Recombinant High Density Lipoprotein As Drug Carriers And Imaging Agents

Lipoproteins are natural macromolecules, in which a hydrophobiclipid core consisting of triglycerides and cholesterol esters surrounded bya phospholipid monolayer shell. Cholesterol and a family of proteins,so-called apolipoproteins, are embedded in the phospholipid monolayer.The apolipoproteins stabilize the phospholipid layer and regulate thelipoprotein’s biological properties such as the receptor-mediated bindingand endocytosis. Lipoproteins are endogenous, biodegradable,biocompatible and do not trigger immune reaction. In addition,lipoproteins avoid the recognition of the mononuclear phagocytic system.Therefore, lipoproteins are potential candidates for target-specificdelivery carrier and attracted much attention in recent years.High density lipoprotein (HDL) is a member of lipoprotein families.HDL play a pivotal role in the process of cholesterol reverse transport(RCT) in which it promotes cholesterol efflux from peripheral cells andreturns it to the liver for biliary secretion, therefore HDL have aprotective role in atherosclerosis. The main apolipoprotein component ofHDL is apolipoprotein A-I (apo A-I). Free apo A-I binds phospholipids and cholesterol from peripheral cells via ATP-binding cassette transporterA1(ABCA1) and subsequently the discoidal HDL is formed.Cholesterols encapsulated in HDL are taken up by liver through severalpathways in RCT: cholesteryl ester selective uptake mediated byscavenger receptor class B type I (SR-BI); or holoparticle uptakeinvolving the extracellular localized β-chain of the mitochondrial F1ATPase as well as P2Y13receptors.On the basis of attractive features such as small size, shieldedhydrophobic core, and the possibility of receptor mediated uptake,recombinant HDL nanoparticle was designed as a liver-targeted deliverysystem and had the potential of transporting different materials to theliver.We transformed a plasmid inserted with optimized apo A-I cDNAcoding sequence into Escherichia coli strain BL21(DE3)pLysS. Largeamounts of recombinant apo A-I were obtained using E. coli expressionsystem. Then we proved that the recombinant apo A-I protein had similarbiological behaviors compared with wild-type apo A-I, which could reactrapidly with phospholipids to form recombinant HDL (rHDL)nanoparticles.We wanted to synthesize cholesterol derivatives which could bedelivered to liver through RCT pathway. We initially conjugated ethylenediamine with cholesteryl chloroformate to generatecholesterylamine, and then the amino group of cholesterylamine reactedwith DTPA-bisanhydride to produce two compounds: monoamide(DTPA-chol) and bis-amide (DTPA-(chol)2). The compounds werepurified by silica gel chromatography. Subsequently the two compoundswere coordinated with gadolinium ions to give the final gadoliniumcomplexes, Gd-DTPA-chol and Gd-DTPA-(chol)2, respectively. Theproducts were confirmed by NMR and high resolution mass spectrometry.The purity of these gadolinium complexes was over99.5%.These gadolinium complexes were incorporated into rHDLnanoparticles using traditional sodium cholate detergent dialysis method.The final products were named Gd-DTPA-chol and Gd-DTPA-(chol)2,respectively. The Gd-HDL nanoparticles were purified by NaBr densitygradient ultracentrifugation. The concentrations of apo A-I proteins andgadolinium in rHDL were determined to calculate the recovery efficiencyof ultracentrifugation. The diameters of Gd-HDL nanoparticles weredetermined using dynamic light scattering method. The diameters were inthe range from20nm to25nm, which were slightly larger than that ofnative HDL. And the morphology of Gd-HDL nanoparticles wasobserved by transmission electron microscope. It was found that bothGd-HDL nanoparticles were discoidal, similar to nascent HDL. Thelongitudinal relaxivities (r1) of two Gd-HDL nanoparticles were higher than that of commercial contrast agent Gd-DTPA.It was found that both Gd-HDL nanoparticles could mimic thewild-type HDL in HepG2hepatocellular carcinoma cell lines in vitro. Wedemonstrated that these Gd-HDL nanoparticles could transportgadolinium complexes into the cells and verified that the delivery wasmediated by the HDL receptors in the surface of the cells. We alsoevaluated the cytotoxicity of the Gd-HDL nanoparticles by CCK-8assayin HepG2cells.To study in vivo MR imaging of Gd-HDL, healthy rats wereadministered with Gd-HDL at a dose of10μmol Gd/kg via tail vein. TheT1-weighted MR images were obtained at various time points using3Tclinical MRI machine. The T1signals of the axial sectional images fromthe middle and inferior slices of the liver were measured and the relativeenhancement (RE) was calculated. The results showed that both Gd-HDLnanoparticles could significantly increase the signal intensities of the liverat low gadolinium concentrations. And the signal intensities remainedhigher than the pre-injection baseline after24h post-injection. It wasconcluded that Gd-chol-HDL and Gd-(chol)2-HDL could target the liverand resulted in prominent signal enhancement of the liver mass.Meanwhile, the signal intensity at the duodenum area steadily increasedup to24h. Therefore, Gd-DTPA-chol and Gd-DTPA-(chol)2were thoughtto be metabolized into the bile through different pathway. We carried out research on the liver-targeted gene delivery usingrHDL nanoparticles. The in vitro experiment demonstrated that rHDL,composed of cationic cholesterol derivative CDAN, could deliver DNA tohepatic carcinoma cell strain SMMC-7721and promote the transfectionefficiency. Then we studied the in vivo distribution of rHDL in mice aftertail vein injection using small animal imaging system. The resultsindicated that rHDL nanoparticles mainly accumulated in the liver.We also studied rHDL as a delivery system for siRNA. siRNA wascomplexed with five cationic polymers, respectively. Then the siRNAcomplexes were incorporated into rHDL nanoparticles which wereprepared with neutral lipid (EPC and cholesterol) and apo A-I proteins.These rHDL nanoparticles were similar to natural HDL in terms of size,zeta potentials and morphology. We demonstrated that rHDLnanoparticles facilitate high efficiency of luciferase gene silencing invitro and the delivery of siRNA was mediated by HDL receptors in thesurface of the cells. In addition, our studies indicated that rHDL had thepotential of the delivery of siRNA in vivo.