CN112546300B - 一种雷洛昔芬改性mof涂层介导局部抗骨质疏松性金属基材植入材料及其制备方法 - Google Patents
一种雷洛昔芬改性mof涂层介导局部抗骨质疏松性金属基材植入材料及其制备方法 Download PDFInfo
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- CN112546300B CN112546300B CN202011333709.5A CN202011333709A CN112546300B CN 112546300 B CN112546300 B CN 112546300B CN 202011333709 A CN202011333709 A CN 202011333709A CN 112546300 B CN112546300 B CN 112546300B
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- raloxifene
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Abstract
本发明涉及一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料及其制备方法，本发明首先通过简单层层自组装途径，于金属基材表面构建负载锌离子的壳聚糖/明胶/氯化锌多层膜，进一步将其浸泡于含2‑甲基咪唑和雷洛昔芬的甲醇溶液后，首次制备获得局部抗骨质疏松性LBL‑MOF/Ral金属基材，MOF涂层经Ral改性后，表面疏水性及水稳定性显著升高，对成骨细胞的生物相容性也得到明显改善，另外，LBL‑MOF/Ral涂层还将在金属植入材料周围局部缓释锌离子和雷洛昔芬，表现出优越的促成骨及抗破骨潜能，具有良好的临床局部抗骨质疏松应用前景。
Description
技术领域
本发明涉及生物应用技术领域，具体来说，涉及一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料及其制备方法。
背景技术
尽管钛材被广泛应用于骨/牙植入物制备，但其生物惰性表面与周围骨组织多为“机械缩合”，难以实现初期机械稳定性向中/后期生物稳定性过渡，所以极易出现植入物松动或移位现象。相较于健康人群，骨质疏松患者的骨骼脆性增加，骨量降低和代谢异常，导致种植体的初期稳定性进一步下降，骨愈合时间延长，进而影响种植长期成功率。因此，提高骨质疏松病理条件下植入物的初期稳定性，加速早期骨结合是迫切解决的难题。近年来，金属有机框架(MOF)材料受到人们的广泛关注。它们具有高度的可调节性，通过改变有机配体的长度、形状、配位点数目以及官能团修饰等手段对其加以设计，从而得到新型的晶体结构和功能化的内部孔环境。然而相关研究显示，常见MOF材料在水溶液稳定性较差，水分子将通过配位键干扰等诱发材料快速降解，降解所产生的大量金属离子及有机配体对骨损伤修复细胞产生差异化的细胞毒性。所以有效改善MOF材料水稳定性成为研究热点之一。
因此提供一种改善MOF材料水稳定性的金属基材植入材料是本领域急需解决的技术问题。
发明内容
针对现有技术存在的不足，本专利旨在于利用雷洛昔芬改性MOF涂层赋予钛材优越的局部抗骨质疏松性能。该制备方法操作简便、重复性好、可控性强。利用该方法制备的新型MOF涂层在水环境中降解速率适中，可同时缓释锌离子和Ral药物，且具有优越的促成骨及抑破骨潜能，拥有良好的临床应用前景。
本发明提供一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法，包括如下步骤：
步骤一、利用层层自组装技术于金属基材表面构建壳聚糖/明胶/氯化锌多层膜；
步骤二、将步骤一制备的金属基材浸泡于2-甲基咪唑/雷洛昔芬/甲醇混合液中，制备获得LBL-MOF/Ral目的材料。
进一步的，所述金属基材包括钛基材或者钛合金基材。
进一步的，所述步骤一中的金属基材为利用热碱途径处理金属基材获得纳米针状表面结构金属基材。
进一步的，所述步骤一中热碱途径为利用100-2000目砂纸对金属基材进行表面打磨处理，并依次用乙醇、丙酮和去离子水各洗5-20min；再次，将洁净的金属基材置于50-100℃的1-10M氢氧化钠溶液中处理12-48h；然后，将碱腐蚀样本置于1-5mg/mL的多巴胺溶液中浸泡3-12小时，然后用双蒸水冲洗。
进一步的，所述步骤一中热碱途径为依次利用100、400、1000和2000目砂纸对金属基材进行表面打磨处理，并依次用乙醇、丙酮和去离子水各洗10min；再次，将洁净的金属基材置于80℃的5M氢氧化钠溶液中处理24h；然后，将碱腐蚀样本置于2mg/mL的多巴胺溶液中浸泡6小时，然后用双蒸水冲洗。
进一步的，步骤一中的层层自组装技术为将金属基材置于旋涂仪基台上，在500-2000rpm条件下依次涂覆1-10mg/mL的2％醋酸配置的壳聚糖溶液、1-10mg/mL离子水配置的明胶溶液、20-100mg/mL离子水配置的氯化锌溶液，每层涂覆10-30秒，n个循环后获得目的涂层(Chi/Gel/ZnCl2)n，5≤n≤10。
进一步的，步骤一中的层层自组装技术为将金属基材置于旋涂仪基台上，在1000rpm条件下依次涂覆5mg/mL的2％醋酸配置的壳聚糖溶液、5mg/mL离子水配置的明胶溶液、5mg/mL离子水配置的氯化锌溶液，每层涂覆15秒，n个循环后获得目的涂层(Chi/Gel/ZnCl2)n，5≤n≤10。
进一步的，所述步骤二中的2-甲基咪唑/雷洛昔芬/甲醇混合液为20-100mg/mL甲醇配置含2-甲基咪唑与0.1-1mg/mL雷洛昔芬的混合反应液，将步骤一制备的金属基材浸泡于pH7.4的磷酸缓冲液中，去除涂层中多余醋酸分子；然后再浸泡在2-甲基咪唑/雷洛昔芬/甲醇混合液中浸泡1-6h，经甲醇/去离子水冲洗后获得LBL-MOF/Ral目的材料。
进一步的，所述步骤二中的2-甲基咪唑/雷洛昔芬/甲醇混合液为45mg/mL甲醇配置含2-甲基咪唑与0.6mg/mL雷洛昔芬的混合反应液，将步骤一制备的金属基材浸泡于pH7.4的磷酸缓冲液中，去除涂层中多余醋酸分子；然后再浸泡在2-甲基咪唑/雷洛昔芬/甲醇混合液中浸泡2h，经甲醇/去离子水冲洗后获得LBL-MOF/Ral目的材料。
本发明还提供上述制备方法所制备的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料。
本发明具有如下优点：本发明的制备方法操作简便、重复性好、可控性强；通过碱腐蚀钛材表面使得MOF多层膜结构能够紧密包覆在金属基材表面，通过疏水性抗骨质疏松药物Ral对MOF材料进行改性成功的构建了MOF/Ral功能涂层。相较于单纯MOF涂层，MOF/Ral的水稳定性显著升高，缓慢释放锌离子及雷洛昔芬药物，使得锌离子不会过量释放，对成骨细胞的相容性也得到明显改善，另外局部缓释的锌离子和雷洛昔芬协同作用下还可同时实现促成骨及抗破骨的目的，利用该方法制备的新型MOF涂层具有理想是水稳定性，可同时，赋予目的材料优越的促成骨及抑破骨能力，在骨损伤修复领域有重要的研究价值和临床意义。
附图说明
图1：1A、5以及10个LBL循环后，所获得样本表面MC3T3细胞的细胞活性；1B、5以及10个LBL循环后，所获得样本表面MC3T3细胞的碱性磷酸酶活性B(置信区间为99.5％)。
图2：不同样本的表面扫描电镜(SEM)图及原子力显微镜(AFM)图。
图3：3A不同样本表面不同元素的XPS曲线；3B不同样本表面S2p3的XPS曲线；3C不同样本表面水接触角(置信区间为99.5％)。
图4：不同样本的锌离子释放曲线。
图5：5A不同样本表面MC3T3细胞荧光形貌及细胞面积统计图(置信区间为99.5％)；5B不同样本表面MC3T3细胞SEM形貌图。
图6：6A不同样本表面MC3T3细胞的细胞活性；6B不同样本表面MC3T3细胞的细胞矿化(置信区间为99.5％)。
图7：不同样本表面MC3T3细胞的成骨相关基因表达(置信区间为99.5％)。
图8：8A不同样本表面RAW264.7细胞TRAP活性；8B同样本表面RAW264.7细胞破骨分化相关基因表达(置信区间为99.5％)。
具体实施方式
下面将结合实施例和效果例对本发明做进一步的详述，而非限制本发明的范围。
实施例1：制备SF/Hb含氧水凝胶
首先利用100、400、1000和2000目砂纸对钛材进行表面打磨处理，并依次用乙醇、丙酮和去离子水各洗10min，这个步骤制备的样品记为Ti。其次，将洁净的钛材(Ti)置于80℃5M氢氧化钠溶液中处理24h，得到碱腐蚀样品记为AT。并进一步将碱腐蚀样本(AT)置于2mg/mL的多巴胺溶液中浸泡6小时。
使用2％醋酸配置浓度为5mg/mL壳聚糖溶液，记为Chi；使用去离子水配置浓度为5mg/mL明胶溶液，记为Gel；使用去离子水配置浓度为50mg/mL氯化锌溶液，记为ZnCl2。
再次，将多巴胺处理AT样本置于旋涂仪基台上，在1000rpm条件下依次将制备的Chi(5mg/mL)、Gel(5mg/mL)、ZnCl2(50mg/mL)涂覆在多巴胺处理AT样本，每层涂覆15秒形成多层膜结构，5或10个循环后获得(Chi/Gel/ZnCl2)5或(Chi/Gel/ZnCl2)10样本，分别记为LBL5-Zn、LBL10-Zn，并设置对照组，将多巴胺处理AT样本置于旋涂仪基台上，在1000rpm条件下依次将制备的Chi(5mg/mL)、Gel(5mg/mL)涂覆在多巴胺处理AT样本，每层涂覆15秒形成多层膜结构，5或10个循环后获得(Chi/Gel)5、(Chi/Gel)10样本，分别记为LBL5、LBL10。然后，将多层膜覆盖样本浸泡于pH7.4的磷酸缓冲液中3次(每次5min)；最后将LBL5-Zn或LBL10-Zn样本置于2-甲基咪唑/Ral/甲醇混合液中浸泡2h，制备获得LBL5-MOF/Ral以及LBL10-MOF/Ral。设置对照组，将LBL5-Zn或LBL10-Zn样本置于2-甲基咪唑/甲醇混合液中浸泡2h，制备获得LBL5-MOF、LBL10-MOF样本。
通过将实施例和对照组的溶出物与细胞进行培养来检测生物毒性，结果如图1表明LBL5-MOF/Ral组细胞活性及碱性磷酸酶活性均最高，5个循环所制备样本的生物相容性明显优于10个循环，所以我们选取5个循环的样本(LBL5、LBL5-Zn、LBL5-MOF及LBL5-MOF/Ral)进行后续材料表征及细胞学实验，且样本分别被重命名为LBL、LBL-Zn、LBL-MOF和LBL-MOF/Ral。
以上步骤制备所得样本的扫描电镜(SEM)和原子力显微镜(AFM)图片如图2所示：纯钛(Ti)样本表面具有无序凹陷结构，粗糙度为94±22nm；AT、LBL及LBL-Zn样本表面呈现出明显的针状结构，粗糙度分别约为61±11nm、46±17nm和60±14nm；LBL-MOF和LBL-MOF/Ral表面针状结构消失，可观测到颗粒状MOF或MOF/Ral形成，粗糙度升高为131±19nm和122±24nm。上述形貌及粗糙度变化表明，MOF或MOF/Ral涂层在碱腐蚀样本表面被成功制备，且该结论得到了X射线光电子能谱(XPS)和水接触角结果的进一步验证。XPS数据(图3A、B)表明：Ti样本表面有明显的Ti2s、Ti2p3、O1s、OKL1、C1s特征峰；经氢氧化钠热处理后，AT样本表面出现Na1s和NaKL1特征峰；与AT样本相比，LBL表面具有明显的N1s特征峰，这显示壳聚糖/明胶多层膜被成功制备；进一步负载氯化锌后，LBL、LBL-MOF、LBL-MOF/Ral表面可观测到Zn2p1及Zn2p3信号；另外，仅在LBL-MOF/Ral组材料表面检测到了S2p3的特征峰，其可归因于涂层中Ral的有效负载。水接触角(图3C)结果显示，LBL-MOF/Ral的表面较LBL-MOF更加疏水，其从侧面进一步证明Ral改性的MOF涂层已被成功的制备。
实验例2、MOF涂层水稳定性检测
通过检测锌离子评估MOF及MOF/Ral涂层的水稳定性。将LBL-Zn、LBL-MOF和LBL-MOF/Ral样本浸泡于5mL pH7.4的磷酸缓冲液中，不同时间点(0、1、3、5、7和14d)时收取释放液，利用电感耦合等离子体质谱仪对Zn2+的释放量进行表征。
Zn2+释放结果(图4)显示：LBL-Zn组Zn2+释放速率最快，约5d即被释放完全，且释放总量最低(样本制备/冲洗过程中Zn2+损失严重)；LBL-MOF样本在浸泡的前7d均可快速释放Zn2+(释放总量达6.4ppm)，但7d后的释放速率显著降低；与LBL-MOF相比，LBL-MOF/Ral涂层在相同时间点释放的Zn2+总量明显减少，且14d后仍有明显的释放趋势。上述结果表明，LBL-MOF/Ral比LBL-MOF涂层具有更强的水稳定性。高水稳定性可归因于疏水Ral在MOF中的有效掺杂，这进一步验证了LBL-MOF/Ral被成功制备。
实验例3、不同样本表面MC3T3-E1细胞的生物相容性检测
通过检测细胞早期形貌及活性评估不同样本表面MC3T3-E1细胞的生物相容性。为了表征不同组MC3T3-E1细胞(初始接种浓度为2×104个/孔)的早期形貌，培养3d后利用4％多聚甲醛固定细胞。然后，一方面使用鬼笔环肽(细胞骨架染色)及H33258(细胞核染色)染液对MC3T3-E1细胞进行染色，并进行荧光观察；另一方面利用梯度乙醇溶液对上述固定细菌进行脱水处理，最后进行SEM观察。为了表征不同组MC3T3-E1细胞(初始接种浓度为2×104个/孔)的活性，培养3和7d后，向各孔中逐个加入含10％CCK8溶液的新鲜培养基，孵育2h后在450nm波长下测量吸光值。
荧光染色结果(图5A)表明：培养3d后，与Ti组相比，AT、LBL、LBL-Zn表面MC3T3-E1细胞的铺展面积显著降低，但LBL-MOF及LBL-MOF/Ral组细胞的铺展性能未受到显著影响。上述细胞铺展趋势在SEM结果(图5B)中进一步得到验证，且LBL-MOF及LBL-MOF/Ral组细胞具有更多的伪足形成(黑色箭头标注)。细胞活性(图6A)结果显示，与Ti组相比，AT组细胞的活性显著降低，但于其表面制备MOF/Ral涂层后，表面细胞的活性显著升高(尤其在第7天)。上述结果表明，MOF/Ral比纯MOF涂层具有更加优越的生物相容性，更利于表面MC3T3-E1细胞的铺展和增殖。
实验例4、不同样本表面MC3T3-E1细胞的成骨分化水平检测
通过检测矿化及成骨相关基因表达评估不同样本表面MC3T3-E1细胞的成骨分化水平。为了检测MC3T3-E1细胞(初始接种浓度为2×104个/孔)的矿化水平，培养14d后利用4％多聚甲醛固定细胞，然后使用商业化茜素红染液进行染色及定量分析，具体流程可参考详细的试剂盒说明书。为了检测MC3T3-E1细胞(初始接种浓度为2×104个/孔)的成骨相关基因表达，培养7d后利用RNA提取试剂盒收集各组细胞内总RNA，然后利用反转录试剂盒获取单链cDNA，并进一步通过定量PCR试剂盒检测各组Runt相关转录因子2(Runx2)、骨桥素(OPN)、骨钙素(OCN)、骨保护素(OPG)及TNF相关激活诱导细胞因子(RANKL)基因的表达，引物序列如表1所示。
矿化结果(图6B)显示：培养14d后，相较于其它五组细胞，LBL-MOF/Ral组MC3T3-E1细胞的矿化水平显著升高(*p＜0.05)；LBL、LBL-Zn和LBL-MOF/Ral组细胞的矿化水平也显著高于AT组(*p＜0.05)，但三组间无显著性差异。成骨相关基因表达结果(图7)也证明，LBL-MOF/Ral组细胞的成骨相关基因Runx2、OPN及OCN表达量最高。上述结果表明，LBL-MOF/Ral材料可显著地促进成骨细胞成骨分化。另外，基因检测结果还表明：LBL-MOF/Ral组OPG基因的表达水平最高，RANKL基因的表达水平最低，OPG/RANKL比值显著高于其它组(*p＜0.05)。因为OPG和RANKL分别为破骨细胞形成的抑制和激活分子，所以OPG/RANKL常被应用于评估生物材料的抗破骨性能。LBL-MOF/Ral组最高的OPG/RANKL比值证明其可能兼具最优越的抗破骨细胞形成潜能。
实验例5、不同样本表面RAW264.7细胞的破骨分化水平检测
通过检测抗酒石酸酸性磷酸酶(TRAP)活性及破骨相关基因表达评估不同样本表面RAW264.7细胞的破骨分化水平。为了检测RAW264.7细胞(初始接种浓度为1×105个/孔)的TRAP活性，培养5d后利用1％的TritonX-100溶液裂解细胞，然后使用商业化TRAP试剂盒对各组酶活性定量检测，具体流程可参考详细的试剂盒说明书。为了检测RAW264.7细胞(初始接种浓度为1×105个/孔)的破骨相关基因表达，培养5d后利用RNA提取试剂盒收集各组细胞内总RNA，然后利用反转录试剂盒获取单链cDNA，并进一步通过定量PCR试剂盒检测各组TRAP、组织蛋白酶K(CTSK)及核因子κB受体活化因子(RANK)基因的表达，包括基因Runx2(上游引物如SEQ ID NO.1所示，下游引物如SEQ ID NO.2所示)、OPN(上游引物如SEQ IDNO.3所示，下游引物如SEQ ID NO.4所示)、OCN(上游引物如SEQ ID NO.5所示，下游引物如SEQ ID NO.6所示)、OPG(上游引物如SEQ ID NO.7所示，下游引物如SEQ ID NO.8所示)、RANKL(上游引物如SEQ ID NO.9所示，下游引物如SEQ ID NO.10所示)、TRAP(上游引物如SEQ ID NO.11所示，下游引物如SEQ ID NO.12所示)、CTSK(上游引物如SEQ ID NO.13所示，下游引物如SEQ ID NO.14所示)、RANK(上游引物如SEQ ID NO.15所示，下游引物如SEQ IDNO.16所示)、GAPDH(上游引物如SEQ ID NO.17所示，下游引物如SEQ ID NO.18所示)；具体的引物序列如表1所示。
TRAP结果(图8A)显示：培养5d后，LBL-MOF/Ral组RAW264.7细胞的TRAP活性最低，显著低于其它五组(*p＜0.05)；与Ti、AT、LBL、LBL-Zn组相比，LBL-MOF组细胞的TRAP活性也显著降低(*p＜0.05)。基因检测结果(图8B)进一步证实，LBL-MOF/Ral组具有最低的TRAP、CTSK、RANK破骨相关基因表达。上述结果表明，LBL-MOF/Ral具有最优越的抗破骨细胞形成能力。
因此，本研究通过层层自组装及2-甲基咪唑/Ral/甲醇浸泡途径，制备获得目的MOF/Ral涂层，赋予材料优越的涂层水稳定性、促成骨及抗破骨性能，进而解决MOF材料水稳定性差及骨质疏松条件下骨形成能力弱的难题。
最后有必要在此说明的是：以上实施例只用于对本发明的技术方案作进一步详细地说明，不能理解为对本发明保护范围的限制，本领域的技术人员根据本发明的上述内容作出的一些非本质的改进和调整均属于本发明的保护范围。
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Claims (9)
1.一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法，其特征在于，包括以下步骤：
步骤一、利用层层自组装技术于金属基材表面构建壳聚糖/明胶/氯化锌多层膜，金属基材为利用热碱途径处理金属基材，获得纳米针状表面结构金属基材，将纳米针状表面结构金属基材置于1-5mg/mL的多巴胺溶液中浸泡3-12小时；
步骤二、将步骤一制备的金属基材浸泡于2-甲基咪唑/雷洛昔芬/甲醇混合液中，制备获得LBL-MOF/Ral目的材料。
2.根据权利要求1所述的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法，其特征在于：所述金属基材包括钛基材或者钛合金基材。
3.根据权利要求1所述的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法，其特征在于，所述步骤一中热碱途径为利用100-2000目砂纸对金属基材进行表面打磨处理，并依次用乙醇、丙酮和去离子水各洗5-20min，再次，将洁净的金属基材置于50-100℃的1-10M氢氧化钠溶液中处理12-48h；
将步骤一中多巴胺溶液处理后的纳米针状表面结构金属基材用双蒸水冲洗。
4.根据权利要求3所述的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法，其特征在于，所述步骤一中热碱途径为依次利用100、400、1000和2000目砂纸对金属基材进行表面打磨处理，并依次用乙醇、丙酮和去离子水各洗10min；再次，将洁净的金属基材置于80℃的5M氢氧化钠溶液中处理24h；然后，将碱腐蚀样本置于2mg/mL的多巴胺溶液中浸泡6小时，然后用双蒸水冲洗。
5.根据权利要求1-4中任意一项所述的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法，其特征在于，步骤一中的层层自组装技术为将金属基材置于旋涂仪基台上，在500-2000rpm条件下依次涂覆1-10mg/mL的2％醋酸配置的壳聚糖溶液、1-10mg/mL离子水配置的明胶溶液、20-100mg/mL离子水配置的氯化锌溶液，每层涂覆10-30秒，n个循环后获得目的涂层(Chi/Gel/ZnCl2)n，5≤n≤10。
6.根据权利要求5所述的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法，其特征在于，步骤一中的层层自组装技术为将金属基材置于旋涂仪基台上，在1000rpm条件下依次涂覆5mg/mL的2％醋酸配置的壳聚糖溶液、5mg/mL离子水配置的明胶溶液、5mg/mL离子水配置的氯化锌溶液，每层涂覆15秒，n个循环后获得目的涂层(Chi/Gel/ZnCl2)n，
5≤n≤10。
7.根据权利要求5所述的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法，其特征在于，所述步骤二中的2-甲基咪唑/雷洛昔芬/甲醇混合液为20-100mg/mL甲醇配置含2-甲基咪唑与0.1-1mg/mL雷洛昔芬的混合金属基材反应液，将步骤一制备的金属基材浸泡于pH7.4的磷酸缓冲液中，去除涂层中多余醋酸分子；然后再浸泡在2-甲基咪唑/雷洛昔芬/甲醇混合液中浸泡1-6h，经甲醇/去离子水冲洗后获得LBL-MOF/Ral目的材料。
8.根据权利要求7所述的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法，其特征在于，所述步骤二中的2-甲基咪唑/雷洛昔芬/甲醇混合液为45mg/mL甲醇配置含2-甲基咪唑与0.6mg/mL雷洛昔芬的混合反应液，将步骤一制备的金属基材浸泡于pH7.4的磷酸缓冲液中，去除涂层中多余醋酸分子；然后再浸泡在2-甲基咪唑/雷洛昔芬/甲醇混合液中浸泡2h，经甲醇/去离子水冲洗后获得LBL-MOF/Ral目的材料。
9.如权利要求1-8任意一项所述的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料的制备方法制备的一种雷洛昔芬改性MOF涂层介导局部抗骨质疏松性金属基材植入材料。
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