【作者】 陈丽；

【导师】 杜永忠；

【作者基本信息】 浙江大学 ， 药剂学， 2018， 硕士

【摘要】 急性肺损伤(acute lung injury,ALI)迄今尚无有效治疗手段,是一种临床常见的危重症,对患者和社会造成严重的负担。核因子KappaB(nuclear factor kappa B,NF-kB)信号通路是启动炎症反应的关键通路,糖皮质激素可以阻断NF-kB信号通路,对急性肺损伤的治疗具有深刻意义,然而其不良反应局限了其应用。本研究以地塞米松(dexamethasone,DEX)为模型药物,构建细胞间粘附分子1(intercellular adhesion molecule 1,ICAM-1)单克隆抗体修饰地塞米松纳米结构脂质载体(nanostructured lipid carriers,NLCs),以期将地塞米松高效递送至病变肺部,在发挥疗效的同时潜在性降低药物的不良反应,为ALI的临床治疗提供理论依据。采用地塞米松为模型药物,单硬脂酸甘油酯(monostearin,MS)为固态脂质,中链脂肪酸甘油脂(medium chain triglycerides,MCT)为液态脂质,聚乙二醇单硬脂酸酯(Polyethylene glycol monostearate,PEG2000-SA)和单氨基终端的聚乙二醇单硬脂酸酯(monoamino-terminated polyethylene glycol monostearate,NH2-PEG2000-SA)为两性脂质材料,以水性溶剂扩散法制备阴离子地塞米松NLCs。以N,N’-琥珀酰亚胺基碳酸酯为链接,制备anti-ICA]M-1单抗修饰阴离子地塞米松NLCs(ICAM/DEX/NLCs)和 anti-IgG 单抗修饰阴离子地塞米松 NLCs(IgG/DEX/NLCs)。基于表面电荷对纳米粒的体内药动学具有重要影响,本研究通过控制脂质总量不变,以3%质量比的硬脂胺(octadecylamine,ODA)代替原处方中3%质量比的MS,采用相同方法制备anti-ICAM-1单抗修饰阳离子地塞米松NLCs(ICAM/DEX/ODA-NLCs)和anti-IgG单抗修饰阳离子地塞米松NLCs(IgG/DEX/ODA-NLCs),进一步考察并比较阴、阳离子地塞米松NLCs的理化性质等特性、以及对ALI的修复作用。制备得到的ICAM/DEX/NLCs,IgG/DEX/NLCs,ICAM/DEX/ODA-NLCs和IgG/DEX/ODA-NLCs纳米粒形态圆整、大小相对均一。纳米粒粒径相近,分别为 249.9±21.5 nm,229.3±17.2 nm,235.9±1.8 nm 和 227.9±7.4 nm;Zeta 电位分别为-30.3±0.5mV,-28.7±1.1mV,37.4±0.7mV 和 34.2±3.3mV;包封率分别为 90.11%±1.34%,85.72%±0.84%,82.93%±0.94%和 81.39%±3.23%;载药量分别为 3.62%±0.05%,3.49%±0.03%,3.34%±0.04%和 3.28%±0.13%。体外药物释放研究结果表明,地塞米松NLCs中药物均可持续释放24 h以上,累积释放率达59.0%以上。采用人血管内皮细胞株(human vascular endothelial cell line,EAhy926)为模型细胞,以脂多糖(lipopolysaccharide,LPS)刺激构建ALI炎症内皮细胞模型,考察地塞米松NLCs的细胞毒性。地塞米松NLCs对活化的EAhy926细胞的抑制率存在剂量依赖性。阴离子ICAM/DEX/NLCs和IgG/DEX/NLCs的细胞半数致死量(IC50)在 600 μg/mL 以上,阳离子 ICAM/DEX/ODA-NLCs 和 IgG/DEX/ODA-NLCs 的 IC50值在600μg/mL以下,IC50值随着ODA的修饰而减小。细胞摄取研究表明,相对于 ICAM/DEX/ODA-NLCs,ICAM/DEX/NLCs 在静息的 EAhy926 细胞上的摄取效率较低,而在活化的EAhy926细胞上的摄取效率较高;相对于IgG/DEX/NLCs,ICAM/DEX/NLCs在活化的EAhy926细胞上同样体现出显著摄取优势。研究结果提示阴离子ICAM/DEX/NLCs可潜在性更高效的将地塞米松转运至活化的内皮细胞、从而提高对病变内皮的干预能力。细胞转运研究表明,ICAM/DEX/NLCs经CAM介导的内吞途径(CAM-mediatedendocytosis)被EAhy926细胞摄取。以Balb/c小鼠为模型动物,采用暴露式气管滴注脂多糖构建ALI小鼠模型,之后经尾静脉静注给药,考察地塞米松NLCs在正常小鼠和ALI小鼠模型体内的肺分布。相对于ICAM/DEX/ODA-NLCs,ICAM/DEX/NLCs在正常小鼠体内的肺分布较低,而在ALI小鼠模型体内的肺分布较高;相对于IgG/DEX/NLCs,ICAM/DEX/NLCs在正常小鼠和ALI小鼠模型体内的肺分布均较高。研究结果提示阴离子ICAM/DEX/NLCs可潜在性更高效的将地塞米松递送至ALI小鼠肺脏、从而提高对病变肺脏的修复效力。此外,通过尾静脉注射给药,考察游离地塞米松、ICAM/DEX/NLCs、IgG/DEX/NLCs、ICAM/DEX/ODA-NLCs 和IgG/DEX/ODA-NLCs对ALI小鼠模型的体内药效。经给药24 h,ICAM/DEX/NLCs对小鼠肺内TNF-α和IL-6炎性因子水平的抑制作用强于其他药物,同样,对炎症细胞浸润也强于其他药物。此外,相对于其他治疗药物,ICAM/DEX/NLCs在给药12h和24h后更显著的修复了 ALI小鼠模型肺内炎性细胞浸润、充血及肺泡隔增厚等病理表征。研究结果表明,相对于阳离子ICAM/DEX/ODA-NLCs,所制备的阴离子ICAM/DEX/NLCs具有较低的细胞毒性、显著的炎症内皮细胞内在化特性和在ALI小鼠体内的肺分布优势。可通过良好的肺血管内皮细胞结合功能,潜在性的改善地塞米松在ALI小鼠模型体内的肺分布,有望减少药物不良反应,实现对ALI的安全、有效治疗。更多还原

【Abstract】 Acute lung injury(ALI)is one of the most common critical illness with few curable treatments available and has become a serious burden for both patients and society.It has been suggested that Nuclear Factor Kappa B(NF-kB)signaling is crucial in the initiation of the inflammatory response.Glucocorticoid can block the NF-kB signaling to prevent or alleviate acute lung injury.However,its application is limited due to the side effects of glucocorticoid.Based on dexamethasone(DEX),this study aimed to construct intercellular adhesion molecule 1 modified DEX conjugated nanostructured lipid carriers(NLCs),to aid the successful and efficient delivery of DEX to the lung.The new drug plays a therapeutic effect with a decreased potential side effect,thus providing a theoretical basis for the clinical treatment of acute lung injury.Employing monostearin(MS)as a solid lipid,medium chain triglycerides(MCT)as liquid lipid,polyethylene glycol monostearate(PEG2000-SA)and monoamino-terminated polyethylene glycol monostearate(NH2-PEG2000-SA)as amphiphilic lipid material,an anionic dexamethasone nanostructured lipid carrier was prepared by aqueous solvent diffusion method.N,N’-succinimidyl carbonate was assigned as a chain bridge to prepare anti-ICAM-1 monoclonal antibody modified anionic dexamethasone nanostructured lipid carriers(ICAM/DEX/NLCs)and anti-IgG monoclonal antibody modified anionic dexamethasone nanostructured lipid carriers(IgG/DEX/NLCs).Because surface charge has a crucial impact on the pharmacokinetics of nanoparticles in the body,a 3%mass of MS was substituted by 3%octadecylamine(ODA)to keep total amount of lipid constant.Same method was applied to prepare anti-ICAM-1 monoclonal antibody modified cationic dexamethasone nanostructured lipid carriers(ICAM/DEX/ODA-NLCs)and anti-IgG monoclonal antibody modified cationic dexamethasone nanostructured lipid carriers(IgG/DEX/ODA-NLCs),in order to further investigate and compare the physical and chemical properties of anionic and cationic DEX/NLCs,as well as the therapeutic response to ALI.The prepared ICAM/DEX/NLCs,IgG/DEX/NLCs,ICAM/DEX/ODA-NLCs and IgG/DEX/ODA-NLCs nanoparticles were round and uniform in size.The particle size of four DEX/NLCs(ICAM/DEX/NLCs,IgG/DEX/NLCs,ICAM/DEX/ODA-NLCs and IgG/DEX/ODA-NLCs)were 249.9 ±21.5 nm,229.3±17.2 nm,235.9±1.8nm,227.9±7.4 nm,respectively.The Zeta potentials of them were 30.3±0.5 mV,-28.7±1.1 mV,37.4±0.7 mV and 34.2±3.3 mV;the encapsulation rates were 90.11%±1.34%,86.72±%10.84%,82.93%±0.94%and 81.39%±3.23%,respectively.Drug loadings were 3.62%±0.05%,3.49%±0.03%,3.34%±0.04%and 3.28%±0.13%,respectively.In vitro release study showed that the DEX loaded in NLCs could be released continuously for more than 24 h,with the cumulative release rate being above 59.0%.To investigate the cytotoxicity of these four DEX/NLCs,human vascular endothelial cell line(EAhy926)was chosen as model cells while lipopolysaccharide(LPS)stimulation was employed to construct ALI inflammatory endothelial cell model.The cellular inhibition rates of all these DEX/NLCs were dose-dependent.The median lethal doses(IC50)of anionic ICAM/DEX/NLCs and IgG/DEX/NLCs were above 600μg/mL,while that of cationic ICAM/DEX/ODA-NLCs and IgG/DEX/ODA-NLCs were less than 600 μg/mL,with the values of IC50 decrease with the addition of ODA.Cell uptake studies indicated that,compared to ICAM/DEX/ODA-NLCs,the uptake efficiency of ICAM/DEX/NLCs was lower at resting EAhy926 cells whereas higher at activated EAhy926 cells;compared to IgG/DEX/NLCs,the uptake efficiency of ICAM/DEX/NLCs on activated EAhy926 cells was significantly higher.These results suggested that anionic ICAM/DEX/NLCs were potentially more efficient in transporting dexamethasone to activated endothelial cells,thereby increasing the ability to target the abnormal endothelium.Cell transport studies indicated that ICAM/DEX/NLCs were taken up by cells via the ICAM-1 receptor-mediated endocytic pathway.Balb/c mice were exploited to construct the ALI animal model by trachea exposure of drip lipopolysaccharide.The lung distribution of DEX/NLCs in healthy mice and ALI mouse models was examined by intravenous administration of the tail vein.Compared with ICAM/DEX/NLCs,IgG/DEX/NLCs had higher lung distribution both in healthy mice and ALI mouse models;compared to ICAM/DEX/NLCs,the lung distribution of ICAM/DEX/ODA-NLCs were lower in healthy mice while higher in the ALI mouse model.These results suggested that anionic ICAM/DEX/NLCs potentially deliver DEX to the lung of the ALI mice in a more efficient way,thereby improving the therapeutic effect on abnormal lung.In addition,in vivo drug administration of DEX,ICAM/DEX/NLCs,IgG/DEX/NLCs,ICAM/DEX/ODA-NLCs,and IgG/DEX/ODA-NLCs in ALI mouse models was investigated by tail vein injection.After 24 h of administration,compared with other drugs,the ICAM/DEX/NLCs had a stronger inhibition on the levels of TNF-a and IL-6 inflammatory factors and inflammatory cell infiltration in the lungs of mice.The pathological morphology of lung tissue was observed by HE staining at 12 h and 24 h after administration.ICAM/DEX/NLCs presented more significantly improvement of pathological characterization in ALI mouse model compared with other drugs,including inflammatory cell infiltration,hyperemia and alveolar septal thickening.Our results showed that compared to cationic ICAM/DEX/ODA-NLCs,anionic ICAM/DEX/NLCs prepared in our study have lower cytotoxicity,improved cell internalization characteristics and lung distribution ability in mice with ALI.They have a potentially improved lung distribution of DEX in the ALI mouse model through a better binding to pulmonary vascular endothelial cell.The administration of ICAM/DEX/NLCs may be expected to reduce drug side effects and achieve safe and effective treatment for ALI.更多还原