New cationic polyprenyl derivative proposed as a lipofecting agent.

Cationic linear poly-cis-isoprenoid prepared from natural plant polyprenol in a mixture with dioleyl phosphatidylethanolamine was found to be an effective lipofection agent for eukaryotic cells. The transfecting activity is related to the poly-cis structure of the polyprenyl chain.


INTRODUCTION
The linear polymers of isoprene residues were found to be lipid components of the systems of glycosylation in prokaryotes and eukaryotes (Robbins et al, 1967;Chojnacki & Dallner, 1988), thus establishing this new class of lipids as specific, crucial elements of cellular metabolism. The discovery of this class of lipids documented in the nineteen sixties (Burgos et al., 1963;Lindgren, 1965;Stone et al., 1967) was followed by testing their effect on membrane structure (Valterson et al., 1985;Janas et al., 1986) and thus approaching the mechanism of their coenzymatic functions. There were also trials to find the possible effect of these substances on eukaryotic cells (Korohoda et al., 1980) and the motility of cells was affected even by low amounts of dolichol (α-saturated poly-cis-prenol). The linear isoprenoids known so far occur in nature as alcohols, esters with acetic acid or fatty acids, phosphates or pyrophosphates and also as diesters with sugars. The cationic form of poly-prenols or dolichols which are most probably absent from living organisms and have never been the subject of interest in bioorganic chemistry may be interesting substances as components of typical lipofecting cocktails allowing the transfer of genetic material into the cells of interest. So far, only non-isoprenoid methylene based cationic lipids have been used in the lipofecting cocktails of Lipofectamine 2000 (cf. leaflet of Lipofectamine Reagent, 2004).
The aim of this report is to present the case a lipofecting activity of heptaprenyltrimethylammonium iodide in a model system of human prostate cancer cells.

MATERIALS AND METHODS
Heptaprenyltrimethylammonium iodide is one of the set of compounds of this type prepared by the method of Sen & Roach (1995) from naturally occuring poly-cis-prenols. The biological results * This paper is dedicated to Professor Tadeusz Chojnacki from the Institute of Biochemistry and Biophysics, Polish Academy of Sciences in Warsaw on the occasion of the 50th anniversary of his scientific activity and 75th birthday.  Correspondence to: Zbigniew Madeja, Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnol-Z. Madeja and others (negative) obtained on using all-trans-nonaprenyltrimethylammonium iodide prepared by the same method are also stated in this report.
The preparation of a range of these compounds from naturally occurring polyprenols and dolichols is described elsewhere (Masnyk et al., in preparation) and these substances are the subject of a patent application (No. PL 382448).
Lipofecting mixtures. The Lipofectamine 2000 Reagent was from Invitrogen Corporation (USA). The lipofecting mixture containing our new cationic isoprenoids ( Fig. 1) was made by making a suspension with dioleyl phosphatidylethanolamine. In the control experiments with Lipofectamine Reagent the polycationic lipid 2,3-dioleyl-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and the neutral lipid dioleyl phosphatidylethanolamine (Invitrogen Corporation, Carlsbad, CA, USA) in a 3 : 1 (w/w) were used. Other proportions of the ingredients were also used with the aim of finding optimal conditions for transfection in the liposome formulation.
Transfection procedure. DU145 cells were seeded one day before transfection at density 0.08 × 10 6 per well of 24-well plate in DMEM-F12 with 10% FCS but without antibiotics and grown to 70-80% confluency. Heptaprenyltrimethylammonium salt and helper lipid dioleylphosphatidylethanolamine (DOPE) were diluted in 99% ethanol to achieve final concentrations of 10 mg/ml and 3.2 mg/ ml, respectively. Next, they were diluted and vor-texed to suspend the lipid in DMEM-F12 medium without serum, at the indicated heptaprenyltrimethylammonium iodide/DOPE ratio (w/w). The plasmid pEGFP-C1 (kindly supplied by Professor A. F. Sikorski, University of Wrocław, Poland) was dissolved in DMEM-F12 without serum to a final concentration of 0.01 µg /µl. Equal volumes of cationic liposome suspension (the final lipid concentration indicated in the text) and plasmid DNA were gently mixed and incubated for 30 min at room temperature and then diluted 4 times with DMEM-F12 without serum. Medium from cells was aspirated and replaced with 200 µl of DMEM-F12 without serum and 200 µl of diluted complexes (transfection mixture). Cells were incubated at 37 o C in 5% CO 2 for 5 h. Then 400 µl DMEM-F12 containing 2 × the normal concentration of serum and antibiotics was added without removing the transfection mixture. The transfection efficiency was calculated after 24 h. Cells were washed with PBS and incubated in the presence of Hoechst 33342 (Sigma, St. Louis, MO, USA) (1 µg/ml in PBS) for 10 min. After this time cells were again washed with PBS and growing medium was added. The proportion of transfected cells was calculated under fluorescence Leica DM IRE2 microscope equipped with Leica DC350 FX digital camera as follows: (Number of cells expressing GFP/Number of cells stained with Hoechst 33342) × 100%.
Lipofectamine 2000 reagent was used as a positive control. Diluted plasmid was combined with diluted lipofectamine 2000 (1 µl of lipofectamine : 0.25 µg of plasmid) total volume (50 µl ) and incubated for 30 min at room temp. Then, the diluted complexes were used for the transfection procedure as described above.

Transfection efficiency on using heptaprenyltrimethylammonium salt
Transfection efficiency of this polyprenyl derivative was estimated by means of GFP-expression assay (Fig. 2). Cationic liposomes were prepared by mixing heptaprenyltrimethylammonium iodide and dioleyl phosphatidylethanolamine in ratio 3 : 1, followed by mixing with a plasmid DNA, pEGFP-C1, which encodes green fluorescent protein (GFP). DU145 cells were transfected with the liposome-DNA complex and the transfection efficiency was evaluated by means of fluorescence microscopy. As shown in Fig. 2, heptaprenyltrimethylammonium salt containing liposomes induced efficient transfection of DU145 cells. The highest transfection efficiency (32% of transfected cells) was observed at 1 µg of lipids per well (Fig. 3).
Interestingly, another tested compound -nonaprenyltrimethylammonium iodide under the same experimental procedure induced transfection with low efficiency, not exceeding 5% (not shown). This result seems to be consistent with previous observations that shorter-chain lipids are more fusogenic than long-chain ones (Tarahovsky et al., 2004). Cationic lipids are usually formulated as liposomes using co-lipids such as DOPE (Rao & Gopal, 2006). In our preliminary research we prepared cationic liposomes by mixing heptaprenyltrimethylammonium salt with DOPE in ratio 3 : 1 (Fig. 3). However, transfection efficiency of such liposomes was lower than with commercially available lipofectamine 2000, used in our experiments as a positive control (Fig. 3). To improve efficiency of transfection we investigated the effect of liposomes prepared in various heptaprenyltrimethylammonium salt and DOPE ratios. The results presented in Fig. 4 show that optimal efficiency (exceeding 40%) was obtained for the lipids in a ratio of 1 : 1. This is comparable to other highly efficient lipofectamines, e.g., lipofectamine 2000 (Fig. 4).

DISCUSSION
The data presented in this report extend the list of cationic lipids that may be used as components of lipofecting mixtures. The idea of constructing cationic lipids built from linear isoprene polymers came from the already known information on the effect of polyprenols and dolichols on artificial lipid membranes  (Valtersson et al., 1985;Janas et al., 1986). It was also known that the phosphate derivatives of these compounds function as lipid coenzymes of transglycosylation in the cell and may play an important role in natural cellular membranes (Chojnacki & Dallner, 1988).
The cationic forms of linear polymers of isoprene units do not occur in living organisms. Data are reported concerning the synthesis of such substances from naturally occurring plant isoprenoid alcohols, heptaprenol of the wood of Betula verrucosa (Lindgren, 1965) and other members of Betulaceae (Chojnacki, unpublished) and nonaprenol from leaves of Nicotiana tabacum (Rowland, 1955) and from several other plants. Several linear poly-cis-prenols occur in many plants as secondary metabolites (e.g.: Stone et al., 1967;Swiezewska et al., 1994).
The lack of the lipofecting activity of nonaprenyltrimethylammonium salt observed in the present study may be due not only to the length of the isoprenoid chain of nonaprenyl radical as postulated by Tarahovsky et al. (2004) but also to the stiff, all-trans structure of this unit. The current studies of our laboratories on derivatizing several linear cationic polyprenoids available in the Collection of Polyprenols of the Institute of Biochemistry and Biophysics, PAS, (Masnyk et al., in preparation) should solve this question.