| Nucleic Acids Research | Pages |
Novel cationic amphiphiles as delivery agents for antisense oligonucleotides
Introduction
Materials And Methods
Cholic acid-polyamine conjugates (molecular umbrellas)
Cell culture
Nucleic acids
Preparation of nucleic acid complexes
Plasmid transfection assay
Toxicity and growth assays
Flow cytometry
Confocal microscopy
Antisense assays
Results
Amphiphile structure
Cytotoxicity studies
Gene expression
Association of oligonucleotide with cells
Intracellular distribution
Oligonucleotide activity assays
Discussion
Acknowledgements
References
Novel cationic amphiphiles as delivery agents for antisense oligonucleotides
Received as resubmission April 19, 1999; Revised and Accepted May 7, 1999
ABSTRACT There has been great interest recently in therapeutic use of nucleic acids including genes, ribozymes and antisense oligonucleotides. Despite recent improvements in delivering antisense oligonucleotides to cells in culture, nucleic acid-based therapy is still often limited by the poor penetration of the nucleic acid into the cytoplasm and nucleus of cells. In this report we describe nucleic acid delivery to cells using a series of novel cationic amphiphiles containing cholic acid moieties linked via alkylamino side chains. We term these agents `molecular umbrellas' since the cationic alkylamino chains provide a `handle' for binding of nucleic acids, while the cholic acid moieties are likely to interact with the lipid bilayer allowing the highly charged nucleic acid backbone to traverse across the cell membrane. Optimal gene and oligonucleotide delivery to cells was afforded by a derivative (amphiphile 5) containing four cholic acid moieties. With this amphiphile used as a constituent in cationic liposomes, a 4-5 log increase in reporter gene delivery was measured. This amphiphile used alone provided a 250-fold enhancement of oligonucleotide association with cells as observed by flow cytometry. A substantial fraction of cells exposed to complexes of amphiphile 5 and fluorescent oligonucleotide showed nuclear accumulation of the fluorophore. Enhanced pharmacological effectiveness of antisense oligonucleotides complexed with amphiphile 5 was observed using an antisense splicing correction assay that activates a Luciferase reporter. Intracellular delivery, nuclear localization and pharmacological effectiveness of oligonucleo-tides using amphiphile 5 were similar to those afforded by commercial cytofectins. However, in contrast to most commercial cytofectins, the umbrella amphiphile showed substantial delivery activity even in the presence of high concentrations of serum.
INTRODUCTION
Currently there is substantial interest in the use of antisense oligonucleotides, ribozymes, high molecular weight DNA and other types of nucleic acids to modify gene expression for therapeutic purposes (1-5). Antisense oligonucleotides are usually administered in vitro or in vivo either in free form or complexed with various cationic lipid-based formulations (6-9). Gene therapy has primarily relied on viral vectors (1), but there is increasing interest in use of non-viral gene delivery systems based on lipids, polymers or combinations of both (10-14).
The ability of a nucleic acid to achieve a desired function within cells is influenced by several critical factors in addition to its own innate biological activity. These include the heterogeneity of uptake within the cell population; the degree to which the nucleic acid reaches appropriate cellular compartments (cytosol and nucleus); and, when a carrier is used, the extent to which the nucleic acid can dissociate from the carrier and attain its biologically active form in the target cell. The commercially available `cytofectins' (cationic liposomes) currently widely used as carriers for genes and oligonucleotides typically form large, complex aggregates with nucleic acids (15). Despite some recent progress (16), there is still relatively little known about the ability of such complexes to enter key cellular compartments or to release biologically active nucleic acids. In addition, most cationic lipid complexes do not function well in the presence of plasma proteins, although there are some exceptions. While some studies with cationic lipids have provided evidence for gene expression in vivo (17,18), there are still many problems with access of the complex to desired tissue sites and with attaining and sustaining appropriate levels of gene expression in the target issue.
In vivo studies with therapeutic antisense oligonucleotides have primarily utilized administration of the oligonucleotide alone, without complexation to carriers (2,19,20). However, a substantial amount of in vitro data suggests that carrier systems, including cytofectins, can substantially increase the delivery of antisense compounds to cells, as well as the consequent pharmacological effects (6,7). Thus it is possible that suitable carrier systems could increase the in vivo efficacy of antisense oligonucleotides as well.
In the studies reported here, we have examined novel cationic amphiphiles as delivery agents for nucleic acids, particularly for antisense oligonucleotides. These amphiphiles utilize one or more cholic acid moieties tethered to alkylamino side chains that are derivatives of spermine and/or spermidine (21). The amphiphiles were designed to act as `molecular umbrellas' where the hydrophobic portion of the cholic acid moiety allows membrane interaction and penetration and the hydrophilic face of the cholic acid moiety, along with the alkylamino side chain, permits interaction with the negatively charged nucleic acid. In studies with cultured cells, we have tested several forms of `molecular umbrella' amphiphiles for their ability to enhance DNA transfection and for their ability to deliver antisense oligonucleotides. These amphiphiles proved to be only moderately effective as DNA transfection agents and were clearly less efficient than the commercially available cytofectins Lipofectin® and Lipofectamine®. However, the umbrella amphiphiles were quite effective in promoting delivery of antisense oligonucleotides to the nucleus, as observed by fluorescence microscopy. Further, we found that the umbrella surfactants could enhance the pharmacological effectiveness of antisense oligonucleotides to a significant extent. However, in contrast to many lipid cytofectins, the umbrella amphiphile/oligonucleotide complexes retained substantial activity in the presence of serum. This suggests that these compounds may have some value for in vivo delivery of antisense oligonucleotides.
MATERIALS AND METHODS
Cholic acid-polyamine conjugates (molecular umbrellas)
Specific procedures that were used to prepared the cholic acid-polyamine conjugates 1, 3, 4 and 5 were essentially those previously described (21). Compound 2, which was prepared by similar methods, exhibited the expected 1H NMR spectrum.
Cell culture
Handling and maintenance of multi-drug resistant NIH 3T3 cells has been previously described (6). Human endothelial cells (ECV304; ATCC) were routinely grown in Medium 199 (Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS; Hyclone), 2 mM L-glutamine and penicillin (50 U/ml)/streptomycin (50 µg/ml) in 5% CO2/95% air at 37°C. ECV304 cultures were grown to confluence in 185 cm2 Nunc tissue culture flasks and typically split at a ratio of 1:6. HeLa cells containing a modified Luciferase gene interrupted by a [beta]-globin intron (HeLa/705) were maintained as described (22).
Nucleic acids
A 2[prime]-O-methyl-phosphorothioate oligonucleotide complementary to an aberrant splice site in the [beta]-globin intron (5[prime]-CCUCUUACCUCAGUUACA-3[prime]) was prepared as decribed (22). FITC or cyanine-5 fluorophore derivatives were coupled to the 5[prime]-end of oligonucleotides as phosphoramidites (Glen Research) during the final step in automated synthesis. Luciferase plasmid pGL3 (Promega), under the control of the SV40 promoter, was maintained as a 1 mg/ml stock in phosphate-buffered saline (PBS) and stored at -4°C.
Preparation of nucleic acid complexes
Amphiphile/liposome/DNA complexes. Amphiphiles 1-5 dissolved in methanol (1 mg/ml), were combined with an equal volume of dioleoylphosphatidyl ethanolamine (DOPE) (1 mg/ml) (Avanti Polar Lipids) in a solution of chloroform. After evaporation of the solvent, the mixture was suspended in sterile water (1 mg/ml). Each lipid-amphiphile solution (1L-5L) was vortexed for 5 min, bath sonicated (30 min) and stored at 4°C under N2. Complexes of plasmid (1 µg/ml) or oligonucleotide (10 nM-1 µM) were formed by combining the DNA stock dissolved in 100 µl of Opti-Mem (Gibco BRL) with an equal volume of the (1L-5L) amphiphile/liposomes (10 µg/ml). The complexes were incubated for 30 min at room temperature and then added directly to cells in 1 ml cell culture medium.
Amphiphile/oligonucleotide complexes. Stocks of oligonucleo-tide (50-100 µM) were diluted to the appropriate concentration for complexation (10 nM-10 µM) with sterile water. The oligo-nucleotide was then stirred within 0.5 ml glass V-vials (Wheaton) in 40-100 µl sterile water. Amphiphile at 5-10 µg/ml in 1% methanol/water was then added rapidly via a 0.5 cc U-100 Insulin Syringe (Becton Dickinson) and stirred for 15 min at room temperature. The complexes were incubated for 30 min at room temperature and then added directly to cells in 1 ml cell culture medium.
Cytofectin complexes. Complexes of oligonucleotides with Lipofectin® or Lipofectamine® were prepared as previously described (6), essentially according to the manufacturer's directions.
Plasmid transfection assay
For transfections, ECV304 or MDR3T3 cells were seeded in 6-well dishes ~24 h prior to transfection and used at 60-80% confluence. Cells were transfected for 4 h in serum-free medium with ~1 µg of pGL3 plasmid (Promega) and either 20 µg/ml of Lipofectamine® (Gibco BRL) or amphiphile (5-10 µg/ml); cells were then placed in serum-containing growth medium and allowed to recover for 24 h. Transfected cells were lysed in a buffer containing 0.25 M Tris (pH 7.8), 1% Triton X-100 and 1 mM dithiothreitol (DTT). Lysate protein concentrations were determined using the bicinchonic acid assay (Pierce Chemical Co.). Cell lysates were stored at -70°C until used. Luciferase activity was measured in cell lysates using reagents from Analytical Luminesence Laboratory (San Diego, CA). Briefly, using the automatic injector of the Monolight® 2010 Luminometer (Analytical Luminescence), 100 µl (~200 µg) of cell extract was mixed sequentially with 100 µl of Luciferase buffer (3 mM ATP, 15 mM MgSO4, 30 mM Tricine, 10 mM DTT, pH 7.8) and 100 µl of luciferase substrate (1 mM D-Luciferin); the light output was measured for 10 s. Determinations of protein concentration were used to convert raw activity values to specific activities for all samples. All transfections were performed in triplicate.
Toxicity and growth assays
Cytotoxicity studies were performed by plating MDR3T3 cells into 24-well plates (Nunc) at 8 × 104 cells/well. Cells were incubated with complexes of nucleic acid and umbrella amphiphile or with nucleic acids and commercial cytofectins, or with control medium for 24 h, rinsed twice with PBS, followed by the addition of 2 ml OPTI-MEM (Gibco BRL) and incubation for a further 24 h. The surviving fraction was determined by the MTT dye assay, measuring absorbance at 540 nm with an automated microplate reader (BioTech EL 340) as described (23).
Flow cytometry
The flow cytometry technique for oligonucleotide-cell association has been previously reported (6,24). Briefly, 500 000 cells/well were plated in a Nunclon 6-well plate and allowed to attach for 18-24 h. Amphiphile-oligonucleotide complexes formed as above were made with FITC-phosphorothioate oligonucleotide (100 nM-1 µM). Complexes were added to cells in a 40-100 µl volume and incubated for 18 h at 37°C. Cells were then rinsed with PBS, trypsinized, pelleted, and resuspended in PBS (100 000 cells/ml) prior to flow cytometry.
Confocal microscopy
For confocal microscopy, cells were grown on glass coverslips (Corning) coated with fibronectin (10 µg/ml) and treated with complexes of cyanine-derivatized oligonucleotide, essentially as previously described (6,24).
Antisense assays
Splicing correction assay ([beta]-globin system). In the thalassemic human [beta]-globin gene, a T->G mutation at position 705 of intron 2 (IVS2-705) improves the match of the surrounding sequence to the consensus donor (5[prime]) splice site (ACTGAT/GTAAGA to ACTGAG/GTAAGA; slash indicates the splice site). In the transcribed IVS2-705 pre-mRNA, the presence of the created 5[prime] splice site activates an acceptor (3[prime]) splice site 126 nt upstream, resulting in incorrectly spliced [beta]-globin mRNA containing a fragment of the intron. In a novel method developed by Sierakowska et al. (25) and extended by Kang et al. 22), HeLa-based cell lines were created that have a human [beta]-globin intron 2 with the 705 mutation inserted into a Luciferase reporter gene. In the absence of treatment, the presence of the intron prevents correct splicing and thus blocks Luciferase expression. When the cells are effectively treated with a 2[prime]-O-methyl-phosphorothioate oligonucleotide targeted to the aberrant splice site, the oligonucleotides restore correct splicing and thus permit expression of the Luciferase protein. This approach provides a basis for an excellent assay of antisense activity and delivery, since only the presence of active oligonucleotide within the nuclei of living cells will permit correct splicing. This assay was used, essentially as described (22), to evaluate the ability of the umbrella amphiphiles to deliver oligonucleotides. Typically a 100 µl aliquot of oligonucleotide at a given concentration in Opti-MEM is mixed with 100 µl of Opti-MEM containing various concentrations of delivery agent (amphiphile 5 or commercial cytofectin). After being briefly mixed, the preparation is left undisturbed at room temperature for ~15 min, followed by dilution to 1 ml with Opti-MEM before being layered on the cells. The cells are incubated for 6 h and subsequently the medium is replaced with 10% FBS/DMEM. An additional 18 h later, the cells are rinsed with PBS and lysed in 100 µl of lysis buffer (200 mM Tris-HCl, pH 7.8, 2 mM EDTA, 0.05% Triton X-100) on ice for 15 min. Following centrifugation (13 000 r.p.m., 2 min), 5 µl of supernatant cell extract is mixed with 100 µl of Luciferase assay buffer (3 mM ATP, 15 mM MgSO4, 30 mM Tricine, 10 mM DTT, pH 7.8) and 100 µl of Luciferase substrate (1 mM D-luciferin). The light emission is quantified for 10 s using a Monolight® 2010 luminometer (Analytical Luminescence Laboratory). Luciferase activity is expressed as relative light units (RLU) per well. Light emission is normalized to the protein concentration of each sample, determined according to the bicinchonic acid assay (Pierce Chemical Co.) for protein concentration.
Oligonucleotide/amphiphile 5 complexes in serum. Effects of serum on the oligonucleotide uptake mediated by umbrella amphiphile was performed by plating cells into a 24-well tray (Nunc) at 4 × 104 cells/well. Cells were incubated for 12 h at 37°C. A 100 µl aliquot of an oligonucleotide at a given concentration in Opti-MEM was mixed with 100 µl of Opti-MEM containing 6.5 µg (3.0 µM) of amphiphile 5. After being briefly mixed, the preparation was left at room temperature for ~15 min, followed by dilution to 1 ml with a given percentage of serum (FBS) in Opti-MEM. A 200 µl aliquot of the complex solution was plated in a Nunclon 24-well tray. The cells were incubated for 6 h and subsequently the medium was replaced with the same percentage of serum containing DMEM. An additional 18 h later, the cells were rinsed with PBS and lysed in 40 µl of lysis buffer on ice for 15 min. Following centrifugation (13 000 r.p.m., 2 min), 10 µl of supernatant cell extract was mixed with 100 µl of luciferase assay buffer to measure the luciferase activity as described in the antisense splicing correction assay.
RESULTS
Amphiphile structure
The structures of the cholic acid-based amphiphiles used in this study are shown in Figure 1 (21). Molecular umbrellas 2-5 contain two or more cholic acid moieties covalently coupled to spermine and/or spermidine moieties to provide a cationic `tail' or `handle' to which anionic oligonucleotides can bind. The design principle involves the linking of two or more rigid amphiphilic `walls' or `ribs' to the cationic `handle' to form an `umbrella'. The flexible nature of the linkages allows the cholate residues to be in a compact conformation (`umbrella`) in a hydrophobic membrane environment, thus possibly shielding the bound oligonucleotide, and in an extended conformation (`inverted umbrella`) in a hydrophilic environment. For purposes of comparison, a `single walled' analog (1) has been included.
Figure 1. Structure of the umbrella amphiphile series. The structures of the five amphiphiles used in this study are shown. The cholic acid ring system is indicated as a gray and black rectangle with the black portion representing the non-polar portion of this so-called `facial amphiphile'. The nitrogens of the spermidine and spermine residues provide positive charge at physiological pH that permits binding of anionic oligonucleotides.
Cytotoxicity studies
Using the MTT assay (23) a systematic comparison of the cytotoxicity of the various umbrella amphiphiles was performed. Figure 2 shows a representative acute toxicity versus concentration plot for amphiphile 5. As shown in Table 1, there was a range of approximate LD10 and LD50 values for compounds 1-5. In general, complexation with oligonucleotides tended to modestly reduce the toxicity of the amphiphiles; however, a variety of effects were observed. In further studies, the various delivery agents were used at equitoxic concentrations.
Figure 2. Toxicity profile of amphiphile 5 alone or amphiphile 5:oligo complex. Data are expressed as percent cell number compared to untreated 3T3 cells based on the MTT assay (n = 3 experiments per concentration).
Table 1. Toxicity of umbrella amphiphiles
| Amphiphile | -Oligo | +Oligo | ||
| LD10 (µg/ml) | LD50 (µg/ml) | LD10 (µg/ml) | LD50 (µg/ml) | |
| 1 | nt | nt | nt | nt |
| 2 | 2.4 | 15.0 | 1.7 | 4.0 |
| 3 | 2.0 | 8.0 | 7.0 | 20.0 |
| 4 | 1.7 | 16.0 | 3.0 | 15.0 |
| 5 | 0.3 | 4.0 | 2.7 | 20.0 |
Gene expression
The ability of the umbrella compounds 1-5 to deliver plasmid DNA was evaluated using luciferase expression in fibroblastic and endothelial cell lines, as shown in Figure 3. When complexed with DNA and formulated with the phospholipid DOPE, several amphiphiles were quite active, giving luminescence readings 4-5 log units higher than DNA alone. Interestingly, a significant amount of expression was also obtained in the absence of added DOPE. However, the best transfections attained with the umbrella/DOPE formulations were still many-fold less than that attained with Lipofectin® or Lipofectamine®. Endothelial cells were consistently less efficiently transfected than fibroblasts either with the Lipofectamine® positive control or with the umbrella compounds and showed a greater dependence on the presence of DOPE.
Figure 3. Reporter gene expression. Transfection activity of the amphiphiles into either fibroblastic (A) or endothelial (B) cells is shown. Relative luciferase gene expression levels was quantitated by the number of luciferase units (RLU)/µg of cellular protein after incubation with DNA complexed with amphiphiles 1-5 either with (1L-5L) or without (1-5) 1:1 wt% DOPE. Cells treated with Lipofectin® or Lipofectamine®:DNA were used as positive controls. All values of means and standard errors reflect three to five independent measurements.
Association of oligonucleotide with cells
The umbrella compounds' ability to enhance oligonucleotide uptake by cells was estimated using flow cytometry, as shown in Figure 4 (24). Relative fluorescence (RFU) from cells treated with amphiphile or amphiphile/lipid complexed with FITC-derivatized phosphorothioate oligonucleotide was measured. Amphiphiles 4 and 5 stimulated oligonucleotide association with cells by as much as 100- to 200-fold relative to oligonucleotide alone. In contrast to gene delivery, formulation with DOPE provided no significant advantage in promoting oligonucleotide-cell association.
Figure 4. Oligonucleotide uptake. Total cell association of oligonucleotide was quantitated by flow cytometry. MDR3T3 cells treated with amphiphile (1-5) or amphiphile/DOPE (1L-5L) complexed with FITC-phosphorothioate oligonucleotide were analyzed for total cell-associated fluorescence. The ordinate expresses the mean values of fluorescence in arbitrary units relative to free oligonucleotide (n [ge] 4 measurements per compound or formulation). Light scatter parameters were used to gate viable cells and exclude dead or damaged cells.
Intracellular distribution
Figure 5 depicts the intracellular distribution of oligonucleotide in live cells visualized by confocal fluorescence microscopy. Red color is derived from a cyanine fluor directly conjugated to the oligonucleotide. The cell is highlighted in green via immunostaining with an FITC anti-MDR antibody. Free oligonucleotide (Fig. 5A) shows only a slight degree of cell association and no evidence for nuclear accumulation. The positive control, a Lipofectin®/oligo complex (Fig. 5B) demonstrates a high level of cell-associated fluor as well as many cells showing nuclear localization. Cells treated with oligonucleotide/amphiphile 5 complexes (Fig. 5C) are seen to have a high degree of cell-associated material and a significant fraction of the cells show nuclear fluorescence. Quantitation of total cell and nuclear fluorescence from fluorescent oligonucleotides complexed with the amphiphile series is summarized in Table 2. Amphiphile 5 produced a consistently higher fraction of cells with oligonucleotide in the nucleus (27%) relative to the other amphiphiles, yet lower than the case for Lipofectin® (59%).
Figure 5. Confocal microscope images of live cells treated with fluorescent phosphorothioate oligonucleotide. MDR3T3 cells were cultured overnight in the presence of 1 µM phosphorothioate oligonucleotide derivatized with a cyanine fluorophore. The fluorescent oligonucleotide was presented either in free form (A), complexed with Lipofectin® (B) or complexed with amphiphile 5 (C). Cells were rinsed and the cell surface was immunostained with anti-P-glycoprotein antibody and an FITC-conjugated secondary antibody (Materials and Methods). The cells were then allowed to recover for 1-2 h in complete medium lacking dye indicator, placed on the microscope stage and imaged within several minutes. The green color shows the cell membrane while the red color indicates the cellular location of the oligonucleotide. Yellow color indicates areas where the cyanine and FITC fluorophores are in close proximity and probably represents binding of the oligonucleotide at the cell surface or in endosomes. Similar results were observed with HeLa cells, but 3T3 cells spread extensively and are better subjects for obtaining fluoresence images.
Table 2. Quantitation of oligonucleotide uptake and nuclear localization by confocal microscopy
| Condition | Nuclear (%) | Nuclear fluorescence | Total cell fluorescence |
| Free oligo, 1 µM | 1 (n = 95) | 0.4 | 3.4 0.26 |
| Lipofectin + oligo | 59 (n = 121) | 5.6 1.6 | 37 7.84 |
| 1 + 1 µM oligo | 1 (n = 107) | 1.6 0.44 | 13 1.1 |
| 2 + 1 µM oligo | 5 (n = 57) | 2.6 0.32 | 15 14.7 |
| 3 + 1 µM oligo | 4 (n = 76) | 2.0 0.92 | 11 1.83 |
| 4 + 1 µM oligo | 13 (n = 50) | 6.9 1.2 | 39 14.7 |
| 5 + 1 µM oligo | 27 (n = 135) | 6.5 2.6 | 49 11.6 |
Oligonucleotide activity assays
The ability of the amphiphiles to deliver oligonucleotides in pharmacologically active form was examined. A powerful test for the biological activity of a 2[prime]-O-methyl-oligonucleotide involves its ability to correct improper splicing of an intron derived from the [beta]-globin gene (25). Using a recently developed reporter assay, this effect is manifested as an increase in Luciferase activity (22). Oligonucleotide complexed with Lipofectin® or Lipofectamine® was quite effective, with a substantial increase in Luciferase activity. Oligonucleotide complexed with amphiphile 5 was also effective, attaining approximately two-thirds the effect seen with Lipofectamine® (Fig. 6A). The Luciferase activity showed progressive dose-response relationships both with respect to oligonucleotide concentration (Fig. 6B) and with respect to the concentration of the amphiphile (Fig. 6C). The response at the highest level of amphiphile 5 was attenuated, probably due to cytotoxicity. The biologically active form of the OD-705/amphiphile 5 complex was shown to be particulate in nature since it could be sedimented by high speed centrifugation (data not shown).
Figure 6. Oligonucleotide activity (correction of splicing). HeLa/Luc 705 cells were treated with various concentrations of a 2[prime]-O-Me phosphorothioate oligonucleotide complementary to the [beta]-globin splice site junction (ON-705). Various complexes were used as delivery formulations. Luciferase activity was measured as described in Materials and Methods. (A and B) Activation of the Luciferase reporter using umbrella 5 to deliver antisense oligonucleotide. (A) Luciferase expression at various concentrations of umbrella amphiphile 5 was compared with Lipofectamine® (8 µg/ml) and Lipofectin® (10 µg/ml); (B) Luciferase expression at constant concentration of the umbrella amphiphile (3.0 µM) with varying concentrations of oligonucleotide; (C) Luciferase expression at constant molar ratio of amphiphile to oligonucleotide. The activity of Luciferase was normalized on total cellular protein and is presented in relative luminescence units (RLU)/µg of protein. Vertical bars indicate means and standard errors (n = 3).
In contrast to most commercial cytofectins, amphiphile 5 was also effective in the presence of substantial amounts of serum protein, as seen in Figure 7. Thus, at 10% serum the effects of Lipofectamine® were diminished almost to baseline, while a reduced but significant effect was still seen with amphiphile 5. The ability of the umbrella molecule to deliver oligonucleotide and thus activate Luciferase expression was detectable even in 50% serum.
Figure 7. Effect of serum on the effectiveness of oligonucleotide/amphiphile 5 complexes. Complexes of ON-75 and amphiphile 5 were formed and added to HeLa cells stably transfected with plasmid pLuc/705 in 24-well trays as described in Materials and Methods. Cells treated with Lipofectamine® (8 µg/ml)/oligonucleotide complex were used for comparison. Additional control samples underwent the same oligonucleotide treatment, but in the absence of any delivery agent. The abcissa indicates the percentage of fetal calf serum present during the incubation with oligonucleotides. Vertical bars indicate means and standard errors (n = 3).
DISCUSSION
Umbrella amphiphiles were designed to complex with the negatively charged backbone of nucleic acids and to provide a shield between the highly polar nucleic acid and the non-polar interior of the lipid bilayer membrane. Initially, it was thought that the umbrella compounds might form bi-molecular complexes with oligonucleotides. However, current investigations have indicated that the first generation umbrella amphiphiles form large complexes with oligonucleotides that can be sedimented by high speed centrifugation; the precise physical characterisation of these complexes is now being pursued in other studies.
The first generation umbrella compounds reported here have displayed significant activity both for transfection of high molecular weight DNA and for delivery of oligonucleotides. In the former case, complexation with the umbrella surfactants alone provided only modest increases in DNA transfection efficiency compared to a `naked' plasmid. The umbrella amphiphiles could be mixed with the non-bilayer forming phospholipid DOPE and in this form reasonably high transfection efficiencies were attained; however, this approach offers little advantage over cationic lipid-based `cytofectins' that are already in use. Presumably the umbrella compounds, acting alone, cannot form a cationic lipid-DNA complex that is efficient in destabilizing membranes sufficiently to permit the delivery of functional high molecular weight DNA complexes.
In contrast, the umbrella amphiphiles seem to work quite well when applied to the delivery of relatively low molecular weight oligonucleotides. Thus complexation of oligonucleotides with the tetra-walled umbrella compound 5 resulted in complexes that could convey oligonucleotides into the nucleus of viable cells. Differences in the ability to deliver small oligonucleotides versus high molecular weight plasmids may not be surprising, since the physical characteristics of the two types of complexes are likely to be different. The evidence for nuclear delivery includes (i) the visualization of fluorescent oligonucleotides in the nucleus by confocal microscopy; and (ii) an increase in the pharmacological effectiveness of an antisense oligonucleotide that modifies splicing of a [beta]-globin intron inserted into a Luciferase reporter gene. These observations, particularly the latter, provide convincing proof that umbrella compounds can deliver oligonucleotides to key target compartments in living cells. The splice site correction assay has some important advantages for evaluating the delivery and effectiveness of oligonucleotides. First, it is very sensitive since it provides a positive readout rather than relying on a difference measurement (the inhibition of message levels) as is the case for standard antisense assays. Second, splicing can only occur within the nucleus of a living cell; thus the effects measured on splice site selection are clearly associated with the oligonucleotide functioning in viable cells and cannot be ascribed to toxicity.
In most experiments the delivery efficiency of the oligonucleotide/umbrella amphiphile complexes in serum-free medium was somewhat less than that provided by commercial cytofectins (cationic liposomes) such as Lipofectin®. However, the materials studied here were first generation compounds; it seems likely that modification of the size/charge of the cationic tail or modification of the cholic acid moieties of the umbrellas might confer more desirable delivery properties on this class of molecules. In addition, our understanding of the most appropriate way to formulate oligonucleotide/umbrella surfactant complexes is very limited; we have not yet systematically optimized these complexes for size, charge or delivery efficiency. Despite this, even the first generation umbrella amphiphiles seem to offer an important advantage in that they retain the ability to deliver oligonucleotides in the presence of substantial amounts of serum proteins, whereas many commerical cytofectins show significantly reduced efficacy in the presence of low concentrations of serum. There have been previous reports of effective oligonucleotide delivery in the presence of serum. For example, Lewis et al. (26) used a novel cationic lipid formulated with the fusogenic lipid DOPE to attain significant antisense effects in 10% serum. In the present case, however, no accesssory lipid was used, but nonetheless significant antisense effects were obtained at up to 50% serum concentration.
The mechanism of action of amphiphile 5 and other umbrella amphiphiles is unclear at this point. Unlike most of the cationic surfactants used in commercial cytofectins, the umbrella compounds do not require a neutral lipid such as DOPE to allow oligonucleotide delivery to cells. Centrifugation experiments indicate that the biologically active form of the oligonucleotide/amphiphile 5 complex is a sedimentable particle and not a low molecular weight moiety. These particles bind to cell membranes, as shown by flow cytometry, and may be taken into endosomes, as suggested by the appearance of material in vesicular structures seen by confocal microscopy. Whether the actual delivery of oligonucleotide to the cytoplasm and nucleus occurs at the plasma membrane or in endosomes and whether it involves particles or soluble complexes of oligonucleotide and umbrella amphiphiles remains to be determined.
An interesting possibility for future investigation will be to link oligonucleotides directly to the cholic acid umbrella framework, rather than using a cationic `handle' to promote ionic interactions. If a bio-reversible covalent linkage were employed, this might allow development of non-cationic delivery agents that could carry the oligonucleotide through the cell membrane as a mono-molecular species and then release it within the cell in biologically active form. This might permit efficient in vivo delivery without some of the problems associated with use of cationic lipids.
ACKNOWLEDGEMENTS
This work was supported by NIH grants CA47044 to R.L.J. and GM51814 to S.L.R. and by an NIH fellowship CA72208 to R.K.D.
REFERENCES
*To whom correspondence should be addressed. Tel: +1 919 966 4383; Fax: +1 919 966 5640; Email: arjay{at}med.unc.edu Present address: R. K. DeLong, Megabios Corp., Burlingame, CA, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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