【作者】 楼滨；

【导师】 吴满平；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 脂蛋白是一种内源性物质,具有疏水性脂核（甘油三酯,胆固醇酯）,外面由磷脂,胆固醇及一些载脂蛋白组成外壳的单层脂质颗粒结构。因此脂蛋白可看作是一种广义的脂质体。载脂蛋白镶嵌在脂蛋白表面具有稳定脂蛋白颗粒,介导脂核形成,调节酶活性,及介导细胞通过受体结合并内吞脂蛋白的作用。由于脂蛋白结构中具有一个脂核,并且CM,VLDL,LDL,HDL能被特定组织通过受体途径迅速吸收,设想如果将疏水性药物掺入到脂蛋白脂核部位,取代其核心脂质而不改变天然脂蛋白的完整性,则脂蛋白可作为药物载体特异传递药物到靶细胞。已有研究证明脂核中的脂质能被亲脂性药物取代,并不影响其细胞识别结合特性,并且由于脂蛋白是内源性物质,可彻底生物降解,不引起免疫反应,也不被网状内皮系统快速清除。所以脂蛋白是一种值得研究的主动靶向药物载体。脂蛋白作为药物载体的优点包括（1）是天然成分并具有相对较长的半衰期,（2）颗粒体积小直径在纳米级范围,易从血管内扩散到血管外,（3）通过受体介导被细胞吸收与内吞,（4）大容量脂核可作为疏水性药物储存的场所,避免所载药物与血浆中成分相互作用而分解破坏。 有研究报道用80nm拟乳糜微粒的乳剂颗粒作为抗病毒药物的肝靶向载体,这类乳剂在肝脏中的吸收高于游离药物。用拟乳糜微粒作为药物载体,药物必须首先形成脂肪酸衍生物,增加亲脂性,并且组成外层的膜材料应使用含有不饱和脂肪链的磷脂,以利于LPL的水解TG及残粒的形成。拟乳糜微粒作为药物载体的更深入研究及在体内确定的代谢过程未见报道。 有研究显示VLDL-药物与游离药物相比对HeLa和MCF-7细胞的生长抑制作用没有差别,并且结果未显示通过受体途径被细胞吸收,所以VLDL不适合作为药物载体。 目前大多数以脂蛋白作为药物载体的研究主要集中于LDL。许多亲脂性药物能储存在LDL的非极性脂核中。LDL通过颗粒表面的载脂蛋白apoB₁₀₀与细胞膜表面的特异受体（LDL受体）结合并被细胞内吞。快速生长的细胞高表达LDL受体。药物与LDL结合不影响其识别LDL受体,并有效抑制体外肿瘤细胞。LDL作为药物靶向载体已申请美国专利。但LDL受体存在大多数正常组织细胞,所以LDL作更多还原

【Abstract】 Lipoproteins are spherical macromolecular particles in which a hydrophobic core containing triglycerides (TG) and cholesteryl esters is emulsified by a shell composed of phospholipids, unesterified cholesterol, and one or several specific apolipoproteins (apos). Apolipoproteins stabilise the particles .regulate enzyme activity, and mediated lipoproteins binding and endocytosis by their receptors. Being endogenous, these particles are completely biodegradable, do not trigger immunological responses, and escape recognition and elimination by the reticuloendothelial system (RES).Advantages of lipoproteins as drug carriers include their (1) being natural components with a relatively long half-life in the circulation, (2)having small particle size, (ranging in nanometer) which allows diffusion from vascular to extravascular compartments, (3)being internalized via receptor-mediated uptake and endocytosis, and (4)having a large lipid core, which provides a store compartment for hydrophobic drugs and protects its carried drug from decomposition by plasma components.P. C. N. Rensen et al explored the possibility to use the 80-nm-sized emulsion particles(chylomicron-like emulsions) for targeting carried antiviral drugs to hepatocytes. The emulsion particles had higer uptake by liver than free drugs. When the emulsion particles are used for drugs carrier , the ’prodrug’ strategy was pursued, in which hydrophilic drugs are chemically modified with lipophilic residues to confer affinity for lipidic particles. As a component of emulsion particle surface unsaturated chains at the 2-position of phosphatidylcholine is imperative for efficient hydrolysis of emulsion TG by LPL and the formation of remnants from these particles. Further studies of emulsion particles as drug carrier have not reported Compared with free drugs, VLDL - drug complexes did not affectedcytotoxicity. No evidence shows the VLDL-drugs are taked up by the cells via a receptor pathway. VLDL is not appropriate drug carrier.A large number of investigations were concentrated in LDL-drug complex. Dividing cells require large amounts of cholesterol for cell membrane. Some carcinoma cell lines had been reported to express more LDL receptors than normal cells. Most of tumor cells readily internalize and degrade LDL by the high-affinity receptor pathway, and then drugs are released from LDL particles into the cells. The core compartment of LDL particles allow a substantial quantity of lipophilic drug to be stored inside. Highly lipophilic drugs can be incorporated into the apolar core without affecting lipoprotein receptor recognition.LDL have been extensively studied as drug carriers and have been shown to be effective in improving efficacy and/ or reducing toxicity of therapeutical agents. LDL as drugs targeting carrier has applied US patent. However LDL receptors present in most normal tissue cells,the approach may also not solve the problem of specificity.Recently HDL has been explored as a drug carrier system for a hydrophobic prodrug of IudR in the cervical and breast cancer chemotherapy. HDL play a major role in the transport of cholesterol from peripheral tissues to the liver(called ’reverse cholesterol transport’ ), HDL transport cholesrterol to liver cell, where they are recognized and taken up via specific receptors. Cholesteryl esters within HDL are selectively uptaked by hepatocytes via the scavenger receptor BI(SR-BI). An interesting feature of SR-BI is that the receptor selectively mediates translocation of HDL-cholesteryl esters from the lipoprotein particle to the cytosol of the liver parenchymal cell without a parallel uptakes of the apolipoproteins and this property may allow the delivery of its loaded drugs to liver cells avoiding lysosomal degradation. The ectopic P-chain of ATP synthase, as an new hepatic apoA-I receptor, and hepatic lipase in the surface of hepatocytes may also take part in the selective uptake ofHDL-cholesteryl esters. The rate of selective cellular uptake of cholesteyl ester by liver can be up to 40-fold higher than the uptake of apolipoprotein. The lysosomol pathway of endocytic LDL may result in destruction of its carried drug. As a drug carrier, nonlysosomal pathway of endocytic HDL may be more desirable. As a drug delivery system, HDL may be better than other lipoprotein in hepatoma chemotherapy. In this paper, we choose HDL as a drug carrier to explore the possibility of HDL-ACM complex in hepatoma chemotherapy.Using HDL reconstitution technique we prepared rHDL-ACM complex, soy phosphatidylcholine, HDL-apos and ACM were sonicated. rHDL-ACM was purified on SephadexG-25. The density range of rHDL-ACM was 1. 063-1. 21g/ml the same as that of native HDL. The purity of all rHDL-ACM preparations are more than 92% . Its encapsulated efficiencies are more than 90%. The mobility of rHDL-ACM on agarose gels was faster than that of native HDL(0. 32 vs 0. 16). The diameter of the rHDL-ACM particles was obtained by Zata Potential/Particle Sizer(Nicomp 380 ZLS). The average diameter is 30. 7 + 3. 9nm in the range of diameter of lipoproteins. The morphology was observed by transmission electron microscope and found that the rHDL-ACM particles is typical sphere model of lipoproteins and heterogeneous in particle size. In order to determine whether rHDL-ACM still has the same cell-binding activity as native HDL, we carried out a competitive binding assay. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed away in the present of excess native HDL, indicating that rHDL-ACM complex still keep native HDL property. To test the drug delivery efficiency, we incubated the SMMC-7721 cells with increasing concentrations of rHDL-ACM or free ACM. rHDL-ACM complex delivered significantly higher amount of ACM into cells than free ACM at concentration range of 0. 5-10iig/ml (P<0. 01) revealing that rHDL-ACM complex had high drug delivery efficiency.We further evaluated the cytotoxicity and 50% inhibitory concentration(IC50) of rHDL-ACM and freeACM on SMMC-7721 cells. It was found that cytotoxicity of rHDL-ACM was significantly higher than that of free ACM at concentration range of 0-5 u g/ml (p<0. 01) and IC50 of rHDL-ACM was lower than of free ACM(1.30u g/ml vs 2. 33 u g/ml). In order to investigate the specificity of rHDL-ACM delivery system, we utilized both SMMC-7721 cells and L02 cells(a normal liver cell line) and found that uptake of rHDL-ACM by SMMC-7721 cell was significantly higher than by L02 cells at concentration range of 0. 5-7. 5 u g/ml (p<0. 01) and with a dose-dependent manner, suggesting that rHDL-ACM has some specificity to target the hepatoma. Finally, we determined cytotoxicity of rHDL-ACM on both SMMC-7721 and L02 cells. Cytotoxicity of the rHDL-ACM on SMMC-7721 cells was siginificantly higher than on L02 cells at concentration range of 0-7. 5u g/ml(p<0. 01). IC50 of rHDL-ACM on SMMC-7721 cells(1.30ug/ml) was lower than that on L02 cells(4. 54y g/ml), showing a preferential cytotoxicity of rHDL-ACM on the SMMC-7721 cells to L02 cells.The plasma clearance of the rHDL-ACM in rats was studied. rHDL-ACM was cleared slower from the circulation than fACM. The half life rHDL-ACM and fACM is 2. 7h and 1. 49h, respectively. At 4h after injection, the hepatic associated rHDL-ACM was considerably higher than kidney, spleen and lung. This indicates that complex of ACM with the hepatotropic carrier HDL indeed enhances its liver uptake.A high-performance liquid chromatographic (HPLC) method for the analysis of rHDL-ACM in mouse plasma was developed. rHDL-ACMwas separated on an Eclipse XDB C8(4.6mm X 150mm, 5u m)Column and detected by spectrofluorimeter at X ex 434nm, X em505nm. The mobile phase consisted of methol-water(64:36, v/v), pH3. 0 and eluted at l.Oml/min.The retention times of fACM in mobile phase, the impurity in plasma and fACM in plasma are 5. 809min, 1. 449-1. 617min, 5. 672min, respectively. The determination of ACM was not interfered with the impurity of plasma. The limit of quantitation was 1.5ng.The half life of rHDL-ACM in mouse更多还原