was expected to control the cardiovascular restenosis by speeding up the endothelialization.
引文
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