CN103462933B - 死亡配体抗体偶联聚乙二醇修饰脂质纳米递药系统及应用 - Google Patents
死亡配体抗体偶联聚乙二醇修饰脂质纳米递药系统及应用 Download PDFInfo
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Abstract
本发明提供一种死亡配体抗体偶联聚乙二醇修饰脂质纳米递药系统,通过制备聚乙二醇(PEG)修饰、死亡配体(Fas ligand)抗体偶联的脂质纳米粒递药系统,结合缺血性脑损伤实验动物模型,从整体动物水平,对偶联Fas ligand抗体的脑靶向纳米载药系统的药效学指标进行评价,证明其良好的脑靶向性及脑缺血区域的靶向性。从梗死面积减少、动物神功能损伤改善等指标,证明本递药系统可以在降低药物剂量的同时提高药物的药效,从而为临床治疗缺血性脑卒中提供更加高效、低毒的载药系统。
Description
技术领域
本发明属于生物医药技术领域,涉及靶向递药系统,具体涉及一种用于治疗缺血性脑卒中及脑卒中后遗症的脑靶向递药系统,尤其是一种死亡配体(Fas ligand)抗体偶联的聚乙二醇(PEG)修饰脂质纳米给药系统及其制备方法和应用。
背景技术
脑血管病是危害人类生命健康的常见病、多发病,由于人口老龄化和心血管危险因素的流行,脑卒中已成为我国第一大致死、致残疾病,且死亡率远高于日本和欧美发达国家,给患者及其家庭和社会带来沉重的医疗经济负担。据统计资料显示,脑血管病以缺血性为多见。缺血性脑血管病是多因素、多层次的复杂病理过程,由各种诱发因素引起脑内血管狭窄、闭塞或破裂而造成的脑血液循环障碍性疾病,临床表现为一过性或永久性功能障碍的症状和体征。临床上一般用溶栓治疗来解除梗塞,建立缺血区域血流再灌注,对于缺血引起的脑组织损伤尚无有效治疗药物。因此, 寻找新型有效的脑缺血治疗药物是目前研究热点。
由于小分子药物透过血脑屏障(BBB)的能力有限,许多研究认为血脑屏障是脑缺血损伤治疗的瓶颈。因此通过设计新型的脑靶向载药系统,克服血脑屏障的阻碍,对于提高传统药物的疗效、增加治疗窗,最终减少缺血引起的神经血管损伤具有重要的意义。
纳米粒是目前研究较多的一类用于脑部靶向治疗的递药载体系统,包括如下优点:具有特异性,靶向性,定量准确、易吸收,具有适当的粒径与粒型,颗粒小;可减少药物降解,提高药物稳定性;具有较高的载药量,具有在靶部位定位的能力,减少给药剂量,进一步减少或避免药物的毒副作用;具有较长的体内循环时间,增加脑靶向效率。
当前的递药系统设计主要关注提高药物的生物利用度和透过血脑屏障的效率,却很少考虑入脑后在缺血病灶区的靶向性。我们前期研究发现(Lu Y M, Tao R R, Huang J Y, et al. P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand[J]. J. Neuroinflammation, 2012, 9: 172.),缺血区附近小胶质细胞激活,释放大量Fas ligand,作为关键的免疫反应参与损伤区域的代偿性修复或组织重塑。我们猜想,利用在缺血损伤区大量表达的分子,作为靶标,设计将Fas ligand的单克隆抗体修饰在脂质纳米粒表面,并包封抗脑血管损伤类药物,提高药物的病灶区域的靶向性,实现脑部疾病的靶向治疗。
发明内容
本发明的一个目的是提供聚乙二醇(PEG)修饰、死亡配体(Fas ligand)抗体偶联的脂质纳米粒递药系统,通过以下制备步骤实现:
将13.5mg单硬脂酸甘油酯,8.5mg中链甘油三酯,1.5mg罗丹明硬脂胺,5mg丁苯酞,3mg聚乙二醇单硬脂酸酯溶于1ml乙醇溶液中,水浴70℃下加热搅拌使其溶解。趁热将上述溶液缓慢注入9ml 体积比为0.1%的吐温80水溶液中,400rpm磁力搅拌5分钟,即得3mg/ml PEG修饰的脂质纳米粒溶液。在脂质纳米粒溶液中加入0.3mgN-N’-二琥珀酰胺亚胺碳酸盐,室温下反应4h。反应后加入1.6ug 死亡受体的单克隆抗体(Fas ligand antibody),室温下再反应4h,制备得到3 mg/ml Fas ligand 抗体偶联、PEG修饰的载药脂质纳米粒。
其中脂质纳米粒的材料不限于单硬脂酸甘油酯、中链甘油三酯等,可以是其他常用的脂肪酸甘油酯。
本发明的另一个目的是提供聚乙二醇(PEG)修饰、死亡配体(Fas ligand)抗体偶联的脂质纳米粒载药系统在制备治疗缺血性脑卒中、脑卒中后遗症等脑血管病变为基础的脑损伤疾病药物中的应用。所载药物为对缺血性脑卒中有治疗作用的药物。
本发明提供的PEG修饰、Fas ligand 抗体偶联载药脂质纳米粒具有很好的脑靶向性,尤其是在缺血病灶区分布量多,在较小的剂量下产生明显的改善梗死面积和动物神经功能缺陷的作用,有利于减少药物在非病灶区的毒副作用;内载药物从载体中释放具有缓释作用,载体材料无毒、可生物降解和生物相容性好,可应用于治疗缺血性脑卒中及相关疾病。
本发明通过制备聚乙二醇(PEG)修饰、死亡配体(Fas ligand)抗体偶联的脂质纳米粒递药系统,结合缺血性脑损伤实验动物模型,从整体动物水平,对偶联Fas ligand抗体的脑靶向纳米载药系统的药效学指标进行评价,证明其良好的脑靶向性及脑缺血区域的靶向性。从梗死面积减少、动物神功能损伤改善等指标,证明本递药系统可以在降低药物剂量的同时提高药物的药效,从而为临床治疗缺血性脑卒中提供更加高效、低毒的载药系统。
本发明的有益之处是:(1)采用纳米技术,可以有效提高不溶性载药的溶解度,增强药效。(2)PEG化长循环纳米粒,增加表面亲水性,不易被巨噬系统吞噬,能延长药物在体内的循环时间,提高纳米粒的入脑效率。(3)Fas-ligand抗体修饰纳米粒表面后,可以提高在缺血病灶区分布量,在较小的剂量下产生明显的改善作用,有利于减少药物在非病灶区的毒副作用。(4)脂质纳米粒载药系统具有缓释作用,延长作用时间。
本发明的突出优点在于,创新性地采用具有临床应用潜力的脑缺血区域靶向头基——Fas ligand 单克隆抗体,修饰载药的脂质纳米粒,实现抗脑卒中药物的主动靶向传递;以亲水性PEG修饰纳米粒,增加在体内长循环,最终使药物在缺血区域内富集并长时间滞留,提高药物的生物利用度,降低药物用量,并明显减少对正常组织器官的伤害。
附图说明
图1:脑靶向死亡受体(Fas ligand)、PEG修饰偶联的载药脂质纳米粒的示意图
图2:载药纳米粒与抗体修饰的载药纳米粒的体外药物累积释放曲线
图3:缺血模型(MCAO)后偶联Fas ligand 抗体的载药脂质纳米粒在小鼠脑部的累积荧光图。分为三组,从左到右分别为丁苯酞组、载丁苯酞的脂质纳米粒组、抗体修饰的载丁苯酞的脂质纳米粒组
图4:Fas ligand偶联、PEG修饰的载药脂质纳米粒对小鼠MCAO模型的神经功能缺陷损伤评分统计图,结果由平均值±S.E.M(n=10)表示,*P < 0.05, **P < 0.01
图5: Fas ligand偶联、PEG修饰的载药脂质纳米粒对小鼠MCAO模型后甲酚紫染色切片图,虚线区域为缺血损伤区
图6:Fas ligand偶联、PEG修饰的载药脂质纳米粒对小鼠MCAO模型后甲酚紫染色切片,同侧缺血区梗死面积统计图
图7:Fas ligand偶联、PEG修饰的载药脂质纳米粒给药后小鼠Fluoro-Jade B染色图
图8:Fluoro-Jade B染色的阳染细胞数计量图。结果由平均值±S.E.M(n=8)表示,;*P < 0.05, **P < 0.01与空载组比;#P < 0.05与正常给丁苯酞比
图9:Fas ligand偶联、PEG修饰的载药脂质纳米粒给药后缓解缺血再灌注导致的血脑屏障损伤的免疫印迹图
图10:Fas ligand偶联、PEG修饰的载药脂质纳米粒给药后缓解缺血再灌注导致的血脑屏障损伤的免疫印迹定量图。结果由平均值±S.E.M(n=4)表示,**P < 0.01 与假手术组比;#P < 0.05, ##P < 0.01与空载组比;§P < 0.05与正常给丁苯酞比
图11:Fas ligand偶联、PEG修饰的载药脂质纳米粒给药后缓解缺血再灌注导致损伤机制研究的免疫印迹图
图12:Fas ligand偶联、PEG修饰的载药脂质纳米粒给药后缓解缺血再灌注导致损伤机制研究的免疫印迹定量图。结果由平均值±S.E.M(n=4)表示,**P < 0.01 与假手术组比;#P < 0.05, ##P < 0.01与空载组比;§P < 0.05与正常给丁苯酞比
具体实施方式
为了便于理解,以下将通过具体的附图和实施例对本发明的Fas ligand偶联、PEG修饰的载药脂质纳米粒进行详细地描述。需要特别指出的是,具体实例和附图仅是为了说明,显然本领域的技术人员可以根据本文说明,在本发明的范围内对本发明做出各种各样的修正和改变,这些修正和改变也纳入本发明的范围内。
以下是实施例所用实验材料:硬脂胺(octadecylamine,Fluka chemie AG,Switzerland),罗丹明B(Rhodamine B isothiocyanate,AMRESCO,USA),单硬脂酸甘油酯(Monostearin,上海化学试剂有限公司),中链甘油三酯(medium chain triglycerides,MCT),聚乙二醇单硬脂酸酯(Polyethylene Glycol Monostearate n=40,日本,东京化成),吐温80(polysorbate 80,上海申宇医药化工有限公司),无水乙醇(ethanol,国药集团化学试剂有限公司)。
实施例的载药为丁苯酞原料药(dl-3-n-butylphthalide,NBP石药集团有限公司)
实施例一 偶联Fas ligand 抗体的脑靶向纳米给药系统建立
1.1罗丹明硬脂胺的合成
将25.14g硬脂胺与50mg罗丹明B溶于6mL乙醇中,在400rpm的磁力搅拌下避光反应24小时,然后加水50mL,冷冻干燥。将冷冻干燥后的样品用5mL蒸馏水洗涤,再用0.45μm的滤膜过滤。室温下干燥,即得罗丹明硬脂胺。
1.2载药脂质纳米粒的制备
将13.5mg单硬脂酸甘油酯,8.5mg中链甘油三酯,1.5mg罗丹明硬脂胺,5mg丁苯酞,溶于1ml乙醇中,水浴70℃下加热搅拌使其溶解。趁热将上述溶液缓慢注入9ml 0.1%的吐温80水溶液中,400rpm磁力搅拌5分钟,即得3mg/ml脂质纳米粒溶液。
1.3 PEG修饰载药脂质纳米粒的制备及Fas ligand 抗体偶联
将13.5mg单硬脂酸甘油酯,8.5mg中链甘油三酯,1.5mg罗丹明硬脂胺,5mg丁苯酞,3mg聚乙二醇单硬脂酸酯溶于1ml乙醇中,水浴70℃下加热搅拌使其溶解。趁热将上述溶液缓慢注入9ml 0.1%的吐温80水溶液中,400rpm磁力搅拌5分钟,即得3mg/ml PEG修饰的脂质纳米粒溶液。
其中脂质纳米粒的材料不限于单硬脂酸甘油酯、中链甘油三酯等,可以是其他常用的脂肪酸甘油酯。
获得稳定PEG修饰的脂质纳米粒后进行Fas ligand 抗体偶联,在脂质纳米粒溶液0.3mg N-N’-二琥珀酰胺亚胺碳酸盐,室温下反应4h。反应后加入1.6ug 死亡受体的单克隆抗体(Fas ligand antibody),室温下再反应4h,制备得到3 mg/ml抗体偶联的PEG修饰载药脂质纳米粒。作为对照,丁苯酞溶解于聚乙二醇/水(1:3)的混合溶剂中使其浓度为1mg/ml。PEG修饰、Fas ligand偶联的载药脂质纳米粒的示意图参见图1。
实施例二 偶联Fas ligand 抗体的脑靶向脂质纳米粒理化性质评价
2.1脂质纳米粒粒径、电位的测定
取上述制备的未修饰、Fas ligand 抗体偶联并且PEG修饰的两种载药脂质纳米粒0.5ml,用去离子水稀释10倍,用微粒粒度与表面电位测定仪(Zetasizer ,3000HS, Malvern Instruments)分别测定两种脂质纳米粒的粒径和电位。
结果显示,抗体修饰的载药纳米粒直径(60.97±7.95nm)大于无抗体修饰的载药纳米粒(38.23±3.22nm),抗体修饰后纳米粒的空间直径增大;而两者的电位大小相似。
2.2 药物包封率和药物装载量
脂质纳米粒中丁苯酞的含量通过228nm下紫外分光光度计(DU640, Beckman-Counter)的吸收度计算;药物包封率和药物装载量通过离心超滤法测量:0.5mL脂质纳米粒混悬液加到离心超滤管(Microcon YM-10, MWCO 3000, Millipore)内,10000rpm离心20分钟,超滤液中丁苯酞的浓度用吸收度法测得。利用以下公式计算药物包封率和药物装载量:
药物包封率=( M0-Mc) / M0×100% (1)
药物装载量=( M0-Mc) / (M0+Mn) ×100% (2)
注:Mc表示超滤液中游离丁苯酞的质量;M0表示纳米粒中丁苯酞总投料量;Mn表示脂质材料的质量
结果如下表,两种脂质纳米粒的药物包封率均大于90%,药物装载量大于15%,载药效果较好。
2.3 体外实验两种脂质纳米粒的丁苯酞释放曲线
pH7.4的磷酸盐缓冲生理盐水(PBS)作为溶出介质,6ml装载丁苯酞的脂质纳米粒溶液装入含有30mlPBS的塑料管内。塑料管放于保持50℃水浴并以60rpm水平震荡的摇床(HZ-8812S, Scientific and Educational Equipment plant)内,在规定的时间间隔内,1ml的溶出介质被取出,并补加等量的空白PBS。丁苯酞的溶出浓度通过紫外分光光度法的标准曲线法求得。药物释放试验重复进行3次,计算累计释放百分率,结果参见图2。
释放曲线结果显示,两种脂质纳米粒的药物释药行为基本相似,都呈现两相释放行为,前10h 快速释放,后面基本呈零级释放,24h基本可以达到丁苯酞的100%释放。结果表明,Fas-ligand抗体修饰并不影响丁苯酞的释放行为。
实施例三 脑靶向载药纳米粒抗缺血性脑损伤药效学研究
3.1 采用小鼠大脑中动脉线栓方法构建缺血性脑损伤病理模型
利用小鼠局灶性大脑中动脉缺血再灌注模型(middle cerebral artery occlusion/reperfusion,MCAO)建立脑微血管缺血损伤模型:将小鼠用3%异氟醚麻醉、仰卧固定后,颈部消毒,颈正中切口,分离左侧颈总动脉、颈内、颈外动脉。颈总动脉近心端和颈外动脉结扎,动脉夹夹住颈内动脉,在颈总动脉剪一小口,将尼龙线栓(光滑球面,线栓直径0.26 mm)水平向颈内动脉端插入8~10mm,致大脑中动脉堵塞引起的脑缺血损伤。45 min后拔出线栓进行脑缺血再灌注,外科用手术封口胶修补动脉,皮肤缝合。假手术组(sham)经历相同的手术过程,手术过程保持室温,小鼠体温用加热毯保持在37℃。小鼠建立MCAO缺血再灌注模型后24 h进行神经功能障碍评分。
3.2 偶联Fas ligand 抗体的脑靶向纳米载药系统抗缺血性脑损伤药效学评价及机制研究
实验分组:正常对照组、MCAO模型组、丁苯酞常规给药组(10 mg/kg)、Fas ligand-PEG-丁苯酞纳米粒组(1mg/kg)、Fas ligand-PEG-丁苯酞纳米粒组(5mg/kg)。MCAO缺血1h后小鼠尾静脉注射给药。在小鼠MCAO手术前60min,药物以0.1ml/10g的剂量静脉注射给药。
评价指标:
A. 脑内缺血区药物累积成像:缺血模型(MCAO)后偶联Fas ligand 抗体的载药脂质纳米粒在小鼠脑部的累积荧光强度成像。分为三组,从左到右分别为丁苯酞组、载丁苯酞的脂质纳米粒组、抗体修饰的载丁苯酞的脂质纳米粒组。静脉给药24h后离体小鼠脑片用于成像,纳米粒用罗丹明标记,利用Xenogen体外成像仪(IVIS 200 imaging)成像。
结果参见图3,直接以化学药物甲给药组,在脑内的药物累积量很少;两种载药脂质纳米粒给药24h后则主要集中在脑区,而载丁苯酞的脂质纳米粒在大脑缺血同侧和异侧都有分布,Fas ligand抗体修饰的载丁苯酞的脂质纳米粒在缺血区的同侧显著累积,其主要的荧光信号定位在缺血区域周围。
由此表明Fas ligand抗体修饰的载丁苯酞的脂质纳米粒不仅具有很好的脑靶向性,并且Fas ligand抗体修饰可以引导药物在缺血区富集,减少对正常组织的毒副作用,提高对缺血区治疗的效果。
B. 神经症状评分:术后24h缺血性脑损伤小鼠神经功能障碍评价:观察动物行为变化、通常动物会出现眼球震颤,共济失调,运动障碍等一系列神经功能损害的表现,评分参照Longa法,标准如下:0分:无神经功能缺损症状;1分:对侧前肢屈曲;2分:拉尾时对侧前肢抓力下降;3分:无方向性运动,抓尾时向对侧转圈;4分:自发向对侧转圈及意识障碍。所有数据以均数±标准差表示,统计学分析采用Dunnett-t检验。评分越高,神经功能缺陷越严重。
结果如图4所示,相较于空载组,丁苯酞直接给药和抗体修饰的载药纳米粒均能改善缺血性脑损伤小鼠的神经功能,但后者在较小的剂量下(5mg/kg)显示出比前者(10mg/kg)更好的治疗效果。
C. 脑梗死面积定量:缺血再灌注23h后,小鼠处死,脑片用甲酚紫染色,扫描后利用Image J软件对脑梗死面积定量,用百分比表示。代表图参见图5,虚线区域为缺血损伤区;定量图参见图6。
结果可见,相较于空载给药组,PEG修饰、Fas ligand连接的载药脂质纳米粒给药可明显减小梗死面积,起到良好的治疗作用,且相较于丁苯酞直接给药组,也能显著减小梗死面积。
D. Fluoro-Jade B染色: 自由浮动的脑切片在0.06%的高锰酸钾溶液浸泡10分钟,然后在蒸馏水中漂洗1分钟,再转移到0.0001%Fluoro-Jade B(Millipore)染色溶液中染色30分钟。染色后脑切片在蒸馏水中漂洗3次,每次1分钟,然后将脑片固定在载玻片上。利用Zeiss LSM 510共聚焦显微镜观察免疫荧光。荧光结果参见图7,定量结果参见图8。
空载组的Fluoro-Jade B阳性染色细胞相较于假手术组显著增加,而丁苯酞直接给药和抗体修饰的载药纳米粒(5mg/kg、10mg/kg)给药后均能减少阳染细胞数量;特别的是,较小剂量的抗体修饰的载药纳米粒(5mg/kg)对神经元退化的抑止作用优于较大剂量的丁苯酞直接给药(10mg/kg)。
E. 免疫印迹检测:MCAO模型造模处理24小时后断头取脑,收集脑组织样本,并用裂解液匀浆裂解组织提取蛋白,Western-blotting法考查紧密连接蛋白spectrin、MMP-9、TIMP1动态表达变化,表征血脑屏障损伤程度,免疫印迹参见图9,定量参见图10;考查p-Akt、p-ERK,表征神经元损伤机制,免疫印迹参见图11,定量参见图12;
Western-blotting法步骤:提取蛋白,样品中含有相同浓度的总蛋白,在10~15%的十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)分离。免疫印迹分析用到以下一抗: TIMP1 (Santa Cruz ); Phospho-ERK and phospho-Akt (Cell Signaling Technology)、spectrin, MMP-9(Millipore)、β-actin (Sigma Chemical)。使用增强的化学发光检测系统ECL(Amersham Life Science)使得免疫反应性蛋白可视化。
结果显示,MCAO后小鼠的MMP-9和spectrin的降解片段增多,缺血损伤导致磷酸化Akt和ERK的量减少,表明缺血损伤后紧密连接蛋白降解、血脑屏障渗漏;而丁苯酞直接给药和抗体修饰的载药纳米粒(5mg/kg、10mg/kg)给药后均能够抑制细胞存活信号通路的下调;较小剂量的抗体修饰的载药纳米粒(5mg/kg)对细胞存活通路下调的抑制作用优于较大剂量的丁苯酞直接给药(10mg/kg),表明Fas ligand偶联、PEG修饰的载药脂质纳米粒对神经血管单元的成分具有一定的保护作用。
Claims (3)
1.一种死亡配体抗体偶联聚乙二醇修饰的脂质纳米递药系统,其特征在于,所述的递药系统为死亡配体抗体偶联、聚乙二醇修饰的脂质纳米粒;通过以下方法制备:
将13.5mg单硬脂酸甘油酯、8.5mg中链甘油三酯、1.5mg罗丹明硬脂胺、5mg丁苯酞、3mg聚乙二醇单硬脂酸酯溶于1ml纯乙醇中,水浴70℃下加热搅拌使其溶解,趁热将上述溶液缓慢注入9ml 体积百分比为0.1%的吐温80水溶液中,400rpm磁力搅拌5分钟,即得聚乙二醇修饰的脂质纳米粒溶液,加入0.3mg的N-N’-二琥珀酰亚胺碳酸盐,室温下反应4h,反应后加入1.6μg的死亡配体的单克隆抗体,室温下再反应4h,制备得到3 mg/ml死亡配体抗体偶联、聚乙二醇修饰的载药脂质纳米粒;
所述罗丹明硬脂胺由以下方法制备:将25.14g硬脂胺与50mg罗丹明B溶于6mL乙醇中,在400rpm的磁力搅拌下避光反应24小时,然后加水50mL,冷冻干燥,然后用5mL蒸馏水洗涤,再用0.45μm的滤膜过滤,室温下干燥,即得。
2. 根据权利要求1所述方法制备的递药系统在制备治疗缺血性脑卒中及脑卒中后遗症药物中的应用。
3. 根据权利要求2所述的应用,其特征在于,所述递药系统用于作为药物的给药载体。
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