CN114524951B - 一种聚多巴胺药物递送载体及其应用 - Google Patents
一种聚多巴胺药物递送载体及其应用 Download PDFInfo
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Abstract
本发明提供了一种聚多巴胺药物递送载体及其应用。具体而言,本发明提供了一种聚多巴胺微凝胶,所述的聚多巴胺微凝胶由主要单体与共聚单体共聚合制备得到,所述的主要单体包括PEGMA和APTAC,所述的共聚单体包括DPMA。本发明还提供了所述的聚多巴胺微凝胶制备预防或治疗眼部疾病的药物中的应用。
Description
技术领域
本发明属于生物医药领域,具体涉及一种聚多巴胺药物递送载体及其应用。
背景技术
眼球是人体的重要器官,眼表上皮细胞作为眼的首道屏障,其对维持眼表面的健康发挥着关键作用。眼表上皮细胞结构异常和/或功能障碍,将导致眼表上皮疾病。眼表直接暴露在外界环境中,仅由7~10μm泪膜将角结膜上皮细胞与周围环境因素隔离,有害物质极易进入眼睛造成流泪、干涩、眼痒、眼红、眼痛、异物感等不适,严重影响人们日常工作和生活。眼表疾病概指损伤眼表结构及功能的疾病,是最常见的眼科疾病,临床上常见的眼表疾病主要有眼表(角膜和结膜)的感染性炎症(如细菌、病毒、衣原体感染)、干眼病、过敏性角结膜炎及眼表面外伤等。
干眼病是一种最常见的多因素眼表慢性疾病,它是由于泪液的量或质的异常引起的泪膜不稳定,并伴有眼表损害、眼部不适、视觉受损等症状的一种眼科常见疾病。引起干眼病的病因有很多,眼表面的病理性改变,免疫因素相关的炎症反应、性激素水平的改变、细胞凋亡以及环境污染的影响是干眼病发生发展的常见因素。轻度的干眼病通常由环境因素、个人习惯(如长期处于空调环境中,长期使用电脑、手机等)等引起,仅出现较轻的症状而没有相关的眼表损害,通过及时改善这些因素,眼部的不适感可减轻;中度或重度的干眼病通常有明确的局部或全身病因(例如干燥综合征、眼表化学及热烧伤、睑缘炎、移植物抗宿主病等),其发生发展的机制较为复杂。与干眼病发病有关联的眼表结构(结膜、角膜、副泪腺和睑板腺)、主泪腺以及这些组织结构之间的神经连接由于其紧密的解剖、功能关系组成了一个整体功能单位,它们能协同产生对泪液分泌的调控作用,继而维护眼表的健康,其中任一方面的损伤或缺失都会引起眼表泪膜的完整性和功能性破坏,从而会导致干眼病的不适症状出现,同时,泪膜的损伤会引起眼表正常的防御及修复机制的损伤,致使眼表组织长期处于慢性炎症状态中。
干眼病发病率高、发病机制复杂、病程反复且严重影响患者的生活质量给个人与社会带来巨大的负担。目前,常用的治疗方法有药物点眼治疗和手术治疗。但药物点眼治疗通常只是补充泪液量、促进泪液分泌或者广谱抗炎,并不能打破干眼的炎症“恶性循环”,使干眼症得到治愈,而且点眼液中含有的防腐剂等成分反而会改变眼表环境加重干眼,手术治疗还会改变原有的眼表结构,而且治疗效果并非十分确切。因此,寻求干眼病的更有效疗法是目前亟待解决的眼科问题之一。
发明内容
针对现有技术的不足,本发明的一个目的在于提供一种用于治疗眼表疾病的药物递送载体;本发明的另一个目的在于提供一种治疗干眼病的方法。
为实现上述目的,本发明采用了如下技术方案:
本发明第一方面提供了一种制备聚多巴胺微凝胶的方法,所述的方法包括:
(1)将主要单体、光引发剂加入反应容器中,溶解于水;
(2)将反应容器封口后置于光照下进行聚合反应;
(3)将共聚单体的乙醇溶液注射进入前面所述的反应容器,继续反应;
所述的主要单体包括PEGMA和APTAC,所述的共聚单体包括DPMA。
本领域技术人员将理解,术语“反应容器”是指适于本发明化学反应并且与其兼容的任何种类和尺寸的试管、桶、培养瓶、壶、箱或容器。反应容器可方便地选自标准实验室设备,例如试管、离心管、单颈或多颈圆底或梨形培养瓶等,其通常由玻璃或合适的耐溶剂聚合物制成。用于扩大规模和生产目的的反应容器可选自中试规模和生产规模的反应器,其可以是玻璃内衬的或由不锈钢或其他合金生产而得。
作为一种优选的实施方式,(2)中所述的光为406nm的可见光。
作为一种更为优选的实施方式,(2)中聚合反应的时间为2小时。
作为一种优选的实施方式,(3)中继续反应的时间为4小时。
作为一种更为优选的实施方式,所述的共聚单体与所述的主要单体的摩尔比为1:10。
本发明第二方面提供了一种聚多巴胺微凝胶,所述的聚多巴胺微凝胶由主要单体与共聚单体共聚合制备得到,所述的主要单体包括PEGMA和APTAC,所述的共聚单体包括DPMA。
作为一种优选的实施方式,所述的聚多巴胺微凝胶由本发明第一方面所述的方法制备获得。
作为一种优选的实施方式,所述的聚多巴胺微凝胶的平均流体动力学直径为356nm。
本文所用的术语“流体动力学直径”是指以扩散速度与颗粒的扩散速度相同的假设硬球的直径。
作为一种更为优选的实施方式,所述的聚多巴胺微凝胶Zeta电位为+15.9mV。
如本文所用,术语“Zeta电位”是指浸在导电液体中的固体颗粒表面之间存在的电位差。
作为一种优选的实施方式,PEGMA、APTAC和DPMA的摩尔比为3:7:1。
术语“摩尔比”用于表示一种化合物相对于另一种化合物的摩尔化学计量。摩尔比可以通过1H NMR确定。
本发明第三方面提供了一种药物体系,所述的药物体系由本发明第二方面所述的聚多巴胺微凝胶包载带负电荷的药物得到。
作为一种优选的实施方式,所述的带负电荷的药物包括MCC950。
本发明第四方面提供了一种制备本发明第三方面所述的药物体系的方法,所述的方法包括:
(1)将带负电荷的药物、主要单体、光引发剂加入反应容器,溶解于水;
(2)将反应容器封口后置于光照下进行聚合反应;
(3)将共聚单体的乙醇溶液注射进入前面所述的反应容器,继续反应;
所述的主要单体包括PEGMA和APTAC,所述的共聚单体包括DPMA。
在一些实施方案中,聚合反应通过自由基引发剂启动。在一些情况下,反应可通过光(例如,紫外光或太阳光)而不用引发剂化合物启动。在优选的实施方案中,所述自由基引发剂可为光引发剂化合物。可使用任何合适的光引发剂,例如Bowman等人使用的那些,例如美国专利7,288,608和WO 2012/103445。在一些实施方案中,所述光引发剂为苯基-2,4,6-三甲基苯甲酰基亚膦酸锂(LAP)、苯基-2,4,6-三甲基苯甲酰基亚膦酸钠(NAP)、或2-羟基-1-[4-(2-羟基乙氧基)苯基]-2-甲基-1-丙酮、1-羟基-环己基-苯基-酮或2,2-二甲氧基-1,2-二苯基乙-1-酮。
作为一种优选的实施方式,选择使用的光的波长以匹配光引发剂的激发波长。因此,在一些实施方案中,通过使光引发剂暴露于具有与光引发剂的激发波长匹配的波长的光来引发聚合反应。
在LAP的情况下,所述光的波长为365nm-405nm,其中,406nm在可接受的范围内。在NAP的情况下,所述光的波长为大约372nm,其中360nm或380nm在可接受的范围内。在2-羟基-1-[4-(2-羟基乙氧基)苯基]-2-甲基-1-丙酮的情况下,所述光的波长为大约276nm或331nm。在1-羟基-环己基-苯基-酮的情况下,所述光的波长为大约246nm、280nm或333nm。在2,2-二甲氧基-1,2-二苯基乙-1-酮的情况下,所述光的波长为大约254nm或337nm。
作为一种优选的实施方式,所述的光引发剂为LAP,更为优选的,(2)中所述的光为406nm的可见光。
作为一种优选的实施方式,(2)中聚合反应的时间为2小时。
作为一种更为优选的实施方式,(3)中继续反应的时间为4小时。
本发明第五方面提供了如下任一项所述的应用:
(1)本发明第二方面所述的聚多巴胺微凝胶在制备抗炎的药物中的应用;
(2)本发明第三方面所述的药物体系在制备抗炎的药物中的应用;
(3)本发明第二方面所述的聚多巴胺微凝胶在制备抗氧化的药物中的应用;
(4)本发明第三方面所述的药物体系在制备抗氧化的药物中的应用
(5)本发明第二方面任一项所述的聚多巴胺微凝胶在制备预防或治疗眼部疾病的药物中的应用;
(6)本发明第三方面所述的药物体系在制备预防或治疗眼部疾病的药物中的应用;
(7)本发明第二方面任一项所述的聚多巴胺微凝胶在制备预防或治疗ROS相关疾病的药物中的应用;
(8)本发明第三方面所述的药物体系在制备预防或治疗ROS相关疾病的药物中的应用。
作为一种优选的实施方式,所述的眼部疾病为包括眼表疾病、眼前节疾病、眼底病变、眼眶疾病。
术语“眼表疾病”(包括“干眼病”或“干眼综合征”)是指与泪液分泌减少或不足或泪膜蒸发增加或过度相关的、或者由其引起的一种或多种疾病,或两者兼而有之,其特征是眼睛发红、发痒和灼热。眼表疾病进一步定义为包括干眼病、神经营养性眼表疾病、睑板腺疾病、眼表感染性炎症、过敏性角结膜炎、眼表面外伤。
术语“眼前节疾病”是指引起或由角膜或结膜上皮中的至少一个的物理损伤、包括角膜或结膜上皮的细胞的再生率降低和结膜上皮中腺体分泌物的减少导致的疾病。在本发明中,所述的眼前节疾病包括但不限于结膜炎、角膜炎、角膜病、虹膜炎、睫状体炎、青光眼、白内障。
在本发明中,眼底病变主要为在视网膜及/或脉络膜显现的病变。具体可例如为:因高血压与动脉硬化衍生的眼底变化;视网膜中央动脉阻塞症、视网膜中央静脉阻塞症、视网膜分支静脉阻塞症等视网膜静脉阻塞症;糖尿病视网膜病变、糖尿病黄斑水肿、糖尿病黄斑病变、伊尔斯氏病、柯氏症等先天性视网膜血管异常;冯希伯氏症、无脉症;黄斑病变(中心性浆液性脉络视网膜病变、黄斑囊样水肿、老年性黄斑病变、黄斑裂孔、近视性黄斑退化、视网膜玻璃体界面黄斑病变、药物毒性黄斑病变、遗传性黄斑病变等);裂孔性、牵引性、渗出性等视网膜剥离、视网膜色素变性症、早产儿视网膜病变等。
在本发明中,眼眶疾病为发生于眼眶内组织的病变,包括但不限于眼眶炎症、眼眶肿瘤、眼眶外伤、眼眶先天性疾病,眼眶代谢和内分泌性疾病、眼眶寄生虫类疾病。
作为一种更为优选的实施方式,所述的眼表疾病为眼表疾病。
在本发明的具体实施例中,所述的眼表疾病为干眼病,包括但不限于偶发性干眼病、顽固性干眼病、年龄相关性干眼病。
本说明书中所使用的“治疗”是指将所述的聚多巴胺微凝胶或所述的药物体系给药到有不期望病症的受试者或系统。病症可能包括疾病或紊乱。“预防”是指将所述的聚多巴胺微凝胶或所述的药物体系给药于处于病症危险中的受试者或系统。病症可能包括疾病或紊乱的倾向。向受试者给药的效果可以是但不限于停止该病症的一个或多个症状、减轻或预防该病症的一个或多个症状、减轻该病症的严重程度、病症完全消融,使某一特定事件或特征的发展或进展稳定或延缓或者使某一特定事件或特征发生的可能性最小化。
术语“受试者”是指任何动物(例如,哺乳动物),包括,但不限于,人类、非人灵长类、犬、猫、啮齿动物等。进一步,受试者是人类受试者。术语“受试者”、“个体”和“患者”在本文中可互换使用。因此,术语“受试者”、“个体”和“患者”涵盖患有疾病(例如,干眼病)的个体。
在某些情况下,ROS是代谢活动(如细胞呼吸)过程中产生的副产物。例如,ROS和钙(如Ca2+)的过量积累可能导致线粒体通透性转换孔(mPTP)的开放和线粒体内膜电位的降低。这可能导致线粒体肿胀和随后的破裂。另外地或可选择地,促死亡信号可能会被释放出来(例如,细胞色素c),这可以导致细胞凋亡。过量的ROS可引起DNA损伤、促进癌细胞增殖、抑制三磷酸腺苷(ATP)的合成等。因此,过量的ROS可能会影响线粒体的功能和某些细胞的功能。在一些实施例中,过量的ROS可能与一种或以上疾病相关。例如,心肌细胞需要大量的能量(由ATP提供)来进行心脏兴奋收缩耦合。线粒体功能失调可能导致心脏疾病或紊乱。
在本发明中,所述的ROS相关疾病包括但不限于心血管疾病、阿尔茨海默病、神经退行性疾病、癌症、糖尿病、不孕症(例如男性不育)等,或其任意组合。具体而言,心血管疾病可包括但不限于心绞痛、心肌梗死、中风、心力衰竭、高血压性心脏病、风湿性心脏病、心肌病、心律失常、先天性心脏病、心肌炎、外周动脉疾病、血栓栓塞疾病、静脉血栓形成等,或其任意组合。癌症可包括但不限于非小细胞肺癌、横纹肌肉瘤、乳腺癌、膀胱癌、结直肠癌、肾癌、白血病、肝癌、肺癌、淋巴瘤、胰腺癌、前列腺癌、皮肤癌、甲状腺癌、黑色素瘤、口腔癌和口咽癌、子宫癌等,或其任意组合。作为另一个例子,本发明所述聚多巴胺微凝胶或药物体系可以用于防止癌症及/或减缓衰老过程。具体地,本发明所述聚多巴胺微凝胶或药物体系可以用于,减缓对象的至少一个身体部位的衰老过程,例如大脑、心脏、肺、肾、皮肤等。
本发明的有益效果:
本发明提供了一种聚多巴胺微凝胶,所述聚多巴胺微凝胶作为一种药物载体,在ROS相关疾病中可以发挥抗炎、抗氧化治疗效果。所述的聚多巴胺微凝胶还可以延长药物在眼表滞留时间。
本发明提供了一种药物体系,所述的药物体系由所述的聚多巴胺微凝胶包载带负电荷的药物得到。在本发明的具体实施例中,所述的聚多巴胺微凝胶为PPAD3-7-1,所述的带负电荷的药物为MCC950。本发明制备的MCC950@PPAD3-7-1微凝胶,可以同时发挥清除ROS与抑制NLRP3炎症小体的作用,从而减少过度生成的ROS所造成的组织损伤作用,以及NLRP3炎症小体激活引起的炎症反应。该协同作用可以减少药物用量,发挥显著的治疗效果。
附图说明
图1为聚多巴胺微凝胶的理化性质测定结果图,其中,图A是微凝胶粒径分析图,图B是微凝胶zeta电位分析图,图C是PPAD5-5-1的形态分析图,图D是PPAD4-6-1的形态分析图,图E是PPAD3-7-1的形态分析图,图F是PPAD2-8-1的形态分析图,图G是PPAD1-9-1的形态分析图;
图2为PPAD3-7-1微凝胶的体外药物释放实验结果图;
图3为MCC950 sodium、PPAD3-7-1及MCC950@PPAD3-7-1对HCECs毒性结果图;
图4为PPAD3-7-1微凝胶降低高渗条件下细胞内ROS水平的实验结果图,其中,图A是对照组(NC)的结果图,图B是高渗应激组(HS)的结果图,图C是HS+0.01μg/mL PPAD3-7-1微凝胶组的结果图,图D是HS+0.1μg/mL PPAD3-7-1微凝胶组的结果图,图E是HS+1μg/mL PPAD3-7-1微凝胶组的结果图,图F是各组的荧光强度统计图;
图5为滴注游离FS或FS@PPAD3-7-1微凝胶后兔泪液中FS的浓度统计图;
图6为角膜荧光素染色结果图,其中图A是对照组的结果图,图B是Scop组的结果图,图C是Scop+PPAD3-7-1组的结果图,图D是Scop+MCC950组的结果图,图E是Scop+MCC950/PPAD3-7-1组的结果图,图F是Scop+MCC950@PPAD3-7-1组的结果图,图G为荧光素染色评分统计图;
图7为角膜上皮细胞凋亡检测结果图;
图8为PCR、WB实验检测炎症相关因子表达结果图,其中,图A是PCR检测IL-1β表达水平结果图,图B是PCR检测NLRP3表达水平结果图,图C是PCR检测IL-6表达水平结果图,图D是WB检测IL-1β表达水平结果图,图E是WB检测NLRP3、IL-6表达水平结果图,图F是WB检测IL-1β表达水平统计图,图G是WB检测NLRP3表达水平统计图,图H是WB检测IL-6表达水平统计图。
具体实施方式
除非提供具体定义,否则本说明书中所述的与分析化学、合成有机化学和无机化学有关的术语以及实验室程序和技术是本领域已知的。标准化学符号可与这些符号代表的全名互换使用。因此,例如,术语“氢”和“H”被理解为具有相同的含义。标准技术可用于化学合成、化学分析、配制和测试成分。上述技术和程序通常可以根据本领域公知的常规方法实施。
应当理解,上述一般性描述和以下详细描述都只是示例性和解释性的,并不限制所要求保护的本发明。如本文所用,除非另有特别说明,否则单数的使用包括复数。本文使用的章节标题仅用于组织目的,不应解释为限制所述主题。
此处使用的“或”是指“和/或”,除非另有说明。此外,术语“包括”的使用不是限制性的。短语“由…组成”排除权利要求中未指定的任何成分、步骤或要素。短语“基本上由…组成”将权利要求的范围限定为指定的材料或步骤,以及那些不会实质性地影响所要求保护的发明的基本和新颖性的材料或步骤。本发明涉及与这些短语中的每一个的范围相对应的本发明组合物和方法的实施例。
在本说明书和所附权利要求书中,除非上下文另有明确规定,否则单数形式“一个”、“一种”包括复数形式。因此,例如,对“方法”的引用包括本文所述类型的一种或多种方法和/或步骤,这些方法和/或步骤对本领域技术人员在阅读本发明等后将变得显而易见。
“大约”是指被称作“大约”的数字包括由所述数字加上或减去所述数字的1-10%。例如,“大约”100度可以表示95-105度或99-101度,具体取决于上下文。每当在本文出现时,数字范围如“1到20”或“1-20”是指给定范围中的每个整数;即仅表示1,仅表示2,仅表示3等,直到并包括仅20。
下面结合具体实施例,进一步阐述本发明,仅用于解释本发明,而不能理解为对本发明的限制。本领域的普通技术人员可以理解:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照厂商所建议的条件实施检测。
实验材料
MCC950 sodium购自MedChemExpress(MCE;China,#HY-12815A);(3,4-dihydroxyphenethyl)methacrylamide(DPMA)购自Macklin Biochemical Co.,Ltd(中国,上海);
Poly(ethylene glycol)methacrylate(PEGMA)从Aladdin Reagent Co.,Ltd购买;
Acrylamidopropyl trimethylammonium chloride(APTAC,75wt%in H2O)和2,2'-azobis(2-methylpropionamidine)dihydrochloride(V-50,97%)从Aladdin ReagentCo.,Ltd购买;
Fluorescein sodium(FS)购自美国Abmole公司;
Scopolamine(Scop)购自Sigma-Aldrich Co.;
人角膜上皮细胞(HCECs)购自ATCC(美国弗吉尼亚州马纳萨斯)。
实施例1聚多巴胺微凝胶的合成以及MCC950的原位包载
一、实验方法
1、聚多巴胺微凝胶的合成方法
聚多巴胺微凝胶是由主要单体[中性单体聚乙二醇甲基丙烯酸酯(poly(ethyleneglycol)methacrylate,PEGMA)和阳离子单体(3-丙烯酰胺丙基)三甲基氯化铵(3-acrylamidopropyl trimethylammonium chloride,APTAC)]与共聚单体[多巴胺单体3,4-dihydroxyphenethyl methacrylamide(DPMA)]在水溶液中通过光诱导自由基共聚合制备的。以固含量为5%的PPAD3-7-1的合成为例进行说明:将PEGMA(0.193g,0.536mmol)、APTAC(0.345g,1.669mmol)和光引发剂LAP加入圆底烧瓶中,溶解于10mL去离子水中。混合溶液在0℃下用氩气鼓泡30分钟后,密封并暴露在406nm的光照下,以500rpm的速度搅拌。聚合2小时后,将40mg DPMA(0.179mmol)溶于0.4mL乙醇中,注入聚合混合物中。几分钟后,观察到乳白色悬浮液,表明形成了聚多巴胺微凝胶,记为PPADx-y-z,其中,其中第一个P代表聚合,第二个P代表中性单体PEGMA,A代表阳离子单体APTAC,D代表多巴胺单体DPMA,x、y和z指PEGMA、APTAC和DPMA的摩尔比。
2、MCC950的原位包载方法
将一定量的MCC950钠盐、中性单体聚乙二醇甲基丙烯酸酯(poly(ethyleneglycol)methacrylate,PEGMA)、阳离子单体(3-丙烯酰胺丙基)三甲基氯化铵(3-acrylamidopropyl trimethylammonium chloride,APTAC)和光引发剂LAP加入反应瓶,再加入双蒸水分散均匀。采用阳离子单体的目的是通过静电作用提高原位包载MCC950钠盐的效率。反应瓶封口后在室温并且406nm的可见光照射下引发聚合反应。聚合约2小时后将多巴胺单体3,4-dihydroxyphenethyl methacrylamide(DPMA)的乙醇溶液注射进入反应瓶,继续反应四个小时,即得到乳白色的聚多巴胺微凝胶分散液。通过改变聚合投料比、反应体系浓度以及反应时间来调节聚多巴胺微凝胶的尺寸、形貌、zeta电位以及MCC950的包载量和包封率。制备的包载了MCC950的微凝胶命名为MCC950@PPAD。
3、聚多巴胺微凝胶表征
采用动态光散射来表征聚多巴胺微凝胶的尺寸、分布以及zeta电位;采用透射电镜来表征聚多巴胺微凝胶的形貌;采用荧光光谱来表征包封率和包载量。
二、实验结果
较高的DPMA含量会导致微凝胶的不稳定。因此,在本研究中,DPMA与主要单体(PEGMA和APTAC)的摩尔比设定为1:10。由于阳离子单体APTAC负责MCC950的原位包载,因此APTAC的含量是一个关键因素。虽然APTAC的高含量有助于MCC950的高包封率,但过多的正电荷聚合物可能会导致细胞毒性和眼表损伤。此外,阳离子PAPTAC聚合物之间的静电斥力也影响微凝胶的尺寸。本发明研究了APTAC含量对PPAD微凝胶的粒径和16Zeta电位的影响。图1A中的DLS结果表明,随着APTAC含量的增加,PPAD微凝胶的平均流体力学直径逐渐增大。随着APTAC含量的增加,微凝胶的Zeta电位逐渐增大。用透射电子显微镜观察微凝胶的形态,如图1C-G所示。
结合粒径、Zeta电位和形貌,选择平均流体动力学直径为356nm、Zeta电位为+15.9mV的PPAD3-7-1微凝胶作为后续体内外实验的最佳样品。
实施例2PPAD3-7-1微凝胶的体外药物释放实验
一、实验方法
FS被用作模型分子来评估MCC950在不同条件下从PPAD3-7-1微凝胶中的释放行为。同时还进行了游离FS在ddH2O中的释放作为对照。
二、实验结果
如图2所示,在第一个小时内,游离FS在ddH2O中的累积释放量迅速达到90%以上。相反,在ddH2O中PPAD3-7-1微凝胶的FS释放几乎可以忽略不计,因为FS通过静电作用被稳定地包裹在PPAD3-7-1微凝胶中。因此,可以合理地预期PPAD3-7-1微凝胶的FS释放可以通过离子强度加速。如图2所示,在离子强度为50mM的条件下,PPAD3-7-1微凝胶在1小时内实现了19.34%的累计FS释放,11小时后达到72.7%。此外,在离子强度为90mM的情况下,PPAD3-7-1微凝胶的FS释放明显改善。泪液高渗是DED的核心致病机制之一。此外,通常利用含有90mMNaCl的培养基来创造一个高渗透性的环境。因此,基于上述释放结果,证明本发明的微凝胶在干眼症模型的眼表高渗条件下,可以实现高效的药物释放。
实施例3体外细胞毒性检测
一、实验方法
细胞毒性:使用Kit-8试剂盒(CCK-8,Dojindo,Japan)对比分析MCC950sodium、PPAD3-7-1及MCC950@PPAD3-7-1的细胞毒性。
三、实验结果
图3为MCC950 sodium、PPAD3-7-1及MCC950@PPAD3-7-1对HCECs毒性的结果图,证明了MCC950@PPAD3-7-1具有良好的生物相容性,可以减少其所负载药物的毒性。
实施例4PPAD3-7-1体外ROS清除性能检测
一、实验方法
将HCECs接种于96孔黑板中,细胞密度为5×103个/孔。细胞孵育24小时后,更换成含有90mM NaCl的无FBS DMEM-F12培养基,以创造一个高渗透环境(500mOsm)。以生理等渗条件培养的HCECs作为对照组。然后,在孔中加入不同浓度的PPAD3-7-1微凝胶,最终的PPAD3-7-1微凝胶浓度分别为0.01mg/mL、0.1mg/mL和1mg/mL。细胞在高渗环境下培养18h后,根据制造商的说明,使用活性氧检测试剂盒(Roche)测定细胞内ROS水平。
二、实验结果
图4为PPAD3-7-1对ROS的清除能力结果图,证明了PPAD3-7-1具有良好的ROS清除能力。
实施例5药物眼表滞留率检测
一、实验方法
选择角膜荧光素染色常用的荧光染料FS作为模型分子,考虑其与MCC950的分子量、水溶性、带负电荷等相似性,可用于研究其药物眼表滞留率。
本实验使用新西兰大白兔,体重2.5-3Kg,实验兔入选标准如下:裂隙灯检查无眼表感染和炎症,无角膜溃疡,无陈旧性的角膜白斑。实验前1h生理盐水冲洗兔双眼结膜囊,兔双眼分别滴入50μL药物,定时取样(5、15、30、45、60、90、120、150和180min),取样时有齿镊夹取1×1cm的滤纸,贴近眼表5s吸取液体后迅速放入500μL ddH2O中,超声10min,过滤(0.22μm)后用荧光光谱仪分析测量FS含量。
二、实验结果
如图5所示,用含10mg/mL FS的50μL PPAD3-7-1微凝胶外用30分钟后,泪液中FS含量明显高于50μL游离FS(10mg/mL)组,证明PPAD3-7-1微凝胶有延长眼表滞留时间的作用。
实施例6干眼小鼠模型治疗效果评价
一、实验方法
本研究所使用的实验动物为6-8周龄、雌性C57BL/6小鼠84只,实验小鼠入选标准如下:裂隙灯检查无眼表感染和炎症,无角膜溃疡,无陈旧性的角膜白斑,角膜荧光素钠染色评分小于10分的健康小鼠。实验动物均由温州医科大学实验动物中心提供,经过温州医科大学实验动物伦理委员会的批准。70只小鼠饲养于智能环境控制系统(相对湿度30±5%,风速2.1±0.2m/s,温度21-23℃)作为实验组(每组14只,共5组),氢溴酸东莨菪碱(0.5mg/0.2ml)皮下注射抑制泪液分泌,每天3次,连续5天。
(1)实验分组:
1)置于正常环境并无干预措施的正常对照组,即NC组;
2)氢溴酸东莨菪碱(0.5mg/0.2mL)皮下注射组,即Scop组;
3)氢溴酸东莨菪碱(0.5mg/0.2mL)皮下注射同时予以PPAD3-7-1(3.5mg/mL,一天3次)滴眼,即Scop+PPAD3-7-1组;
4)氢溴酸东莨菪碱(0.5mg/0.2mL)皮下注射同时予以MCC950(10μM,一天3次)滴眼,即Scop+MCC950组;
5)氢溴酸东莨菪碱(0.5mg/0.2mL)皮下注射同时予以MCC950与PPAD3-7-1简单混合(等量于MCC950@PPAD3-7-1,一天3次)滴眼,即Scop+MCC950/PPAD3-7-1组;
6)氢溴酸东莨菪碱(0.5mg/0.2mL)皮下注射同时予以MCC950@PPAD3-7-1(按MCC950浓度计算,MCC950浓度为10μM,PPAD3-7-1浓度为3.5mg/mL,一天3次)滴眼,即Scop+MCC950@PPAD3-7-1组。
(2)角膜荧光素染色:
实验第6天,对全部实验小鼠进行裂隙灯角膜荧光素钠染色情况观察评估。角膜荧光素钠染色方法:1mg的荧光素钠溶解于0.5mL生理盐水中,吸取荧光素钠溶液2μL滴入小鼠结膜囊,人工辅助眨眼2-3次,2min后在裂隙灯显微镜下钴蓝光观察小鼠角膜的荧光素钠染色情况并进行总体评分。选用美国国立眼科研究所标准分级作为评分标准:小鼠角膜分为上方、下方、鼻侧、颞侧和中央五个区域,每个区域染色情况分别评1-4分(0分,无染色;1分,1-5个点状染色;2分,6-15个点状染色;3分,16-30个染色;4分,大于30个染色点。5个区域评分完毕后计算其总和。以固定人员,单盲方式评估并记录结果。
(3)角膜上皮细胞凋亡检测
麻醉后颈椎脱臼法处死小鼠,快速取出带眼球,立即使用生理盐水冲洗,滤纸吸干水分后放入OCT(冷冻保护剂)中(角膜垂直包埋,切面为冠状面),液氮速冻后(15min),放入-20℃冰箱保存。冷冻切片机切片(矢状面,厚度10μm),直接实验或-80℃恒温冰箱保存。将准备好的冰冻切片取出行角膜凋亡标志物检测。采用原位末端转移酶标记技术(TUNEL)免疫荧光染色法检测。
(4)炎症因子mRNA水平检测
利用RNA提取试剂盒(RNeasyMiniKit,QIAGEN)提取2只小鼠4眼球的全部结膜组织中的RNA,用M-MLV逆转录试剂盒合成cDNA,将模板RNA溶液和随机引物加到200μLPCR管中,混匀后放入PCR仪加热70℃,5min,迅速取出冰上放置5min。取出PCR管,每管加入上述表格中余下四种成分的混合溶液。混匀,37℃孵育1h后,95℃5min,-20℃保存样品用于PCR的扩增。
(5)炎症因子蛋白水平检测
蛋白水平表达分析:按1:100哺乳动物总蛋白裂解液比PMSF(100mM)的比例配制100μL工作液于1.5mL离心管中,处死小鼠后,冰上操作取出小鼠的角结膜剪碎置于管内,电动球磨组织匀浆器匀浆约8min后,16 000g,4℃,离心10min。取上清液转移至1.5mL离心管中。按Western Blot实验方法进行蛋白表达分析。
二、实验结果
图6为角膜荧光素染色结果,证明了MCC950@PPAD3-7-1对比单纯使用MCC950与PPAD3-7-1简单混合以及单纯使用MCC950或PPAD3-7-1具有更好的减少干眼小鼠角膜上皮损伤作用;
图7为角膜上皮细胞凋亡检测结果,证明了PPAD3-7-1可以减少干眼小鼠眼表上皮细胞凋亡,同时对比各组可以发现MCC950@PPAD3-7-1具有更好地抑制上皮细胞凋亡的作用;
图8为PCR与WB实验结果,证明了MCC950@PPAD3-7-1可以显著抑制炎症相关因子表达(mRNA水平与蛋白水平)。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
Claims (15)
1.一种制备用于眼部疾病的聚多巴胺微凝胶的方法,其特征在于,所述的方法包括:
(1)将主要单体、光引发剂加入反应容器中,溶解于水;
(2)混合溶液在0℃下用氩气气体鼓泡30分钟;
(3)将反应容器封口后置于光照下进行聚合反应;所述的光为406 nm的可见光;所述聚合反应的时间为2小时;
(4)将共聚单体的乙醇溶液注射进入前面所述的反应容器,继续反应;
所述的主要单体包括PEGMA和APTAC,所述的共聚单体包括DPMA;所述的共聚单体与所述的主要单体的摩尔比为1:10,所述继续反应的时间为4小时,PEGMA、APTAC和DPMA的摩尔比为3:7:1。
2.根据权利要求1所述的方法,其特征在于,所述的眼部疾病为包括眼表疾病、眼前节疾病、眼底病变、眼眶疾病。
3.根据权利要求2所述的方法,其特征在于,所述的眼部疾病为眼表疾病。
4.根据权利要求3所述的方法,其特征在于,所述的眼表疾病为干眼病、神经营养性眼表疾病、睑板腺疾病、眼表感染性炎症、过敏性角结膜炎、眼表面外伤。
5.根据权利要求4所述的方法,其特征在于,所述的眼表疾病为干眼病。
6.一种用于眼部疾病的聚多巴胺微凝胶,其特征在于,所述的聚多巴胺微凝胶由主要单体与共聚单体共聚合制备得到,所述的主要单体包括PEGMA和APTAC,所述的共聚单体包括DPMA,所述的聚多巴胺微凝胶由权利要求1-5任一项所述的方法制备获得。
7.根据权利要求6所述的聚多巴胺微凝胶,其特征在于,所述的聚多巴胺微凝胶的平均流体动力学直径为356 nm。
8.根据权利要求6所述的聚多巴胺微凝胶,其特征在于,所述的聚多巴胺微凝胶Zeta电位为+15.9 mV。
9.一种药物体系,其特征在于,所述的药物体系由权利要求6-8任一项所述的聚多巴胺微凝胶包载带负电荷的药物得到。
10.根据权利要求9所述的药物体系,其特征在于,所述的带负电荷的药物包括MCC950。
11.一种制备权利要求9或10所述的药物体系的方法,其特征在于,所述的方法包括:
(1)将带负电荷的药物、主要单体、光引发剂加入反应容器,溶解于水;
(2)将反应容器封口后置于光照下进行聚合反应;
(3)将共聚单体的乙醇溶液注射进入前面所述的反应容器,继续反应;
所述的主要单体包括PEGMA和APTAC,所述的共聚单体包括DPMA。
12.根据权利要求11所述的方法,其特征在于,所述的光引发剂包括LAP。
13.根据权利要求11所述的方法,其特征在于,所述的光为406 nm的可见光。
14.根据权利要求11所述的方法,其特征在于,所述聚合反应的时间为2小时。
15.根据权利要求11所述的方法,其特征在于,所述继续反应的时间为4小时。
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