CN115054579B - 一种活性氧响应性梯度递药的纳米粒子关节炎药物及其制备方法 - Google Patents
一种活性氧响应性梯度递药的纳米粒子关节炎药物及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种活性氧响应性梯度递药的纳米粒子关节炎药物及其制备方法。该纳米粒子材料制备步骤包括:采用超声乳化‑溶剂挥发法制备包覆有疏水软骨修复类药物的聚富马酸丙二醇酯‑聚酮缩硫醇(PPF‑PTK)纳米粒子,并在PPF‑PTK纳米粒子表面利用PPF残留双键修饰超支化聚赖氨酸,并利用粒子表面富含正电荷的超支化聚赖氨酸吸附负电亲水性药物。该纳米粒子由于表面的正电性,可以吸附在负电的软骨基质表面,实现在关节腔的长时间驻留,且具有活性氧消除作用,并实现内外层药物的梯度响应释放。该纳米粒子具有良好的生物相容性,降解产物对人体无害,在鼠骨关节炎模型中具有良好的治疗效果,可以用于活性氧水平偏高的病理组织环境,能广泛应用于生物医药及组织工程等领域。
Description
技术领域
本发明涉及一种活性氧响应性的梯度释药聚合物纳米粒子的制备方法及应用,属于生物医用材料技术领域。
背景技术
骨关节炎是一种非常普遍的退行性关节疾病,多发于老年人群和肥胖人群,给病人生活和社会经济造成极大的负担。骨关节炎的特征在于软骨细胞外基质的降解、滑膜炎症、软骨下骨的重塑以及关节功能丧失。由于软骨的自我再生能力有限,目前关节炎的临床治疗主要集中在缓解疼痛和延缓关节炎的发展。其临床药物治疗主要为口服或关节腔注射非甾体抗炎药(如双氯芬酸和布洛芬)和特异性环氧合酶抑制剂(如塞来昔布)。然而,口服药物会导致在非目标部位的积累和全身毒性,关节注射由于滑液的快速交换,药物在关节中的保留时间不足,因此需要过量服用,这可能会导致胃肠道和心血管并发症、骨质疏松等。
近年来,越来越多的研究将重点放在纳米药物上,纳米药物具有载药率高、保留时间长、靶向精准、治疗效果好等显着优势。目前临床研究的纳米药物主要是载有已上市用于治疗骨关节炎的药物的非功能性纳米/微米级材料,主要目的是延长药物在关节腔的滞留时间,减少全身扩散。在这方面,纳米药物载体在全身或关节内给药方面仍存在一些障碍,包括在其他器官中的非特异性积累、药物过早释放和暴释。因此,目前许多研究致力于开发能够响应于疾病部位特异性特征(如微环境中高表达信号)的纳米药物载体,实现治疗剂的刺激响应性递送,并同时实现对疾病微环境的调控。关节炎往往伴有过氧化氢、羟基自由基等活性氧(ROS)物质的过表达,因此可以消耗ROS的纳米载体被广泛用于对关节炎的治疗。但响应释放药物,实现药物简易、高效负载是纳米载体亟需解决的问题。
赖氨酸是AB2型不对称氨基酸,能够通过热均聚形成聚阳离子聚合物。作为一种天然氨基酸,赖氨酸的主要优点之一是毒性较低。在生理条件下,L-赖氨酸及其手性形式D-赖氨酸处于两性离子形式,可以发生静电相互作用。聚赖氨酸因其具有优异的广谱抗菌性,在日本等国家已被用于产业化的食品添加剂中,而α型的聚赖氨酸(PLL)常用于增强细胞粘附。超支化聚赖氨酸同时具有以上两种结构单元。由于其聚阳离子的特性,目前超支化聚赖氨酸在基因传递、药物载体领域的研究较为广泛,用于组织修复领域的研究则较少见。
本发明将超支化聚赖氨酸固定于ROS响应粒子表面,通过物理包埋和静电吸附两种简单的制备手段,实现两种药物的负载和梯度释放、响应释放。
发明内容
本发明的目的在于针对现有技术的不足,提供一种具有活性氧响应性的梯度释药的聚合物纳米粒子的制备方法。
本发明的另一目的是提供上述具有活性氧响应性的载药聚合物纳米粒子在关节炎中的应用。具有活性氧响应性的载药聚合物纳米粒子可以消耗骨关节炎中过表达的ROS,调控炎症微环境,实现骨关节炎的治疗,有望于应用于关节炎药物中。
可以通过以下技术方案实现本发明的目的。
本发明提供的具有活性氧响应性的梯度释药的聚合物纳米粒子的制备方法,包括以下步骤:
(1)将两种聚合物和药物充分溶解在油性有机溶剂中,配制成均一溶液;将乳化剂溶于去离子水中,配制成透明的乳化剂水溶液;将光引发剂溶解在去离子水中,配制成光引发剂水溶液;
(2)活性氧响应性载药聚合物纳米粒子的制备:将上述溶解有两种聚合物和药物的油相溶液与乳化剂水溶液混合,并通过磁力搅拌均匀混合,得到粗乳液;将装有粗乳液的容器置于冰水浴中,用超声破碎仪对混合溶液超声乳化即制得均匀的活性氧响应性聚合物乳液;将聚合物乳液加入至光引发剂水溶液中,紫外辐射;
(3)将上述活性氧响应性聚合物乳液常温搅拌使有机溶剂充分挥发,之后离心弃去上清再用去离子水离心洗涤,最后用去离子水分散离心沉淀物,即可得到去除表面活性剂及游离药物的活性氧响应性聚合物载药纳米粒子的水分散液;
(4)超支化聚赖氨酸水溶液的制备;
(5)将超支化聚赖氨酸固定在纳米粒子表面;
(6)将亲水性药物吸附在接枝超支化聚赖氨酸的纳米粒子表面;
步骤(1)中两种聚合物分别为聚富马酸丙二醇酯(PPF)和活性氧响应性聚合物聚酮缩硫醇(PTK),数均分子量分别为1.6~3.0kDa、1.0~1.8kDa;
步骤(1)中两种聚合物PPF和PTK的质量比为1:1~1:3;
步骤(1)中配得的聚合物油性溶液的浓度是5~30mg/mL;
步骤(1)中所述的乳化剂为聚乙烯吡咯烷酮(PVP)或聚乙烯醇(PVA);
步骤(1)中配得的表面活性剂水相溶液的浓度是5~40mg/mL;
步骤(1)中所述的光引发剂为苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐(LAP);
步骤(1)中配得的光引发剂水相溶液的浓度是1~20mg/mL;
步骤(1)中所述的药物为疏水性促软骨形成药物,如1-甲基-8-[4-(2-quinolinylmethoxy)苯氧基]-4,5-二氢-1H-噻吩并[3,4-g]吲唑-6-甲酰胺(TD-198946)、2-([1,1-联苯]-4-基氨基甲酰)苯甲酸(Kartogenin,KGN);
步骤(1)中配得的药物浓度为3~20mg/mL;
步骤(1)中溶解聚合物和药物的油性有机溶剂可以是二氯甲烷或三氯甲烷;
步骤(2)中水油两相的体积比为3:1~12:1;
步骤(2)中用超声破碎仪进行超声乳化,冰浴中进行,功率30~100W,时间1~5min;
步骤(2)中紫外固化系统的UVA强度50~100mW cm-2,时间2~8min;
步骤(3)中高速离心的转速为11,000rpm;
步骤(4)中超支化聚赖氨酸溶液的固含量为1~10wt%;
步骤(5)中加热的温度50~80℃,时间3~12h;
步骤(6)中亲水性药物为抗炎类药物,如地塞米松磷酸钠、双氯酚酸、乙酰水杨酸、盐酸多西环素。
步骤(6)中药物水溶液浓度为3~20mg/mL。
所述的活性氧响应性的梯度释药聚合物纳米粒子的活性氧响应性来源于其中PTK聚合物主链中大量的酮缩硫醇基团;
所述的活性氧响应性的梯度释药聚合物纳米粒子的粒径大小为200~700nm;
所述的活性氧响应性的梯度释药聚合物纳米粒子的载药率为3~15%。
所述的活性氧响应性的梯度释药聚合物纳米粒子适用于骨关节炎、肺炎、心肌梗死等炎症疾病中的一种。
所述的活性氧响应性的梯度释药聚合物纳米粒子的制备方法为超声乳化-溶剂挥发法。其原理为,将聚合物和疏水药物溶解到与水不互溶且易挥发的有机溶剂中,加入到含有分散剂的连续相中,并通过超声细胞破碎仪得到均匀的聚合物乳化液滴,形成离散的水包油液滴,疏水药物包在聚合物液滴中。通过调节超声时间和超声探头的振幅可以控制液滴的大小和均匀程度。有机溶剂会缓慢扩散到连续相中,并在界面处挥发,此过程中,聚合物微球逐渐得到固化,经过离心洗涤除去粒子表面的乳化剂后,经过干燥处理即可得到一定尺寸的聚合物载药粒子。
本发明将含有活性氧响应性基团的聚合物制备为活性氧响应的聚合物纳米粒子,具有良好的生物相容性和降解性能。该纳米粒子内部包载疏水促软骨修复类药物,粒子表面接枝富含正电荷的超支化聚赖氨酸,并吸附亲水负电抗炎药物。该纳米药物载体能够被动释放表面药物,并响应于炎症性疾病部位过量表达的ROS缓释内部药物,从而实现不同功能药物的梯度释放。并且该纳米粒子能够消耗ROS,调控炎症微环境中ROS水平,在释放药物的同时发挥治疗作用。载体表面的超支化聚赖氨酸,赋予其软骨表面的结合能力,提高载体在关节腔的驻留。因此,该抗炎粒子在关节炎治疗中具有广阔的应用前景。
所述的活性氧响应性的梯度释药聚合物纳米粒子的应用原理是,粒子表面正电的HBPL使得粒子被动靶向至负电的损伤暴露的软骨基质,表面吸附的负电抗炎药物释放,活性氧响应性纳米粒子在活性氧作用下逐渐降解,内部包载的软骨修复药物缓慢释放。实现快速抗炎,后期促修复的双重功能。
与现有的骨关节炎治疗聚合物纳米粒子相比,本发明制备的活性氧响应性的梯度释药聚合物纳米粒子具有以下优点:
1)本发明中,纳米粒子主体材料中的活性氧响应性聚合物,其活性氧响应基团数量多,可以大量消耗炎症疾病微环境中的活性氧,调控炎症微环境,材料本身具有治疗效果。
2)本发明中,纳米粒子表面的正电超支化聚赖氨酸,可以被动靶向粒子至负电损伤的软骨基质,实现靶向治疗软骨,靶向递送药物,并延长粒子在关节腔的驻留时间,避免重复注射。
3)本发明中,纳米粒子表面结合抗炎药物,可以快速被动释放;内部包载软骨修复药物,随着纳米粒子消除活性氧发生降解,可以缓慢响应性释放。活性氧响应性的梯度释药聚合物纳米粒子在炎症早期快速释放抗炎药物,后期缓慢释放修复类药物,实现梯度释药以及抗炎促修复双重功能。
4)本发明中,纳米粒子的形貌规则、载药率高,具有良好的生物相容性。载药纳米粒子的关节腔注射,可以降低药物在非靶向部位的积累和毒性,减少药物带来的胃肠道和心脏疾病的风险。
附图说明
图1为聚酮缩硫醇(PTK),聚富马酸丙二醇酯(PPF)和超支化聚赖氨酸(HBPL)的结构;
图2为聚酮缩硫醇(PTK)和聚富马酸丙二醇酯(PPF)形成纳米粒子的原理及接枝超支化聚赖氨酸(HBPL)的原理示意图;
图3为实施例1所制得的活性氧响应性的梯度释药聚合物纳米粒子的粒径分布图;
图4为实施例2所制得的活性氧响应性的梯度释药聚合物纳米粒子的透射电镜图;
图5为实施例1所制得的活性氧响应性的梯度释药聚合物纳米粒子的DPPH自由基清除数据;
图6为实施例1所制得的活性氧响应性的梯度释药聚合物纳米粒子在水中释放磷酸地塞米松和在100mM羟基自由基中释放地塞米松的释放曲线;
具体实施方式
以下结合实施例进一步说明本发明的技术方案,但这些实施例并不用于限制本发明。
实施例1:
将400μL溶解有4mg PPF、8mg PTK和1.5mg KGN的二氯甲烷溶液加入到5mL 1%PVP水溶液中,所用聚合物结构如图1。超声波探头底部放在水-油界面处,在冰浴条件下,以输出功率65W超声分散1分钟。所得乳液在搅拌下被立即转移到30mL的5‰(w/v)LAP水溶液,并在紫外辐射箱中以75mW cm-2紫外固化4分钟,迈克尔加成反应原理如图2所示。所得纳米粒子水溶液在室温下搅拌挥发有机溶剂一夜后,以11,000rpm转速高速离心粒子,加入超纯水洗涤粒子,离心弃去上清,共洗涤三次。将所得的PPF-PTK纳米粒子与5mg/mL HBPL水溶液共孵育,加入10μL三乙胺,于50度下孵育12小时,将HBPL接枝到粒子表面。离心水洗PPF-PTK-HBPL粒子,所得粒子与5mg/mL地塞米松磷酸钠水溶液孵育4小时,离心水洗粒子。通过动态光散射技术测得粒子的粒径大小为531nm(如图3),电位为10.7mV,扫描电镜和透射电镜显示粒子呈均匀的球形。如图5所示,通过检测0-1000μg/mL纳米粒子与广谱自由基1,1-二苯基-2-三硝基苯肼(DPPH)孵育24h后上清液的紫外吸收,显示粒子有较好的DPPH清除能力且其自由基清除能力随粒子浓度增加而增加。通过紫外光谱检测到纳米粒子内部疏水药物的载药率为4.4%,外部吸附药物的载药率为3.5%,并分别在水和100mM羟基自由基中检测磷酸地塞米松和地塞米松的释放速率,其释放曲线分别如图6(a)、(b)所示。外部吸附药物在水中逐渐释放,内部疏水药物在响应性粒子在ROS下逐渐结构松散、崩解时释放,实现内外药物的层级释放。
实施例2:
将400μL溶解有6mg PPF、6mg PTK和1.5mg KGN的二氯甲烷溶液加入到5mL 1%PVP水溶液中。超声波探头底部放在水-油界面处,在冰浴条件下,以输出功率70W超声分散2分钟。所得乳液在搅拌下被立即转移到30mL的5‰(w/v)LAP水溶液,并在紫外辐射箱中以75mWcm-2紫外固化6分钟。所得纳米粒子水溶液在室温下搅拌挥发有机溶剂一夜后,以11,000rpm转速高速离心粒子,加入超纯水洗涤粒子,离心弃去上清,共洗涤三次。将所得的PPF-PTK纳米粒子与7mg/mL HBPL水溶液共孵育,加入10μL三乙胺,于50度下孵育10小时,将HBPL接枝到粒子表面。离心水洗PPF-PTK-HBPL粒子,所得粒子与10mg/mL地塞米松磷酸钠水溶液孵育4小时,离心水洗粒子。通过动态光散射技术测得粒子的粒径大小为507nm,电位为12.3mV,透射电镜显示粒子呈均匀的球形,如图4所示。通过紫外光谱检测到纳米粒子内部疏水药物的载药率为4.4%,外部吸附药物的载药率为4%。
Claims (10)
1.一种活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于,所述的制备方法,包括以下步骤:
(1)将聚富马酸丙二醇酯、活性氧响应性的聚酮缩硫醇和疏水药物充分溶解在油性有机溶剂中,配制成均一溶液;将乳化剂溶于去离子水中,配制成透明的乳化剂水溶液;将光引发剂苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐(LAP)溶解在去离子水中,配制成光引发剂水溶液;
(2)将所述有机溶液与所述乳化剂水溶液混合,并通过搅拌均匀混合,得到粗乳液;将装有粗乳液的容器置于冰水浴中,用超声破碎仪对粗乳液超声乳化即制得均匀的活性氧响应性聚合物乳液;将聚合物乳液加入至光引发剂水溶液中,紫外辐射;
(3)将(2)处理后的聚合物乳液常温搅拌使有机溶剂充分挥发,之后离心弃去上清再用去离子水离心洗涤,最后用去离子水分散离心沉淀物,即得到去除乳化剂及游离药物的活性氧响应性聚合物载药纳米粒子的水分散液;
(4)制备超支化聚赖氨酸水溶液;并将超支化聚赖氨酸固定在(3)所得纳米粒子表面;
(5)将亲水性药物吸附在(4)所得接枝超支化聚赖氨酸的纳米粒子表面;得到活性氧响应性梯度递药的纳米粒子关节炎药物。
2.根据权利要求1所述的活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于,步骤(1)所述的聚富马酸丙二醇酯和活性氧响应性聚合物聚酮缩硫醇数均分子量分别为1.6~3.0kDa和1.0~1.8kDa;所述的疏水药物为疏水性促软骨形成药物,为1-甲基-8-[4-(2-quinolinylmethoxy)苯氧基]-4,5-二氢-1H-噻吩并[3,4-g]吲唑-6-甲酰胺(TD-198946)、2-([1,1-联苯]-4-基氨基甲酰)苯甲酸(Kartogenin,KGN)中的至少一种;所述的油性有机溶剂为二氯甲烷、三氯甲烷中的至少一种;所述的乳化剂为聚乙烯吡咯烷酮(PVP)、聚乙烯醇(PVA)中的至少一种。
3.根据权利要求1所述的活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于:步骤(1)中所述的均一溶液中聚合物的浓度为5~30mg/mL;所述的疏水药物浓度为3~20mg/mL;所述的乳化剂水溶液的浓度为5~40mg/mL;所述的光引发剂水溶液的浓度为1~20mg/mL。
4.根据权利要求1所述的活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于:步骤(1)中聚富马酸丙二醇酯和活性氧响应性聚合物聚酮缩硫醇的质量比为1:1~1:3。
5.根据权利要求1所述的活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于:步骤(2)中水油两相的体积比为3:1~12:1。
6.根据权利要求1所述的活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于:步骤(2)中用超声破碎仪进行超声乳化,冰浴中进行,功率30~100W,时间1~5min。
7.根据权利要求1所述的活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于:步骤(2)中用紫外固化系统进行紫外辐照,强度50~100mW cm-2,时间2~8min。
8.根据权利要求1所述的活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于:步骤(4)中超支化聚赖氨酸溶液的固含量为1~10wt%。
9.根据权利要求1所述的活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于:步骤(4)中将步骤(3)处理得到的活性氧响应性载药聚合物纳米粒子浸泡在超支化聚赖氨酸水溶液中,加入催化剂三乙胺,加热50~80℃,时间3~12h。
10.根据权利要求1所述的活性氧响应性梯度递药的纳米粒子关节炎药物的制备方法,其特征在于,步骤(5)中所述的亲水性药物为抗炎类药物:塞米松磷酸钠、双氯酚酸、乙酰水杨酸、盐酸多西环素中的至少一种,所述的药物溶液浓度为3~20mg/mL。
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