CN111607844A - 一种基于改性透明质酸的超分子纳米纤维的制备方法 - Google Patents
一种基于改性透明质酸的超分子纳米纤维的制备方法 Download PDFInfo
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Abstract
本发明提供了一种基于改性透明质酸的超分子纳米纤维的制备方法。由甲基丙烯酸酐和Phe‑Cys‑Cys‑Phe四肽改性的透明质酸与葫芦脲发生主客体相互作用形成超分子结构再结合静电纺丝技术制得。本发明通过对透明质酸改性而赋予的共价键和非共价键,可以解决透明质酸在组织工程和医药领域中极易溶于水、在组织中停留时间短、降解速度快等涉及稳定性和机械强度的问题。另外改性透明质酸与静电纺丝技术相结合制备的纳米纤维,对于像神经修复领域需要的高比表面积纤维状结构也是具有重大意义的,因此,有望替代神经修复领域常用的如PCL,PLA,PU等高分子聚合物材料。
Description
技术领域
本发明涉及组织工程、药物释放等生物医用材料制备技术领域,具体涉及一种基于改性透明质酸的超分子纳米纤维的制备方法。
背景技术
透明质酸又名玻尿酸,是一种天然高分子酸性粘多糖,广泛存在于人体组织间、眼球内和关节头的滑液中,起到粘合、保湿和润滑的保护作用。同时由于水凝胶材料具有内在的生物相容性、高吸水性、可注射性以及与天然细胞外基质结构相似等优异性质近年来在生物医用领域得到广泛关注。(Giovanna Pitarresi,Paola Pierro.Biomacromolecules2006,7,1302-1310)指出传统的透明质酸水凝胶是由透明质酸大分子无规交联形成的本体胶,存在力学性能低、水解速度快、在组织内停留时间短等缺点,导致在组织工程应用中受到了很大的限制。为了解决这一问题Antunes J C,Oliveira J,Reis R,etc.JournalofBiomedical Materrials Research PartA,2010,94,856-869.;Ibrahim S,Kang Q K,RamamurthiA.Journal of biomedical materials Reserch partA,2010,94,355-370.;Ohri R,Hahn S K,HoffmanA S,etc.Research PartA,2004,70,328-334.文献指出采用包括戊二醛、二乙烯基砜(DVS)、己二酸二酰肼(ADH)等交联剂以提升透明质酸的性能,虽然达到了提升力学性能的作用。但是存在生物相容性问题,文献【Dong C-M,WuX,Caves J,etc.Photomediated crosslinking ofC6-cinnamate derivatized type Icollagen.Biomaterials,2005,26,4041-4049.】指出交联残留物会造成细胞毒性和细胞钙化,并且不能被人体完全吸收。此外,透明质酸表面具有丰富羧基和羟基,可以通过对透明质酸表面的官能团进行改性以达到自交联的目的,来解决透明质酸稳定差的缺点。此外纳米纤维结构对于像神经修复、伤口愈合等领域表现出更好的细胞粘附、增殖和分化的效果。
发明内容
本发明的目的是为了改进现有技术的不足而提供一种基于改性透明质酸的超分子纳米纤维的制备方法;该方法具有普适性、高效性、技术成熟等优点,为实现改性纳米纤维的快速制备提供了路径,在组织工程及生物医用领域具有较高的应用价值。
本发明的技术方案为:一种基于改性透明质酸的超分子纳米纤维的制备方法,其具体步骤如下:
(1)透明质酸的初步改性:将透明质酸与甲基丙烯酸酐在碱性条件下发生酯化反应,反应完成后用有机溶剂析出、冷冻干燥收集,获得具有双键的初步改性透明质酸,低温保存;
(2)透明质酸的最终改性:将初步改性的透明质酸溶于去离子水中,在惰性气体的保护下,加入Phe-Cys-Cys-Phe四肽,再加入还原剂进行反应,待反应完成后,将反应产物透析,冷冻干燥,获得具有双键与Phe-Cys二肽的最终改性透明质酸;
(3)将步骤(2)制得得最终改性透明质酸溶解于水中,并加入葫芦脲、光引发剂,搅拌混合均匀后,获得用于制备纳米纤维的纺丝液;
(4)将步骤(3)制得的纺丝液通过静电纺丝方法制成纳米纤维,然后将纳米纤维置于紫外光下进行交联,得到超分子纳米纤维。
本发明步骤(1)中Phe-Cys-Cys-Phe四肽的合成可参照文献【Matthew J.Rowland,Marina Atgie,Dominique Hoogland,and Oren A.Scherman.Biomacromolecules 2015,16,2436-2443.】并优化部分参数制得;具体制备步骤为:L-胱氨酸与BOC-Phe-OSU酰胺化反应得到粗产品,再通过盐酸的四氧六环溶液除去保护基团,待反应完成后,然后洗涤、过滤获得Phe-Cys-Cys-Phe四肽,低温保存;优选L-胱氨酸与BOC-Phe-OSU的摩尔比为1:(2.1-2.25)。Phe-Cys-Cys-Phe四肽的结构式如下:
上述制备的改性透明质酸制备过程反应式如下:
优选步骤(1)中所述的透明质酸的分子量在100-150万。
优选步骤(1)中甲基丙烯酸酐的添加量为透明质酸质量的10-15%。
优选步骤(1)中所述的碱性条件为控制反应的PH维持在8-12之间。
优选步骤(1)中所述的机溶剂为无水乙醇、丙酮或二甲基亚砜。
优选步骤(2)中所述的Phe-Cys-Cys-Phe四肽的用量为初次改性透明质酸质量的0.1-1%。
优选步骤(2)中所述的还原剂为二硫苏糖醇、2-巯基乙醇或三(2-羰基乙基)磷盐酸盐;还原剂的添加量为初次改性透明质酸质量的10-15%。
优选步骤(2)中反应时间为3-4小时。
优选步骤(3)中葫芦脲的添加量为最终改性透明质酸的质量的0.1-0.5%。
优选步骤(3)中所述的光引发剂为偶氮二异丁脒盐酸盐、偶氮二异丁咪唑啉盐酸盐、2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮中的一种;光引发剂的添加量为最终改性透明质酸的质量的0.25-1%。
优选步骤(3)中最终改性透明质酸溶解于水中后最终改性透明质酸的质量浓度为1-5%。
优选步骤(4)中所述的静电纺丝方法的参数为:电压为15--20kv,纺丝液流量为0.5-2ml/h,接收距离为15-20cm。
优选所述紫外光的波长为365-395nm;其照射时间为1-3min。
本发明根据所需要纳米纤维膜的厚度来调节纺丝时间,一般控制在5-6h后即可获得透明的超分子纳米纤维膜。
有益效果:
本发明采用透明质酸为原料,通过对透明质酸进行改性并结合静电纺丝技术制备成纳米纤维。单纯的透明质酸在实际应用时存在着机械强度低,水解速度快等问题,难以满足临床应用中对材料的要求。此外,对于像神经修复等领域需要材料具有微纳米纤维结构,常规的透明质酸凝胶不能满足要求,所以于静电纺丝技术的结合显得尤为重要。因此,我们通过对透明质酸的改性,使透明质酸大分子上同时具备备得到更为理想的生物医学材料,并将改性的透明质酸制备成纳米纤维膜。得到的产共价键和非共价健制品具有更好的机械强度并且更有利于细胞粘附和促进细胞生长,从而提升了透明质酸在生物医学领域的应用范围。
附图说明
图1为本发明静电纺丝设备示意图;
图2为实施例1制备的初次改性透明质酸、最终改性透明质酸和纯透明质酸的实物示意图;
图3为实施案例1中通过静电纺丝技术制备的超分子纳米纤维SEM图;
图4为实施案例2中通过静电纺丝技术制备的超分子纳米纤维SEM图;
图5为实施案例3中通过静电纺丝技术制备的超分子纳米纤维SEM图;
图6为实施例1-3制备的超分子透明质酸与纯透明质酸在透明质酸酶下的降解速率图。
具体实施方式
以下通过具体实施例说明本发明,但本发明并不仅仅限定于这些实施例。本发明静电纺丝设备示意图如图1所示。
实施例1
(1)称取1.5g L-胱氨酸与4.2g BOC-Phe-OSU溶解于DMF中,酰胺化反应制备而成,在得到粗产品后用4mol/L的四氧六环的盐酸溶液除去保护基团。待反应完成后,再用乙醚进行洗涤、过滤、收集得到Phe-Cys-Cys-Phe四肽(结构式如图1所示)。称取5g分子量为100万的透明质酸溶解于去离子水中,待完全溶解之后。往溶液中缓慢滴加4mol/L的氢氧化钠溶液,调节PH至8,再加入0.5g甲基丙烯酸酐反应9h,在反应过程中通过滴加4mol/L的氢氧化钠溶液使溶液的PH值维持在8,待反应完成后用无水乙醇析出后再用去离子水溶解,反复洗涤3次除去未反应的甲基丙烯酸酐,最后冷冻干燥收集。获得具有双键的初步改性透明质酸。称取2g初步改性的透明质酸溶解于去离子水中,待完全溶解完成后,往溶液中加入0.002g Phe-Cys-Cys-Phe四肽,并加入0.2g二硫苏糖醇,在氮气的保护下,混合溶液在常温条件下充分反应3h,待反应完成后,将反应产物置于透析袋中用去离子水透析7d,冷冻干燥。得到同时具有双键与Phe-Cys二肽的最终改性透明质酸。所制备的初次改性透明质酸、最终改性透明质酸的交联效果图和纯透明质酸溶液图如图2所示。从图中可以看出初次改性的透明质酸在紫外光固化之后由溶液态变成了凝胶态,但是机械强度较弱。在通过进一步改性并将最终改性的透明质酸在交联后,获得的既有共价和非共价相互作用下的凝胶相较于初次改性透明质酸拥有更佳的机械性能。
(2)取1g步骤(1)中最终改性透明质酸,溶解于水中制备为1wt%的透明质酸基溶液,并加入0.001g葫芦脲、0.0025g偶氮二异丁脒盐酸盐光引发剂,搅拌1h。获得用于制备纳米纤维的纺丝液。将纺丝液静置除去气泡。配制好纺丝液后注入注射泵中置于微流泵上,调节电压为15kV;喷射针头到收集板的距离为15cm;纺丝液流量为0.5mL/h;收集器为圆筒收集器,开启静电纺丝装置,静电纺丝5小时后将纳米纤维膜置于365nm紫外光下交联1min,最终得到超分子透明质酸纳米纤维膜,所制备的纳米纤维膜的扫描电子显微镜(SEM)图如图3所示,从图上可以看出纤维直径分布均匀,且纤维直径在20-80nm。
实施例2
(1)称取1.5g L-胱氨酸与4.3g BOC-Phe-OSU溶解于DMF中,酰胺化反应制备而成,在得到粗产品后用4mol/L的四氧六环的盐酸溶液除去保护基团。待反应完成后,再用乙醚进行洗涤、过滤、收集得到Phe-Cys-Cys-Phe四肽。称取5g分子量为125万的透明质酸溶解于去离子水中,待完全溶解之后。往溶液中缓慢滴加4mol/L的氢氧化钠溶液,调节PH至10,再加入0.65g甲基丙烯酸酐反应10h,在反应过程中通过滴加4mol/L的氢氧化钠溶液使溶液的PH值维持在10,待反应完成后用丙酮析出后再用去离子水溶解,反复3次除去未反应的甲基丙烯酸酐,最后冷冻干燥收集。获得具有双键的初步改性透明质酸。称取2g初步改性的透明质酸溶解于去离子水中,待完全溶解完成后,往溶液中加入0.01g Phe-Cys-Cys-Phe四肽,并加入0.25g 2-巯基乙醇,在氮气的保护下,混合溶液在常温条件下充分反应3.5h,待反应完成后,将反应产物置于透析袋中用去离子水透析7d,冷冻干燥。得到同时具有双键与Phe-Cys二肽的最终改性透明质酸。
(2)取1g步骤(1)中最终改性透明质酸,溶解于水中制备为2.5wt%的透明质酸基溶液,并加入0.0025g葫芦脲、0.005g偶氮二异丁咪唑啉盐酸盐光引发剂,搅拌1h。获得用于制备纳米纤维的纺丝液。将纺丝液静置除去气泡。配制好纺丝液后注入注射泵中置于微流泵上,调节电压为17.5kV;喷射针头到收集板的距离为17.5cm;纺丝液流量为1.5mL/h;收集器为圆筒收集器,开启静电纺丝装置,静电纺丝5.5小时后后将纳米纤维膜置于365nm紫外光下交联2min,最终得到超分子透明质酸纳米纤维膜,所制备的纳米纤维膜的扫描电子显微镜(SEM)图如图4所示,从图上可以看出纤维直径分布均匀,且纤维直径在200-400nm。
实施例3
(1)称取1.5g L-胱氨酸与4.4g BOC-Phe-OSU溶解于DMF中,酰胺化反应制备而成,在得到粗产品后用4mol/L的四氧六环的盐酸溶液除去保护基团。待反应完成后,再用乙醚进行洗涤、过滤、收集得到Phe-Cys-Cys-Phe四肽。称取5g分子量为150万的透明质酸溶解于去离子水中,待完全溶解之后。往溶液中缓慢滴加4mol/L的氢氧化钠溶液,调节PH至12,再加入0.75g甲基丙烯酸酐反应12h,在反应过程中通过滴加4mol/L的氢氧化钠溶液使溶液的PH值维持在12,待反应完成后用二甲基亚砜析出后再用去离子水溶解,反复3次除去未反应的甲基丙烯酸酐,最后冷冻干燥收集。获得具有双键的初步改性透明质酸。称取2g初步改性的透明质酸溶解于去离子水中,待完全溶解完成后,往溶液中加入0.02g Phe-Cys-Cys-Phe四肽,并加入0.3g三(2-羰基乙基)磷盐酸盐,在氮气的保护下,混合溶液在常温条件下充分反应4h,待反应完成后,将反应产物置于透析袋中用去离子水透析7d,冷冻干燥。得到同时具有双键与Phe-Cys二肽的最终改性透明质酸。
(2)取1g实施例1中,步骤(1)中最终改性透明质酸,溶解于水中制备为5wt%的透明质酸基溶液,并加入0.005g葫芦脲0.01g 2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮光引发剂,搅拌1h。获得用于制备纳米纤维的纺丝液。将纺丝液静置除去气泡。配制好纺丝液后注入注射泵中置于微流泵上,调节电压为20kV;喷射针头到收集板的距离为20cm;纺丝液流量为2mL/h;收集器为圆筒收集器,开启静电纺丝装置,静电纺丝6小时后后将纳米纤维膜置于365nm紫外光下交联3min,最终得到超分子透明质酸纳米纤维膜,所制备的纳米纤维膜的扫描电子显微镜(SEM)图如图5所示,从图上可以看出纤维直径分布均匀,且纤维直径在400-600nm。
将上述实施例1~3制备的超分子透明质酸与纯透明质酸浸入37℃的透明质酸酶溶液(50U/ml的PBS溶液中)中。每天更换溶液,并在指定的时间间隔内监控重量变化,得到透明质酸酶下的降解速率图如图6示;
剩余重量(%)的计算如下:
剩余重量(%)=Wt/Wo×100(Wo是初始重量,Wt是降解时间t之后的重量)
从酶降解失重曲线图(图5)中可以看出实施例1、实施例2和实施例3水凝胶随降解时间的延长,失重速率逐渐减慢,纯透明质酸在第3天时就完全降解,而实施例1、实施例2和实施例3分别在第9天、10天、10天时完全溶解。这是因为实施例1、实施例2、实施例3中的水凝胶发生了交联作用而引起的,实施例2和实施例3中的水凝胶的交联密度较案例一高,导致酶降解破坏交联点引起失重变化需要更长的时间。相反,纯透明质酸的交联密度最低,最容易被降解发生失重变化。这是因为交联密度低的高分子聚合物的交联点之间的平均分子质量大,酶可以更容易靠近高分子主链,从而使透明质酸酶的活性位点能更好的束缚六糖环使其被降解。因此通过改性获得的透明质酸具有减缓降解速度和增加在组织内停留时间的效果。
Claims (10)
1.一种基于改性透明质酸的超分子纳米纤维的制备方法,其具体步骤如下:
(1)透明质酸的初步改性:将透明质酸与甲基丙烯酸酐在碱性条件下发生酯化反应,反应完成后用有机溶剂析出、冷冻干燥收集,获得具有双键的初步改性透明质酸,低温保存;
(2)透明质酸的最终改性:将步骤(1)制得的初步改性的透明质酸溶于去离子水中,在惰性气体的保护下,加入Phe-Cys-Cys-Phe四肽,再加入还原剂进行反应,待反应完成后,将反应产物透析,冷冻干燥,获得具有双键与Phe-Cys二肽的最终改性透明质酸;
(3)将步骤(2)制得的最终改性透明质酸溶解于水中,并加入葫芦脲、光引发剂,搅拌混合均匀后,获得用于制备纳米纤维的纺丝液;
(4)将步骤(3)制得的纺丝液通过静电纺丝方法制成纳米纤维,然后将纳米纤维置于紫外光下进行交联,得到超分子纳米纤维。
2.根据权利要求1所述的制备方法,其特征在于:步骤(1)中所述的透明质酸的分子量在100-150万。
3.根据权利要求1所述的制备方法,其特征在于:步骤(1)中甲基丙烯酸酐的添加量为透明质酸质量的10-15%;步骤(1)中所述的碱性条件为控制反应的PH维持在8-12之间。
4.根据权利要求1所述的制备方法,其特征在于:步骤(1)中所述的机溶剂为无水乙醇、丙酮或二甲基亚砜。
5.根据权利要求1所述的制备方法,其特征在于:步骤(2)中所述的Phe-Cys-Cys-Phe四肽的用量为初次改性透明质酸质量的0.1-1%;步骤(2)中所述的还原剂为二硫苏糖醇、2-巯基乙醇或三(2-羰基乙基)磷盐酸盐;还原剂的添加量为初次改性透明质酸质量的10-15%。
6.根据权利要求1所述的制备方法,其特征在于:步骤(2)中反应时间为3-4小时。
7.根据权利要求1所述的制备方法,其特征在于:步骤(3)中葫芦脲的添加量为最终改性透明质酸的质量的0.1-0.5%;步骤(3)中最终改性透明质酸溶解于水中后最终改性透明质酸的质量浓度为1-5%。
8.根据权利要求1所述的制备方法,其特征在于:步骤(3)中所述的光引发剂为过硫酸钾、偶氮二异丁脒盐酸盐、偶氮二异丁咪唑啉盐酸盐、过硫酸铵或2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮中的一种;光引发剂的添加量为最终改性透明质酸的质量的0.25-1%。
9.根据权利要求1所述的制备方法,其特征在于:步骤(5)中所述的静电纺丝方法的参数为:电压为15--20kv,纺丝液流量为0.5-2ml/h,接收距离为15-20cm。
10.根据权利要求1所述的制备方法,其特征在于:所述紫外光的波长为365-395nm;其照射时间为1-3min。
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CN112853622A (zh) * | 2020-12-31 | 2021-05-28 | 苏州国纳思新材料科技有限公司 | 一种自愈合水凝胶微纤维织物的制备方法 |
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CN114351289A (zh) * | 2021-12-31 | 2022-04-15 | 中国科学院苏州纳米技术与纳米仿生研究所 | 一种透明质酸纤维及其制备方法和应用 |
CN114351289B (zh) * | 2021-12-31 | 2024-03-19 | 中国科学院苏州纳米技术与纳米仿生研究所 | 一种透明质酸纤维及其制备方法和应用 |
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