Tissue and intrahepatic distribution and subcellular localization of a mannosylated lipoplex after intravenous administration in mice
Introduction
The success of in vivo gene therapy relies on the development of a vector that achieves target cell-specific, efficient, and prolonged transgene expression following its application. Nonviral vectors are considered to be less toxic, less immunogenic, and easier to prepare than viral vectors and are, therefore, attractive vectors for clinical application. One of the most promising class of nonviral vectors developed so far is the cationic liposome-based transfection system. The lipoplex formation via electrostatic interaction of cationic liposomes and plasmid DNA (pDNA) facilitates the interaction of pDNA with cell membranes, leading to transgene expression in the cells [1]. In an attempt to increase the efficiency of transgene expression as well as to reduce cytotoxicity, several kinds of cationic lipids, such as quaternary ammonium detergents, cationic derivatives of cholesterol [2], diacylglycerol [1], [3], and alkyl derivative of polyamines [4] have been developed. Among them, some lipoplex have been used in clinical trials for the treatment of cancer and cystic fibrosis [5], [6].
The lipoplex is a useful nonviral vector, but it lacks specificity in delivery and transfection after systemic administration. Although the levels of gene expression vary from study to study, the lung invariably shows the highest gene expression. The attachment of a ligand that can be recognized by a specific mechanism would endow a vector with the ability to target a specific population of cells. In the search for macromolecule-based nonviral vectors, several ligands including galactose [7], [8], mannose [9], [10], transferrin [11], and antibodies [12] have been used to improve the delivery of pDNA to target cells. Therefore, the incorporation of such ligands into cationic liposomes would improve the cell specificity of in vivo gene transfer by lipoplex.
Mannose receptor-mediated targeting is a promising approach to achieve cell-specific delivery after systemic administration because (i) the expression of mannose receptors is restricted to the liver NPC and other macrophages, (ii) a complex entering the systemic circulation has easy access to the liver NPC, and (iii) the liver has a high blood flow. These physical and biological features give a mannosylated vector an opportunity to deliver pDNA to the liver NPC via mannose receptor-mediated endocytosis. Liver nonparenchymal cells (NPC), including sinusoidal endothelial cells and Kupffer cells, can be the targets for gene therapy because they have been implicated in a wide variety of diseases [13], [14].
In a previous study, we developed a novel mannosylated derivative of cholesterol, cholesten-5-yloxy-N-(4-((1-imino-2-d-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol), and used it to prepare a cationic liposome formulation (Man liposome) [10]. Man-C4-Chol possesses multi-functional properties, that is, (i) a lipophilic anchor moiety (cholesterol) for stable incorporation into liposomes, (ii) a mannose moiety for recognition by the mannose receptors, and (iii) an imino group for binding to pDNA via electrostatic interaction [15]. Furthermore, low-molecular-weight glycolipids are more promising due to their low immunogenicity, high reproducibility, and ease of mass production. Although, a high gene expression in the liver and spleen after intravenous injection was observed for Man lipoplex via mannose receptor-mediated endocytosis compared with the lung, its transfection efficiency was relatively low and, consequently, further improvements in the efficiency of transgene expression are required.
In order to obtain a theoretical strategy to develop an efficiently targetable gene carrier to the liver by mannosylation, therefore, detailed information on the distribution of a Man lipoplex needs to be obtained. In the present study, we studied the tissue, intrahepatic distribution, and subcellular localization of a [32P]- or [111In]-labeled Man lipoplex after intravenous administration. The results were compared with those for a 3β[N′,N′,N′-dimethylaminoethane]-carbamoyl]cholesterol liposomes (DC liposome), which is a cationic cholesterol derivative, based lipoplex [2].
Section snippets
Chemicals
N-(4-Aminobutyl)carbamic acid tert-butyl ester was purchased from Tokyo Chemical Industry (Tokyo, Japan). Cholesteryl chloroformate was obtained from Sigma (St. Louis, MO, USA), dioleoylphosphatidylethanolamine (DOPE) was purchased from Avanti Polar-Lipids (Alabaster, AL, USA). [α-32P]dCTP was obtained from Amersham (Tokyo, Japan). 111Indium chloride ([111In]InCl3) was supplied by Nihon Medi-Physics (Hyogo, Japan). Diethylenetriaminepentaacetic acid (DTPA) anhydride and 4-[p
Physicochemical properties and gene expression of the lipoplex
The zeta potential of lipoplex and Man lipoplex was 9.78±3.5 (n=3) and 12.5±4.11 (n=3), respectively. The mean particle size of lipoplex and Man lipoplex was 287.2±1.2 nm (n=3) and 285.4±18.3 nm (n=3), respectively. Thus, physicochemical properties of both lipoplex were almost the same.
Fig. 1A demonstrates the gene expression after intravenous administration of Man lipoplex at 3 and 6 h. High gene expression was observed in the liver and spleen, which is expressed mannose receptor on cell
Discussion
Transgene expression in target cells after intravenous administration of the Man lipoplex involves a number of distribution processes for pDNA: delivery to the target cells (tissue distribution), internalization, intracellular sorting, and nuclear entry [26]. In particular, the data presented in this study show the importance of intracellular sorting for efficient gene transfection of the Man lipoplex.
Since lipoplex was taken up the cell by the mechanism of endocytosis, pDNA needs to avoid
Acknowledgements
This work was supported in part by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by Health and Labour Sciences Research Grants for Research on Hepatitis and BSE from the Ministry of Health, Labour and Welfare of Japan.
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