Polycation liposomes, a novel nonviral gene transfer system ... - Nature

2 downloads 0 Views 306KB Size Report
1Department of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka; 2Department of ..... obtaining high efficiency of gene transfer by polylysine- .... (Midland, MI, USA) and from Nippon Shokubai (Osaka,.

Gene Therapy (2000) 7, 1148–1155  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt

NONVIRAL TRANSFER TECHNOLOGY

RESEARCH ARTICLE

Polycation liposomes, a novel nonviral gene transfer system, constructed from cetylated polyethylenimine Y Yamazaki1, M Nango2, M Matsuura1, Y Hasegawa1, M Hasegawa3 and N Oku1 1

Department of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka; 2Department of Applied Chemistry, Nagoya Institute of Technology, Nagoya; and 3DNAVEC Research Inc, Kannondai, Tsukuba, Ibaraki, Japan

A novel gene transfer system was developed by using liposomes modified with cetylated polyethylenimine (PEI, MW 600). This polycation liposome, PCL, showed remarkable transfection efficiency as monitored by the expression of the GFP reporter gene. Most conventional cationic liposomes require phosphatidylethanolamine or cholesterol as a component, although PCLs did not. Egg yolk phosphatidylcholine- and dipalmitoylphosphatidylcholine-based PCL were as effective as dioleoylphosphatidylethanolaminebased PCLs for gene transfer. Concerning the cytotoxicity against COS-1 cells and hemolytic activity, the PCL was superior to conventional cationic liposome preparations. Fur-

thermore, the transfection efficacy of PCLs was enhanced, instead of being diminished, in the presence of serum. Effective gene transfer was observed in all eight malignant and two normal cells line tested, as well as in COS-1 cells. We also examined the effect of the molecular weight of PEI on PCL-mediated gene transfer, and observed that PEI with a MW of 1800 Da was as effective as that with one of 600, but that PEI of 25 000 was far less effective. Finally, an in vivo study was done in which GFP was effectively expressed in mouse liver after injection of PCL via the portal vein. Thus, PCL represents a new system useful for transfection and gene therapy. Gene Therapy (2000) 7, 1148–1155.

Keywords: gene transfer; transfection; liposome; polycation; polyethylenimine

Introduction Efficient and safe gene transfer systems are the fundamental basis for gene therapy, as well as for laboratory applications.1–3 Viral systems are, in general, quite effective for gene transfer, although there are arguments about their safety and immunogenicity.4,5 Therefore, a number of nonviral systems, especially cationic liposomes, have been developed.6–9 Cationic liposomes form a complex with anionic DNA molecules and are thought to deliver DNA through endosomes after endocytosis of the complex,10 although the precise mechanism for gene transfection mediated by cationic liposomes is still unclear. The cationic liposomal system, however, has some disadvantages such as low efficiency of transfection due to DNA degradation in lysosomes and strong cyototoxicity.11 Polycations have been also used for nonviral systems. Among them, polyethylenimine (PEI) has been revealed to be effective to deliver genes,12–20 where genes may be delivered to the cytoplasm via endosomes due to the proton-sponge effect of PEI. In this study, we developed an effective nonviral gene transfer system by combining the advantages of both liposomes and polycations. Polycation liposomes (PCLs) were prepared by the modification of liposomes with cetylated PEI. We originally reported that liposomes modified with cetylated PEI derivative might deliver agents into the cytosol via Correspondence: N Oku, Department of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan Received 17 August 1999; accepted 12 March 2000

the endosomal pathway.21 The present paper indicates that PCLs actually deliver genes effectively with low cytotoxicity. Furthermore, the PCL is, unlike many cationic liposomal formulations,22–24 effective in the presence of serum. This characteristic is favorable for in vivo use of gene delivery systems.

Results Transfection by PCL At first, we grafted 22 mol% cetyl groups on to polyethyleneimine-600 (PEI600 with an average molecular weight (MW) of 600); thus one polymer may contain 14 ethylene units and three cetyl groups. PCLs were prepared with this tricetyl-PEI600 (P6C22) and dioleoylphosphatidylethanolamine (DOPE) (0.65:1 as a molar ratio). We confirmed the modification of liposomes with the polycation, ie PEI, by the determination of the liposomal ␨-potential. The ␨-potential of the PCL in phosphate-buffered saline (PBS, pH 7.4) was +41.6 ± 0.1 mV, whereas that of DOPE liposomes was −3.4 ± 2.9 mV, indicating that the liposomal surface of the PCL was positively charged. Then, we transfected COS-1 cells with pEGFP bearing the green fluorescent protein (GFP) reporter gene by means of these PCLs. The GFP gene was expressed 1 day after transfection, and the highest expression was observed during the second to fourth day. Figure 1 shows a typical image of transfected COS-l cells seen by fluorescence microscopy after 48 h, at which time the cells were still subconfluent. As shown in this Figure, many cells were fluorescence positive. The gene expression was increased dose dependently (data not shown).

Gene transfer system by polycation liposomes Y Yamazaki et al

location of the original plasmid was decreased by increasing the amount of PCL, suggesting the complex formation between PCL and DNA; and a drastic decrease was observed at 9 equivalents. The DNA band at the location of the original plasmid was completely diminished at 12 equivalents (data not shown). This result suggests that PCLs neutralized DNA between 9 and 12 equivalents and that the PCL/DNA complex assumed a net positive charge at least when the PCL was present as more than 12 equivalents against the DNA.

a

b

Figure 1 GFP expression in COS-1 cells after transfection with PCL. PCLs composed of P6C22 and DOPE (0.65:1 as molar ratio) was complexed with 1 ␮g GFP plasmid (PCL/DNA = 1:1 as unit ratio) and incubated with COS-1 cells for 3 h at 37°C. The cells were incubated for an additional 48 h and observed by bright field microscopy (a), fluorescence microscopy (b).

The efficiency of transfection mediated by PCLs was evaluated by monitoring GFP fluorescence intensity and comparing this efficiency with that of transfection mediated by conventional cationic liposomes, namely, liposomes containing 1,2-dimyristyloxypropyl-3dimethyl-hydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-3- trimethylammonium propane (DOTAP), or 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,Ndimethyl-1-propanaminium trifluoroacetate (DOSPA, lipofectamine). The DNA concentration was fixed as 1 ␮g/35-mm dish in this and subsequent experiments. As shown in Figure 2a, the efficiency was higher for PCLmediated transfection than for that by DMRIE- or DOTAP- liposomes. The DNA/carrier ratio for obtaining adequate fluorescence intensity was also broad for PCLs. Furthermore, the PCLs did not require DOPE or cholesterol as a liposomal component, since COS-1 cells could also be transfected with egg yolk phosphatidylcholine (EPC)or dipalmitoyl-PC (DPPC)-based PCLs (Figure 2b). Figure 3 summarizes the efficiency of GFP gene transfection of COS-1 cells when the PCL composition or PCL/DNA ratio was varied. Appropriate transfection efficiency was observed when the molar ratio of P6C22 and DOPE was changed from 0.65 to 3.0, although the optimum transfection was observed at the ratio of 0.65 or 1.0 depending on the PCL/DNA ratio. PCL and DNA ratio was expressed in terms of ethylenimine units (14 units per one P6C22 molecule) and DNA phosphate. When the P6C22/DOPE ratio was 0.65 or 1.0, in both cases the highest transfection was observed at 9.5 equivalents (9.5-fold excess of total ethylenimine units in PCL against total DNA-phosphorus number). To determine the optimal complex formation between PCL and plasmid DNA, we conducted agarose gel electrophoresis of samples having various PCL/DNA unit ratios. The amount of DNA at the electrophoretic

1149

Cytotoxic action of PCL Since conventional cationic liposomes are known to have marked cytotoxic activity, we next determined the cytotoxic activity of PCL against COS-1 cells. PCL and control cationic liposomes showed cytotoxic activity in a dosedependent manner (data not shown). PCL and control cationic liposomes containing LipofectAMINE also showed similar cytotoxic activities after complexation with DNA. Table 1 summarizes the most effective dose for transfection and the 50% cytotoxic doses (CD50) against COS-1 cells. As is apparent from the Table, PCL caused the least cytotoxic action as evaluated by the difference between transfection dose and CD50. We also determined the hemolytic activity of PCL and cationic liposomes toward chicken erythrocytes, and observed that PCL was the least hemolytic (data not shown). Transfection efficiency of PCL in the presence of serum It is well known that the transfection efficiency of conventional cationic liposomes is suppressed in the presence of serum. This tendency, however, is unfavorable especially if the carrier is to be used in vivo. Thus, we determined the transfection efficiency of PCL in the presence of serum. As shown in Figure 4, LipofectAMINE showed the highest transfection efficiency in the absence of serum, although the effect was markedly suppressed in the presence of serum. The amount of GFP expression in the presence of 50% serum was only 27% and 17% for LipofectAMINE and DOTAP liposome, respectively, in comparison with that in the absence of serum. On the contrary, the transfection efficiency of PCL was mildly increased in the presence of serum. DMRIE liposomes also showed serum resistance in terms of transfection activity. The amount of GFP expression in the presence of 50% serum was 129% and 136% of that in its absence for PCL and DMRIE liposomes, respectively. To clarify the reason for serum activation of PCLmediated transfection, we examined the formation of DNA/PCL complexes under the microscope. PCL and DNA appeared as rather heterogeneous aggregates in the absence of serum, but formed smaller and rather homogeneous ones in the presence of serum (data not shown). Therefore, it is possible that the aggregation status of DNA/PCL in the presence of serum is favorable for transfection, although further experiments relating to the transfection mechanism must be done before an explanation can be given for the effect of serum on PCLmediated transfection. PCL-mediated gene transfer to various cells The data shown above indicate that PCL-mediated GFP gene transfer to COS-1 cells was quite effective without the requirement of nonbilayer lipids, less toxic than other conventional cationic liposomes against COS-1 cells, and Gene Therapy

Gene transfer system by polycation liposomes Y Yamazaki et al

1150

Figure 2 Transfection efficiency by PCL and cationic liposomes. (a) PCL (䊉), DMRIE liposomes (䊊), DOTAP liposomes (䊐), or LipofectAMINE (왕) were complexed with 1 ␮g of plasmid DNA at various cationic lipid-to-DNA unit ratios. COS-1 cells were incubated with the liposome/plasmid DNA complexes for 3 h at 37°C. At 48 h after transfection, the cells were solubilized and fluorescence intensity was then determined (Ex 493 nm, Em 510 nm). Data represent the mean ± s.d. (n = 3). (b) PCLs composed of P6C22/DOPE (0.65/1), P6C22/eggPC (0.65/1), or P6C22/DPPC/cholesterol (0.65/1/1) were prepared. P6C22 alone or PCLs were complexed with 1 ␮g of plasmid DNA at a 1:1 unit ratio. COS-1 cells were incubated with P6C22/plasmid DNA complexes or PCL/DNA complexes for 3 h at 37°C. GFP expression was determined fluorometrically after additional incubation for 48 h. Data represent the mean ± s.d. (n = 3).

Table 1 Efficiency and cytotoxicity of PCL and cationic liposomes Dosea (nmol) PCL (P6C22) LipofectAMINEc DOTAP DMRIE

2.03 12.9 6.25 3.13

GFP expression 2.47 1.91 0.42 1.24

(0.03) (0.11) (0.10) (0.04)

CD50b 223 143 113 63

a

Optimum dose for GFP expression is shown. CD50 represents the 50% cytotoxic dose of P6C22 or cationic lipid (nmol). c One microgram of plasmid DNA was complexed with 12.5 ␮l of LipofectAMINE reagent. b

brain endothelial cells. The experiment was done in the absence or presence of 50% serum. As shown in Figure 5, the PCL was quite efficient for gene transfer to all cells tested. The enhancement of efficiency in the presence of serum or resistance against serum was also observed in all cells tested, although a slight decrease of efficiency was observed in PANC-1 and C2C12 cells. Figure 3 Optimization of PCL-mediated transfection. PCLs composed of P6C22 and DOPE at the indicated ratios were complexed with 1 ␮g of plasmid DNA at various unit ratios (PEI nitrogen/DNA phosphate ratio). At 48 h after transfection of COS-1 cells with these PCL/DNA complexes, GPF expression was determined.

resistant against serum. Next we investigated the ability of PCL to transfer another gene to other kinds of cells. LacZ gene, pCAG-LZ15, was mixed with PCLs and transfected to MCF-7 breast adenocarcinoma, MCA-MB-435S breast ductal carcinoma, BxPC-3 pancreatic adenocarcinoma, PANC-1 pancreatic epithelioid carcinoma, U87 glioblastoma, U-20S osteosarcoma and HepG2 hepatoma cells. We also examined the gene transfer to normal cell lines, namely, C2C12 mouse myoblast and BBMC bovine Gene Therapy

Molecular weight effect of PEI on PCL-mediated gene transfer To investigate the effect of the molecular weight of the polymer on PCL-mediated gene transfer, we grafted cetyl groups on to PEI with molecular weights of 1800 and 25 000, and prepared PCLs from them. GFP expression after transfection mediated by these PCLs is summarized in Figure 6 in relation to the PEI/DNA unit ratio (ethyleneimine unit/DNA phosphorus ratio) and PEI/DOPE molar ratio for PEI 1800, or in relation to the PEI/DNA unit ratio and mol% of cetyl groups grafted onto PEI 25 000. As shown in Figure 6a, DOPE liposomes modified with 5 mol% P18C18 was the most efficient for gene transfer at 18–36 equivalents (PEI/DNA unit ratio) among P18C18-PCL-mediated gene transfers. On the

Gene transfer system by polycation liposomes Y Yamazaki et al

PCL is more stable than DOPE-based PCL although DOPE-based PCL could also deliver GFP gene into hepatic cells (data not shown). Acute toxicity on mice (five per group) was not observed by the injection of PCL, and the fluorescent colonies were observed in all five mice injected with PCL although none of the fluorescent colonies was observed in mice administered GFP plasmid alone.

1151

Discussion

Figure 4 Effect of serum on the transfection efficiency by PCL and cationic liposomes. PCL (䊉), DMRIE liposomes (䊊), DOTAP liposomes (䊐), or LipofectAMINE (왕) were complexed with 1 ␮g of plasmid DNA. COS1 cells were incubated with liposome/plasmid DNA complexes for 3 h at 37°C in the presence of various concentrations of serum. At 48 h after transfection, the cells were solubilized and fluorescence intensity was then determined (Ex 493 nm, Em 510 nm). Data represent the mean ± s.d. (n = 3). *, Significantly different from the data without serum (P ⬍ 0.05) evaluated by a Student’s t test.

other hand, DOPE liposomes modified with PEI 25 000 did not show comparable gene transfer activity at any cetyl density tested (Figure 6b).

PCL-mediated gene transfer in vivo Finally, we tested PCL for gene delivery in vivo. GFP plasmid was mixed with PCL and injected into mice via the portal vein. After 1 day the liver was removed and examined under fluorescence microscopy. The number of fluorescent colonies was observed on the surface of the liver. Figure 7 shows a typical fluorescence photomicrograph by use of DPPC-based PCL, since DPPC-based

As an indication of the rapid progress in molecular biology, gene therapies for various diseases have been attempted. For gene transfer, effective and safe delivery of the desired gene into the target cells is the most important issue. For gene delivery systems, both virusmediated and nonviral systems have been considered. Some of virus-mediated gene transfer systems have been found to be quite effective for gene transfer, although there are arguments about their safety and preparation homogeneity. Nonviral carriers for gene transfer might overcome these objections. Cationic liposomes are thought to be potential nonviral carriers, however, present formulations are not satisfactory due to their low transfection efficiency and potent cytotoxicity.6–9 On the other hand, polycations, such as spermine,25 polylysine,26 and polycationic polymers,27,28 have been used as tools of gene transfer. The main reason for the usage of these polycations is that polycations enable compaction of DNA. As presented here, we have developed a novel carrier for transfection by combining these two modalities, namely, cationic liposomes and polycations. A similar attempt was made by using lipopolylysine and DOPE,29 although treatment for destabilized endosomal function, such as chloroquine treatment, was essential for obtaining high efficiency of gene transfer by polylysinemodified liposomes.30 At first, we grafted hydrophobic anchors on PEI, a

Figure 5 PCL-mediated transfection of various cells. PCLs composed of P6C22 and DOPE (0.65:1 as molar ratio) were complexed with 0.29 ␮g LacZ plasmid (PCL/DNA = 1:1 as unit ratio), or 84 ␮g LipofectAMINE was complexed with 0.29 ␮g LacZ plasmid. Then, PCL– or LipofectAMINE–DNA complexes were incubated with MCF-7 breast adenocarcinoma, MCA-MB-435S breast ductal carcinoma, BxPC-3 pancreatic adenocarcinoma, PANC1 pancreatic epithelioid carcinoma, U87 glioblastoma, U-20S osteosarcoma, HepG2 hepatoma, C2C12 mouse myoblast, and BBMC bovine brain endothelial cells (3 × 105 cells) for 3 h at 37°C in the absence or presence of 50% serum. After having been washed, the cells were incubated for an additional 48 h in DMEM supplemented with 10% serum. ␤-Galactosidase activity is expressed as relative light units per microgram protein per second ± s.d. (n = 3). Open bar, PCL in the absence of serum; closed bar, PCL in the presence of serum; hatched bar, LipofectAMINE in the absence of serum; and gray bar, LipofectAMINE in the presence of serum. Gene Therapy

Gene transfer system by polycation liposomes Y Yamazaki et al

1152

Figure 6 Effect of molecular weight of PEI on PCL-mediated transfection. (a) PCLs were prepared with P18C18 and DOPE at various PCL/DOPE ratios, and complexed with 1 ␮g of plasmid DNA at various PEI nitrogen/DNA phosphate ratios. Transfection efficiency was determined as described in the experimental protocol. (b) PCL was prepared with polycetyl PEI250 (P250C5, P250C14, and P250C23) and DOPE (0.01:1 as molar ratio). After complex formation with 1 ␮g pEGFP, transfection efficiency was determined as described in the experimental protocol.

Figure 7 Effect of molecular weight of PEI on PCL-mediated transfection. PCLs composed of P6C22, DPPC, and cholesterol (0.65:1:1 as molar ratio) were mixed with 0.1 mg pEGFP (PCL/DNA = 1:1 as unit ratio) and injected into 5-week-old ddY male mice via the portal vein. After 24 h, mice were killed, and GFP expression in the liver was observed under a fluorescence microscope.

polycationic polymer known to have transfection activity, to modify liposomes with the polycation. Modification of liposomes with PEI was confirmed by the determination of ␨-potential of the liposomes, and the polycation-modified liposomes actually showed high positive charges. For gene transfer, PEI of high molecular weight, namely 25 000 or above, is commonly used.13 Since liposomalization may concentrate polycations on the liposomal surface, polycations with high molecular weight might not be required. Furthermore, the liposomalization effect, such as anchoring of polymers, may be strongly observed with polymers of low molecular weight. Thus, in the present study we mainly used PEI of low molecular weight, namely PEI 600, which has 14 ethylene units in one molecule. Actually, PEIs of low molecular weight, ie 600 and 1800, were shown to be more effective for gene transfer than the PEI of high molecular weight, 25 000 (Figure 6) after liposomalization. Gene Therapy

In the present study, the GFP gene was mainly used as a reporter, since the amount of the gene product after transfection can be directly measured in terms of fluorescence intensity. The GFP gene was markedly expressed in COS-1 cells after incubation of the cells with pEGFP/PCL (Figures 1 and 2). In the case of cationic liposomes, it is often observed that a narrow range of DNA/cationic lipids ratio is effective,31,32 although comparable expression of GFP was observed over a wide range of pEGFP/PCL ratios. Furthermore, PCL did not require PE or cholesterol as a liposome component. Most cationic liposomes require PE with unsaturated fatty acyl chains, which is well known as a nonbilayer lipid.33–37 Although the mechanism of gene transfer by cationic liposomes is not fully understood, the PE requirement may be explained by the possibility that DNA/cationic liposomes may destabilize the endosomal membrane after endocytosis by target cells. Nonbilayer lipid is thought to be suitable for such destabilization at acidic pH. On the contrary, PEI can destabilize the endosomal membrane by protonation of PEI itself without the requirement of nonbilayer lipids.38 PEI has many protonation sites, which may cause influx of ions into endosomes, and as a result of this proton-sponge effect, endosomes are broken to release their internal contents. Thus, PEI-modified PCL could deliver the gene without the requirement of PE as a liposomal component. Interestingly, DPPC-based PCLs were also effective for gene transfer. Liposomes formed by unsaturated phospholipids are unstable in plasma, and these lipids are easily transferred to lipoproteins. On the other hand, DPPCbased liposomes are stable in the bloodstream and may be suitable for in vivo usage. To obtain high efficiency of gene transfer, various investigators have attempted to modify carriers with ligands specific for cell-surface receptors, such as the transferrin receptor.39,40 PCL also might be useful for such attempts, since both PEI and liposomes can be easily modified with various ligands. Since cell membranes are negatively charged, cationic liposomes and polycations are known to cause cell damage. Thus we examined the action of PCLs and conventional cationic liposomes on erythrocytes and COS-1 cells.

Gene transfer system by polycation liposomes Y Yamazaki et al

The PCL actually showed hemolytic and cytotoxic action, although the doses of PCL for inducing these unfavorable actions were high compared with the effective dose of PCL for gene transfer. Furthermore, the PCL was resistant against serum. Besides COS-1 cells, not only malignant but also normal cell lines were quite effectively transfected by the PCLs. Finally, we investigated the effect of PCL in vivo. GFP fluorescence was observed on the surface of the liver after transfection of mice with the GFP gene complexed with PCL. Thus, PCLs may be suitable as an in vivo carrier of genes as well as for in vitro usage.

Materials and methods Synthesis of cetylated polyethylenimines (P6C22, P18C18, P250C5, P250C14 and P250C23) PEI with an average molecular weight of about 600 (PEI 600) and that with one of about 1800 (PEI 1800) or 25 000 (PEI 25 000) was purchased from Dow Chemical (Midland, MI, USA) and from Nippon Shokubai (Osaka, Japan) respectively. The polymers were purified by ultrafiltration with an Amicon ultrafiltration apparatus (Beverly, MA, USA) equipped with a Toyo Roshi (Tokyo, Japan) ultrafilter. For the preparation of P6C22 as an example, cetyl bromide (1.62 g) was added to purified PEI 600 (1 g) in chloroform (20 ml). The solution was refluxed in the presence of triethylamine (1 ml). The polymer-containing solution was dialyzed against 40% ethanol in water and then against water at room temperature, after which the aqueous solution was lyophilized. Integration of the proton magnetic resonance (1H NMR) spectrum of the product in D2O indicated 22 mol% of cetyl groups (C16H33) per residue mol of ethylenimine unit (C2H4N) in the polymer. The modified polymer may be represented by the stoichiometric formula (C2H4N)m(C16H33)0.22m (P6C22), m = 14. Similar modification was done with PEI 1800 or PEI 25 000 as described above. The modified polymers may be represented by the following stoichiometric formulas: (C2H4N)m(C16H33)0.18m (P18C18), m = 43, (C2H4N)m (C16H33)0.05m (P250C5), m = 595, (C2H4N)m(C16H33)0.14m (P250C14), m = 595, or (C2H4N)m(C16H33)0.23m (P250C23), m = 595. Preparation of liposomes Liposomes were prepared as follows: for PCL, P6C22 and DOPE (0.65/1 as molar ratio) were dissolved in chloroform, dried under reduced pressure, and stored in vacuo for at least 1 h. The liposomes were produced by hydration of the thin lipid film with Dulbecco’s modified Eagle’s (DME) medium (1 mm as final concentration of lipids). This liposomal solution was freeze–thawed by using liquid nitrogen, and sonicated for 15 min with a bath-type sonicator. For cationic liposomes, DMRIE or DOTAP (Avanti Polar Lipids, Alabaster, AL, USA) solution was mixed with DOPE (1/1 as molar ratio) and the mixture was dried to produce a thin lipid film. Liposomes were prepared similarly as in the P6C22-liposome preparation except that the hydration was done in distilled water instead of medium. Liposomes were prepared just before use, although they remain stable for a long period of time. In the case of lipofectamine, LipofectAMINE reagent (Life Technologies, Gaithersburg, MD, USA) was used, which contains lipofectamine and PE (3/1 as weight ratio).

␨-Potential measurement ␨-Potential of liposomes was determined in PBS at 25°C by use of an ELS-800 apparatus (Otsuka Densi, Osaka, Japan).

1153

Preparation liposome/DNA complex A plasmid encoding the GFP gene, pEGFP-C1 (Clontech Laboratories, Palo Alto, CA, USA) was amplified in E. coli HB101 and purified by CsCl density gradient centrifugation. The purity of the plasmid was confirmed by agarose gel electrophoresis. Plasmids were dissolved in Tris-EDTA buffer, pH 7.5 and the liposome/DNA complexes were prepared as follows: an appropriate amount of plasmid was mixed with liposomal solution (1 mm as DOPE). After addition of DMEM, the mixture was incubated for 20 min. A plasmid encoding ␤-galactosidase, pCMVbeta, was purchased from Clontech Laboratories and the CAG promoter was inserted to form pCAGLZ15. Then the LacZ plasmids were mixed with PCL similarly as for the GFP gene. Transfection COS-1 cells were cultured in DME medium supplemented with 10% fetal bovine serum (FBS, JRH Biosciences, Lenexa, KS, USA) under a humidified atmosphere of 5% CO2 in air. One day before transfection, 1 × 105 COS-1 cells were seeded on to a 35-mm dish (Corning) and incubated overnight in a CO2 incubator. Then the cells were washed twice with DME medium, after which PCL/DNA or cationic liposome/DNA complexes (0.25 ml, 1 ␮g DNA) were added to them. After incubation for 3 h at 37°C, the cells were washed twice with DMEM and cultured for another 48 h in 2 ml of DMEM supplemented with 10% FBS. Expression of the GFP-gene in COS-1 cells was observed under a fluorescence microscope (Olympus IMT-2, Tokyo, Japan). Quantitative assay was done as follows: the cells were washed with PBS in a 35-mm dish, solubilized with 1% reduced Triton X-100 (Aldrich Chemical, Milwaukee, WI, USA) for 30 min, and centrifuged at 3000 r.p.m. for 10 min. Fluorescence intensity of the supernatant was measured with an excitation wavelength of 493 nm and an emission one of 510 nm by a fluorescence spectrophotometer (Hitachi F-4010; Tokyo, Japan). LacZ gene transfer to various cells was performed as follows: cells were seeded on to 24-well plates (0.3 × 105 cells per well) and incubated for 1 day. Then, 0.2 ml of PCL mixture with 0.29 ␮g pCAG-LZ15 was added to each well. After incubation for 3 h at 37°C, the cells were washed twice with DMEM and cultured another 48 h in 1 ml DMEM supplemented with 10% FBS. After having been washed, the cells were solubilized with 0.1 ml PicaGene LC␤ (Wako Pure Chemical, Osaka, Japan). ␤Galactosidase activity was measured by a Galacto-light system (Tropix, Bedford, MA, USA) and expressed as relative light units per microgram protein per second. Cytotoxic assay COS-1 cells were seeded on to 24-well plates (2 × 104 cells per well, Corning) and incubated overnight in a CO2 incubator. Then, the cells were washed twice with DME medium, and PCL or cationic liposomes were added thereafter. After incubation for 3 h at 37°C, the cells were washed twice with DME medium, and cultured another 1 h in 0.25 ml DMEM containing AlamarBlue reagent Gene Therapy

Gene transfer system by polycation liposomes Y Yamazaki et al

1154

(AccuMed International, Westlake, OH, USA). After dilution with PBS, viable cells were monitored fluorometrically (excitation wavelength of 535 nm and emission wavelength of 583 nm). Cytotoxic assay of PCL or cationic liposomes complexed with DNA (0.2 ml, 0.2 ␮g DNA) was also done by a similar procedure.

Hemolytic assay PCL and cationic liposomes were diluted serially (0.1 ml) with PBS in a 96-well plate. Then, 0.1 ml of freshly prepared 2% chicken erythrocytes was added to each well, and the plate was incubated at 37°C for 30 min. After centrifugation at 1500 r.p.m. for 10 min, absorbance of an aliquot of each well was monitored at 570 nm. In vivo study Five-week-old ddY mice (five per group) were anesthetized with sodium pentobarbital (0.05 mg/g mouse body weight). For the injection of plasmid into the hepatic portal system, an incision was made along the midline of the abdomen to expose the large vein located in the mesentery. Then, PCL-complex of 0.1 mg pEGFP (PCL/DNA = 1:1 as unit ratio) or plasmid alone was injected into the mice via the portal vein. The animals were killed 24 h after injection, and the liver was perfused with saline. The liver was then examined under a fluorescence microscope equipped with CCD camera (Olympus IMT-2).

Acknowledgements We thank Drs K Takeda and H Miyazaki and Mr Y Suzuki at DNAVEC Research, for their helpful discussions; Mr T Iwasaki and Mr H Ori for technical assistance, and Dr Y Sadzuka of the University of Shizuoka for measurement of ␨-potentials. We also thank Dr Y Namba at Nippon Fine Chemical for the supply of phospholipids.

References 1 Kay MA, Liu D, Hoogerbrugge PM. Gene therapy. Proc Natl Acad Sci USA 1997; 94: 12744–12746. 2 Anderson WF. Human gene therapy. Nature 1998; 392: 25–30. 3 Verma IM, Somia N. Gene therapy – promises, problems and prospects. Nature 1997; 389: 239–242. 4 Jooss K, Yang Y, Fisher KJ, Wilson JM. Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J Virol 1998; 72: 4212–4223. 5 Fisher KJ et al. Recombinant adeno-associated virus for muscle directed gene therapy. Nature Med 1997; 3: 306–312. 6 Gao X, Huang L. Cationic liposome-mediated gene transfer. Gene Therapy 1995; 2: 710–722. 7 Meyer O et al. Cationic liposomes coated with polyethylene glycol as carriers for oligonucleotides. J Biol Chem 1998; 273: 15621–15627. 8 Behr JP. Gene transfer with synthetic cationic amphiphiles. Prospects for gene therapy. Bioconj Chem 1994; 5: 382–389. 9 Vigneron JP et al. Guanidinium-cholesterol cationic lipids: efficient vectors for the transfection of eukaryotic cells. Proc Natl Acad Sci USA 1996; 93: 9682–9686. 10 Wrobell I, Collins D. Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochem Biophys Acta 1995; 1235: 296–304. 11 Cortesi R et al. Effect of cationic liposome composition on in vitro cytotoxicity and protective effect on carried DNA. Int J Pharm 1996; 139: 69–78. Gene Therapy

12 Boussif O et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 1995; 92: 7297–7301. 13 Abdallah B et al. A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum Gene Ther 1996; 7: 1947–1954. 14 Lambert RC et al. Polyethylenimine-mediated DNA transfection of peripheral and central neurons in primary culture. Probing Ca2+ channel structure and function with antisense oligonucleotides. Mol Cell Neurosci 1996; 7: 239–246. 15 Zanta MA, Boussif O, Adib A, Behr JP. In vitro gene delivery to hepatocytes with galactosylated polyethylenimine. Bioconj Chem 1997; 8: 839–844. 16 Kircheis R et al. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Therapy 1997; 4: 409–418. 17 Tang MX, Szoka FC. The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Therapy 1997; 4: 823–832. 18 Boletta A et al. Nonviral gene delivery to the rat kidney with polyethylenimine. Hum Gene Ther 1997; 8: 1243–1251. 19 Ferrari S et al. Exgen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Therapy 1997; 4: 1100–1106. 20 Pollard H et al. Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J Biol Chem 1998; 273: 7507–7511. 21 Oku N et al. Low pH induced membrane fusion of lipid vesicles containing proton-sensitive polymer. Biochemistry 1987; 26: 8145–8150. 22 Hofland HEJ, Shephard L, Sullivan SM. Formation of stable cationic lipid/DNA complexes for gene transfer. Proc Natl Acad Sci USA 1996; 93: 7305–7309. 23 Escriou V et al. Cationic lipid-mediated gene transfer. Effect of serum on cellular uptake and intracellular fate of lipopolyamine/DNA complexes. Biochem Biophys Acta 1998; 1368: 276–288. 24 Sternberg B, Hong K, Zheng W, Papahadjopoulos D. Ultrastructural characterization of cationic liposome-DNA complexes showing enhanced stability in serum and high transfection activity in vivo. Biochem Biophys Acta 1998; 1375: 23–35. 25 Kichler A, Mechtler K, Behr JP, Wagner E. Influence of membrane-active peptide on lipospermine/DNA complex mediated gene transfer. Bioconj Chem 1997; 8: 213–221. 26 Zhou X, Klibanov AL, Huang L. Lipophilic polylysines mediate efficient DNA transfection in mammalian cells. Biochem Biophys Acta 1991; 1065: 8–14. 27 Sorgi FL, Bhattacharya S, Huang L. Protamine sulfate enhances lipid-mediated gene transfer. Gene Therapy 1997; 4: 961–968. 28 Goldman CK et al. In vitro and in vivo gene delivery mediated by a synthetic polycationic amino polymer. Nat Biotechnol 1997; 15: 462–466. 29 Zhou X, Huang L. DNA transfection mediated by cationic liposomes containing lipopolylysine. Characterization and mechanism of action. Biochem Biophys Acta 1994; 1189: 195–203. 30 Plank C et al. The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. J Biol Chem 1994; 269: 12918–12924. 31 Felnger JH et al. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 1994; 269: 2550–2561. 32 Xu Y, Szoka FC Jr. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 1996; 35: 5615–5623. 33 Farhood H, Serbina N, Huang L. The role of dioleoylphosphatidyl-ethanolamine in cationic liposome mediated gene transfer. Biochem Biophys Acta 1995; 1235: 289–295. 34 Reimer DL et al. Formation of novel hydrophobic complexes between cationic lipid and plasmid DNA. Biochemistry 1995; 39: 12877–12883. 35 Hui SW et al. The role of helper lipid in cationic liposomemediated gene transfer. Biophys J 1996; 71: 590–599.

Gene transfer system by polycation liposomes Y Yamazaki et al

36 Bailey AL, Cullis PR. Membrane fusion with cationic liposomes. Effect of target membrane lipid composition. Biochemistry 1997; 36: 1628–1634. 37 Noguchi A, Furuno T, Kawaura C, Nakanishi M. Membrane fusion plays an important role in gene transfection mediated by cationic liposomes. FEBS Lett 1998; 433: 169–173. 38 Demeneix BA, Behr JP. The proton sponge. A trick the viruses did not exploit. In: Felgner PL et al (eds). Artificial Self-Assembling Systems for Gene Delivery. ACS Conference Proc, 1996, pp 146– 151.

39 Cheng PW. Receptor ligand-facilitated gene transfer enhancement of liposome-mediated gene transfer and expression by transfection. Hum Gene Ther 1996; 7: 275–282. 40 Harbottle RP et al. An RGD-oligolysine peptide. A prototype construct for integrin-mediated gene delivery. Hum Gene Ther 1998; 9: 1037–1047.

1155

Gene Therapy

Suggest Documents