CN116251229A - 一种复合凝胶微球及其制备方法 - Google Patents
一种复合凝胶微球及其制备方法 Download PDFInfo
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- CN116251229A CN116251229A CN202211619393.5A CN202211619393A CN116251229A CN 116251229 A CN116251229 A CN 116251229A CN 202211619393 A CN202211619393 A CN 202211619393A CN 116251229 A CN116251229 A CN 116251229A
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- magnesium silicate
- aqueous solution
- gelma
- nano particles
- composite gel
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Abstract
本发明公开一种复合凝胶微球及其制备方法,包括以下步骤:采用stober法制备二氧化硅胶体球;采用水热法将二氧化硅胶体球合成硅酸镁纳米粒子;利用物理吸附的方式在硅酸镁纳米粒子上负载可溶药物或促生长因子,得到负载材料;将负载材料水溶液加入GelMA/海藻酸钠水溶液中,得到生物墨水;其中,硅酸镁纳米粒子和GelMA质量比为(1:200)‑(1:25);将生物墨水加入光固化3D打印机中进行打印,得到复合凝胶微球。本发明的复合凝胶微球既包含无机纳米粒子,又包含有机水凝胶,并负载可溶药物或促生长因子叠加生物学效应,将生物材料、种子细胞、可溶药物或促生长因子三者完美结合,旨在骨组织工程领域充分发挥作用,为骨再生和重建提供了良好方案。
Description
技术领域
本发明涉及凝胶微球技术领域,尤其涉及一种复合凝胶微球及其制备方法。
背景技术
纳米材料理化性质可控且易于改性,一直是医学领域的一个热门话题。纳米结构在医学领域中一直被广泛应用,常被用于药物输送、分子成像、废物清除和肿瘤杀伤等领域。其中硅基纳米粒子在生物医学和生物工程领域取得了巨大的成功,在骨重建领域也表现出了巨大潜力。早在1968年stober成功合成不同粒径的均质二氧化硅胶体,后来学者在此基础上改良,制备出不同形态的纳米粒子,如球状、花状、树枝状。本发明首先通过经典stober法合成二氧化硅胶体,再以此作为前体材料通过水热法制备中空多孔硅酸镁纳米粒子。硅酸镁纳米粒子具有较大的比表面积和丰富的介孔结构,在生物分子递送方面具有广阔的应用前景。
但是纳米粒子具有较高的表面能,极易发生团聚,且聚集形态难以控制,从而失去了其优良性能。它是以粉状等形式存在的生物玻璃材料,不适于硬组织修复,可能会面临固定困难、塑型困难等问题。另外将纳米粒子局部应用于骨缺损处,纳米粒子作为异物首先被吞噬系统内化,大大降低其生物利用度。因此选择合适的载体对硅酸镁纳米粒子进行负载可以有效的解决这一问题。目前国内外研究学者多将纳米粒子负载到水凝胶、胶束、金属有机骨架等材料上。其中水凝胶由于其多孔性、结构可调节性等性质备受瞩目。
理想的骨修复材料还应该具有骨传导性、骨诱导、成骨性、可降解性以及良好的生物相容性等性能。作为骨缺损的移植材料,水凝胶一方面需要提供力学上的结构支持,另一方面需提供成骨细胞的长入空间。甲基丙烯酸酐化明胶(GelMA)水凝胶通过引入甲基丙烯酸酯基团,其机械强度可通过光交联过程进行改进,具有很好的生物相容性,在骨再生方面显示出强大的应用优势。GelMA在室温、中性pH、水溶液环境下即可聚合的宽泛条件非常有利于细胞的存活,可光交联的性征使其有灵活的成型性,不仅可以作为细胞二维培养的底物,促进其黏附、增殖;还可构建三维立体环境或特定图案结构,起到类似于细胞外基质的作用,从而制造各类仿生组织结构与类器官然而由于甲基丙烯酸化程度滴,结构的快速分解影响了机械结构的稳定性。研究表明在GelMA生物墨水中加入纳米硅酸盐可以增强GelMA的生物学特性和物理强度。值得强调的是,硅酸镁纳米粒子具有骨诱导特性,其硅烷醇基团和钙离子、磷酸根离子可相互作用,并能持续释放镁离子,因此是一种促进骨成熟的有效材料。将二者结合已被印证能够提高细胞在水凝胶内或表面的初始粘附、细胞增殖和分化。
组织工程三要素分别为种子细胞、支架与细胞因子。拥有了适宜的组织种子细胞与生物材料,还需要先进的生物制造技术将其结合,以制造出匹配修复目标部位结构、特性的植入物。近年来,3D打印技术因其快速、个性化、成本低廉、制造精度高等特点,被广泛应用于生物制造。目前在组织工程中应用较多的3D打印技术主要可分为挤出式、喷墨式和光固化打印技术三类。其中,本文使用的电喷/光固化打印机可打印200-1000um的凝胶微球,微球细胞微载体不但可以在体外大量扩增细胞,还可以作为细胞和药物的载体,通过注射的方法把细胞输送到缺损部位,既能够提供充足的空间满足细胞体外三维培养的需求,又可以保护其内部细胞在往组织注射的过程中免于挤压和摩擦的伤害。基于上述优异的性能,微球已经被用于骨缺损修复、软骨再生和心肌修复等诸多领域。但是,微球往往缺乏生物活性,在体外细胞增殖以及注射到体内以后,没法直接促进细胞的增殖和分化。添加生长因子等方式,往往也会面临生长因子脆弱易失活、释放快等问题。所以,迫切需要一种具有良好生物活性的微球。
发明内容
为了克服现有技术的不足,本发明提供一种复合凝胶微球及其制备方法,该复合凝胶微球既包含无机纳米粒子,又包含有机水凝胶,并负载可溶药物或促生长因子叠加生物学效应,将生物材料、种子细胞、可溶药物或促生长因子三者完美结合,旨在骨组织工程领域充分发挥作用,为骨再生和重建提供了良好方案。进一步地,本发明将负载贝尼地平的硅酸镁纳米粒子与高分子材料复合,在高分子基质包裹下,纳米粒子可以缓释活性硅、镁离子,就能够获得既有良好生物活性和骨诱导性,又具有足够强度支架结构的微载体,在体外快速扩增细胞,满足组织修复过程当中对细胞扩增的需求。
本发明第一方面提供一种复合凝胶微球的制备方法,包括以下步骤:
S10、二氧化硅胶体球制备步骤:采用stober法制备二氧化硅胶体球;
S20、硅酸镁纳米粒子合成步骤:采用水热法将二氧化硅胶体球合成硅酸镁纳米粒子;
S30、负载步骤:利用物理吸附的方式在硅酸镁纳米粒子上负载可溶药物或促生长因子,得到负载材料;
S40、生物墨水配置步骤:
将甲基丙烯酰化明胶(GelMA)加入海藻酸钠水溶液配成第一原液,加入光引发剂,充分溶解混匀,得到GelMA/海藻酸钠水溶液,备用;
将负载材料超声分散于去水中,得到负载材料水溶液,备用;
配置氯化钙水溶液作为交联剂,备用;
将负载材料水溶液、交联剂加入GelMA/海藻酸钠水溶液中,得到生物墨水;其中,硅酸镁纳米粒子和GelMA质量比为(1:200)-(1:25);
S50、打印步骤:将生物墨水加入光固化3D打印机中进行打印,打印出的微球落入交联剂后初步固化,再经紫外光二次固化,最终得到复合凝胶微球。
在本发明中,作为一种优选的实施例,S30、负载步骤中,可溶药物为贝尼地平。
在本发明中,作为一种优选的实施例,S10、二氧化硅胶体球制备步骤的具体过程如下:将2mol/L氨水、10mol/L去离子水加入70ml无水乙醇中搅拌25min,混合均匀后在上述溶液中快速滴入正硅酸乙酯0.2mol,10min溶液逐渐变成乳白色,继续于400rpm搅拌4h,8000rpm离心后分别用去离子水和无水乙醇清洗胶体球3次,60℃干燥12h。
在本发明中,作为一种优选的实施例,S20、硅酸镁纳米粒子合成步骤的具体过程如下:称取200mg二氧化硅胶体球放入40ml去离子水中超声分散均匀;将1.5mmol六水合氯化镁、20mmol氯化铵和2ml氨水加入40ml水中搅拌混匀;然后将上二者混匀,放入马弗炉中160℃高温持续12h;室温冷却后离心收集沉淀,分别用水和无水乙醇清洗3次,60℃干燥12h;备用。
在本发明中,作为一种优选的实施例,S20、硅酸镁纳米粒子合成步骤中,硅酸镁纳米粒子为多孔的中空结构。
在本发明中,作为一种优选的实施例,S30、负载步骤的具体过程如下:称取6mg盐酸贝尼地平溶于10ml无水乙醇,逐滴加入10ml含10mg硅酸镁纳米粒子的水溶液中,500rpm搅拌24h,水清洗3次,离心收集沉淀,60℃干燥。
在本发明中,作为一种优选的实施例,S40、生物墨水配置步骤的具体过程如下:
取15ml离心管称取GelMA冻干海绵1g加入浓度为0.5%的海藻酸钠水溶液配成浓度为10%的第一原液,50℃充分溶解后引入质量体积比0.5%的光引发剂LAP,充分溶解混匀。
称取40mg负载贝尼地平的硅酸镁纳米粒子超声分散于4ml去离子水中配成10mg/mL的负载材料水溶液,备用;
配置5%的氯化钙水溶液100ml作为交联剂。
在本发明中,作为一种优选的实施例,S40、生物墨水配置步骤中,硅酸镁纳米粒子和GelMA质量比为1:200、1:100、1:50或1:25。
在本发明中,作为一种优选的实施例,S50、打印步骤中,打印参数设置为气压20KPa,电压6.66kv;将生物墨水加入注射器中固定恒温加热带,连接气压泵,开通直流电电源,检查打印设备状态,交联剂氯化钙水溶液于50ml小烧杯承接微球,紫外灯照射气液界面使凝胶球快速固化;离心收集沉淀于电镜下观察,-4℃保存
本发明第二方面还提供一种复合凝胶微球,采用第一方面的复合凝胶微球的制备方法制备而成。
相比现有技术,本发明的有益效果在于:
按本发明制备出的复合凝胶微球既包含无机纳米粒子,又包含有机水凝胶,并负载可溶药物或促生长因子叠加生物学效应,将生物材料、种子细胞、可溶药物或促生长因子三者完美结合,在骨组织工程领域充分发挥作用,为骨再生和重建提供了良好方案。进一步地,本发明将负载贝尼地平的硅酸镁纳米粒子与高分子材料复合,在高分子基质包裹下,纳米粒子可以缓释活性硅、镁离子,就能够获得既有良好生物活性和骨诱导性,又具有足够强度支架结构的微载体,在体外快速扩增细胞,满足组织修复过程当中对细胞扩增的需求。
本发明制备出的载贝尼地平的硅酸镁纳米粒子复合凝胶微球,其粒径可控,表面圆整,具有很好的生物相容性。硅酸镁纳米粒子平均粒径为380nm,为多孔的中空结构,具有很高的比表面积,载药率高达340.1mg/g,电位为-25mv,贝尼地平24小时内释放率小于10%,硅镁元素持续释放时间长达28d。复合凝胶微球直径为230nm,机械强度增加,具有更好的生物相容性,与单纯GelMA微球相比,于大鼠骨髓间充质干细胞培养1d,3d,7d,本发明的生物活性明显提高,黏附于微球表面的细胞数量增加,黏着斑增多,成骨分化能力更强。
附图说明
图1-1为实施例1-5的复合凝胶微球于大鼠骨髓间充质干细胞共培养1天后细胞的增殖结果对比图;
图1-2为实施例1-5的复合凝胶微球于大鼠骨髓间充质干细胞共培养4天后细胞的增殖结果对比图;
图1-3为实施例1-5的复合凝胶微球于大鼠骨髓间充质干细胞共培养7天后细胞的增殖结果对比图;
图2-1为WD=8.6时硅酸镁纳米粒子的扫描电镜图;
图2-2为WD=9.4时硅酸镁纳米粒子的扫描电镜图;
图3-1为MAG=500X时单纯GelMA微球的扫描电镜图;
图3-2为MAG=3.00KX时单纯GelMA微球的扫描电镜图;
图4-1为硅酸镁纳米粒子和GelMA质量比为1:100组(实施例2)的复合凝胶微球的扫描电镜图;MAG=2.90KX;
图4-2为硅酸镁纳米粒子和GelMA质量比为1:100组(实施例2)的复合凝胶微球的扫描电镜图;MAG=1.99KX;
图5为硅酸镁纳米粒子和GelMA质量比为1:100组(实施例2)的复合凝胶微球的扫描电镜图;MAG=5.0KX;
图6-1为单纯GelMA凝胶球的剖面扫描电镜图;
图6-2为复合凝胶微球的剖面扫描电镜图;
图7-1、7-2为载贝尼地平的硅酸镁纳米粒子复合凝胶微球在GFP(绿色荧光蛋白)下的激光共聚焦图。
图8-1为单纯GelMA(对比例1)的复合凝胶微球于细胞共培养的激光共聚焦显微图片;
图8-2为硅酸镁纳米粒子和GelMA质量比为1:50组(实施例3)复合凝胶微球于细胞共培养的激光共聚焦显微图片;
图8-3为硅酸镁纳米粒子和GelMA质量比为1:100组(实施例2)复合凝胶微球于细胞共培养的激光共聚焦显微图片;
图8-4为硅酸镁纳米粒子和GelMA质量比为1:200组(实施例1)复合凝胶微球于细胞共培养的激光共聚焦显微图片。
具体实施方式
下面,结合附图以及具体实施方式,对发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。除特殊说明的之外,本实施例中所采用到的材料及设备均可从市场购得。
本发明提供一种复合凝胶微球的制备方法,包括以下步骤:
S10、二氧化硅胶体球制备步骤:采用stober法制备二氧化硅胶体球;
S20、硅酸镁纳米粒子合成步骤:采用水热法将二氧化硅胶体球合成硅酸镁纳米粒子;
S30、负载步骤:利用物理吸附的方式在硅酸镁纳米粒子上负载可溶药物或促生长因子,得到负载材料;
S40、生物墨水配置步骤:
将甲基丙烯酰化明胶(GelMA)加入海藻酸钠水溶液配成第一原液,加入光引发剂,充分溶解混匀,得到GelMA/海藻酸钠水溶液,备用;
将负载材料超声分散于去水中,得到负载材料水溶液,备用;
配置氯化钙水溶液作为交联剂,备用;
将负载材料水溶液加入GelMA/海藻酸钠水溶液中,得到生物墨水;其中,硅酸镁纳米粒子和GelMA质量比为(1:200)-(1:25);
S50、打印步骤:将生物墨水加入光固化3D打印机中进行打印,打印出的微球落入交联剂后初步固化,再经紫外光二次固化,最终得到复合凝胶微球。
作为一种优选的实施例,S30、负载步骤中,可溶药物为贝尼地平。
作为一种优选的实施例,S10、二氧化硅胶体球制备步骤的具体过程如下:将2mol/L氨水、10mol/L去离子水加入70ml无水乙醇中搅拌25min,混合均匀后在上述溶液中快速滴入正硅酸乙酯0.2mol,10min溶液逐渐变成乳白色,继续于400rpm搅拌4h,8000rpm离心后分别用去离子水和无水乙醇清洗胶体球3次,60℃干燥12h。
在本发明中,作为一种优选的实施例,S20、硅酸镁纳米粒子合成步骤的具体过程如下:称取200mg二氧化硅胶体球放入40ml去离子水中超声分散均匀;将1.5mmol六水合氯化镁、20mmol氯化铵和2ml氨水加入40ml水中搅拌混匀;然后将上二者混匀,放入马弗炉中160℃高温持续12h;室温冷却后离心收集沉淀,分别用水和无水乙醇清洗3次,60℃干燥12h;备用。
作为一种优选的实施例,S20、硅酸镁纳米粒子合成步骤中,硅酸镁纳米粒子为多孔的中空结构。
作为一种优选的实施例,S30、负载步骤的具体过程如下:称取6mg盐酸贝尼地平溶于10ml无水乙醇,逐滴加入10ml含10mg硅酸镁纳米粒子的水溶液中,500rpm搅拌24h,水清洗3次,离心收集沉淀,60℃干燥。
作为一种优选的实施例,S40、生物墨水配置步骤的具体过程如下:
取15ml离心管称取GelMA冻干海绵1g加入浓度为0.5%的海藻酸钠水溶液配成浓度为10%的第一原液,50℃充分溶解后引入质量体积比0.5%的光引发剂LAP,充分溶解混匀。
称取40mg负载贝尼地平的硅酸镁纳米粒子超声分散于4ml去离子水中配成10mg/mL的负载材料水溶液,备用;
配置5%的氯化钙水溶液100ml作为交联剂。
作为一种优选的实施例,S40、生物墨水配置步骤中,硅酸镁纳米粒子和GelMA质量比为1:200、1:100、1:50或1:25。
作为一种优选的实施例,S50、打印步骤中,打印参数设置为气压20KPa,电压6.66kv;将生物墨水加入注射器中固定恒温加热带,连接气压泵,开通直流电电源,检查打印设备状态,交联剂氯化钙水溶液于50ml小烧杯承接微球,紫外灯照射气液界面使凝胶球快速固化;离心收集沉淀于电镜下观察,-4℃保存。
本发明还提供一种复合凝胶微球,采用复合凝胶微球的制备方法制备而成。
下面通过参考附图描述的实施例是示例性的,仅用于解释本申请,而不能理解对本申请的限制。
实施例1:
本实施例提供一种复合凝胶微球的制备方法,包括以下步骤:
S10、二氧化硅胶体球制备步骤:采用stober法制备二氧化硅胶体球;具体过程如下:将2mol/L氨水、10mol/L去离子水加入70ml无水乙醇中搅拌25min,混合均匀后在上述溶液中快速滴入正硅酸乙酯0.2mol,10min溶液逐渐变成乳白色,继续于400rpm搅拌4h,8000rpm离心后分别用去离子水和无水乙醇清洗胶体球3次,60℃干燥12h;
S20、硅酸镁纳米粒子合成步骤:采用水热法将二氧化硅胶体球合成硅酸镁纳米粒子;具体过程如下:称取200mg二氧化硅胶体球放入40ml去离子水中超声分散均匀;将1.5mmol六水合氯化镁、20mmol氯化铵和2ml氨水加入40ml水中搅拌混匀;然后将上二者混匀,放入马弗炉中160℃高温持续12h;室温冷却后离心收集沉淀,分别用水和无水乙醇清洗3次,60℃干燥12h;备用;
S30、负载步骤:利用物理吸附的方式在硅酸镁纳米粒子上负载可溶药物或促生长因子,得到负载材料;具体过程如下:称取6mg盐酸贝尼地平溶于10ml无水乙醇,逐滴加入10ml含10mg硅酸镁纳米粒子的水溶液中,500rpm搅拌24h,水清洗3次,离心收集沉淀,60℃干燥;
S40、生物墨水配置步骤:
取15ml离心管称取GelMA冻干海绵1g加入浓度为0.5%的海藻酸钠水溶液配成浓度为10%的第一原液,50℃充分溶解后引入质量体积比0.5%的光引发剂LAP,充分溶解混匀。
称取40mg负载贝尼地平的硅酸镁纳米粒子超声分散于4ml去离子水中配成10mg/mL的负载材料水溶液,备用;
配置5%的氯化钙水溶液100ml作为交联剂;
将负载材料水溶液加入GelMA/海藻酸钠水溶液中,得到生物墨水;其中,硅酸镁纳米粒子和GelMA质量比为1:200;
S50、打印步骤:将生物墨水加入光固化3D打印机中进行打印,得到复合凝胶微球;具体过程为:打印参数设置为气压20KPa,电压6.66kv;将生物墨水加入注射器中固定恒温加热带,连接气压泵,开通直流电电源,检查打印设备状态,交联剂氯化钙水溶液充满于50ml小烧杯中承接微球,紫外灯照射气液界面使凝胶球快速固化;离心收集沉淀于电镜下观察,-4℃保存。
实施例2:
本实施例的特点在于,S40、生物墨水配置步骤中,将负载材料水溶液加入GelMA/海藻酸钠水溶液中,得到生物墨水;其中,硅酸镁纳米粒子和GelMA质量比为1:100;其它与实施例1相同。
实施例3:
本实施例的特点在于,S40、生物墨水配置步骤中,将负载材料水溶液加入GelMA/海藻酸钠水溶液中,得到生物墨水;其中,硅酸镁纳米粒子和GelMA质量比为1:50;其它与实施例1相同。
实施例4:
本实施例的特点在于,S40、生物墨水配置步骤中,将负载材料水溶液加入GelMA/海藻酸钠水溶液中,得到生物墨水;其中,硅酸镁纳米粒子和GelMA质量比为1:25;其它与实施例1相同。
实施例5:
本实施例的特点在于,S40、生物墨水配置步骤中,将负载材料水溶液加入GelMA/海藻酸钠水溶液中,得到生物墨水;其中,硅酸镁纳米粒子和GelMA质量比为1:400;其它与实施例1相同。
对比例1:
本实施例的特点在于,S40、生物墨水配置步骤中,不添加负载材料水溶液,仅仅使用GelMA/海藻酸钠水溶液,得到生物墨水;其它与实施例1相同。
图1-1为实施例1-5的复合凝胶微球于大鼠骨髓间充质干细胞共培养1天后细胞的增殖结果对比图;
图1-2为实施例1-5的复合凝胶微球于大鼠骨髓间充质干细胞共培养4天后细胞的增殖结果对比图;
图1-3为实施例1-5的复合凝胶微球于大鼠骨髓间充质干细胞共培养7天后细胞的增殖结果对比图;
从图1-1、1-2、1-3可以看出,不同浓度硅酸镁纳米粒子掺入GelMA后,微球表面的细胞明显扩增,其中硅酸镁纳米粒子和GelMA质量比为1:50组(实施例3)显示出了最高的细胞活力。
图2-1为WD=8.6时硅酸镁纳米粒子的扫描电镜图;
图2-2为WD=9.4时硅酸镁纳米粒子的扫描电镜图;
图中,WD为工作距离,指的的是样品成像表面到物镜的距离;
从图2-1、2-2可以看出,硅酸镁纳米粒子成中空介孔结构,其粒径约为380nm。
图3-1为MAG=500X时单纯GelMA微球的扫描电镜图;
图3-2为MAG=3.00KX时单纯GelMA微球的扫描电镜图;
从图3-1、3-2可以看出,微球表面粘附少量细胞,表面光滑圆整。
图4-1为硅酸镁纳米粒子和GelMA质量比为1:100组(实施例2)的复合凝胶微球的扫描电镜图;MAG=2.90KX;
图4-2为硅酸镁纳米粒子和GelMA质量比为1:100组(实施例2)的复合凝胶微球的扫描电镜图;MAG=1.99KX;
从图4-1、4-2可以看出,相同时间内掺杂纳米粒子的凝胶微球表面粘附的细胞量更多,局部放大表面凸凹不平,呈现出多孔且又纳米粒子的微纳表面。
图5为硅酸镁纳米粒子和GelMA质量比为1:100组(实施例2)的复合凝胶微球的扫描电镜图;MAG=5.0KX;
从图5可以看出,纳米粒子含量高,有机高分子基质相对减少,含水量减少,脆性增加,微球易碎。
图6-1为单纯GelMA凝胶球的剖面扫描电镜图;
图6-2为复合凝胶微球的剖面扫描电镜图;
从图6-1可以观察到单纯GelMA凝胶微球疏松多孔,细胞易于进入并生长分化。从图6-2可以观察到载贝尼地平的硅酸镁纳米粒子复合凝胶微球孔隙减小,降解更慢。
图7-1、7-2为载贝尼地平的硅酸镁纳米粒子复合凝胶微球在GFP(绿色荧光蛋白)下的激光共聚焦图,印证了纳米粒子在微球内部均匀分布,较为分散。
图8-1为单纯GelMA(对比例1)的复合凝胶微球于细胞共培养的激光共聚焦显微图片;
图8-2为硅酸镁纳米粒子和GelMA质量比为1:50组复合凝胶微球于细胞共培养的激光共聚焦显微图片;
图8-3为硅酸镁纳米粒子和GelMA质量比为1:100组复合凝胶微球于细胞共培养的激光共聚焦显微图片;
图8-4为硅酸镁纳米粒子和GelMA质量比为1:200组复合凝胶微球于细胞共培养的激光共聚焦显微图片。
从图8-1、8-2、8-3、8-4可以看出,绿色荧光显示纳米粒子分布,蓝色为细胞核染色,红色为鬼笔环肽骨架染色,FITC标记粘着斑。结果显示随着纳米粒子配比增加,微球表面细胞数量增多,铺展在表面,粘着斑染色增多,具有更好的生物相容性。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (10)
1.一种复合凝胶微球的制备方法,其特征在于,包括以下步骤:
S10、二氧化硅胶体球制备步骤:采用stober法制备二氧化硅胶体球;
S20、硅酸镁纳米粒子合成步骤:采用水热法将二氧化硅胶体球合成硅酸镁纳米粒子;
S30、负载步骤:利用物理吸附的方式在硅酸镁纳米粒子上负载可溶药物或促生长因子,得到负载材料;
S40、生物墨水配置步骤:
将GelMA加入海藻酸钠水溶液配成第一原液,加入光引发剂,充分溶解混匀,得到GelMA/海藻酸钠水溶液,备用;
将负载材料超声分散于去水中,得到负载材料水溶液,备用;
配置氯化钙水溶液作为交联剂,备用;
将负载材料水溶液加入GelMA/海藻酸钠水溶液中,得到生物墨水;其中,硅酸镁纳米粒子和GelMA质量比为(1:200)-(1:25);
S50、打印步骤:将生物墨水加入光固化3D打印机中进行打印,打印出的微球落入交联剂后初步固化,再经紫外光二次固化,最终得到复合凝胶微球。
2.如权利要求1所述的制备方法,其特征在于,S30、负载步骤中,可溶药物为贝尼地平。
3.如权利要求1所述的制备方法,其特征在于,S10、二氧化硅胶体球制备步骤的具体过程如下:将2mol/L氨水、10mol/L去离子水加入70ml无水乙醇中搅拌25min,混合均匀后在上述溶液中快速滴入正硅酸乙酯0.2mol,10min溶液逐渐变成乳白色,继续于400rpm搅拌4h,8000rpm离心后分别用去离子水和无水乙醇清洗胶体球3次,60℃干燥12h。
4.如权利要求1所述的制备方法,其特征在于,S20、硅酸镁纳米粒子合成步骤的具体过程如下:称取200mg二氧化硅胶体球放入40ml去离子水中超声分散均匀;将1.5mmol六水合氯化镁、20mmol氯化铵和2ml氨水加入40ml水中搅拌混匀;然后将上二者混匀,放入马弗炉中160℃高温持续12h;室温冷却后离心收集沉淀,分别用水和无水乙醇清洗3次,60℃干燥12h;备用。
5.如权利要求1所述的制备方法,其特征在于,S20、硅酸镁纳米粒子合成步骤中,硅酸镁纳米粒子为多孔的中空结构。
6.如权利要求1所述的制备方法,其特征在于,S30、负载步骤的具体过程如下:称取6mg盐酸贝尼地平溶于10ml无水乙醇,逐滴加入10ml含10mg硅酸镁纳米粒子的水溶液中,500rpm搅拌24h,水清洗3次,离心收集沉淀,60℃干燥。
7.如权利要求1所述的制备方法,其特征在于,S40、生物墨水配置步骤的具体过程如下:
取15ml离心管称取GelMA冻干海绵1g加入浓度为0.5%的海藻酸钠水溶液配成浓度为10%的第一原液,50℃充分溶解后引入质量体积比0.5%的光引发剂LAP,充分溶解混匀。
称取40mg负载贝尼地平的硅酸镁纳米粒子超声分散于4ml去离子水中配成10mg/mL的负载材料水溶液,备用;
配置5%的氯化钙水溶液100ml作为交联剂。
8.如权利要求1所述的制备方法,其特征在于,S40、生物墨水配置步骤中,硅酸镁纳米粒子和GelMA质量比为1:200、1:100、1:50或1:25。
9.如权利要求1所述的制备方法,其特征在于,S50、打印步骤中,打印参数设置为气压20KPa,电压6.66kv;将生物墨水加入注射器中固定恒温加热带,连接气压泵,开通直流电电源,检查打印设备状态,交联剂氯化钙水溶液于50ml小烧杯承接微球,紫外灯照射气液界面使凝胶球快速固化;离心收集沉淀于电镜下观察,-4℃保存。
10.一种复合凝胶微球,其特征在于,它采用如权利要求1-9任意一项所述的复合凝胶微球的制备方法制备而成。
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