CN111303871B - 一种磺化糖胺聚糖仿生启迪的碳量子点及其制备方法和应用 - Google Patents
一种磺化糖胺聚糖仿生启迪的碳量子点及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种磺化糖胺聚糖仿生启迪的碳量子点的制备方法，包括以下步骤：1)将含有糖环结构的单糖分子和含有磺酸基团的有机小分子加入水中混合均匀；2)在水热反应釜中进行水热反应，反应温度为100℃～240℃，反应时间为4h～8h；3)反应结束后，所得产物先进行离心，取上清液透析，干燥，得到磺化糖胺聚糖仿生启迪的碳量子点。本发明的磺化糖胺聚糖仿生启迪的碳量子点在细胞标记以及促进骨髓间充质干细胞的分化上具有显著效果。
Description
技术领域
本发明属于生物医用材料领域，涉及一种磺化糖胺聚糖仿生启迪的碳量子点及其制备方法和应用。
背景技术
糖胺聚糖(GAGs)，有着磺化多糖结构，广泛存在于细胞外基质(ECM)和骨组织结构中，对细胞的生理行为及相关的骨代谢过程具有显著的调节作用。但是，其本身纯度不高，而且生物活性易受外界条件影响，因此不易进行物理或化学修饰，从而限制了其生物应用。另一方面，越来越多的研究致力于将生物医用材料与骨髓间充质干细胞(BMSCs)相结合，利用干细胞的自我更新和多分化潜能，使材料在组织工程、疾病治疗等领域得到了广泛的应用。然而，目前的生物医用材料大多只能实现单一的调控细胞行为的作用，如何实现对移植干细胞的精准定位尤其是植入生物体后的实时成像依然是生物医用材料所面临的一个难点。
发明内容
有鉴于此，本发明提供一种磺化糖胺聚糖仿生启迪的碳量子点及其制备方法和应用。
本发明具体提供了如下的技术方案：
1、一种磺化糖胺聚糖仿生启迪的碳量子点的制备方法，包括以下步骤：
1)将含有糖环结构的单糖分子和含有磺酸基团的有机小分子加入水中混合均匀；
2)在水热反应釜中进行水热反应，反应温度为100℃～240℃，反应时间为4h～8h；
3)反应结束后，所得产物先进行离心，取上清液透析，干燥，得到磺化糖胺聚糖仿生启迪的碳量子点。
进一步，所述的含有糖环结构的单糖分子为葡萄糖、甘露糖、阿拉伯糖、来苏糖、果糖、半乳糖中的一种。
进一步，所述的含有磺酸基团的有机小分子为乙烯类磺酸盐。
进一步，所述的含有糖环结构的单糖分子为氨基葡萄糖盐酸盐，所述的含有磺酸基团的有机小分子为对苯乙烯磺酸钠。
进一步，所述的含有糖环结构的单糖分子和含有磺酸基团的有机小分子的摩尔比1:2～2:1。
进一步，所述的含有糖环结构的单糖分子和含有磺酸基团的有机小分子的摩尔比为1：1。
进一步，步骤2)为在160℃反应6h。
2、根据上述制备方法制备得到的一种磺化糖胺聚糖仿生启迪的碳量子点。
3、上述一种磺化糖胺聚糖仿生启迪的碳量子点在细胞标记上的应用。
4、上述一种磺化糖胺聚糖仿生启迪的碳量子点在促进骨髓间充质干细胞分化上的应用。
本发明的有益效果在于：
1、本发明从化学仿生合成的角度出发，通过水热反应成功制备了一系列不同糖和磺酸基团含量的磺化糖胺聚糖仿生启迪的碳量子点，通过在荧光分光光度计上测试荧光性能，从而选出具有较高发光强度的碳量子点，并将这些碳量子点与细胞共培养后，可实现较好的细胞成像效果。
2、由于制备的碳量子点保留了磺化糖胺聚糖的某些结构和功能，因此可作为培养基的外来补充物促进骨髓间充质干细胞的定向分化。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图：
图1是磺化糖胺聚糖仿生启迪的碳量子点制备及应用示意图；
图2是磺化糖胺聚糖仿生启迪的碳量子点的透射电镜图及高倍透射电镜图；
图3是磺化糖胺聚糖仿生启迪的碳量子点的发光强度结果；
图4是磺化糖胺聚糖仿生启迪的碳量子点CDs-GA1SS1的细胞成像结果；
图5是磺化糖胺聚糖仿生启迪的碳量子点CDs-GA1SS1分别在基础培养基(BM)和成骨诱导培养基(OIM)中调控骨髓间充质干细胞成骨分化的实验测试结果；
图6是磺化糖胺聚糖仿生启迪的碳量子点CDs-GA1SS1分别在基础培养基和成软骨诱导培养基(CIM)中调控骨髓间充质干细胞成软骨分化的实验测试结果。
具体实施方式
下面结合附图，对本发明的优选实施例进行详细的描述。
实施例1磺化糖胺聚糖仿生启迪的碳量子的制备
图1是磺化糖胺聚糖仿生启迪的碳量子点制备及应用示意图，磺化糖胺聚糖仿生启迪的碳量子点的制备包括以下步骤：
将单糖分子、乙烯类磺酸小分子和水在搅拌下混合均匀。其中，单糖分子为氨基葡萄糖盐酸盐(GA)，乙烯类磺酸小分子为对苯乙烯磺酸钠(SS)，两者总物质的量为0.5mmol。通过改变GA和SS的初始投料比，设计所得的磺化糖胺聚糖仿生的碳量子点含有不同的糖和磺酸单元。其中当GA与SS初始投料比为1:1时(即GA:0.25mmol,SS:0.25mmol)，得到的糖胺聚糖仿生的碳量子点命名为CDs-GA1SS1；当GA与SS初始投料比为2:1时(即GA:1/3mmol,SS:1/6mmol)，得到的糖胺聚糖仿生的碳量子点命名为CDs-GA2SS1；当GA与SS初始投料比为1:2时(即GA:1/6mmol,SS:1/3mmol)，得到的磺化糖胺聚糖仿生的碳量子点命名为CDs-GA1SS2。然后将水加入分别上述三组不同投料比的反应中，均匀混合，在高温水热条件下，160℃反应6h，得到具有不同投料比的磺化糖胺聚糖仿生启迪的碳量子点。溶剂水的用量为10mL。反应结束后，所得产物先进行离心(10000rpm,5min)，然后取上清液置于透析袋中(MWCO＝200Da),透析2h，最后冷冻干燥，得到具有不同投料比的磺化糖胺聚糖仿生启迪的碳量子点，分别命名为CDs-GA1SS1、CDs-GA1SS2和CDs-GA2SS1，其中1和2代表氨基葡萄糖盐酸盐(GA)和对苯乙烯磺酸钠(SS)的原始投料比。
实施例2磺化糖胺聚糖仿生启迪的碳量子的表征
将实施例1中合成的磺化糖胺聚糖仿生启迪的碳量子点CDs-GA1SS1分散到水中(碳量子点浓度为0.1mg/mL),超声30min,然后用滴管取一滴滴到铜网上，待铜网烘干后，置于透射电镜下观察碳量子点的形貌。图2a是实施例1中所合成的碳量子点CDs-GA1SS1的透射电镜图片，图2b是所合成的磺化糖胺聚糖仿生启迪的碳量子点CDs-GA1SS1的高倍透射电镜图片，由图2a可知，所合成的碳量子点大小均一，呈球状,由图2b可知，碳量子点的晶格间距为0.21nm,对应于石墨结构中的(100)晶面，由图2可证明，磺化糖胺聚糖仿生启迪的碳量子点已经成功合成。
实施例2荧光强度测试
将实施例1中所得到的三种碳量子点在超声条件下分别分散到水中，配成浓度为0.2mg/mL的碳量子点溶液，各取2mL加入荧光比色皿中，然后分别置于荧光分光光度计中进行荧光测试。其中荧光分光光度计的狭缝宽度设为3nm,光谱扫描速度设为600nm/min。通过测试所得到的三种碳量子点CDs-GA1SS1、CDs-GA1SS2和CDs-GA2SS1的荧光强度如图3所示，从图3中可以看出，当GA与SS的原始投料比例为1:1时，所得到的碳量子点的荧光强度最大，故以下实施例均采用原始投料比为GA:SS＝1:1时所合成的磺化糖胺聚糖仿生启迪的碳量子点CDs-GA1SS1进行测试。
实施例3细胞成像功能测试
将实施例1制备的碳量子点CDs-GA1SS1(碳量子点浓度为200μg/mL)与细胞培养12h后，置于激光共聚焦显微镜下观察细胞成像功能。图4是磺化糖胺聚糖仿生启迪的碳量子点CDs-GA1SS1的细胞成像结果，其中对照组和实验组分别是细胞直接在基础培养基(BM)条件下以及基础培养基+碳量子点条件下(BM+CDs-GA1SS1,其中碳量子点浓度为200μg/mL)所得到的激光共聚焦图片，从图4中可以看出，该量子点具有很高发光强度，可以用于细胞成像，由此说明，本发明制备的磺化糖胺聚糖仿生启迪的碳量子点具有较好的细胞成像效果。
实施例4细胞分化效果测试
1、将具有最高发光强度的碳量子点CDs-GA1SS1与基础培养基(BM)混合，配成含有碳量子点的基础培养基(碳量子点浓度为25μg/mL),命名为BM+CDs-GA1SS1。
2、将具有最高发光强度的碳量子点CDs-GA1SS1与成骨诱导培养基(OIM)混合，配成含有碳量子点的成骨诱导培养基(碳量子点浓度为25μg/mL),命名为OIM+CDs-GA1SS1。
3、将具有最高发光强度的碳量子点CDs-GA1SS1与成软骨诱导培养基(CIM)混合，配成含有碳量子点的成软骨诱导培养基(碳量子点浓度为25μg/mL),命名为CIM+CDs-GA1SS1。
4、将以上含有或不含有碳量子点的三种培养基(BM,BM+CDs-GA1SS1,OIM,OIM+CDs-GA1SS1,CIM,CIM+CDs-GA1SS1)分别与骨髓间充质干细胞培养3天，7天，14天,并测试其相应分化指标(ALP活性或者糖胺聚糖含量)验证细胞分化效果。
图5是磺化糖胺聚糖仿生启迪的碳量子点CDs-GA1SS1分别在基础培养基(BM)和成骨诱导培养基(OIM)中调控骨髓间充质干细胞成骨分化的实验测试结果，由图5可知，骨髓间充质干细胞BMSCs在BM和BM+CDs-GA1SS1培养条件下所分泌的ALP没有显著性差异，而在OIM+CDs-GA1SS1条件下，BMSCs所分泌的ALP与其他组相比，均具有显著性差异，证明此磺化糖胺聚糖仿生启迪的碳量子点可以作为成骨诱导培养基的外来补充物，从而促进BMSCs向成骨向分化。
图6是磺化糖胺聚糖仿生启迪的碳量子点CDs-GA1SS1分别在基础培养基和成软骨诱导培养基(CIM)中调控骨髓间充质干细胞成软骨分化的实验测试结果。由图6可知，骨髓间充质干细胞BMSCs在BM和BM+CDs-GA1SS1培养条件下所分泌的GAG没有显著性差异，而在CIM+CDs-GA1SS1条件下，BMSCs所分泌的GAG与其他组相比，均具有显著性差异，证明磺化糖胺聚糖仿生启迪的碳量子点可以作为成软骨诱导培养基的外来补充物，从而促进BMSCs向成软骨方向分化。
综上，磺化糖胺聚糖仿生启迪的碳量子点自身在基础培养基下很难促进BMSCs分化，但是可以在相应的诱导培养基存在下，加速BMSCs向指定方向分化，同时又具有良好的成像功能，因此在生物医学领域具有重要的意义。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
Claims (3)
1.一种磺化糖胺聚糖仿生启迪的碳量子点在以非疾病诊断和治疗方法为目的细胞标记上或在促进骨髓间充质干细胞分化上的应用，其特征在于，所述的一种磺化糖胺聚糖仿生启迪的碳量子点的制备方法包括以下步骤：
1）将含有糖环结构的单糖分子和含有磺酸基团的有机小分子加入水中混合均匀；
2）在水热反应釜中进行水热反应，反应温度为100 ℃~240℃，反应时间为4 h~8 h；
3）反应结束后，所得产物先进行离心，取上清液透析，干燥，得到磺化糖胺聚糖仿生启迪的碳量子点；
所述的含有糖环结构的单糖分子为氨基葡萄糖盐酸盐，所述的含有磺酸基团的有机小分子为对苯乙烯磺酸钠，含有糖环结构的单糖分子和含有磺酸基团的有机小分子的摩尔比为1:2~2:1。
2.根据权利要求1所述的一种磺化糖胺聚糖仿生启迪的碳量子点在以非疾病诊断和治疗方法为目的细胞标记上或在促进骨髓间充质干细胞分化上的应用，其特征在于，所述的含有糖环结构的单糖分子和含有磺酸基团的有机小分子的摩尔比为1：1。
3.根据权利要求1所述的一种磺化糖胺聚糖仿生启迪的碳量子点在以非疾病诊断和治疗方法为目的细胞标记上或在促进骨髓间充质干细胞分化上的应用，其特征在于，步骤2）为在160 ℃反应6 h。
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