CN112107733B - 一种注射型负载血小板裂解液的温敏水凝胶-高分散纳米颗粒体系及其应用 - Google Patents
一种注射型负载血小板裂解液的温敏水凝胶-高分散纳米颗粒体系及其应用 Download PDFInfo
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Abstract
本发明涉及一种注射型负载血小板裂解液的温敏水凝胶‑高分散纳米颗粒体系,体系包括:温敏水凝胶;以及嵌入所述温敏水凝胶中负载血小板裂解液的肝素/ε‑聚赖氨酸纳米颗粒。本发明首次设计双重缓释的微球水凝胶体系来负载血小板裂解液并使其应用于软骨组织工程,单次注射,长效缓释,持续起效;首次根据血小板裂解液本身的特性来设计材料,且是首次发现血小板裂解液这种不均质的颗粒分布会导致水凝胶的物理特性受影响,而通过微球的搭载可以很好的改善这种情况,让构建的材料更合理。
Description
技术领域
本发明涉及骨关节炎治疗领域,尤其涉及一种注射型负载血小板裂解液的温敏水凝胶-高分散纳米颗粒体系及其应用。
背景技术
骨关节炎(OA)是一种退行性关节疾病,其病理特征是关节软骨肥大钙化,软骨下骨重塑以及滑膜炎症。全世界60岁以上的人群中,有超过10%的患者遭受着OA的困扰。据报道,尽管多种危险因素,包括年龄、性别、遗传因素、创伤和肥胖等,被证明与OA相关,但其确切的病因和有效的治疗选择仍然是目前临床上的一大难题。目前FDA批准的OA药物,包括广泛用于早期患者的非甾体类抗炎药,关节腔注射的透明质酸钠等,都只能帮助缓解症状,但不能阻止疾病的发展。而随着疾病的进展,后期患侧关节会出现严重的软骨剥脱,骨赘形成,关节畸形等,此时关节置换术则成为唯一的选择。但是长期的口服药物治疗和手术带来的副作用往往也会加剧患者的痛苦,因此新的OA治疗手段亟待开发。近年来,关节腔注射富血小板血浆(PRP)及其衍生物血小板裂解液(PL)用于OA的治疗得到了大量研究支持,被认为能延缓OA的进展,但其仍存在局部突释,原位降解快,组织在位性差等不足。因此,如何将其有效负载于生物材料中以提高关节腔注射的效率,是更好的促进PRP及PL临床应用前景的重要手段。
在本发明中,首先将血小板裂解液(PL)包载在肝素/ε-聚赖氨酸纳米颗粒(Hep/EPL NP)中以改善其分散性和释放行为。而后,将该负载PL的纳米粒进一步负载于聚(d,L-丙交酯)-聚(乙二醇)-聚(d,L-丙交酯)(PDLLA-PEG-PDLLA,PLEL)三嵌段水凝胶中以构建多功能水凝胶/纳米颗粒/PL复合体系。与单纯的PLEL@PL混合物不稳定的胶凝状态相比,PLEL@PL-NPs体系表现出与空白PLEL相似的胶凝能力。同时,通过分子模拟和体外释放实验证实了该体系对PL中生长因子的高亲和力和持久释放。此外,PLEL@PL-NPs体系在体内关节腔原位注射治疗后,能促进大鼠骨软骨缺损的修复。
发明内容
本发明的目的是针对现有技术中的不足,提供一种注射型负载血小板裂解液的温敏水凝胶-高分散纳米颗粒体系及其应用。
为实现上述目的,本发明采取的技术方案是:
本发明的第一方面是提供一种注射型负载血小板裂解液的温敏水凝胶-高分散纳米颗粒体系,体系包括:
温敏水凝胶;以及分散于所述温敏水凝胶中负载血小板裂解液的纳米颗粒。
优选地,所述纳米颗粒为肝素/ε-聚赖氨酸纳米颗粒。
优选地,所述温敏水凝胶为三嵌段高分子共聚物聚(D,L-丙交酯)-聚(乙二醇)-聚(D,L-丙交酯)纳米胶束水凝胶。
优选地,所述温敏水凝胶在室温或者相对低温的环境中呈现液体状的溶胶态;在人体温度37℃或者相对高温的环境中呈现固体状的凝胶态。
优选地,其制备步骤包括:
S1、制备负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒;
S2、将S1所制备的纳米颗粒分散于温敏水凝胶中,即得温敏水凝胶-血小板裂解液-高分散纳米颗粒复合体系。
优选地,所述制备步骤S1包括:
通过肝素和ε-聚赖氨酸的静电自组装和肝素与血小板裂解液之间的高亲和力制备负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒。
优选地,所述制备步骤S2包括:
采用Sn(Oct)2作为催化剂,引发聚乙二醇和D,L-丙交酯开环共聚,合成聚(D,L-丙交酯)-聚(乙二醇)-聚(D,L-丙交酯)三嵌段高分子共聚物;
将负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒嵌入聚(D,L-丙交酯)-聚(乙二醇)-聚(D,L-丙交酯)纳米胶束水凝胶中构建温敏水凝胶-血小板裂解液-高分散纳米颗粒复合体系。
本发明的第二方面是提供一种如前所述体系在软骨组织工程中的应用,所述体系在体内关节腔原位注射治疗后,能促进大鼠骨软骨缺损的修复。
本发明采用以上技术方案,与现有技术相比,具有如下技术效果:
本发明首次设计双重缓释的微球水凝胶体系来负载血小板裂解液并使其应用于软骨组织工程,单次注射,长效缓释,持续起效;首次根据血小板裂解液本身的特性来设计材料,且是首次发现血小板裂解液这种不均质的颗粒分布会导致水凝胶的物理特性受影响,而通过纳米粒的搭载可以很好的改善这种情况,让构建的材料更合理。
本发明设计的注射型温敏水凝胶-高分散微球体系负载血小板裂解液,在软骨应用上具有多功能,一方面对于关节炎这种退行性变,可以做为一种关节液的补充剂,另一方面对于软骨缺损的病人又可作为一种无缝的填充物。
附图说明
图1为本发明实施例1动态光散射结果;
图2为本发明实施例1动态光散射结果;
图3为本发明实施例1Zeta电位结果;
图4为本发明实施例1透射电子显微镜结果;
图5为本发明实施例2核磁共振波谱结果;
图6为本发明实施例2傅里叶红外光谱结果;
图7为本发明实施例2动态光散射结果;
图8为本发明实施例2临界胶凝温度结果;
图9为本发明实施例2流变检测结果;
图10为本发明检测实施例亲和力结果;
图11为本发明检测实施例体外降解结果;
图12为本发明检测实施例总蛋白和生长因子释放结果;
图13为本发明应用实施例ICRS大体观评分结果;
图14为本发明应用实施例ICRS组织学评分结果;
图15为本发明应用实施例II型胶原光密度总和结果。
具体实施方式
提供一种注射型负载血小板裂解液的温敏水凝胶-高分散纳米颗粒体系,体系包括:
温敏水凝胶;以及嵌入所述温敏水凝胶中负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒。
优选地,所述温敏水凝胶为三嵌段高分子共聚物聚(D,L-丙交酯)-聚(乙二醇)-聚(D,L-丙交酯)纳米胶束水凝胶。
优选地,所述温敏水凝胶在室温或者相对低温的环境中呈现液体状的溶胶态;在人体温度37℃或者相对高温的环境中呈现固体状的凝胶态。
还提供一种如前所述体系的制备方法,步骤包括:
S1、制备负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒;
S2、制备温敏水凝胶-血小板裂解液-高分散纳米颗粒复合体系。
优选地,所述制备步骤S1包括:
通过肝素和ε-聚赖氨酸的静电自组装和肝素与血小板裂解液之间的高亲和力制备负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒。
优选地,所述制备步骤S2包括:
采用Sn(Oct)2作为催化剂,引发聚乙二醇和D,L-丙交酯开环共聚,合成聚(D,L-丙交酯)-聚(乙二醇)-聚(D,L-丙交酯)三嵌段高分子共聚物;
将负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒嵌入聚(D,L-丙交酯)-聚(乙二醇)-聚(D,L-丙交酯)纳米胶束水凝胶中构建温敏水凝胶-血小板裂解液-高分散纳米颗粒复合体系。
再提供一种如前所述体系在软骨组织工程中的应用,所述体系具有延缓骨关节炎的进展和骨软骨缺损的修复作用。
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。
需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。
下面结合具体实施例对本发明作进一步说明,但不作为本发明的限定。
实施例1
负载血小板裂解液的肝素(Hep)/ε-聚赖氨酸(EPL)纳米颗粒(NPs)的制备和表征:
众所周知,PL含有复杂的蛋白质成分,其中带负电荷的白蛋白占大部分,而PL中起主要生物活性的生长因子,包括PDGF-BB、TGF-β1、bFGF均为带负电,其等电点,分别为9.8、8.9和9.6。这种不同的带电属性和不均匀的成分构成可能导致其聚集甚至沉淀。动态光散射(DLS)结果(图1和图2)显示PL的粒径分布相对分散且无规则(10nm至几微米,平均2089.96±391.23nm)。为改善PL的分散性并以温和的负载方式延缓PL中生长因子的释放,我们利用Hep与GFs之间的高亲和力以及Hep和EPL的静电自组装方式制备了负载PL的NPs溶液。空白Hep/EPL NPs的粒径表现为正态分布,平均大小为72.87±0.86nm,而负载PL的NPs的粒径为387.81±65.16nm,也呈现正态分布。此外,根据Zeta电位结果,PL溶液呈现相对电中性(-5.567±0.64mV),而Hep/EPL NPs和Hep/EPL/PL NPs溶液分别为-15.167±3.41mV和-12.333±0.47mV(图3)。不同种类蛋白质的电荷中和作用可能进一步导致PL颗粒的聚集,但是带负电荷的NPs有助于彼此间相互排斥进而提高分散性。透射电子显微镜(TEM)结果证实了相似的发现,PL颗粒的形态为微米级的聚集体,且粒径分布散乱。相比之下,Hep/EPL NPs呈现出均匀的球状,直径分布均一,为30nm。此外,尽管负载PL的NPs的颗粒大小显著低于PL(图4)。这些数据表明,通Hep/EPL NPs负载的方式可以显著改善PL颗粒的团聚和无序分布,有助于其保持均一性和稳定性。
实施例2
温敏水凝胶-血小板裂解液-高分散纳米颗粒复合体系的制备和表征:
使用Sn(Oct)2作为催化剂,对PEG引发的D,L-丙交酯进行开环共聚反应,以合成PLEL三嵌段共聚物。核磁共振波谱(1H-NMR)(图5)显示,5.10ppm处和1.55ppm处的峰分别表示质子峰(-CH-)和甲基质子峰(-CH3)。在4.30ppm处的峰表示D,L-丙交酯和PEG之间的亚甲基质子峰(-O-CH2-CH2-),在3.65ppm处的峰对应于PEG的亚甲基质子(-CH2-)。此外,傅里叶红外光谱(FTIR)图显示(图6),在1757cm-1处出现了一个强C=O峰,在1097cm-1处的吸收峰则是代表性的C-O-C扩展PEG中–OCH2CH2重复单元的振动,在3664cm-1处的吸收峰是PLEL共聚物的末端羟基(-OH)。同时,DLS测试结果证明低浓度(0.1wt%)的PLEL溶液可以单独分散球形纳米颗粒的粒径为40~50nm(图7)。以上这些结果证实,通过上述共聚方法可成功制备得到PLEL三嵌段共聚物。
为了确定不同种类的复合PLEL凝胶体系(PLEL,PLEL@PL,PLEL@NP和PLEL@PL-NP)的特性。我们首先采用试管倒置法测试凝胶体系的相变。加热时的相变过程由三种基本物理状态组成,包括溶胶,凝胶和沉淀。具体来说,溶胶-凝胶转变对应于较低的临界胶凝温度(LCGT),凝胶-沉淀转变对应于较高临界胶凝温度(UCGT),这与胶束的堆积和聚集有关,LCGT和UCGT间的温度范围即凝胶的温度区间。尽管随着浓度的增加,所有组的LCGT和UCGT之间的温度范围变宽,但在相同浓度下它们彼此不同。该范围宽度的大小排序为PLEL>PLEL@NPs>PLEL@PL-NPs>PLEL@PL(图8)。更高的浓度意味着降解速度更慢以及更长的药物释放时间。此外,为了确保所有组的凝胶都可以在低于体温(37℃)的温度下凝胶化,以便于实际的临床应用,后续的流变检测和体内外实验均选择了25%的浓度。流变结果中溶胶-凝胶转变被定义为存储模量(G')增加到大于损耗模量(G″)的点。在图9中可以观察到,所有四种水凝胶在室温(25℃)时都呈溶胶态,并在体温(37℃)附近变成凝胶态,这进一步证明了所有这些水凝胶体系室温下的可注射性及其在体内应用的原位形成凝胶的潜力。与试管倒置结果相似,与空白的PLEL凝胶(29.7℃–42.3℃)相比,直接掺入PL的PLEL@PL体系的凝胶态温度范围明显窄,为36.4℃-38.9℃。但通过将NPs引入凝胶中,可以纠正这一缺点。PLEL@NPs(33.4℃–41.2℃)和PLEL@PL-NPs(34.8℃–41.8℃)组均显示出凝胶状态与PLEL组的相近的凝胶态温度范围。
检测实施例
在本实施例中,首先通过分子对接模拟计算Hep和PL中生长因子的亲和力,结果显示Hep对三种主要的生长因子都具有较高亲和力。且亲和力排序bFGF>TGF-β1>PDGF-BB(图10)。而后分别在PBS和弹力蛋白酶溶液中研究了体外凝胶的降解以及总蛋白和三种主要生长因子的释放行为。如图11所示,四种凝胶体系在体外降解时间均达到35天以上。总体上,水凝胶在弹力蛋白溶液中比单纯的PBS中降解更快,而其中PLEL@PL凝胶降解最快,其次是PLEL@PL-NP,PLEL@NP和PLEL凝胶,这与水凝胶的表征结果相呼应,与水凝胶的稳定性相吻合。由此推测,凝胶中共混的颗粒的粒径和整体的均质性决定了凝胶的稳定性,并因此影响了它们的体外降解。另外,通过对于总蛋白和三种生长因子的释放检测(图12),发现由于不均相结构带来的不稳定性,PLEL@PL体系的释放在初期呈现出一种突释现象,符合一级释放动力学。相反,PLEL@PL-NPs凝胶体系的释放曲线则相对稳定,而没有明显的突释现象。特别是对于三种生长因子的释放,其曲线符合零级释放动力学。综上所述,即使在添加弹性蛋白酶的条件下,所有水凝胶的降解和生长因子的释放仍超过1个月,而由于骨关节炎中软骨退变和缺损的修复是一种慢性而长久的过程,这表明这种基于PLEL的水凝胶复合体系,非常适用于软骨组织工程应用。而且,与直接负载PL相比,构建PLEL@PL-NPs体系效果更优,一方面,NPs改善了PL的均相性,从而提高了PLEL凝胶的稳定性。另一方面,从PLEL@PL-NPs体系在PL控释方面呈现为一种双重缓释的形式:首先从凝胶中释放PL-NP,然后从PL-NP释放PL。
应用实施例
在大鼠股骨髁间钻孔构建骨软骨缺损模型,以模拟OA中后期的骨软骨缺损情况。将四种水凝胶体系分别植入缺损处,于手术后6周和12周,收集股骨髁标本进行分析。大体观评估发现(图13),缺损组术后6-12周仍表现为明显的不愈合(ICR大体观评分从1.125升高到2.375)。而PLEL和PLEL@NPs组的缺损在术后第6周和第12周时则被观察到有大量纤维样组织覆盖,其ICR评分与缺损组相比,有明显提高(p<0.05)。在PL负载的凝胶治疗组中,可观察到缺陷部位被大量组织替代,表面光滑。特别是在PLEL@PL-NPs组中,缺损处被紧密的软骨样组织所替代,再生的软骨样组织与周围的软骨完全整合(ICR大体观评分从6.125增至10.75)。
组织学上可见缺损组第6和12周关节表面无明显软骨再生,仅仅被覆一层薄薄的纤维组织。在PLEL和PLEL@NPs组中,可见缺损区域在第6周时被大量纤维组织充满,而在第12周时几乎没有观察到明显的胶原沉积。而在PLEL@PL和PLEL@PL-NPs组中,缺损处在第6周时被大量纤维组织和软骨样组织所混合填充。然而,在第12周时,两组均出现了透明软骨样组织。特别是PLEL@PL-NPs治疗的缺损部位表现为,几乎完全被透明软骨替代。组织学评分评估显示,从6周到12周,PLEL@PL-NPs组的平均ICR组织学评分最高(10.63至15.38),其次是PLEL@PL(9.375至11.88),PLEL@NPs(7.5至9.75)和PLEL组(2.125至3.125)(图14)。另外,根据免疫组织化学染色定量结果显示,在PLEL@PL-NPs组中观察到高密度的软骨特异性胶原蛋白(II型胶原蛋白),其次是PLEL@PL,PLEL@NPs和PLEL组(图15)。综上所述,体内实验结果表明,PLEL@PL-NP体系可有效修复软骨缺损。
以上所述仅为本发明较佳的实施例,并非因此限制本发明的实施方式及保护范围,对于本领域技术人员而言,应当能够意识到凡运用本发明说明书所作出的等同替换和显而易见的变化所得到的方案,均应当包含在本发明的保护范围内。
Claims (1)
1.一种注射型负载血小板裂解液的温敏水凝胶-高分散纳米颗粒体系在制备治疗骨关节炎和骨软骨缺损制剂中的应用,其特征在于,所述体系包括:
温敏水凝胶;以及分散于所述温敏水凝胶中负载血小板裂解液的纳米颗粒;所述温敏水凝胶在室温或者相对低温的环境中呈现液体状的溶胶态;在人体温度37℃或者相对高温的环境中呈现固体状的凝胶态;
所述纳米颗粒为肝素/ε-聚赖氨酸纳米颗粒;
所述温敏水凝胶为三嵌段高分子共聚物聚(D,L-丙交酯)-聚(乙二醇)-聚(D,L-丙交酯)纳米胶束水凝胶;
所述体系的制备步骤包括:
S1、制备负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒;
S2、将S1所制备的纳米颗粒分散于温敏水凝胶中,即得温敏水凝胶-血小板裂解液-高分散纳米颗粒复合体系;
所述制备步骤S1包括:
通过肝素和ε-聚赖氨酸的静电自组装和肝素与血小板裂解液之间的高亲和力制备负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒;
所述制备步骤S2包括:
采用Sn(Oct)2作为催化剂,引发聚乙二醇和D,L-丙交酯开环共聚,合成聚(D,L-丙交酯)-聚(乙二醇)-聚(D,L-丙交酯)三嵌段高分子共聚物;
将负载血小板裂解液的肝素/ε-聚赖氨酸纳米颗粒嵌入聚(D,L-丙交酯)-聚(乙二醇)-聚(D,L-丙交酯)纳米胶束水凝胶中构建温敏水凝胶-血小板裂解液-高分散纳米颗粒复合体系。
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