High-Density Lipoprotein As A Potential Carrier Of An Lipophilic Antitumoral Drug

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...