CN110551296B - 一种果胶基双物理交联水凝胶及制备方法和应用 - Google Patents
一种果胶基双物理交联水凝胶及制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种果胶基双物理交联水凝胶及制备方法和应用,该凝胶制备过程分为三步:首先制备凝胶前驱液:将疏水单体增溶到十二烷基硫酸钠胶束溶液,果胶粉末、丙烯酰胺及光引发剂均匀分散在胶束溶液后获得前驱液;其次紫外光引发自由基聚合使凝胶前驱液转变为单交联水凝胶;最后是浸泡处理:将单交联水凝胶浸泡于Fe3+溶液中后,取出再置于去离子水中浸泡,得到具有高强韧性的果胶基双物理交联水凝胶。该水凝胶具有可调节的力学性能:拉伸强度0.60‑2.0MPa,断裂应变130‑1500%,弹性模量0.3‑2.0MPa,韧性1.0‑12.0MJ m‑3。该水凝胶具有良好的生物相容性,可用于3D细胞培养。
Description
技术领域
本发明涉及一种果胶基双物理交联水凝胶及制备方法和应用,尤其是涉及制备一种具有优异生物相容性的双物理交联可用于3D细胞培养的果胶基水凝胶的方法,属于功能材料领域。
背景技术
水凝胶是由亲水性聚合物通过物理或化学交联作用,物理交联作用有疏水缔合,配位络合,氢键等作用,化学交联主要是形成各种共价键交联而成的三维立体网状结构,通过自由扩散和亲水基团的吸引将水分子结合并保留在网络内部,使水凝胶质地柔软弹性并具有一定形状。其独特的“软湿”性质与天然组织细胞外基质相似,是开发新型骨及软骨组织修复用支架的理想材料。天然多糖具有良好的生物相容性、可降解性及免疫原性,成为构建组织工程用水凝胶的极佳原料。然而传统多糖基水凝胶固有的脆性及弱稳定性限制了他们在组织工程领域的应用。
针对上述问题,人们从能量耗散或应力分散角度出发,开发了一系列高强高韧性水凝胶:纳米复合水凝胶,大分子微球水凝胶,滑环结构水凝胶,双网络水凝胶,四臂PEG水凝胶等。双网络水凝胶硬而脆的第一网络在承受应力时首先断裂以分散应力,软而韧的第二网络则受力伸展变形并负责维持凝胶的完整性,这种高效能量耗散机制使双网络水凝胶成为近年来构建高强高韧水凝胶广泛采取的策略。然而由共价键作用交联而成的双网络水凝胶存在抗疲劳性差的问题,用于耗散能量扮演“牺牲键”角色的共价键一经断裂再难恢复,而在实际应用场景中,通常要求水凝胶可以忍受多次重复拉伸或压缩。构建基于纯物理交联的高强韧性双网络水凝胶是解决上述问题有效手段之一,物理交联作用的动态性使其在断裂后可以实现重新恢复。因此,在凝胶网络中引入动态物理交联键如氢键,离子键,疏水缔合等构建具有自恢复、抗疲劳甚至自愈合特性的高强韧性双网络水凝胶成为研究热点。目前已有弹性模量或拉伸强度达到MPa水平物理交联水凝胶的报道,但多专注于力学性能的增强,缺乏生物相容性方面的探究,限制了所开发水凝胶的应用领域。本发明中以来源广泛,价廉易得,兼具生物相容性与可降解性的天然聚合物为原料,采取“双网络”策略,开发出兼具优异力学性能和生物相容性的高强韧性水凝胶,体外细胞培养证明该凝胶可用作3D细胞培养支架,在组织工程领域具有广阔的应用前景。
发明内容
本发明的目的在于克服现有多糖基双网络高强韧性水凝胶缺乏力学自恢复性、抗疲劳性弱、生物相容性差等缺点,提供一种制备过程简单、具有力学自恢复性和抗疲劳性,兼具优异生物相容性的果胶基物理交联水凝胶及其制备方法。
本发明的技术方案如下:
一种果胶基双物理交联水凝胶;本发明的技术方案如说明书附图1所示;首先制备凝胶前驱液:将疏水单体甲基丙烯酸十八烷基酯(SMA)增溶到十二烷基硫酸钠(SDS)胶束溶液,随后将果胶粉末均匀分散在含有疏水单体的胶束溶液中,加入丙烯酰胺及光引发剂;其次光引发条件下将水凝胶前驱液转变为单交联的单网络水凝胶:紫外光照射下引发原位自由基聚合形成丙烯酰胺与甲基丙烯酸甲酯共聚链poly(AM-co-SMA),该共聚链以SMA之间疏水缔合形成的疏水微域为交联点形成单网络水凝胶;随后是两步浸泡处理:将单网络水凝胶浸泡于三价铁离子溶液中后,果胶链上的-COO-与Fe3+与形成多齿配位络合,得到双网络水凝胶;将该双网络水凝胶置于去离子水中浸泡,除去不稳定交联的Fe3+,得到含有稳定三齿配位作用的高强韧性果胶基双物理交联水凝胶。
本发明的一种果胶基双物理交联水凝胶;具有力学性能自恢复性和抗疲劳性,兼具优异生物相容性的高强韧性水凝胶,通过分析比较原料及各水凝胶红外图谱,可知该凝胶由物理交联的丙烯酰胺网络及果胶网络组成,总固含量为12%-25%,果胶及丙烯酰胺的质量比为1:1.5-1:20,除果胶和丙烯酰胺外,水凝胶其余组分为水。
本发明的一种果胶基双物理交联水凝胶的制备方法,具体制备步骤如下:
(1)制备质量分数为7%的阴离子型表面活性剂十二烷基硫酸钠胶束溶液;
(2)将疏水单体甲基丙烯酸十八烷基酯加入步骤(1)所得胶束溶液中,25-70℃下磁力搅拌获得透明混合溶液;
(3)在步骤(2)所得混合溶液中加入果胶粉末,25-70℃磁力搅拌获得均匀混合溶液
(4)在步骤(3)所得溶液中加入丙烯酰胺和光引发剂I2959,25-70℃下磁力搅拌得到均匀溶液;
(5)将步骤(4)所得均匀溶液注入模具,紫外光引发后得到单交联单网络水凝胶;
(6)将步骤(5)所得单交联单网络水凝胶浸入三价铁离子溶液中,取出后浸泡于去离子水中,得到双交联双网络水凝胶;
本发明所得的双网络水凝胶高压灭菌后可直接用于3D细胞培养。
在步骤(2)-(4)过程中每一次加料完成后都采用磁力搅拌以期获得均匀的混合溶液,保证最后所得水凝胶结构均匀,避免因为凝胶结构不均匀而造成的力学性能损失。
水凝胶制备过程优选条件如下:
所述步骤(2)中甲基丙烯酸十八烷基酯相对丙烯酰胺摩尔比例为1-4mol%。
所述步骤(3)中果胶相对丙烯酰胺的质量比为1:1.5-1:20。
所述步骤(3)中所用果胶优选添加高甲氧基化(>65%)柑橘或苹果来源的果胶。
所述步骤(4)中I2959相对丙烯酰胺和疏水单体甲基丙烯酸十八烷基酯总量的1mol%所述步骤(5)中光引发时间为1-5h。
所述步骤(5)(5)中采用紫外光波长为250-420nm,光强度为5-8W。
所述步骤(6)中浸泡铁离子溶液摩尔浓度为0.02-0.2M。
所述步骤(6)中在铁离子溶液中浸泡时间为10min-16h。
所述步骤(6)中在去离子水中浸泡时间为1-72h。
水凝胶应用于体外细胞培养时处理方案如下:
所述果胶基水凝胶经高压灭菌后可直接用于3D细胞培养;灭菌温度为121℃,灭菌时间30min;所用细胞模型为小鼠前成软骨细胞ATDC5,将ATDC5细胞种植于水凝胶表面,该细胞表现出正常的增殖行为,并逐渐浸润到细胞内部。
与现有技术相比,本发明具有以下突出特点:
(1)本发明在整个制备过程中具有耗能低,生物安全性高,价格低廉,操作简单方便,对环境友好等优势。
(2)通过该发明制备的果胶基物理交联高强韧性水凝胶具有良好的力学性能。通过疏水缔合丙烯酰胺网络与离子配位络合的果胶网络协同作用,所述果胶基水凝胶拉伸强度在0.60-2.0MPa,断裂应变在130%-1500%,弹性模量在0.3-2.0MPa,韧性在1.0-12.0MJm-3范围内可调节。
(3)通过该发明制备的果胶基物理交联高强韧性水凝胶具有良好的力学自恢复性及抗疲劳性能。
(4)通过该发明制备的果胶基物理交联高强韧性水凝胶具有优异的细胞相容性,水凝胶可用于三维细胞培养,粘附于该水凝胶表面的多种细胞可维持正常的增殖行为并可逐步浸润到水凝胶内部。
附图说明
图1:水凝胶制备流程实物图及相应交联原理示意图;
图2:水凝胶制备原料(果胶;丙烯酰胺)及单/双网络水凝胶红外图谱;
图3:果胶基高强韧性水凝胶在不同疏水单体浓度下的拉伸应力-应变曲线;
图4:果胶基高强韧性水凝胶在不同果胶丙烯酰胺质量比下的拉伸应力-应变曲线;
图5:果胶基高强韧性水凝胶用于ATDC5细胞体外3D培养,细胞正常增殖并表现出浸润行为。
具体实施方式
下面结合具体实施例进一步说明本发明的技术方案。本发明的技术方案如说明书附图1所示;首先制备凝胶前驱液:将疏水单体甲基丙烯酸十八烷基酯(SMA)增溶到十二烷基硫酸钠(SDS)胶束溶液,随后将果胶粉末均匀分散在含有疏水单体的胶束溶液中,加入丙烯酰胺及光引发剂;其次光引发条件下将水凝胶前驱液转变为单交联的单网络水凝胶:紫外光照射下引发原位自由基聚合形成丙烯酰胺与甲基丙烯酸甲酯共聚链poly(AM-co-SMA),该共聚链以SMA之间疏水缔合形成的疏水微域为交联点形成单网络水凝胶;随后时两步浸泡处理:将单网络水凝胶浸泡于三价铁离子溶液中后,果胶链上的-COO-与Fe3+与形成多齿配位络合,得到双网络水凝胶;将该双网络水凝胶置于去离子水中浸泡,除去不稳定交联的Fe3+,得到含有稳定三齿配位作用的高强韧性果胶基双物理交联水凝胶。
下面实施例中使用的试剂主要包括以下几种:十二烷基硫酸钠,果胶,六水合三氯化铁,丙烯酰胺,甲基丙烯酸十八烷基酯。按照优选条件说明如下:
(1)制备质量分数为7%的阴离子型表面活性剂十二烷基硫酸钠胶束溶液;
(2)按摩尔比例1-4mol%将疏水单体增溶至步骤(1)中所得胶束溶液中,25-70℃磁力搅拌至透明;
(3)在步骤(2)所得溶液中按质量比1:1.5~1:20加入果胶粉末,25-70℃磁力搅拌获得均匀混合溶液
(4)在步骤(3)所得溶液中按步骤(3)中所选用比例加入丙烯酰胺,按1mol%添加光引发剂,25-70℃磁力搅拌获得均匀溶液;
(5)将步骤(4)所得均匀溶液注射入模具,光强度5~8W光波长为250~420nm的紫外光下引发1-5h得到单网络水凝胶;
(6)将步骤(5)所得单网络水凝胶摩尔浓度在0.02-0.2M的三价铁离子溶液中浸泡10min~16h,取出后再浸泡于去离子水中1~72h,得到结构均匀的高强韧性双网络水凝胶。
实施例1
(1)制备质量分数为7%的阴离子型表面活性剂十二烷基硫酸钠胶束溶液10ml;
(2)将1mol%疏水单体,亦即68μL SMA增溶到步骤(1)中所得胶束溶液,35℃磁力搅拌至透明;
(3)在步骤(2)所得溶液中0.1364g加入果胶粉末,70℃磁力搅拌至透明;
(4)在步骤(3)所得溶液中按1.2273g加入丙烯酰胺,添加光引发剂I 29590.0395g,70℃磁力搅拌均匀;
(5)将步骤(4)所得均匀溶液注射入模具,光强度为8W的365nm波长紫外光下引发1h得到单网络水凝胶,;
(6)将步骤(5)所得单网络水凝胶依次浸入摩尔浓度在0.06M的三价铁离子溶液8h和去离子水中72h,得到结构均匀的高强韧性双网络水凝胶。通过单轴拉伸试验测得所得果胶基双物理交联高强韧性水凝胶拉伸强度可达1.568±0.026MPa。
该实施实例所制备水凝胶总固含量为12%,果胶与丙烯酰胺的质量比为1:9.0,水凝胶制备过程中的形态变化(从前驱液到双网络水凝胶)如说明书附图1所示,水凝胶前驱液注入模具中在紫外光下引发3h得到白色偏透明的单网络水凝胶,该凝胶刚性较弱,使用镊子斜持可观察到受重力作用的明显下垂;将其浸泡在三价铁离子溶液后变为黄色的双网络水凝胶,该凝胶刚性得到极大提升,使用镊子斜持不受重力影响无下垂;进一步浸泡于去离子水中得到黄色加深的水凝胶,其刚性得到进一步提高,该过程中多余三价铁离子逐渐浸泡除去,凝胶内部铁离子与果胶上羧基形成稳定三齿配位。利用傅里叶红外光谱分析技术分析各原料(果胶与丙烯酰胺)与各步骤所得凝胶成分,如附图2所示,a,b光谱分别为果胶和丙烯酰胺的红外吸收光谱,c、d、e光谱为各步骤制得水凝胶红外光谱,相对于水凝胶制备原料,除特征峰偏移外无新的特征吸收峰出现,证明凝胶过程中没有共价键生成,所得水凝胶为纯物理交联的水凝胶。
实施例2
(1)制备质量分数为7%阴离子型表面活性剂十二烷基硫酸钠胶束溶液10mL;
(2)将2mol%疏水单体,亦即90μL SMA增溶至步骤(1)中所得胶束溶液中,25℃磁力搅拌至透明;
(3)在步骤(2)所得溶液加入0.5454g果胶粉末,45℃磁力搅拌至均匀;
(4)在步骤(3)所得溶液中加入0.8181g丙烯酰胺,添加光引发剂I 2959 0.0263g,70℃磁力搅拌均匀;
(5)将步骤(4)所得均匀溶液注射入模具,光强度为6W的420nm波长紫外光下引发3h得到单网络水凝胶;
(6)将步骤(5)所得单网络水凝胶浸入摩尔浓度在0.02M的三价铁离子溶液16h,去离子水中浸泡36h,得到结构均匀的高强韧性双网络水凝胶。该凝胶总固含量为12%,果胶与丙烯酰胺的质量比为1:1.5跟据单轴拉伸试验,所得果胶基双物理交联高强韧性水凝胶弹性模量可达1.662±0.025MPa,韧性高达5.1MJ m-3。在实验过程中,固定水凝胶固含量为12%,果胶与丙烯酰胺比例为1:2.25,改变疏水单体的摩尔浓度可得到不同力学性能的水凝胶,如图3所示,疏水单体SMA含量为1mol%时,水凝胶断裂强度为0.75MPa,增加SMA含量至4mol%,在拉伸应变为390%情况下水凝胶的拉伸强度即可达1.0MPa,该果胶基水凝胶展示出受SMA含量影响的可调控的机械性能。
实施例3
(1)制备质量分数为7%的阴离子型表面活性剂十二烷基硫酸钠胶束溶液10mL;
(2)将4mol%疏水单体,亦即233μL SMA增溶至步骤(1)中所得胶束溶液中,70℃磁力搅拌至透明;
(3)在步骤(2)所得溶液中加入0.0529g果胶粉末,70℃磁力搅拌至透明;
(4)在步骤(3)所得溶液中加入1.0582g丙烯酰胺,添加光引发剂I 2959 0.0347g,45℃磁力搅拌均匀;
(5)将步骤(4)所得均匀溶液注射入模具,光强度为5W的250nm波长紫外光下引发3h得到单网络水凝胶;
(6)将步骤(5)所得单网络水凝胶浸入摩尔浓度在0.20M的三价铁离子溶液16h,去离子水中1h,得到结构均匀的高强韧性双网络水凝胶。该凝胶总固含量为10%,果胶与丙烯酰胺质量比为1:20,单轴拉伸试验表明所得果胶基双物理交联高强韧水凝胶弹性模量可达300±48KPa。
实施例4
(1)制备质量分数为7%阴离子型表面活性剂十二烷基硫酸钠胶束溶液10ml;
(2)将2mol%疏水单体,亦即186μL SMA增溶至步骤(1)中所得胶束溶液中,25℃磁力搅拌至透明;
(3)在步骤(2)所得溶液中加入0.6754g果胶粉末,25℃磁力搅拌至均匀
(4)在步骤(3)所得溶液中加入1.6885g丙烯酰胺,添加0.0543g光引发剂I2959,25℃磁力搅拌均匀;
(5)将步骤(4)所得均匀溶液注射入模具,光强度为8W的365nm波长紫外光下引发5h得到单网络水凝胶;
(6)将步骤(5)所得单网络水凝胶浸入摩尔浓度在0.06M的三价铁离子溶液浸泡10min,在去离子水中浸泡72h,得到结构均匀的高强韧双网络水凝胶。该凝胶总固含量为18%,果胶与丙烯酰胺的质量比为1:2.25,单轴拉伸试验表明所得果胶基双物理交联高强韧水凝胶弹性模量可达1.527±76MPa。
将所得高强韧水凝胶高压灭菌处理后参考国家标准GB/T 16886.12-2000,按0.1g/mL比例制备水凝胶浸提液并用于细胞培养,共培养24h后L929细胞活性可达132.704±13.010%,该水凝胶具有良好的生物相容性。
实施例5
(1)制备质量分数为7%阴离子型表面活性剂十二烷基硫酸钠胶束溶液10mL;
(2)将2mol%疏水单体,亦即96μL SMA增溶至步骤(1)中所得胶束溶液中,35℃磁力搅拌至透明;
(3)在步骤(2)所得溶液中加入0.4870果胶粉末,70℃磁力搅拌至均匀;
(4)在步骤(3)所得溶液中加入0.8766g丙烯酰胺,添加0.0282g光引发剂I2959,70℃磁力搅拌均匀;
(5)将步骤(4)所得均匀溶液注射入模具,光强度为8W的365nm波长紫外光下3h得到单网络水凝胶;
(6)将步骤(5)所得单网络水凝胶依次浸入摩尔浓度在0.06M的三价铁离子溶液8h和去离子水中72h,得到结构均匀的高强韧性双网络水凝胶。该凝胶总固含量为12%,果胶与丙烯酰胺的质量比为1:2.25。单轴拉伸试验测定所得果胶基双物理交联高强韧性水凝胶弹性模量可达1662±25KPa,拉伸强度为0.970±0.040MPa,韧性高达1.043±0.202MJ m-3。改变果胶与丙烯酰胺的比例也可调控水凝胶的力学性能,果胶含量增加凝胶刚性增强,断裂伸长率降低,减少果胶含量将增加水凝胶柔性,如图4所示,在总固含量为12%条件下,果胶与丙烯酰胺质量比为1:1.8时,所制得水凝胶的断裂伸长率为575%,将低果胶含量使其与丙烯酰胺质量比为1:9.0时,断裂伸长率增加至1289%,该双网络水凝胶机械性能可调性使其适用于多种场景。
将所得高强韧性水凝胶高压灭菌处理后用于ATDC5细胞培养,体外培养3d、14d后通过高分辨率共聚焦显微镜获得荧光图像,如图5所示,分析Z轴扫描结果可知初始粘附于细胞表面的细胞维持增殖行为并逐步浸润至水凝胶内部,该水凝胶可用于细胞3D培养。
值得注意的是,以上列举的仅是本发明的几个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (9)
1.一种果胶基双物理交联水凝胶;其特征是凝胶通过分析傅里叶红外光谱确定组分为物理交联的丙烯酰胺和果胶,总固含量为12%-25%,果胶及丙烯酰胺的质量比为1:1.5-1:20;制备方法包括如下步骤:
(1)制备质量分数为7%的阴离子型表面活性剂十二烷基硫酸钠胶束溶液;
(2)将疏水单体甲基丙烯酸十八烷基酯加入步骤(1)所得胶束溶液中,25-70℃下磁力搅拌获得透明混合溶液;
(3)在步骤(2)所得混合溶液中加入果胶粉末,25-70℃磁力搅拌获得均匀混合溶液;
(4)在步骤(3)所得溶液中加入丙烯酰胺和光引发剂I2959,25-70℃下磁力搅拌得到均匀溶液;
(5)将步骤(4)所得均匀溶液注入模具,紫外光引发后得到单交联单网络水凝胶;
(6)将步骤(5)所得单交联单网络水凝胶浸入三价铁离子溶液中,取出后再浸入去离子水中,得到双交联双网络水凝胶。
2.权利要求1的果胶基双物理交联水凝胶的制备方法,其特征是包括如下步骤:
(1)制备质量分数为7%的阴离子型表面活性剂十二烷基硫酸钠胶束溶液;
(2)将疏水单体甲基丙烯酸十八烷基酯加入步骤(1)所得胶束溶液中,25-70℃下磁力搅拌获得透明混合溶液;
(3)在步骤(2)所得混合溶液中加入果胶粉末,25-70℃磁力搅拌获得均匀混合溶液;
(4)在步骤(3)所得溶液中加入丙烯酰胺和光引发剂I2959,25-70℃下磁力搅拌得到均匀溶液;
(5)将步骤(4)所得均匀溶液注入模具,紫外光引发后得到单交联单网络水凝胶;
(6)将步骤(5)所得单交联单网络水凝胶浸入三价铁离子溶液中,取出后再浸入去离子水中,得到双交联双网络水凝胶。
3.如权利要求2所述的方法,其特征是所述步骤(2)中甲基丙烯酸十八烷基酯相对丙烯酰胺摩尔比例为1-4mol%。
4.如权利要求2所述的方法,其特征是所述步骤(3)中果胶相对丙烯酰胺的质量比为1:1.5-1:20。
5.如权利要求2所述的方法,其特征是所述步骤(4)中光引发剂I 2959相对丙烯酰胺和疏水单体甲基丙烯酸十八烷基酯总量的1mol%。
6.如权利要求2所述的方法,其特征是所述步骤(5)中光引发时间为1-5h;采用紫外光波长为250-420nm,光强度为5-8W。
7.如权利要求2所述的方法,其特征是所述步骤(6)中浸泡铁离子溶液摩尔浓度为0.02-0.2M。
8.如权利要求2所述的方法,其特征是所述步骤(6)中在铁离子溶液中浸泡时间为10min-16h;在去离子水中浸泡时间为1-72h。
9.权利要求1的果胶基双物理交联水凝胶用于3D细胞培养。
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