CN112516330A - 丝素蛋白与甲状旁腺激素偶联接枝的方法及其应用 - Google Patents
丝素蛋白与甲状旁腺激素偶联接枝的方法及其应用 Download PDFInfo
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Abstract
本发明涉及医药领域,特别是涉及丝素蛋白与甲状旁腺激素偶联接枝的方法及其应用,所述方法包括将丝素蛋白在交联剂的情况下与甲状旁腺激素偶联接枝,获得的产物水凝胶可作为3D生物打印的水凝胶材料。该制备方法操作简单,切实可行,控释时间和浓度较以往方法更长更有效。本发明的水凝胶3D生物打印成多孔生物支架后植入体内可进一步提高骨软骨损伤的修复效果,具有重要的临床意义和巨大的社会经济价值。
Description
技术领域
本发明涉及医药领域,特别是涉及丝素蛋白与甲状旁腺激素偶联接枝的方法及其应用。
背景技术
凝胶是介于固体与液体之间的一种物理状态,具有独特的性质,尤其是水凝胶,性质柔软,能保持一定的形状,在其中保持有大量的水份,由于聚合物链间的物理交联和化学交联作用而不会溶解于水中,只能溶涨且保持一定的形状。水凝胶具有良好的生物相容性、水渗透性,目前被广泛用于人工皮肤、组织工程材料、药物释放、人造肌肉、生物传感器、分离装置、重金属离子回收等方面。
丝素蛋白(Silk fibroin)是一种理想的水凝胶材料,不但具有优异的力学性能,还有很好的生物相容性,同时具有可吸收降解的特质。丝素蛋白无毒、无刺激性,具有良好的生物相容性,能够促进人体细胞的生长,具有一定的生物可降解性。因此,丝素蛋白是制造生物医学材料的较理想原料。丝素蛋白水凝胶可由丝素蛋白溶液制得。
重组人甲状旁腺激素PTH可抑制骨髓间充质干细胞软骨细胞的肥大和钙化,目前其用于骨软骨损伤治疗。可见的方法是关节腔内注射或者用乳酸/乙醇酸共聚物做成微球包裹甲状旁腺激素后控制其释放,这些方法主要存在的问题是:因甲状旁腺激素体内半衰期太短(1h)以及乳酸/乙醇酸共聚物微球降解太快,从而导致其有效释放时间只能维持1周左右,无法达到缓慢控释的效果用于骨软骨损伤的治疗。目前尚未见有丝素蛋白与PTH化学接枝方法的报道。
发明内容
鉴于以上所述现有技术的缺点,本发明的目的在于提供丝素蛋白与甲状旁腺激素偶联接枝的方法及其应用,用于解决现有技术中的问题。
为实现上述目的及其他相关目的,本发明首先提供丝素蛋白与甲状旁腺激素偶联接枝的方法,所述方法包括以下步骤:
1)将丝素蛋白与交联剂混合;
2)步骤1)反应结束后,将Cys-PTH加入上述反应产物中,反应结束后即获得水凝胶。
本发明还提供一种水凝胶,所述水凝胶通过前述方法制备获得。
本发明还提供所述水凝胶在制备生物墨水中的用途。
本发明还提供一种生物墨水,所述生物墨水包括所述水凝胶与甲基丙烯酸酐化明胶。
本发明还提供所述水凝胶在制备骨软骨修复产品中的用途。
本发明还提供一种耗材,所述耗材为生物支架,所述生物支架为利用所述生物墨水通过3D生物打印得到的。
如上所述,本发明的一种丝素蛋白与甲状旁腺激素偶联接枝的方法及其应用,具有以下有益效果:本发明能够实现丝素蛋白与甲状旁腺激素的偶联接枝,通过Sulfo-SMCC双异官能团作为交联剂,可作为3D生物打印的水凝胶材料。3D生物打印成多孔生物支架后植入体内,用于抑制骨髓间充质干细胞软骨细胞的终末分化,比如肥大和钙化,维持关节透明软骨表型,以更好的提高骨软骨损伤的修复质量。
该方法操作简单,切实可行,控释时间和浓度较以往方法更长更有效。如果本发明能够在临床上大量应用,将会进一步提高骨软骨损伤的修复效果,具有重要的临床意义和巨大的社会经济价值。
附图说明
图1显示为本发明的丝素蛋白与甲状旁腺激素偶联接枝的反应原理图。
图2显示为甲状旁腺激素与丝素蛋白水凝胶的接枝结果验证。
图3显示为本发明的生物墨水的表征结果图。
图4显示为本发明的生物支架体内植入与评价结果。
图5显示为本发明的生物支架内甲状旁腺激素体外累积释放结果。
具体实施方式
本发明提供一种丝素蛋白与甲状旁腺激素偶联接枝的方法,所述方法包括以下步骤:
1)将丝素蛋白与交联剂混合;
2)步骤1)反应结束后,将Cys-PTH加入上述反应产物中,反应结束后即获得水凝胶。
所述丝素蛋白选自天然蛋白。所述丝素蛋白由重链、轻链及糖蛋白P25组成,分子比为重链:轻链:P25=6:6:1。其中重链约5112个氨基酸残基,分子量约为300~350kD,轻链约244个氨基酸残基,分子量约为25kD,P25包括203个氨基酸残基和3个寡糖链,分子量约为23kD,丝素蛋白的分子量约为2286kD。重链和轻链由各自C末端的二硫键相互连接,形成重链-轻链复合体,P25糖蛋白以非共价键的相互作用加入重链-轻链复合体中构成丝素蛋白的基本单位。丝素中存在两种区域,一种是由侧链小的氨基酸排列成的紧密、整齐、有序的结晶区,另一种是由侧链较大的氨基酸排列成的疏松、散乱、无序的非结晶区。结晶区主要由甘氨酸、丙氨酸和丝氨酸的残基组成,非结晶区主要由甘氨酸、丙氨酸以外的残基组成。所述丝素蛋白上的游离氨基可与交联剂发生反应。所述丝素蛋白可从商业化渠道购买得到,例如从苏州丝美特生物技术有限公司的可溶性冻干丝素蛋白。
步骤1)中,所述交联剂的投入量为使其足量反应的量。在一种实施方式中,所述丝素蛋白与交联剂的质量比为100:0.5~100:2。较佳的,质量比为100:1。
步骤1)中,所述交联剂选自水溶性的双官能团的交联剂。
在一种实施方式中,所述交联剂为Sulfo-SMCC。所述Sulfo-SMCC是指4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐,是一种常见且成熟的水溶性含有双异官能团的交联剂。Sulfo-SMCC一端的N-羟基琥珀酰亚胺(NHS)活性酯可与丝素蛋白的伯胺在pH 7-9的环境形成酰胺键。其另外一端的马来酰亚胺与巯基在pH 6.5-7.5的环境下可形成稳定的硫醚键。
在一种实施方式中,步骤1)的反应pH为7.5~8.5。
在一种实施方式中,步骤1)的反应温度为1~10℃。
在一种实施方式中,步骤1)的反应时间为1.5~2.5h。
所述Cys-PTH中的“PTH”是指甲状旁腺激素,氨基端为其生物活性部位,完整的人PTH为一单链蛋白质,含84个氨基酸残基,分子量9500Da。循环血中的PTH具有显著的不均一性,包括多种PTH肽段,如PTH(1-84)、PTH(1-34)或PTH(1-27),即含有第1~84个氨基酸残基片段、第1~34个氨基酸残基片段或第1~27个氨基酸残基片段的肽段。在一种实施方式中,所述甲状旁腺激素选自PTH(1-34)或PTH(1-84)。优选的,选自PTH(1-34)。PTH(1-34)即NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-COOH。
所述Cys-PTH是指连接含巯基的半胱氨酸(Cys)的甲状旁腺激素;较佳的,是指在甲状旁腺激素的羧基端连接有一个含巯基的半胱氨酸,即NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Cys-COOH。
在一种实施方式中,所述甲状旁腺激素选自重组甲状旁腺激素。所述甲状旁腺激素选自哺乳动物甲状旁腺激素;在一种实施方式中,所述哺乳动物甲状旁腺激素为人甲状旁腺激素。
在一种实施方式中,步骤2)中Cys-PTH的量与步骤1)中丝素蛋白的质量比为1:100~1:5000。
在一种实施方式中,步骤2)的反应pH为6.5~7.5。
在一种实施方式中,步骤2)的反应温度为1~10℃。
在一种实施方式中,步骤2)的反应时间为1.5~2.5h。
一般的,步骤1)和2)反应结束后均需要去除未反应的反应物和其他副产物。在一种实施方式中,通过透析去除未反应的交联剂和其他副产物。
在一种实施方式中,将步骤2)获得的水凝胶冷冻干燥,冻干粉末可作为3D生物打印的材料。
在一种实施方式中,所述水凝胶的制备方法原理如图1所示。
本发明还提供一种水凝胶,所述水凝胶通过前述方法制备获得。
所述水凝胶为结合了甲状旁腺激素PTH(1-34)和丝素蛋白的优势,制成的偶联接枝PTH的改性丝素蛋白水凝胶。
本发明还提供所述水凝胶在制备生物墨水中的用途。
所述生物墨水是指用于3D生物打印的墨水。
本发明还提供一种生物墨水,所述生物墨水为通过将所述水凝胶与甲基丙烯酸酐化明胶、光引发剂反应后,再与细胞混合后制备获得。
在一种实施方式中,首先将所述冷冻干燥的水凝胶溶解后,再与甲基丙烯酸酐化明胶和光引发剂混合反应。
甲基丙烯酸酐化明胶(GelMA)是一种光敏性的生物材料,配合光引发剂使用时能在蓝光或紫外光下迅速交联固化,形成具有一定强度的三维结构。甲基丙烯酸酐化明胶可通过购买获得。
所述生物墨水中水凝胶与甲基丙烯酸酐化明胶的重量比为1:1~0.1:1。
所述光引发剂选自水溶性的双官能团交联剂。例如Sulfo-SMCC。
在一种更佳实施方式中,所述细胞选自骨髓间充质干细胞和关节软骨细胞。
本发明还提供所述水凝胶在制备骨软骨修复产品中的用途。
所述骨软骨修复产品是指可用于修复骨软骨损伤的产品。
所述产品可以为单成分物质,亦可为多成分物质。
所述产品包括生物耗材、药品。在一种实施方式中,所述产品为药品,所述药品还包括药学上可接受的辅料。
“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时,它们不会产生不利的、过敏的或其它不良反应。
更进一步的,药学上可接受的辅料应当与所述有效成分相容,即能与其共混而不会在通常情况下大幅度降低药物的效果。可作为药学上可接受的载体或辅料的一些物质的具体例子是糖类,如乳糖、葡萄糖和蔗糖;淀粉,如玉米淀粉和土豆淀粉;纤维素及其衍生物,如甲基纤维素钠、乙基纤维素和甲基纤维素;西黄蓍胶粉末;麦芽;明胶;滑石;固体润滑剂,如硬脂酸和硬脂酸镁;硫酸钙;植物油,如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油;多元醇,如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇;海藻酸;乳化剂,如Tween;润湿剂,如月桂基硫酸钠;着色剂;调味剂;压片剂、稳定剂;抗氧化剂;防腐剂;无热原水;等渗盐溶液;和磷酸盐缓冲液等。这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味。
在一种实施方式中,所述骨软骨修复产品为耗材。较佳的,所述耗材为生物支架。具体的,首先用所述水凝胶制备生物墨水,再用所述生物墨水制备生物支架。
本发明还提供一种耗材,所述耗材为生物支架,所述生物支架为利用所述生物墨水通过3D生物打印得到的。此含细胞的仿生多孔生物支架可植入体内骨软骨缺损处,延长PTH的缓释时间,提高骨软骨损伤再生修复的质量,修复治疗骨软骨损伤,维持透明软骨的表型和功能。
本发明还提供一种制备生物支架的方法,所述方法包括利用所述生物墨水进行3D生物打印获得生物支架。
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围;在本发明说明书和权利要求书中,除非文中另外明确指出,单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例1甲状旁腺激素与丝素蛋白水凝胶的接枝
⑴200mg冷冻干燥的丝素蛋白溶解于4mL去离子水中,37℃,磁力子搅拌30min;
⑵待⑴中丝素蛋白完全溶解后,加入2mg 4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐(Sulfo-SMCC),搅拌均匀后,在pH 8.0,4℃反应2h;
⑶⑵反应完成后,上述混合液用截留分子量8000的透析袋透析三天,以去除未反应的Sulfo-SMCC和其它副产物,每天更换三次去离子水透析液;
⑷待⑶混合液透析完成后加入1mg羧基端接上一个半胱氨酸甲状旁腺激素(Cys-PTH),搅拌均匀后,在pH 7.0,4℃反应2h。
⑸待⑷反应完成后,上述混合液用截留分子量8000的透析袋透析三天,以去除未反应的Cys-PTH和其它副产物,每天更换三次去离子水透析液;
⑹待⑸透析完成后,上述混合液在冷冻机内干燥2天。冻干粉末即可作为后续3D生物打印的水凝胶材料备用。
丝素蛋白接枝绿色荧光标记的甲状旁腺激素后激光扫描共聚焦图片如图2所示,表明丝素蛋白与甲状旁腺激素化学接枝成功。
实施例2 3D生物打印仿生分层的支架
⑴0.5g上述冷冻干燥的丝素蛋白溶解于10mL去离子水中,37℃,磁力子搅拌10min;
⑵待⑴中丝素蛋白完全溶解后,加入1g冻干的甲基丙烯酸酐化明胶和0.05g光引发剂,37℃,磁力子搅拌30min;
⑶待⑵反应完成后,将上述混合液用0.22μm的滤嘴过滤除菌备用;
⑷过滤后的混合液中以2×107/mL的细胞密度向软骨相加入软骨细胞、骨相加入骨髓间充质干细胞。
⑸⑷中含细胞的混合液利用3D生物打印机制备圆柱型的仿生双相支架。其中打印喷头温度设置为18℃,打印速度为4mm/s,打印气压设置为0.2kPa。
实施例3水凝胶的表征
1.力学性能检测
通过试验机(HY-940FS;中国衡阳)测定了四种水凝胶(10%GM;10%GM-5%SF;10%GM-5%SF-MA;10%GM-5%SF-PTH)(其中GM代表甲基丙烯酸酐化明胶,SF代表丝素蛋白,SF-MA代表甲基丙烯酸酐化丝素蛋白)的机械性能。每组分别制备5个直径9mm、高度15mm的圆柱形支架。所有试验均以0.5mm/min的恒定速度进行。结果如图3A、3B所示,较单纯的甲基丙烯酸酐化明胶或未改性的丝素蛋白复合水凝胶,甲基丙烯酸酐化丝素蛋白的复合水凝胶其弹性模量提高了2-4倍。
2.流变学检测
使用旋转流变仪(美国ThermoFisher HAAKE)对上述生物墨水的流变特性进行了评估。首先,将所有样品放置在40℃的板上,以完全填充两个板之间的1mm间隙。通过在25℃下旋转试验将剪切速率从1变化到100s-1,进行粘度和剪切应力测试。然后在温度从5℃以2℃min-1的速率从5℃渐变到37℃时测量粘度。剪切速率保持在1s-1。储能模量(G’)和损耗模量(G”)在1Hz的恒定频率和0.1%的恒定应变下进行测试,水凝胶样品在37℃下平衡,然后以2℃min-1的速率从37℃冷却到5℃。结果如图3C、3D、3E所示,上述四种水凝胶材料为温敏性材料,粘度随温度的升高而降低。单纯的甲基丙烯酸酐化明胶打印温度在18℃左右,其余3组复合水凝胶的打印温度在20-25℃范围内。
3.体外降解检测
将上述四组支架冷冻干燥后称重,记录为W0,然后将各组支架浸入pH为7.42μg/mL的2型胶原酶溶液中。37℃下于第1、3、5、7、14、21、28天,4组每个时间点取出3个样本,再次冷冻干燥后称重,记录为Wt,最后按如下公式计算各组支架的降解率D:D=W0-Wt/W0X100%。结果如图3F所示,单纯的甲基丙烯酸酐化明胶在体外7天完全降解,其余3组复合水凝胶在体外28天降解了50%。
4.微观形态观察
上面提到的四组支架冻干后安装在铝桩上并镀金。使用Mira3扫描电子显微镜(SEM)(TESCAN;捷克共和国)在5kV的加速电压下观察4种生物墨水的微观形态。结果如图3G所示,扫描电子显微镜下4组水凝胶都形成了大小较均一的多孔结构。
实施例4关节骨软骨缺损模型
本研究选用成年雄性新西兰大白兔40只,体重2.8~3.2kg。为评价不同治疗方法对兔骨软骨缺损的修复效果,将兔随机分为4组:(1)假手术(不治疗);(2)GM+SF/GM+SF支架;(3)GM+SF/GM+SF-MA支架;(4)GM+SF-PTH/GM+SF-MA支架(每组10个)。在麻醉和常规准备后,兔髌骨脱位后暴露膝关节,用电环钻在股骨远端滑车沟处形成一个直径5mm、深5mm的全层骨软骨缺损。然后将3D生物打印支架植入骨软骨缺损处。在确认支架固定于缺损处后,逐层缝合筋膜、皮肤闭合伤口。最后,肌注青霉素预防感染。手术后,让兔子在单独的笼子里自由活动,并喂以标准食物和水。在各个预定的时间点(1、2、6和12周)处死动物,从耳静脉中抽取血液,并在4℃下以3000rpm的速度离心10分钟。然后收集上清液并冷冻在-80℃。肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)的浓度根据说明书使用ELISA试剂盒检测(Rabbit TNF-αELISA Kit,CSB-E06998Rb;Rabbit IL-1βELISA Kit,CSB-E06900Rb;CUSABIO,China)。并行组织学分析、骨软骨缺损修复效果的评价。结果如图4所示。
实施例5甲状旁腺激素在体外的释放
用PTH(1-34)酶联免疫吸附试剂盒(Mlbio,China)测定GM+SF-PTH/GM+SF-MA双相支架中PTH的释放。将支架置于含5mL磷酸盐缓冲液(PBS)的15mL离心管中并在37℃下以100rpm转速摇晃离心管。在第1、3、5、7、14、21、28、35、42、49、56天,离心管以2500rpm离心3min,分别收集上清液200μL,同时补充PBS 200μL。最后根据PTH(1-34)酶联免疫吸附试剂盒说明书计算PTH的浓度。结果如图5所示。
以上的实施例是为了说明本发明公开的实施方案,并不能理解为对本发明的限制。此外,本文所列出的各种修改以及发明中方法的变化,在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述,但应当理解,本发明不应仅限于这些具体实施例。事实上,各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
Claims (12)
1.丝素蛋白与甲状旁腺激素偶联接枝的方法,其特征在于,所述方法包括以下步骤:
1)将丝素蛋白与交联剂混合;
2)步骤1)反应结束后,将Cys-PTH加入上述反应产物中,反应结束后获得水凝胶。
2.根据权利要求1所述的方法,其特征在于,步骤1)还具备以下条件中的一种或几种:
a)所述交联剂选自水溶性的双官能团交联剂;优选的,所述交联剂为Sulfo-SMCC;
b)反应pH为7.5~8.5;
c)反应温度为1~10℃;
d)反应时间为1.5~2.5h。
3.根据权利要求1所述的方法,其特征在于,所述Cys-PTH中半胱氨酸连接于甲状旁腺激素的羧基端;优选的,所述Cys-PTH中甲状旁腺激素选自PTH(1-34)或PTH(1-84)。
4.根据权利要求1所述的方法,其特征在于,所述方法中步骤2)还具备以下条件中的一种或几种:
a)Cys-PTH的量与步骤1)中丝素蛋白的质量比为1:100~1:5000;
b)反应pH为6.5~7.5;
c)反应温度为1~10℃;
d)反应时间为1.5~2.5h。
6.一种水凝胶,其特征在于,所述水凝胶通过权利要求1-5任一所述的方法制备获得。
7.权利要求6所述的水凝胶在制备生物墨水中的用途、或在制备骨软骨修复产品中的用途。
8.根据权利要求7所述的用途,所述骨软骨修复产品包括耗材和药品。
9.一种生物墨水,其特征在于,所述生物墨水为通过权利要求6所述的水凝胶与甲基丙烯酸酐化明胶、光引发剂反应后,再与细胞混合制备获得。
10.根据权利要求9所述的生物墨水,其特征在于,所述生物墨水制备时水凝胶与甲基丙烯酸酐化明胶的重量比为1:1~0.1:1。
11.根据权利要求9所述的生物墨水,其特征在于,所述细胞为骨髓间充质干细胞和关节软骨细胞。
12.一种耗材,所述耗材为生物支架,其特征在于,所述生物支架为利用权利要求9所述的生物墨水通过3D生物打印得到的。
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