CN116059400A - 调节髓核氧代谢平衡的水凝胶微球的制备方法及其应用 - Google Patents
调节髓核氧代谢平衡的水凝胶微球的制备方法及其应用 Download PDFInfo
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Abstract
本发明属于水凝胶微球领域,尤其涉及一种调节髓核氧代谢平衡的水凝胶微球的制备方法及其应用。具体的,本发明公开了一种调节髓核氧代谢平衡的水凝胶微球的制备方法,包括:S1:制备黑磷量子点;S2:将S1制备得到的黑磷量子点加入壳聚糖纳米粒中,得到壳聚糖纳米粒‑黑磷量子点;S3:制备GelMA水凝胶微球;S4:将S3得到的GelMA水凝胶微球、EDC和NHS加到MES中活化后,将S2中制备得到的壳聚糖纳米粒‑黑磷量子点加入其中,孵育后得到GM@CS‑BP。本发明的水凝胶微球GM@CS‑BP(氧代谢平衡工程化水凝胶微球),能够为以黑磷为还原剂延缓椎间盘退变提供新的理论基础,同时为局部氧化应激微环境下生物材料的再生应用提供新思路,给椎间盘退变患者带来了福音。
Description
技术领域
本发明属于水凝胶微球领域,尤其涉及一种调节髓核氧代谢平衡的水凝胶微球的制备方法及其应用。
背景技术
活性氧(ROS)是一种线粒体内膜呼吸链中的代谢副产物,包括超氧阴离子(O2-)、羟自由基(OH-)、过氧化氢(H2O2)等,在细胞信号传导和体内平衡中具有重要作用。活性氧簇(ROS)产生与清除的失平衡是机体受氧化应激损伤的重要根源,组织损伤时,细胞线粒体呼吸链传递受损,产生大量ROS溢出内源性清除系统。氧代谢的失衡引起琥珀酸盐堆积,升高组织脂质过氧化水平,从而下调蛋白聚糖的合成以及引发细胞外基质的降解。此外,ROS作为第二信使与富集在线粒体内Ca2+协同刺激线粒体通透性转化孔(MTPTP)的开放,导致Cytc、AIF、SMAC等大量凋亡因子释放,诱导细胞凋亡。并进一步破坏线粒体功能,活化NF-κB、MAPK等炎症信号通路,分泌IL-1β、TNF-α炎症因子导致炎症瀑布,诱导多种继发性疾病。椎间盘(IVD)由于邻近椎体上下终板、前纵和后纵韧带的围绕,形成严重缺血缺氧、乳酸和ROS堆积的密闭微环境,构成椎间盘退变(IVDD)的结构基础。过氧化氢引发的酸性微环境加重IVD氧化应激损伤。髓核细胞(NP)炎症信号的激活将导致基质金属蛋白酶MMP表达,降解COL-II,加速椎间盘退变。
近年,精准医学促进靶向性治疗快速发展,采用局部注射中和ROS的方式,阻断了ROS链式恶性负反馈作用。但常见的抗氧化药物具有不易溶于有机溶剂、半衰期较短等缺陷,限制了这些药物的临床应用。因此,寻找稳定高效的中和ROS途径,抑制损伤组织病理生理发展是亟需突破的瓶颈。
最近,应用纳米材料深入研究组织病理生理发展和重塑再生打开了新大门。黑磷(BP)由磷原子构成,作为二维半导体纳米结构的模拟物在生物医学领域有着广泛的应用前景。考虑到黑磷的强还原性,黑磷纳米片可以作为活性氧清除剂用于减轻ROS触发的急性损伤,且其抗氧化能力远远优于传统的抗氧化剂N-乙酰半胱氨酸(NAC)。此外,黑磷量子点由于其粒径较小,相较于纳米片增大了表面积,能够更加高效清除退变椎间盘内过量的ROS。且黑磷在体内被降解为无毒的磷酸根阴离子,具有较强的生物安全性。因此,应用黑磷量子点可能为椎间盘退变提供新的治疗平台。壳聚糖纳米粒由于操作简单、生物相容性良好,特别是表面带有大量正电荷氨基官能团,能够有效与呈负电荷的黑磷量子点结合,是一种适宜的黑磷保护材料利用壳聚糖纳米颗粒(CS)作为黑磷量子点载体,有望达到提高其还原性和包封率的目标,进而使黑磷量子点更稳定发挥维持IVDD氧代谢平衡作用。
椎间盘是脊柱椎体衔接的组织架构,在传递人体工程学应力方面具有举足轻重作用,导致其长期处于高压状态,直接局部注射纳米颗粒可能伴随针道渗漏、扩散的问题,而且缺乏机械应力无法维持椎间盘高度。因此,纳米颗粒仍然依赖转运载体,以提高注射效率和靶向性。甲基丙烯酸酐化明胶(GelMA)拥有优秀的生物相容性,且能够通过微流控技术精确制造为均匀的水凝胶微球,冻干后形成的表面多孔结构可有效接枝纳米颗粒,成为椎间盘退变中注射疗法的重要选择。然而,关于可用于调节髓核氧代谢平衡的水凝胶微球目前还鲜有报道。
发明内容
本发明中申请人根据IVDD中ROS激发的链式病理改变,将静电力装载BPQDs(黑磷量子点)的CS通过酰胺键接枝到GelMA多孔微球表面,制备了氧代谢平衡工程化可注射水凝胶微球GM@CS-BP。复合微球实现了纳米颗粒的高效均匀负载,能够通过微量泵注射器定向植入椎间盘微环境,持续释放强还原性BPQDs,原位中和ROS,减轻氧化应激损伤,改善椎间盘氧代谢微环境。GM@CS-BP抑制细胞凋亡的同时改善细胞外酸中毒,阻断下游炎症通路活化引发的炎症风暴。基于此,申请人希望构建一种氧代谢平衡工程化水凝胶微球,能够为以黑磷为还原剂延缓椎间盘退变提供新的理论基础,同时为局部氧化应激微环境下生物材料的再生应用提供新思路。
具体的,本发明的技术方案如下:
本发明第一个方面公开了一种调节髓核氧代谢平衡的水凝胶微球的制备方法,包括:
S1:制备黑磷量子点;
S2:将S1制备得到的黑磷量子点加入壳聚糖纳米粒中,得到壳聚糖纳米粒-黑磷量子点;
S3:制备GelMA水凝胶微球;
S4:将S3得到的GelMA水凝胶微球、EDC和NHS加到MES中活化后,将S2中制备得到的壳聚糖纳米粒-黑磷量子点加入其中,孵育后得到GM@CS-BP。
优选的,在S1中,将BP晶体分散于NMP溶液中,并在冰水浴中超声2-4h,之后将获得的溶液再次于冰水浴中超声10-14h得到分散液,并将分散液离心10-30分钟以除去未分散的BP,得到上清液;取上清液于新的容器中,离心1-3h,弃上清,并用去离子水重悬得到黑磷量子点。
在本发明的一些具体实施例中,通过已有的液相剥离技术制备黑磷量子点(全程于氮气保护下进行)。首先将20mgBP晶体分散于20mL的NMP溶液中,并在冰水浴中以1200W的功率超声3h(南京赛飞有限公司,中国,超声波频率:19-25kHz,2秒开/3秒关),之后将获得的溶液再次于冰水浴中超声12h,并将分散液以7000rpm的转速离心20分钟以除去未分散的BP。取上清液于新试管中,以15000rpm的转速离心2h,弃上清,并用去离子水重悬。
优选的,在S2中,将壳聚糖溶解于醋酸溶液中,当溶剂澄清透明时,pH调节至4.5-5.5,过滤后以BPQDs/CS质量比为(1:8)-(1:12)的黑磷量子点重悬于壳聚糖纳米粒中;逐滴添加TPP,使得CS/TPP的质量比为(3:1)-(5:1);超声后将获得的壳聚糖纳米粒再次过滤得到壳聚糖纳米粒-黑磷量子点。
更优选的,在S2中,将壳聚糖溶解于醋酸溶液中,当溶剂澄清透明时,pH调节至4.5-5.5,通过0.45μm滤膜滤过后以BPQDs/CS质量比为(1:8)-(1:12)的黑磷量子点重悬于壳聚糖纳米粒中。
在本发明的一些具体实施例中,将壳聚糖(20mg,200-400mPa.s)于室温环境下在磁力搅拌中溶解于20mL1%(w/v)醋酸溶液中,当溶剂澄清透明时,使用10M NaOH溶液将pH调节至5.0,并用0.45μm的滤膜过滤三次(如制备壳聚糖纳米粒-黑磷量子点,则以BPQDs/CS质量比1:10的黑磷量子点重悬于壳聚糖纳米粒中)。随后在720rpm的磁力搅拌下逐滴添加三聚磷酸盐(TPP)溶液(0.7mg/mL),使得CS/TPP的质量比为4:1。此时溶液产生透明乳光色的特征,表明TPP和CS发生了离子交联聚合。随后使用900W功率的超声波破碎仪超声5分钟,重复2-3次。之后将获得的壳聚糖纳米粒再次通过0.22μm的滤膜过滤三次,得到壳聚糖纳米粒-黑磷量子点。
优选的,在S3中,制备甲基丙烯酸酐化明胶溶液后,使用微控流技术制备GelMA水凝胶微球。
在本发明的一些具体实施例中,甲基丙烯酸酐化明胶溶液制备方法包括:
首先,称取20g的明胶加入200ml的PBS中,并将其置于水浴锅中于60°下搅拌1h直至完全溶解,获得10%(w/v)的透明淡黄色的明胶溶液。之后,于避光条件下抽取16ml的甲基丙烯酸酐,使用微量泵推注器以0.25mL/min的速度将其滴加于明胶溶液中。之后维持60°温度连续搅拌2h,将预热的800mlPBS加入上述反应物中,继续搅拌15min。之后将溶液用12-14kDa的透析袋分装透析2周,每2天更换一次PBS,最终得到GelMA溶液,冷冻干燥后保存于-80℃冰箱。
在本发明的一些具体实施例中,GelMA水凝胶微球的制备方法包括:
通过油包水形式的微流控技术制备。首先,将同轴电纺喷头(内针与外针的直径分别是30G和21G)通过硅胶管分别连接于两个微量泵推注器。同轴电纺喷头的外针作为共流剪切的连续相。连接含有配好的10%(w/w)Span80的肉豆蔻酸异丙酯溶液,内针作为分散相,连接的溶液是7%(w/v)GelMA水溶液和0.5%(w/v)的I2595光引发剂。结合本组已有研究,调节微量泵推注器使得水相(即GelMA相)与油相的流速比为15:500μl/min,打开开关,分别排气直至于接口处产生连续单分散球形微滴。待微滴大小均匀后于避光条件下置入预先装有油性溶液的烧杯中。每10-20min取出使用紫外光充分照射并更换烧杯(整个环境下维持针道系统一定温度以防止针道内发生交联堵塞)。之后将收集到的固化水凝胶微球通过75%乙醇和PBS反复洗涤(75%乙醇3次,PBS5次),之后每3h更换一次PBS,以去除光引发剂和油。最后将纯化后的微球于-80°冰箱中冷冻过夜,取出后立即冷冻干燥48h,得到GelMA水凝胶微球。
优选的,在S4中,将S3得到的GelMA水凝胶微球、EDC和NHS加到MES中恒温摇床活化10-20分钟后,将S2中制备得到的壳聚糖纳米粒-黑磷量子点加入其中,恒温摇床孵育10-15h后得到GM@CS-BP。
在本发明的一些具体实施例中,将GelMA水凝胶微球、16mg EDC和24mg NHS依次添加到2mL MES(pH=6.0)缓冲液中。在37℃恒温摇床中活化15分钟。之后将制备的壳聚糖纳米颗粒-黑磷量子点溶液以不同体积加入微球(使得微球溶液与壳聚糖纳米粒-黑磷量子点的体积比分别为1:1、1:2、1:4、1:8、1:16)。在37℃恒温摇床下孵育过夜,离心后用去离子水冲洗三次,得到GM@CS-BP。
本发明第二个方面公开了上述方法制备得到的水凝胶微球。
本发明第三个方面公开了一种纳米材料,所述纳米材料包括上述的水凝胶微球。
本发明第四个方面公开了上述的方法或上述的水凝胶微球在调节髓核氧代谢平衡领域中的应用。
本发明受椎间盘退变环境中氧代谢失衡导致的髓核细胞外基质的降解及ROS和酸敏感复合体之间存在的正反馈交互作用所启发,构建氧代谢平衡工程化水凝胶微球,通过均匀有效的装载并靶向缓释具备强抗氧化特性的黑磷量子点,精准调控椎间盘退变中ROS的失衡。通过一系列材料学测试、细胞实验及动物研究发现,复合微球在高强度ROS刺激下能够下调髓核细胞酸敏感复合体的表达,阻断下游炎症通路的活化,切断氧化应激和炎症之间的恶性循环。从而实现椎间盘退变环境中ECM(细胞外基质)的稳定,恢复组织功能,促进髓核再生。
与现有技术相比,本发明至少具有以下有益效果:
本发明将静电力装载BPQDs的CS通过酰胺键接枝到GelMA多孔微球表面,制备了氧代谢平衡工程化可注射水凝胶微球GM@CS-BP。该复合微球实现了纳米颗粒的高效均匀负载,能够通过微量泵注射器定向植入椎间盘微环境,持续释放强还原性BPQDs,原位中和ROS,减轻氧化应激损伤,改善椎间盘氧代谢微环境。GM@CS-BP抑制细胞凋亡的同时改善细胞外酸中毒,阻断下游炎症通路活化引发的炎症风暴。该水凝胶微球GM@CS-BP(氧代谢平衡工程化水凝胶微球),能够为以黑磷为还原剂延缓椎间盘退变提供新的理论基础,同时为局部氧化应激微环境下生物材料的再生应用提供新思路,给椎间盘退变患者带来了福音。
附图说明
图1为本发明的构思示意图:A)黑磷量子点、壳聚糖纳米粒、GelMA微球的制备;B)GM及CS-BP纳米粒的活化及接枝;C)氧代谢平衡工程化水凝胶微球的大鼠椎间盘靶向注射及治疗机制。
图2为黑磷量子点与壳聚糖纳米粒的制备和表征示意图:A)液相剥离法超声制备黑磷量子点示意图;B)黑磷量子点超声前后大体图;C)黑磷量子点的TEM下形貌;D)CS-BP的TEM下形貌;E)拉曼光谱图(曲线图从上往下依次为CS-BP、CSNP、BPQDs);F)黑磷量子点的粒径大小;G)CS-BP的粒径大小;H)CS、BP、CS-BP的zeta电位分析(柱形图从上往下依次为CS、BP、CS-BP)。
图3为GelMA微球、GM@CS-BP的制备和表征示意图:A)CS-BP与GelMA接枝示意图;B)GelMA与CS-BP大体图;C)GelMA微球SEM下形貌;D)GM@CS-BP微球SEM下形貌;E)GM@CS-BP局部放大示意图;F)微量泵注射器注射GM@CS-BP微球;G)光镜下油相GM@CS-BP形貌;H)红外光谱分析分析(曲线图从上往下依次为GM、CS、GM@CS、GM@CS-BP);I)EDS分析;J)黑磷量子点的ICP释放检测;K)GM@CS-BP微球表面孔隙大小;L)GM@CS-BP微球粒径大小。
图4为GM@CS-BP的生物相容性及抗氧化、抗凋亡实验示意图:A)GM@CS-BP抗髓核细胞氧化应激示意图;B)不同浓度过氧化氢刺激髓核细胞6、24h后的CCK-8分析(柱形图从左往右依次为6H、24H);C)DCFH-DA荧光探针检测不同GM与CS-BP体积比下材料抗ROS能力半定量分析;D)总抗氧化能力分析(柱形图从左往右依次为CS-BP、GM、GM-BP、GM@CS-BP);E)过氧化氢刺激后材料与细胞共培养的JC-1线粒体膜电位相对荧光强度半定量分析(柱形图从左往右依次为Control、H2O2、CS-BP、GM、GM-BP、GM@CS-BP);F)DCFH-DA荧光探针检测细胞内ROS;G)微球与细胞共培养活死染色;H)过氧化氢刺激后材料与细胞共培养的JC-1线粒体膜电位检测;黄色圆圈代表不同微球。
图5:A)过氧化氢干预后细胞与微球共培养示意图;B)ASIC-3及COL-II的免疫荧光半定量分析(柱状图从左往右依次为H2O2、CS-BP、GM、GM-BP、GM@CS-BP);C-D)ASIC-3及COL-II免疫荧光染色。
图6为过氧化氢刺激条件下髓核细胞蛋白的表达示意图;A)ASIC-3、TNF-α、cleaved IL-1β、pro IL-1β、IL-6、MMP13、COL-II及NF-κB和MAPK信号通路的Westren Blot结果;B-G)ASIC-3、TNF-α、cleaved IL-1β/pro IL-1β、IL-6、MMP13、COL-II蛋白表达的半定量分析(柱形图从左往右依次为H2O2、CS-BP、GM、GM-BP、GM@CS-BP);I)p65、p-p65、p38和p-p38蛋白表达的半定量分析(柱形图从左往右依次为H2O2、CS-BP、GM、GM-BP、GM@CS-BP)。
图7为椎间盘退变模型构建的示意图及影像学分析示意图:A)动物模型示意图及手术照片;B)X-ray照片;C)MRI扫描;D-E)4周和8周的DHI变化(图7E中柱形图从左往右依次为Control、NC、CS-BP、GM、GM-BP、GM@CS-BP);F)术后4W和8W的MRI分级(柱形图从左往右依次为Control、NC、CS-BP、GM、GM-BP、GM@CS-BP)。
图8为动物实验的组织学评价示意图:A)4W和8W的H&E染色结果;B)番红快绿染色;C)HO-1免疫荧光染色;D-E)COL-II、ASIC-3免疫组化染色;F)IL-1β免疫荧光染色;G)4W和8W的组织学评级(柱形图从左往右依次为Control、NC、CS-BP、GM、GM-BP、GM@CS-BP);H)HO-1半定量分析(柱形图从左往右依次为Control、NC、CS-BP、GM、GM-BP、GM@CS-BP);I-J)COL-II及ASIC-3免疫组化的半定量分析(柱形图从左往右依次为Control、NC、CS-BP、GM、GM-BP、GM@CS-BP);K)IL-1β免疫荧光的半定量分析(柱形图从左往右依次为Control、NC、CS-BP、GM、GM-BP、GM@CS-BP)。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细描述,但并不因此将本发明限制在所述的实施例范围之中。
下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。本发明所用试剂和原料均市售可得。
实施例1
本实施例公开了一种制备GM@CS-BP的方法,包括:
1.黑磷量子点的制备:
通过已有的液相剥离技术制备黑磷量子点(全程于氮气保护下进行)。首先将20mgBP晶体分散于20mL的NMP溶液中,并在冰水浴中以1200W的功率超声3h(南京赛飞有限公司,中国,超声波频率:19-25kHz,2秒开/3秒关),之后将获得的溶液再次于冰水浴中超声12h,并将分散液以7000rpm的转速离心20分钟以除去未分散的BP。取上清液于新试管中,以15000rpm的转速离心2h,弃上清,并用去离子水重悬。
2.壳聚糖纳米粒-黑磷量子点的制备:
将壳聚糖(20mg,200-400mPa.s)于室温环境下在磁力搅拌中溶解于20mL1%(w/v)醋酸溶液中,当溶剂澄清透明时,使用10M NaOH溶液将pH调节至5.0,并用0.45μm的滤膜过滤三次,以BPQDs/CS质量比1:10的黑磷量子点重悬于壳聚糖纳米粒中。随后在720rpm的磁力搅拌下逐滴添加三聚磷酸盐(TPP)溶液(0.7mg/mL),使得CS/TPP的质量比为4:1。此时溶液产生透明乳光色的特征,表明TPP和CS发生了离子交联聚合。随后使用900W功率的超声波破碎仪超声5分钟,重复2-3次。之后将获得的壳聚糖纳米粒再次通过0.22μm的滤膜过滤三次。
3.甲基丙烯酸酐化明胶(GelMA)的制备:
首先,称取20g的明胶加入200ml的PBS中,并将其置于水浴锅中于60°下搅拌1h直至完全溶解,获得10%(w/v)的透明淡黄色的明胶溶液。之后,于避光条件下抽取16ml的甲基丙烯酸酐,使用微量泵推注器以0.25mL/min的速度将其滴加于明胶溶液中。之后维持60°温度连续搅拌2h,将预热的800mlPBS加入上述反应物中,继续搅拌15min。之后将溶液用12-14kDa的透析袋分装透析2周,每2天更换一次PBS,最终得到GelMA溶液,冷冻干燥后保存于-80℃冰箱。
4.GelMA水凝胶微球与GM-BP的制备:
通过油包水形式的微流控技术制备。首先,将同轴电纺喷头(内针与外针的直径分别是30G和21G)通过硅胶管分别连接于两个微量泵推注器。同轴电纺喷头的外针作为共流剪切的连续相。连接含有配好的10%(w/w)Span80的肉豆蔻酸异丙酯溶液,内针作为分散相,连接的溶液是7%(w/v)GelMA水溶液和0.5%(w/v)的I2595光引发剂。结合本组已有研究,调节微量泵推注器使得水相(即GelMA相)与油相的流速比为15:500μl/min,打开开关,分别排气直至于接口处产生连续单分散球形微滴。待微滴大小均匀后于避光条件下置入预先装有油性溶液的烧杯中。每10-20min取出使用紫外光充分照射并更换烧杯(整个环境下维持针道系统一定温度以防止针道内发生交联堵塞)。之后将收集到的固化水凝胶微球通过75%乙醇和PBS反复洗涤(75%乙醇3次,PBS5次),之后每3h更换一次PBS,以去除光引发剂和油。最后将纯化后的微球于-80°冰箱中冷冻过夜,取出后立即冷冻干燥48h,得到GelMA水凝胶微球。
通过物理共混构建GM-BP微球以对照验证CS对黑磷的保护作用(GM与BP之间没有特殊的化学键存在,使用的是单纯物理共混方法,设计本组的目的是为了作为对照以验证CS对黑磷的包封及缓释作用)。具体方法:将制备的冻干GelMA微球复溶溶液与BPQDs溶液(0.2mg/mL)混合,并在37℃下搅拌,在氮气保护下保持5分钟,得到GM-BP微球。
5.微球与纳米粒的接枝:
将冻干GelMA水凝胶微球、16mg EDC和24mg NHS依次添加到2mL MES(pH=6.0)缓冲液中。在37℃恒温摇床中活化15分钟。之后将制备的壳聚糖纳米颗粒-黑磷量子点溶液以不同体积加入微球(使得微球溶液与壳聚糖纳米粒-黑磷量子点的体积比分别为1:1、1:2、1:4、1:8、1:16)。在37℃恒温摇床下孵育过夜,离心后用去离子水冲洗三次,得到水凝胶微球GM@CS-BP。
实施例2
一、黑磷量子点、CS-BP的制备和表征:
1.透射电镜(TEM)
使用透射电子显微镜(TEM,日本日立HT7700)在200kV电压下检测壳聚糖纳米粒、黑磷量子点、壳聚糖纳米粒-黑磷量子点的形貌。
2.粒径及表面电荷检测
使用纳米粒度仪(英国马尔文ZetasizerNano zs90)测量壳聚糖纳米粒的粒径和Zeta电位。
3.包封率检测
将最终制成的GM@CS-BP及GM-BP材料置于10ml纯水中,并以60转/分的速度置于37°恒温摇床中。在0、6、12、24、48、72、120、168、336h后取上清并再次用10ml纯水重悬(氮气保护下,取最终BP质量为0.25mg)。所得上清液于电感耦合等离子发射光谱仪(ICP,赛默飞,7400DUO)测量每种溶液中磷元素的浓度。
4.拉曼光谱检测
拉曼光谱(Renishaw,InVia Spectrometer)检测纳米粒、黑磷量子点的元素构成。
二、GelMA水凝胶微球及氧代谢平衡工程化水凝胶微球的表征:
1.扫描电镜(SEM)
使用导电胶固定GM微球和GM@CS-BP微球,镀金45秒(Quorum Technologies,SC7620,英国)。然后,在10kV的加速电压下,使用SEM观察其形态(日本日立S-4800SEM)。
2.红外和EDS分析
使用EDS(Oxford)对微球进行表面元素分析,通过红外光谱(Thermo Scientific,Nicolet 6700,USA)检测微球、纳米粒、黑磷量子点的元素构成。
3.微球与纳米粒的接枝浓度筛选
将制备的壳聚糖纳米颗粒-黑磷量子点溶液以不同体积加入微球(使得微球溶液与壳聚糖纳米粒-黑磷量子点的体积比分别为1:1、1:2、1:4、1:8、1:16)。在37℃恒温摇床下孵育过夜,离心后用去离子水冲洗三次。为了探索拥有最适宜的ROS清除能力的微球与纳米粒体积比,将所得到的纯化的微球约200个放入24孔板中,酒精及紫外灭菌后用PBS清洗3次,用培养基浸泡6h。将约4*104个用200μM的H2O2刺激后的髓核细胞接种入微球共培养过夜。按照1:1000用无血清培养基稀释DCFH-DA荧光探针,用PBS清洗微球-细胞共培养体系后加入适当体积稀释后的DCFH-DA荧光探针,于37°细胞培养箱中孵育30min,之后通过荧光显微镜进行形态学观察,用ImageJ软件进行半定量荧光分析。
实施例3
氧代谢平衡工程化水凝胶微球的体内效果探究
1.体外实验的准备
实验中使用的NPC从SD大鼠尾椎提取。在无菌条件下分离大鼠尾椎,使用眼科钳精细移除各个阶段的NP组织,加入0.25%的II型胶原酶,在37°细胞培养箱中孵育2h。使用细胞过滤器过滤细胞悬液,离心弃上清,使用无菌PBS洗涤三次后加入10%胎牛血清的DMEM/F12培养基。在37℃,5%CO2的细胞培养箱中孵育,每2-3天更换培养基,一周左右传代或冻存细胞。
2.微球与细胞的共培养及H2O2浓度的筛选
将所得到的纯化的微球约200个放入24孔板中,酒精及紫外灭菌后用PBS清洗3次,用培养基浸泡6h。将约4*104个预先分别被0、50、100、200、400μM的H2O2刺激30min后的髓核细胞接种入微球共培养过夜。再于37℃细胞培养箱中继续孵育6h、24h。使用CCK8检测试剂盒(日本Dojindo)以10%(v/v)的比例添加至培养基中培养4h。使用枪头将100μL的培养基移入96孔板。使用酶标仪测量450nm的吸光度。
3.材料总抗氧化能力检测
将约200个GM、GM-BP、GM@CS-BP微球重悬于2mlPBS中静置6h,取上清液按照T-AOC检测试剂盒(Beyotime,中国上海)制造商的说明,采用2,2′-叠氮双(3-乙基苯并噻唑啉-6-磺酸(ABTS)法检测材料的Trolox-Equivalent Antioxidant Capacity,并以mmol/L表示。
4.细胞活性检测
进行活/死染色测定。按照上述共培养方法,在微球和细胞共培养4天、7天后通过活/死试剂盒(美国Invitrogen)添加至24孔板中,室温培养30min,使用倒置荧光显微镜观察细胞形态。
5.免疫荧光
按照前述微球、细胞共培养方法使用200μM的H2O2干预,继续培养3天后,经过固定、渗透、封闭后将其与一抗ASIC-3和COL-II孵育过夜,洗涤后再与二抗室温下孵育2h。依次用鬼笔环肽(Yearsen,中国)和DAPI(Abcam,美国)染色,然后用共聚焦显微镜观察。用ImageJ软件进行半定量荧光分析。
6.线粒体膜电位检测
通过上述方法得到微球-细胞共培养体系。按照JC-1试剂盒(Solarbio,中国)的说明书制备JC-1染色工作液。加入适量工作液,与37℃细胞培养箱中孵育20min后将JC-1染色缓冲液1X洗涤2次,使用培养基洗涤2次,于倒置荧光显微镜下观察,使用ImageJ软件进行半定量荧光分析。
7.WesternBlot分析
按照上述共培养体系,在200uM的H2O2刺激后继续孵育4天,通过前述WesternBlot方法检测ASIC-3,IL-1β(ab9722),IL-6(ab9324),TNF-α(ab66579),MMP-13(ab39012),COL-II(ab34712)蛋白,以及p65(ab16502)、p-p65(ab28856)、p38(ab31828)、p-p38(ab45381)和GAPDH(ab8245)蛋白表达量。所得的图片通过ImageJ进行条带的灰度值定量分析。
实施例4
氧代谢平衡工程化水凝胶微球的体内效果探究
1.体内实验及动物筛选
雄性SD大鼠,平均体重300-350克,购自苏州大学实验动物中心。手术和治疗效果良好。经苏州大学第一附属医院伦理委员会批准。
2.大鼠尾椎椎间盘退变模型的建立
大鼠腹腔注射10%(wt%)水合氯醛。完全麻醉后,对大鼠尾部进行消毒,然后用针头连续穿刺大鼠7-9IVD以诱导退化。为确保诱发退变,针刺后静置抽吸损毁椎间盘内部组织。之后使用微量泵注射器注射GM、GM@CS-BP微球溶液约15μL,阴性对照组注射PBS。手术后,将大鼠置于温暖通风的地方。
3.X线和MR影像学评估
术后4周和8周,从每组随机选择3只大鼠,在处死前进行X线和MRI检查。将每只大鼠置于仰卧位,并将其尾巴置于钼靶放射成像装置上。获取的X-ray图片由通过ImageJ软件测量并计算椎间盘高度指数(DHI%)。使用1.5T系统(GE)进行MRI检查。在冠状面获得T2加权图像。不知道分组的影像学临床医生根据改进的Thomson分类法,评估T2加权信号强度,将MRI评分为I至IV级。
4.组织学评价和免疫荧光、免疫组化
术后4周和8之后,将大鼠处死,并去除IVD放入福尔马林中浸泡。在10%EDTA中脱钙30天,包埋于石蜡中。将标本切片约5μm。之后分别通过苏木精-伊红染色(H&E染色)、番红O-快绿染色(S-O-FastGreen染色)观察椎间盘内组织结构和胶原的变化。组织学分级采用Masuda建立的量表,将其评分为4分(正常)到12分(严重变性)。并对切片进行HO-1荧光及COL-II、ASIC-3免疫组织化学染色,最后进行IL-1β免疫荧光染色。
具体实验结果描述如下所述:
一、黑磷量子点及壳聚糖纳米粒的表征
图1为本发明的构思示意图:A)黑磷量子点、壳聚糖纳米粒、GelMA微球的制备;B)GM及CS-BP纳米粒的活化及接枝;C)氧代谢平衡工程化水凝胶微球的大鼠椎间盘靶向注射及治疗机制。
黑磷量子点(BP)在空气中易被氧化,过于微小的粒径决定其在制备过程中存在不稳定性。壳聚糖(CS)由于表面丰富的氨基基团,以及优秀的生物相容性被申请人选为黑磷量子点(BPQDs)的保护剂。通过液相剥离法制备黑磷量子点(Figure 2A),在高强度超声操作前后可见(Figure 2B),单纯黑磷纳米片在空气中呈现深黑色,经过不断地离心超声后黑磷量子点为浅黄色。按照离子交联法制备CS-BP,用TEM观测黑磷量子点及CS-BP形貌(Figure2C)。黑磷量子点普遍呈现为大小不等的圆形结构,而在纳米粒-量子点复合结构中(Figure2D),黑磷量子点均匀分布在壳聚糖纳米粒的内部和周围,为了验证纳米粒和量子点的成功连接,申请人对CS-BP进行了拉曼散射光谱分析(Figure 2E)。在拉曼光谱中,单纯黑磷量子点的代表峰为A1g:358.4、B2g:438.2、A2g:467.1cm-1,在CS-BP纳米粒中申请人检测到了黑磷的代表峰值。之后,申请人分析了黑磷量子点及CS的纳米粒径大小(Figure 2F、2G),黑磷量子点直径分布于4.8±2.2nm,纳米粒度仪显示CS与CS-BP颗粒的粒径普遍分布在78.56±21.45nm,这与TEM的形貌结果一致。最后,申请人对纳米颗粒进行了Zeta电位检测(Figure 2H),黑磷量子点的电势表现为-19.63±2.86mV,由其表面氧化形成的磷酸根阴离子而来,而壳聚糖纳米粒的电势为34.43±3.82mV,源于其丰富的氨基。离子交联形成的的CS-BP电位表现为19.37±2.46mV,相对壳聚糖有较为明显的下降,这也符合申请人的预期。
二、GelMA微球及氧代谢平衡工程化水凝胶微球的表征
GelMA微球由于优秀的生物相容性及丰富的表面孔隙成为还原剂注射治疗的理想载体。但是,简单的物理共混可能存在分布不均,负载药物在环境中不稳定等问题。因此,申请人选择通过酰胺键实现纳米粒的接枝,以提高微球持续释放黑磷量子点的能力。按照本组已有的微流控制备技术,通过同轴电纺喷头的连续相和分散相形成的共流剪切作用制备GelMA微球,在EDC(1-乙基-3-[3-二甲氨基丙基]碳二亚胺盐酸盐)/NHS(N-羟基琥珀酰亚胺)体系下,使壳聚糖中的氨基与微球的羧基反应(Figure 3A),完成纳米粒子的接枝,形成GM@CS-BP微球,在SEM下观测其形貌(Figure 3C、3D)。GM@CS-BP微球拥有较为均匀的表面孔隙,分布在5.6±4.05μm(Figure 3K),这使得GM微球能够充分的与CS-BP纳米粒相结合,成为GM微球有效负载载药纳米粒的形态基础。微球大小分布在68.7±21.25μm(Figure 3L),这与单纯的GM微球和GM-BP微球没有明显改变,微球表面的孔隙局部放大图可以看到表面存在均匀密布的CS-BP纳米粒(红色箭头标注),证明了二者的连接和有效负载。能否通过针道进行有效注射,是申请人治疗方案的结构基础。申请人选择针尖较细小的微量泵注射器,充分模拟大鼠椎间盘环境的注射器具,申请人发现微球能够在保持形态完好的前提下,均匀分散的完成注射过程(Figure 3F)。光镜下检测GelMA微球形貌发现(Figure 3G),微球在油相中均匀排列,30min内大小、结构均无明显改变。之后,申请人进行了红外光谱(FT-IR)和EDS分析以验证GM微球和纳米粒之间的结合。如FT-IR光谱所示(Figure 3H),在GM@CS-BP光谱中均观察到GelMA和壳聚糖的特定化学基团,这表明GelMA和壳聚糖形成了良好混合。具体来说,相对于单纯的GelMA微球,GM@CS-BP在3300cm-1峰值处有明显的下移,表明-OH的存在,而在1640cm-1处峰值的下降代表着C=C振动的存在。另一边,与壳聚糖的红外光谱进行比较,可以发现1620cm-1和1530cm-1两处的峰值下降,这可能与壳聚糖上NH3相关。而GM@CS-BP与GM@CS组无明显变化。接下来,申请人通过能量色散光谱(EDS)比较GM和GM@CS-BP元素含量。正如预期的那样,与单纯GM组相比,GM@CS-BP中的P元素大量升高,占比达10.2%,这表明了壳聚糖纳米粒于微球表面成功接枝,及其释放的黑磷量子点的存在。同时申请人发现GelMA微球表面仍能检测到少量N元素存在,考虑GelMA制备中部分氨基没有完全取代。综上所述,申请人通过傅里叶红外光谱和能量色散光谱成功证明了GelMA微球和CS-BP纳米颗粒的接枝。
GelMA微球与CS-BP纳米粒溶液示意图如Figure 3B所示。
此外,材料释放黑磷的能力也是本研究中非常重要的内容。由于黑磷并非传统的药物,绘制其释放曲线成为了一项难题。为了验证材料能否持续有效的释放黑磷,申请人通过电感耦合等离子体原子发射光谱法(ICP-AES)测量溶液中的磷元素以代表材料释放黑磷的能力(Figure 3J)。简而言之,申请人取0、6、12、24、48、72、120、168、336h的微球上清液,计算累计的磷元素释放量,以此推断黑磷的释放规律。根据结果所示,微球和黑磷单纯通过物理吸附的释放曲线在120h之内表现出快速的突释(4.46±0.43ppm),且336h之后释放的黑磷总和较低(5.77±0.17ppm)。而通过壳聚糖纳米粒包封后的黑磷虽然在120h之内的释放相近(4.37±0.27ppm),但其后半程仍能够维持较为规律的持续释放,且最终释放的黑磷总和(8.34±0.15ppm)要远超过单纯使用物理吸附,证实包封率提高了1.45倍。这是因为壳聚糖有效使黑磷免于制备过程中的氧化损耗,同时微球和壳聚糖形成的酰胺键能够通过化学键的断裂持续有效的释放纳米颗粒。这极其有意义的证明了氧代谢平衡功能化水凝胶微球的包封和缓释能力。
三、氧代谢平衡工程化水凝胶微球抗氧化、抗凋亡能力及生物相容性实验
1.氧代谢平衡功能化水凝胶微球抗凋亡、抗氧化能力及生物相容性实验
由于椎间盘内部长期处于缺血缺氧的微环境中,活性氧的产生和清除的这一动态平衡被打破,引发椎间盘内部大量的活性氧簇产物堆积。这些产物的堆积一方面会引起组织脂质过氧化水平升高,引发线粒体膜电位变化,导致线粒体外膜通透性增加,大量凋亡因子外流,最终导致细胞的凋亡。另一方面也激活了下游炎症通路,导致椎间盘内部大量炎症因子的释放。申请人选择ROS中最具代表性的过氧化氢来模拟椎间盘内部的氧化应激环境,并通过CCK8法验证需要的过氧化氢浓度。结果如图4所示,其中Figure4A为GM@CS-BP微球抗髓核细胞氧化应激的示意图。
Figure 4B中显示,当以50μM的过氧化氢刺激后,髓核细胞活性受抑制程度不显著,当以200μM的过氧化氢刺激后,相对于Control组来说,细胞的生长能力表现出明显的抑制,但仍能维持接近控制组半数的活性。而400μM的过氧化氢刺激将导致髓核细胞死亡超过半数。结合文献,申请人最终筛选出200μM的过氧化氢浓度作为模拟椎间盘氧化应激环境的干预条件。在本研究中,材料的抗ROS能力是申请人最为关注的一项指标。申请人选用DCFH-DA探针来验证材料的清除过氧化氢能力,并验证材料适宜的配比。简而言之,申请人将不同体积比(1:1、1:2、1:4、1:8、1:16)的GM微球溶液和CS-BP溶液,通过酰胺键进行化学连接,并将复合微球与髓核细胞共培养。使用过氧化氢刺激共培养体系6h后使用荧光探针来验证环境内ROS的水平。结果如Figure 4F所示,当微球溶液与CS-BP纳米粒溶液体积比为1:2(**p<0.01,与Control、1:8、1:16组相比),1:4(***p<0.001,与Control、1:8、1:16组相比)时,环境内活性氧荧光强度明显降低。其中,体积比为1:4时,DCFH-DA荧光探针检测到过氧化氢强度最低(**p<0.01,与体积比1:2组相比),此时材料的抗ROS能力达到最高峰(Figure 4C),相较Control组的荧光探针强度下降2.29倍。同时申请人发现,升高或者降低二者的体积比都会使得材料的清除ROS能力下降。申请人考虑,这可能是由于当黑磷量子点较低时,材料的抗氧化能力不足,而当黑磷量子点过量时,复合微球可能过度还原了环境内的O2,导致缺氧环境并未改善。申请人还验证了GM、GM-BP、GM@CS-BP制备后6h的总抗氧化能力(T-AOC)(Figure 4D),发现GM微球几乎没有抗氧化能力,而GM-BP和GM@CS-BP的抗氧化能力大幅提高。由于GM-BP存在快速的突释,因此6h的T-AOC结果表明GM-BP抗氧化能力更强。结合DCFH-DA探针的荧光半定量结果,申请人不仅成功证明了材料拥有较为满意的ROS清除能力,同时筛选出了GM微球和CS-BP纳米粒溶液的体积比,为后续实验建立基础。
之后申请人进行了活/死细胞检测,如图所示(Figure 4G),细胞均匀分布于微球之上,而在第7天时,可以观察到微球中的活细胞数量有所增加,而死细胞增多不明显,充分表明复合微球支架是适合于髓核细胞生长的生物支架。线粒体是产生ROS的主要场所,在线粒体呼吸链复合物部位,ROS诱导线粒体跨膜电位降低,使得凋亡相关蛋白跨膜转运至细胞质中,继而引发细胞凋亡。申请人通过JC-1试剂盒对线粒体的膜电位进行了验证,当线粒体膜电位较高时,JC-1探针以聚合物形式(J-aggregates)存在于线粒体中,荧光显微镜下展现为红色,而线粒体膜电位较低时,则其单体(Monomer)表现为绿色荧光(Figure 4H)。将单纯使用过氧化氢干预设立为Control组,而通过微球和细胞的共培养及过氧化氢干预后的GM、GM-BP、GM@CS-BP与其进行比较(Figure 4E),GM和Control组主要以绿色荧光为主,其RFI(相对荧光强度)=J-aggregates/Monomer比值较Control组无明显差异,这表明Control组与GM组其线粒体膜电位较低,细胞处于凋亡早期状态,而GM-BP组与GM@CS-BP组中主要以红色荧光为主,且相对荧光强度接近Control组。申请人认为,这是由于水凝胶微球支架释放的黑磷量子点成功阻断ROS对线粒体膜的过氧化作用,是阻断线粒体凋亡通路的重要环节。
四、氧代谢平衡工程化水凝胶微球对酸敏感通道及炎症的调节
酸敏感离子通道(ASIC)是由细胞外H+直接激活的阳离子通道。ASIC不仅在触觉、痛觉、学习和记忆等神经功能中发挥重要作用,还参与炎症、缺血和缺氧等病理生理过程。ASIC参与IVD过程,其特征是NP细胞减少和ECM破坏(Figure 5A)。最近的一项研究表明,ASIC-3在退化的NP细胞中显著增加,即被细胞外酸激活后续信号通路并调节基因表达,通过核因子-κB(NF-κB)信号通路刺激多种促炎因子的产生,如TNF-α、IL-6、IL-1β等,进一步增强基质金属蛋白酶(MMP)活性,从而诱导炎症。在椎间盘组织中,基质金属蛋白酶13(MMP13)能够特异性的降解椎间盘ECM的主要成分的二型胶原(COL-II),而ECM的产生和降解的失平衡是导致椎间盘退变的主要原因。因此,通过生物材料阻断氧化应激过程,并进一步抑制酸敏感通道的激活及下游炎症级联反应的发生,促进细胞外基质的重建是IVD重要的治疗方案。申请人首先通过免疫荧光染色验证了GM@CS-BP微球阻断ASIC-3的能力(Figure 5C),在单纯GM微球中,ASIC-3的表达量较高,而单纯负载有BP的GM微球能够使得ASIC-3的荧光强度降低22%(Figure 5B),GM@CS-BP微球能够抑制ASIC-3的荧光强度53%,这证明复合微球能够负性调节ASIC-3的表达。而在COL-II的免疫荧光染色中(Figure 5D),GM-BP组与GM@CS-BP组的COL-II表达量相对于GM组分别提高29%、61%(Figure 5B,p<0.001)。
五、通过Westernblot来确定蛋白质水平上的表达情况
条带灰度值的半定量结果正如预期一样(Figure 6A),ASIC-3的表达明显受到抑制(Figure 6B),同时BP分别使得TNF-α、IL-1β、IL-6等促炎因子的表达下降了2.89、3.76、2.16倍(Figure 6C、6D、6E),下调了MMP13的合成(Figure 6F),从而减少髓核中ECM的降解,使得COL-II的含量增强3.76倍(Figure 6G)。申请人进一步验证了丝裂原活化蛋白激酶(MAPK)和核因子κB(NF-κB)等炎症相关的信号通路的激活情况,结果如图所示,在GM@CS-BP组中,p38和p65的磷酸化程度分别相对Control组被抑制2.63、3.87倍(Figure 6H、6I)。这些结果证明了氧代谢平衡工程化水凝胶微球释放的抗氧化型黑磷量子点的介入成功减轻了酸性环境引起的椎间盘内的炎症风暴,从而逆转了ECM的合成和分解的失衡。综上所述,GM@CS-BP能够应对退变椎间盘内的氧化应激微环境,在调节局部炎症的基础上,为髓核细胞提供良好黏附和增殖能力,实现髓核的细胞外基质重塑。
六、氧代谢平衡工程化水凝胶微球的体内效果探究
改善椎间盘内部氧化应激环境,促进髓核内ECM重建是申请人的最终目标。为了研究氧代谢平衡工程化水凝胶微球体内延缓椎间盘退变的能力,申请人建立了大鼠尾椎穿刺模型(Figure 7A)。影像学结果及组织学切片是反映椎间盘内部退变及再生的有效指标。申请人对置入复合微球支架4周、8周的大鼠进行了X线检测(Figure 7B),如Figure 7D、7E所示,4周、8周的空白对照组和单纯穿刺组NC的椎间盘高度有明显差异(p<0.001),且8周相对于4周下降明显,证明了椎间盘退变模型的成功构建。4周和8周的GM组和单纯穿刺组无明显差异,而GM-BP于GM@CS-BP两组相对于GM和NC两组椎间盘高度有明显恢复(p<0.001)。而GM@CS-BP组相对于GM-BP组有更为明显的上升(p<0.05),这是由于CS包封黑磷量子点使得负载率和缓释率上升。磁共振成像(MRI)中较高的T2加权信号表明髓核含水量较高(Figure7C)。随着椎间盘退变的发展,蛋白多糖的大量丢失会损害IVD中的水分,椎间盘会逐渐“黑化”,与MRI分析中显示为白色的健康含水组织形成对比。在4周和8周时,NC组的髓核显示出明显的低T2加权信号(Figure 7F),而Control组仍保持髓核的高含水量(p<0.001),证明了椎间盘穿刺后的不可逆损伤。MRI分级(Thomson分类法)表示,GM@CS-BP和GM-BP两组相对于GM组和NC组椎间盘内的分级有较为明显的改善((p<0.001),且在8周更为明显。而GM@CS-BP组相对于GM-BP组分级改善(p<0.05),同样证明了黑磷量子点能够调节局部氧化应激环境修复椎间盘的含水量和塌陷程度,延缓椎间盘退变,而CS的引入进一步加强了氧代谢平衡工程化水凝胶微球的修复作用。
术后4周和8周收集组织切片。苏木精-伊红(H&E)染色可以观察髓核的形貌(Figure8A)。GM@CS-BP组发现髓核的面积较Contorol组有所减少,但可以观察到较为明显的组织边缘。GM组相对NC组,其NP的萎缩无明显改善,NP边界难以区分。而GM-BP组虽然可以观测到一定面积的NP,但不足以代表NP的再生。番红/快绿染色用于评估IVD中的蛋白多糖(橙色)和胶原(蓝色)含量(Figure 8B)。如图所示,NC组椎间盘髓核完全断裂,在8周时仍无改善。GM@CS-BP相对于NC组表现出明显的橙色聚集,代表着蛋白多糖的重塑。GM-BP虽然表达一定量的蛋白多糖,但并没有形成完整的结构,椎间盘的组织变性和结构损失无明显修复。Masuda量表观察到4周和8之后的GM@CS-BP和GM-BP组评分均相对GM组和NC组显著降低(p<0.001),且随着时间的推移进一步下降,表明再生过程的发生。(Figure 8G)而GM@CS-BP组相对于GM-BP组有更为明显的修复(p<0.05)。HO-1易受多种刺激诱导,其降解血红素产生的产物对细胞起重要的保护作用。申请人观察到,NC组相对Control组的HO-1免疫荧光染色强度稍有上升(Figure 8C),可能是由于氧化应激环境激活了Nrf/HO-1通路,引发髓核自我防御,而通过GM-BP和GM@CS-BP治疗后,HO-1荧光强度相对单纯穿刺组明显增强,证明复合微球能够上调HO-1的表达,减轻氧化应激损伤(Figure 8H)。为了验证材料阻断ROS引发的酸敏感通道活化及抑制局部ECM的降解能力,申请人对COL-II、ASIC-3进行了免疫组织化学分析(Figure 8D、8E)。结果如图,4周和8周的COL-II的强度在GM@CS-BP组相对于NC、GM、GM-BP组均有较为明显的改善,且相对于NC组分别提高10.22和17.47倍(Figure 8I),证明了材料持续缓释黑磷量子点引发的ECM重塑。而4周和8周的ASIC-3的免疫组化中,GM@CS-BP相对于NC组分别下降了1.91和4.31倍(Figure 8J)。4周内GM@CS-BP与GM-BP组均相对于GM组和NC组有明显的下降(p<0.001),而GM@CS-BP与GM-BP组ASIC-3表达量不显著。申请人进一步发现,8周内GM@CS-BP组不仅相对于GM、NC组有明显ASIC-3的抑制(p<0.001),而对GM-BP组也有明显的下降(p<0.05),IL-1β在椎间盘退变中发挥重要作用,通过免疫荧光染色申请人发现(Figure 8F),4W和8W的GM@CS-BP微球组相对NC组IL-1β荧光强度下降(Figure 8K)。申请人认为,这是由于GM@CS-BP材料相对微球负载黑磷量子点的结构能够均一有效的缓释抗氧化型黑磷量子点,并通过抑制氧化应激阻断酸敏感复合体的表达,随着时间的推移这一趋势更为明显。综上所述,体内实验结果证明,虽然GM-BP组的DHI%和MRI信号优于GM组和NC组,但仍然弱于GM@CS-BP组,且在8周后更为明显。组织学染色表明GM@CS-BP组髓核组织的残留和蛋白多糖的沉积最为明显,且ROS清除能力以及HO-1的表达均超过其他微球。免疫组化中COL-II强度增高及ASIC-3强度的下降同样提示抗ROS治疗能够调控ECM分解和合成代谢的失衡,为抑制酸敏感通道及阻断后续炎症爆发提供了证据。这些结果证明,使用GelMA微球和壳聚糖纳米粒形成的微球支架能够持续的将黑磷量子点靶向释放至IVD内,通过阻断氧化应激抑制局部过度活跃的炎症反应,促进髓核细胞再生。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (8)
1.一种调节髓核氧代谢平衡的水凝胶微球的制备方法,其特征在于,包括:
S1:制备黑磷量子点;
S2:将S1制备得到的黑磷量子点加入壳聚糖纳米粒中,得到壳聚糖纳米粒-黑磷量子点;
S3:制备GelMA水凝胶微球;
S4:将S3得到的GelMA水凝胶微球、EDC和NHS加到MES中活化后,将S2中制备得到的壳聚糖纳米粒-黑磷量子点加入其中,孵育后得到GM@CS-BP。
2.根据权利要求1所述的方法,其特征在于,在S1中,将BP晶体分散于NMP溶液中,并在冰水浴中超声2-4h,之后将获得的溶液再次于冰水浴中超声10-14h得到分散液,并将分散液离心10-30分钟以除去未分散的BP,得到上清液;取上清液于新的容器中,离心1-3h,弃上清,并用去离子水重悬得到黑磷量子点。
3.根据权利要求1所述的方法,其特征在于,在S2中,将壳聚糖溶解于醋酸溶液中,当溶剂澄清透明时,pH调节至4.5-5.5,过滤后以BPQDs/CS质量比为(1:8)-(1:12)的黑磷量子点重悬于壳聚糖纳米粒中;逐滴添加TPP,使得CS/TPP的质量比为(3:1)-(5:1);超声后将获得的壳聚糖纳米粒再次过滤得到壳聚糖纳米粒-黑磷量子点。
4.根据权利要求1所述的方法,其特征在于,在S3中,制备甲基丙烯酸酐化明胶溶液后,使用微控流技术制备GelMA水凝胶微球。
5.根据权利要求1所述的方法,其特征在于,在S4中,将S3得到的GelMA水凝胶微球、EDC和NHS加到MES中恒温摇床活化10-20分钟后,将S2中制备得到的壳聚糖纳米粒-黑磷量子点加入其中,恒温摇床孵育10-15h后得到GM@CS-BP。
6.根据权利要求1-5任一项所述方法制备得到的水凝胶微球。
7.一种纳米材料,其特征在于,所述纳米材料包括权利要求6所述的水凝胶微球。
8.根据权利要求1-5任一项所述的方法或权利要求6所述的水凝胶微球在调节髓核氧代谢平衡领域中的应用。
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