CN116474165A - 一种具有近红外和酶双重响应功能的gmp/gp智能水凝胶骨膜及其制备方法 - Google Patents
一种具有近红外和酶双重响应功能的gmp/gp智能水凝胶骨膜及其制备方法 Download PDFInfo
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- CN116474165A CN116474165A CN202310359956.XA CN202310359956A CN116474165A CN 116474165 A CN116474165 A CN 116474165A CN 202310359956 A CN202310359956 A CN 202310359956A CN 116474165 A CN116474165 A CN 116474165A
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Abstract
本发明公开了一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜及其制备方法。其制备方法包括:1)制备金纳米棒GNR,2)在GNR表面引入带有氨基的MSN层,得到GNR@MSN‑NH2,并在MSN内负载成骨因子;3)将羧基和巯基修饰的聚乙二醇接枝到MSN表面得到GNR@MSN‑PEG‑SH;4)将末端丙烯酸修饰的成血管多肽与GNR@MSN‑PEG‑SH结合得到GMP;5)将甲基丙烯酸明胶溶解于引发剂/PBS溶液,加入聚N‑异丙基丙烯酰胺、GMP分散均匀,浸渍并光固化制得所述骨膜。该智能水凝胶骨膜可通过近红外(NIR)、酶双重响应实现促成血管多肽和促成骨因子的智能控制释放,以满足骨折不同时间段的修复需求,在骨折修复及大段骨缺损同种异体骨整合等领域展现出巨大的应用潜力。
Description
技术领域
本发明属于组织工程材料领域,具体涉及一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜及其制备方法。
背景技术
大段节段性骨缺损的修复一直是骨科面临的重大难题。当缺损超过临界尺寸时,无法凭借人体自身的自愈能力对其实现完全修复。在这种情况下,通常使用自体移植物或同种异体移植物来填充缺损。然而,单纯自体或异体骨移植物在制备过程中放入液氮中或用辐射灭活,导致移植物后骨缺损表面缺乏骨膜,使其对骨的修复效果大大减弱。
骨膜是一种致密的结缔组织膜,覆盖了除关节外的大部分骨骼外表面。骨膜对于预防骨不连和治疗严重骨缺损至关重要,为骨折愈合提供生长因子和募集骨骼干细胞,促进骨愈合和骨周围血管生成,特别是对于骨折部位稳定和血液供应受损的患者更为重要。针对上述骨移植物表面缺乏骨膜的问题,很多研究通过将生物材料与生物活性因子结合或携带细胞构建来构建仿生骨膜,但是现有的大多数仿生骨膜存在生长因子不可控释放,载细胞产生免疫反应等缺陷,导致骨折附近血管畸形或异位骨化。
为此,本发明设计了一种通过近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜。其可结合自然骨折愈合不同阶段中对于生长因子种类、用量的实际需求,精准、按需控制不同生长因子的释放。在骨折早期控制释放促成血管多肽促进血管生成,中期控制释放成骨因子促进成骨。仿生骨膜中的金纳米棒包裹在介孔硅中,介孔硅覆盖有NIR响应可逆开放的PEG阀。PEG尾端用促血管多肽修饰,在体内酶的作用下,成血管多肽脱落,可促进骨折早期血管生成。用近红外激光照射后,GNR产生的光热效应破坏PEG与硅壳之间的静电相互作用,使阀门处于“打开”状态,并从纳米载体中释放成骨因子。PEG末端用促血管多肽修饰,在酶的剪切下成血管多肽序列释放,可促进骨折早期血管生成。GelMA-PNIPAM水凝胶具有热敏性,当外部温度超过低临界溶解温度(LCST)时,疏水收缩状态,水凝胶内包裹的“货物”释放增多,当温度降低到LCST或以下时,亲水溶胀,“货物”释放减少。NIR触发的纳米载体释放的成骨因子可以顺序精准地促进骨折愈合第二阶段骨缺损的成骨。
本发明设计的仿生骨膜具有巨大的临床潜力,可在大面积骨缺损骨折情况下与移植物相结合,动态调节骨折愈合不同阶段所需的成血管和成骨因子,通过成血管促进骨愈合实现两者有机协同,展现出优异的骨折修复及促进大段骨缺损同种异体骨移植物整合的疗效。
发明内容
本发明针对使用传统负载因子的骨组织工程材料治疗大段骨缺损时,其搭载的因子存在不可控释放,导致局部异位骨化、血管畸形和骨缺损修复效果差的问题,设计了一种同时具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜。其作用机理为:第一阶段利用酶响应响应性释放促成血管多肽,促进血管生成,从而促进骨折部位免疫细胞、干细胞募集,抑制炎症反应及为后续成骨提供有利内环境;第二阶段NIR响应控制纳米填料“阀门”开关以及水凝胶温敏效应,实现成骨因子的智能释放,在骨折修复的骨痂期促进骨缺损部位新生骨痂的形成,避免局部纤维机化导致成骨不良,最终导致骨不连。
本发明具体通过以下步骤实现:
一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜的制备方法,包括如下:1)利用种子介导方法生成金纳米棒GNR;2)通过共缩合法在GNR表面引入带有氨基改性的介孔二氧化硅MSN层,得到GNR@MSN-NH2,并结合浸渍工艺在MSN内负载成骨因子;3)通过酰胺化反应将羧基和巯基修饰的聚乙二醇HOOC-PEG-SH接枝到MSN表面得到GNR@MSN-PEG-SH;4)将分子链末端丙烯酸修饰的成血管多肽与GNR@MSN-PEG-SH通过点击反应结合得到GNR@MSN-PEG-peptides即GMP;5)将甲基丙烯酸明胶GelMA溶解于引发剂/PBS溶液中,水浴加热溶解,冷却后加入聚N-异丙基丙烯酰胺PNIPAM、GMP分散均匀,离心脱泡后浇筑,结合循环原位浸渍紫外光固化工艺制得具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜。具体为:
1)GNR的制备:
①金种子溶液的制备:依次在水中加入一定量的四氯金酸(HAuCl4)溶液和十六烷基三甲基溴化铵(CTAB)溶液混合均匀,再将冰硼氢化钠(NaBH4)水溶液加入到上述混合溶液中形成亮棕黄色溶液,该溶液持续反应至得到咖啡色金种子溶液备用。
②生长溶液的制备:首先在轻微搅拌条件下,将CTAB添加到HAuCl4中得到混合溶液;再将硝酸银(AgNO3)、盐酸(HCl)和抗坏血酸依次添加到上述混合溶液中;然后将步骤①中制得的金种子溶液添加到生长溶液中,反应数小时;最后将溶液高速离心洗涤以去除多余的CTAB,并重新分散在水中,得到GNR分散液。
2)向步骤1)中制得的GNR分散液中加入适量氢氧化钠,使用pH计测量pH在合适范围,再分次间隔相同时间加入硅酸四乙酯(TEOS)甲醇溶液、3-氨丙基三乙氧基硅烷(APTES)甲醇溶液,搅拌条件下反应充分以在GNR表面形成具有介孔结构的二氧化硅壳层,即GNR@MSN-NH2;使用甲醇和水在高速离心条件下洗涤以去除残留在GNR@MSN-NH2内的CTAB;将GNR@MSN-NH2分散于含有成骨因子的PBS溶液中,原位浸渍以实现成骨因子的负载,离心洗涤后得到负载促成骨因子的GNR@MSN-NH2纳米粒子;
3)取步骤2)中得到的负载促成骨因子的GNR@MSN-NH2纳米粒子重新分散于PBS溶液中,加入羧基聚乙二醇巯基(HOOC-PEG-SH)、N-羟基丁二酰亚胺(NHS)搅拌实现活化,再加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)搅拌,通过GNR@MSN-NH2的氨基和HOOC-PEG-SH的羧基发生偶联反应。再在高速离心条件下使用甲醇、去离子水洗涤以去除多余的HOOC-PEG-SH、NHS和EDC等杂质,制得GNR@MSN-PEG-SH;
4)将步骤3)中制得的GNR@MSN-PEG-SH重新使用PBS溶液分散,加入含有三乙胺的二甲基亚砜(DMSO)溶液、带有双键的成血管多肽,磁力搅拌条件下GNR@MSN-PEG-SH和带有双键的成血管多肽反应;再在高速离心条件下使用甲醇、去离子水洗涤以去除多余的DMSO、三乙胺和成血管多肽,制得GMP;
5)将GelMA与引发剂依次加入PBS溶液中,水浴加热,冷却后将步骤4)中制得的GMP与PNIPAM加入混匀,离心脱泡后,将待修复骨样品浸泡于所得凝胶溶液中数秒后取出,紫外光固化数秒,循环上述操作数次,最后紫外长时间照射固化,在待修复骨样品表面制成具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜。
上述方案中,进一步地,
步骤1)中所述金种子溶液中HAuCl4、CTAB和NaBH4浓度分别为:10-50mM、0.1-0.5M和0.01-0.1M,用量比为0.1-1mL:5-50mL:0.1-1mL;所述生长溶液中CTAB、HAuCl4、AgNO3、HCl和抗坏血酸的浓度分别为:0.1-1M、5-20mM、0.01-0.1M、1.0-5M、和0.1 -1M,用量比为10-50mL:1-5mL:0.1-1mL:0.1-1mL:0.1-1mL;。种子溶液反应温度为25-35℃,反应时间为2-6小时;生长溶液反应温度为27-35℃,搅拌反应速度为200-500rpm,反应时间为6-10小时;离心洗涤10-15分钟,3-5次,速度10000-15000rpm。
步骤2)中的NaOH、GNR分散液、TEOS甲醇溶液、APTES甲醇溶液和GNR@MS-NH2的PBS溶液浓度分别为0.1-0.5M、0.5-1mM、0.8-1.5mM、8.5-20mM、0.5-1mM,用量比为:0.1-1mL:10-50mL:10-30μL:5-10μL:5-10mL;所述pH范围为10-12;间隔30-60分钟,分3-5次加入TEOS、APTES,反应温度25-35℃,搅拌反应速度为500-750rpm;反应时间为24-48小时;成骨因子为骨成型蛋白(BMP)中的BMP2、BMP4和BMP7模拟肽;原位浸渍时间为24-36小时;离心洗涤10-15分钟,3-5次,速度10000-15000rpm。
步骤3)中的GNRs@MS-NH2分散于PBS溶液中浓度为0.5-1mM;HOOC-PEG-SH分子量为2000-10000,HOOC-PEG-SH、NHS和EDC的纯度均≥98%,GNRs@MS-NH2的PBS溶液、HOOC-PEG-SH、NHS和EDC的用量比为5-10mL:1-10mg:1-10mg:10-20mg;搅拌活化时间30-60分钟,搅拌反应时间24-36小时,搅拌速度500-1500rpm;离心洗涤10-15分钟,3-5次,速度10000-15000rpm。
步骤4)中的GNR@MSN-PEG-SH分散于PBS溶液中浓度为:0.5-1mM;三乙胺、DMSO和带有双键的成血管多肽的纯度均≥98%,成血管多肽为血管内皮生长因子包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E和胎盘生长因子(PGF)模拟肽中的一种或几种,GNR@MSN-PEG-SH的PBS溶液、三乙胺、DMSO和带有双键的成血管多肽用量比为1-5mL:0.01-0.1mg:1-5mL:10-50mg。所述反应时间为24-36小时,搅拌速度为500-750rpm;离心洗涤10-15分钟,3-5次,速度10000-15000rpm。
步骤5)凝胶溶液中GMP、PNIPAM、GelMA和引发剂的浓度分别为:0.2-1wt%、10-15wt%、2.5-5wt%和0.5-1wt%;PNIPAM数均分子量Mn为40000-100000,GelMA取代率为20%-90%;冷却至25-30℃,离心脱泡速度为3000-4000rpm,3-5分钟;所述浸泡时间1-10秒;紫外光波长为350-405nm,紫外光循环固化时间1-10秒,循环操作5-10次,最后紫外光固化10-30分钟。
本发明制备的仿生骨膜具有以下功能:
1)本发明中使用GelMA、PNIPAM和GNR作为原料,运用晶种生长法硬模板法生成GNR@MSN-NH2,通过浸渍工艺在纳米填料孔隙种负载成骨因子、通过酰胺化反应和点击反应在纳米粒子表面接枝具有酶响应切割序列的成血管多肽,制得GMP;同时在同种异体骨表面运用循环浇铸GelMA、PNIPAM和GMP的混合物,结合原位循环紫外光固化组合工艺制得GMP/GP智能水凝胶骨膜,此工艺是本发明的创新。通过上述组合工艺,制得具有多级核壳结构的GMP:以金纳米棒为核心,核外具有介孔硅包壳,壳外包裹NIR和酶响应的PEG-多肽链(阀门)。智能凝胶的结构为多孔疏松的生物“海绵”纳米复合结构;凝胶在NIR照射时凝胶内GMP的存在使凝胶温度升高,当温度控制在LCST以上时,水凝胶像“海绵”被挤压一样疏水收缩,多孔疏松结构变致密,将生物“海绵”内搭载的因子挤出来,制成一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜,此纳米复合结构是本发明的创新。
2)本发明中GelMA具有良好的生物相容性,理化性质稳定,PNIPAM具有优异的热敏性,GNR具有近红外响应的光热效应,GNR@MSN-PEG-SH搭载的成骨因子具有近红外响应释放的功能,成血管多肽具有酶响应切割的序列,它们的组合水凝胶可以实现NIR触发多肽和因子的智能控释,并且使成血管多肽和成骨因子对于骨缺损修复过程发挥协同增效功能。
所述的仿生骨膜具有NIR和酶双重响应促进成骨作用:当仿生骨膜植入到骨缺损部位时,GNR@MSN表面的PEG阀另一端搭载的成血管多肽具有酶响应,骨折缺损部位发生血肿机化,周围的脂肪和肌肉细胞产生大量的基质金属蛋白酶以降解细胞外基质,参与细胞的分解与重组。在这种酶的存在下,多肽中的特定成血管序列被剪切下来,促进血管生成。对应解决了骨折修复第一阶段,血肿机化期血管重塑不佳,局部炎症细胞浸润产生炎症反应,在此阶段按需释放成血管多肽,有助于早期建立血运,募集干细胞及免疫细胞参与免疫微环境的调节。另外脱落的多肽序列包裹在水凝胶内,水凝胶缓慢且持续向周围释放成血管多肽,骨折修复全阶段中都需要适量促成血管因子的参与,为此本发明设计成血管多肽在整个骨折修复全阶段中缓慢释放,避免局部成血管作用太强,导致血管畸形;
然后,GNR可以在NIR的照射下产生热效应,使包裹GNR@MSN的PEG阀门打开,从而使介孔硅中吸附的成骨因子迅速释放,对应解决了骨折修复的第二阶段骨痂期需要大量新生骨痂生成,填补缺损部位,传统同种异体骨移植缺乏生长因子,极少有新骨痂形成,然而骨组织工程移植物搭载的成骨因子未进行控释,短期一过性成骨因子突释对于新生骨痂形成作用甚微,为此我们设计的NIR响应释放成骨因子,NIR控释系统可以在照射的骨缺损特定区域释放成骨因子,并且在骨痂期内特定所需的成骨因子给予精确的的释放,于第一阶段的血肿机化期,成骨因子的大量存在反而影响早期血管形成及募集免疫细胞参与免疫调节,破坏局部微环境,从而影响骨缺损的早期愈合。NIR控释系统在NIR不照射情况下载体释放的因子量几乎可忽略不计,优化了传统骨组织工程搭载的因子难以控制具体缓释时间的问题。NIR选择性的在骨折愈合第二阶段开始时照射,载体释放成骨因子,以促进骨缺损的愈合。当没有NIR照射时,PEG阀门可以可逆的关闭,介孔硅内的成骨因子停止释放,储存在介孔硅内的少量的成骨因子可以在成骨周期内可控释放,达到脉冲释放的效果,实现成骨因子促新生骨痂效率最大化。纳米载体可以高负载量和高效率地封装药物,同时减少副作用。文献报道中存在很多刺激触发纳米载体,它们可以有效地调节负载药物的释放以响应给定的刺激,例如pH变化、温度变化、氧化还原活化、竞争结合和光辐照等。在这些方法中,光触发药物递送不依赖于环境特定化学性质的变化。已经设计了多种基于紫外光或可见光激发的光响应纳米载体,以释放包埋的货物分子,这可能对生物样品造成损害,并且由于其在组织中的快速衰减而仅适用于体外研究。NIR触发纳米载体由于对组织和器官的穿透力最大且损伤最小,为提高药物输送效率带来了新的机会。并且,包裹纳米载体的温敏水凝胶也会对NIR同时进行响应:当NIR照射时GMP释放因子存于凝胶的多孔内,同时GMP产热致水凝胶温度升高;当温度超过LCST时,多孔疏松生物“海绵”从亲水溶胀转变为疏水收缩,“海绵”孔内的因子被“挤”出释放至周围组织,当NIR关闭,载体停止释放因子,同时温度降低到LCST或以下,凝胶又恢复到原来的亲水状态,以达到协同精准促进成骨因子释放的作用。
本发明中的复合水凝胶可以实现在骨折修复的早期持续释放成血管多肽促进血管生成,骨折部位血管生成增多有利于骨折部位血供的恢复,募集周围的干细胞参与骨缺损修复,近红外触发成骨因子在骨修复中期成骨阶段精准释放,在成骨因子的刺激下促进软骨内成骨,促进大段骨缺损处同种异体骨的整合。成骨因子在和成血管多肽的协同作用下,相比于单独应用成血管或成骨因子,其促进骨修复的成功率明显提高。
3)本发明创新性地仿生水凝胶骨膜以NIR和酶双重响应促进成血管和成骨功能协同作用,有望应用于组织工程中大段骨缺损修复等领域。所述仿生水凝胶骨膜的功能是NIR和酶协同响应发挥作用:当PEG尾端搭载的成血管多肽被酶剪切,成血管多肽序列释放到水凝胶中,NIR不存在的情况下,缓慢持续的释放到周围组织中,促进成血管,NIR照射时GNR热效应使MSN内成骨因子释放到水凝胶中,同时水凝胶的热敏性协同促使水凝胶中的成骨因子释放到周围组织,从而加快成骨因子和成血管多肽协同释放的效率。凭借NIR和酶的共同响应,对大段骨缺损后同种异体骨移植后的整合具有良好的效果,早期促进骨折周围成血管,晚期促进成骨,模拟生理条件下的骨折愈合阶段中生长因子作用的顺序,更大程度上提高了大段骨缺损愈合的机率;
附图说明
图1本发明制备的GNR、GNR@MSN和GNR@MSN-PEG-peptides的粒径测试结果图;
图2本发明制备的水凝胶材料在高于低临界溶解温度(LCST)时疏水收缩示意图。
图3本发明制备的在同种异体骨表面的具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜实物图;
图4本发明制备的GMP/GP智能水凝胶骨膜在体外细胞实验中的促成血管结果图;
图5本发明制备的GMP/GP智能水凝胶骨膜在体外细胞实验中的促成骨结果图(体外成骨茜素红染色)。
具体实施方式
以下结合实例进一步说明本发明。
实施例1:
1)GNR的制备:
①金种子溶液的制备:
依次在水中加入HAuCl4(10mM 0.5mL)溶液和CTAB(0.1M 20mL)溶液混合均匀,再将冰NaBH4(0.01M 1.2mL)水溶液加入到上述混合溶液中形成亮棕黄色溶液,该溶液在25℃下以500rpm搅拌持续反应至少2小时得到咖啡色金种子溶液备用。
②生长溶液的制备:首先在500rpm搅拌下,将CTAB(0.1M 80mL)添加到HAuCl4(10mM 4mL)中;再将AgNO3(0.01M 640μL)、HCl(1.0M 1.6mL)和抗坏血酸(0.1M,0.64mL)依次添加到上述混合溶液中;然后将步骤①中制得的50μL种子溶液添加到生长溶液中,27℃下静置反应6小时;最后将溶液以10000rpm离心洗涤3次以去除多余的CTAB,并重新分散在100mL水中,得到GNR。
2)向步骤1)中制得的30mL GNR分散液中加入氢氧化钠(0.1M 300μL),使用pH计测量pH为10、每隔30分钟加入一次,共三次,加入TEOS(0.8mM45μL)甲醇溶液、APTES(8.5mM 15μL)甲醇溶液。在500rpm搅拌条件下反应24小时,以在GNR表面形成具有介孔结构的二氧化硅壳层,即GNR@MSN-NH2;使用甲醇和水在10000rpm离心条件下洗涤3次以去除残留在GNR@MSN-NH2内的CTAB;将GNR@MSN-NH2分散于BMP2模拟肽/PBS(400μM)溶液中,原位浸渍以实现BMP2模拟肽的负载,10000rpm离心洗涤3次后得到负载BMP2模拟肽的GNR@MSN-NH2纳米粒子;
3)取步骤2)中得到的负载BMP2模拟肽的1mg GNR@MSN-NH2纳米粒子重新分散于5mLPBS中,加入2mg HOOC-PEG-SH(Mn 2000)、5mg NHS以500rpm速度搅拌30分钟实现活化,再加入10mg EDC以1000rpm搅拌,通过GNR@MSN-NH2的氨基和HOOC-PEG-SH的羧基发生偶联反应24小时。再在10000rpm离心条件下使用甲醇、去离子水洗涤3次以去除多余的HOOC-PEG-SH、NHS和EDC等杂质,制得GNR@MSN-PEG-SH;
4)依次将步骤3)中制得的2mg GNR@MSN-PEG-SH重新使用2mL PBS分散,加入含有三乙胺的DMSO(50μM 2mL)溶液、10mg VEGF-A模拟肽,750rpm速度搅拌反应条件下GNR@MSN-PEG-SH和VEGF-A模拟肽反应24小时;再在10000rpm离心条件下使用甲醇、去离子水洗涤3次以去除多余的DMSO、三乙胺和VEGF-A模拟肽,制得GMP。
5)将取代率20%的GelMA(2.5wt%)与LAP引发剂(0.5wt%)依次加入5mL PBS溶液中,水浴加热15分钟,冷却至25℃后将步骤4)中制得的GMP(0.2wt%)与PNIPAM(Mn4000010wt%)加入混匀,2000rpm离心脱泡后,将同种异体骨浸泡于凝胶溶液中2秒后取出,405nm紫外光固化1秒,循环上述操作5次,最后405nm紫外长时间照射10分钟固化,在同种异体骨表面制成GMP/GP智能水凝胶骨膜。
6)所制得的水凝胶在第一个时间点的释放“货物”率为6.5±2.00%。该值持续缓慢增加,最终在最后一个时间点达到20.00±1%;结合茜素红染色和成管实验可知,该水凝胶可以促进血管生成,在NIR照射后具有良好促成骨作用。
实施例2:
1)GNR的制备:
①金种子溶液的制备:
依次在水中加入HAuCl4(5mM 1mL)溶液和CTAB(0.05M 40mL)溶液混合均匀,再将冰NaBH4(0.02M 0.6mL)水溶液加入到上述混合溶液中形成亮棕黄色溶液,该溶液在25℃下以400rpm搅拌持续反应至少2小时得到咖啡色金种子溶液备用。
②生长溶液的制备:首先在400rpm搅拌下,将CTAB(0.05M 160mL)添加到HAuCl4(5mM 8mL)中;再将AgNO3(0.02M 320μL)、HCl(0.5M 3.2mL)和抗坏血酸(0.2M,0.32mL)依次添加到上述混合溶液中;然后将步骤①中制得的25μL种子溶液添加到生长溶液中,27℃下静置反应8小时;最后将溶液以11000rpm离心洗涤3次以去除多余的CTAB,并重新分散在100mL水中,得到GNR。
2)向步骤1)中制得的15mL GNR分散液中加入氢氧化钠(0.1M 150μL),使用pH计测量pH为11、每隔45分钟加入一次,共4次,加入TEOS(1mM 25μL)甲醇溶液、APTES(15mM 7.5μL)甲醇溶液。在600rpm搅拌条件下反应36小时,以在GNR表面形成具有介孔结构的二氧化硅壳层,即GNR@MSN-NH2;使用甲醇和水在11000rpm离心条件下洗涤2次以去除残留在GNR@MSN-NH2内的CTAB;将GNR@MSN-NH2分散于BMP4模拟肽/PBS(500μM)溶液中,原位浸渍以实现BMP4模拟肽的负载,11000rpm离心洗涤2次后得到负载BMP4模拟肽的GNR@MSN-NH2纳米粒子;
3)取步骤2)中得到的负载BMP2模拟肽的2mg GNR@MSN-NH2纳米粒子重新分散于7.5mL PBS中,加入4mg HOOC-PEG-SH(Mn 4000)、10mg NHS以750rpm速度搅拌45分钟实现活化,再加入20mg EDC以750rpm搅拌,通过GNR@MSN-NH2的氨基和HOOC-PEG-SH的羧基发生偶联反应36小时。再在11000rpm离心条件下使用甲醇、去离子水洗涤2次以去除多余的HOOC-PEG-SH、NHS和EDC等杂质,制得GNR@MSN-PEG-SH;
4)依次将步骤3)中制得的4mg GNR@MSN-PEG-SH重新使用4mL PBS分散,加入含有三乙胺的DMSO(75μM 1mL)溶液、15mg VEGF-B模拟肽,500rpm速度搅拌反应条件下GNR@MSN-PEG-SH和VEGF-B模拟肽反应36小时;再在11000rpm离心条件下使用甲醇、去离子水洗涤2次以去除多余的DMSO、三乙胺和VEGF-B模拟肽,制得GMP。
5)将取代率40%的GelMA(5wt%)与LAP引发剂(1wt%)依次加入10mL PBS溶液中,水浴加热30分钟,冷却至30℃后将步骤4)中制得的GMP(0.5wt%)与PNIPAM(Mn500005wt%)加入混匀,2000rpm离心脱泡后,将同种异体骨浸泡于凝胶溶液中5秒后取出,405nm紫外光固化3秒,循环上述操作10次,最后405nm紫外长时间照射15分钟固化,在同种异体骨表面制成GMP/GP智能水凝胶骨膜。
6)所制得的水凝胶在第一个时间点的释放“货物”率为5.0±2.00%。该值持续缓慢增加,最终在最后一个时间点达到15.00±1%;结合茜素红染色和成管实验可知,该水凝胶可以促进血管生成,在NIR照射后具有良好促成骨作用。
实施例3:
1)GNR的制备:
①金种子溶液的制备:
依次在水中加入HAuCl4(7.5mM 0.5mL)溶液和CTAB(0.075M 60mL)溶液混合均匀,再将冰NaBH4(0.06M 0.3mL)水溶液加入到上述混合溶液中形成亮棕黄色溶液,该溶液在27℃下以600rpm搅拌持续反应至少8小时得到咖啡色金种子溶液备用。
②生长溶液的制备:首先在600rpm搅拌下,将CTAB(0.15M 125mL)添加到HAuCl4(10mM 5mL)中;再将AgNO3(0.05M 150μL)、HCl(1.0M 1.6mL)和抗坏血酸(0.5M,0.1mL)依次添加到上述混合溶液中;然后将步骤①中制得的100μL种子溶液添加到生长溶液中,25℃下静置反应12小时;最后将溶液以12000rpm离心洗涤3次以去除多余的CTAB,并重新分散在50mL水中,得到GNR。
2)向步骤1)中制得的20mL GNR分散液中加入氢氧化钠(0.1M 200μL),使用pH计测量pH为12、每隔45分钟加入一次,共3次,加入TEOS(1.5mM15μL)甲醇溶液、APTES(15mM 7.5μL)甲醇溶液。在750rpm搅拌条件下反应48小时,以在GNR表面形成具有介孔结构的二氧化硅壳层,即GNR@MSN-NH2;使用甲醇和水在12000rpm离心条件下洗涤4次以去除残留在GNR@MSN-NH2内的CTAB;将GNR@MSN-NH2分散于BMP2模拟肽/PBS(600μM)溶液中,原位浸渍以实现BMP2模拟肽的负载,12000rpm离心洗涤4次后得到负载BMP2模拟肽的GNR@MSN-NH2纳米粒子;
3)取步骤2)中得到的负载BMP2模拟肽的4mg GNR@MSN-NH2纳米粒子重新分散于20mL PBS中,加入5mg HOOC-PEG-SH(Mn 2000)、15mg NHS以750rpm速度搅拌60分钟实现活化,再加入15mg EDC以1500rpm搅拌,通过GNR@MSN-NH2的氨基和HOOC-PEG-SH的羧基发生偶联反应36小时。再在12000rpm离心条件下使用甲醇、去离子水洗涤4次以去除多余的HOOC-PEG-SH、NHS和EDC等杂质,制得GNR@MSN-PEG-SH;
4)依次将步骤3)中制得的5mg GNR@MSN-PEG-SH重新使用10mL PBS分散,加入含有三乙胺的DMSO(100μM 1mL)溶液、15mg VEGF-E模拟肽,1000rpm速度搅拌反应条件下GNR@MSN-PEG-SH和VEGF-E模拟肽反应48小时;再在12000rpm离心条件下使用甲醇、去离子水洗涤4次以去除多余的DMSO、三乙胺和VEGF-E模拟肽,制得GMP。
5)将取代率50%的GelMA(7.5wt%)与LAP引发剂(0.75wt%)依次加入7.5mL PBS溶液中,水浴加热20分钟,冷却至27℃后将步骤4)中制得的GMP(0.75wt%)与PNIPAM(Mn100000 7.5wt%)加入混匀,2500rpm离心脱泡后,将同种异体骨浸泡于凝胶溶液中3秒后取出,405nm紫外光固化2秒,循环上述操作5次,最后405nm紫外长时间照射15分钟固化,在同种异体骨表面制成GMP/GP智能水凝胶骨膜。
6)所制得的水凝胶在第一个时间点的释放“货物”率为3.5±1.50%。该值持续缓慢增加,最终在最后一个时间点达到10.00±1.5%;结合茜素红染色和成管实验可知,该水凝胶可以促进血管生成,在NIR照射后具有良好促成骨作用。
Claims (9)
1.一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜的制备方法,其特征在于:包括如下步骤:
1)利用种子介导方法生成金纳米棒GNR;2)通过共缩合法在GNR表面引入带有氨基改性的介孔二氧化硅MSN层,得到GNR@MSN-NH2,并结合浸渍工艺在MSN内负载成骨因子;3)通过酰胺化反应将羧基和巯基修饰的聚乙二醇HOOC-PEG-SH接枝到MSN表面得到GNR@MSN-PEG-SH;4)将分子链末端丙烯酸修饰的成血管多肽与GNR@MSN-PEG-SH通过点击反应结合得到GNR@MSN-PEG-peptides即GMP;5)将甲基丙烯酸明胶GelMA溶解于引发剂/PBS溶液中,水浴加热溶解,冷却后加入聚N-异丙基丙烯酰胺PNIPAM、GMP分散均匀,离心脱泡后浇筑,结合循环原位浸渍紫外光固化工艺制得具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜。
2.如权利要求1所述的一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜的制备方法,其特征在于:具体步骤如下:
1)GNR的制备:
①金种子溶液的制备:依次在水中加入一定量的四氯金酸(HAuCl4)溶液和十六烷基三甲基溴化铵(CTAB)溶液混合均匀,再将冰硼氢化钠(NaBH4)水溶液加入到上述混合溶液中形成亮棕黄色溶液,该溶液持续反应至得到咖啡色金种子溶液备用;
②生长溶液的制备:首先在轻微搅拌条件下,将CTAB添加到HAuCl4中得到混合溶液;再将硝酸银(AgNO3)、盐酸(HCl)和抗坏血酸依次添加到上述混合溶液中;然后将步骤①中制得的金种子溶液添加到生长溶液中,反应数小时;最后将溶液高速离心洗涤以去除多余的CTAB,并重新分散在水中,得到GNR分散液;
2)向步骤1)中制得的GNR分散液中加入适量氢氧化钠,使用pH计测量pH在合适范围,再分次间隔相同时间加入硅酸四乙酯(TEOS)甲醇溶液、3-氨丙基三乙氧基硅烷(APTES)甲醇溶液,搅拌条件下反应充分以在GNR表面形成具有介孔结构的二氧化硅壳层,即GNR@MSN-NH2;使用甲醇和水在高速离心条件下洗涤以去除残留在GNR@MSN-NH2内的CTAB;将GNR@MSN-NH2分散于含有成骨因子的PBS溶液中,原位浸渍以实现成骨因子的负载,离心洗涤后得到负载促成骨因子的GNR@MSN-NH2纳米粒子;
3)取步骤2)中得到的负载促成骨因子的GNR@MSN-NH2纳米粒子重新分散于PBS溶液中,加入羧基聚乙二醇巯基(HOOC-PEG-SH)、N-羟基丁二酰亚胺(NHS)搅拌实现活化,再加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)搅拌,通过GNR@MSN-NH2的氨基和HOOC-PEG-SH的羧基发生偶联反应,再在高速离心条件下使用甲醇、去离子水洗涤以去除多余的HOOC-PEG-SH、NHS和EDC,制得GNR@MSN-PEG-SH;
4)将步骤3)中制得的GNR@MSN-PEG-SH重新使用PBS溶液分散,加入含有三乙胺的二甲基亚砜(DMSO)溶液、带有双键的成血管多肽,磁力搅拌条件下GNR@MSN-PEG-SH和带有双键的成血管多肽反应;再在高速离心条件下使用甲醇、去离子水洗涤以去除多余的DMSO、三乙胺和成血管多肽,制得GMP;
5)将GelMA与引发剂依次加入PBS溶液中,水浴加热,冷却后将步骤4)中制得的GMP与PNIPAM加入混匀,离心脱泡后,将待修复骨样品浸泡于所得凝胶溶液中数秒后取出,紫外光固化数秒,循环上述操作数次,最后紫外长时间照射固化,在待修复骨样品表面制成具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜。
3.如权利要求2所述的一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜的制备方法,其特征在于:步骤1)所述金种子溶液中HAuCl4、CTAB和NaBH4浓度分别为:10-50mM、0.1-0.5M和0.01-0.1M,用量比为0.1-1mL:5-50mL:0.1-1mL;所述生长溶液中CTAB、HAuCl4、AgNO3、HCl和抗坏血酸的浓度分别为:0.1-1M、5-20mM、0.01-0.1M、1.0-5M、和0.1-1M,用量比为10-50mL:1-5mL:0.1-1mL:0.1-1mL:0.1-1mL;种子溶液反应温度为25-35℃,反应时间为2-6小时;生长溶液反应温度为27-35℃,搅拌反应速度为200-500rpm,反应时间为6-10小时;离心洗涤10-15分钟,3-5次,速度10000-15000rpm。
4.如权利要求2所述的一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜的制备方法,其特征在于:步骤2)中的NaOH、GNR分散液、TEOS甲醇溶液、APTES甲醇溶液和GNR@MS-NH2的PBS溶液浓度分别为0.1-0.5M、0.5-1mM、0.8-1.5mM、8.5-20mM、0.5-1mM,用量比为:0.1-1mL:10-50mL:10-30μL:5-10μL:5-10mL;所述pH范围为10-12;间隔30-60分钟,分3-5次加入TEOS、APTES,所述反应温度25-35℃,搅拌反应速度为500-750rpm;反应时间为24-48小时;所述成骨因子为骨成型蛋白(BMP)中的BMP2、BMP4和BMP7模拟肽;原位浸渍时间为24-36小时;离心洗涤10-15分钟,3-5次,速度10000-15000rpm。
5.如权利要求2所述的一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜的制备方法,其特征在于:
步骤3)中的GNRs@MS-NH2分散于PBS溶液中浓度为0.5-1mM;HOOC-PEG-SH分子量为2000-10000,HOOC-PEG-SH、NHS和EDC的纯度均≥98%,GNRs@MS-NH2的PBS溶液、HOOC-PEG-SH、NHS和EDC的用量比为5-10mL:1-10mg:1-10mg:10-20mg;搅拌活化时间30-60分钟,搅拌反应时间24-36小时,搅拌速度500-1500rpm;离心洗涤10-15分钟,3-5次,速度10000-15000rpm。
6.如权利要求2所述的一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜的制备方法,其特征在于:步骤4)中的GNR@MSN-PEG-SH分散于PBS溶液中浓度为0.5-1mM;三乙胺、DMSO和带有双键的成血管多肽的纯度均≥98%,成血管多肽为血管内皮生长因子包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E和胎盘生长因子(PGF)模拟肽中的一种或几种,GNR@MSN-PEG-SH的PBS溶液、三乙胺、DMSO和带有双键的成血管多肽用量比为1-5mL:0.01-0.1mg:1-5mL:10-50mg;所述反应时间为24-36小时,搅拌速度500-1000rpm;离心洗涤10-15分钟,3-5次,速度10000-15000rpm。
7.如权利要求2所述的一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜的制备方法,其特征在于:步骤5)中凝胶溶液中GMP、PNIPAM、GelMA和引发剂的浓度分别为:0.2-1wt%、10-15wt%、2.5-5wt%和0.5-1wt%;PNIPAM数均分子量Mn为40000-100000,GelMA取代率为20%-90%;冷却至25-30℃,离心脱泡速度为3000-4000rpm,3-5分钟;所述浸泡时间1-10秒;紫外光波长为350-405nm,紫外光循环固化时间1-10秒,循环操作5-10次,最后紫外光固化10-30分钟。
8.一种具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜,其特征在于,采用如权利要求1-7任一项所述的方法制备得到。
9.根据权利要求8所述的具有近红外和酶双重响应功能的GMP/GP智能水凝胶骨膜,其特征在于,所述水凝胶为多孔疏松的生物“海绵”纳米复合结构,水凝胶内均匀搭载的纳米复合材料结构为多级核壳结构,壳外包裹可控PEG-多肽链形成阀门,具有近红外和酶双重响应功能,在酶的剪切下释放成血管多肽序列,可促进骨折早期血管生成,用近红外激光照射后,则阀门处于“打开”状态,释放成骨因子。
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