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Langmuir 1996, 12, 1879-1883
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Divalent Cation Mediated Binding of Oligonucleotides to Langmuir Monolayers of Charged Lipids David G. Rhodes*,† and Jie Liu‡ Department of Pharmaceutical Sciences, University of Connecticut School of Pharmacy, Storrs, Connecticut 06269-2092, and Pharmaceutics Division, College of Pharmacy, University of Texas at Austin, Austin, Texas 78717-1074 Received August 7, 1995. In Final Form: January 19, 1996X Antisense oligonucleotides inhibit the expression of complementary target sequences with extraordinary selectivity and thus have great potential for development of novel therapeutics in many critical areas, including cancer and infectious disease. However, the mechanism by which oligonucleotides pass through the cell membrane and reach the site of action is not well understood. In this study, phospholipid monolayers were used as models of cell membranes. Using a Langmuir film balance, the effects of ions, oligonucleotides, and other polyions (polylysine) on the isotherms of phospholipid monolayers were determined. Differences were observed in the coexistence region between liquid expanded and condensed phases. These results indicate that calcium or other divalent cations may mediate interaction of oligonucleotides with DPPG or other anionic membrane components.
Introduction Antisense oligonucleotides are short nucleic acid polymer fragments, usually single-stranded DNA or a related analog 15-30 bases in length, designed to complement a target “sense” sequence.1 The selective binding to a complementary mRNA target sequence can inhibit expression of the target sequence by blocking translation or by inducing RNAse-H activity. Thus, one should be able to specifically design “drugs” directed to a designated active site and have a very high probability of inhibiting activity. There is tremendous potential for application of this approach in many therapeutic areas, such as viral infection, other infectious diseases, and cancer. However, because oligonucleotides are bulky polyanions, the cell membrane might be expected to act as an effective barrier against entry to the cytoplasm or nucleus. In many cases, oligonucleotides may enter cells by a receptor-mediated endocytotic pathway.2 Work by Wu-Pong et al.3-5 has suggested that, in at least some systems, there are other pathways in effect which are trypsin insensitive and thus not receptor-mediated. Recent results indicate that the trypsin-insensitive pathway is sensitive to divalent cations and, in particular, that certain cations (e.g. Ca2+) are effective promotors, while others (e.g. Mg2+) are ineffective. The basic chemistry of this process is not well understood. There is some published data which suggest that DNA or DNA fragments associate with lipid vesicles6 and with cell membranes. For example, Budker et al.7 found that, in the presence of Mg2+, DNA coprecipitated with mitochondrial membranes or liposomes. The mechanism of this association is not clear, particularly since cell * Corresponding author. Phone: 860-486-5413. Fax: 860-4861553. Internet:
[email protected]. † University of Connecticut School of Pharmacy. ‡ University of Texas at Austin. X Abstract published in Advance ACS Abstracts, March 15, 1996. (1) Knorr, D. G.; Vlassov, V. V.; Zarytova, V. F.; Lebedev, A. V.; Federova, O. S. Design and Targeted Reactions of Oligonucleotide Derivatives; CRC Press: Boca Raton, FL, 1994. (2) Loke, S.; Stein, C.; Zhang, X.; Mori, K.; Nakanishi, M.; Subasinghe, C.; Cohen, J.; Neckers, L. Proc. Natl. Acad. Sci. USA 1989, 86, 34743478. (3) Wu-Pong, S.; Weiss, T. L.; Hunt, C. A. Pharm. Res. 1992, 9, 1010-1017. (4) Wu-Pong, S.; Weiss, T.; Hunt, C. Cell Mol. Biol. 1994, 40, 843-850. (5) Wu-Pong, S. Pharm. Res. 1994, 11, S77. (6) Budker, V.; Kazatchkov, Y.; Naumova, L. F.E.B.S. Lett. 1978, 95, 143-146. (7) Budker, V. G.; Kazatchkov, Y. A.; Naumova, L. P. F.E.B.S. Lett. 1978, 95, 143-146.
membranes typically carry a net negative charge due to constituents like phosphatidylglycerol (PG), phosphatidylserine (PS), and fatty acids. We propose here that calcium or other divalent cations mediate the association between the anionic oligonucleotide and anions in the lipid bilayer. Moreover, it is suggested that the presence of the polyanion associated with the lipid bilayer induces local perturbation to the bilayer structure that permits the oligonucleotide molecule to cross the membrane. Because of the complex composition of cell membranes, bilayers of synthetic lipids have long been used as membrane analogs in a wide variety of experiments. There are risks in this simplification (see Discussion), but the ability to isolate specific effects is sufficient incentive to continue to utilize model systems. In the work described here, a Langmuir monolayer of anionic phospholipids has been used to provide a system which is, in some ways, even simpler than a bilayer. In this system, an oligonucleotide may bind to the monolayer at the air/water interface but cannot penetrate to the other (air) side. Thus, the system is “trapped” in the state we wish to investigate. The drawback to such investigations is that the precise phase behavior of the monolayer is distinct from that of the assembled bilayer, but it is possible to determine, as in the work reported here, whether association occurs and whether this association affects the lipid phase behavior. The results presented here were obtained using Langmuir compression isotherms of the dipalmitoyl-PG (DPPG) monolayers in the presence of divalent cations, monovalent cations, polycations, or oligonucleotides in the subphase. Compression isotherms are direct representations of the pressure/area phase behavior of the monolayer and can be interpreted in terms of the specific intermolecular interactions between molecules at the air/water interface. Although data from compression isotherms are often interpreted qualitatively, one may infer the presence of phase transitions and the compressibility of the monolayer in a particular phase and may evaluate, on the basis of these criteria, whether monolayer systems are equivalent. Compression isotherms are affected by anything that affects the intermolecular interaction, including the structure of the surfactant, the electrostatic properties of the surfactant, the interaction of the surfactant with the subphase, and any adsorption of subphase constituents to the monolayer. This approach thus provides a sensitive probe of the effect of the composition of aqueous phases on the phase behavior of lipid assemblies.
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Materials and Methods Materials. CaCl2, NaCl, MgCl2, poly-L-lysine‚HBr (MW 4000-15 000), and buffer compounds were obtained from Sigma. Phosphorothioate and phosphodiester oligodeoxynucleotides were generously donated by ISIS Pharmaceuticals Inc. (Carlsbad, CA). Subphase additives, CaCl2, NaCl, poly-L-lysine‚HBr, phosphodiester oligodeoxynucleotides, and phosphorothioate oligodeoxynucleotides were used without further purification and were added to the subphase buffer prior to film deposition. DPPG was purchased as a lyophilized powder from Avanti Polar Lipids and used without further purification. The lipid was dissolved in chloroform to the desired concentration. Monolayers. The Teflon trough (323.0 × 150.0 mm2) of the Langmuir film balance (KSV “Mini-trough”) was filled with buffer (either Tris or Hepes, titrated to pH 7.4, at concentrations specified in the text) to approximately 1 mm below the trough edge. The surface was then cleaned by repeated “blank” runs, aspirating the surface if any increase (g1 mN/m) in surface pressure was observed. Monolayers were formed by depositing 17 µL of 1 mg/mL DPPG in chloroform onto the buffered subphase using a Hamilton microsyringe, so that the molecular area was ∼1.75 nm2. The solvent was allowed to evaporate for 20 min prior to compression. Isotherms. The surface pressure/molecular area (π/A) isotherms were obtained by symmetric compression with two hydrophilic Delrin barriers. Compression isotherms were performed using a constant gradient of surface pressure of dπ/dt ) 5.0 mN/min with a maximum linear barrier velocity of dx/dt ) 5 mm/min. This means that at large areas and high compressibility, dA/dt was 0.055 nm2/(molecule min but became significantly smaller as the film became less compressible. Surface pressure was monitored with an etched platinum Wilhelmy plate. Data were collected, archived, and presented as surface pressure as a function of mean molecular area (MMA). All isotherms were run at least three times under the same conditions with freshly prepared monolayers and subphases. Replicate (3×) compression isotherms that did not superimpose (indistinguishable from a single isotherm at the line width illustrated except at the collapse point) were not acceptable and usually were taken to represent an effect of contamination. All measurements were made at 20.0 ( 0.1 °C and pH ) 7.40 ( 0.01.
Results Because DPPG is charged, one expects the repulsive interactions between headgroups to be a significant factor in the compression isotherm. Accordingly, one expects that the ionic composition of the subphase would have a significant effect on the monolayer phase behavior, and this may explain the apparent diversity of DPPG compression isotherms in the journal literature.8-10 Increasing the ionic strength of the subphase would decrease this interaction energy, and more specific condensation effects might be expected for divalent or multivalent cations. The compression isotherm of DPPG on 10 mM Tris buffer (trace A in Figure 1) indicates that the DPPG monolayer has moderate compressibility over the range 1.20-0.50 nm2, with a weak phase transition at π ≈ 22 mN/m, and becomes fairly incompressible at smaller areas, collapsing at ∼70 mN/m. DPPC under identical conditions shows a well defined liquid expanded (LE)/liquid condensed (LC) transition at approximately 5 mN/m. Addition of 10 mM NaCl to the buffered subphase apparently decreases electrostatic repulsion between DPPG headgroups, as all points on the compression isotherm obtained in the presence of added NaCl (regardless of molecular area) are at lower surface pressure (trace B, Figure 1) than is found in the presence of buffer alone. There is a well (8) El Mashak, E. M.; Tocanne, J. F. Biochim. Biophys. Acta 1980, 596, 165-179. (9) El Mashak, E. M.; Tocanne, J. F. Eur. J. Biochem. 1980, 105, 593-601. (10) Mingotaud, A. F.; Mingotaud, C.; Patterson, L. K. Handbook of Monolayers; Academic Press: San Diego, CA, 1993.
Figure 1. Small cations affect the compression isotherms. Compression isotherms were obtained with (A) Tris buffer (pH 7.4, 10 mM) alone, (B) buffer with 10 mM NaCl, (C) buffer with 1 µM CaCl2, or (D) 10 µM CaCl2. Note the isotherms have been truncated at π ≈ 40 mN/m for the purposes of clarity in the lower π region of the curve. Most isotherms continued to higher π with the same compressibility and then indicated collapse at π ≈ 70 mN/m.
defined transition at approximately 14 mN/m when the buffered subphase contains 10 mM added NaCl. The effect of divalent cations on the phase behavior of DPPG was found to be specific and quite distinct from that of the monovalent ions tested. In the presence of 1 µM CaCl2 (trace C, Figure 1), the DPPG transition is very well defined and occurs at π ≈ 10 mN/m. At 10 µM CaCl2 (trace D, Figure 1), the condensation was nearly complete even at π < 2 mN/m, resulting in an isotherm similar to those obtained from simple long-chain fatty acids.10 MgCl2 had a much weaker effect (see data below); in the presence of 1 µM MgCl2 the isotherm was similar to trace B. The increment of ionic strength (see footnote 13) due to addition of 10 mM NaCl was over 300-fold higher than that due to addition of 10 µM CaCl2, but the effect of 10 µM CaCl2 on the isotherm was greater. Thus, the effect of CaCl2 is not due to ionic strength alone. This conclusion is supported by the differences in the effects of Ca2+ and Mg2+. Since divalent cations strongly affect the phase behavior of DPPG monolayers, it should not be surprising that polyvalent cations (cationic polymers) have a substantial effect. When polylysine was added to the subphase at various concentrations, significant concentration-dependent condensation was observed (Figure 2).11 Addition of polylysine at a ratio of 1:1 or 2:1 (Traces B and C) condensed the monolayer and addition of higher concentrations (Traces D, E) resulted in a clearly defined transition at a lower surface pressure. The effect of the polyvalent cations was significant, stronger (per mole) than that of monovalent or divalent cations, and distinct from that of the divalent or monovalent ions. The importance of the specific ionic composition of the subphase extends to the buffer composition as well. X-ray diffraction data by Wilkinson et al.12 have demonstrated (11) The concentration of polymeric ions is given as an ion ratio, specifically, the ratio of charges on the polymer to charges on the anionic lipid. In practice, this is the ratio of the total number of lysine residues (i.e. the total number of cationic amines) or the total number of nucleotide bases (i.e. the number of anionic phosphates) to the number of lipid molecules. The final polymer subunit concentration in the subphase for most experiments is in the unit micromolar range. The monolayer consists of 22.8 nmol of lipid, and the subphase volume is approximately 125 mL. Thus, at a ratio of 10:1, for example, the subphase concentration of charges due to polymer is 1.8 µM. However, because the subphase depth in this trough is about 5 mm, the “local” environment, immediately adjacent to the lipid headgroups, would be expected to have a ratio of bases to lipids much lower than 10:1. (12) Wilkinson, D. A.; Tirrell, D. A.; Turek, A. B.; McIntosh, T. J. Biochim. Biophys. Acta 1987, 905, 447-453.
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Figure 2. Polylysine condenses DPPG monolayers. Trace A is DPPG in the absence of added cation. Other data were obtained in the presence of poly-L-lysine‚HBr (MW 4000-15 ,000) added with a lysine residue-to-lipid ratio of (B) 1:1, (C) 2:1, (D) 4:1, and (E) 10:1. All data were obtained using a Tris buffer subphase (pH 7.4, 10 mM).
Figure 3. Buffer composition of the subphase affects DPPG phase behavior. All data were obtained on buffered subphases at pH 7.4. The buffer concentrations are (A) 10 mM Tris, (B) 5 mM Tris, and (C) 5 mM HEPES.
that Tris buffer causes acyl chain interdigitation in DPPG bilayers. Because ionized Tris is a cation, association of Tris with DPPG monolayers might interfere with the interaction of DPPG with cations of interest. In this system, at a Tris concentration of 1 mM, the condensation effect of 1 µM Ca2+ is similar to that of 10 µM Ca2+ with 10 mM Tris. DPPG monolayers behave differently on similar (pH 7.4, 5 mM) subphases of Tris or HEPES buffer. Figure 3 (and other data obtained with more complex subphase composition, not shown) shows that the phase behavior of DPPG monolayers on a subphase with a cationic buffer is distinct from that observed on a zwitterionic buffer. Traces B and C were obtained with subphases at pH 7.4 with 5 mM Tris or HEPES, respectively. The apparent expansion of the monolayer in the presence of increased Tris concentration is consistent with the structure data obtained by Wilkinson et al.12 These data suggest that the effect is not simply one of ionic strength. For equimolar buffers, the calculated ionic strength of a Tris buffer should be less than that of a HEPES buffer.13 The isotherm obtained with HEPES buffer (trace C) indicates a more condensed monolayer than that obtained with an equimolar Tris buffer (trace B), and this difference is contrary to that one would expect due to effects of ionic strength; it is likely that the observed buffer effect is due to Tris/DPPG interaction. To minimize possible effects of cation competition, HEPES buffer was used for all subsequent experiments involving oligonucleotide binding described in this report. As expected, oligonucleotides themselves did not affect the phase behavior of anionic monolayers (data not shown).
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Figure 4. Phosphorothioate oligonucleotides affect the DPPG compression isotherm in the presence of Ca2+. Trace A is from DPPG on a 1 mM HEPES buffer subphase. Trace B is from DPPG on the same buffered subphase with 0.1 µM CaCl2 added. Traces C, D, and E were obtained with 0.15, 0.2, and 0. 3µM added CaCl2, respectively. The bold trace was obtained by adding a 21-base phosphorothioate oligonucleotide to a 0.3 µM CaCl2containing subphase at a base-to-lipid ratio of 50:1.
Compression isotherms of DPPG with moderate concentrations of oligonucleotides added to the subphase were not statistically distinct from those without oligonucleotide in the subphase, even at relatively high ratios of phosphodiester oligonucleotides. Isotherms of DPPG on a subphase containing anionic oligonucleotides and divalent cations were found to be expanded relative to those obtained on a subphase with divalent cations alone. Figure 4 shows that when a 21base phosphorothioate oligonucleotide (14% A, 19% T, 33% G, 33% C) was included in a subphase containing 0.3 µM CaCl2, DPPG monolayers were expanded relative to monolayers on a subphase containing 0.3 µM CaCl2 alone. (The sequence was not palindromic and was not expected to form hairpins or dimers.) The expansion was concentration dependent. It is important to note that the shape of isotherms in the presence of 0.3 µM CaCl2 and anionic oligonucleotides was distinct from the shape of isotherms obtained with concentrations of CaCl2 less than 0.3 µM. Because the compressibility of DPPG monolayers on 0.3 µM CaCl2/oligonucleotide-containing buffer was distinct from that of DPPG monolayers on buffer with
