CN116462879A - 一种明胶微球载体及其应用 - Google Patents
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Abstract
本申请公开了一种明胶微球载体及其应用,以明胶为主体,制成水溶液,加入柠檬酸调节pH至酸性可抑制交联剂京尼平的反应,再加入京尼平为交联剂,通过滴注进入液氮冷冻一步成型,再与碳酸氢钠反应将pH调节至碱性,促进京尼平进行交联固化,得到明胶微球载体。本申请的明胶微球载体制备工艺简单,稳定性强利于储藏、且制备成本低,对细胞无害、能有效负载细胞并扩增细胞,可用于细胞3D培养。
Description
技术领域
本申请涉及生物医药材料技术领域,具体而言,涉及一种明胶微球载体及其应用。
背景技术
与传统的平面细胞培养技术相比,三维细胞培养技术能够较为真实地模拟生物体内细胞的存活环境,更逼真的还原出细胞间相互作用和生理生化反应,以及细胞对内源性和外源性刺激如温度、pH、转运和分化等方面的改变所作出的实际应答。3D细胞培养可以用于构建类器官模型或是肿瘤模型,以更为准确贴合的方式进行如药物的代谢动力学研究、药效评估以及更加直观的细胞生物学方面的研究,使得3D细胞培养成为必不可少的研究工具。
目前,以明胶作为细胞负载基质或是制成球型载体是当前3D细胞培养的研究热点。明胶是动物胶原蛋白部分水解后的主要产物,具有良好的生物降解性、生物相容性、无毒性、亲水性和非免疫原性等生物特性,在药品、食品、组织工程、农业领域都是广泛应用的功能性生物高分子材料。
明胶微球的制备技术较为成熟,目前有乳化交联、溶剂挥发、喷雾干燥和相分离等多种制备方法。大部分方法都是通过油相和水相乳化搅拌剪切成球,再使用交联剂如甲醛、戊二醛固化成明胶微球。也有使用其他交联剂进行反应的,如CN109793717A公开了一种明胶微球的制备方法与应用,以明胶水溶液为水相,石蜡为油相乳化搅拌成球经过冷冻后,再由京尼平进行交联。或者是通过微流控制芯片成型的方法,如CN110200922B公开了一种明胶微球的制备方法及应用,以明胶水溶液为水相,油为油相经过微流控装置成液滴进入收集相,然后在4-12℃下成胶,最后使用京尼平进行交联。这两个制备方法所使用的交联剂京尼平是一种植物天然交联剂,可以与蛋白质、胶原、明胶和壳聚糖等交联制作生物材料,其毒性远低于戊二醛和其他常用化学交联剂,但是这两个制备方法在微球成型过程中需要使用油相。再如CN112250892A先将明胶溶液进行喷雾干燥或是喷雾冻干处理获得微球前体,微球前体再于不良溶剂如甲醇、丙酮及乙腈等溶剂或水溶液中进行交联固化,但是CN112250892A在固定阶段使用有机溶剂固化或有机溶剂进行保护球体形状,在成球后必须设法除去使得清洗步骤较为麻烦,加大成本。也有未使用交联剂进行固化微球的,如CN112266487A是以壳聚糖为骨架固定明胶,将壳聚糖明胶溶液冷冻喷雾处理,再于赋形剂如糖、葡聚糖、明胶、甘露醇等保护下冻干处理得到微球,但是该方法需要喷雾干燥以及多次冷冻干燥处理,制备过程复杂时间较长,涉及不同设备的使用,加大成本。因此,需要提供一种制备过程简单安全,绿色环保且不伤害细胞的活性,能够有利于干细胞贴附以及扩增,且节省成本方便大规模生产的微球的制备方法。
发明内容
本发明提供了一种一种明胶微球载体,采用如下方法制备:
步骤一,取明胶溶于温水中,搅拌均匀,得到明胶水溶液;
步骤二,将柠檬酸加入步骤一制备的明胶水溶液调节pH≤3,溶解拌匀,得到柠檬酸明胶溶液;使用柠檬酸调节明胶水溶液的pH≤3可以抑制交联剂的反应活性,延长明胶、柠檬酸和京尼平混合溶液的使用时间。
步骤三,将京尼平粉末加入到步骤二制备的柠檬酸明胶溶液,溶解拌匀,制成明胶混合溶液;
步骤四,将步骤三制备的明胶混合溶液滴注至液氮中,凝固成型,得到明胶微球;
步骤五,将步骤四制备的明胶微球放入碳酸氢钠溶液中,反应3h或待球体变为浅蓝色提示反应完成后,用水洗净获得明胶微球载体。
进一步的,所述的明胶微球载体的制备方法还包括如下(1)-(7)中的一项或多项:
(1)每升所述明胶水溶液中明胶的添加量为50-300g;
(2)每升所述柠檬酸明胶溶液中柠檬酸的添加量为10-50g;
(3)每升所述明胶混合溶液中京尼平的添加量为3-10g;
(4)通过针头将明胶混合溶液滴注到液氮中;
(5)所述碳酸氢钠溶液pH为8-9;
(6)所述碳酸氢钠溶液的浓度为10-50g/L;
(7)所述温水的温度为55-60℃。
所述明胶为胶强度为250g bloom的明胶和100g bloom的明胶混合物,其质量比为0.5-1:0-0.5。所述明胶的来源为鱼皮、猪皮、猪骨、牛皮、牛骨中的一种或是几种。
所述针头型号为4号或5号针头。
可替换的,在所述步骤一制备的明胶水溶液中可以先添加京尼平粉末再加入柠檬酸调节pH≤3,获得所述明胶混合溶液。
本发明还提供一种前述的明胶微球载体在细胞3D培养产业链中的应用。
进一步的,所述明胶微球载体用于干细胞3D培养。
所述明胶微球载体使用乙醇灭菌和/或辐照灭菌后,用于干细胞3D培养。具体的,所述乙醇灭菌是将所述明胶微球载体放入无水乙醇中浸泡30min;和/或,所述辐射灭菌是将所述明胶微球载体在紫外灯下辐射过夜。
本发明的有益效果包括:本发明提供的明胶微球载体,其制备工艺简单,只需原材料明胶、柠檬酸和京尼平混合再经过冷冻塑型即可,减少了有机溶剂如各种与水不混溶的油相和油相清洗剂的使用;固化工艺采用了天然交联剂京尼平,对人体无害,对细胞的活性不构成影响,不必像使用甲醛或是戊二醛交联剂需要有除去的步骤;通过控制pH低于3可以抑制京尼平反应活性,明胶柠檬酸京尼平混合液的保存或是使用时间可延长数天,在调节pH高于8时仍可以恢复反应活性,且京尼平具有一定的抑菌性能保护原料液被细菌分解;明胶微球使用液氮冷冻预先成型,可以使得明胶微球前体在常温水中形状不会变化,因此也不需要使用赋形剂或是不良溶剂进行保护;所使用的碳酸氢钠洗涤剂,可以将微球前体中柠檬酸中和,同时产生气泡在球体表面制造孔隙,且碳酸氢钠pH相对于氢氧化钠或是碳酸钠更为温和,更适合于京尼平进行交联反应;微球的尺寸可以通过选择不同型号的针孔和流速的大小进行调节;微球制备工艺简单,相较其他制备方法更为节省成本,具备大规模生产的潜力;明胶微球具有稳定性和一定的耐酸碱能力,利于贮藏;明胶微球能被胶原酶特异分解而不损伤细胞,具备一定的机械强度,可以有效负载并扩增细胞,且对细胞无害。
附图说明
图1为显微镜四倍镜头下的实施例1制备的明胶微球载体图片;
图2为扫描电镜下实施例1制备的明胶微球载体表面形貌图片;
图3为毛囊干细胞在明胶微球载体表面的贴附情况;
图4为明胶微球载体表面附着细胞的活力情况。
具体实施方式
下面结合实施例对本发明进行进一步说明和描述,但所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明和实施例中,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他发明和实施例,都属于本发明保护的范围。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、明胶微球载体的制备
具体制备方法如下:
(1)分别称取250g bloom的明胶0.7g以及100g bloom的明胶0.3g,混合加入10mL55℃的去离子水中,搅拌10min完全溶解。
(2)称取无水柠檬酸0.2g加入制备的明胶水溶液中搅拌溶解,调节pH至3以下,得到柠檬酸明胶溶液。
(3)称取京尼平0.05g加入步骤(2)制备的柠檬酸明胶水溶液中,搅拌溶解,制成明胶混合溶液。
(4)吸取步骤(3)得到的明胶混合溶液,经过4号针头滴注至装有液氮的保温壶中,凝固定型。
(5)称取5g碳酸氢钠加入500mL的去离子水中,完全溶解;将步骤(4)制备好的明胶微球放入碳酸氢钠水溶液中浸泡3h,待球体颜色变成浅蓝色后,取出水洗,得到明胶微球载体。
获得的明胶微球载体,于显微镜和扫描电镜下观察粒径大小和表面形貌。图1为显微镜四倍镜头下的明胶微球载体图片,可以看出明胶微球载体是尺寸在500μm左右,呈浅蓝色的半透光颗粒。图2为扫描电镜下明胶微球载体表面形貌图片,其中,图2中A是的放大倍数为1000倍,可以看出明胶微球载体表面存在大量的沟壑型褶皱,使微球的比表面积增加。图2中B是放大倍数为2000倍的视野,可以观察到沟壑的凹陷处宽度在10μm左右,凸起处部分的宽度大于20μm,可供细胞贴附。
实施例2明胶微球载体用于毛囊干细胞3D培养
取实施例1制备的明胶微球载体12颗,放入10ml的无水乙醇浸泡30min,重复三次。再置于紫外灯下辐照过夜,待酒精完全挥发。用PBS缓冲液漂洗明胶微球载体,取出微球载体加入培养基在37℃培养箱中孵育30min。孵育结束后,加入数量为10万的毛囊干细胞细胞悬液,在37℃培养箱培养48h。
将孵育48h的载有毛囊干细胞的明胶微球载体用戊二醛固定,与普通明胶微球一起进行冷冻干燥处理,在扫描电镜下观察微球载体的表面形貌和表面细胞贴附情况。结果如图3所示。其中图3中A和B分别为放大倍数1000倍下和2000倍下的明胶微球表面细胞附着情况。从图3中A可以观察到毛囊干细胞能够贴附在明胶微球载体表面的沟壑中。从图3中B可以观察到毛囊干细胞的大小在10μm左右,毛囊干细胞通过伸出突触在明胶微球载体表面粘附,并且抱团增殖扩增。
实施例3明胶微球载体细胞毒性检测
取实施例1制备的明胶微球载体24颗,放入10ml的无水乙醇浸泡30min,重复三次。再置于紫外灯下辐照过夜,待酒精完全挥发。用PBS缓冲液漂洗明胶微球载体,取出微球载体加入培养基在37℃培养箱中孵育30min。孵育结束后,分成两组分别加入数量为10万的毛囊干细胞细胞悬液和非小细胞肺癌细胞悬液,在37℃培养箱培养48h。
分别在24h和48h时从两组培养体系中各取出4颗微球,用PBS溶液漂洗,加入CCK-8试剂于37℃培养箱中孵育2h,后取出用酶标仪测定450nm处的吸光度。图4为附着在明胶微球表面的细胞活力情况,结果反映出48h的OD值高于24h,说明在明胶微球载体表面的毛囊干细胞和非小细胞肺癌细胞是呈增殖态,其细胞数量是在增加的。由于毛囊干细胞的贴附能力比较弱,且肺癌细胞的增殖速度更快,所以毛囊干细胞在24h和48h两个时间段OD值是低于非小细胞肺癌细胞,但是总体都是呈现增长趋势。说明本发明制备的明胶微球载体,对细胞无害,并且有利于细胞增殖。
Claims (9)
1.一种明胶微球载体,其特征在于,所述明胶微球载体采用如下方法制备:
步骤一,取明胶溶于温水中,搅拌均匀,得到明胶水溶液;
步骤二,将柠檬酸加入步骤一制备的明胶水溶液调节pH≤3,溶解拌匀,得到柠檬酸明胶溶液;
步骤三,将京尼平粉末加入到步骤二制备的柠檬酸明胶溶液,溶解拌匀,制成明胶混合溶液;
步骤四,将步骤三制备的明胶混合溶液滴注至液氮中,得到明胶微球;
步骤五,将步骤四制备的明胶微球放入碳酸氢钠溶液中,反应3h或待球体变为浅蓝色后,用水洗净获得明胶微球载体。
2.根据权利要求1所述的明胶微球载体,其特征在于,包括如下(1)-(7)中的一项或多项:
(1)每升所述明胶水溶液中明胶的添加量为50-300g;
(2)每升所述柠檬酸明胶溶液中柠檬酸的添加量为10-50g;
(3)每升所述明胶混合溶液中京尼平的添加量为3-10g;
(4)通过针头将明胶混合溶液滴注到液氮中;
(5)所述碳酸氢钠溶液pH为8-9;
(6)所述碳酸氢钠溶液的浓度为10-50g/L;
(7)所述温水的温度为55-60℃。
3.根据权利要求2所述的明胶微球载体,其特征在于,所述明胶为胶强度为250gbloom的明胶和100g bloom的明胶混合物,其质量比为0.5-1:0-0.5。
4.根据权利要求2所述的明胶微球载体,其特征在于,所述针头型号为4号或5号针头。
5.根据权利要求1所述的明胶微球载体,其特征在于,可替换的,在所述步骤一制备的明胶水溶液中先添加京尼平粉末再加入柠檬酸调节pH≤3,获得所述明胶混合溶液。
6.权利要求1-5任一项所述的明胶微球载体在细胞3D培养领域中的应用。
7.根据权利要求6所述的应用,其特征在于,所述明胶微球载体用于干细胞3D培养。
8.根据权利要求7所述的应用,其特征在于,所述明胶微球载体使用乙醇灭菌和/或辐照灭菌后,用于干细胞3D培养。
9.根据权利要求8所述的应用,其特征在于,所述乙醇灭菌是将所述明胶微球载体放入无水乙醇中浸泡30min;和/或,所述辐射灭菌是将所述明胶微球载体在紫外灯下辐射过夜。
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