微波辅助下羟乙基壳聚糖/聚乳酸接枝共聚物的合成与生物相容性评价
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摘要
本文采用微波辅助合成法,以羟乙基壳聚糖为大分子引发剂引发丙交酯开环聚合,成功合成了一系列不同组成的羟乙基壳聚糖／聚乳酸接枝共聚物(HECS-g-PLA)。
为了降低壳聚糖的结晶性能以及其分子间或分子内较强的氢键作用,提高壳聚糖的反应活性,获得接枝率高的共聚物,本研究分别以羟乙基壳聚糖和壳聚糖为大分子引发剂引发丙交酯开环聚合,系统研究和比较了两者引发丙交酯开环聚合的反应活性,结果显示羟乙基化改性可明显提高壳聚糖的反应活性,壳聚糖／聚乳酸接枝共聚物的接枝率为225%,而相应的羟乙基壳聚糖／聚乳酸共聚物提高到307%。进一步以羟乙基壳聚糖为大分子引发剂,分别采用传统封管热聚合法和微波辅助法实施该共聚合反应,考察了聚合方法对该接枝共聚合反应的影响,结果表明,微波辅助聚合15min获得的共聚物和封管热聚合24h获得的共聚物具有相近的接枝率,这表明微波辅助可有效提高聚合反应速率,大大缩短聚合反应时间。
在上述研究基础上,以羟乙基壳聚糖为大分子引发剂,通过正交试验确定最佳的聚合条件：催化剂按照nD,L-LA：n辛酸亚锡为1000：1投入,微波辐射功率10W,120℃下反应时间为15min。在最佳的聚合条件下,采用不同的原料投料比(nD,L-LA：nHECS糖单元：20：1,30：1,40：1),通过微波辅助聚合方法制备了一系列不同组成的HECS-g-PLA,通过FTIR、1H-NMR、X-RD、元素分析、DSC、TG等测试方法,系统研究了nD,L-LA：nHECS糖单元投料比对共聚物的组成与结构、产率、接枝率和性能的影响。结果表明共聚物的接枝率可控,接枝率随着投料比的增加而增加,其中投料比为40：1,可得接枝率最大为307%的共聚物,该共聚物的单位糖环平均乳酰单元数为24.1。产物颜色随着投料比的增加,由深黄色向淡黄色变化,可溶于DMSO,锡残留量为0.658mg/g; X-RD分析结果表明随着投料比的增大,产率增大,共聚物的接枝率增大,结晶性能下降。DSC、TG分析结果表明随着投料比的增大,玻璃化转变温度升高,裂解温度下降。表面接触角和吸水率测试显示,HECS的含量对PDLLA的亲水性有改善作用,HECS的含量越高,HECS的亲水性越好。
从体外降解、细胞毒性、溶血试验、动态凝血、QCM等手段考察了材料的生物学性能。降解结果表明所得共聚物的降解速率和产物的接枝率密切相关,接枝率越高降解速率越快,失重曲线显示HECS可以减缓PDLLA的降解速率。FTIR谱图显示共聚物前8w的降解主要发生在聚乳酸链段上,pH值变化曲线显示HECS对PDLLA降解带来的酸性有缓冲作用；溶血试验结果显示,该材料的溶血率为1.04,无溶血作用；动态凝血和QCM蛋
白吸附结果显示该材料有利于白蛋白吸附,BCI曲线坡度较PDLLA平缓,这些表明了HECS-g-PLA的抗凝血性能比PDLLA好。体外细胞培养结果显示,HECS-g-PLA的细胞毒性较PDLLA小,细胞毒性级别为1,浸提体液中的细胞生长情况较好。
Using a new method—microwave-irradiation to get the Hydroxyethylated chitosan-graft-poly(D,L-lactide) (HECS-g-PLA)copolymer, the graft copolymerization was carried out by ring opening polymerisation(ROP) using stannous octoate (Sn(oct)2) as catalyst without any organic solvent.
It is showed that hydroxyethylated chitosan(HECS) have higher reaction activity than CS as initiator for ROP of D,L-lactide at same condition.The grafting percentage of HECS-g-PLA is 307%, while CS-g-PLA is 225%. Because hydroxyethylation can decrease the crystallinity and hydrogen bond interaction of CS, increase the reaction activity. The grafting percentage of HECS-g-PLA synthesized by microwave-assisted ROP(MAROP) for 24h approximately equal to closed-tube polymerization(CTROP) for 15min.The result show MAROP would significantly increase polymerization rate and decrease polymerization time using same initiator compare with using CTROP.Base on those result, orthogonal experiment was used to conform the best synthesis condition, which the copolymer was synthesized at 120℃under 15min microwave radian,with the dosage of Sn(oct)2 1000:1.Using this reaction condition and changing the nD,L-LA/nHECS feed ratio(D,L-lactide/aminoglucoside units molar ratio) from 20:1 to 40:1, get a serial of HECS-g-PLA copolymer.The structure of the copolymer was characterized by FTIR, 1HNMR and EA, its crystal property was studied by WXRD, its thermostability was studied by DSC and TG, the effect of feed ratio on productivity and grafting percentage was also studied. The copolymers'grafting percentage can be controlled. the HECS-g-PLA40 copolymer's grafting percentage was 307%, the average degree of polymerization of polylactide branch chains on the graft copolymer is 24.1. The result of X-RD show the higher feed ratios led to copolymer with lower crystallinity, while the result of DSC and TG show the higher feed ratios led to copolymer with higer Tg but lower temperature of degradation.The colour of copolymer change from deep orange to light orange.The copolymer can resolve in DMSO.The Residue of Sn in the copolymer is 0.658mg/g. The conten of HECS in copolymer can improve the hydrophilic nature,the high content of HECS,the high hydrophilic nature copolymer obtained.
The results of vitro degradation test by physiologicalsaline indicated that we can adjust the degradation velocity of the products and PH value by change of the feed ratio, which can reduce acidity of the degradation product of the pure PDLLA and decrease the degradation rate of PDLLA. FTIR spectra of degradation at 8w show that degradation react in
polylactide branch chains.Cell culture of vitro was assessed using the MTT assay, the results show that in vitro the copolymers' biocompatibility is more better than PDLLA, and the HECS-g-PLA system's cell viabilitys was much better than the PDLLA systerm. The results of hemolysis test and dynamic blood-clotting test indicated that the copolymers consistent with the standard of test's requirement, and the HECS-g-PLA system was much better than the PDLLA.The QCM-D analytic results of HECS-g-PLA show that the content of HECS can increase the adsorption of BSA,which can improve the anti-coagulant ability.
为了降低壳聚糖的结晶性能以及其分子间或分子内较强的氢键作用,提高壳聚糖的反应活性,获得接枝率高的共聚物,本研究分别以羟乙基壳聚糖和壳聚糖为大分子引发剂引发丙交酯开环聚合,系统研究和比较了两者引发丙交酯开环聚合的反应活性,结果显示羟乙基化改性可明显提高壳聚糖的反应活性,壳聚糖／聚乳酸接枝共聚物的接枝率为225%,而相应的羟乙基壳聚糖／聚乳酸共聚物提高到307%。进一步以羟乙基壳聚糖为大分子引发剂,分别采用传统封管热聚合法和微波辅助法实施该共聚合反应,考察了聚合方法对该接枝共聚合反应的影响,结果表明,微波辅助聚合15min获得的共聚物和封管热聚合24h获得的共聚物具有相近的接枝率,这表明微波辅助可有效提高聚合反应速率,大大缩短聚合反应时间。
在上述研究基础上,以羟乙基壳聚糖为大分子引发剂,通过正交试验确定最佳的聚合条件：催化剂按照nD,L-LA：n辛酸亚锡为1000：1投入,微波辐射功率10W,120℃下反应时间为15min。在最佳的聚合条件下,采用不同的原料投料比(nD,L-LA：nHECS糖单元：20：1,30：1,40：1),通过微波辅助聚合方法制备了一系列不同组成的HECS-g-PLA,通过FTIR、1H-NMR、X-RD、元素分析、DSC、TG等测试方法,系统研究了nD,L-LA：nHECS糖单元投料比对共聚物的组成与结构、产率、接枝率和性能的影响。结果表明共聚物的接枝率可控,接枝率随着投料比的增加而增加,其中投料比为40：1,可得接枝率最大为307%的共聚物,该共聚物的单位糖环平均乳酰单元数为24.1。产物颜色随着投料比的增加,由深黄色向淡黄色变化,可溶于DMSO,锡残留量为0.658mg/g; X-RD分析结果表明随着投料比的增大,产率增大,共聚物的接枝率增大,结晶性能下降。DSC、TG分析结果表明随着投料比的增大,玻璃化转变温度升高,裂解温度下降。表面接触角和吸水率测试显示,HECS的含量对PDLLA的亲水性有改善作用,HECS的含量越高,HECS的亲水性越好。
从体外降解、细胞毒性、溶血试验、动态凝血、QCM等手段考察了材料的生物学性能。降解结果表明所得共聚物的降解速率和产物的接枝率密切相关,接枝率越高降解速率越快,失重曲线显示HECS可以减缓PDLLA的降解速率。FTIR谱图显示共聚物前8w的降解主要发生在聚乳酸链段上,pH值变化曲线显示HECS对PDLLA降解带来的酸性有缓冲作用；溶血试验结果显示,该材料的溶血率为1.04,无溶血作用；动态凝血和QCM蛋
白吸附结果显示该材料有利于白蛋白吸附,BCI曲线坡度较PDLLA平缓,这些表明了HECS-g-PLA的抗凝血性能比PDLLA好。体外细胞培养结果显示,HECS-g-PLA的细胞毒性较PDLLA小,细胞毒性级别为1,浸提体液中的细胞生长情况较好。
Using a new method—microwave-irradiation to get the Hydroxyethylated chitosan-graft-poly(D,L-lactide) (HECS-g-PLA)copolymer, the graft copolymerization was carried out by ring opening polymerisation(ROP) using stannous octoate (Sn(oct)2) as catalyst without any organic solvent.
It is showed that hydroxyethylated chitosan(HECS) have higher reaction activity than CS as initiator for ROP of D,L-lactide at same condition.The grafting percentage of HECS-g-PLA is 307%, while CS-g-PLA is 225%. Because hydroxyethylation can decrease the crystallinity and hydrogen bond interaction of CS, increase the reaction activity. The grafting percentage of HECS-g-PLA synthesized by microwave-assisted ROP(MAROP) for 24h approximately equal to closed-tube polymerization(CTROP) for 15min.The result show MAROP would significantly increase polymerization rate and decrease polymerization time using same initiator compare with using CTROP.Base on those result, orthogonal experiment was used to conform the best synthesis condition, which the copolymer was synthesized at 120℃under 15min microwave radian,with the dosage of Sn(oct)2 1000:1.Using this reaction condition and changing the nD,L-LA/nHECS feed ratio(D,L-lactide/aminoglucoside units molar ratio) from 20:1 to 40:1, get a serial of HECS-g-PLA copolymer.The structure of the copolymer was characterized by FTIR, 1HNMR and EA, its crystal property was studied by WXRD, its thermostability was studied by DSC and TG, the effect of feed ratio on productivity and grafting percentage was also studied. The copolymers'grafting percentage can be controlled. the HECS-g-PLA40 copolymer's grafting percentage was 307%, the average degree of polymerization of polylactide branch chains on the graft copolymer is 24.1. The result of X-RD show the higher feed ratios led to copolymer with lower crystallinity, while the result of DSC and TG show the higher feed ratios led to copolymer with higer Tg but lower temperature of degradation.The colour of copolymer change from deep orange to light orange.The copolymer can resolve in DMSO.The Residue of Sn in the copolymer is 0.658mg/g. The conten of HECS in copolymer can improve the hydrophilic nature,the high content of HECS,the high hydrophilic nature copolymer obtained.
The results of vitro degradation test by physiologicalsaline indicated that we can adjust the degradation velocity of the products and PH value by change of the feed ratio, which can reduce acidity of the degradation product of the pure PDLLA and decrease the degradation rate of PDLLA. FTIR spectra of degradation at 8w show that degradation react in
polylactide branch chains.Cell culture of vitro was assessed using the MTT assay, the results show that in vitro the copolymers' biocompatibility is more better than PDLLA, and the HECS-g-PLA system's cell viabilitys was much better than the PDLLA systerm. The results of hemolysis test and dynamic blood-clotting test indicated that the copolymers consistent with the standard of test's requirement, and the HECS-g-PLA system was much better than the PDLLA.The QCM-D analytic results of HECS-g-PLA show that the content of HECS can increase the adsorption of BSA,which can improve the anti-coagulant ability.
引文
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