O-Alkyl dioleoylphosphatidylcholinium Compounds - American

306

Bioconjugate Chem. 2000, 11, 306−313

O-Alkyl dioleoylphosphatidylcholinium Compounds: The Effect of Varying Alkyl Chain Length on Their Physical Properties and in Vitro DNA Transfection Activity Howard S. Rosenzweig,† Vera A. Rakhmanova, Thomas J. McIntosh,‡ and Robert C. MacDonald* Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208, and The Department of Cell Biology, Duke University, Durham, North Carolina 27710. Received September 1, 1999; Revised Manuscript Received January 3, 2000

1,2-Dioleoyl-sn-3-ethylphosphocholine (EDOPC) has been previously shown be a highly effective DNA transfection reagent in vitro. To assess the effect of alkyl chain length on transfection efficiency, the O-methyl, O-propyl, O-hexyl, O-decyl, and O-octadecyl derivatives have been prepared from dioleoylphosphatidylcholine using the corresponding alkyl trifluoromethylsulfonate. The methyl, ethyl, and propyl derivatives formed liposomes which were very large and unilamellar. The ethyl and propyl derivatives were equally efficient at mediating transfection (even in the presence of serum) of BHK cells, but the chemically labile methyl derivative was a much weaker transfection agent. The O-decyl and O-octadecyl compounds, which assume the inverted hexagonal phase in excess water (as determined by X-ray diffraction), were almost inactive after manual agitation in both water and in saline; however, after sonication, these compounds exhibited good transfection activity. The O-hexyl derivative displayed novel behavior, assuming the lamellar phase at low and a cubic phase at high ionic strength. All compounds, whether lamellar or not, formed lamellar structures when complexed with DNA. In water, where the hexyl compound dispersed well, sonication diminished transfection activity, whereas at physiological ionic strength, which led to poor manual dispersion, sonication was essential for good transfection. These results emphasize the importance of optimal dispersion of a cationic lipid: too little, and interaction with DNA is handicapped, too much, and the resultant particle transfects poorly. Lipid dispersibility is thus an important variable in assessing lipid transfection agents, and caution is advised in attributing too much significance to chemical structure until interaction with DNA has been optimized.

INTRODUCTION

Transfection, the introduction of plasmid DNA into cultured mammalian cells for the transient expression of a desired protein, has become a standard technique in molecular biology. Earlier methods have involved the use of DNA coprecipitation with calcium phosphate, soluble DEAE, or Polybrene resins (1), as well as electroporation (2). Following the demonstration by Felgner et al. that the cationic amphipath, DOTMA (dioleoloxypropyl-trimethylammonium bromide), is a simple and generally effective method to transfect DNA into cultured cells (3), use of “cationic lipids” has become widespread. A number of other cationic lipids with transfection activity have been developed in recent years, e.g., refs 4-12. One of the potentially most important applications of cationic lipids is as DNA delivery agents for gene therapy (13). Another potential clinical application is administration of antisense oligonucleotides (14). Other molecules can be delivered to cells as cationic complexes, such as proteins (15, 16), and small pharmacological agents (17). It is likely that additional applications will be developed soon. * To whom correspondence should be addressed. Phone: (847) 491-5062. Fax: (847) 467-1380. E-mail: [email protected]. † Current address: Wesley Jessen, Inc., Des Plaines, IL 60018. ‡ Duke University.

Although with few exceptions (e.g., refs 5 and 12), most of the compounds described in the literature as “cationic lipids” differ chemically from lipid natural products, it is readily possible to modify phospholipids to convert them into cationic derivatives. We have previously described the reaction which involves the alkylation of the phosphate oxygen of phosphatidylcholine such as to generate O-alkyl phosphatidylcholinium compounds (18). The ethyl ester has been shown to be an effective transfection agent both in vitro (19) and in vivo (20). These compounds consist of an essentially unperturbed phospholipid scaffold linked with metabolizable ester linkages. Since they exhibit low toxicity against cells in culture, they could be well suited for clinical applications involving gene and drug therapy. To explore the possibility that other alkyl derivates besides the ethyl esters are also potent transfection agents, we have synthesized a series of O-alkyl cationic analogues ranging from methyl to octadecyl, tested them for transfection activity, and examined the nature of their lyotropic phases. EXPERIMENTAL PROCEDURES

Materials. Lipids were obtained from Avanti Polar Lipids (Alabaster, AL) except for the cationic lipids, which were synthesized according to procedures described below. Most other biochemicals were purchased from Sigma (St. Louis, MO). Organic reagents were purchased from Aldrich (Milwaukee, WI). When commercially available methyl and ethyl triflates were used in synthesis,

10.1021/bc9901144 CCC: $19.00 © 2000 American Chemical Society Published on Web 03/24/2000

O-Alkyl Cationic Phospholipids

as much as possible, they were used very soon after opening the ampule; however, they could be stored for over a month at a time with negligible degradation in a desiccator at -20 °C. Triflic anhydride could be successfully stored after opening for over a month in a desiccator kept at room temperature. Tissue culture reagents were obtained from Life Technologies (Grand Island, NY). A plasmid with the β-galactosidase gene driven by the cytomegalovirus promoter (pCMV-β-gal) was purchased from Life Technologies (Grand Island, NY). It was propagated in DH5 cells and purified with a Plasmid Mega Kit from Qiagen (Valencia, CA). Synthetic Methods. Higher Homologues of O-Alkylated DOPC Triflates. In a 50 mL, one-necked roundbottom flask was placed anhydrous CH2Cl2 (3 mL), followed by 250 mmol of the required anhydrous alcohol (e.g., propyl, hexyl, decyl or octadecyl alcohol), and 250 mmol of triethylamine. The reaction flask was sealed with a rubber septum, placed under a positive argon atmosphere, and then cooled in an ice-water bath. A solution of triflic anhydride (250 mmol in 2 mL of anhydrous CH2Cl2) was slowly (ca. 5 min) added through a syringe into the cooled reaction solution. The reaction mixture was stirred for 1-2 h at 0 °C under a positive argon atmosphere, after which it was transferred (within 2-3 min) with a Pasteur pipet to a 20 mL screwcap vial containing 5 mL of ice-cold 10% HCl and the mixture was extracted. The lower organic layer was removed and transferred to another 20 mL screwcap vial containing 5 mL of ice-cold H2O and extracted again. The organic layer was then filtered twice through anhydrous granular sodium sulfate. The dried organic solution was then placed into a 100-mL round-bottom flask, which was sealed with a rubber septum and then placed under a positive argon atmosphere. To this triflate solution, 10 mL of a room-temperature phosphatidylcholine CHCl3 solution (20 mg/mL, 250 mmol) was added through a syringe, and the reaction mixture was allowed to stir at room temperature under argon for at least 2 h. The reaction was monitored by analytical silica gel TLC (CHCl3:MeOH:H2O; 65:25:4, v:v). When the reaction appeared to be over, the crude product was purified on a dry silica gel column, using at least 10 times the mass of DOPC contained in the reaction (>2 mL of silica). After adding the reaction mixture to the column, the column was washed with several volumes of CHCl3 and then with solvents containing increasing amounts of methanol in chloroform. The product began eluting with 9:1 CHCl3: MeOH and was completely removed from the column with 5:1 or 4:1 CHCl3:MeOH. Slight positive pressure with argon gas (“flash” conditions) accelerated the purification. Yields ranged from 20 to 70%. O-Methyl and O-Ethyl DOPC. These compounds were synthesized using commercially available ethyl and methyl triflate, following the procedure above from the point where the triflate was synthesized. Product Characterization. Liquid secondary ion mass spectra were obtained on a VG70 mass spectrometer (V. G. Analytical) with Cs ion as a primary source. Proton NMR spectra were obtained on a Varian Gemini 300 MHz spectrometer. The 1H NMR spectra and mass spectra obtained were all consistent with the structure of the expected product. All lipids gave a single spot by thin-layer chromatography with an Rf of 0.5 when developed in chloroform:methanol:water, 65:25:1. Lipid Dispersions. Lipids were stored at -20 °C in chloroform. When needed, aliquots were transferred to vials where the bulk of the solvent was evaporated with a gentle stream of argon gas. The vial was then placed

Bioconjugate Chem., Vol. 11, No. 3, 2000 307

under a high vacuum for at least 30 min to remove residual chloroform. Next, water or Dulbecco’s phosphatebuffered saline (D-PBS; with calcium and magnesium) was added to obtain a 1 mg/mL concentration, and the vial was either gently vortexed or sonicated for 2 min in an 80 kHz bath sonicator (Laboratory Supply Co., Inc, Hicksville, NY). The bath was tuned by adjusting the height of the water so as to obtain maximum agitation of the surface. X-ray Diffraction of Cationic Lipids and Their Complexes with DNA. Cationic lipids were hydrated in 10-fold excess water or D-PBS for several hours at room temperature. When the lipids dispersed readily, a portion of the suspension was simply concentrated by centrifugation and a portion of the pellet transferred to thin-walled X-ray capillary tubes. In the case of those lipids that dispersed less well, a portion of the hydrated lipid was scraped from its container and transferred to a capillary tube with a spatula. The capillary tubes were then sealed and mounted in a point-collimation X-ray camera or a mirror-mirror X-ray camera. Complexes of lipid and DNA were prepared from salmon sperm DNA which had been sonicated to reduce the length of the strands and reduce the solution viscosity. Gel electrophoresis on preparations such as this revealed a broad band corresponding to 400-700 base pairs. Dispersed lipid was mixed with DNA in the same proportions as used for cell transfection (see below), namely 3 parts lipid with 1 part by weight of DNA to generate a near-neutral particle. The complex was collected, by centrifugation if necessary, and transferred to X-ray tubes as described above. X-ray diffraction patterns were recorded on Kodak DEF X-ray film at ambient temperature. X-ray films were processed by standard techniques and densitometered with a Joyce-Loebl microdensitometer as described previously (21, 22). Cell Transfection. BHK cells (CCL-10) were obtained from American Type Culture Collection (Rockville, MD) and were cultured according to the recommended conditions. BHK cells were seeded in 96-well microplates at 48 h before transfection at densities to give approximately 80% confluence at the time of transfection. Complete medium was replaced with serum-free medium or medium containing 10% fetal bovine serum immediately prior to transfection. The complex for transfection was prepared by mixing a cationic lipid dispersion (1 mg/mL, normally in D-PBS, but sometimes in water) and β-galactosidase plasmid DNA solution (0.1 mg/mL) at a lipid: DNA mass ratio of 3:1. The lipid-DNA mixture was incubated for 20 min at room temperature. Cells in each well of the 96-well plate were treated with lipid-DNA complexes and then incubated for 4 h, at which time a 1/10 volume of fetal bovine serum was added to the cells. The total volume of medium was 100 µL/well. Replicates of three were used in each experiment. Cells were tested for β-galactosidase activity 20 h after transfection using a microplate fluorimetric assay that employed the substrate fluorescein digalactoside (23). RESULTS

O-Alkylation of Phospholipids Proceeds Readily with Alkyl Trifluoromethylsulfonates (Triflates). Triflates are among the most potent alkylating agents available for preparative organic chemistry, and they have proven to be very useful reagents for preparing O-alkyl derivatives of phospholipids. The reaction, for the

308 Bioconjugate Chem., Vol. 11, No. 3, 2000

Figure 1. Reaction for preparation of alkyl esters of phosphatidylcholine.

case of dioleoylphosphatidylcholine, is shown in Figure 1. The alkyl triflate was produced in anhydrous methylene chloride, and then the reaction mixture was extracted with cold 10% HCl solution to remove both the triethylamine and triflic acid byproduct, as well as any unreacted triflic anhydride. No attempt was made to isolate the intermediate, due to the thermal instability of long-chain alkyl triflates. Instead, the alkyl triflate solution was dried with anhydrous sodium sulfate, after which it was reacted with DOPC added as a chloroform solution. The appearance of the product was easily monitored by analytical silica gel thin-layer chromatography using either general staining procedures such as charring or standard lipid detection reagents such as those used for visualizing compounds containing phosphate or choline (ref 24, pp 119-121). The structure of the product was verified by proton NMR and mass spectral analysis. Mass spectra of all these compounds synthesized to date have been unambiguous; for essentially all of the O-alkylated lipids, the parent ion has been by far the most intense peak in the spectrum. A representative example of a 1H NMR spectrum is shown in Figure 2 for the O-propyl derivative. The key diagnostic features of it are (a) the slightly obscured hextet centered at ca. 1.72 ppm, which corresponds to the β-methylene carbon protons of the appended propyl group, and especially (b) the triplet centered at 0.97 ppm, which corresponds to the terminal methyl group protons of the appended propyl group. These two features verify that the propyl group is bonded to the DOPC as proposed. Further interpretation of the spectrum is provided in the legend to Figure 2. Cationic Phospholipids with Short Alkyl Groups Hydrate Well and Disperse Readily as Liposomes. Longer Chain Homologues Disperse Poorly in Aqueous Phases. As observed previously (18), O-ethyl-DOPC readily hydrates at room temperature and forms large, usually unilamellar vesicles, easily visible under the light microscope. The same behavior was observed with both O-methyl- and O-propyl-DOPC. In contrast, neither the O-decyl- nor O-octadecyl-DOPC formed liposomes, in either water or salt solutions, based on light microscopy. The O-hexyl compound exhibited unusual and unex-

Rosenzweig et al.

Figure 2. NMR spectrum of O-propylDOPC triflate. The 300 MHz proton NMR spectrum shows that the alkylation of DOPC with propyl triflate has been achieved. The quintet centered at 5.34 ppm (4H, from integration) corresponds to the vinyl protons of the oleoyl ester groups, while the broad signal at 5.26 ppm (1H) corresponds to the single proton of the central carbon of the glycerol backbone. The broad signals found at 4.51 ppm (2H) and 3.89 ppm (2H) correspond to the protons of the two methylene carbons of the choline group (the more downfield resonance, 4.51 ppm, derives from the protons of the carbon connected to the phosphate ester group, while the other protons belong to the carbon adjacent to the trimethylammonium group, respectively). The multiplet between 4.37 and 4.00 ppm (6H) results from the overlap of the double triplet arising from the diastereomeric R-methylene protons of the propyl group attached to the third phosphate oxygen (the site of alkylation with propyl triflate) plus the two triplets arising from the two remaining methylene carbon protons of the glycerol backbone. The sharp singlet at 3.32 ppm (9H) represents the protons of the trimethylammonium group. The pseudo quartet centered at 2.33 ppm (4H) corresponds to the R-methylene protons of the oleoyl ester carbonyl groups. The broad doublet centered at ca. 2.00 ppm (8H) is due to the allylic protons, while the slightly obscured hextet centered at ca. 1.72 ppm (2H) represents the β-methylene protons of the appended propyl group. Finally, the triplets centered at 0.97 ppm (3H) and 0.87 ppm (6H) correspond to the terminal methyl group protons of the propyl and both oleoyl ester groups, respectively. The solvent was deuteriochloroform, with tetramethylsilane as the internal standard.

pected behavior. In water, it hydrated as a bilayer phase to form liposomes, whereas in salt solution, it remained as a viscous, nonbirefringent liquid. X-ray Diffraction of Hydrated Lipid Phases and of Their Complexes with DNA. According to X-ray diffraction, both O-decyl-DOPC and O-octadecyl-DOPC definitely formed hexagonal phases in excess water. Both compounds gave broad, wide-angle bands centered at 4.5 Å. The C10 compound gave spacings of 59.6, 34.4, 29.8, and 22.5 Å, which index as orders of a hexagonal phase with a spacing of d ) 59.6 Å. The C18 compound gave spacings of 61.4, 35.4, and 30.7, which index as orders of a hexagonal phase with d ) 61.4 Å. Essentially the same structure was observed when the lipid was dispersed in D-PBS. In excess D-PBS, the hexyl compound behaved quite differently than in excess water. During preparation of this compound for X-ray analysis, the hydrated material clung to the vessel wall. After scraping it away, a very viscous, clear mass was obtained. This material was not birefringent. It gave an X-ray pattern with a broad wideangle band at 4.5 Å (consistent with a liquid crystalline phase) and six strong, sharp low-angle reflections at


spacings of 60.6, 49.8, 43.1, 35.3, 30.3, and 29.4 Å. These six reflections index quite closely to orders (110), (111), (200), (211), (220), and (300) of a cubic phase with a fundamental repeat period of 85.7 Å. The only reflections that are missing for the expected first eight reflections of a 85.7 Å cubic phase are the (100) and (210) reflections. Moreover, no observed reflections are unaccounted for by this cubic phase. Cubic phases are viscous and nonbirefringent. So, at least under the ionic strength conditions for phosphate basic saline and at moderately high concentration of lipid (10%), O-hexylphosphatidylcholine forms a cubic phase. This conclusion was verified by the observation of a bicontinuous cubic array by freezefracture electron microscopy (Y. Tarahovsky et al., unpublished material). The complexes formed by some of the lipids with DNA were examined by X-ray diffraction. As previously found, the complex of DNA with the ethyl compound was lamellar in both water with salmon sperm DNA and D-PBS with plasmid DNA (R.C.M. and Ashley et al., unpublished material). HexylDOPC, dispersed in water and combined with DNA was not highly ordered, giving two reflections that indexed as d and d/2 of a repeating unit of 59 Å. Since there was no indication of a reflection at d/x3, which would indicate a hexagonal phase or at any repeats expected for cubic phases, this complex was evidently lamellar. Octadecyl-DOPC, although it formed a hexagonal complex by itself, gave a lamellar complex with salmon sperm DNA at a 4:1 ratio of lipid to DNA. The initial complex, diffracted immediately after formation, revealed a mixture of lamellar and hexagonal phases, but after standing overnight, the hexagonal reflections disappeared, leaving only the lamellar reflections for a repeating unit of 70 Å. Cationic Phospholipids Efficiently Mediate DNA Transfection, even in the Presence of Serum. To establish the effect of varying the alkyl chain length on transfection efficiency, we assessed the ability of the O-alkyl derivatives, namely, O-methyl, O-propyl, O-hexyl, O-decyl, and O-octadecyl-DOPC, to mediate DNA transfection in BHK cells. Unlike most cationic lipids available commercially, these cationic phospholipids do not require any DOPE co-lipid in order to mediate transfection. Consequently, the transfection complexes were typically prepared simply by combining DNA with the cationic phospholipid, since, from prior experience with the ethyl derivative (25), we expected that these cationic phospholipids might also mediate transfection efficiently in the presence of serum proteins, and therefore tested transfection both in the presence and absence of 10% serum. As shown in Figure 3, the O-ethyl (Figure 3B) and O-propyl (Figure 3C) compounds were the most active compounds in the series with alkyl chains of 1, 2, 3, and 6 carbons, and essentially equivalent in transfection mediation efficiency. The methyl derivative exhibited significantly lower activity (Figure 3A). The O-hexyl derivative mediated transfectionsat least at one lipid: DNA ratioswith nearly the same efficiency as the Oethyl and O-propyl compounds, but only if the lipid had been dispersed in water (Figure 3D). Although O-hexyl-PC was virtually devoid of transfection activity when prepared according to the standard hydration procedure involving vigorous vortexing in saline, it exhibited good activity when it was sonicated before complex formation (Figure 4A). The long-chain O-alkyl derivatives, O-decyl-PC and O-octadecyl-PC, exhibited little transfection activity when prepared in either water or in saline by manual agitation (Figure 4,


Figure 3. Transfection efficiency of O-alkyl compounds in presence of serum. BHK cells were transfected with (A) OmethyDOPC/DNA complex, (B) O-ethylDOPC/DNA complex, (C) O-propylDOPC/DNA complex, and (D) O-hexylDOPC/DNA complex. Transfection was performed in medium without supplements (white bars) and in medium containing 10% FBS (gray bars). All lipids were dispersed in D-PBS with the exception of O-hexyl-DOPC, which was dispersed in water. Error ranges indicate the highest and the lowest values of replicates.


Rosenzweig et al.

sonication, the O-hexyl-PC loses activity if dispersed in water. Loss of transfection activity was also seen with the two longer derivatives, but it required longer exposures; at sonication times of 10 min or more, the tranfection efficiency dropped by 90% or more. Thus, in D-PBS, these three compounds require sonication for proper dispersion, but excessive sonication clearly leads to a loss of activity. In general, it appears that the more easily dispersed the lipid, the more quickly sonication reduces transfection activity. In the case of the ethyl compound, which generates liposomes spontaneously without agitation, only a few tens of seconds of sonication eliminated activity. In that case, there was no chemical decomposition of the lipid detectable by thin-layer chromatography even after minutes of sonication (26). A related test has been done on octadecyl-PC; it was sonicated to produce a uniform dispersion in water and then allowed to stand at room temperature for a week. By thin-layer chromatography there was no detectable degradation (A. Hashimoto, unpublished). It is thus clear that the effect of sonication is physical. Our preliminary evidence suggests that small lipid particles give rise to small transfection complexes (E. Pozharski, V.A.R., and R.C.M., unpublished material) and that these, probably because they remain in suspension longer than larger complexes, are less effective at transfection. Others have observed that multilamellar dispersions of lipids are more effective transfection agents, at least under some conditions, than sonicated preparations (11, 27-29). DISCUSSION

Figure 4. Effect of solvent and dispersion on transfection activity of medium and long chain O-alkyl derivatives. BHK cells were transfected with (A) O-Hexyl-DOPC/DNA complex, (B) O-decyl-DOPC/DNA complex; (C) O-Octadecyl-DOPC/DNA complex. O-alkyl compounds were dispersed in water or in PBS with or without sonication. Lipid-DNA complexes were used in amounts 1.5/0.5 µg/well (black bars), 3.0/1.0 µg/well (gray bars), 6.0/2.0 µg/well (white bars). Error ranges indicate the highest and the lowest values of replicates.

panels B and C). As mentioned earlier, the long-chain derivatives are very difficult to disperse in aqueous media compared to O-methyl, ethyl, or propyl versions, and even when they are first solubilized in ethanol and injected into the aqueous phase, they do not give efficient transfection. After sonication, however, O-decyl- and O-octadecyl-DOPC exhibit significant transfection activity (Figure 4, panels B and C), although typically less than the other O-alkyl compounds mentioned above (except Omethyl). The decyl and octadecyl compounds have also been tested for transfection at ratios of lipid to DNA of 4:1 and 5:1, with no marked difference in transfection efficiency from those found for the 3:1 ratio. The sonication time for the lipids used for the transfection assays of Figure 4 was 2 min. At this duration of

Alkylation of Zwitterionic Phospholipids Provides a Facile Route to Cationic Phospholipids. Amphipathic molecules have a multitude of important medical, technical, and cosmetic applications. In some cases, the properties of natural compounds are an advantage, either because of diminished toxicity or because of the characteristic required for the natural function. Aside from modifying the fatty acid substituents and derivatizing the headgroup to incorporate markers such as fluorophores, there has been relatively little research involving basic changes in the structure of natural phospholipids. We found that it is straightforward to change zwitterionic phospholipids to their cationic counterparts. It is clear from the present work that such modifications of the structure of phosphatidylcholine can produce molecules with some very unusual properties that were not anticipated. Furthermore, because cationic phospholipids are very rare and those of the alkyl triester type described here are unknown in nature, they are anticipated to have unusual interactions with cells. Physical Properties of Cationic Phospholipids. The conversion of a phosphatidylcholine to an O-alkyl phosphatidylcholinium ion not only eliminates the negative charge but also drastically reduces the hydrogen bond accepting potential of the molecule. In addition, the size and polarity of the headgroup may be altered, depending upon the length of the alkyl chain appended and the size and hydrophobicity of the resulting headgroup. The alkyl group may be varied from the minimum, a methyl group, to a chain as long or potentially longer than the fatty acid groups already present in the molecule. The methyl, ethyl, and propyl derivatives are evidently only modestly affected with respect to size and hydrophobicity, whereas the longer chain compounds are sufficiently changed that they prefer to assume the cubic or hexagonal phase than to reside in the lamellar phase. The methyl phosphatidylcholinium is, as expected of methyl phosphate esters, somewhat unstable due to


susceptibility to SN2 nucleophilic attack. However, the ethyl and, it may be presumed, the longer chain members of the family are quite stable. The ethyl compound is susceptible to hydrolysis by some purified lipases and in cells, although loss of the ethyl group was not observed (25). The rate of hydrolysis appears to decrease the closer the O-alkyl group is to the bond attacked by the lipase. This susceptibility to hydrolysis may contribute to the relatively low toxicity that these compounds exhibit toward mammalian cells. They may also be able to integrate into the various cellular membranes and coexist peacefully with their zwitterionic neighbors. These characteristics, in addition to their ability to mediate transfection efficiently in the presence of serum proteins, make them attractive candidates for agents to deliver nucleic acids and other pharmacological and physiological agents (17) to cells, both in the laboratory and the clinic. The O-methyl, O-ethyl, and O-propyl derivatives hydrate very well, forming a lamellar phase with large spacings, whereas the O-decyl and O-octadecyl derivatives do not. The latter compounds assume a hexagonal phase (30) presumably because the alkyl chains are so long that they must fold back among the acyl chains, where they increase the tail cross-sectional area to the point where lamellar phase packing is not possible. As is typical of inverted hexagonal phases, because they do not exhibit extended hydrophilic surfaces with which to make low-energy interfaces with water, the long-chain O-alkyl phosphatidylcholine derivatives are very difficult to disperse in aqueous phases, especially at higher ionic strength where ionization and hence hydration would be repressed. While the O-hexyl derivative can assume a lamellar phase in water, it seems to be on the edge of stability such that it prefers to reside in a cubic phase upon hydration in saline. This behavior is not surprising, though, since cubic phases are typically found in phase diagrams between lamellar and hexagonal phases (31) and, structurally, the hexyl derivative stands between shorter chain lamellar phase-forming compounds and longer chain hexagonal phase-forming compounds. Presumably, this behavior of the hexyl compound can be attributed to a folding back of the hexyl group into the region of the acyl chains, which generates enough tailto-head asymmetry in the molecule for it to favor a cubic phase, but not enough for preference of the inverted hexagonal phase. Structure and Transfection Efficiency of O-Alkyl Derivatives. The newly synthesized O-alkyl derivatives were examined for their capacity to mediate transfection in mammalian cells. The first compound in this set, O-methyl, hydrated and dispersed well, but its reduced chemical stability, perhaps even after uptake into cells, may explain why it exhibited much less activity compared to the next short-chain compounds. The short-chain derivatives, O-ethyl-PC and O-propyl-PC, which formed a lamellar phase after hydration, were the most effective in DNA transfection. The former compound is known to form a sandwich-type complex with DNA in which the nucleic acid binds together bilayers of lipid (26), as occurs with some other cationic lipid transfection agents (32-35). The intermediate chain compound, O-hexyl, which is also lamellar in water, had approximately the same transfection efficiency as O-ethyl and O-propyl. However, in saline, O-hexyl assumed a cubic phase and became capable of mediating transfection only when vigorously dispersed by sonication. When combined with DNA, this compound gave a lamellar phase complex, presumably


because, topologically, a bicontinuous cubic phase would be incompatible with maximal interaction of lipid and DNA. Transformation to a hexagonal phase complex, while possible, is evidently not energetically favorable with compounds of this type, as evidenced by the behavior of the octadecyl derivative (see next paragraph). As particularly well illustrated by the hexyl derivative, there are critical aspects of formulation with plasmids that impose clear limits on the efficiency of transfection mediated by cationic amphipaths. In water, where Ohexyl-PC dispersed fairly well, sonication was detrimental, presumably because the small vesicles so generated give rise to small DNA complexes which transfected poorly, either because of poor uptake or reduced contact with cells (29, 36). In contrast, in D-PBSsin which the compound assumed a cubic phase and dispersed much less well than in watersthe lipid had to be sonicated in order to disperse it well enough to form productive interactions with DNA. The transfection complex that O-hexyl-PC forms with DNA at high ionic strength is lamellar, suggesting that there is a smaller structural penalty involved in the transition from a cubic array to a lamellar complex than to a hexagonal complex. Since there is no obvious way in which DNA-lipid surface electrostatic interactions could be maximized given the limited structural parameters of the cubic phase, we would not expect to see a stable cubic array of DNA and lipid. It should be appreciated, however, that the mode of formation of transfection complexes can influence their size, transfection activity, and possibly structure (37). The longest chain compounds in the series, O-decylPC and O-octadecyl-PC, assumed the inverted hexagonal phase in water (and presumably also in saline) and were also essentially inert as transfection agents when prepared according to the standard hydration procedure. These compounds only exhibited significant activity if they were dispersed by sonication. The structure of the complex they formed with DNA was lamellar (26). DNA can form hexagonal complexes with cationic lipid mixtures (38), and although we were initially surprised that a hexagonal phase cationic phospholipid formed a lamellar phase complex with DNA, theoretical analyses have affirmed that, unless the intrinsic curvature of the lipid is very high, lamellar phases are more stable than hexagonal arrays (39-41). Consistent with these analyses, we have found that when the long-chain cationic phospholipids are formulated with an equal amount of DOPE, the resulting lipid mixture does indeed generate a hexagonal phase complex with DNA (19). In contrast to other investigators (38), we found that there hexagonal phase complexes had no advantages over lamellar phase complexes and, indeed, were generally less effective than lamellar phase complexes (19). Implications for Assessment of New Cationic Transfection Mediators. Transfection mediators of the cationic lipid type may exist in a variety of different phase states which have intrinsically much different abilities to be uniformly dispersed in an aqueous phase. It is therefore essential that surveys of new compounds be constructed such as to ensure that the lipid is present in the aqueous phase in a form allowing near-complete exposure to DNA in solution. Otherwise, there is the risk that potentially valuable compounds will be overlooked and that conclusions about particular chemical classes could be erroneous. Our results suggest that with a given chemical type of transfection agent, there is no inherent advantage in either lamellar or hexagonal phases, but


that optimal conditions for DNA complex formation may well differ greatly. ACKNOWLEDGMENT

We are grateful to Joseph Tucker, Jaime Stearns, and Akihiro Hashimoto for contributions to optimization of reaction conditions and to Jaime Stearns and Akihiro Hashimoto for the synthesis of some of the lipids used here. We are also grateful to Ruby MacDonald for carefully reading and commenting on the manuscript and for support in the laboratory. Funded by grants from the NIH: GM52329 and GM57305 to R.C.M. and GM27278 to T.J.M. We are also grateful to Avanti Polar Lipids for lipid starting materials. LITERATURE CITED (1) Keown, W. A., Campbell, C. R., and Kucherlapati R. S. (1990) Methods for introducing DNA into mammalian cells. Methods Enzymol. 185, 527-537. (2) Forster W., and Neumann E. (1989) Gene Transfer by Electroporation- -A Practical Guide. In Electroporation and Electrofusion in Cell Biology (E. Neumann, A. E. Sowers, and C. A. Jordan, Eds.) pp 299-318, Plenum Press, New York. (3) Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofection: highly efficient, lipidmediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417. (4) Leventis, R., and Silvius, J. R. (1990) Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles. Biochim. Biophys. Acta 1023, 124-132. (5) Gao X., and Huang L. (1991) A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem. Biophys. Res. Commun. 179, 280-285. (6) Rose, J. K., Buonocore, L., and Whitt, M. A. (1991) A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques 10, 520-525. (7) Barthel, F., Remy, J. S., Loeffler, J. P., and Behr, J. P. (1993) Gene transfer optimization with lipospermine-coated DNA. DNA Cell Biol. 12, 553-560. (8) Solodin, I., Brown, C. S., Bruno, M. S., Chow, C. Y., Jang, E. H., Debs, R. J., and Heath, T. D. (1995) A novel series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery. Biochemistry 34, 13537-13544. (9) Akao, T., Nakayama, T., Takeshia, K., and Ito, A. (1994) Design of a new cationic amphiphile with efficient DNAtransfection ability. Biochem. Mol. Biol. Int. 34, 915-920. (10) Bichko, V., Netter, H. J., and Taylor, J. (1994) Introduction of hepatitis delta virus into animal cell lines via cationic liposomes. J. Virol. 68, 5247-5252. (11) Felgner, J. H., Kumar, R., Sridhar, C. N., Wheeler, C. J., Tsai, Y. J., Border, R., Ramsey, P., Martin, M., and Felgner, P. L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550-2561. (12) Wang, J., Guo, X., Xu, Y., Barron, L., and Szoka, F. C. Jr. (1998) Synthesis and characterization of long chain alkyl acyl carnitine esters. Potentially biodegradable cationic lipids for use in gene delivery. J. Med. Chem. 41, 2207-2215. (13) Morgan, R. A., and Anderson, W. F. (1993) Human gene therapy. Annu. Rev. Biochem. 62, 191-217. (14) Bennett, C. F., Chiang, M. Y., Chan, H., Shoemaker, J. E., and Mirabelli, C. K. (1992) Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharmacol. 41, 1023-1033. (15) Debs, R. J., Freedman, L. P., Edmunds, S., Gaensler, K. L., Du¨zgu¨nes¸ N., and Yamamoto, K. R. (1990) Regulation of gene expression in vivo by liposome-mediated delivery of a purified transcription factor. J. Biol. Chem. 265, 1018910192. (16) Huang, L., Farhood, H., Serbina, N., Teepe, A. G., and Barsoum, J. (1995) Endosomolytic activity of cationic liposomes enhances the delivery of human immunodeficiency

Rosenzweig et al. virus-1 trans-activator protein (TAT) to mammalian cells. Biochem. Biophys. Res. Commun. 217, 761-768. (17) Barber, K., Mala, R. R., Lambert, M. P., Qiu, R., MacDonald, R. C., and Klein, W. L. (1996) Delivery of membraneimpermeant fluorescent probes into living neural cell populations by lipotransfer. Neurosci. Lett. 207, 17-20. (18) Ashley, G. W., Shida, M. M., Qiu, R., Lahiri, M. K., Levisay, P. C., Jones, R. D., Baker, K. A., and MacDonald, R. C. (1996) Phosphatidylcholinium compounds: A new class of phospholipids with transfection activity and unusual physical properties. Biophys. J. 70, A88. (19) Rakhmanova, V. A., McIntosh, T. J., and MacDonald, R. C. (2000) Effects of dioleoylphosphatidylethanolamine on the activity and structure of O-alkylphosphatidylcholine-DNA transfection complexes. Cell Mol. Biol. Lett. (in press). (20) Gorman, C. M., Aikawa, M., Fox, B., Fox, E., Lapuz, C., Michaud, B., Nguyen, H., Roche, E., Sawa, T., and WienerKronish, J. P. (1997) Efficient in vivo delivery of DNA to pulmonary cells using the novel lipid EDMPC. Gene Ther. 4, 983-992. (21) McIntosh, T. J., Magid, A. D., and Simon, S. A. (1987) Steric repulsion between phosphatidylcholine bilayers. Biochemistry 26, 7325-7332. (22) McIntosh, T. J., Magid, A. D., and Simon, S. A. (1989) Cholesterol modifies the short-range repulsive interactions between phosphatidylcholine membranes. Biochemistry 28, 17-25. (23) Rakhmanova, V. A., and MacDonald, R. C. (1998) A microplate fluorimetric assay for transfection of the β-galactosidase reporter gene. Anal. Biochem. 257, 234-237. (24) Christie, W. W. (1982) Lipid Analysis, pp 161-166, Pergammon, Oxford. (25) MacDonald, R. C., Rakhmanova, V. A., Choi, K. L., Rosenzweig, H. S., and Lahiri, M. K. (1999) O-ethylphosphatidylcholine: A metabolizable cationic phospholipid which is a serum-compatible DNA transfection agent. J. Pharm. Sci. 88, 896-904. (26) MacDonald, R. C., Ashley, G. W., Shida, M. M., Rakhmanova, V. A., Tarahovsky, Y. S., Pantazatos, D. P., Kennedy, M. T., Pozharski, E. V., Baker, K. A., Jones, R. D., Rosenzweig, H. S., Choi, K. L., Qiu, R., and McIntosh, T. J. (1999) Physical and biological properties of cationic triesters of phosphatidylcholine. Biophys. J. 77, 2612-2629. (27) Kojima, H., Ohishi, N., Takamori, M., and Yagi, K. (1995) Cationic multilamellar liposome-mediated gene transfer into primary myoblasts. Biochem. Biophys. Res. Commun. 207, 8-12. (28) Holmen, S. L., Vanbrocklin, M. W., Eversole, R. R., Stapleton, S. R., and Ginsberg, L. C. (1995) Efficient lipidmediated transfection of DNA into primary rat hepatocytes. In Vitro Cell. Dev. Biol. 31, 347-351. (29) Ross, P. C., and Hui, S. W. (1999) Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Ther. 6, 651-659. (30) Seddon, J. M. (1990) Structure of the inverted hexagonal (HII) phase, and non-lamellar phase transitions of lipids. Biochim. Biophys. Acta 1031, 1-69. (31) Larsson, K. (1994) Lipids-Molecular Organization, Physical Functions and Technical Applications, The Oily Press, Dundee. (32) Xu, Y., Hui, S. W., Frederik, P., and Szoka, F. C., Jr. (1999) Physicochemical characterization and purification of cationic lipoplexes. Biophys. J. 77, 341-353. (33) Ra¨dler, J. O., Koltover, I., Salditt, T., and Safinya, C. R. (1997) Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes. Science 275, 810-814. (34) Lasic, D. D., Strey, H., Stuart, M. C. A., Podgornik, R., and Frederik, P. M. (1997) The structure of DNA-liposome complexes. J. Am. Chem. Soc. 119, 832-833. (35) Boukhnikachvili, T., Aguerre-Chariol, O., Airiau, M., Lesieur, S., Ollivon, M., and Vacus, J. (1997) Structure of inserum transfecting DNA-cationic lipid complexes. FEBS Lett. 409, 188-194. (36) Zuidam, N. J., Hirsch-Lerner, D., Margulies, S., and Barenholz, Y. (1999) Lamellarity of cationic liposomes and

O-Alkyl Cationic Phospholipids mode of preparation of lipoplexes affect transfection. Biochim. Biophys. Acta 1419, 207-220. (37) Kennedy, M. T., Pozharski, E. V., Rakhmanova, V. A., and MacDonald, R. C. (2000) Factors governing the assembly of cationic phospholipid-DNA complexes. Biophys. J. 78, 16201633. (38) Koltover, I., Salditt, T., Ra¨dler, J. O., and Safinya, C. R. (1998) An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science 281, 78-81.

Bioconjugate Chem., Vol. 11, No. 3, 2000 313 (39) Dan, N. (1998) The structure of DNA complexes with cationic liposomes-cylindrical or flat bilayers? Biochim. Biophys. Acta 1369, 34-38. (40) Harries, D., May, S., Gelbart, W. M., and Ben Shaul, A. (1998) Structure, stability, and thermodynamics of lamellar DNA-lipid complexes. Biophys. J. 75, 159-173. (41) Bruinsma, R. (1998) Electrostatics of DNA cationic lipid complexes: Isoelectric instability. Eur. Phys. J. B4, 75-88.

BC9901144

O-Alkyl dioleoylphosphatidylcholinium Compounds - American

Recommend Documents