CN115044092A - 一种三维多孔磁性纳米水凝胶及其制备方法和应用 - Google Patents
一种三维多孔磁性纳米水凝胶及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种三维多孔磁性纳米水凝胶及其制备方法和应用,属于生物材料技术领域,所述三维多孔磁性纳米水凝胶的制备方法,包括以下步骤:1)将明胶溶液与磁性纳米粒子混合,超声分散获得磁性纳米粒子‑明胶混合液;2)将所述磁性纳米粒子‑明胶混合液预冷降温至14~18℃后,进行3D打印获得三维材料;3)将所述三维材料浸泡在转谷酰胺酶溶液中,进行二次交联,获得稳定的三维多孔磁性纳米水凝胶。本发明获得的三维多孔的磁性纳米水凝胶具备超顺磁性作用,与细胞相容性好,更适合干细胞在材料表面粘附生长,三维多孔结构有利于营养物质的输送,材料表面的凹凸结构能够使干细胞与材料表面较为紧密地结合。
Description
技术领域
本发明属于生物材料技术领域,尤其涉及一种三维多孔磁性纳米水凝胶及其制备方法和应用。
背景技术
通常的细胞培养都是采用2D培养,但是由于机体内的环境较为复杂,2D培养不能很好的模拟体内情况,不能更真实的反应细胞在体内的存在及变化状态。而最近兴起的3D细胞培养模型比2D更接近体内微环境,培养的细胞更接近机体内部细胞的存在状态,因此,相对于2D培养,可以提供更准确的细胞信息,是研究细胞功能的理想选择,为细胞基础研究、药物筛选等领域提供更优的选择。
第一代3D细胞培养基质水凝胶主要是从鼠尾胶或从小鼠肉瘤细胞基底膜提取的ECM,主要成分是胶原、层粘连蛋白以及生长因子。第一代3D水凝胶使用广泛,是目前基础研究中普遍使用的材料。但也存在一些问题,如含有动物来源的成分,特别是蛋白和生长因子的种类和数量变化无法控制,对细胞或类组织生长有很大的影响,在此基础上建立的3D细胞模型也存在不稳定的情况。另外胶原形成凝胶后不可逆,从凝胶中无法分离出细胞来。实验过程中,需要保持基质胶及操作耗材的低温,难以高通量操作。
第二代水凝胶主要是人工合成多肽支架,在温度或pH/离子强度变化下高度交联,形成三维结构,支持细胞和类组织生长。这类基质胶性能稳定。但是,多肽运输保存必须维持在-20度以下,还需要避免反复冻融,存在操作不方便的问题。
发明内容
有鉴于此,本发明的目的在于提供一种三维多孔磁性纳米水凝胶及其制备方法和应用,原材料为磁性纳米粒子和明胶,原材料不需要低温保存;制备获得的三维多孔磁性纳米水凝胶,具备超顺磁性作用,与细胞相容性好,更适合干细胞在材料表面粘附生长。
本发明提供了一种三维多孔磁性纳米水凝胶的制备方法,包括以下步骤:
1)将明胶溶液与磁性纳米粒子混合,超声分散获得磁性纳米粒子-明胶混合液;
2)将所述磁性纳米粒子-明胶混合液预冷降温至14~18℃后,进行3D打印获得三维材料;
3)将所述三维材料浸泡在转谷酰胺酶溶液中,进行二次交联,获得稳定的三维多孔磁性纳米水凝胶。
优选的,步骤1)中所述明胶溶液的浓度为8wt%~12wt%。
优选的,步骤1)中所述明胶溶液与磁性纳米粒子混合的比例为10ml:(0.1~0.5)g。
优选的,步骤1)中所述超声分散的频率≥20KHz,功率密度≥0.3w/cm2;所述超声分散的时间为20~40S。
优选的,步骤2)中所述3D打印过程中,打印底板温度为14~18℃,喷头大小为0.1~0.2um,喷头的填充速度为10~15mm/s,喷头的移动速度为15~20mm/s。
优选的,步骤3)中转谷酰胺酶溶液的浓度为1.5~2.5wt%;所述浸泡的温度为15~20℃,所述浸泡的时间为8~12min。
本发明提供了所述的制备方法制备获得的三维多孔磁性纳米水凝胶。
优选的,所述三维多孔磁性纳米水凝胶的孔径为500-700μm,所述三维多孔磁性纳米水凝胶的表面有凹凸不平的微孔,所述微孔的孔径为5~20μm。
本发明还提供了所述三维多孔磁性纳米水凝胶在细胞培养中的应用。
本发明还提供了所述三维多孔磁性纳米水凝胶在制备医用体内植入材料中的应用。
与现有技术相比,本发明具有如下有益效果:本发明提供的三维多孔磁性纳米水凝胶,原材料为磁性纳米粒子和明胶,原材料不需要低温保存;在制备过程中,利用凝胶的温度敏感性效应,实现了3D打印,并且经过转谷酰胺酶溶液的浸泡,凝胶进行了二次交联,明胶中的氨基、羧基、酰胺基等基团,通过利用转谷酰胺酶作为交联剂,产生酰胺反应而形成稳定的水凝胶结构;解决了温度升高至37℃以上,凝胶可逆为溶液状态的技术问题,可用于细胞培养实验以及后续的动物体内植入实验。
本发明获得的三维多孔的磁性纳米水凝胶具备超顺磁性作用,与细胞相容性好,磁性水凝胶三维多孔的结构更适合干细胞在材料表面粘附生长,三维多孔结构有利于营养物质的输送,扫描电镜显示磁性纳米水凝胶的材料表面的凹凸结构能够使干细胞与材料表面较为紧密地结合。根据本发明实施例的记载,脐血干细胞在三维多孔磁性纳米水凝胶材料表面生长良好,增殖迅速,第7天的细胞基本覆盖在材料孔的表面。
附图说明
图1为3D生物打印构建的三维多孔磁性纳米水凝胶材料;
图2为三维多孔磁性纳米水凝胶材料扫描电镜观察结果;
图3为培养的P3代脐血间充质干细胞;
图4为死活细胞染色结果体现的干细胞在三维多孔磁性纳米水凝胶表面生长的情况;
图5为三维多孔磁性纳米水凝胶材料与普通的细胞培养板培养细胞的CCK-8测试结果。
具体实施方式
本发明提供了一种三维多孔磁性纳米水凝胶的制备方法,包括以下步骤:
1)将明胶溶液与磁性纳米粒子混合,超声分散获得磁性纳米粒子-明胶混合液;
2)将所述磁性纳米粒子-明胶混合液预冷降温至14~18℃后,进行3D打印获得三维材料;
3)将所述三维材料浸泡在转谷酰胺酶溶液中,进行二次交联,获得稳定的三维多孔磁性纳米水凝胶。
在本发明中,将明胶溶液与磁性纳米粒子混合,超声分散获得磁性纳米粒子-明胶混合液。在本发明中,所述明胶溶液的浓度优选为8~12wt%,进一步优选为9~11wt%,更进一步优选为10wt%。在本发明中,所述明胶溶液的溶剂优选的超纯水。在本发明中,所述磁性纳米粒子优选为Fe3O4磁性纳米粒子,所述磁性纳米粒子的粒径优选为15~25nm,进一步优选为20nm,在本发明的具体实施过程中,所述磁性纳米粒子优选的购置于深圳欣城生物科技有限公司。在本发明中,所述明胶溶液与磁性纳米粒子混合的比例优选为10ml:(0.1~0.5)g;所述混合过程中伴随搅拌,所述搅拌的转速优选为500~700rpm,进一步优选为550~650rpm,更进一步优选为600rpm。在本发明中,所述超声分散的频率≥20KHz,功率密度≥0.3w/cm2;所述超声分散的时间优选为20~40S,进一步优选为25~35S,更进一步优选为30S。
本发明在获得所述磁性纳米粒子-明胶混合液后,将所述磁性纳米粒子-明胶混合液预冷降温至14~18℃后,进行3D打印获得三维材料。在本发明中,优选的预冷降温至15~17℃,更优选的预冷降温至16℃,在上述温度范围内,所述磁性纳米粒子-明胶混合液呈凝胶状态,能够被3D打印。在本发明所述3D打印过程中,打印底板温度优选为14~18℃,进一步优选为15~17℃,更优选为16℃。喷头大小优选为0.1~0.2μm,喷头的填充速度优选为10~15mm/s,喷头的移动速度优选为15~20mm/s。
本发明在获得三维材料后,将所述三维材料浸泡在转谷酰胺酶溶液中,进行二次交联,获得稳定的三维多孔磁性纳米水凝胶。在本发明中,转谷酰胺酶溶液的浓度优选为1.5~2.5wt%,进一步优选为1.8~2.2wt%,更进一步优选为2wt%;所述浸泡的温度优选为15~20℃,所述浸泡的时间优选为8~12min,进一步优选为9~11min,更进一步优选为10min。经过转谷酰胺酶溶液的浸泡,凝胶进行了二次交联,明胶中的氨基、羧基、酰胺基等基团,通过利用转谷酰胺酶作为交联剂,产生酰胺反应而形成稳定的水凝胶结构。
本发明还提供了所述的制备方法制备获得的三维多孔磁性纳米水凝胶。在本发明中,所述三维多孔磁性纳米水凝胶的孔径优选为500-700μm,所述三维多孔磁性纳米水凝胶的表面有凹凸不平的微孔,所述微孔的孔径优选为5~20μm;所述微孔的存在对于干细胞在材料表面的粘附生长起到非常关键的作用。
本发明还提供了所述三维多孔磁性纳米水凝胶在细胞培养中的应用。在本发明中,对于细胞的种类没有特殊限定,任意种类的细胞均可,在本发明具体实施过程中,以干细胞为例,进一步的以脐血间充质干细胞为例。
本发明还提供了所述三维多孔磁性纳米水凝胶在制备医用体内植入材料中的应用。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
磁性纳米粒子(Fe3O4),粒径为20nm,购置于深圳欣城生物科技有限公司。
将明胶加入超纯水中溶解得到10%的明胶水溶液,取1g明胶加入10ml超纯水,在600rpm搅拌下,向明胶溶液中加入磁性纳米粒子0.1g,并使用超声分散获得磁性粒子分散较好的磁性纳米粒子-明胶混合液,超声频率:F=20KHz,超声功率密度:p=发射功率(W)/发射面积(cm2);通常p=0.3w/cm2;超声振荡时间30S。
将磁性纳米粒子-明胶混合液加入到3D打印机的物料槽中进行预冷降温,物料槽温度调节为16℃,明胶在低温下处于凝胶状态,可被打印,打印底板温度也调节为16℃,通过参数设定,对3D打印机喷头大小选择(0.1μm),喷头的填充速度(10mm/s),喷头的移动速度(15mm/s),进行打印,打印后得到三维多孔的磁性纳米水凝胶。将打印的磁性纳米水凝胶浸泡在2wt%的转谷酰胺酶溶液(18℃)中10min,进行二次交联,从而得到稳定的磁性纳米水凝胶结构。
获得的三维多孔磁性纳米水凝胶材料如图1和图2所示,扫描电镜观察显示材料孔径在500~700μm大小,而且在材料表面有凹凸不平的微孔(5~20μm大小),微孔的存在对于干细胞在材料表面的粘附生长起到非常关键的作用。
实施例2
磁性纳米粒子(Fe3O4),粒径为20nm,购置于深圳欣城生物科技有限公司。
将明胶加入超纯水中溶解得到11%的明胶水溶液,取1.1g明胶加入10ml超纯水,在550rpm搅拌下,向明胶溶液中加入磁性纳米粒子(0.3g),并使用超声分散获得磁性粒子分散较好的磁性纳米粒子-明胶混合液,超声频率:F=25KHz,超声功率密度:p=发射功率(W)/发射面积(cm2);通常p=0.4w/cm2;超声振荡时间30S。
将磁性纳米粒子-明胶混合液加入到3D打印机的物料槽中进行预冷降温,物料槽温度调节为15℃,明胶在低温下处于凝胶状态,可被打印,打印底板温度也调节为15℃,通过参数设定,对3D打印机喷头大小选择(0.15μm),喷头的填充速度(13mm/s),喷头的移动速度(17mm/s),进行打印,打印后得到三维多孔的磁性纳米水凝胶。将打印的磁性纳米水凝胶浸泡在2.2wt%的转谷酰胺酶溶液(20℃)中8min,进行二次交联,从而得到稳定的磁性纳米水凝胶结构。
实施例3
磁性纳米粒子(Fe3O4),粒径为20nm,购置于深圳欣城生物科技有限公司。
将明胶加入超纯水中溶解得到8%的明胶水溶液,取0.8g明胶加入10ml超纯水,在650rpm搅拌下,向明胶溶液中加入磁性纳米粒子(0.5g),并使用超声分散获得磁性粒子分散较好的磁性纳米粒子-明胶混合液,超声频率:F=30KHz,超声功率密度:p=发射功率(W)/发射面积(cm2);通常p=0.5w/cm2;超声振荡时间40S。
将磁性纳米粒子-明胶混合液加入到3D打印机的物料槽中进行预冷降温,物料槽温度调节为16℃,明胶在低温下处于凝胶状态,可被打印,打印底板温度也调节为16℃,通过参数设定,对3D打印机喷头大小选择(0.2μm),喷头的填充速度(15mm/s),喷头的移动速度(20mm/s),进行打印,打印后得到三维多孔的磁性纳米水凝胶。将打印的磁性纳米水凝胶浸泡在1.8wt%的转谷酰胺酶溶液(15℃)中12min,进行二次交联,从而得到稳定的磁性纳米水凝胶结构。
实验例1
脐血间充质干细胞培养:
脐血间充质干细胞的体外培养(购买商品化冻存的原代脐血间充质干细胞)。
①把水浴锅加热至37℃,再将DMEM液置入水浴锅内预热。
②从液氮罐中取出细胞盒核对冻存管编号,取出原代脐血间充质干细胞;迅速将冻存管在37℃水浴锅内解冻1-2min,边解冻边摇晃冻存管。
③冻存管内液体完全融化后,用75%酒精消毒冻存管外壁,并将冻存管置于超净台内操作。向冻存管内加入培养基,吹打制备成细胞悬液。再将细胞悬液转移至15ml离心管中。然后于高速冷冻离心机中1200rpm,离心10min。
④离心完毕后,倒出上清液,向离心管中加入少量培养基,将细胞重新制备成细胞悬液;再将上述细胞悬液分装至25cm2的培养瓶中,置于细胞培养箱中培养,之后每3天换液一次。
⑤待培养瓶内细胞长满整个培养瓶的80%-90%时,将原培养基倒净,加入适量无菌PBS冲洗2次,再加入1ml 0.25%胰蛋白酶进行消化,放置在培养箱中孵育2min后,取出培养瓶在倒置显微镜下观察细胞形态变化;当细胞由长梭形变成圆形时,加入2mlDMEM培养基终止消化过程,并反复吹打将细胞制成细胞悬液。然后将细胞悬液转移到离心管内,1200rpm下离心10min;之后弃去上清,再加入少量培养基重悬细胞,按1:2的比例将细胞悬液分装传代。
P3代的脐血间充质干细胞的生长状态如图3所示。
将第三代的脐血间充质干细胞(数量为1×106个)与三维多孔磁性纳米水凝胶材料(大小为5mm×5mm×3mm,)复合,干细胞在三维多孔磁性纳米水凝胶材料表面可以紧密的结合,黏附生长,稳定增殖。
分别在培养的1天,3天和7天进行死活细胞染色,结果如图4所示,脐血间充质干细胞在三维多孔磁性纳米水凝胶材料表面生长良好,增殖迅速,第7天的细胞基本覆盖在材料孔的表面。圆圈为材料孔的位置。说明三维多孔磁性纳米水凝胶材料具有良好的细胞相容性,多孔结构为干细胞的营养物质输送提供好的途径。
CCK-8实验定量检测分析细胞增殖效果:
3D打印制备三维多孔磁性纳米水凝胶材料5mm×5mm×3mm。实验组:将P3代脐血间充质干细胞接种至三维多孔磁性纳米水凝胶材料表面上,接种细胞数量为1×106个,对照组直接在空白细胞培养板中加入同数量的细胞,每个组重复3个样;置于5%CO2、37℃的细胞培养箱中培养,每3天更换一次细胞培养基。分别于接种后的1d,3d,7d,进行CCK-8细胞增殖试验测定。
按说明书配制体积比培养基:CCK-8=10:1的CCK-8染液,吸干实验组以及对照组的原培养基,更换成配制好的CCK-8染液,每个样加入400μl混合液,放置在37℃下孵育2h;孵育完成后实验组的材料取出,并将上述两组的CCK-8染液依次转移至96孔板中(每孔100μl);选定450nm的波长,用酶标仪测定吸光度。
测试结果如图5所示:通过CCK-8的测试结果显示磁性纳米水凝胶中细胞增殖率达到与对照组相当的水平,说明磁性纳米水凝胶的细胞相容性很好,是一种良好的生物医用支架材料。
由上述实施例可知,本发明提供的三维多孔磁性纳米水凝胶,具备超顺磁性作用,与细胞相容性好,更适合干细胞在材料表面粘附生长。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种三维多孔磁性纳米水凝胶的制备方法,其特征在于,包括以下步骤:
1)将明胶溶液与磁性纳米粒子混合,超声分散获得磁性纳米粒子-明胶混合液;
2)将所述磁性纳米粒子-明胶混合液预冷降温至14~18℃后,进行3D打印获得三维材料;
3)将所述三维材料浸泡在转谷酰胺酶溶液中,进行二次交联,获得稳定的三维多孔磁性纳米水凝胶。
2.根据权利要求1所述的制备方法,其特征在于,步骤1)中所述明胶溶液的浓度为8~12wt%。
3.根据权利要求1或2所述的制备方法,其特征在于,步骤1)中所述明胶溶液与磁性纳米粒子混合的比例为10ml:(0.1~0.5)g。
4.根据权利要求3所述的制备方法,其特征在于,步骤1)中所述超声分散的频率≥20KHz,功率密度≥0.3w/cm2;所述超声分散的时间为20~40S。
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3D打印过程中,打印底板温度为14~18℃,喷头大小为0.1~0.2μm,喷头的填充速度为10~15mm/s,喷头的移动速度为15~20mm/s。
6.根据权利要求1所述的制备方法,其特征在于,步骤3)中转谷酰胺酶溶液的浓度为1.5~2.5wt%;所述浸泡的温度为15~20℃,所述浸泡的时间为8~12min。
7.权利要求1~6任意一项所述的制备方法制备获得的三维多孔磁性纳米水凝胶。
8.根据权利要求7所述的三维多孔磁性纳米水凝胶,其特征在于,所述三维多孔磁性纳米水凝胶的孔径为500-700μm,所述三维多孔磁性纳米水凝胶的表面有凹凸不平的微孔,所述微孔的孔径为5~20μm。
9.权利要求7或8所述三维多孔磁性纳米水凝胶在细胞培养中的应用。
10.权利要求7或8所述三维多孔磁性纳米水凝胶在制备医用体内植入材料中的应用。
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