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Cell-specific delivery of genes with glycosylated carriers
Advanced Drug Delivery Reviews 52 (2001) 187–196 www.elsevier.com / locate / drugdeliv
Cell-specific delivery of genes with glycosylated carriers Mitsuru Hashida*, Makiya Nishikawa, Fumiyoshi Yamashita, Yoshinobu Takakura Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606 -8501, Japan
Abstract Cationic liposomes and polymers have been accepted as effective non-viral vectors for gene delivery with low immunogenicity unlike viral vectors. However, the lack of organ or cell specificity sometimes hampers their application and the development of a cell-specific targeting technology for them attracts great interest in gene therapy. In this review, the potential of cell-specific delivery of genes with glycosylated liposomes or polymers is discussed. Galactosylated liposomes and poly(amino acids) are selectively taken up by the asialoglycoprotein receptor-positive liver parenchymal cells in vitro and in vivo after intravenous injection. DNA–galactosylated cationic liposome complexes show higher DNA uptake and gene expression in the liver parencymal cells in vitro than DNA complexes with bare cationic liposomes. In the in vitro gene transfer experiment, galactosylated liposome complexes are more efficient than DNA–galactosylated poly(amino acids) complexes but they have some difficulties in their biodistribution control. On the other hand, introduction of mannose residues to carriers resulted in specific delivery of genes to non-parenchymal liver cells. These results suggest advantages of these glycosylated carriers in cell-specific targeted delivery of genes. 2001 Elsevier Science B.V. All rights reserved. Keywords: Plasmid DNA delivery; Galactosylated liposomes; Galactosylated polymers; Hepatocyte specific delivery; Mannosylated carriers; Gene therapy
Contents 1. Introduction ............................................................................................................................................................................ 2. Biodistribution of naked plasmid DNA ..................................................................................................................................... 3. Biodistribution and gene expression of plasmid DNA complexed with bare cationic liposomes ..................................................... 4. Cell-specific gene transfection by plasmid DNA–glycosylated cationic liposome complexes ........................................................ 5. Galactosylated cationic poly(amino acids) for gene delivery ....................................................................................................... 6. Mannosylated carriers for macrophage-specific gene transfection................................................................................................ 7. Conclusions ............................................................................................................................................................................ References ..................................................................................................................................................................................
1. Introduction The use of non-viral vectors in gene delivery *Corresponding author. Tel.: 1 81-75-753-4525; fax: 1 81-75753-4575. E-mail address: [email protected] (M. Hashida).
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attracts great interests because they lack some of the risks inherent in viral vector systems [1,2]. Among various types of non-viral vector systems, cationic liposomes or polymers seem to be promising because of their high gene expression efficiency [1]. In the case of cationic liposomes, however, the highest gene expression is observed in the lung after in-
0169-409X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 01 )00209-5
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travenous injection of their plasmid DNA complexes in most cases because the lung capillaries are the first traps to be encountered [3–6]. Therefore, development of carrier systems that can escape from undesired tissue uptake and exhibits target cell-specific gene expression is highly required. For cell specific delivery, the receptor-mediated endocytosis (RME) systems endowed to various cell types would be useful and a number of gene delivery systems have been developed to introduce the foreign DNA into specific cells with RME. Ligands currently being investigated include galactose [7– 11], mannose [12–16], lactose [17], transferrin [18,19], epidermal growth factor [20], and antibodies [21]. Among these receptors, asialoglycoprotein receptor is the most promising for gene targeting since it exhibits high affinity and a rapid internalization rate [22]. In this series of investigations, we have developed various kinds of macromolecular and particulate carrier systems with carbohydrate recognition devices for cell-selective delivery of low-molecular weight drugs [23–26], proteins [27–29], and genes [30–34]. In these approaches, we emphasized the importance of the pharmacokinetic consideration for the rational design of drug delivery systems. The physicochemical properties, such as molecular weight, particle size, and electrical charge are also demonstrated to determine the biodistribution profile of carriers after intravenous injection [5,25,28]. Even in the case of glycosylated carriers, the findings indicate that not only ligand structure grafted to carriers but also the overall physicochemical properties of the carriers themselves determine the amount delivered to receptor-positive cells after their systemic administration [30–32]. In this review, we attempt to assess the glycosylated cationic liposomes and polymers for in vivo gene delivery in relation to their physicochemical and pharmacokinetic properties.
nated from plasma due to extensive uptake by the liver after intravenous injection and the uptake clearance is almost identical to plasma flow rate in the liver as shown in Fig. 1 [35]. Uptake of [ 32 P] DNA is preferentially proceeded by the liver nonparenchymal cells and Takagi et al. demonstrated that the binding and uptake of [ 32 P] DNA in cultured mouse peritoneal macrophages were inhibited by polyinosinic acid (poly [I]) and dextran sulfate [36]. Thus, the scavenger receptor which recognizes a wide variety of the anionic macromolecules expected to be responsible for this phenomenon, although Takakura et al. reported that the peritoneal macrophages from class A scavenger receptor-knockout mice [37] still exhibit uptake of [ 32 P] DNA. Degradation of DNA by nuclease is another route of elimination from blood circulation but its rate (clearance) is about one-tenth of that of hepatic uptake (Fig. 1).
3. Biodistribution and gene expression of plasmid DNA complexed with bare cationic liposomes Cationic liposomes condense plasmid DNA to form particles based on the electrostatic interaction
2. Biodistribution of naked plasmid DNA To construct a strategy for the design of cellspecific carrier systems of plasmid DNA, a thorough understanding of their in vivo disposition characteristics is required. Kawabata et al. reported that [ 32 P] labeled plasmid DNA ([ 32 P] DNA) is rapidly elimi-
Fig. 1. Hepatic uptake and urinary excretion clearances for plasmid DNA in comparison with degradation clearances in plasms.
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and protect it from degradation. Mahato et al. demonstrated disposition characteristics of [ 32 P] DNA–cationic liposomes complexes after intravenous injection in mice [4,5]. Rapid clearance of plasmid DNA from the circulation was observed with extensive accumulation in the lung and liver. In addition, [ 32 P] DNA–cationic liposome complexes were predominantly taken up by the liver non-parenchymal cells and the uptake was inhibited by the preceeding administration of dextran sulfate, suggesting the involvement of a phagocytic process. Intravenous injection of naked plasmid DNA actually shows no gene expression even in the liver where the highest uptake is observed [4,38,39]. In most cases, the highest gene expression is observed in the lung after intravenous injection of DNA– lipopsome complexes because the lung capillaries are the first traps to be encountered (Fig. 2). Li et al. compared intravenous and intraportal injection of
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cationic liposome–protamine–DNA complexes and demonstrated that higher gene expression was observed in the lung, even if the intraportal route was employed [40]. These results suggest difficulty of transfection in the liver using simple cationic liposomes by both intravenous and intraportal administration. On the other hand, it was proposed that dioleoylphosphatidylethanolamine (DOPE) promotes the fusion with the endosomal membrane followed by the release of plasmid DNA into the cytoplasm [41,42]. Therefore, DOPE is frequently formulated in commercially available cationic liposome preparations as a co-lipid. However, it was shown that the cationic liposomes containing cholesterol as a neutral lipid, such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) / Chol liposomes exhibit greater in vivo transfection activity than those containing DOPE [43,44]. The use of the
Fig. 2. In vitro and in vitro transfection efficiency of plasmid DNA–cationic liposome complexes with different neutral lipids. In vitro transfection: HUVEC cells were transfected with DNA–cationic liposome complexes. In vivo transfection: DNA–cationic liposome complexes were intravenously injected and luciferase activities in various organs were determined.
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cholesterol improved the in vivo transfection efficiency, but did not increase transfection efficiency in vitro. Cholesterol-containing complexes maintain the small liposomal structures under in vivo condition [45], and results in efficient transfection in the lung (Fig. 2). Mahato et al. reported that the effects of the size of cationic liposome complexes on the in vivo transfection efficiency after intravenous injection [46]. When the sizes of DOTMA liposomes increased from below 100–400 nm or greater, the increased transfection efficiency was observed in the lung. The critical time period for gene delivery is considered to be first 60 min after intravenous injection of cationic liposome–DNA complexes [47] and the retention of plasmid DNA–cationic liposome complexes in the lung plays an important role [48].
4. Cell-specific gene transfection by plasmid DNA–glycosylated cationic liposome complexes Receptors for carbohydrates such as the asialoglycoprotein receptor on hepatocytes and the mannose receptor on macrophages and liver endothelial cells produce opportunities for cell-specific gene delivery with liposomal carriers. In their delivery, however, not only the receptor recognition but also the accessibility to the cell surface plays an important role. The structure of the blood capillary wall varies among organs and tissues [49]. Discontinuous capillaries of the liver, spleen, and bone marrow sinusoid show large inter-endothelial junctions, i.e. fenestrations of up to 150 nm [49]. In addition, sinusoids of the liver are linked to highly phagocytic Kupffer cells. Thus, the size of plasmid DNA–galactosylated cationic liposome complexes for hepatocytes targeting must be condensed to 150 nm in diameter while the size of plasmid DNA–mannosylated cationic liposome complexes for Kuppfer cells targeting are allowed to exceed over this size. The charge of the plasmid DNA–liposome complexes is also important factor for receptor-mediated gene transfection and the cationic charge of complexes should not be too high to allow specific interaction with receptor. The lipid compositions of cationic liposomes also affect the in vivo and in vitro transfection efficiency. Thus,
control of these properties play important roles in gene transfer with glycosylated liposome carriers in vivo. Glycosylation of liposomes can be achieved through coating with glycoproteins or incorporation of synthetic glycolipids on the surface of liposomes. Hara et al. reported that asialofetuin-labeled liposome encapsulating plasmid DNA were taken up by the asialoglycoprotein receptor-mediated endocytosis in cultured hepatocytes and exhibited the highest hepatic gene expression after intraportal injection with a preload of EDTA [50]. However, the introduction of asialoglycoproteins to liposomes is rather complicated, and a number of problems are associated with the carriers in such as reproducibility and immunogenicity. From these viewpoints, the low molecular weight glycolipids would be more promising. Remy et al. reported the feasibility of galactosepresenting lipopolyamine vectors towards targeted gene transfer into hepatoma cells [9]. Inclusion of galactose residues in the electrically neutral complex increased transgene expression by avoiding interaction with serum proteins because of their electric neutrality. The chemical structure and physicochemical properties of glycolipids seems to be also crucial for successful targeting using liposomes. If the incorporated glycolipids are easily removed from the liposomal membrane via interaction with lipoproteins and so under in vivo conditions, targeting should be ineffective. Based on these considerations, we synthesized cholesten-5-yloxy-N -(4-((1-imino-2-b- D -thiogalactosylethyl)amino)alkyl)formamide (Gal-C4-Chol), a novel cholesterol derivative possessing the cationic charge necessary for DNA binding and galactose residues as a targetable ligand for liver parenchymal cells as shown in Fig. 3 [31,32]. Introduction of many hydrophilic galactose moieties to a lipid anchor would result in their removal from liposomes by interaction with lipoproteins or other lipid compartments [51]. In our formulation, cholesterol was chosen as a hydrophobic anchor because of its stable association with the liposomal membrane [52]. The developed galactosylated liposomes were shown to be very useful with a high biological affinity and a wide range of pharmaceutical applications. Gal-C4-Chol containing cationic liposome / DNA complexes showed low cytotoxicity in human hepa-
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Fig. 3. Chemical structures of Gal-C4-Chol and Man-C4-Chol.
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toma HepG2 cells [31]. Plasmid DNA complexed with Gal-C4-Chol / 3[N-(N9,N9-dimethylaminoethan)carbamoyl] cholesterol (DC-Chol) / DOPE (3:3:4) liposomes showed higher transfection activity and [ 32 P] DNA uptake than that with DC-Chol / DOPE (6:4) liposomes. The presence of 20 mM galactose significantly inhibited both transfection efficiency and uptake of DNA with Gal-C4-Chol liposomes, but not those of bare liposomes. These results indicate that the Gal-C4-Chol containing cationic liposome–DNA complexes are efficiently recognized by asialoglycoprotein receptors, internalized, and lead to gene expression in HepG2 cells. Thus superior in vivo delivery and expression of genes in the liver via asialoglycoprotein receptor-mediated endocytosis was achieved using Gal-C4-Chol containing cationic liposomes as shown in Fig. 4. Optimization of the co-lipid type of cationic liposomes, the charge ratio of the liposome–DNA complexes, and other factors based on the physicochemical considerations should lead to further improvements in hepatic gene delivery using galactosylated liposomes.
5. Galactosylated cationic poly(amino acids) for gene delivery
Fig. 4. Transfection activity of DNA–galactosylated liposome complexes after intravenous administration in mice. Plasmid DNA (50 mg) was complexed with cationic lipids at a charge ratio of 2:3. Luciferase activity was determined 6 h post-injection in the lung (h), kidney (j), spleen (j), and heart (j). Each value represents the mean6S.D. values (n 5 3).
Poly(amino acids) are considered to possess advantages as drug carriers since they are soluble in water, biodegradable, not significantly immunogenic, and have multiple functional groups that can be chemically modified. Among various poly(amino acids) available, poly( L-glutamic acid) (PLGA) and poly( L-lysine) (PLL) have been widely used as carrier backbones. PLGA is an anionic polymer and PLL is a cationic one, so their biodistribution properties are rather different. After intravenous injection into mice, PLL is largely delivered to liver parenchymal cells due to its positive charge, whereas PLGA is partially taken up by liver non-parenchymal cells, probably via a scavenger receptor-like mechanism [53]. Glycosylation changes the biodistribution properties of these polymers especially PLGA and galactosylated (Gal-) PLGA was recovered mainly in the liver after intravenous injection. Furthermore, uptake of Gal-PLGA was observed selectively in liver parenchymal cells, indicating their receptor-
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mediated uptake. It was also supported by the competitive inhibition observed by co-injection of Gal- or Man-bovine serum albumin (BSA) [54]. On the other hand, both Gal-PLL and Man-PLL accumulated largely in parenchymal cells. However, these glycosylated PLLs are also shown to be taken up through receptor-mediated recognition as glycosylated PLGAs. From a pharmacokinetic point of view, PLL seems to be a promising hepatocyte-specific carrier, but galactosylated polymers have some advantages over cationic PLL. Since the hepatotropic nature of cationic polymers is likely due to their positive charge, neutralization of this charge abolishes its affinity for the cells. Complex formation with nucleic acids like plasmid DNA reduces the positive charge of PLL. This, in turn, makes the recognition of glycosylated PLLs via receptors more evident. In addition, the internalization of galactosylated carriers is much faster than that of cationic ones, which could be an advantage for DNA and some drugs [55]. The biodistribution of DNA complexes with macromolecular carriers was more easily controlled than that of DNA–liposome complexes. We have developed various Gal-PLLs with different molecular weights and different numbers of galactose units [16]. Hepatocyte-specific delivery of DNA was successfully achieved by selecting the most suitable size of PLL and by controlling the galactose density on PLL. If a short PLL with a molecular weight of 1800 was used, a larger amount of PLL was required for complex formation than with PLLs with molecular weights of 13 000 and 29 000. In addition, there was hardly any DNA condensation by the short PLL. Intravenously administered DNA–Gal 13 -PLL 13000 and DNA–Gal 26 -PLL 29000 , could reach hepatocytes, indicating that these DNA complexes have suitable biodistribution characteristics for targeting. Compared with these complexes, DNA–Gal 5 -PLL 1800 and DNA–Gal 5 -PLL 13000 had a smaller hepatic uptake clearance, suggesting that both the molecular weight and the degree of galactose modification of PLL determine the hepatic targeting of DNA [16]. In vitro and in vivo gene expression studies revealed that DNA–Gal 13 -PLL 13000 and DNA– Gal 26 -PLL 29000 complexes are superior to the DNA– Gal 5 -PLL 1800 complex. However, the level of gene expression was low compared with that obtained
with DNA–cationic liposome complexes. To improve the efficiency of galactosylated cationic poly(amino acids)-based gene transfer, the use of compounds that can enhance gene expression such as viruses or viral proteins, fusogenic lipids, and membrane-disruptive peptides can be considered. Fusogenic peptides could be promising materials for this purpose because the addition of these peptides to carrier systems strongly enhanced the in vitro gene transfer by several DNA–carrier complexes [56]. However, their application to in vivo gene transfer has not been reported so far. As far as their biodistribution is concerned, they should be firmly attached to carriers. Our study suggested that galactosylated polymeric carrier conjugated with a fusogenic peptide was very effective in improving the level of gene transfer after intravenous injection of DNA– carrier complexes [57]. Fig. 5 gives conceptual explanation about hepatocyte-specific gene delivery with multi-functional polymeric carriers.
6. Mannosylated carriers for macrophagespecific gene transfection Macrophages are important targets for the gene therapy of diseases such as Gaucher’s disease [58] and human immunodeficiency virus (HIV) infection [59], but gene transfection for treatment of such cases is not easy. Application of DEAE-dextran is one of the methods used for gene delivery to macrophages in vitro [60,61]. However, this method is generally not suitable for in vivo application due to problems associated with cellular toxicity, low efficiency, or non-specific biodistribution. Erbacher et al. investigated the suitability of various glycosylated poly( L-lysine) derivatives for introducing plasmid DNA into human monocyte-derived macrophages and found that mannosylated poly( L-lysine) exhibited high transfection activity among various glycosylated poly( L-lysine) [16]. They also reported that the transfection activity was markedly enhanced in the presence of chloroquine due to prevention of endosomal and / or lysosomal degradation of plasmid DNA after mannose receptor-mediated endocytosis. However, the transfection efficiency of poly( Llysine) is inferior to that of cationic liposomes in
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Fig. 5. Design of multi-functional polymeric carriers with galactose moiety for hepatocyte-specific gene delivery and its performance in vivo.
most cases and further chloroquine cannot be used for in vivo gene transfection. We synthesized a novel mannosylated cholesterol
derivative, cholesten-5-yloxy-N-(4-((1-imino-2-b-Dthiomannosylethyl)amino)alkyl)formamide (ManC4-Chol), for gene delivery to macrophages which
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are known to express large numbers of mannose receptors on their surface (Fig. 3) [14,15]. Four types of liposomes were prepared with various molar ratios and their particle size was confirmed to be about 200 nm. Plasmid DNA complexed with cationic liposomes, consisting of a 6:4 mixture of Man-C4-Chol and DOPE, showed higher transfection activity than that complexed with DC-Chol:DOPE (6:4) and DOTMA:DOPE (1:1) liposomes in mouse peritoneal macrophages. The presence of 20 mM mannose significantly inhibited the transfection efficiency of plasmid DNA complexed with Man-C4-Chol:DCChol:DOPE (3:3:4) and Man-C4-Chol:DOPE (6:4) liposomes. These results suggest that the complexes of plasmid DNA and mannosylated cationic liposomes is recognized and taken up by the mannose receptors on mouse peritoneal macrophages. In the in vitro gene delivery, the liver non-parenchymal cells seem to be more efficient target cells than liver parenchymal cells because DNA–cationic liposome complexes can more easily access the target cells in this case. After intravenous injection of DNA–Man-C4-Chol:DOPE (6:4) liposome complexes in mice, the highest gene expression was
observed in the liver, whereas DNA–DC-Chol:DOPE (6:4) liposome complexes showed marked expression only in the lung (Fig. 6). In addition, the gene expression with Man-C4-Chol:DOPE (6:4) liposome–DNA complexes in the liver was observed preferentially in the non-parenchymal cells and was significantly reduced by predosing with mannosylated BSA. These results suggest that plasmid DNA complexed with mannosylated liposomes exhibits the high transfection activity in liver nonparenchymal cells due to recognition by mannose receptors. This may be due to Kuppfer cells were existed around the sinusoidal membranes and selection of the administration route must be considered for the efficient targeted delivery of plasmid DNA. The lipid composition of mannosylated cationic liposome was suggested to affect on both pulmonary and hepatic uptake as well as gene expression based on mannose receptor-mediated endocytosis.
7. Conclusions In conclusion, the superior in vivo delivery and expression of genes in the liver via asialoglycoprotein and mannose receptor-mediated endocytosis is achieved using Gal-C4-Chol and Man-C4-Chol containing cationic liposomes, respectively. The adequate selection of the co-lipid type of liposomes and cationic charge ratio of cationic liposomes–DNA complexes, and administration route, etc. would form basis of design of total carrier systems. Polymeric carriers offer another possibility in gene delivery utilizing RME.
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Fig. 6. Transfection activity of DNA–mannosylated liposome complexes after intraportal administration in mice. Plasmid DNA (50 mg) was complexed with cationic lipids at a charge ratio of 2:3. Luciferase activity was determined 6 h post-injection in the lung (empty square), liver (filled square), kidney (hatched box), spleen (diagonally hatched box), and heart (horizontally hatched box). Each value represents the mean6S.D. values (n 5 3).
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M. Hashida et al. / Advanced Drug Delivery Reviews 52 (2001) 187 – 196 scavenger receptors in its hepatic uptake, Pharm. Res. 12 (1995) 825. T. Takagi, M. Hashiguchi, R.I. Mahato, H. Tokuda, Y. Takakura, M. Hashida, Involvment of specific mechanism in plasmid DNA uptake by mouse peritoneal macrophages, Biochem. Biophys. Res. Commun. 245 (1998) 729. Y. Takakura, T. Takagi, M. Hashiguchi, M. Nishikawa, F. Yamashita, T. Doi, T. Imanishi, H. Suzuki, T. Kodama, M. Hashida, Characterization of plasmid DNA binding and uptake by peritoneal macrophages from class A scavenger receptor knockout mice, Pharm. Res. 16 (1999) 503. Y. Liu, D. Liggitt, W. Zhong, G. Tu, K. Gaensler, R. Debs, Cationic liposome-mediated intravenous gene delivery, J. Biol. Chem. 270 (1995) 24864. J.W. McLean, E.A. Fox, P. Baluk, P.B. Bolton, A. Haskell, R. Pearlman, G. Thurston, E.Y. Umemoto, D.M. Mcdonals, Organ-specific endothelial cell uptake of cationic liposomeDNA complexes in mice, Am. J. Physiol. 273 (1997) H387. S. Li, L. Huang, In vivo gene transfer via intravenous administration of cationic lipid–protamine–DNA (LPD) complexes, Gene Ther. 4 (1997) 891. J.H. Felgner, R. Kumar, C.N. Sridhar, C.J. Wheeler, Y.J. Tsai, R. Border, P. Ramsey, M. Martin, P.L. Felgner, Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations, J. Biol. Chem. 269 (1994) 2550. H. Farhood, N. Serbina, L. Huang, The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer, Biochim. Biophys. Acta 1235 (1995) 289. H. Farhood, R. Bottega, R.M. Epand, L. Huang, Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity, Biochim. Biophys. Acta 1111 (1992) 239. Y.K. Song, F. Liu, S. Chu, D. Liu, Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration, Hum. Gene Ther. 8 (1997) 1585. F. Sakurai, R. Inoue, Y. Nishino, A. Okuda, O. Matsumoto, T. Taga, F. Yamashita, Y. Takakura, M. Hashida, Effect of DNA / liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular and trafficking of plasmid DNA / cationic liposome complexes and subsequent gene expression, J. Control. Release 66 (2000) 255. R.I. Mahato, K. Anwer, F. Tagliaferri, C. Meaney, P. Leonard, M.S. Wadhwa, M. Logan, M. French, A. Rolland, Biodistribution and gene expression of lipid / plasmid complexes after systemic administration, Hum. Gene Ther. 9 (1998) 2083. L.G. Barron, L. Gagne, F.C. Szoka Jr., Lipoplex-mediated gene delivery to the lung occurs within 60 min of intravenous administration, Hum. Gene Ther. 10 (1999) 1683. F. Sakurai, T. Nishioka, H. Saito, T. Baba, A. Okuda, O. Matsumoto, T. Taga, F. Yamashita, Y. Takakura, M. Hashida, Interaction between DNA–cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: The role of neutral helper lipid, Gene Ther. 8 (2001) 677–686.
[49] Y. Takakura, R.I. Mahato, M. Hashida, Extravasation of macromolecules, Adv. Drug Del. Rev. 34 (1998) 93. [50] T. Hara, Y. Aramaki, S. Takada, K. Koike, S. Tsuchiya, Receptor-mediated transfer of pSV2CAT DNA to mouse liver cells using asialofetuin-labeled liposomes, Gene Ther. 2 (1995) 284. [51] L.A.J.M. Sliedregt, P.C.N. Rensen, E.T. Rump, P.J. van Santbrink, M.K. Bijsterbosch, A.R.P.M. Valentijn, G.A. van der Marel, J.H. van Boom, T.J.C. van Berkel, E.A.L. Biessen, Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor, J. Med. Chem. 42 (1999) 609. [52] Y. Tokunaga, T. Iwasa, J. Fujisaki, S. Sawai, A. Kagayama, Liposomal sustained-release delivery systems for intravenous injection II. Design of liposome carriers and blood disposition of lipophilic mitomycin C prodrug-bearing liposomes, Chem. Pharm. Bull. 36 (1988) 3557. [53] M. Nishikawa, Y. Takakura, M. Hashida, Pharmacokinetic evaluation of polymeric carriers, Adv. Drug Del. Rev. 21 (1996) 135. [54] M. Hashida, K. Akamatsu, M. Nishikawa, F. Yamashita, H. Yoshikawa, Y. Takakura, Design of polymeric prodrugs of PGE 1 for cell-specific hepatic targeting, Die Pharmazie 55 (2000) 202. [55] K. Nishida, T. Takino, Y. Eguchi, F. Yamashita, M. Hashida, H. Sezaki, Pharmacokinetic analysis of uptake process of lactosaminated albumin in rat liver constant infusion experiments, Int. J. Pharm. 80 (1992) 101–108. [56] G. Ashwell, J. Harford, Carbohydrate-specific receptors of the liver, Annu. Rev. Biochem. 51 (1982) 531–554. [57] M. Nishikawa, M. Yamauchi, K. Morimoto, E. Ishida, Y. Takakura, M. Hashida, Hepatocyte-targeted in vivo gene expression by intravenous injection of plasmid DNA complexed with synthetic multi-functional gene delivery system, Gene Ther. 7 (2000) 548. [58] T. Ohashi, S. Boggs, P. Robbins, A. Bahnson, K. Patrene, F. Wei, J. Wei, J. Li, L. Lucht, Y. Fei, S. Clark, M. Kimak, H. He, P. Mowery-Rushton, J.A. Barranger, Efficient transfer and sustained high expression of the human glucocerebrosidase gene in mice and their functional macrophages following transplantation of bone marrow transduced by a retroviral vector, Proc. Natl. Acad. Sci. USA 89 (1992) 11332. [59] D.B. Kohn, N. Sarver, Gene therapy for HIV-1 infection, Adv. Exp. Med. Biol. 394 (1996) 421. [60] A.P. Rupprecht, D.L. Coleman, Transfection of adherent murine peritoneal macrophages with a reporter gene using DEAE-dextran, J. Immunol. Methods 144 (1991) 157. [61] K.D. Mack, R. Wei, A. Elbagarri, N. Abbey, M.S. McGrath, A novel method for DEAE-dextran mediated transfection of adherent primary cultured human macrophages, J. Immunol. Methods 211 (1998) 79.
Cell-specific delivery of genes with glycosylated carriers Mitsuru Hashida*, Makiya Nishikawa, Fumiyoshi Yamashita, Yoshinobu Takakura Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606 -8501, Japan
Abstract Cationic liposomes and polymers have been accepted as effective non-viral vectors for gene delivery with low immunogenicity unlike viral vectors. However, the lack of organ or cell specificity sometimes hampers their application and the development of a cell-specific targeting technology for them attracts great interest in gene therapy. In this review, the potential of cell-specific delivery of genes with glycosylated liposomes or polymers is discussed. Galactosylated liposomes and poly(amino acids) are selectively taken up by the asialoglycoprotein receptor-positive liver parenchymal cells in vitro and in vivo after intravenous injection. DNA–galactosylated cationic liposome complexes show higher DNA uptake and gene expression in the liver parencymal cells in vitro than DNA complexes with bare cationic liposomes. In the in vitro gene transfer experiment, galactosylated liposome complexes are more efficient than DNA–galactosylated poly(amino acids) complexes but they have some difficulties in their biodistribution control. On the other hand, introduction of mannose residues to carriers resulted in specific delivery of genes to non-parenchymal liver cells. These results suggest advantages of these glycosylated carriers in cell-specific targeted delivery of genes. 2001 Elsevier Science B.V. All rights reserved. Keywords: Plasmid DNA delivery; Galactosylated liposomes; Galactosylated polymers; Hepatocyte specific delivery; Mannosylated carriers; Gene therapy
Contents 1. Introduction ............................................................................................................................................................................ 2. Biodistribution of naked plasmid DNA ..................................................................................................................................... 3. Biodistribution and gene expression of plasmid DNA complexed with bare cationic liposomes ..................................................... 4. Cell-specific gene transfection by plasmid DNA–glycosylated cationic liposome complexes ........................................................ 5. Galactosylated cationic poly(amino acids) for gene delivery ....................................................................................................... 6. Mannosylated carriers for macrophage-specific gene transfection................................................................................................ 7. Conclusions ............................................................................................................................................................................ References ..................................................................................................................................................................................
1. Introduction The use of non-viral vectors in gene delivery *Corresponding author. Tel.: 1 81-75-753-4525; fax: 1 81-75753-4575. E-mail address: [email protected] (M. Hashida).
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attracts great interests because they lack some of the risks inherent in viral vector systems [1,2]. Among various types of non-viral vector systems, cationic liposomes or polymers seem to be promising because of their high gene expression efficiency [1]. In the case of cationic liposomes, however, the highest gene expression is observed in the lung after in-
0169-409X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 01 )00209-5
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travenous injection of their plasmid DNA complexes in most cases because the lung capillaries are the first traps to be encountered [3–6]. Therefore, development of carrier systems that can escape from undesired tissue uptake and exhibits target cell-specific gene expression is highly required. For cell specific delivery, the receptor-mediated endocytosis (RME) systems endowed to various cell types would be useful and a number of gene delivery systems have been developed to introduce the foreign DNA into specific cells with RME. Ligands currently being investigated include galactose [7– 11], mannose [12–16], lactose [17], transferrin [18,19], epidermal growth factor [20], and antibodies [21]. Among these receptors, asialoglycoprotein receptor is the most promising for gene targeting since it exhibits high affinity and a rapid internalization rate [22]. In this series of investigations, we have developed various kinds of macromolecular and particulate carrier systems with carbohydrate recognition devices for cell-selective delivery of low-molecular weight drugs [23–26], proteins [27–29], and genes [30–34]. In these approaches, we emphasized the importance of the pharmacokinetic consideration for the rational design of drug delivery systems. The physicochemical properties, such as molecular weight, particle size, and electrical charge are also demonstrated to determine the biodistribution profile of carriers after intravenous injection [5,25,28]. Even in the case of glycosylated carriers, the findings indicate that not only ligand structure grafted to carriers but also the overall physicochemical properties of the carriers themselves determine the amount delivered to receptor-positive cells after their systemic administration [30–32]. In this review, we attempt to assess the glycosylated cationic liposomes and polymers for in vivo gene delivery in relation to their physicochemical and pharmacokinetic properties.
nated from plasma due to extensive uptake by the liver after intravenous injection and the uptake clearance is almost identical to plasma flow rate in the liver as shown in Fig. 1 [35]. Uptake of [ 32 P] DNA is preferentially proceeded by the liver nonparenchymal cells and Takagi et al. demonstrated that the binding and uptake of [ 32 P] DNA in cultured mouse peritoneal macrophages were inhibited by polyinosinic acid (poly [I]) and dextran sulfate [36]. Thus, the scavenger receptor which recognizes a wide variety of the anionic macromolecules expected to be responsible for this phenomenon, although Takakura et al. reported that the peritoneal macrophages from class A scavenger receptor-knockout mice [37] still exhibit uptake of [ 32 P] DNA. Degradation of DNA by nuclease is another route of elimination from blood circulation but its rate (clearance) is about one-tenth of that of hepatic uptake (Fig. 1).
3. Biodistribution and gene expression of plasmid DNA complexed with bare cationic liposomes Cationic liposomes condense plasmid DNA to form particles based on the electrostatic interaction
2. Biodistribution of naked plasmid DNA To construct a strategy for the design of cellspecific carrier systems of plasmid DNA, a thorough understanding of their in vivo disposition characteristics is required. Kawabata et al. reported that [ 32 P] labeled plasmid DNA ([ 32 P] DNA) is rapidly elimi-
Fig. 1. Hepatic uptake and urinary excretion clearances for plasmid DNA in comparison with degradation clearances in plasms.
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and protect it from degradation. Mahato et al. demonstrated disposition characteristics of [ 32 P] DNA–cationic liposomes complexes after intravenous injection in mice [4,5]. Rapid clearance of plasmid DNA from the circulation was observed with extensive accumulation in the lung and liver. In addition, [ 32 P] DNA–cationic liposome complexes were predominantly taken up by the liver non-parenchymal cells and the uptake was inhibited by the preceeding administration of dextran sulfate, suggesting the involvement of a phagocytic process. Intravenous injection of naked plasmid DNA actually shows no gene expression even in the liver where the highest uptake is observed [4,38,39]. In most cases, the highest gene expression is observed in the lung after intravenous injection of DNA– lipopsome complexes because the lung capillaries are the first traps to be encountered (Fig. 2). Li et al. compared intravenous and intraportal injection of
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cationic liposome–protamine–DNA complexes and demonstrated that higher gene expression was observed in the lung, even if the intraportal route was employed [40]. These results suggest difficulty of transfection in the liver using simple cationic liposomes by both intravenous and intraportal administration. On the other hand, it was proposed that dioleoylphosphatidylethanolamine (DOPE) promotes the fusion with the endosomal membrane followed by the release of plasmid DNA into the cytoplasm [41,42]. Therefore, DOPE is frequently formulated in commercially available cationic liposome preparations as a co-lipid. However, it was shown that the cationic liposomes containing cholesterol as a neutral lipid, such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) / Chol liposomes exhibit greater in vivo transfection activity than those containing DOPE [43,44]. The use of the
Fig. 2. In vitro and in vitro transfection efficiency of plasmid DNA–cationic liposome complexes with different neutral lipids. In vitro transfection: HUVEC cells were transfected with DNA–cationic liposome complexes. In vivo transfection: DNA–cationic liposome complexes were intravenously injected and luciferase activities in various organs were determined.
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cholesterol improved the in vivo transfection efficiency, but did not increase transfection efficiency in vitro. Cholesterol-containing complexes maintain the small liposomal structures under in vivo condition [45], and results in efficient transfection in the lung (Fig. 2). Mahato et al. reported that the effects of the size of cationic liposome complexes on the in vivo transfection efficiency after intravenous injection [46]. When the sizes of DOTMA liposomes increased from below 100–400 nm or greater, the increased transfection efficiency was observed in the lung. The critical time period for gene delivery is considered to be first 60 min after intravenous injection of cationic liposome–DNA complexes [47] and the retention of plasmid DNA–cationic liposome complexes in the lung plays an important role [48].
4. Cell-specific gene transfection by plasmid DNA–glycosylated cationic liposome complexes Receptors for carbohydrates such as the asialoglycoprotein receptor on hepatocytes and the mannose receptor on macrophages and liver endothelial cells produce opportunities for cell-specific gene delivery with liposomal carriers. In their delivery, however, not only the receptor recognition but also the accessibility to the cell surface plays an important role. The structure of the blood capillary wall varies among organs and tissues [49]. Discontinuous capillaries of the liver, spleen, and bone marrow sinusoid show large inter-endothelial junctions, i.e. fenestrations of up to 150 nm [49]. In addition, sinusoids of the liver are linked to highly phagocytic Kupffer cells. Thus, the size of plasmid DNA–galactosylated cationic liposome complexes for hepatocytes targeting must be condensed to 150 nm in diameter while the size of plasmid DNA–mannosylated cationic liposome complexes for Kuppfer cells targeting are allowed to exceed over this size. The charge of the plasmid DNA–liposome complexes is also important factor for receptor-mediated gene transfection and the cationic charge of complexes should not be too high to allow specific interaction with receptor. The lipid compositions of cationic liposomes also affect the in vivo and in vitro transfection efficiency. Thus,
control of these properties play important roles in gene transfer with glycosylated liposome carriers in vivo. Glycosylation of liposomes can be achieved through coating with glycoproteins or incorporation of synthetic glycolipids on the surface of liposomes. Hara et al. reported that asialofetuin-labeled liposome encapsulating plasmid DNA were taken up by the asialoglycoprotein receptor-mediated endocytosis in cultured hepatocytes and exhibited the highest hepatic gene expression after intraportal injection with a preload of EDTA [50]. However, the introduction of asialoglycoproteins to liposomes is rather complicated, and a number of problems are associated with the carriers in such as reproducibility and immunogenicity. From these viewpoints, the low molecular weight glycolipids would be more promising. Remy et al. reported the feasibility of galactosepresenting lipopolyamine vectors towards targeted gene transfer into hepatoma cells [9]. Inclusion of galactose residues in the electrically neutral complex increased transgene expression by avoiding interaction with serum proteins because of their electric neutrality. The chemical structure and physicochemical properties of glycolipids seems to be also crucial for successful targeting using liposomes. If the incorporated glycolipids are easily removed from the liposomal membrane via interaction with lipoproteins and so under in vivo conditions, targeting should be ineffective. Based on these considerations, we synthesized cholesten-5-yloxy-N -(4-((1-imino-2-b- D -thiogalactosylethyl)amino)alkyl)formamide (Gal-C4-Chol), a novel cholesterol derivative possessing the cationic charge necessary for DNA binding and galactose residues as a targetable ligand for liver parenchymal cells as shown in Fig. 3 [31,32]. Introduction of many hydrophilic galactose moieties to a lipid anchor would result in their removal from liposomes by interaction with lipoproteins or other lipid compartments [51]. In our formulation, cholesterol was chosen as a hydrophobic anchor because of its stable association with the liposomal membrane [52]. The developed galactosylated liposomes were shown to be very useful with a high biological affinity and a wide range of pharmaceutical applications. Gal-C4-Chol containing cationic liposome / DNA complexes showed low cytotoxicity in human hepa-
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Fig. 3. Chemical structures of Gal-C4-Chol and Man-C4-Chol.
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toma HepG2 cells [31]. Plasmid DNA complexed with Gal-C4-Chol / 3[N-(N9,N9-dimethylaminoethan)carbamoyl] cholesterol (DC-Chol) / DOPE (3:3:4) liposomes showed higher transfection activity and [ 32 P] DNA uptake than that with DC-Chol / DOPE (6:4) liposomes. The presence of 20 mM galactose significantly inhibited both transfection efficiency and uptake of DNA with Gal-C4-Chol liposomes, but not those of bare liposomes. These results indicate that the Gal-C4-Chol containing cationic liposome–DNA complexes are efficiently recognized by asialoglycoprotein receptors, internalized, and lead to gene expression in HepG2 cells. Thus superior in vivo delivery and expression of genes in the liver via asialoglycoprotein receptor-mediated endocytosis was achieved using Gal-C4-Chol containing cationic liposomes as shown in Fig. 4. Optimization of the co-lipid type of cationic liposomes, the charge ratio of the liposome–DNA complexes, and other factors based on the physicochemical considerations should lead to further improvements in hepatic gene delivery using galactosylated liposomes.
5. Galactosylated cationic poly(amino acids) for gene delivery
Fig. 4. Transfection activity of DNA–galactosylated liposome complexes after intravenous administration in mice. Plasmid DNA (50 mg) was complexed with cationic lipids at a charge ratio of 2:3. Luciferase activity was determined 6 h post-injection in the lung (h), kidney (j), spleen (j), and heart (j). Each value represents the mean6S.D. values (n 5 3).
Poly(amino acids) are considered to possess advantages as drug carriers since they are soluble in water, biodegradable, not significantly immunogenic, and have multiple functional groups that can be chemically modified. Among various poly(amino acids) available, poly( L-glutamic acid) (PLGA) and poly( L-lysine) (PLL) have been widely used as carrier backbones. PLGA is an anionic polymer and PLL is a cationic one, so their biodistribution properties are rather different. After intravenous injection into mice, PLL is largely delivered to liver parenchymal cells due to its positive charge, whereas PLGA is partially taken up by liver non-parenchymal cells, probably via a scavenger receptor-like mechanism [53]. Glycosylation changes the biodistribution properties of these polymers especially PLGA and galactosylated (Gal-) PLGA was recovered mainly in the liver after intravenous injection. Furthermore, uptake of Gal-PLGA was observed selectively in liver parenchymal cells, indicating their receptor-
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mediated uptake. It was also supported by the competitive inhibition observed by co-injection of Gal- or Man-bovine serum albumin (BSA) [54]. On the other hand, both Gal-PLL and Man-PLL accumulated largely in parenchymal cells. However, these glycosylated PLLs are also shown to be taken up through receptor-mediated recognition as glycosylated PLGAs. From a pharmacokinetic point of view, PLL seems to be a promising hepatocyte-specific carrier, but galactosylated polymers have some advantages over cationic PLL. Since the hepatotropic nature of cationic polymers is likely due to their positive charge, neutralization of this charge abolishes its affinity for the cells. Complex formation with nucleic acids like plasmid DNA reduces the positive charge of PLL. This, in turn, makes the recognition of glycosylated PLLs via receptors more evident. In addition, the internalization of galactosylated carriers is much faster than that of cationic ones, which could be an advantage for DNA and some drugs [55]. The biodistribution of DNA complexes with macromolecular carriers was more easily controlled than that of DNA–liposome complexes. We have developed various Gal-PLLs with different molecular weights and different numbers of galactose units [16]. Hepatocyte-specific delivery of DNA was successfully achieved by selecting the most suitable size of PLL and by controlling the galactose density on PLL. If a short PLL with a molecular weight of 1800 was used, a larger amount of PLL was required for complex formation than with PLLs with molecular weights of 13 000 and 29 000. In addition, there was hardly any DNA condensation by the short PLL. Intravenously administered DNA–Gal 13 -PLL 13000 and DNA–Gal 26 -PLL 29000 , could reach hepatocytes, indicating that these DNA complexes have suitable biodistribution characteristics for targeting. Compared with these complexes, DNA–Gal 5 -PLL 1800 and DNA–Gal 5 -PLL 13000 had a smaller hepatic uptake clearance, suggesting that both the molecular weight and the degree of galactose modification of PLL determine the hepatic targeting of DNA [16]. In vitro and in vivo gene expression studies revealed that DNA–Gal 13 -PLL 13000 and DNA– Gal 26 -PLL 29000 complexes are superior to the DNA– Gal 5 -PLL 1800 complex. However, the level of gene expression was low compared with that obtained
with DNA–cationic liposome complexes. To improve the efficiency of galactosylated cationic poly(amino acids)-based gene transfer, the use of compounds that can enhance gene expression such as viruses or viral proteins, fusogenic lipids, and membrane-disruptive peptides can be considered. Fusogenic peptides could be promising materials for this purpose because the addition of these peptides to carrier systems strongly enhanced the in vitro gene transfer by several DNA–carrier complexes [56]. However, their application to in vivo gene transfer has not been reported so far. As far as their biodistribution is concerned, they should be firmly attached to carriers. Our study suggested that galactosylated polymeric carrier conjugated with a fusogenic peptide was very effective in improving the level of gene transfer after intravenous injection of DNA– carrier complexes [57]. Fig. 5 gives conceptual explanation about hepatocyte-specific gene delivery with multi-functional polymeric carriers.
6. Mannosylated carriers for macrophagespecific gene transfection Macrophages are important targets for the gene therapy of diseases such as Gaucher’s disease [58] and human immunodeficiency virus (HIV) infection [59], but gene transfection for treatment of such cases is not easy. Application of DEAE-dextran is one of the methods used for gene delivery to macrophages in vitro [60,61]. However, this method is generally not suitable for in vivo application due to problems associated with cellular toxicity, low efficiency, or non-specific biodistribution. Erbacher et al. investigated the suitability of various glycosylated poly( L-lysine) derivatives for introducing plasmid DNA into human monocyte-derived macrophages and found that mannosylated poly( L-lysine) exhibited high transfection activity among various glycosylated poly( L-lysine) [16]. They also reported that the transfection activity was markedly enhanced in the presence of chloroquine due to prevention of endosomal and / or lysosomal degradation of plasmid DNA after mannose receptor-mediated endocytosis. However, the transfection efficiency of poly( Llysine) is inferior to that of cationic liposomes in
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Fig. 5. Design of multi-functional polymeric carriers with galactose moiety for hepatocyte-specific gene delivery and its performance in vivo.
most cases and further chloroquine cannot be used for in vivo gene transfection. We synthesized a novel mannosylated cholesterol
derivative, cholesten-5-yloxy-N-(4-((1-imino-2-b-Dthiomannosylethyl)amino)alkyl)formamide (ManC4-Chol), for gene delivery to macrophages which
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are known to express large numbers of mannose receptors on their surface (Fig. 3) [14,15]. Four types of liposomes were prepared with various molar ratios and their particle size was confirmed to be about 200 nm. Plasmid DNA complexed with cationic liposomes, consisting of a 6:4 mixture of Man-C4-Chol and DOPE, showed higher transfection activity than that complexed with DC-Chol:DOPE (6:4) and DOTMA:DOPE (1:1) liposomes in mouse peritoneal macrophages. The presence of 20 mM mannose significantly inhibited the transfection efficiency of plasmid DNA complexed with Man-C4-Chol:DCChol:DOPE (3:3:4) and Man-C4-Chol:DOPE (6:4) liposomes. These results suggest that the complexes of plasmid DNA and mannosylated cationic liposomes is recognized and taken up by the mannose receptors on mouse peritoneal macrophages. In the in vitro gene delivery, the liver non-parenchymal cells seem to be more efficient target cells than liver parenchymal cells because DNA–cationic liposome complexes can more easily access the target cells in this case. After intravenous injection of DNA–Man-C4-Chol:DOPE (6:4) liposome complexes in mice, the highest gene expression was
observed in the liver, whereas DNA–DC-Chol:DOPE (6:4) liposome complexes showed marked expression only in the lung (Fig. 6). In addition, the gene expression with Man-C4-Chol:DOPE (6:4) liposome–DNA complexes in the liver was observed preferentially in the non-parenchymal cells and was significantly reduced by predosing with mannosylated BSA. These results suggest that plasmid DNA complexed with mannosylated liposomes exhibits the high transfection activity in liver nonparenchymal cells due to recognition by mannose receptors. This may be due to Kuppfer cells were existed around the sinusoidal membranes and selection of the administration route must be considered for the efficient targeted delivery of plasmid DNA. The lipid composition of mannosylated cationic liposome was suggested to affect on both pulmonary and hepatic uptake as well as gene expression based on mannose receptor-mediated endocytosis.
7. Conclusions In conclusion, the superior in vivo delivery and expression of genes in the liver via asialoglycoprotein and mannose receptor-mediated endocytosis is achieved using Gal-C4-Chol and Man-C4-Chol containing cationic liposomes, respectively. The adequate selection of the co-lipid type of liposomes and cationic charge ratio of cationic liposomes–DNA complexes, and administration route, etc. would form basis of design of total carrier systems. Polymeric carriers offer another possibility in gene delivery utilizing RME.
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Fig. 6. Transfection activity of DNA–mannosylated liposome complexes after intraportal administration in mice. Plasmid DNA (50 mg) was complexed with cationic lipids at a charge ratio of 2:3. Luciferase activity was determined 6 h post-injection in the lung (empty square), liver (filled square), kidney (hatched box), spleen (diagonally hatched box), and heart (horizontally hatched box). Each value represents the mean6S.D. values (n 5 3).
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