Polycation Liposomes, a Novel Nonviral Gene Transfer System, Constructed from Cetylated Polyethylenimine
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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 fluor- escence 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 dimin- ished 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 cyto- toxic 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 incu- bated 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.