RGD介导脑靶向阿魏酸脂质体的研究
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摘要
炎症在许多神经退行性疾病的发病机理中起着关键性的作用，其中包括局部脑缺血、帕金森病、阿尔茨海默病以及与艾滋病相关的脑水肿。炎症反应过程主要是白细胞(单核细胞和中性粒细胞)通过细胞渗出和趋化性作用跨过血脑屏障(BBB)向炎症部位迅速大量的迁移。在白细胞表面，具有高度表达的整合素受体，RGD肽可以与之特异性地结合。利用这一性质，可将RGD肽与脂质体偶联，使RGD-脂质体被携带跨过BBB并到达脑部的病灶部位。据此，本研究以抗氧化药物阿魏酸(FA)为模型药，通过羧基胆固醇将RGD肽偶联到FA脂质体表面，利用RGD肽与白细胞表面的整合素受体特异性地结合，不仅实现了脑靶向给药，而且将药物直接输送到脑部的病灶区，显著提高了FA的抗氧化效果，达到预期设计目的。相关研究工作的主要内容及结果如下：
采用醋酸钙梯度法制备阿魏酸脂质体(FAL)，包封率80.2±5.20％，5h药物释放80％。应用电子顺磁共振技术、电位滴定技术从分子水平研究了药物与磷脂之间的作用，对醋酸钙载药的机理进行初步的探讨。提出从pH梯度和电势梯度两个方面解释其载药机理。选用与FA分子结构相似的水杨酸(SA)为模型药物，制备不同盐梯度脂质体，测定不同盐溶液中FA及SA饱和溶解度，发现内水相中高溶液度也可以获得高包封率。
通过胆固醇衍生物将RGD偶联到脂质体表面，以体外细胞亲和力为指标，筛选了RGD与脂质体的连接臂，结果由羧基胆固醇连接的RGD脂质体与白细胞具有更高的亲和力，并确定RGD在脂质体表面的连接密度为2.5％。在人全血中，偶联RGD的脂质体与白细胞结合率达72.4％。考察了羧基胆固醇与RGD偶联工艺的温度、pH值、磷脂浓度等因素对结合率的影响。结果表明98％以上的RGD肽可在2h内反应完全，且催化剂没有引起脂质体的聚集。对偶联RGD的FAL(RGD-FAL)及FAL进行了理化性质评价，冷冻蚀刻电镜观察其显微形态。电镜观察结果为单室脂质体，两种脂质体获得的包封率分别为80.2±5.20％和81.0±3.7％，稳定性良好。
采用脑部微量注射IL-1β建立大鼠脑部炎症模型，考察了RGD-FAL脂质体在炎症大鼠体内的分布。结果表明由RGD介导的脑靶向脂质体可以在炎症大鼠的脑组织中以高于FA溶液组6倍，高于FA普通脂质体3倍的浓度分布。且由于RGD脂质体在血中与白细胞的结合，避免了被肝、脾组织的吞噬，与溶液组相比，其半衰期延长了2.6倍，与普通脂质体比较半衰期延长了1.43倍，消除速率明显下降。此外，将荧光标记的RGD脂质体注射入脑部炎症的大鼠体内，不同时间点采用荧光显微镜观察了其脑组织切片，研究RGD脂质体在脑内的分布。荧光照片显示，在炎症反应期脑组织切片的荧光强度持续增加，且出现了荧光脂质体聚集的光斑，证实RGD脂质体在炎症大鼠体内不仅可以随白细胞通过BBB，而且可聚集在脑部的炎症反应病灶。
建立U937细胞株氧化损伤模型，以细胞活度、细胞线粒体膜电位的改变及形态学改变为指标评价了RGD-FAL的体外抗氧化活性。结果表明，将FA制成脂质体后，其体外抗氧化能力显著高于FA溶液。
建立大鼠脑缺血／再灌注损伤模型，以超氧化物歧化酶、丙二醛以及总抗氧化能力为指标，以川芎嗪为阳性对照药物，评价RGD-FAL在体内的抗氧化效果。结果表明，RGD-FAL在体内抗氧化效果明显高于FA溶液、普通FA脂质体组、阳性对照药物组。结果表明体内药效的结果与体内分布结果具有良好的相关性。
Inflammatory response plays an important role in the pathogenesis of neurodegenerative diseases such as ischemia in the brain, Alzheimer's and Parkinson's. Leukocytes including monocytes and neutrophils can cross an intact blood-brain barrier (BBB) and be delivered to the site of injury or infection in the brain. RGD peptide (Arg-Gly-Asp) can combine with integrin receptors which are expressed on the surface of leukocytes (neutrophils and monocytes). Then, RGD-liposomes can be devised for selective and preferential presentation to blood monocytes/neutrophils, and taken into the brain in response to the inflammation recruitment. Thus, in the present study, FA was used as model drug and loaded into liposomes. RGD peptide was coupled with FA liposomes for binding to monocytes and neutrophils in peripheral blood for brain targeting in response to leukocyte recruitment. The pharmacodynamic of FA in vivo was greatly enhanced and brain targeted delivery was achieved.
FA was loaded into liposome via calcium acetate gradient with 80.2% entrapment efficiency (EE) and 80% FA released form liposomes within 5h. However, the application of the pH gradient theory to account for the remote loading of the calcium acetate gradient did not appear to be satisfactory. Hence, the potentiometric technique and EPR were used to investigate the interactions between drugs and liposomes. The chemical potential gradient and electrical potential were used to account to the mechanism of loading drugs. The EEs of salicylic acid liposomes via a variety of salt gradients was determined to verify the explanation. It was found that high solubility in interior could contribute to high EE.
The CHS was used as coupling arm according to the affinity ability to leukocytes with 2.5% coupling density. 72.4% of RGD-liposomes could combine with integrin receptors which were expressed on the surface of leukocytes. The temperature, pH, lipid concentration were studied where it was found that 98% of RGD peptide was coupled with liposomes within 2h. The drug leakage and liposome aggregation were not observed. The physicochemical properties of RGD-liposome were valued and morphology was observed by freeze-fracture electron microscopy. A good stability and high EEs were obtained and unilamellar vesicles were present in the micrographs.
The rats were subjected to intrastriatal microinjections of 100μl of human recombinant IL-1βto produce brain inflammation to study the body distribution. For RGD coated liposomes, the concentration of FA in brain was 6-fold higher than that of FA solution and 3-fold higher than that of uncoated liposomes. The half life was prolonged with 2.6-fold compared to FA solution and 1.43-fold in comparison with FA liposome. Thus, the uptake of RGD FA liposome into liver and spleen was avoided. Furthermore, the rats were subjected to an administration of fluorescence-labeled RGD-liposome. The result of fluorescent microscopy showed that RGD-liposome could be mediated into brain and accumulated in the focus of infection.
Mitochondrial transmembrane potential alternation, cell viability assay and morphology were assayed to value the antioxidant activity of RGD-FAL. It was found that FA liposomes exhibited greater antioxidant activity than FA solution on U937 cell.
Animal model of ischemia/reperfusion was set up to value the antioxidant activity in vivo and the activity of SOD, T-AOC and the content of MDA were used as index. The results of pharmacodynamic studies had a good correlation with that of body distribution. The antioxidant activity in vivo of RGD-FAL was significantly greater than that of FA injection, liposomal FA and Ligustrazine. Thus, this strategy is a promising approach due to it can deliver drug directly to the inflammatory site in the brain following the recruitment of leukocytes and allow a better protective effect in vivo.
采用醋酸钙梯度法制备阿魏酸脂质体(FAL)，包封率80.2±5.20％，5h药物释放80％。应用电子顺磁共振技术、电位滴定技术从分子水平研究了药物与磷脂之间的作用，对醋酸钙载药的机理进行初步的探讨。提出从pH梯度和电势梯度两个方面解释其载药机理。选用与FA分子结构相似的水杨酸(SA)为模型药物，制备不同盐梯度脂质体，测定不同盐溶液中FA及SA饱和溶解度，发现内水相中高溶液度也可以获得高包封率。
通过胆固醇衍生物将RGD偶联到脂质体表面，以体外细胞亲和力为指标，筛选了RGD与脂质体的连接臂，结果由羧基胆固醇连接的RGD脂质体与白细胞具有更高的亲和力，并确定RGD在脂质体表面的连接密度为2.5％。在人全血中，偶联RGD的脂质体与白细胞结合率达72.4％。考察了羧基胆固醇与RGD偶联工艺的温度、pH值、磷脂浓度等因素对结合率的影响。结果表明98％以上的RGD肽可在2h内反应完全，且催化剂没有引起脂质体的聚集。对偶联RGD的FAL(RGD-FAL)及FAL进行了理化性质评价，冷冻蚀刻电镜观察其显微形态。电镜观察结果为单室脂质体，两种脂质体获得的包封率分别为80.2±5.20％和81.0±3.7％，稳定性良好。
采用脑部微量注射IL-1β建立大鼠脑部炎症模型，考察了RGD-FAL脂质体在炎症大鼠体内的分布。结果表明由RGD介导的脑靶向脂质体可以在炎症大鼠的脑组织中以高于FA溶液组6倍，高于FA普通脂质体3倍的浓度分布。且由于RGD脂质体在血中与白细胞的结合，避免了被肝、脾组织的吞噬，与溶液组相比，其半衰期延长了2.6倍，与普通脂质体比较半衰期延长了1.43倍，消除速率明显下降。此外，将荧光标记的RGD脂质体注射入脑部炎症的大鼠体内，不同时间点采用荧光显微镜观察了其脑组织切片，研究RGD脂质体在脑内的分布。荧光照片显示，在炎症反应期脑组织切片的荧光强度持续增加，且出现了荧光脂质体聚集的光斑，证实RGD脂质体在炎症大鼠体内不仅可以随白细胞通过BBB，而且可聚集在脑部的炎症反应病灶。
建立U937细胞株氧化损伤模型，以细胞活度、细胞线粒体膜电位的改变及形态学改变为指标评价了RGD-FAL的体外抗氧化活性。结果表明，将FA制成脂质体后，其体外抗氧化能力显著高于FA溶液。
建立大鼠脑缺血／再灌注损伤模型，以超氧化物歧化酶、丙二醛以及总抗氧化能力为指标，以川芎嗪为阳性对照药物，评价RGD-FAL在体内的抗氧化效果。结果表明，RGD-FAL在体内抗氧化效果明显高于FA溶液、普通FA脂质体组、阳性对照药物组。结果表明体内药效的结果与体内分布结果具有良好的相关性。
Inflammatory response plays an important role in the pathogenesis of neurodegenerative diseases such as ischemia in the brain, Alzheimer's and Parkinson's. Leukocytes including monocytes and neutrophils can cross an intact blood-brain barrier (BBB) and be delivered to the site of injury or infection in the brain. RGD peptide (Arg-Gly-Asp) can combine with integrin receptors which are expressed on the surface of leukocytes (neutrophils and monocytes). Then, RGD-liposomes can be devised for selective and preferential presentation to blood monocytes/neutrophils, and taken into the brain in response to the inflammation recruitment. Thus, in the present study, FA was used as model drug and loaded into liposomes. RGD peptide was coupled with FA liposomes for binding to monocytes and neutrophils in peripheral blood for brain targeting in response to leukocyte recruitment. The pharmacodynamic of FA in vivo was greatly enhanced and brain targeted delivery was achieved.
FA was loaded into liposome via calcium acetate gradient with 80.2% entrapment efficiency (EE) and 80% FA released form liposomes within 5h. However, the application of the pH gradient theory to account for the remote loading of the calcium acetate gradient did not appear to be satisfactory. Hence, the potentiometric technique and EPR were used to investigate the interactions between drugs and liposomes. The chemical potential gradient and electrical potential were used to account to the mechanism of loading drugs. The EEs of salicylic acid liposomes via a variety of salt gradients was determined to verify the explanation. It was found that high solubility in interior could contribute to high EE.
The CHS was used as coupling arm according to the affinity ability to leukocytes with 2.5% coupling density. 72.4% of RGD-liposomes could combine with integrin receptors which were expressed on the surface of leukocytes. The temperature, pH, lipid concentration were studied where it was found that 98% of RGD peptide was coupled with liposomes within 2h. The drug leakage and liposome aggregation were not observed. The physicochemical properties of RGD-liposome were valued and morphology was observed by freeze-fracture electron microscopy. A good stability and high EEs were obtained and unilamellar vesicles were present in the micrographs.
The rats were subjected to intrastriatal microinjections of 100μl of human recombinant IL-1βto produce brain inflammation to study the body distribution. For RGD coated liposomes, the concentration of FA in brain was 6-fold higher than that of FA solution and 3-fold higher than that of uncoated liposomes. The half life was prolonged with 2.6-fold compared to FA solution and 1.43-fold in comparison with FA liposome. Thus, the uptake of RGD FA liposome into liver and spleen was avoided. Furthermore, the rats were subjected to an administration of fluorescence-labeled RGD-liposome. The result of fluorescent microscopy showed that RGD-liposome could be mediated into brain and accumulated in the focus of infection.
Mitochondrial transmembrane potential alternation, cell viability assay and morphology were assayed to value the antioxidant activity of RGD-FAL. It was found that FA liposomes exhibited greater antioxidant activity than FA solution on U937 cell.
Animal model of ischemia/reperfusion was set up to value the antioxidant activity in vivo and the activity of SOD, T-AOC and the content of MDA were used as index. The results of pharmacodynamic studies had a good correlation with that of body distribution. The antioxidant activity in vivo of RGD-FAL was significantly greater than that of FA injection, liposomal FA and Ligustrazine. Thus, this strategy is a promising approach due to it can deliver drug directly to the inflammatory site in the brain following the recruitment of leukocytes and allow a better protective effect in vivo.
引文
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