CN115025723A - 经神经生长因子及多巴胺改良的peg-plga微球 - Google Patents
经神经生长因子及多巴胺改良的peg-plga微球 Download PDFInfo
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Abstract
本发明涉及一种经神经生长因子及多巴胺改良的PEG‑PLGA微球，属于生物材料领域。将PVA溶液与神经生长因子溶液混合均匀；聚乳酸‑羟基乙酸共聚物、聚乙二醇‑聚乳酸‑羟基乙酸共聚物分别溶解于二氯甲烷中，混合，制得白色微球，将多巴胺加入三羟甲基氨基甲烷溶液溶解，向其中加入白色微球，搅拌。本发明能解决现存干细胞移植治疗中细胞体外扩增效率低、耗时长的问题，对骨髓间充质干细胞的增殖起到显著促进作用，提高干细胞移植治疗的安全性，在促进骨髓间充质干细胞增殖的同时，对其旁分泌功能也有显著提升，bFGF能够参与血管生成、损伤修复，并且能促进神经组织的生长发育，骨髓间充质干细胞旁分泌bFGF增多将提高干细胞移植治疗的效率。
Description
技术领域
本发明涉及生物材料领域，尤其涉及一种经神经生长因子及多巴胺改良的PEG-PLGA微球。
背景技术
间充质干细胞(Mesenchymal Stem Cell,MSC)具有自我更新能力，并具有成骨、成脂、成纤维等多向分化潜能，同时由于其表面缺乏主要组织相容性复合物II(MHCII)的表达，从而在移植过程中不存在免疫排斥反应，成为近年来再生医学领域的关注点。间充质干细胞MSC来源丰富，从最初被发现时的骨髓，至今已可从包括脐带、脂肪组织、扁桃体、牙髓、嗅觉粘膜等组织中提取。尽管间充质干细胞MSC具有多种来源，且在组织修复、抗炎和抗氧化方面具有显著作用，但其分离培养过程耗时长、细胞离体后在体外增殖缓慢、不同来源细胞随传代进行，其细胞活性及分化潜能明显下降。如何加速间充质干细胞MSC在体外培养过程中的增殖效率，使其能较早达到临床应用所需的细胞密度和数量成为当下丞待解决的问题。
发明内容
本发明提供一种经神经生长因子及多巴胺改良的PEG-PLGA微球，以解决间充质干细胞MSC在体外培养过程中的增殖效率低的问题。
本发明采取的技术方案是，包括下列步骤：
(1)称取一定量聚乙烯醇PVA 124，加入到去离子水中，制成浓度为5mg/mL的溶液，放入80℃的水浴锅中，搅拌至PVA全部溶解，得到均一的PVA溶液，将所得PVA溶液与浓度为20μg/ml的神经生长因子NGF溶液按50：1混合均匀；
(2)称取聚乳酸-羟基乙酸共聚物PLGA12k溶解于二氯甲烷制得A溶液；称取聚乙二醇-聚乳酸-羟基乙酸共聚物PEG5k-PLGA45k溶于二氯甲烷制得B溶液；A、B两溶液浓度均为0.5g/mL，将A、B溶液按2：1混合，于800rpm的磁力搅拌条件下逐滴滴加至第一步得到的溶液中，AB混合溶液与步骤(1)溶液比例为1：2，至二氯甲烷全部挥发，所得分散液经冻干处理得到白色微球；
(3)将多巴胺DA加入浓度为0.25mmol/L的三羟甲基氨基甲烷Tris溶液溶解，制得0.5mg/mL溶液，称取步骤(2)得到的白色微球，以1g/100mL比例加入到上述DA溶液中，以800rpm速度在磁力搅拌器上搅拌12h，随后将溶液进行冻干处理，得到聚多巴胺PDA包覆的经神经生长因子NGF改性的微球。
本发明的积极效果是，经实验验证，本发明很好地解决了现存干细胞移植治疗中细胞体外扩增效率低、耗时长的问题，在发明材料的干预下，将二维细胞培养转化为三维立体培养，并且通过对微球进一步改性，使其表面凹凸不平，增加间充质干细胞MSC可贴附表面积，从而提高其增殖效率，对骨髓间充质干细胞BM-MSC的增殖起到显著促进作用。同时，增殖效率的提升减少了体外扩增至特定数量级所需传代的代数，降低了干细胞癌变的可能，提高了干细胞移植治疗的安全性。除此之外，还对发明材料干预下骨髓间充质干细胞BM-MSC对生长因子bFGF(碱性成纤维生长因子)的旁分泌水平进行测定，结果表明材料在促进骨髓间充质干细胞BM-MSC增殖的同时，对其旁分泌功能也有显著提升，bFGF能够参与血管生成、损伤修复，并且能促进神经组织的生长发育，骨髓间充质干细胞BM-MSC旁分泌bFGF增多无疑将提高干细胞移植治疗的效率。
附图说明
图1是材料表征测定中a,b,c三种材料ATR-FTIR谱图；
图2是材料表征测定中a,b,c三种材料XPS谱图；
图3是材料表征测定中a材料SEM图；
图4是材料表征测定中b材料SEM图，为10um比例尺；
图5是材料表征测定中b材料SEM图，为2um比例尺；
图6是材料表征测定中c材料SEM图；
图7是材料表征测定中a材料DLS测定图；
图8是材料表征测定中b材料DLS测定图；
图9是材料表征测定中c材料DLS测定图；
图10是不同材料处理后细胞增殖比较图；
图11是不同材料处理后p3代BM-MSC旁分泌bFGF数量对比图。
具体实施方式
实验仪器与材料
包括下列步骤：
(1)称取一定量聚乙烯醇PVA 124，加入到去离子水中，制成浓度为5mg/mL的溶液，放入80℃的水浴锅中，搅拌至PVA全部溶解，得到均一的PVA溶液，将所得PVA溶液与浓度为20μg/ml的神经生长因子NGF(Nerve Growth Factor)溶液按50：1混合均匀；
(2)称取聚乳酸-羟基乙酸共聚物PLGA12k溶解于二氯甲烷制得A溶液；称取聚乙二醇-聚乳酸-羟基乙酸共聚物PEG5k-PLGA45k溶于二氯甲烷制得B溶液；A、B两溶液浓度均为0.5g/mL，将A、B溶液按2：1混合，于800rpm的磁力搅拌条件下逐滴滴加至第一步得到的溶液中，AB混合溶液与步骤(1)溶液比例为1：2，至二氯甲烷全部挥发，所得分散液经冻干处理得到白色微球；
(3)将多巴胺DA加入浓度为0.25mmol/L的三羟甲基氨基甲烷Tris溶液溶解，制得0.5mg/mL溶液，称取步骤(2)得到的白色微球，以1g/100mL比例加入到上述DA溶液中，以800rpm速度在磁力搅拌器上搅拌12h，随后将溶液进行冻干处理，得到聚多巴胺PDA包覆的经神经生长因子NGF改性的微球。
下边通过实验来进一步说明本发明。
实验例1、材料表征测定
将得到的PDA包覆经神经生长因子NGF改性的微球记为c，同时以不加神经生长因子NGF和多巴胺DA的微球产物记为a，不加NGF只加DA的产物记为b作为对照。分别进行红外(ATR-FTIR)表征，x射线光电子能谱(XPS)表征，扫描电镜(SEM)表征以及纳米粒度电位仪(DLS)表征。
1、ATR-FTIR谱图见图1，结果说明：
a样品在3290cm-1，2933cm-1，1749cm-1以及1084cm-1处均有吸收峰出现，分别对应-OH,C-H,C＝O以及C-O的吸收峰；
b样品与a样品相比，因为表面包覆了一层PDA，所以3290cm-1处-OH的吸收峰强度有所增强，并且1749cm-1处对应的C＝O的吸收峰减弱并右移至1700cm-1处，可能是C＝O在碱性条件下水解生成羧基导致；
c样品与b样品相比，原位于1749cm-1处对应于C＝O的吸收峰又重新出现，对应于生长因子中的C＝O,说明生长因子成功复合上去。
2、XPS谱图见图2，结果说明：
a样品仅有C,O两种元素，分别出现在285eV与532eV处，b样品因有PDA的包覆，因此在400eV左右出现了N元素的吸收峰。
3、a材料SEM图见图3(2um比例尺)，结果说明：
a样品大致呈球形，表面较为光滑，仅有一些因干燥产生的裂纹存在，粒径约在16μm左右。
4、b材料SEM图见图4、图5，结果说明：
b样品大致也呈现为球形，但表面并不光滑，存在很多纳米级的小凸起，为DA聚合生成的PDA颗粒；另外，b样品的粒径约为23μm左右，比a样品的粒径大，说明PDA成功包覆在a样品表面；
5、c材料SEM图(10um比例尺)见图6，结果说明：
c样品同样呈现为球形，表面也有很多纳米级的小凸起且粒径更大，约33μm左右，其凹凸不平的沟槽可能是由于干燥过程导致。
6、a材料DLS测定图见图7，b材料DLS测定图见图8，c材料DLS测定图见图9，结果说明：
a样品平均粒径为17.3μm，b样品平均粒径为26.87μm，c样品平均粒径为36.07μm。DLS结果与SEM结果基本吻合。由于DLS测试得到的是水合直径，所以结果与SEM测出来的结果相比会偏大。
实验例2对骨髓间充质干细胞增值促进效果验证
1、材料与设备
2、实验流程
向DMEM低糖培养基中加入10％FBS，1％青/链霉素，用以进行BM-MSC培养。本实验所用小鼠骨髓来源MSC(BM-MSC)购自上海中乔新舟生物科技有限公司，按生产商说明进行细胞复苏及传代。
将前述所得a、b、c三种微球于液相转移至方形容器内，置于通风橱中使其溶剂挥发制成薄膜，按24孔板孔径大小裁剪后平铺于孔板底部，并进行灭菌处理。
BM-MSC达到80％饱和时，用移液枪将上清培养基移除，加入1ml胰酶消化1min，待贴壁细胞全部悬浮，加入培养基终止消化过程，1500r，离心5min，弃上清，将细胞沉淀用培养基重悬，并计数。每个24孔板的孔中加入1ml含2.5*104BM-MSC的培养基，于37℃，5％CO2培养箱内培养。用CCK8试剂盒于第1，4，7天进行细胞增殖测定。使用GraphPadPrism5制图。
3、实验结果
见图10，结果说明：所制得a、b、c三种微球对于BM-MSC无毒性，a组无添加微球材料细胞生长情况与无材料对照组无明显差异，b、c两组对BM-MSC增殖有明显促进作用，且c组效果更好。
实验例3对骨髓间充质干细胞bFGF(碱性成纤维生长因子，basicfibroblastgrowth factor)分泌情况影响
1、材料与设备
2、实验流程
将p3代BM-MSC计数，按1*105/孔接种至分别含有a、b、c三种微球的96孔板(微球薄膜制备同5.1.2)中，向对照组(不含任何材料)及a、b、c各组分别加入培养基，培养48h后取细胞上清进行上清液中bFGF含量测定，测定方法严格按照试剂盒所提供说明书进行。
3、实验结果
见图11，结果说明：以对照组bFGF表达量为参照，将对照组平均bFGF表达量设定为1，与对照组及a组相比，b组和c组BM-MSC分泌出更多的bFGF，结果有统计学意义，且c组较b组的促进作用更加明显。
Claims (1)
1.一种经神经生长因子及多巴胺改良的PEG-PLGA微球，其特征在于，包括下列步骤：
(1)称取一定量聚乙烯醇PVA 124，加入到去离子水中，制成浓度为5mg/mL的溶液，放入80℃的水浴锅中，搅拌至PVA全部溶解，得到均一的PVA溶液，将所得PVA溶液与浓度为20μg/ml的神经生长因子NGF溶液按50：1混合均匀；
(2)称取聚乳酸-羟基乙酸共聚物PLGA12k溶解于二氯甲烷制得A溶液；称取聚乙二醇-聚乳酸-羟基乙酸共聚物PEG5k-PLGA45k溶于二氯甲烷制得B溶液；A、B两溶液浓度均为0.5g/mL，将A、B溶液按2：1混合，于800rpm的磁力搅拌条件下逐滴滴加至第一步得到的溶液中，AB混合溶液与步骤(1)溶液比例为1：2，至二氯甲烷全部挥发，所得分散液经冻干处理得到白色微球；
(3)将多巴胺DA加入浓度为0.25mmol/L的三羟甲基氨基甲烷Tris溶液溶解，制得0.5mg/mL溶液，称取步骤(2)得到的白色微球，以1g/100mL比例加入到上述DA溶液中，以800rpm速度在磁力搅拌器上搅拌12h，随后将溶液进行冻干处理，得到聚多巴胺PDA包覆的经神经生长因子NGF改性的微球。
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