CN117819530A - 一种基于钙离子掺杂的碳点和自触发缓释体系及其制备方法和应用 - Google Patents
一种基于钙离子掺杂的碳点和自触发缓释体系及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及纳米药物技术领域，提供一种基于钙离子掺杂的碳点和自触发缓释体系及其制备方法和应用；具体涉及一种真空环境下将阿司匹林、二甲双胍及氯化钙水溶液混合加热形成的碳点，并在此基础上利用碳点中所含钙离子正电荷与海藻酸钠羧基负电荷相互吸引交联，实现自触发式凝胶化过程，形成碳点基水凝胶微球缓释体系。该方法通过调节原料配比，可获得高含量钙离子掺杂的荧光碳点及其微球缓释体系，并进一步应用在炎症及骨缺损等生物医学领域中，解决现有非甾体类抗炎药水溶性差及易突释无法长时间驻留于炎症缺损部位等问题。
Description
技术领域
本发明涉及纳米药物技术领域，特别涉及一种基于钙离子掺杂的碳点和自触发缓释体系及其制备方法和应用。
背景技术
因外伤、感染、肿瘤等疾病造成的骨缺损后重建，是临床治疗中面临的重大挑战之一。骨再生是一个缓慢、复杂的生理过程，控制感染消除炎症重塑免疫微环境，并实现组织结构再生和功能重建是炎症性骨缺损治疗的最终目标。传统骨替代材料及生物制剂生长因子的应用虽可以为骨组织再生提供引导支架，但存在价格昂贵、制备复杂、只具有骨引导性不具备抗炎及骨诱导性能等问题，并有待于进一步研发缓释体系可控有序的释放药物。
目前，碳点纳米材料具有低制备成本、低生物毒性及易于功能化修饰等优点，在生物医学领域尤其是生物成像、生物功能调控及药物递送等方面有着重大应用前景。各种药物也为碳点的制备提供了丰富的原料，它们不仅保留其前体的药理活性，还具有其独特的特征。但非甾体类抗炎药物如阿司匹林存在水溶性差难溶解等问题，仍然是纳米药物制备过程中需要克服的技术问题之一。
发明内容
为了解决上述技术问题，本发明提供一种基于钙离子掺杂的碳点，通过以下方法制得：
将二甲双胍与氯化钙的混合水溶液加入至含有阿司匹林的容器中，阿司匹林溶解后，产物在真空环境下200-300℃反应110-150min，得到黄色蓬松多孔的碳点球；经纯化后获得基于钙离子掺杂的碳点溶液；
其中，阿司匹林、二甲双胍、氯化钙的质量比为1：(0.01-20)：(0.01-4)。
作为优选，阿司匹林、二甲双胍、氯化钙的质量比为1：2：4。
所合成的基于钙离子掺杂的碳点中钙元素所占原子比例高达9％以上。
本发明进一步将基于钙离子掺杂的碳点应用于制备自触发缓释体系即水凝胶微球缓释体系。
本发明提供的一种基于钙离子掺杂的碳点自触发缓释体系，通过以下方法制备：
取上述所合成的基于钙离子掺杂的碳点纯化后制成的溶液匀速加入至海藻酸钠溶液中，持续搅拌待其凝胶化，用去离子水和乙醇交替清洗，离心弃上清留沉淀获得碳点基水凝胶微球缓释体系，即基于钙离子掺杂的碳点自触发缓释体系；
其中，碳点与海藻酸钠的质量比为1：(1-100)。
作为优选，海藻酸钠溶液的质量分数为1％。
本发明的工作原理：
本发明提供的基于钙离子掺杂的碳点在保留其药理活性发挥抗炎作用同时合并钙离子掺杂促进骨组织再生，在炎症性骨缺损微环境中能够发挥抗炎及促成骨生物效应；
基于钙离子掺杂的碳点溶液与海藻酸钠溶液混合，通过钙离子正电荷与海藻酸钠羧基负电荷相互吸引交联，利用其掺杂金属离子的螯合作用构建自触发式碳点基水凝胶微球缓释体系，达到延长药物驻留时间的目的，使其更适合应用于骨再生缓慢复杂的生理过程。可应用于炎症性骨缺损微环境中，发挥抗炎及促成骨生物效应。
基于上述原理，本发明提供一种基于钙离子掺杂的碳点在制备抗炎药物中的应用。
进一步的，本发明提供一种基于钙离子掺杂的碳点在制备治疗炎性骨缺损药物中的应用。
本发明还提供一种基于钙离子掺杂的碳点自触发缓释体系在制备治疗炎性骨缺损药物中的应用。
进一步的，本发明提供一种基于钙离子掺杂的碳点自触发缓释体系在制备促进骨再生药物中的应用。
本发明的有益效果：
本发明的合成过程简单且成本低廉，钙掺杂比例高，有利于促进骨再生；一方面利用碳点在炎症性骨缺损微环境中发挥抗炎及促成骨生物效应，另一方面通过构建的水凝胶微球缓释体系达到延长药物作用时间的目的，两者协同调控骨组织再生。
附图说明
图1为本发明实施例1碳点球日光下照片(a)，UV光下照片(b)以及碳点光致发光光谱(c)；
图2为本发明实施例2碳点溶液紫外可见吸收光谱(黑线)和光致发光光谱(红线)，插图：左上为日光下碳点溶液照片，右上为紫外光下碳点溶液照片(a)以及碳点的EDS能谱图(b)；
图3为本发明实施例3碳点溶液处理后RAW264.7细胞炎症因子在基因及蛋白水平上的表达：iNOS的mRNA水平(a1)，IL-1β的mRNA水平(a2)，TNF-α的mRNA水平(a3)以及Western blot检测iNOS，IL-1β，TNF-α的表达(b)和定量分析(c)；
图4为本发明实施例4碳点处理正常状态下rBMSC细胞7天ALP染色；
图5为本发明实施例5碳点基水凝胶微球缓释体系显微照片及大鼠牙周炎动物模型Micro-CT三维重建图像：a1为600μm显微照片，a2为150μm显微照片；b1为正常大鼠牙周动物模型，b2为大鼠牙周炎动物模型，b3为单纯碳点溶液处理组大鼠牙周炎动物模型，b4为碳点水凝胶微球缓释体系处理组大鼠牙周炎动物模型；
图6为本发明实施例6碳点光致发光光谱；
图7为本发明实施例7碳点光致发光光谱。
具体实施方式
实施例1
称取1g二甲双胍和2g氯化钙溶于3mL去离子水中得到透明溶液；加入至含有0.5g阿司匹林的球瓶中，120℃加热110min溶解，产物在真空环境下250℃反应120min，得到黄色蓬松多孔的碳点球(图1a)，球体在405nm激发下可发出绿色荧光(图1b)，发射峰位处于524nm处(图1c)。
实施例2
按实施例1步骤制备出碳点球后，用100mL去离子水溶解碳点球，超声加速溶解后10000rpm室温离心10min，去除大颗粒杂质，旋蒸后转移至100-500Da渗析袋渗析12h，纯化后得到淡黄色碳点溶液，在紫外光激发下可发出蓝色荧光，呈现单一发射峰，发射峰位处于475nm处。本碳点溶液的紫外可见吸收光谱表现出位于270nm和300nm的吸收峰(图2a)。通过EDS能谱的表征观察到所合成的碳点的元素组成包括C、N、O、Cl和Ca元素，其中Ca元素所占原子比例为9.1％(图2b)，证实碳点中有钙元素掺杂，所得碳点为基于钙离子掺杂的碳点。
实施例3
按实施例1和2步骤制备出基于钙离子掺杂的碳点溶液，首先进行细胞毒性的检测，表明所合碳点在300μg/mL浓度以下对RAW264.7细胞的存活率无明显影响。用100ng/mL脂多糖LPS诱导RAW264.7细胞，建立了体外炎症细胞模型，同时用100、200、300μg/mL的碳点溶液处理，24小时后通过qPCR检测了促炎细胞因子在mRNA水平上的表达，结果发现，在加入LPS刺激后细胞中iNOS、IL-1β、TNF-α的mRNA表达水平明显上升，提示炎症细胞模型建立的成功，采用相对定量法将加入碳点后相关炎症因子mRNA的水平与炎症模型组相比，结果表明加入了碳点后以上促炎细胞因子mRNA表达水平明显下调，且呈现明显的浓度依赖性(图3的a1、a2、a3)，并且与单纯加入浓度为300μg/mL阿司匹林和二甲双胍原料药物组相比，碳点具有更强的抗炎效果。在碳点溶液处理24小时后收集细胞总蛋白，通过Western blot检测iNOS、IL-1β、TNF-α在蛋白水平的表达(如图3b)，结果表明炎症模型组的iNOS、IL-1β、TNF-α相比Control组蛋白水平明显增高，提示炎症模型建立的成功。加入本碳点后炎症因子表达水平则明显下降。
实施例4
按实施例1和2步骤制备出基于钙离子掺杂的碳点溶液，通过碱性磷酸酶ALP染色确定碳点溶液处理后对于正常状态下大鼠骨髓间充质细胞(rBMSCs)影响，处理细胞7天和14天后，与空白对照组相比，加入碳点组中ALP阳性细胞的数量显著增多，染色变深，且呈现浓度依赖性(图4a)。
实施例5
按实施例1和2步骤制备出基于钙离子掺杂的碳点溶液，通过注射泵以0.1mL/min速度将1mL浓度为3mg/mL的基于钙离子掺杂的碳点溶液匀速滴至10mL质量分数1％海藻酸钠溶液中，500rpm持续搅拌30min待其凝胶化，用去离子水和乙醇交替清洗3-5遍，10000rpm离心10min弃上清留沉淀即可获得碳点水凝胶微球缓释体系(图5a)。并进一步将其应用于大鼠牙周炎动物模型中，与单纯碳点溶液处理组相比，水凝胶微球组可延缓碳点驻留时间，有效控制牙周炎症，促进组织修复再生(图5b)。
实施例6
称取1g二甲双胍和1g氯化钙溶于3mL去离子水中得到透明溶液；加入至含有0.5g阿司匹林的球瓶中，120℃加热110min溶解，产物在真空环境下250℃反应120min，得到黄色蓬松的碳点未成球形，在405nm激发下呈现单一发射峰，峰位处于548nm处(图6)，证实改变原料氯化钙投料量依旧可以合成荧光碳点。
实施例7
称取0.5g二甲双胍和2g氯化钙溶于3mL去离子水中得到透明溶液；加入至含有0.5g阿司匹林的球瓶中，120℃加热110min溶解，产物在真空环境下250℃反应120min，得到黄色蓬松的碳点球，在405nm激发下呈现单一发射峰，峰位处于520nm处(图7)证实改变原料二甲双胍投料量依旧可以合成荧光碳点。
Claims (10)
1.一种基于钙离子掺杂的碳点，其特征在于：通过以下方法制得：
将二甲双胍与氯化钙的混合水溶液加入至含有阿司匹林的容器中，阿司匹林溶解后，产物在真空环境下200-300℃反应110-150min，得到黄色蓬松的碳点球；经纯化后获得基于钙离子掺杂的碳点溶液；
其中，阿司匹林、二甲双胍、氯化钙的质量比为1：0.01-20：0.01-4。
2.根据权利要求1所述的一种基于钙离子掺杂的碳点，其特征在于：阿司匹林、二甲双胍、氯化钙的质量比为1：2：4。
3.根据权利要求1所述的一种基于钙离子掺杂的碳点，其特征在于：所合成的基于钙离子掺杂的碳点中钙元素所占原子比例为9％以上。
4.根据权利要求1-3任一项所述的一种基于钙离子掺杂的碳点，其特征在于：应用于制备自触发缓释体系即水凝胶微球缓释体系。
5.根据权利要求1-3任一项所述的一种基于钙离子掺杂的碳点的应用，其特征在于：应用于制备抗炎药物。
6.根据权利要求1-3任一项所述的一种基于钙离子掺杂的碳点的应用或权利要求5所述的应用，其特征在于：应用于制备治疗炎性骨缺损药物。
7.一种基于钙离子掺杂的碳点自触发缓释体系，其特征在于：通过以下方法制备：
取权利要求1-3任一项所述的一种基于钙离子掺杂的碳点纯化后制成溶液，匀速加入至海藻酸钠溶液中，持续搅拌待其凝胶化，用去离子水和乙醇交替清洗，离心弃上清留沉淀获得碳点基水凝胶微球缓释体系，即基于钙离子掺杂的碳点自触发缓释体系；
其中，碳点与海藻酸钠的质量比为1：1-100。
8.根据权利要求7所述的一种基于钙离子掺杂的碳点自触发缓释体系，其特征在于：制备方法中，海藻酸钠溶液的质量分数为1％。
9.根据权利要求7或8所述的一种基于钙离子掺杂的碳点自触发缓释体系的应用，其特征在于：应用于制备治疗炎性骨缺损药物。
10.根据权利要求7或8所述的一种基于钙离子掺杂的碳点自触发缓释体系的应用，其特征在于：应用于制备促进骨再生药物。
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