CN114605676A - 一种退变髓核修复可注射水凝胶及其用途 - Google Patents
一种退变髓核修复可注射水凝胶及其用途 Download PDFInfo
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Abstract
本发明提供了一种含有8~12重量份的苯硼酸和环糊精接枝的明胶、1重量份的单宁酸和80~120重量份的水的水凝胶前驱体,以及该水凝胶前驱体固化而成的水凝胶,具有自愈合、可注射性能,在注射入退变髓核内部时,具有抑制炎症、促进髓核修复的作用,进一步复合载姜黄素胶束和胆固醇修饰的miRNA‑21抑制剂,可以根据髓核内部的不同炎症程度,因地制宜的释放抗炎药物姜黄素及促进ECM再生的miRNA‑21抑制剂,双管齐下,促进退变髓核再生,具有很好的应用前景。
Description
技术领域
本发明属于生物医用材料领域,具体涉及一种退变髓核修复可注射水凝胶及其用途。
背景技术
随着社会老龄化的不断加重,脊柱退变性疾病(如腰椎间盘突出症、腰椎管狭窄症等)发病率不断增高,腰腿痛已成为发病率最高的疼痛性疾病之一。据报道,全球腰腿痛患病率高达12%;一项WHO调查显示,37%的青少年每月会至少出现一次腰痛。脊柱的退变是源于椎间盘的退变(变性、失水),椎间盘由内部的髓核和周围包围的纤维环组成。随着脊柱退变性疾病的不断进展,常规保守治疗仅能部分缓解临床症状,大部分患者不得不最终接受手术治疗。虽然目前针对脊柱退变性疾病的手术方式众多,并且不断趋近微创化,但无论何种手术方式都无法克服如手术损伤、术后复发、邻近节段退变加速、远期疗效不确切等问题,这无疑给个人和社会带来沉重的经济、社会负担。针对椎间盘退变初期的患者,尤其是仅表现为盘源性腰痛的患者,如果能够通过某种方式延缓甚至逆转椎间盘退变进程,将从源头上减少脊柱退变性疾病的发生。随着生物工程技术的飞速发展,通过髓核组织工程技术,实现椎间盘髓核再生、修复已成为可能。
近年来,通过构建可以作为细胞和生物大分子(药物、细胞因子、miRNA等)转运载体的髓核替代生物诱导支架材料,结合再生医学为代表的生物医学工程技术,使退变髓核组织再生成为可能。髓核生物医学工程策略的目标是抑制内部炎症反应、促进髓核细胞再生、恢复ECM合成/分解代谢平衡,最终真正恢复椎间盘的生理功能并满足生物力学需求。在这个过程中,髓核替代支架材料的选择处于核心地位。理想的髓核替代材料应当具有良好的生物安全性、生物相容性、与天然髓核相似的生物力学特性、与自体生物化速度匹配的降解性能,最终实现自动生物化,更重要的是可以通过微创的方式植入椎间盘内部:可注射性。研究者们一直希望通过材料筛选和改性等方面的研究,研制出理想的髓核替代支架材料,但是大量研究结果显示,由于椎间盘内组织结构复杂、血供极差、力学环境复杂,现有材料如天然材料:脱细胞基质、藻酸盐、多肽水凝胶,以及人工合成材料:透明质酸、PLGA等,都不能很好地满足体外功能化髓核的构建要求。在国际上,已有大量科学家通过一些包括纤维蛋白凝胶(GelfoamTM),透明质酸海绵(HYAFF)以及多肽水凝胶等在内的可以搭载细胞生长的髓核替代支架材料产品进行临床前期研究,但是目前,仍然没有一款成熟的功能化髓核可替代产品应用于临床。
近年来,人们不断的尝试利用微球、纳米胶束、外泌体、脱细胞基质等递药体系作为髓核替代支架材料,但由于缺乏力学属性、制备工艺复杂、原材料昂贵等局限,始终无法满足上述全部需求。水凝胶材料近年来持续受到关注,并广泛应用于各种组织修复领域。可注射水凝胶固有的渗透性、吸水性、可降解性、良好的生物相容性,以及巨大的载药控释能力和力学支撑潜力,在髓核替代支架材料的选择中具有巨大的应用潜力。
一方面,椎间盘退变是一系列包括炎症介导、ECM合成/分解代谢紊乱和纤维化等在内的复杂序贯过程,将水凝胶仅仅作为细胞或药物载体简单的植入退变椎间盘内部远无法适应其复杂的微环境,因此,需要构建一种能够全程参与退变椎间盘修复过程、并根据不同微环境变化而改变理化特性的“智能响应型水凝胶递药体系”,即具有pH和ROS双相应的“炎症响应型水凝胶递药***”可以迅速对炎症微环境的变化和刺激进行感知和响应,精准可控的将药物释放于疾病部位。
另一方面,MicroRNA(miRNA)是一类由18-24个核苷酸序列组成的小型非编码RNA,广泛存在于真核生物中,并参与基因调节。成熟的miRNA在细胞质中引导RISC复合体(miRISC)靶向3’-UTR区域中带有部分互补序列的mRNA,导致转录沉默或mRNA降解,进而调控细胞的多种生理活动。miRNA调节失衡在椎间盘退变的发生过程中扮演着重要角色。大量的研究表明,多种miRNAs表达异常将导致髓核内部细胞炎症、ECM降解、髓核细胞凋亡等,并通过不同作用机制加速椎间盘髓核退变。因此,寻找并探索miRNAs作用靶点和规律,并通过miRNAs基因治疗的方法调节髓核细胞功能,在退变椎间盘髓核修复中具有巨大的应用前景。
miRNAs基因治疗可根据miRNAs自身表达情况分为miRNAs缺乏时的miRNA替代疗法以及过表达时的miRNA抑制疗法。然而,无论哪种方法,如何将外源miRNA或miRNA抑制剂精准、稳定的转运到靶区域是miRNA基因治疗退变髓核成败的关键。基于椎间盘内无血管的特殊结构特点,以及miRNAs全身递送的诸多局限(如靶器官外异常聚集、靶器官局部低生物活性及多次用药等),miRNAs全身递送的方法并不适用于髓核的miRNAs基因治疗。然而,由于椎间盘髓核特殊的封闭结构,单纯局部注射miRNAs又会导致局部浓度过高、转染效率不足、清除率过快等一系列问题。因此,寻找一种适合椎间盘盘内局部递送miRNAs的转运载体,并结合炎症响应型水凝胶共同构建智能miRNAs递药***,在退变髓核miRNAs基因治疗中具有重大的科学意义。
发明内容
本发明的目的在于提供一种可制备退变髓核修复水凝胶的可注射水凝胶前驱体,以及该水凝胶前驱体固化得到的退变髓核修复水凝胶。
本发明提供了一种水凝胶前驱体,它含有如下重量份数的组分:
苯硼酸和环糊精接枝的明胶8~12份、单宁酸1份、水80~120份。
进一步地,它含有如下重量份数的组分:
苯硼酸和环糊精接枝的明胶10份、单宁酸1份、水100份。
更进一步地,它还含有如下重量份数的组分:
胆固醇修饰的miRNA抑制剂0.003~0.004份,和/或包载姜黄素的纳米胶束0.006~0.008份。
优选地,上述胆固醇修饰的miRNA抑制剂为0.0033份,和/或包载姜黄素的纳米胶束0.0066份。
更进一步地,上述苯硼酸和环糊精接枝的明胶中,苯硼酸的接枝率为17±2.4%,环糊精的接枝率为3.13±0.87%。
更进一步地,上述苯硼酸和环糊精接枝的明胶是以按如下方法制备而成:
(1)明胶和3-羧基苯硼酸在活化剂和缩合剂作用下反应得到苯硼酸接枝的明胶;
(2)苯硼酸接枝的明胶和氨基化β-环糊精在活化剂和缩合剂作用下反应得到苯硼酸和环糊精接枝的明胶。
优选地,上述步骤(1)所述明胶和3-羧基苯硼酸的重量比为1:(0.1~0.5),优选为1:0.4;
和/或步骤(2)所述苯硼酸接枝的明胶和氨基化β-环糊精的重量比为1:(0.1~0.5),优选为1:0.4。
进一步地,上述活化剂是NHS,所述缩合剂是EDC。
进一步地,上述胆固醇修饰的miRNA抑制剂是胆固醇修饰的miRNA-21抑制剂。
进一步地,上述包载姜黄素的纳米胶束中,姜黄素的含量为10%~20%。
更进一步地,上述纳米胶束是聚乙二醇-酮缩硫醇-聚乳酸-羟基乙酸共聚物自组装形成。
更进一步地,上述聚乙二醇-酮缩硫醇-聚乳酸-羟基乙酸共聚物是聚乙二醇和聚乳酸-羟基乙酸共聚物与酮缩硫醇反应制备而成;所述酮缩硫醇的结构为:
更进一步地,上述聚乙二醇的分子量为2000,所述聚乳酸-羟基乙酸共聚物的分子量为1000。
进一步地,上述包载姜黄素的纳米胶束是如下重量份数的原料自组装形成:
聚乙二醇-酮缩硫醇-聚乳酸-羟基乙酸共聚物8~12份、姜黄素1份;
优选为聚乙二醇-酮缩硫醇-聚乳酸-羟基乙酸共聚物10份、姜黄素1份。
本发明还提供了一种水凝胶,它是上述的水凝胶前驱体固化反应而成。
进一步地,上述固化反应条件是在20~30℃静置。
本发明还提供了上述的水凝胶在退变髓核修复材料中的应用。
进一步地,上述退变髓核修复材料是髓核替代材料和/或促髓核再生材料。
本发明的有益效果:首先接枝苯硼酸BA和环糊精CD到明胶Gel侧链,通过BA与单宁酸分子(TA)交联的作用自组装成水凝胶,同时包裹包载姜黄素的胶束MIC@Cur和胆固醇修饰的miRNA抑制剂。此种水凝胶可以通过注射的方式植入退变椎间盘内部,在内部pH和ROS刺激下,水凝胶裂解,释放出MIC@Cur,MIC@Cur在ROS存在下继续裂解释放出姜黄素Cur,从而发挥抗炎作用。同时,被CD分子通过亲疏水作用包裹的miRNA抑制剂可以缓慢的释放出来,并转染到髓核细胞中,从而持续发挥促进髓核细胞ECM再生的作用。
本发明通过将可注射水凝胶植入退变髓核内部,具有抑制炎症、促进髓核修复的作用,进一步复合载姜黄素胶束和胆固醇修饰的miRNA-21抑制剂,可以根据髓核内部的不同炎症程度,因地制宜的释放抗炎药物姜黄素及促进ECM再生的miRNA-21抑制剂(Antagomir),双管齐下,促进退变髓核再生。为退变髓核修复提供新思路,最终避免手术带来的风险和巨大的经济花费。
本发明所述“水凝胶前驱体”是一种以制备水凝胶的原料为组分的组合物,这些组分可能是各自独立分别包装的,也可能是混合在一起的。当各组分是混合在一起的状态时,特指其还未固化形成固态水凝胶时的液态组合物形式,具备可注射性能。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为a)3-羧基苯硼酸和b)氨基化β-环糊精的标准曲线。
图2为姜黄素的标准曲线。
图3为Gel-BA-CD聚合物及MIC@Cur的理化特性;a为Gel-BA-CD和水凝胶制备分子图示。b为具有ROS响应的双亲分子mPEG-TK-PLGA制备过程。c,d为NMR扫描结果。e为FTIR扫描结果。f为UV吸收峰结果。g为制备的MIC@Cur及MIC(空载的MIC)的粒径和电位结果。h为粒径分析结果。i为MIC@Cur的UV分析结果。J为MIC@Cur透射电镜(TEM)扫描结果。
图4为Gel-BA-CD/TA水凝胶的理化性能及微观结构表征结果。a为水凝胶成胶示意图。b为加入TA前的Gel-BA-CD溶液,及交联后的水凝胶。c为SEM扫描的水凝胶内部结构。d为本发明水凝胶的可注射性能。e为本发明水凝胶的自愈性能。F-h为水凝胶的流变学结果。
图5为装载两种药物的水凝胶体外降解及pH/ROS响应性能表征结果;a为水凝胶体外降解结果。b为不同水凝胶DPPH抗氧化实验。c为水凝胶在不同环境下的裂解情况。d为MIC@Cur构建示意图。e Cur不同环境下释放曲线。f为罗丹明荧光淬灭实验结果。gAntagomir缓释示意图。h Cy3荧光标记的Antagomir释放曲线图。i,j为大鼠体内荧光显像。
图6为水凝胶髓核细胞ECM再生效果评估。a为不同水凝胶浸提液培养TBHP刺激后的髓核细胞,细胞增殖情况。B为不同水凝胶浸提液培养TBHP刺激后的髓核细胞Col II,Aggrecan,MMP-13基因表达差异结果。c,d为不同水凝胶浸提液培养TBHP刺激后的髓核细胞Col II,MMP-13蛋白表达免疫荧光结果。
图7为水凝胶促进巨噬细胞极化检测结果。a-c为不同组治疗巨噬细胞后光镜观察结果。d为不同组刺激巨噬细胞后RT-PCR检测结果。E为不同组刺激巨噬细胞后M2特异性蛋白CD 206表达情况。f为细胞流式结果。g为细胞内ROS清除实验结果。
图8为水凝胶抗炎效果测定结果。a为巨噬细胞RAW264.7经过LPS处理后,用不同组水凝胶浸提液孵育,RT-PCR抗炎因子CD 206,IL-10及TNF-α表达情况。B为Western-blot检测IL-10及TNF-α表达情况。c,d为免疫荧光检测IL-10及TNF-α表达情况。e为细胞迁移Transwell实验。f为Cur促进巨噬细胞极化作用示意图。
图9为水凝胶对大鼠椎间盘退变治疗效果体内鉴定结果。a为通过建立大鼠椎间盘退变模型,并植入不同水凝胶,4,8周观察退变髓核修复效果。b,d,e为术后4,8周X光扫描结果。c,f为术后4,8周MRI扫描结果。
图10为水凝胶对大鼠椎间盘退变治疗效果组织学检测结果。a,b,c是HE染色,番红O-固绿染色及Col II免疫组化染色结果。d,e,f是定量观察组织学分级结果。
具体实施方式
本发明所用原料与设备均为已知产品,通过购买市售产品所得。
实施例1、本发明具有pH/ROS双响应的水凝胶的制备
1、苯硼酸接枝明胶(Gel-BA)的合成
称取5g明胶溶解于500ml MES缓冲液中。称取EDC(4.8g,25mmol),NHS(1.15g,10mmol/L)和3-羧基苯硼酸(2g,12.5mmol/L)并溶解与明胶溶液中,37℃充分搅拌48h。过滤杂质后,加入透析袋持续透析3d,每天换水3-6次。透析完成后冻干。图1A上
2、苯硼酸和环糊精接枝明胶(Gel-BA-CD)的合成
称取5g Gel-BA粉末充分溶解,称取与上述反应等量EDC,NHS及2g氨基化β-环糊精分子,再次通过酰胺缩合反应合成Gel-BA-CD,反应时间及步骤同上。
3、具有pH/ROS双响应的水凝胶
Gel-BA-CD分子充分溶解于去离子水中(10%w/v),调整pH为中性。溶解单宁酸(TA)制备质量浓度为10%w/v的TA溶液。Gel-BA-CD溶液及其1/10体积量的TA溶液充分搅拌,即刻制成空白水凝胶Gel-BA-CD/TA(记为Control Hydrogel或Hydrogel)。
实施例2、本发明具有pH/ROS双响应的载药水凝胶的制备
1、按照实施例1的步骤1、2的方法制得苯硼酸和环糊精接枝明胶。
2、制备装载姜黄素(Cur)的胶束(MIC@Cur)
称取双亲分子聚乙二醇-酮缩硫醇-聚乳酸-羟基乙酸共聚物(mPEG-TK-PLGA)20mg和2mg Cur溶解于1mL DMSO溶液中,加热至37℃,超声搅拌同时逐滴加入10mL去离子水中,持续透析48h,直径为0.22um的滤嘴过滤后制得MIC@Cur。图5d为MIC@Cur构建示意图。
mPEG-TK-PLGA的来源自行制备,通过分子量2000的PEG与分子量1000的PLGA与TK反应制备而成:TK分子、二环己基碳二亚胺(DCC)及4-二甲氨基吡啶(DMAP)按照1:6:0.6的摩尔比例混合溶解于15ml二甲亚砜(DMSO)中。后溶解463mgPLGA分子(1kDA)溶解于4mlDMSO中,氮气保护下充分搅拌反应24h后加入618mg mPEG(2kDA)分子,继续反应24h。透析冻干备用。反应式见图1b。
3、制备具有pH/ROS双响应的载药水凝胶
Gel-BA-CD分子充分溶解于去离子水中(10%w/v),调整pH为中性,加入已制备成功的66μg MIC@Cur及33μg Antagomir(购自GenePharma公司(上海,中国).分子量:7998.268.碱基序列为:5’-UAGCUUAUCAGACUGAUGUUGA-chol-3’)。溶解单宁酸(TA)制备质量浓度为10%w/v的TA溶液。Gel-BA-CD溶液及其1/10体积量的TA溶液充分搅拌,即刻制成载药水凝胶(记为Hydrogel&MIC@Ant)。
实施例3、本发明具有pH/ROS双响应的载药水凝胶的制备
参照实施例2的制备方法,在步骤(3)制备过程中不加入Antagomir,制备得到仅包载姜黄素的Hydrogel@MIC水凝胶。
实施例4、本发明具有pH/ROS双响应的载药水凝胶的制备
参照实施例2的制备方法,在步骤(3)制备过程中不加入MIC@Cur,制备得到仅包载Antagomir的Hydrogel@Ant水凝胶。
以下通过实验例证明本发明的有益效果。
实验例1、本发明羧基苯硼酸及环糊精接枝率和MIC@Cur载药量
1、羧基苯硼酸及环糊精接枝率的计算方法:
紫外光谱检测不同3-羧基苯硼酸浓度下275nm紫外吸收峰高度并绘制标准曲线(如图1a),实验测得实施例1制备的Gel-BA溶液(浓度625ug/ml)在275nm处紫外吸收峰为0.83,通过标曲计算BA含量约为110.1ug/ml,因此接枝率约为110.1/625=17.6%。
紫外光谱检测不同氨基化β-环糊精浓度下490nm紫外吸收峰高度并绘制标准曲线(如图1b),通过苯酚-硫酸法裂解实施例1制备的Gel-BA-CD溶液(20mg/ml)后,测得在490nm处紫外吸收峰为1.57,通过标曲计算CD含量约为626.2ug/ml,因此接枝率约为626.2/20000=3.1%。
2、MIC@Cur的载药量
紫外光谱检测不同姜黄素溶于DMSO/H2O(1:1)的溶液浓度下430nm紫外吸收峰高度并绘制标准曲线(如图2),实验测得实施例2制备的MIC@Cur溶液(浓度146ug/ml)在430nm处紫外吸收峰为1.427,通过标曲计算Cur含量约为22.10ug/ml,因此接枝率约为22.10/146=15.13%。
实验例2、Gel-BA-CD及MIC@Cur的理化特性表征
1、实验方法:采用NMR、FTIR、UV、DLS、TEM等手段对本发明实施例1制备的Gel-BA-CD聚合物及实施例2制备的MIC@Cur的理化性质进行了一系列表征。
2、实验结果:
结果如图3所示。
图3为Gel-BA-CD聚合物及MIC@Cur的理化特性;从图1a中Gel-BA-CD和水凝胶制备分子图示,反映出水凝胶通过Gel-BA-CD分子和TA分子交联形成,形成的硼酯键在pH和ROS存在的条件下发生断裂,Antagomir通过与CD的主客体自组装作用包裹于CD中,并缓慢释放。
图3c,1d为NMR扫描结果,提示通过酰胺反应,3-羧基苯硼酸及CD分子已成功接枝于明胶分子侧链;并且已成功制备mPEG-TK-PLGA双亲分子。
图3e为FTIR扫描结果,结果提示合成的Gel-BA-CD分子在C=O C=C B-O C-H特征峰都有明显趋势,提示Gel-BA-CD分子合成成功。
图3f为UV吸收峰结果,提示合成的Gel-BA在275nm处有明显的吸收峰,提示Gel-BA分子合成成功。
图3g为制备的MIC@Cur及MIC(空载的MIC)的粒径和电位结果,结果提示装载药物后MIC的粒径变大及电位变小。
图3h为粒径分析结果,结果提示在中性环境下,粒子直径表现为均一的单锋(正态分布),在ROS环境下,表现为多峰(即大小分布不均)。
图3i为MIC@Cur的UV分析结果,结果提示装载药物后的MIC@Cur表现出与Cur相同的特征吸收峰,提示药物装载成功。
图3J为MIC@Cur透射电镜(TEM)扫描结果,结果提示MIC@Cur在PBS(pH 7.4)中表现为均一的圆形颗粒,在ROS环境下粒子裂解,释放出装载药物。
实验例3、Gel-BA-CD/TA水凝胶的理化性能及微观结构表征
1、实验方法:通过SEM观测实施例1制备得到的Gel-BA-CD/TA水凝胶的内部结构,以并对实施例1制备的Gel-BA-CD/TA水凝胶进行自愈合性能和可注射性能验证;此外,对实施例1制备的Gel-BA-CD/TA水凝胶(Control Hydrogel)和实施例2制备的载药水凝胶(Hydrogel&MIC@Ant)进行了流变学测试,比较二者的流变行为。
2、实验结果:
结果如图4所示。
图4a为水凝胶成胶示意图,图4b为加入TA前的Gel-BA-CD溶液,及交联后的水凝胶,可以看出本发明实施例1成功制备得到了固化的水凝胶。
图4c为SEM扫描的水凝胶内部结构,提示水凝胶内部为大小均一的多孔结构,有利于药物的包载。
图4d可以看出水凝胶具有优秀的可注射性能,可以通过粗细针头。
图4e可以看出本发明水凝胶还具有的良好的自愈性能,在通过针头注射后,在缺损部位仍能自愈合形成完整的凝胶结构。
图4f-h为水凝胶的流变学结果,结果提示加入两种药物的水凝胶(实施例2)流变性能与空白水凝胶(实施例1)表现出相似的流变属性,具有良好的剪切变稀和自愈性能,弹性模量可达到1000Pa左右,说明本发明实施例1制备的水凝胶进一步包载药物后,不会影响其可注射性能。
实验例4、载药水凝胶体外降解及pH/ROS响应性能表征
1、实验方法:
(1)将实施例1制备的空白水凝胶,以及实施例2制备的载药水凝胶,置于PBS溶液中,在37℃恒温摇床中进行降解实验,同时将实施例1制备的空白水凝胶在pH=5,H2O2(1mM)条件下进行加速降解实验。
(2)对实施例1~4制备的水凝胶,以及作为对照的PVA水凝胶进行DPPH抗氧化实验。
(3)取实施例1的水凝胶分别加入PBS(pH7.4)、pH=5的溶液(pH5)、H2O2溶液(1mM)(H2O2)、H2O2溶液且pH=5的溶液(pH5/H2O2)中,观察其在不同环境条件下的裂解情况。
(4)取实施例2的载药水凝胶,在PBS(pH7.4)、pH=5的溶液(pH5.0)、H2O2溶液(1mM)(H2O2)、H2O2溶液且pH=5的溶液(pH5+H2O2)中,37℃温度条件下进行姜黄素缓释释放试验。
(5)为证明Gel-BA-CD与Antagomir的组装,进行罗丹明淬灭试验:
不同浓度Gel-BA-CD溶液(0,2.5,5mg/ml)中加入罗丹明B(50mg/ml)溶液(共2ml),UV观察550nm吸收峰变化。而后,选取已加入罗丹明B的5mg/ml的Gel-BA-CD混合溶液,分别加入不同浓度Antagomir-21(0,2,5uM),再次UV观察550nm吸收峰变化。
(6)采用实施例4的水凝胶(为便于观测,Antagomir用Cy3荧光标记),在37℃摇床条件下进行缓释试验,并使用未接枝环糊精,只有苯硼酸修饰的明胶与单宁酸形成的凝胶包载Antagomir(Cy3标记)作为对比,在同等条件下进行缓释试验。
同时,将上述两种凝胶注射植入大鼠体内,通过荧光观测Antagomir在体内的缓释情况。
2、实验结果:
结果如图5所示。
(1)图5a为水凝胶体外降解结果,结果提示水凝胶在PBS中降解速率慢,完全降解约需要21d,当调整pH及ROS含量后,水凝胶将在4d内快速降解,说明本发明制备的空白水凝胶、载药水凝胶均具有很好的可降解性能,特别是在酸性、ROS条件下能够加速降解,具有pH、ROS响应性。
(2)图5b为不同水凝胶DPPH抗氧化实验结果,提示加入MIC的水凝胶抗氧化能力最强。
(3)图5c为水凝胶在不同环境下的裂解情况,结果显示在pH及ROS存在时,几乎完全裂解,呈液态,进一步印证了本发明水凝胶的pH、ROS响应性。
(4)图5e为姜黄素在不同环境下释放曲线,可以看出,本发明载有姜黄素的水凝胶可以有效实现姜黄素的缓释,并且具有pH、ROS响应性,在酸性、ROS条件下姜黄素释放增加。
(5)图5f为罗丹明荧光淬灭实验,结果提示随着加入Gel-BA-CD浓度不断增加,罗丹明荧光不断淬灭,说明罗丹明被Gel-BA-CD中的CD分子包裹而使荧光消失,加入Antagomir后,荧光恢复,说明Antagomir替换罗丹明分子进入CD分子内部,证明了本发明Antagomir的成功包载。
(6)图5g为Antagomir缓释示意图。图5h为Cy3荧光标记的Antagomir释放曲线图。图5i,j为大鼠体内荧光显像,提示Cy3标记的Antagomir包裹于Gel-BA-CD/TA水凝胶后,可以实现长达21d的缓释。
上述实验结果说明,本发明制备的水凝胶具有很好的pH、ROS响应性,有利于所包载的药物在待修复部位进行缓释治疗。
实验例5、水凝胶髓核细胞ECM再生效果评估
1、实验方法:
使用实施例1、实施例2、实施例4的水凝胶培养用过氧化氢叔丁醇(TBHP)刺激后的髓核细胞,观测细胞增值情况以及Col II、Aggrecan、MMP-13基因表达结果。
本部分实验室拿control hydrogel,hydrogel@ant,hydrogel@MIC&Ant(所以应该不是实例4吧,是不是应该有一个实例5,包含两种药物的)三种水凝胶PBS浸泡24h后,取浸提液治疗被TBHP刺激的髓核细胞,最后观测细胞增值情况以及Col II、Aggrecan、MMP-13基因表达结果。
2、实验结果:
结果如图6所示,图6a为不同水凝胶浸提液培养TBHP刺激后的髓核细胞增殖情况,可以看出实施例1的空白水凝胶具有一定的促进髓核细胞增殖的效果,且包载Antagomir后,促进髓核细胞增殖的效果显著提升。
图6b不同水凝胶浸提液培养TBHP刺激后的髓核细胞Col II,Aggrecan,MMP-13基因表达差异结果。可以看出,实施例1制备的空白凝胶本身就具有改善Col II、Aggrecan、MMP-13基因表达,负载了Antagomir的凝胶改善作用显著提升,姜黄素的复合还可进一步提升对上述基因表达的改善作用。图6c,d为不同水凝胶浸提液培养TBHP刺激后的髓核细胞Col II,MMP-13蛋白表达免疫荧光结果。
以上结果表明本发明水凝胶可以有效促进Col II、Aggrecan表达,抑制MMP-13蛋白表达,促髓核细胞ECM再生。
试验例6、水凝胶促进巨噬细胞极化检测
1、实验方法:
采用本发明实施例1制备的空白水凝胶、实施例3制备的载有姜黄素的水凝胶,以及作为对照的单独使用的姜黄素纳米胶束MIC@Cur,对巨噬细胞进行处理,并进行Transwell实验验证细胞迁移情况。
培养巨噬细胞RAW264.7并给与不同组干预24h,显微镜下观察细胞形态,RT-PCR技术检测M2极化特异指标CD 206、CD163,M1极化特异指标TNF-α,IL-1β,细胞流式技术及免疫荧光结果检测CD206蛋白表达,DFCH-DA染色检测细胞内ROS清除情况。
2、实验结果:
结果如图7所示。
图7a-c为不同组治疗巨噬细胞后,光镜观察结果,测量长宽比值,发现实施例1的空白凝胶和纳米胶束单独使用均具有一定的刺激巨噬细胞计划的用作用,而相比于空白的凝胶,和单独使用的姜黄素纳米胶束MIC@Cur,包裹MIC的水凝胶组可以更显著有效地刺激巨噬细胞极化。
图7d为不同组刺激巨噬细胞后RT-PCR检测结果,结果提示装载MIC的水凝胶相较于其他组,有效促进巨噬细胞M2极化,充分发挥抗炎效果。
图7e为不同组刺激巨噬细胞后M2特异性蛋白CD 206表达情况,结果提示实施例1的空白凝胶和纳米胶束单独使用均具有一定的促进巨噬细胞M2极化的作用,而Hydrogel@MIC组可以更加显著有效地促进巨噬细胞M2极化。图7f的细胞流式结果进一步验证了此结论。图8e为细胞迁移Transwell实验结果,图8f为Cur促进巨噬细胞极化作用示意图。提示本发明水凝胶处理后,M2极化的巨噬细胞可以有效促进髓核细胞迁移。
图7g为细胞内ROS清除实验,验证Hydrogel@MIC组可以有效抑制LPS刺激巨噬细胞后ROS生成。
试验例7、水凝胶抗炎效果鉴定
1、实验方法:
采用本发明实施例1制备的空白水凝胶、实施例2制备的双载药水凝胶以及实施例3制备的载有姜黄素的水凝胶,对LPS处理后的巨噬细胞RAW264.7的抗炎因子CD 206、IL-10及TNF-α表达情况进行表征。
2、实验结果:
图8a为巨噬细胞RAW264.7经过LPS处理后,用不同组水凝胶浸提液孵育,RT-PCR抗炎因子CD 206、IL-10及TNF-α表达情况,图8b为Western-blot检测IL-10及TNF-α表达情况,图8c,d为免疫荧光检测IL-10及TNF-α表达情况。结果提示本发明实施例1制备的空白水凝胶,即具有显著的抗炎效果,装载MIC@Cur及Antagomir-21后的水凝胶具有更为显著提升的抗炎效果。
试验例8、水凝胶大鼠椎间盘退变治疗效果体内鉴定
1、实验方法:
建立大鼠椎间盘退变模型,并植入不同水凝胶,4,8周通过X光、MRI、组织学染色(HE、番红O-固绿、Col II及MMP-13免疫组织化学染色)等结果观察退变髓核修复效果,示意图如图9a所示。
2、实验结果:
图9b,d,e为术后4,8周X光扫描结果,说明本发明制备的空白水凝胶可显著恢复椎间隙高度,进一步包载药物后,能够进一步提升椎间隙高度的恢复,双载药的Hydrogel&MIC@Ant恢复效果最佳。
图9c,f为术后4,8周MRI扫描结果,提示本发明制备的空白水凝胶可有效增加髓核含水量,恢复髓核MRI信号,进一步包载药物后,能够进一步提升修复效果,双载药的Hydrogel&MIC@Ant组修复效果最佳。
图10a,b,c是HE染色,番红O-固绿染色及Col II免疫组化染色结果,图10d,e,f是定量观察组织学分级结果,提示Hydrogel&MIC@Ant组退变髓核修复效果最佳。
综上,本发明提供了一种以苯硼酸和环糊精接枝的明胶对基体,在单宁酸作用下交联而成的可注射水凝胶,在注射入退变髓核内部时,具有抑制炎症、促进髓核修复的作用,进一步复合载姜黄素胶束和胆固醇修饰的miRNA-21抑制剂,可以根据髓核内部的不同炎症程度,因地制宜的释放抗炎药物姜黄素及促进ECM再生的miRNA-21抑制剂(Antagomir),双管齐下,促进退变髓核再生,具有很好的应用前景。
Claims (18)
1.一种水凝胶前驱体,其特征在于,它含有如下重量份数的组分:
苯硼酸和环糊精接枝的明胶8~12份、单宁酸1份、水80~120份。
2.如权利要求1所述的水凝胶前驱体,其特征在于,它含有如下重量份数的组分:
苯硼酸和环糊精接枝的明胶10份、单宁酸1份、水100份。
3.如权利要求1或2所述的水凝胶前驱体,其特征在于,所述苯硼酸和环糊精接枝的明胶中,苯硼酸的接枝率为17±2.4%,环糊精的接枝率为3.13±0.87%。
4.如权利要求3所述的水凝胶前驱体,其特征在于,所述苯硼酸和环糊精接枝的明胶是以按如下方法制备而成:
(1)明胶和3-羧基苯硼酸在活化剂和缩合剂作用下反应得到苯硼酸接枝的明胶;
(2)苯硼酸接枝的明胶和氨基化β-环糊精在活化剂和缩合剂作用下反应得到苯硼酸和环糊精接枝的明胶。
5.如权利要求4所述的水凝胶前驱体,其特征在于,步骤(1)所述明胶和3-羧基苯硼酸的重量比为1:(0.1~0.5),优选为1:0.4;
和/或步骤(2)所述苯硼酸接枝的明胶和氨基化β-环糊精的重量比为1:(0.1~0.5),优选为1:0.4。
6.如权利要求4所述的水凝胶前驱体,其特征在于,所述活化剂是NHS,所述缩合剂是EDC。
7.如权利要求1或2所述的水凝胶前驱体,其特征在于,它还含有如下重量份数的组分:
胆固醇修饰的miRNA抑制剂0.003~0.004份,和/或包载姜黄素的纳米胶束0.006~0.008份。
8.如权利要求7所述的水凝胶前驱体,其特征在于,所述胆固醇修饰的miRNA抑制剂为0.0033份,和/或包载姜黄素的纳米胶束0.0066份。
9.如权利要求7或8所述的水凝胶前驱体,其特征在于,所述胆固醇修饰的miRNA抑制剂是胆固醇修饰的miRNA-21抑制剂。
10.如权利要求7或8所述的水凝胶前驱体,其特征在于,所述包载姜黄素的纳米胶束中,姜黄素的含量为10%~20%w/w。
11.如权利要求10所述的水凝胶前驱体,其特征在于,所述纳米胶束是聚乙二醇-酮缩硫醇-聚乳酸-羟基乙酸共聚物自组装形成。
13.如权利要求12所述的水凝胶前驱体,其特征在于,所述聚乙二醇的分子量为2000,所述聚乳酸-羟基乙酸共聚物的分子量为1000。
14.如权利要求11所述的水凝胶前驱体,其特征在于,所述包载姜黄素的纳米胶束是如下重量份数的原料自组装形成:
聚乙二醇-酮缩硫醇-聚乳酸-羟基乙酸共聚物8~12份、姜黄素1份;
优选为聚乙二醇-酮缩硫醇-聚乳酸-羟基乙酸共聚物10份、姜黄素1份。
15.一种水凝胶,其特征在于,它是权利要求1~14任一项所述的水凝胶前驱体固化反应而成。
16.如权利要求15所述的水凝胶,其特征在于,所述固化反应条件是在20~30℃静置。
17.权利要求15或16所述的水凝胶在退变髓核修复材料中的应用。
18.如权利要求17所述的应用,其特征在于,所述退变髓核修复材料是髓核替代材料和/或促髓核再生材料。
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