Polycation Liposomes, a Novel Nonviral Gene Transfer System, Constructed from Cetylated Polyethylenimine

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 1Department 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 lipo- thermore, the transfection efficacy of PCLs was enhanced, somes modified with cetylated polyethylenimine (PEI, MW instead of being diminished, in the presence of serum. Effec- 600). This polycation liposome, PCL, showed remarkable tive gene transfer was observed in all eight malignant and transfection efficiency as monitored by the expression of the two normal cells line tested, as well as in COS-1 cells. We GFP reporter gene. Most conventional cationic liposomes also examined the effect of the molecular weight of PEI on require phosphatidylethanolamine or cholesterol as a PCL-mediated gene transfer, and observed that PEI with a component, although PCLs did not. Egg yolk phosphatidyl- MW of 1800 Da was as effective as that with one of 600, but choline- and dipalmitoylphosphatidylcholine-based PCL that PEI of 25 000 was far less effective. Finally, an in vivo were as effective as dioleoylphosphatidylethanolamine- study was done in which GFP was effectively expressed in based PCLs for gene transfer. Concerning the cytotoxicity mouse liver after injection of PCL via the portal vein. Thus, against COS-1 cells and hemolytic activity, the PCL was PCL represents a new system useful for transfection and superior to conventional cationic liposome preparations. Fur- gene therapy. Gene Therapy (2000) 7, 1148–1155.

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

Introduction the endosomal pathway.21 The present paper indicates that PCLs actually deliver genes effectively with low Efficient and safe gene transfer systems are the funda- cytotoxicity. Furthermore, the PCL is, unlike many cat- mental basis for gene therapy, as well as for laboratory ionic liposomal formulations,22–24 effective in the presence 1–3 applications. Viral systems are, in general, quite effec- of serum. This characteristic is favorable for in vivo use tive for gene transfer, although there are arguments of gene delivery systems. about their safety and immunogenicity.4,5 Therefore, a number of nonviral systems, especially cationic lipo- Results somes, have been developed.6–9 Cationic liposomes form a complex with anionic DNA molecules and are thought Transfection by PCL to deliver DNA through endosomes after endocytosis of At first, we grafted 22 mol% cetyl groups on to poly- 10 the complex, although the precise mechanism for gene ethyleneimine-600 (PEI600 with an average molecular transfection mediated by cationic liposomes is still weight (MW) of 600); thus one polymer may contain 14 unclear. The cationic liposomal system, however, has ethylene units and three cetyl groups. PCLs were pre- some disadvantages such as low efficiency of transfection pared with this tricetyl-PEI600 (P6C22) and dioleoylphos- due to DNA degradation in lysosomes and strong cyoto- phatidylethanolamine (DOPE) (0.65:1 as a molar ratio). 11 toxicity. Polycations have been also used for nonviral We confirmed the modification of liposomes with the systems. Among them, polyethylenimine (PEI) has been polycation, ie PEI, by the determination of the liposomal 12–20 revealed to be effective to deliver genes, where genes ␨-potential. The ␨-potential of the PCL in phosphate-buff- may be delivered to the cytoplasm via endosomes due to ered saline (PBS, pH 7.4) was +41.6 ± 0.1 mV, whereas the proton-sponge effect of PEI. In this study, we that of DOPE liposomes was −3.4 ± 2.9 mV, indicating developed an effective nonviral gene transfer system by that the liposomal surface of the PCL was positively combining the advantages of both liposomes and polyca- charged. Then, we transfected COS-1 cells with pEGFP tions. Polycation liposomes (PCLs) were prepared by the bearing the green fluorescent protein (GFP) reporter gene modification of liposomes with cetylated PEI. We orig- by means of these PCLs. The GFP gene was expressed inally reported that liposomes modified with cetylated 1 day after transfection, and the highest expression was PEI derivative might deliver agents into the cytosol via 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 Correspondence: N Oku, Department of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, were still subconfluent. As shown in this Figure, many Japan cells were fluorescence positive. The gene expression was Received 17 August 1999; accepted 12 March 2000 increased dose dependently (data not shown). Gene transfer system by polycation liposomes Y Yamazaki et al 1149 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.

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 dose- dependent manner (data not shown). PCL and control cationic liposomes containing LipofectAMINE also a b showed similar cytotoxic activities after complexation with DNA. Table 1 summarizes the most effective dose for transfection and the 50% cytotoxic doses (CD50) Figure 1 GFP expression in COS-1 cells after transfection with PCL. against COS-1 cells. As is apparent from the Table, PCL PCLs composed of P6C22 and DOPE (0.65:1 as molar ratio) was com- caused the least cytotoxic action as evaluated by the dif- plexed 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 ference between transfection dose and CD50. We also additional 48 h and observed by bright ﬁeld microscopy (a), ﬂuorescence determined the hemolytic activity of PCL and cationic microscopy (b). liposomes toward chicken erythrocytes, and observed that PCL was the least hemolytic (data not shown).

The efficiency of transfection mediated by PCLs was Transfection efficiency of PCL in the presence of serum evaluated by monitoring GFP fluorescence intensity and It is well known that the transfection efficiency of con- comparing this efficiency with that of transfection ventional cationic liposomes is suppressed in the pres- mediated by conventional cationic liposomes, namely, ence of serum. This tendency, however, is unfavorable liposomes containing 1,2-dimyristyloxypropyl-3- especially if the carrier is to be used in vivo. Thus, we dimethyl-hydroxyethylammonium bromide (DMRIE), determined the transfection efficiency of PCL in the pres- 1,2-dioleoyl-3- trimethylammonium propane (DOTAP), ence of serum. As shown in Figure 4, LipofectAMINE or 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N- showed the highest transfection efficiency in the absence dimethyl-1-propanaminium trifluoroacetate (DOSPA, of serum, although the effect was markedly suppressed lipofectamine). The DNA concentration was fixed as in the presence of serum. The amount of GFP expression 1 ␮g/35-mm dish in this and subsequent experiments. As in the presence of 50% serum was only 27% and 17% for shown in Figure 2a, the efficiency was higher for PCL- LipofectAMINE and DOTAP liposome, respectively, in mediated transfection than for that by DMRIE- or DOT- comparison with that in the absence of serum. On the AP- liposomes. The DNA/carrier ratio for obtaining contrary, the transfection efficiency of PCL was mildly adequate fluorescence intensity was also broad for PCLs. increased in the presence of serum. DMRIE liposomes Furthermore, the PCLs did not require DOPE or choles- also showed serum resistance in terms of transfection terol as a liposomal component, since COS-1 cells could activity. The amount of GFP expression in the presence also be transfected with egg yolk phosphatidylcholine of 50% serum was 129% and 136% of that in its absence (EPC)- or dipalmitoyl-PC (DPPC)-based PCLs for PCL and DMRIE liposomes, respectively. (Figure 2b). To clarify the reason for serum activation of PCL- Figure 3 summarizes the efficiency of GFP gene trans- mediated transfection, we examined the formation of fection of COS-1 cells when the PCL composition or DNA/PCL complexes under the microscope. PCL and PCL/DNA ratio was varied. Appropriate transfection DNA appeared as rather heterogeneous aggregates in the efficiency was observed when the molar ratio of P6C22 absence of serum, but formed smaller and rather homo- and DOPE was changed from 0.65 to 3.0, although the geneous ones in the presence of serum (data not shown). optimum transfection was observed at the ratio of 0.65 Therefore, it is possible that the aggregation status of or 1.0 depending on the PCL/DNA ratio. PCL and DNA DNA/PCL in the presence of serum is favorable for ratio was expressed in terms of ethylenimine units (14 transfection, although further experiments relating to the units per one P6C22 molecule) and DNA phosphate. transfection mechanism must be done before an expla- When the P6C22/DOPE ratio was 0.65 or 1.0, in both nation can be given for the effect of serum on PCL- cases the highest transfection was observed at 9.5 equiva- mediated transfection. lents (9.5-fold excess of total ethylenimine units in PCL against total DNA-phosphorus number). PCL-mediated gene transfer to various cells To determine the optimal complex formation between The data shown above indicate that PCL-mediated GFP PCL and plasmid DNA, we conducted agarose gel gene transfer to COS-1 cells was quite effective without electrophoresis of samples having various PCL/DNA the requirement of nonbilayer lipids, less toxic than other unit ratios. The amount of DNA at the electrophoretic 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 (᭹), DMRIE liposomes (᭺), DOTAP liposomes (ᮀ), or LipofectAMINE (̅) 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

a b Dose (nmol) GFP expression CD50

PCL (P6C22) 2.03 2.47 (0.03) 223 LipofectAMINEc 12.9 1.91 (0.11) 143 DOTAP 6.25 0.42 (0.10) 113 DMRIE 3.13 1.24 (0.04) 63

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

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 ␮ Molecular weight effect of PEI on PCL-mediated gene P6C22 and DOPE at the indicated ratios were complexed with 1 gof transfer 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, To investigate the effect of the molecular weight of the GPF expression was determined. 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 resistant against serum. Next we investigated the ability after transfection mediated by these PCLs is summarized of PCL to transfer another gene to other kinds of cells. in Figure 6 in relation to the PEI/DNA unit ratio LacZ gene, pCAG-LZ15, was mixed with PCLs and trans- (ethyleneimine unit/DNA phosphorus ratio) and fected to MCF-7 breast adenocarcinoma, MCA-MB-435S PEI/DOPE molar ratio for PEI 1800, or in relation to the breast ductal carcinoma, BxPC-3 pancreatic adenocarcin- PEI/DNA unit ratio and mol% of cetyl groups grafted oma, PANC-1 pancreatic epithelioid carcinoma, U87 onto PEI 25 000. As shown in Figure 6a, DOPE liposomes glioblastoma, U-20S osteosarcoma and HepG2 hepatoma modiﬁed with 5 mol% P18C18 was the most efﬁcient for cells. We also examined the gene transfer to normal cell gene transfer at 18–36 equivalents (PEI/DNA unit ratio) lines, namely, C2C12 mouse myoblast and BBMC bovine among P18C18-PCL-mediated gene transfers. On the

Gene Therapy Gene transfer system by polycation liposomes Y Yamazaki et al 1151 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.

Discussion 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 virus- Figure 4 Effect of serum on the transfection efficiency by PCL and cat- mediated and nonviral systems have been considered. ionic liposomes. PCL (᭹), DMRIE liposomes (᭺), DOTAP liposomes (ᮀ), Some of virus-mediated gene transfer systems have been or LipofectAMINE (̅) were complexed with 1 ␮g of plasmid DNA. COS- found to be quite effective for gene transfer, although 1 cells were incubated with liposome/plasmid DNA complexes for 3 h at there are arguments about their safety and preparation 37°C in the presence of various concentrations of serum. At 48 h after homogeneity. Nonviral carriers for gene transfer might transfection, the cells were solubilized and fluorescence intensity was then overcome these objections. Cationic liposomes are determined (Ex 493 nm, Em 510 nm). Data represent the mean ± s.d. (n = 3). *, Significantly different from the data without serum (P Ͻ 0.05) thought to be potential nonviral carriers, however, evaluated by a Student’s t test. 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 other hand, DOPE liposomes modified with PEI 25 000 and polycationic polymers,27,28 have been used as tools did not show comparable gene transfer activity at any of gene transfer. The main reason for the usage of these cetyl density tested (Figure 6b). polycations is that polycations enable compaction of DNA. As presented here, we have developed a novel car- PCL-mediated gene transfer in vivo rier for transfection by combining these two modalities, Finally, we tested PCL for gene delivery in vivo. GFP namely, cationic liposomes and polycations. A similar plasmid was mixed with PCL and injected into mice via attempt was made by using lipopolylysine and DOPE,29 the portal vein. After 1 day the liver was removed and although treatment for destabilized endosomal function, examined under fluorescence microscopy. The number of such as chloroquine treatment, was essential for fluorescent colonies was observed on the surface of the obtaining high efficiency of gene transfer by polylysine- liver. Figure 7 shows a typical fluorescence photomicro- modified liposomes.30 graph by use of DPPC-based PCL, since DPPC-based 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, PANC- 1 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.

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 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) thought to be suitable for such destabilization at acidic were mixed with 0.1 mg pEGFP (PCL/DNA = 1:1 as unit ratio) and pH. On the contrary, PEI can destabilize the endosomal injected into 5-week-old ddY male mice via the portal vein. After 24 h, membrane by protonation of PEI itself without the mice were killed, and GFP expression in the liver was observed under a requirement of nonbilayer lipids.38 PEI has many fluorescence microscope. protonation sites, which may cause influx of ions into endosomes, and as a result of this proton-sponge effect, polycationic polymer known to have transfection activity, endosomes are broken to release their internal contents. to modify liposomes with the polycation. Modification of Thus, PEI-modified PCL could deliver the gene without liposomes with PEI was confirmed by the determination the requirement of PE as a liposomal component. of ␨-potential of the liposomes, and the polycation-modi- Interestingly, DPPC-based PCLs were also effective for fied liposomes actually showed high positive charges. For gene transfer. Liposomes formed by unsaturated phos- gene transfer, PEI of high molecular weight, namely pholipids are unstable in plasma, and these lipids are eas- 25 000 or above, is commonly used.13 Since liposomaliz- ily transferred to lipoproteins. On the other hand, DPPC- ation may concentrate polycations on the liposomal sur- based liposomes are stable in the bloodstream and may face, polycations with high molecular weight might not be suitable for in vivo usage. To obtain high efficiency be required. Furthermore, the liposomalization effect, of gene transfer, various investigators have attempted to such as anchoring of polymers, may be strongly observed modify carriers with ligands specific for cell-surface with polymers of low molecular weight. Thus, in the receptors, such as the transferrin receptor.39,40 PCL also present study we mainly used PEI of low molecular might be useful for such attempts, since both PEI and weight, namely PEI 600, which has 14 ethylene units in liposomes can be easily modified with various ligands. one molecule. Actually, PEIs of low molecular weight, ie Since cell membranes are negatively charged, cationic 600 and 1800, were shown to be more effective for gene liposomes and polycations are known to cause cell dam- transfer than the PEI of high molecular weight, 25 000 age. Thus we examined the action of PCLs and conven- (Figure 6) after liposomalization. tional cationic liposomes on erythrocytes and COS-1 cells.

Gene Therapy Gene transfer system by polycation liposomes Y Yamazaki et al 1153 The PCL actually showed hemolytic and cytotoxic action, ␨-Potential measurement although the doses of PCL for inducing these unfavorable ␨-Potential of liposomes was determined in PBS at 25°C actions were high compared with the effective dose of by use of an ELS-800 apparatus (Otsuka Densi, Osaka, PCL for gene transfer. Furthermore, the PCL was resist- Japan). ant against serum. Besides COS-1 cells, not only malignant but also normal cell lines were quite effectively Preparation liposome/DNA complex transfected by the PCLs. A plasmid encoding the GFP gene, pEGFP-C1 (Clontech Finally, we investigated the effect of PCL in vivo. GFP Laboratories, Palo Alto, CA, USA) was amplified in E. fluorescence was observed on the surface of the liver after coli HB101 and purified by CsCl density gradient centri- transfection of mice with the GFP gene complexed with fugation. The purity of the plasmid was confirmed by PCL. Thus, PCLs may be suitable as an in vivo carrier of agarose gel electrophoresis. Plasmids were dissolved in genes as well as for in vitro usage. Tris-EDTA buffer, pH 7.5 and the liposome/DNA complexes were prepared as follows: an appropriate amount Materials and methods of plasmid was mixed with liposomal solution (1 mm as DOPE). After addition of DMEM, the mixture was incu- Synthesis of cetylated polyethylenimines (P6C22, bated for 20 min. A plasmid encoding ␤-galactosidase, P18C18, P250C5, P250C14 and P250C23) pCMVbeta, was purchased from Clontech Laboratories PEI with an average molecular weight of about 600 (PEI and the CAG promoter was inserted to form pCAG- 600) and that with one of about 1800 (PEI 1800) or 25 000 LZ15. Then the LacZ plasmids were mixed with PCL (PEI 25 000) was purchased from Dow Chemical similarly as for the GFP gene. (Midland, MI, USA) and from Nippon Shokubai (Osaka, Japan) respectively. The polymers were purified by Transfection ultrafiltration with an Amicon ultrafiltration apparatus COS-1 cells were cultured in DME medium sup- (Beverly, MA, USA) equipped with a Toyo Roshi (Tokyo, plemented with 10% fetal bovine serum (FBS, JRH Biosci- Japan) ultrafilter. For the preparation of P6C22 as an ences, Lenexa, KS, USA) under a humidified atmosphere × 5 example, cetyl bromide (1.62 g) was added to purified of 5% CO2 in air. One day before transfection, 1 10 PEI 600 (1 g) in chloroform (20 ml). The solution was COS-1 cells were seeded on to a 35-mm dish (Corning) refluxed in the presence of triethylamine (1 ml). The poly- and incubated overnight in a CO2 incubator. Then the mer-containing solution was dialyzed against 40% etha- cells were washed twice with DME medium, after which nol in water and then against water at room temperature, PCL/DNA or cationic liposome/DNA complexes after which the aqueous solution was lyophilized. Inte- (0.25 ml, 1 ␮g DNA) were added to them. After incu- gration of the proton magnetic resonance (1H NMR) spec- bation for 3 h at 37°C, the cells were washed twice with trum of the product in D2O indicated 22 mol% of cetyl DMEM and cultured for another 48 h in 2 ml of DMEM groups (C16H33) per residue mol of ethylenimine unit supplemented with 10% FBS. Expression of the GFP-gene (C2H4N) in the polymer. The modified polymer may in COS-1 cells was observed under a fluorescence micro- be represented by the stoichiometric formula scope (Olympus IMT-2, Tokyo, Japan). Quantitative = (C2H4N)m(C16H33)0.22m (P6C22), m 14. Similar modifi- assay was done as follows: the cells were washed with cation was done with PEI 1800 or PEI 25 000 as described PBS in a 35-mm dish, solubilized with 1% reduced Triton above. The modified polymers may be represented by the X-100 (Aldrich Chemical, Milwaukee, WI, USA) for 30 following stoichiometric formulas: (C2H4N)m(C16H33)0.18m min, and centrifuged at 3000 r.p.m. for 10 min. Fluor- = (P18C18), m 43, (C2H4N)m (C16H33)0.05m (P250C5), escence intensity of the supernatant was measured with = = m 595, (C2H4N)m(C16H33)0.14m (P250C14), m 595, or an excitation wavelength of 493 nm and an emission one = (C2H4N)m(C16H33)0.23m (P250C23), m 595. of 510 nm by a fluorescence spectrophotometer (Hitachi F-4010; Tokyo, Japan). Preparation of liposomes LacZ gene transfer to various cells was performed as Liposomes were prepared as follows: for PCL, P6C22 and follows: cells were seeded on to 24-well plates (0.3 × 105 DOPE (0.65/1 as molar ratio) were dissolved in chloro- cells per well) and incubated for 1 day. Then, 0.2 ml of form, dried under reduced pressure, and stored in vacuo PCL mixture with 0.29 ␮g pCAG-LZ15 was added to for at least 1 h. The liposomes were produced by each well. After incubation for 3 h at 37°C, the cells were hydration of the thin lipid film with Dulbecco’s modified washed twice with DMEM and cultured another 48 h in Eagle’s (DME) medium (1 mm as final concentration of 1 ml DMEM supplemented with 10% FBS. After having lipids). This liposomal solution was freeze–thawed by been washed, the cells were solubilized with 0.1 ml Pica- using liquid nitrogen, and sonicated for 15 min with a Gene LC␤ (Wako Pure Chemical, Osaka, Japan). ␤- bath-type sonicator. For cationic liposomes, DMRIE or Galactosidase activity was measured by a Galacto-light DOTAP (Avanti Polar Lipids, Alabaster, AL, USA) sol- system (Tropix, Bedford, MA, USA) and expressed as ution was mixed with DOPE (1/1 as molar ratio) and the relative light units per microgram protein per second. mixture was dried to produce a thin lipid film. Lipo- somes were prepared similarly as in the P6C22-liposome Cytotoxic assay preparation except that the hydration was done in dis- COS-1 cells were seeded on to 24-well plates (2 × 104 cells tilled water instead of medium. Liposomes were pre- per well, Corning) and incubated overnight in a CO2 pared just before use, although they remain stable for a incubator. Then, the cells were washed twice with DME long period of time. In the case of lipofectamine, Lipo- medium, and PCL or cationic liposomes were added fectAMINE reagent (Life Technologies, Gaithersburg, thereafter. After incubation for 3 h at 37°C, the cells were MD, USA) was used, which contains lipofectamine and washed twice with DME medium, and cultured another PE (3/1 as weight ratio). 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 12 Boussif O et al. A versatile vector for gene and oligonucleotide dilution with PBS, viable cells were monitored fluoro- transfer into cells in culture and in vivo: polyethylenimine. Proc metrically (excitation wavelength of 535 nm and emission Natl Acad Sci USA 1995; 92: 7297–7301. wavelength of 583 nm). Cytotoxic assay of PCL or cat- 13 Abdallah B et al. A powerful nonviral vector for in vivo gene ionic liposomes complexed with DNA (0.2 ml, 0.2 ␮g transfer into the adult mammalian brain: polyethylenimine. Hum Gene Ther 1996; 7: 1947–1954. DNA) was also done by a similar procedure. 14 Lambert RC et al. Polyethylenimine-mediated DNA transfection of peripheral and central neurons in primary culture. Probing Hemolytic assay Ca2+ channel structure and function with antisense oligonucleo- PCL and cationic liposomes were diluted serially (0.1 ml) tides. Mol Cell Neurosci 1996; 7: 239–246. with PBS in a 96-well plate. Then, 0.1 ml of freshly pre- 15 Zanta MA, Boussif O, Adib A, Behr JP. In vitro gene delivery to pared 2% chicken erythrocytes was added to each well, hepatocytes with galactosylated polyethylenimine. Bioconj Chem and the plate was incubated at 37°C for 30 min. After cen- 1997; 8: 839–844. trifugation at 1500 r.p.m. for 10 min, absorbance of an 16 Kircheis R et al. Coupling of cell-binding ligands to polyethylen- aliquot of each well was monitored at 570 nm. imine 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 In vivo study the resulting complexes. Gene Therapy 1997; 4: 823–832. Five-week-old ddY mice (five per group) were anesthet- 18 Boletta A et al. Nonviral gene delivery to the rat kidney with ized with sodium pentobarbital (0.05 mg/g mouse body polyethylenimine. Hum Gene Ther 1997; 8: 1243–1251. weight). For the injection of plasmid into the hepatic 19 Ferrari S et al. Exgen 500 is an efficient vector for gene delivery portal system, an incision was made along the midline to lung epithelial cells in vitro and in vivo. Gene Therapy 1997; 4: of the abdomen to expose the large vein located in the 1100–1106. mesentery. Then, PCL-complex of 0.1 mg pEGFP 20 Pollard H et al. Polyethylenimine but not cationic lipids pro- (PCL/DNA = 1:1 as unit ratio) or plasmid alone was motes transgene delivery to the nucleus in mammalian cells. J injected into the mice via the portal vein. The animals Biol Chem 1998; 273: 7507–7511. 21 Oku N et al. Low pH induced membrane fusion of lipid vesicles were killed 24 h after injection, and the liver was per- containing proton-sensitive polymer. Biochemistry 1987; 26: fused with saline. The liver was then examined under 8145–8150. a fluorescence microscope equipped with CCD camera 22 Hofland HEJ, Shephard L, Sullivan SM. Formation of stable cat- (Olympus IMT-2). ionic 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 Acknowledgements serum on cellular uptake and intracellular fate of We thank Drs K Takeda and H Miyazaki and Mr Y lipopolyamine/DNA complexes. Biochem Biophys Acta 1998; Suzuki at DNAVEC Research, for their helpful dis- 1368: 276–288. 24 Sternberg B, Hong K, Zheng W, Papahadjopoulos D. Ultra- cussions; Mr T Iwasaki and Mr H Ori for technical assist- structural characterization of cationic liposome-DNA complexes ance, and Dr Y Sadzuka of the University of Shizuoka for ␨ showing enhanced stability in serum and high transfection measurement of -potentials. We also thank Dr Y Namba activity in vivo. Biochem Biophys Acta 1998; 1375: 23–35. at Nippon Fine Chemical for the supply of phospho- 25 Kichler A, Mechtler K, Behr JP, Wagner E. 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