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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
Received December 26, 2005; accepted March 3, 2006.
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Abstract |
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were much lower after the administration of polyplex than lipoplex irrespective of the N/P ratio, dose of pDNA, or structure and molecular weight of PEI, although these factors affected the transfection efficacy in vivo. We demonstrated that the amount of activated nuclear factor-
B, which contributes substantially to the production of cytokines, was comparable with the control (no treatment) level, and significantly less than that obtained with lipoplex. Although the production of proinflammatory cytokines (TNF-, interferon-
, and interleukin-12) was reduced on the administration of the linear PEI polyplex, serum alanine aminotransferase levels were significantly enhanced by pDNA in a dose-dependent manner, suggesting that such hepatic damage is not induced by proinflammatory cytokines.
). Viral vectors, although highly efficient, have inherent drawbacks such as immunogenicity; therefore, nonviral vectors have increasingly been receiving attention (Yang et al., 1994; Knowles et al., 1995). Of the various types of nonviral vectors, polyethylenimine (PEI) and cationic liposomes are most effective vectors in transfecting pDNA into target cells in vivo (Boussif et al., 1995; Boletta et al., 1997; Goula et al., 1998; Tranchant et al., 2004; Neu et al., 2005). PEI and cationic liposome-mediated gene transfer efficiently delivers a gene to the pulmonary endothelium after an i.v. administration. The factors that enhance the transfection efficacy have been well studied, but it is also important to analyze the side effects of nonviral vectors for clinical applications.
As for the cationic liposome/pDNA complex (lipoplex), side effects have been documented. The CpG motifs in the pDNA sequence up-regulate the expression of transcription factors such as nuclear factor (NF)-B, which contributes substantially to the production of cytokines (Krieg et al., 1995; David et al., 2000; Klinman, 2004). Consequently, lipoplex could induce the production of large quantities of proinflammatory cytokines, such as tumor necrosis factor (TNF)-, interferon (IFN)-, and interleukin (IL)-12 (Freimark et al., 1998; Li et al., 1999; Yew et al., 1999; Sakurai et al., 2002). It was suggested that these cytokines cause liver damage (Tan et al., 2001). In addition, these cytokines cause gene inactivation-inducing transient gene expression after a single injection and a refractory period on repeated dosing (Li et al., 1999; Tan et al., 2001). Therefore, these studies demonstrated that the immune response could influence the hepatic toxicity as well as the gene expression period.
To date, the transfection efficacy of the PEI/pDNA complex (polyplex) has been determined based on the N/P ratio of PEI polyplex, the dose of pDNA, and the structure and molecular weight of PEI (Goula et al., 1998; Bragonzi et al., 2000; Zou et al., 2000; Wightman et al., 2001). In contrast, not much has been done to clarify the cytokine response to PEI polyplex after its i.v. administration. Therefore, these three factors need to be related with cytokine levels for the optimization of gene therapy using PEI polyplex.
In this study, the transfection efficacy, production of cytokines, and toxicity in the liver after the i.v. administration of linear and branched PEI polyplexes were evaluated in relation to the N/P ratio of PEI polyplex, the dose of pDNA, and the structure and molecular weight of PEI. As a control, a N-[1-(2, 3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride (DOTMA)/cholesterol liposome-based lipoplex was selected because of its high transfection efficacy in vivo (Song et al., 1997; Sakurai et al., 2001).
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Materials and Methods |
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-32P]dCTP (3000 Ci/mmol) was obtained from Amersham Biosciences Co. (Piscataway, NJ). QIA-GEN Endofree Plasmid Giga Kit was purchased from QIAGEN GmbH (Hilden, Germany). All other chemicals were of the highest purity available. Animals. Five-week-old female ICR mice (20–23 g) were purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka, Japan). Animals were maintained under conventional housing conditions. All animal experiments were carried out in accordance with the Guidelines for Animal Experiments of Kyoto University.
Preparation of pDNA. pCMV-Luc was constructed by subcloning the HindIII/XbaI firefly luciferase cDNA fragment from a pGL3-control vector (Promega Co., Madison, WI) into the polylinker of a pcDNA3 vector (Invitrogen, Co., Carlsbad, CA). pDNA was amplified in the Escherichia coli strain DH5, isolated, and purified using a QIAGEN Endofree Plasmid Giga Kit. The concentration of DNA was determined by measuring UV absorption at 260 nm. The pDNA was labeled with [-32P]dCTP by nick translation.
Preparation of PEI Polyplex. PEI polyplex was prepared as reported (Morimoto et al., 2003). In brief, linear PEI or branched PEI was dissolved in a 5% dextrose solution and adjusted to pH 7.4. PEI polyplex was formed by adding an equal volume of PEI to pDNA in 5% dextrose at various ratios and left at 37 °C for 30 min. The ratio of PEI to pDNA was expressed as the N/P ratio, which is the molar ratio of PEI nitrogen to DNA phosphate (Goula et al., 1998).
Preparation of Cationic Liposomes. DOTMA/cholesterol liposomes were prepared as reported (Kawakami et al., 2000a). DOTMA and cholesterol were dissolved in chloroform at a molar ratio of 1:1. The mixture was vacuum-desiccated and resuspended in 5% dextrose. After hydration, the suspension was sonicated on ice for 3 min, and the resulting liposomes were extruded through a 220-nm polycarbonate filter.
Preparation of Lipoplex. Lipoplex was prepared as reported (Kawakami et al., 2000a,b; Sakurai et al., 2001). In brief, it was formed by adding an equal volume of cationic liposomes to pDNA in 5% dextrose at a mixing ratio (–:+) of 1.0:3.1 and stored at room temperature for 30 min. The ratio of liposomes to pDNA was expressed as a charge ratio (–:+), which is the molar ratio of cationic lipids to DNA phosphate (Yang and Huang, 1997).
Measurement of 
Potential and Particle Size. PEI and pDNA were mixed in 5% dextrose as above and concentrated for i.v. administration. After 30 min, the potential and size of PEI polyplex were measured using Nano ZS (Malvern Instruments, Ltd., Malvern, Worcestershire, UK).
Gene Expression Experiments. Gene expression was measured as described previously (Liu et al., 1997; Kawakami et al., 2000a,b). Mice were administered i.v. with 300 µl of lipoplex or PEI polyplex. At specific time points, mice were sacrificed, the lung and liver were harvested, and homogenates were prepared by adding lysis buffer (0.05% Triton X-1000, 2 mM EDTA, and 0.1 M Tris, pH 7.8) using homogenizer (OMNI TH; Yamato Scientific Co. Ltd., Tokyo, Japan) at 4°C. The volume of lysis buffer added was 4 µl/mg for lung and 5 µl/mg for liver. To lyse cells, the homogenates were treated with three cycles of freezing and thawing. The homogenates were centrifuged at 12,000g for 7 min at 4°C. Twenty microliters of each supernatant was analyzed for luciferase activity with 100 µl of luciferase assay buffer (Picagene; Toyo Ink Mfg. Co. Ltd., Tokyo, Japan), using a luminometer (Lumat LB 9507; Berthold Technologies GmbH and Co. KG, Bad Wildbad, Germany).
Measurements of Cytokines and ALT. Serum was prepared as outlined in our previous study (Sakurai et al., 2001). At specific time points after the i.v. administration of PEI polyplex and lipoplex, blood was collected from the vena cava and left to stand for 3 h at 37°C and then overnight at 4°C. Samples were centrifuged, and the supernatants were collected for serum. Serum TNF-, IFN-, and IL-12 concentrations were determined with enzyme-linked immunosorbent assay kits according to the manufacturer's (Genzyme Co., Cambridge, MA) instructions. The serum ALT concentration was measured with kits using the UV-Rate method according to the manufacturer's direction (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
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B. Three hours after the administration of PEI polyplex and lipoplex, mice were scarified, and their livers were collected. A nuclear extract of liver cells was prepared using the Nuclear/Cytosol Fractionation Kit (BioVision, Inc., CA). To analyze the extract, protein concentrations were prepared at 0.25 µg/µl. The amount of activated NFB was measured by using an Enzyme Immunoassay for NFB (human, mouse and rat) (Oxford Biomedical Research, Inc., Oxford, MI).
Experiments on in Vivo Distribution. Radioactivity was measured as reported previously (Kawakami et al., 2000a). Mice received an i.v. injection of 10 kBq [32P]pDNA and pDNA (30 µg) in a complex with linear PEI25 or cationic liposomes in 5% dextrose (300 µl) and were killed at a given time point. The liver and lung were removed, washed with saline, blotted dry, and weighed. Ten microliters of blood and 20 to 30 mg of each tissue were digested with 700 µl of Soluene-350 by incubation overnight at 45 °C. After the digestion, 200 µl of isopropanol, 200 µl of 30% hydrogen peroxide, 100 µl of 5 N HCl, and 5.0 ml of Clear-sol I were added. The samples were stored overnight, and radioactivity was measured in a scintillation counter (LSA-500; Beckman, Tokyo, Japan).
Statistical Analysis. Statistical analysis was performed using Student's paired t test for two groups. Multiple comparisons among different groups were performed with the Turkey-Kramer test. P < 0.05 was considered to be indicative of statistical significance.
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Results |
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Potential and Particle Size. Figure 1A shows the potential of the linear and branched PEI with various molecular weights and N/P ratios. PEI polyplex was negatively charged at an N/P ratio of 3 except the branched PEI70 polyplex which was positive, and increasing the N/P ratio from 5 resulted in a positive charge for all polyplexes (approximately 30–40 mV). Increasing the N/P ratio between 5 and 25 hardly changed the potential of PEI polyplex (approximately 30–50 mV). As for the size of particles, it was approximately 400 nm at an N/P ratio of 3, except for the branched PEI10 polyplex (Fig. 1B). At an N/P ratio of from 5 to 25, the size of all PEI polyplexes appeared to be reduced to approximately 80 to 90 nm, and this value was constant as the N/P ratio increased. In the present study, the potential and particle size of lipoplex (–:+ of 1.0:3.1) were 60 ± 3.1 mV (n = 3) and 103 ± 0.78 nm (n = 3), respectively. Effect of the N/P Ratio of Polyplex and Structure and Molecular Weight of PEI on Cytokines. After the i.v. administration of PEI polyplex in mice, the gene expression was much stronger in lung than liver, heart, spleen, or kidney (data not shown). Figure 2 shows the gene expression in lung and liver after the i.v. administration of the PEI polyplex and lipoplex preparations. The higher the N/P ratio of the branched PEI polyplex and molecular weight, the higher the transfection efficiency observed (Fig. 2, A–C). As far as the branched PEI polyplex is concerned, an N/P ratio of 10 yielded the highest level of expression in lung and liver, but the gene expression was much lower than that obtained with lipoplex. However, this was the maximal tolerated N/P ratio since a further increase to 15 was lethal for 6-h evaluation. In contrast, the linear PEI polyplex yielded similar levels to lipoplex at N/P ratios of 20 and 25 (Fig. 2D). The maximal tolerated N/P ratio of the linear PEI polyplex was 30.
Intravenously injected lipoplex induced the production of proinflammatory cytokines such as TNF-, IFN-, and IL-12 (Whitmore et al., 1999; Sakurai et al., 2002). Among these cytokines, TNF- is the primary source of toxicity because it induces septic shock in animals at high concentrations (Tan et al., 2002). Then, the response to an i.v. administration of a preparation of PEI polyplex varying in N/P ratio and the structure and molecular weight of PEI was investigated. After the injection, a significantly high TNF- concentration was observed in serum (Fig. 3). The branched PEI was not lethal at an N/P ratio of up to 15 as of 3 h after the injection. After the i.v. injection of PEI polyplex, however, the TNF- concentration was compatible with the control value (no treatment) and was significantly lower than that after the injection of lipoplex (P < 0.01). Moreover, this reduction in response was independent of the N/P ratio of the polyplex or structure and molecular weight of PEI (Fig. 3, A–D), although these factors could affect the gene expression in the lung and liver. When a linear PEI polyplex was prepared at an N/P ratio of 20, the efficacy of transfection increased without lethal toxicity. Accordingly, a linear PEI polyplex with an N/P ratio of 20 was selected for further investigation.
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concentration obtained with PEI polyplex at a pDNA dose of 30, 50, and 80 µg was significantly lower than that obtained with lipoplex at a pDNA dose of 30 µg (Fig. 4B). To investigate the cytokine response in detail, serum levels of not only TNF- but also IFN- and IL-12 were measured for 12 h (Fig. 5, A–C). These proinflammatory cytokines were significantly induced by the administration of lipoplex. This characteristic was consistent with the previous results about lipoplex (Whitmore et al., 1999; Sakurai et al., 2002). However, TNF-, IFN-, and IL-12 concentrations were much lower after the injection of PEI polyplex than that of lipoplex (Fig. 5).
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Serum ALT Activity Triggered by PEI Polyplex. To evaluate the toxicity in the liver, the serum ALT level was determined. Raising the dose of pDNA (30 and 50 µg) in lipoplex increased the serum ALT level, and 80 µg of pDNA was lethal (Fig. 6A). Increasing the dose of pDNA (30, 50, and 80 µg) in the linear PEI polyplex also increased the serum ALT level (Fig. 6B). When we checked the liver surface after abdominal operation, the damage of hepatic lobule was observed after mice administered the linear PEI25 polyplex at a pDNA dose of 50 and 80 µg.
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B Activated by Linear PEI Polyplex. To investigate the mechanism of the cytokine response by linear PEI polyplex, the amount of activated NFB was measured. After i.v. administration of lipoplex, significantly more activated NFB was detected (P < 0.05). In contrast, the amount activated by the PEI polyplex was compatible with the control (no treatment group) and was significantly lower than that in response to lipoplex (P < 0.05) (Fig. 7).
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; Tan et al., 2001). To investigate whether the linear PEI polyplex provides long-term gene expression, its effect was compared with that of lipoplex at various time points. However, there was little difference in gene expression between linear PEI polyplex and lipoplex (Fig. 9).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Bragonzi et al., 2000; Wightman et al., 2001; Zou et al., 2001). In this study, we selected DOTMA/cholesterol liposomes as cationic liposomes to prepare the lipoplex because of their high transfection efficacy in vivo (Song et al., 1997; Kawakami et al., 2000a; Sakurai et al., 2001). As shown in Fig. 2, the linear PEI polyplex induced the highest level of gene expression among the polyplexes under optimized conditions and was equal in efficacy to lipoplex. These observations are consistent with those of Bragonzi et al. (2000), who found that the gene expression efficacy of linear PEI polyplex was equal to lipoplex under optimal conditions.
Then, cytokine response characteristics after the i.v. administration of PEI polyplex were evaluated based on the N/P ratio of PEI polyplex, the dose of pDNA, and the structure and molecular weight of PEI. Interestingly, serum TNF- levels were low irrespective of the N/P ratio of PEI polyplex and structure and molecular weight of PEI (Fig. 3) or the dose of pDNA (Fig. 4B), although these factors affected the transfection efficacy in vivo (Figs. 2 and 4A). These findings are partly supported by the report that the serum TNF- level was much lower in mice administered linear PEI22 polyplex (N/P ratio, 6; pDNA dose, 25 µg/mouse) than those given lipoplex (Tan et al., 2001). To confirm whether this response is specific to TNF- at 3 h, TNF-, IFN-, and IL-12 concentrations were measured until 12 h; consequently, TNF-, IFN-, and IL-12 concentrations by linear PEI polyplex were also much lower than that by lipoplex at various time points (Fig. 5). All of these results provide evidence that PEI polyplex hardly induced the production of proinflammatory cytokines irrespective of the N/P ratio of PEI polyplex, dose of pDNA, or structure and molecular weight of PEI, although these are important factors for transfection efficacy in vivo (Figs. 2 and 4A).
NFB is a central regulator of inflammatory and immune responses (Barnes and Karin, 1997) and is crucial for the transcription of multiple proinflammatory molecules, including TNF-, IL-1
, IL-2, IL-6, IL-8, IL-12, and IFN- (Lenardo and Baltimore, 1989). To investigate further the production of cytokines in response to PEI polyplex, the amount of activated NFB in liver was measured. As shown in Fig. 7, we demonstrated that the amount of NFB activated by linear PEI polyplex was comparable with the control level (untreated group) and was significantly lower than that activated by lipoplex (P < 0.05). In contrast, the amount of hepatic NFB activated by lipoplex was significantly enhanced. These results are well consistent with the concentrations of proinflammatory cytokines produced when lipoplex and linear PEI polyplex were administered (Fig. 5). Thus, these observations lead us to conclude that lower levels of proinflammatory cytokines are produced in response to PEI polyplex after i.v. administration.
The immunostimulatory response observed in mammalian cells has been shown to arise in part from the recognition of the unmethylated CpG dinucleotides present in bacterial DNA or pDNA. Yi et al. (1998) reported that the activation of leukocyte by CpG DNA might occur in association with the acidification of endosomes since chloroquine, which is an inhibitor of endosomal acidification, blocks CpG DNA-induced IB and IB degradation and the subsequent activation of NFB; consequently, the response to produce proinflammatory cytokines is reduced. Likewise, Yew et al. (2000) demonstrated that two such inhibitors, chloroquine and quinacrine, greatly reduced the production of IL-12 by mouse spleen cells in vitro and inhibited cytokine production in the lung by approximately 50% without affecting gene expression. It has been reported that the transfection efficiency of PEI polyplex is due to its capacity to buffer the endosome (proton sponge effect) (Boussif et al., 1995; Kichler et al., 2001; Akinc et al., 2005); therefore, such a property might abolish the pDNA (CpG DNA)-induced activation of NFB (Fig. 7).
We previously investigated the distribution of lipoplex and demonstrated that when i.v. injected, it was predominantly taken up by Kupffer cells via the phagocytic process that is responsible for the production of proinflammatory cytokines (Sakurai et al., 2002). Taking this into consideration, we hypothesized that the hepatic uptake of PEI polyplex is less than that of lipoplex. However, the biodistribution study demonstrated that much more [32P] linear PEI polyplex than [32P] lipoplex accumulates in the liver (Fig. 8). These results suggested that the difference in the response induced by PEI polyplex and lipoplex could not explain their distribution.
As far as the pulmonary accumulation of lipoplex is concerned, we and other groups have reported that lipoplex-induced hemagglutination is an important factor in the localization of lipoplex to the lung (Sakurai et al., 2001; Fumoto et al., 2005). It should be considered that the hemagglutination is caused by electrostatic interaction between the erythrocytes and lipoplex. In the distribution study, the potential of PEI polyplex (approximately 40 mV) (Fig. 1) was lower than that of lipoplex (approximately 60 mV), suggesting less electrostatic interaction between the erythrocytes and linear PEI polyplex. This hypothesis may be partly supported by the report that the aggregation of erythrocytes caused by linear PEI polyplex was minimal (Kircheis et al., 2001). Such potential characteristics may reflect a more hepatic-selective distribution of the linear PEI polyplex (Fig. 8).
To evaluate hepatic damage, serum ALT activity was measured. As shown in Fig. 6A, raising the dose of pDNA in the lipoplex preparation increased the serum level of ALT. This finding regarding toxicity was consistent with the previous reports (Tousignant et al., 2000; Loisel et al., 2001). Recently, Tan et al. (2001) suggested that such hepatic damage was caused by the proinflammatory cytokines secreted when lipoplex was injected i.v. In this study, we demonstrated that PEI polyplex hardly induced the production of any proinflammatory cytokines (Figs. 3, 4, 5), but hepatic toxicity was observed (Fig. 6B). These observations provide evidence that the hepatic damage is not mediated by the proinflammatory cytokines, suggesting that the mechanisms behind the toxicity of lipoplex and linear PEI polyplex are different. Recently, Moghimi et al. (2005) reported PEI polyplex (branched and linear), by using calf thymus DNA, induced the cytotoxicity (necrosis and/or apoptosis) in several cultured human cell lines. These results also suggested hepatic damage might be induced by PEI itself.
In this study, we examined the effect of the dose of pDNA in the PEI polyplex on hepatic damage. As shown in Fig. 6B, serum ALT activity at 30 µg of pDNA was compatible with the control (no treatment). However, at higher doses (50 and 80 µg), the serum ALT level increased, suggesting that i.v. injected PEI polyplex causes hepatic damage in a pDNA dose-dependent manner. These observations are consistent with those of Chollet et al. (2002), whose histological analysis revealed necrosis in the liver after the i.v. administration of a linear polyplex containing 100 µg of pDNA.
As shown in Fig. 6, lipoplex at pDNA dose of 80 µg was lethal. This observation corresponded with Hofland et al. (1997), who reported lipoplex at the pDNA dose of approximately 80 µg was the maximal tolerated dose in mice. It is expected that the hepatic toxicity between lipoplex and polyplex at pDNA dose of 80 µg is to the same extent since hepatic toxicity was nearly similar between lipoplex and PEI polyplex at pDNA dose of 30 and 50 µg (Fig. 6). Therefore, the lethal effect of lipoplex at pDNA dose of 80 µg might be explained by cytokine response, hematologic and serologic changes typified by leukopenia and thrombocytopenia (Tousignant et al., 2000).
Goula et al. (1998) reported that the branched PEI25 polyplex was lethal within a few minutes even when used at a low N/P ratio, although how it was prepared is not clear. As shown in Fig. 2, the maximal tolerated N/P ratio of branched PEI10, PEI25, and PEI70 was 10 since a further increase to 15 was lethal in 6 h. Thus, this observation is consistent with the report by Goula et al. (1998). Our results also suggested that the lethal toxicity of the branched PEI polyplex does not depend on the molecular weight of PEI.
In conclusion, the concentration of proinflammatory cytokines, such as TNF-, produced were much lower when PEI polyplex rather than lipoplex was administered irrespective of the N/P ratio of the polyplex, dose of pDNA used, or structure and molecular weight of PEI, although these factors affected the transfection efficacy in vivo. We demonstrated that the amount of NFB activated by the linear PEI polyplex was comparable with the control (untreated group) and was significantly lower than that when lipoplex was administered. Although the production of proinflammatory cytokines (TNF-, IFN-, and IL-12) was reduced by the administration of the linear PEI polyplex, serum ALT levels were significantly enhanced by pDNA in a dose-dependent manner, suggesting that the hepatic damage is not induced by proinflammatory cytokines. This information will be valuable for the development of nonviral vectors for clinical applications.
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Footnotes |
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ABBREVIATIONS: PEI, polyethylenimine; pDNA, plasmid DNA; lipoplex, cationic liposome/pDNA complex; NFB, nuclear factor B; TNF, tumor necrosis factor; IFN, interferon; IL, interleukin; N/P ratio, the ratio of PEI nitrogen and DNA phosphate; polyplex, PEI/pDNA complex; DOTMA, N-[1-(2, 3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride; –:+, charge ratio; ALT, alanine aminotransferase.
Address correspondence to: Dr. Mitsuru Hashida, Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan. E-mail: hashidam{at}pharm.kyoto-u.ac.jp
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References |
|---|
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Akinc A, Thomas M, Klibanov AM, and Langer R (2005) Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. J Gene Med 7: 657–663.[CrossRef][Medline]
Barnes PJ and Karin M (1997) Nuclear factor-B: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066–1071.
Boletta A, Benigni A, Lutz J, Remuzzi G, Soria MR, and Monaco L (1997) Nonviral gene delivery to the rat kidney with polyethylenimine. Hum Gene Ther 8: 1243–1251.[Medline]
Boussif O, Lezoualc HF, Zanta AM, Mergny DM, Scherman D, Demeneix B, and Behr JP (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 92: 7297–7301.
Bragonzi A, Dina G, Villa A, Calori G, Biffi A, Bordignon C, Assael BM, and Conese M (2000) Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs. Gene Ther 7: 1753–1760.[CrossRef][Medline]
Chollet P, Favrot MC, Hurbin A, and Coll JL (2002) Side-effects of a systemic injection of linear polyethylenimine-DNA complexes. J Gene Med 4: 84–91.[CrossRef][Medline]
David PS, Shalin N, Shannon JB, David AH, and Katryn JS (2000) Phosphorothioate backbone modification modulates macrophage activation by CpG DNA. J Immunol 165: 4165–4173.
Freimark BD, Blezinger HP, Florack VJ, Nordstrom JL, Long SD, Deshpande DS, Nochumson S, and Petrak KL (1998) Cationic lipids enhance cytokine and cell influx levels in the lung following administration of plasmid: cationic lipid complexes. J Immunol 160: 4580–4586.
Fumoto S, Kawakami S, Shigeta K, Higuchi Y, Yamashita F, and Hashida M (2005) Interaction with blood components plays a crucial role in asialoglycoprotein receptor-mediated in vivo gene transfer by galactosylated lipoplex. J Pharmacol Exp Ther 315: 484–493.
Goula D, Benoist C, Mantero S, Merlo G, Levi G, and Demeneix BA (1998) Polyethylenimine-based intravenous delivery of transgenes to mouse lung. Gene Ther 5: 1291–1295.[CrossRef][Medline]
Hofland HEJ, Nagy D, Liu JJ, Spratt K, Lee YL, Danos O, and Sullivan SM (1997) In vivo Gene transfer by intravenous administration of stable cationic lipid/DNA complex. Pharm Res (N Y) 14: 742–749.
Kawakami S, Fumoto S, Nishikawa M, Yamashita F, and Hashida M (2000a) In vivo gene delivery to the liver using novel galactosylated cationic liposomes. Pharm Res (N Y) 17: 306–313.
Kawakami S, Sato A, Nishikawa M, Yamashita F, and Hashida M (2000b) Mannose receptor-mediated gene transfer into macrophages using novel mannosylated cationic liposomes. Gene Ther 7: 292–299.[CrossRef][Medline]
Kircheis R, Wightman L, Schreiber A, Robitza B, Rossler V, Kursa M, and Wagner E (2001) Polyethylenimine/DNA complexes shielded by transferrin target gene expression to tumors after systemic application. Gene Ther 8: 28–40.[CrossRef][Medline]
Kichler A, Leborgne C, Coeytaux E, and Danos O (2001) Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med 3: 135–144.[CrossRef][Medline]
Klinman DM (2004) Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat Rev Immunol 4: 1–10.[CrossRef][Medline]
Knowles MR, Hohneker KW, Zhou Z, Olsen JC, Noah TL, Hu PC, Leigh MW, and Boucher RC (1995) A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N Engl J Med 333: 823–831.
Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, and Klinman DM (1995) CpG motif in bacterial DNA trigger direct B-cell activation. Nature (Lond) 374: 546–549.[CrossRef][Medline]
Lenardo MJ and Baltimore D (1989) NF-B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 58: 227–229.[CrossRef][Medline]
Li S, Wu SP, Whitmore M, Loeffert EJ, Wang L, Watkins SC, Pitt BR, and Huang L (1999) Effect of immune response on gene transfers to the lung via systemic administration of cationic vectors. Am J Physiol 276: L796–L804.
Liu F, Qi H, Huang L, and Liu D (1997) Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration. Gene Ther 6: 517–523.
Loisel S, Le Gall C, Doucet L, Ferec C, and Floch V (2001) Contribution of plasmid DNA to hepatotoxicity after systemic administration of lipoplexes. Hum Gene Ther 12: 685–696.[CrossRef][Medline]
Moghimi SM, Symonds P, Murray JC, Hunter AC, Debska G, and Szewczyk A (2005) A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther 11: 990–995.[CrossRef][Medline]
Morimoto K, Nishikawa M, Kawakami S, Nakano T, Hattori Y, Fumoto S, Yamashita F, and Hashida M (2003) Molecular weight-dependent gene transfection activity of unmodified and galactosylated polyethylenimine on hepatoma cells and mouse liver. Mol Ther 7: 254–261.[Medline]
Neu M, Fischer D, and Kissel T (2005) Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives. J Gene Med 7: 992–1009.[CrossRef][Medline]
Ross G, Erickson R, Knorr D, Motulsky AG, Parkman R, Samulski J, Straus SE, and Smith BR (1996) Gene therapy in the United States: a five-year status report. Hum Gene Ther 7: 1781–1790.[Medline]
Sakurai F, Nishioka T, Saito H, Baba T, Okuda A, Matsumoto O, Taga T, Yamashita F, Takakura Y, and Hashida M (2001) 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 the neutral helper lipid. Gene Ther 8: 677–686.[CrossRef][Medline]
Sakurai F, Terada T, Yasuda K, Yamashita F, Takakura Y, and Hashida M (2002) The role of tissue macrophages in the induction of proinflammatory cytokine production following intravenous injection of lipoplexes. Gene Ther 9: 1120–1126.[CrossRef][Medline]
Song YK, Liu F, Chu SY, and Liu D (1997) Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration. Hum Gene Ther 8: 1585–1594.[Medline]
Tan Y, Liu F, Li Z, Li S, and Huang L (2001) Sequential injection of cationic liposome and plasmid DNA effectively transfects the lung with minimal inflammatory toxicity. Mol Ther 3: 673–682.[CrossRef][Medline]
Tan Y, Zhang JS, and Huang L (2002) Codelivery of NF-B decoy-related oligodeoxynucleotide improves LPD-mediated systemic gene transfer. Mol Ther 6: 804–812.[CrossRef][Medline]
Tousignant JD, Gates AL, Ingram LA, Johnson CL, Nietupski JB, Cheng SH, Eastman SJ, and Scheule RK (2000) Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid: plasmid DNA complexes in mice. Hum Gene Ther 11: 2493–2513.[CrossRef][Medline]
Tranchant I, Thompson B, Nicolazzi C, Mignet N, and Scherman D (2004) Physicochemical optimisation of plasmid delivery by cationic lipids. J Gene Med 6: 24–35.
Whitmore M, Li S, and Huang L (1999) LPD lipopolyplex initiates a potent cytokine response and inhibits tumor growth. Gene Ther 6: 1867–1875.[CrossRef][Medline]
Wightman L, Kircheis R, Rossler V, Carotta S, Ruzicka R, Kursa M, and Wagner E (2001) Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med 3: 362–372.[CrossRef][Medline]
Yang JP and Huang L (1997) Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Ther 4: 950–960.[CrossRef][Medline]
Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, and Wilson JM (1994) Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci USA 91: 4407–4411.
Yew NS, Wang KX, Przybylska M, Bagley RG, Stedman M, Marshall J, Scheule RK, and Cheng SH (1999) Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid: pDNA complexes. Hum Gene Ther 10: 223–234.[CrossRef][Medline]
Yew NS, Zhao H, Wu IH, Song A, Tousignant JD, Przybylska M, and Cheng SH (2000) Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs. Mol Ther 1: 255–262.[CrossRef][Medline]
Yi AK, Tuetken R, Redford T, Waldschmidt M, Kirsch J, and Krieg AM (1998) CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species. J Immunol 160: 4755–4761.
Zou SM, Erbacher P, Remy JS, and Behr JP (2000) Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse. J Gene Med 2: 128–134.[CrossRef][Medline]
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