GM1修饰重组高密度脂蛋白装载的洛伐他汀药物的研制和初步药效检测

发布时间：2018-06-14 22:58

本文选题：动脉粥样硬化 + GM1　；参考：《南昌大学》2017年硕士论文

【摘要】：随着社会的进步,生活节奏的加快,人们对身体健康的忽视,导致不良生活方式的产生和体育锻炼时间的减少,以及高脂高糖高蛋白的摄取,致使“三高”症状、甚至心血管疾病(如动脉粥样硬化等)的频发和早发。对于健康生活意识的传播和相关药物的研发,已经得到越来越多的科研团队的重视。在他汀类降血脂药物中,洛伐他汀(Lovastatin,LT)是比较普遍的缓解心血管疾病的药物,但由于其脂溶性的特性,在血液中的溶药率低,被人体吸收量少;没有靶向性,不能特异性针对血管受损区,增加了肝脏的负担;没有药物缓释的特性,不能持续给药,在血液当中停留时间短等问题。为此,科学家们已提出了多种策略来解决其所存在的缺陷,利用脂蛋白来包裹他汀类药物即是比较有潜力的一种策略。脂蛋白是存在于人体血液中的一类内源性的脂质-蛋白质复合物,具有磷脂单分子层,构成了疏水性的核心和亲水性的外壳;外表镶嵌的载脂蛋白具有识别作用,在血管内能靶向性的运输胆固醇。其中高密度脂蛋白(high-density lipoprotein,HDL)具有载脂蛋白A-1(ApoA-1)能被清道夫受体识别,将周围组织中的胆固醇运送到肝脏代谢并排出到体外,从而降低血脂,是一种抗动脉粥样硬化的脂蛋白,俗称“血管清道夫”。利用这一特性,人工构建重组高密度脂蛋白(rHDL),加以其他修饰,可以达到靶向性、缓释性和同源性等目的。以rHDL为药物载体运送他汀类药物是近几年兴起的研究热点,但在血液滞留时间和缓释性等方面还有待提高。利用单唾液酸神经节苷脂(GM1)修饰rHDL有望实现这个目标。神经节苷脂GM1是内源性的细胞膜组成成分之一。已有研究报道,脂质体表面修饰GM1能延长其在血液循环中的停留时间,也能减少肝脏对脂质体的摄取。本课题首次使用GM1修饰的rHDL作为药物载体来运送洛伐他汀,对该药物-载体复合物(GM1-LT-r HDL)进行了制备和鉴定以及初步的抗动脉粥样硬化的药效检测。(1)利用薄膜分散法制取脂质运载体,进一步用GM1和ApoA-1修饰,并添加药物后得到GM1-LT-rHDL。外源添加的ApoA-1可以给予重组脂蛋白一定的靶向性,将药物运送到受损斑块区。外源添加的GM1能够插入到脂质层,形成类细胞膜表面结构,起到缓慢释放药物等的作用,同时减少肝脏的摄取。(2)通过Zeta粒径仪和透射电子显微镜对GM1-LT-rHDL的形貌和尺寸进行了检测,测得其粒径117.1±3.6 nm,Zeta电位41.97±1.57 mV。(3)以硅珠和巨噬细胞为模型,利用分子间的特异性结合(GM1/CTB,ApoA-1/anti-ApoA-1),通过激光共聚焦显微镜、流式细胞仪和荧光显微镜等仪器确证了GM1-LT-rHDL中含有GM1和ApoA-1分子,进一步验证了GM1-LT-rHDL的成功构建。(4)通过高效液相色谱仪,测得其包封率为76.55±0.41%,载药量为3.83±0.02%。(5)体外药物释放的实验,验证了GM1-LT-rHDL具有缓慢释放药物的能力。(6)体外细胞实验证实,GM1-LT-rHDL能够通过ApoA-1被巨噬细胞表面的清道夫受体SR-BI识别,且对巨噬细胞吞噬Ox-LDL转变成泡沫细胞的过程(动脉粥样硬化发生和发展的关键步骤)有抑制作用。(7)药物注射动脉粥样硬化模式小鼠(高脂喂食的ApoE-/-小鼠),通过全自动生化仪检测血脂,以及对动脉用油红O染色的横向切片和纵向解剖观察斑块区大小,证实GM1-LT-rHDL不仅能够降低小鼠的血脂,而且还能抑制动脉斑块的形成。本课题成功实现了该药物-载体复合物(GM1-LT-rHDL)的制备和鉴定以及初步的抗动脉粥样硬化的药效检测,但对于其在动物体内的代谢、组织中的分布等有待后续深入研究。基于GM1修饰的rHDL药物载体系统不但可运送洛伐他汀等治疗心血管疾病(如动脉粥硬化)的他汀类药物,还可作为运送治疗其他疾病的脂溶性药物的载体系统,后续可在这方面开展进一步研究。
[Abstract]:With the progress of the society, the quickening of the rhythm of life, the neglect of the health of the people, the production of bad lifestyle and the decrease of physical exercise time, and the intake of high fat and high protein, and the frequent occurrence and early onset of the "three high" symptoms, even the cardiovascular disease (such as atherosclerosis, etc.). The research and development of sowing and related drugs has been paid more and more attention. In statins, Lovastatin (LT) is the most common drug to alleviate cardiovascular disease. But because of its fat solubility, the drug rate in the blood is low and the body is absorbed less in the human body; there is no target and no specific needle. In the area of vascular damage, the burden of the liver is increased; no drug release characteristics, no continuous delivery and short stay in the blood, scientists have proposed a variety of strategies to solve the defects it exists. The use of lipoprotein to encapsulate statins is a more potential strategy. Lipoprotein is the existence of a strategy. A class of endogenous lipid protein complexes in the human blood, having a phosphatidylcholine single molecular layer that forms a hydrophobic core and a hydrophilic shell; the inlaid apoprotein has a recognition role in targeting the transport of cholesterol in the blood vessels. High-density lipoprotein (HDL) has a fat loaded egg. White A-1 (ApoA-1) can be identified by the scavenger receptor, transporting cholesterol in the surrounding tissue to the liver and expelled to the body, thereby reducing blood lipids. It is a kind of anti atherosclerotic lipoprotein, known as the "blood vessel scavenger,". Using this characteristic, the recombinant high density lipoprotein (rHDL) is artificially constructed and other modifications can be used to target the target. It is a hot spot in recent years to transport statins with rHDL as a drug carrier, but it still remains to be improved in the period of blood retention and sustained release. The use of monaloside Ganglioside (GM1) to modify rHDL is expected to achieve this target. Ganglioside GM1 is an endogenous cell membrane composition. One of the ingredients. It has been reported that the surface modification of liposome GM1 can prolong its retention time in the blood circulation and reduce the liver's uptake of liposomes. For the first time, the GM1 modified rHDL was used as a drug carrier to transport lovastatin, and the drug carrier complex (GM1-LT-r HDL) was prepared and identified as well as preliminary. The anti atherosclerotic efficacy detection. (1) using the thin film dispersion method to take the lipid carrier, further modified by GM1 and ApoA-1, and adding the GM1-LT-rHDL. exogenous ApoA-1 can give the recombinant lipoprotein a certain target and transport the drug to the damaged patch area. The exogenous GM1 can be inserted into the lipid layer. Form the surface structure of the cell membrane, play the role of slow release of drugs and reduce the uptake of liver. (2) the morphology and size of GM1-LT-rHDL were detected by Zeta particle size meter and transmission electron microscope, and the size of the particle was 117.1 + 3.6 nm, the Zeta potential was 41.97 + 1.57 mV. (3), and the silicon beads and macrophages were used as the model. GM1/CTB (ApoA-1/anti-ApoA-1), the GM1 and ApoA-1 molecules in GM1-LT-rHDL were confirmed by laser confocal microscopy, flow cytometry and fluorescence microscopy, and the successful construction of GM1-LT-rHDL was further verified. (4) the encapsulation efficiency was 76.55 + 0.41% and the drug loading was 3.83 + 0.02%. by high performance liquid chromatography. (5) the experiment of drug release in vitro demonstrated that GM1-LT-rHDL has the ability to release drugs slowly. (6) in vitro cell experiments confirmed that GM1-LT-rHDL can be identified by ApoA-1 by the scavenger receptor SR-BI on the surface of macrophages and the process of macrophage phagocytosis of Ox-LDL into a foam cell (a key step in the occurrence and development of atherosclerosis. (sudden) inhibition. (7) the drug injection of atherosclerosis model mice (high fat fed ApoE-/- mice), using a full automatic biochemical analyzer to detect blood lipids, and the transverse section and longitudinal anatomy of the artery oil red O staining to observe the size of the plaque area, GM1-LT-rHDL can not only reduce the blood lipid in mice, but also inhibit atherosclerotic plaque. The preparation and identification of the drug carrier complex (GM1-LT-rHDL) and the preliminary detection of anti atherosclerotic efficacy have been successfully realized. However, the metabolism in animals and the distribution of tissue in the animals need to be further studied. The GM1 modified rHDL drug carrier system can not only transport lovastatin and so on. Statins for the treatment of cardiovascular diseases, such as atherosclerosis, can also be used as a carrier system for the transport of fat soluble drugs for other diseases, and further research in this respect can be carried out in this respect.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

【参考文献】