CN112023118A - 一种负载全细胞活性成分的修复膜片及其制备方法和用途 - Google Patents
一种负载全细胞活性成分的修复膜片及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种负载全细胞活性成分的修复膜片及其制备方法和用途;本发明充分利用细胞的活性成分将细胞直接微纳化处理,在膜材料制备过程中通过物理或化学方法进行负载,在保证负载成分的生物活性前提下,对提高膜材料的修复效率有显著的提升作用。制备方法包括配置工作液和负载生物活性成分修复膜片制备。用于药物原位释放、延长药物作用时间、促进组织损伤修复。
Description
技术领域
本发明所属领域为组织工程与再生医学,具体说是一种负载全细胞活性成分的修复膜片及其制备方法和用途。
技术背景
在对皮肤创伤、子宫内膜损伤、牙周损伤类疾病进行修复时,膜材料的覆盖有利于创伤修复,防止细菌感染等作用,在大面积损伤修复方面意义重大。理想的膜材料应该具有以下基本特点:(1)良好的生物相容性,材料本身及降解产物对细胞无毒副作用;(2)可降解性,降解速率与组织再生速度相匹配;(3)适宜的孔径、孔隙率以及良好的贯通性;(4)良好的机械性能,与植入部位相匹配的力学强度;(5)良好的稳定性,不易与组织发生反应。但是单纯的膜材料不能充分发挥促进组织再生的作用,因此在膜材料制备的过程中负载生物活性物质,并保证负载成分的生物活性对提高膜材料的修复效率有显著的提升作用。生物活性物质以细胞功能最为全面有效,但是体内应用受限于细胞活性的不稳定性。本发明将细胞全部有效成分微纳化处理后进行膜材料负载,不仅有效利用了细胞的生物调控功能,还避免了细胞凋亡引发的副作用。并将该负载细胞活性物质的膜材料用于创伤修复,实现活性物质原位靶向发挥治疗作用并得以缓慢释放,提升组织愈合效率。
发明内容
本发明负载全细胞活性成分的修复膜片是由天然高分子载体与细胞来源的生物活性物质构成的;所述天然高分子载体,可以是胶原、透明质酸、海藻酸钠、普鲁兰以及明胶中的一种或几种组成的混合物;所述全细胞活性成分包括:蛋白质、脂质、DNA、RNA、糖类;可以来源于间充质干细胞、胚胎干细胞及诱导性多能干细胞中的一种或几种组成的混合物。
其制备方法包括以下步骤:
1)制备全细胞活性成分微纳颗粒:
用PBS以1-10×106个/mL的浓度重悬细胞;使用微型挤出机(Avanti PolarLibers)将悬浮的MSCs依次挤出通过10μm、5μm、1μm和400nm孔径的聚酯纤维膜(Whatman),每个尺度各5次,根据需求选择孔径大小制备不同尺度微纳颗粒;使用密度梯度离心法将挤出后的悬液离心,将碘烷醇(10%(v/v))小心地覆盖在50%(v/v)碘烷醇上,挤出后的悬液加载到最上层,100000g离心2h;收集50%和10%碘烷醇之间的溶液层,并在100000g下再次超速离心2h,将离心后的颗粒重新悬浮在PBS中,得到微纳米生物活性颗粒;
2)负载细胞活性成分修复膜片制备:
将微纳米生物活性颗粒以1-50μg/mL蛋白浓度溶于1-10mg/mL天然高分子原材料,再加入0.5%(v∶v)戊二醛交联剂到天然高分子溶液后形成工作液,将工作液倒入平板容器或者模型容器中,用1mL注射器排出浇铸过程中产生的气泡,37℃静置30-60min,形成固体膜片,拆除模具,即得到负载细胞活性物质的可降解修复膜片。
所得修复膜片可用于药物原位释放、延长药物作用时间、促进组织损伤修复。
本发明与现有技术相比突出优点在于:
1、本发明比传统的修复膜片相比,增加了生物活性物质,该活性物质包含了细胞发挥作用的所有有效成分,可以针对损伤部位的特点及修复需求添加一种或几种细胞来源的活性物质混合物,为个体化医疗提供可能性;
2、本发明应用的负载方式依据生物活性物质本身特性,充分保护负载物质的活性,能够充分保证治疗效果;
3、在制备工艺上,所需设备简单常规、反应温和,形成的修复膜片纤维形貌和力学性能良好,加之活性物质的负载更加模拟细胞外基质环境,更有力与细胞的粘附、迁移和增殖;
4、在功能上,负载细胞活性物质的修复膜片能够原位缓慢释放治疗药物,分发挥治疗作用,促进损伤修复和组织再生,防止药物一次性大量聚集在肝脏或肾脏造成的肝肾损伤。
具体实施方式
实施例1:负载间充质干细胞活性成分的可降解胶原膜片及其制备
1)制备全细胞活性成分微纳颗粒:
用PBS以2×106个/mL的浓度重悬细胞;使用微型挤出机(Avanti Polar Libers)将悬浮的MSCs依次挤出通过10μm、5μm、1μm和400nm孔径的聚酯纤维膜(Whatman),每个尺度各5次,制备尺度为400nm的纳米颗粒,将碘烷醇(10%(v/v))小心地覆盖在50%(v/v)碘烷醇上,挤出后的悬液加载到最上层,100000g离心2h;收集50%和10%碘烷醇之间的溶液层,并在100000g下再次超速离心2h,将离心后的颗粒重新悬浮在PBS中,得到微纳米生物活性颗粒;
2)负载细胞活性成分修复膜片制备:
将微纳米生物活性颗粒以2μg/mL蛋白浓度溶于2mg/mL胶原溶液中,加入胶原体积0.023%体积1M NaOH调整胶原溶液pH,后迅速将工作液倒入平板容器或者模型容器中,用1mL注射器排出浇铸过程中产生的气泡,37℃静置30-60min,形成固体膜片,拆除模具,即得到负载间充质干细胞活性成分的可降解胶原膜片。
实施例2:负载间充质干细胞活性成分的可降解明胶膜片及其制备
1)制备全细胞活性成分微纳颗粒:
用PBS以5×106个/mL的浓度重悬细胞;使用微型挤出机(Avanti Polar Libers)将悬浮的MSCs依次挤出通过10μm、5μm、1μm和400nm孔径的聚酯纤维膜(Whatman),每个尺度各5次,制备尺度为400nm的纳米颗粒,将碘烷醇(10%(v/v))小心地覆盖在50%(v/v)碘烷醇上,挤出后的悬液加载到最上层,100000g离心2h;收集50%和10%碘烷醇之间的溶液层,并在100000g下再次超速离心2h,将离心后的颗粒重新悬浮在PBS中,得到微纳米生物活性颗粒;
2)负载细胞活性成分修复膜片制备:
将微纳米生物活性颗粒以2μg/mL蛋白浓度溶于4%(m/v)明胶溶液中,于1mL明胶溶液中加入20μL 5%(v/v)戊二醛作为交联剂,后迅速将工作液倒入平板容器或者模型容器中,用1mL注射器排出浇铸过程中产生的气泡,37℃静置30-60min,形成固体膜片,拆除模具,即得到负载间充质干细胞活性成分的可降解明胶膜片。
实施例3:负载微米尺度全细胞活性物质的可降解透明质酸修复膜片及其制备
1)制备全细胞活性成分微纳颗粒:
用PBS以8×106个/mL的浓度重悬细胞;使用微型挤出机(Avanti Polar Libers)将悬浮的MSCs依次挤出通过10μm、5μm和1μm孔径的聚酯纤维膜(Whatman),每个尺度各5次,制备尺度为1μm的微米颗粒,将碘烷醇(10%(v/v))小心地覆盖在50%(v/v)碘烷醇上,挤出后的悬液加载到最上层,100000g离心2h;收集50%和10%碘烷醇之间的溶液层,并在100000g下再次超速离心2h,将离心后的颗粒重新悬浮在PBS中,得到微纳米生物活性颗粒;
2)负载细胞活性成分修复膜片制备:
将微纳米生物活性颗粒以2μg/mL蛋白浓度溶于5mg/mL透明质酸溶液中,于1mL溶液中加入20μL 5%(v/v)戊二醛作为交联剂,后迅速将工作液倒入平板容器或者模型容器中,用1mL注射器排出浇铸过程中产生的气泡,37℃静置30-60min,形成固体膜片,拆除模具,即得到微米尺度全细胞活性物质的可降解透明质酸膜片。
实施例4:负载间充质干细胞活性成分的复合膜片及其制备
1)制备全细胞活性成分微纳颗粒:
用PBS以5×106个/mL的浓度重悬细胞;使用微型挤出机(Avanti Polar Libers)将悬浮的MSCs依次挤出通过10μm、5μm、1μm和400nm孔径的聚酯纤维膜(Whatman),每个尺度各5次,制备尺度为400nm的纳米颗粒,将碘烷醇(10%(v/v))小心地覆盖在50%(v/v)碘烷醇上,挤出后的悬液加载到最上层,100000g离心2h;收集50%和10%碘烷醇之间的溶液层,并在100000g下再次超速离心2h,将离心后的颗粒重新悬浮在PBS中,得到微纳米生物活性颗粒;
2)负载细胞活性成分修复膜片制备:
将微纳米生物活性颗粒以2μg/mL蛋白浓度溶于1mg/mL胶原、4mg/mL明胶、5mg/mL透明质酸溶液中,加入0.5%(v/v)戊二醛作为交联剂,后迅速将工作液倒入平板容器或者模型容器中,用1mL注射器排出浇铸过程中产生的气泡,37℃静置30-60min,形成固体膜片,拆除模具,即得到负载间充质干细胞活性成分的复合膜片。
实施例5:负载胚胎干细胞全细胞活性物质的可降解胶原修复膜片及制备
1)制备全细胞活性成分微纳颗粒:
用PBS以2×106个/mL的浓度重悬胚胎干细胞;使用微型挤出机(Avanti PolarLibers)将悬浮的MSCs依次挤出通过10μm、5μm、1μm和400nm孔径的聚酯纤维膜(Whatman),每个尺度各5次,制备尺度为400nm的纳米颗粒,将碘烷醇(10%(v/v))小心地覆盖在50%(v/v)碘烷醇上,挤出后的悬液加载到最上层,100000g离心2h;收集50%和10%碘烷醇之间的溶液层,并在100000g下再次超速离心2h,将离心后的颗粒重新悬浮在PBS中,得到微纳米生物活性颗粒;
2)负载细胞活性成分修复膜片制备:
将微纳米生物活性颗粒以2μg/mL蛋白浓度溶于2mg/mL胶原溶液中,加入胶原体积0.023%体积1M NaOH调整胶原溶液pH,后迅速将工作液倒入平板容器或者模型容器中,用1mL注射器排出浇铸过程中产生的气泡,37℃静置30-60min,形成固体膜片,拆除模具,即得到负载胚胎干细胞活性成分的可降解胶原膜片。
实施例6:负载全间充质干细胞活性物质的可降解胶原修复膜片的缓释作用
取0.1g包裹间充质干细胞活性物质的膜片、间充质干细胞活性物质与空白胶原膜片混合悬液加入到4mL PBS中置于垂直混悬仪上,37℃条件下检测GAPDH释放效率,分别于4h、12h、24h、2d、4d、7d取反应液200μL补充PBS 200μL。反应结束后,用GAPDH ELISA试剂盒检测反应液中GAPDH浓度。结果如表1显示,GAPDH经修复膜片包裹后具有缓释作用,释放作用能够持续7d。
实施例7:负载全间充质干细胞活性物质的可降解胶原修复膜片对血管新生的促进作用
取直径为2.7cm的含有全间充质干细胞活性物质和不含全间充质干细胞活性物质的胶原膜片,置于六孔板中,每个条件三个重复,将1×105个血管内皮细胞种植于膜片上,加入内皮细胞培养基,放置在37℃,5%CO2的细胞培养箱中培养48小时,之后用胰蛋白酶将细胞从胶原膜片上消化下来进行细胞计数。结果如表1显示,负载全间充质干细胞活性物质的胶原膜片比未负载组对血管内皮细胞的增殖起到了明显的促进作用。
实施例8:负载全间充质干细胞活性物质的可降解胶原修复膜片对成纤维细胞的促进作用
取直径为2.7cm的含有全间充质干细胞活性物质和不含全间充质干细胞活性物质的胶原膜片,置于六孔板中,每个条件三个重复,将1×105个成纤维细胞种植于膜片上,加入成纤维细胞培养基,放置在37℃,5%CO2的细胞培养箱中培养48小时,之后用胰蛋白酶将细胞从胶原膜片上消化下来进行Ki67染色,之后进行流式分析。结果如表1显示,负载细胞外囊泡的明胶膜片比未负载组对成纤维细胞的增殖起到了明显的促进作用。
实施例9:负载全间充质干细胞活性物质的可降解胶原修复膜片对皮肤损伤恢复的促进作用
0.25mL/kg陆眠宁肌肉注射麻醉家兔,耳朵腹侧去毛,消毒后用直径1cm打孔器在皮肤层大孔,后用镊子移除皮肤,将含有全间充质干细胞活性物质和不含全间充质干细胞活性物质的可降解胶原修复膜片分别覆盖于伤口上,再覆盖上3M胶固定膜片,分别于2d、4d、7d、10d、14d观察皮肤愈合情况。
结果:负载有全间充质干细胞活性物质的修复膜片对家兔耳部皮肤创伤修复结果发现,包裹有细胞外囊泡的修复膜片能够加速皮肤创伤愈合,CD31免疫荧光染色结果显示血管新生良好,HE染色显示皮肤结构恢复更优。证明本发明的负载全细胞活性物质的修复膜片通过在病灶部位缓释药物发挥了更好的治疗效果。
表1.载药修复膜片和未载药膜片性质比较
Claims (3)
1.一种负载全细胞活性成分的修复膜片及其制备方法和用途,膜片是由生物可降解天然高分子材料和细胞来源的生物活性物质构成的;所采用的可降解天然高分子原材料包括胶原、透明质酸、海藻酸钠、普鲁兰以及明胶中的一种或几种组成的混合物;所述全细胞活性成分,包括:蛋白质、脂质、DNA、RNA、糖类;可以来源于间充质干细胞、胚胎干细胞、诱导性多能干细胞中的一种或几种细胞活性成分混合物。
2.一种负载全细胞活性成分的修复膜片的制备方法,包括如下步骤:
1)制备全细胞活性成分微纳颗粒:用PBS以1-10×106个/mL的浓度重悬细胞;使用微型挤出机(Avanti Polar Libers)将悬浮的MSCs依次挤出通过10μm、5μm、1μm和400nm孔径的聚酯纤维膜(Whatman),每个尺度各5次,根据需求选择孔径大小制备不同尺度微纳颗粒;使用密度梯度离心法将挤出后的悬液离心,将碘烷醇(10%(v/v))小心地覆盖在50%(v/v)碘烷醇上,挤出后的悬液加载到最上层,100000g离心2h;收集50%和10%碘烷醇之间的溶液层,并在100000g下再次超速离心2h,将离心后的颗粒重新悬浮在PBS中,得到微纳米生物活性颗粒;
2)负载细胞活性成分修复膜片制备:将微纳米生物活性颗粒以1-50μg/mL蛋白浓度溶于1-10mg/mL天然高分子原材料中,加入交联剂后将工作液倒入平板容器或者模型容器中,用1mL注射器排出浇铸过程中产生的气泡,37℃静置30-60min,形成固体膜片,拆除模具,即得到负载细胞活性物质的可降解修复膜片。
3.所得修复膜片用于药物原位释放、延长药物作用时间、促进组织损伤修复。
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