Langmuir 1999, 15, 1923-1928
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Solubilization of DNA-Cationic Lipid Complexes in Hydrophobic Solvents. A Single-Molecule Visualization by Fluorescence Microscopy Sergey M. Mel’nikov* and Bjo¨rn Lindman Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund, Sweden Received September 15, 1998. In Final Form: December 10, 1998 The solubilization of DNA-lipid complexes in low-polar organic solvents was studied with the use of the fluorescence microscopy technique. It was demonstrated that the transfer of large T4 DNA complexed with cetyltrimethylammonium bromide (CTAB) and didodecyldimethylammonium bromide (DDAB) is not possible from the aqueous DNA-CTAB and DNA-DDAB solutions. On the other hand, dry DNA-CTAB and DNA-DDAB complexes were successfully dissolved and visualized in some of the studied organic solvents. DNA-DDAB complexes were found to be better soluble, compared to the DNA-CTAB complexes, due to the higher hydrophobicity of DDAB. The ability of the organic solvents to solubilize DNA-lipid complexes was also found to be different, as follows: chlorobenzene > toluene > chloroform, cyclohexane. It was shown that an increase of temperature enhances the solubilization of DNA-lipid complexes. The hydrodynamic radii of T4 DNA-lipid complexes in organic solvents were calculated from the corresponding translational diffusion constants. The obtained data implied that T4 DNA-lipid complexes in hydrophobic liquids have a highly compacted globular conformation and consist of a single T4 DNA macromolecule.
1. Introduction The controlled delivery of genetic material into the patient’s cells has gained an increased interest as a new therapy against genetic and acquired diseases. One of the requirements for such medical therapy is an efficient and harmless DNA delivery system.1 Several types of ex vivo and in vivo DNA delivery systems are presently under clinical development. Some of them, including cationic lipids and positively charged liposomes, have been found to be relatively effective.2,3 In the application of the controlled DNA delivery systems, the limiting stage is often a DNA penetration through the cellular membrane. Thus, the investigation of the mechanism of this process is of especial importance in both fundamental and applied medical sciences. In the studies of this subject, hydrophobic organic solvents were used as a “liquid membranes” model.4 It was shown that penetration of both proteins and nucleic acids through the organic solvents cannot be observed for the free compounds, but their complexation with a cationic lipid leads to a substantial enhancement of the transfer.4 Therefore, the studies of the solubility of DNA-lipid complexes in organic solvents are of especial importance for the understanding of the mechanisms of DNA migration through the cell membrane during the transfection process. The solubilization of DNA-lipid complexes in chloroform was first reported by Ijiro and Okahata for the relatively short DNA fragments (salmon testes DNA).5 (1) Non-Viral Genetic Therapeutics: Advances, Challenges and Applications for Self-Assembling Systems; Walsh, B., Ed.; IBC Biomedical Library Series, International Business Communications: Southborough, MA, 1996. (2) Lasic, D. D. Liposomes in Gene Delivery; CRC Press: Boca Raton, FL, 1997. (3) Handbook of Nonmedical Applications of Liposomes: From Gene Delivery and Diagnostics to Ecology; Lasic, D. D., Barenholz, Y., Eds.; CRC Press: Boca Raton, FL, 1996. (4) Bromberg, L. E.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 143-147. (5) Ijiro, K.; Okahata, Y. J. Chem. Soc., Chem. Commun. 1992, 13391341.
Moreover, they demonstrated with the use of the circular dichroism (CD) spectroscopy that the double-stranded helical structure of DNA remains undisturbed after the introduction of complexes into organic liquids. Recently the transfer of DNA-lipid complexes to the low-polar organic solvents, i.e., chloroform, hexane, and cyclohexane, was comprehensively studied with the use of UV and CD spectroscopy and sedimentation, as well as atomic force and scanning tunneling microscopy.6-10 It was found that the successful solubilization of DNA-lipid complexes in organic liquids depends on the degree of the desiccation of complex, i.e., presence of trace amounts of water, as well as on the molecular weight of DNA. Although the solubilization of DNA-lipid complexes was detected for the short (up to 2 × 106 Da) DNA molecules, large DNA fragments complexed with cationic lipids were insoluble in the studied organic solvents at room temperature. In this connection, it has been recently shown by Sergeyev and Mel’nikov that stoichiometric complexes between CTAB and large T4 DNA and λ-DNA are practically insoluble in chloroform at room temperature.11 It must be noted that the solubilization of DNA-lipid complexes in organic solvents is interesting not only for its medical applications but as a general phenomenon in colloid and polymer chemistry. Highly charged polyelectrolyte molecules are known to be incompatible with the low-polar organic solvents. However, the complexation of (6) Pyshkina, O. A.; Sergeyev, V. G.; Zezin, A. B.; Kabanov, V. A. Dokl. Chem. 1996, 348, 496-498. (7) Pyshkina, O. A.; Sergeyev, V. G.; Lezov, A. V.; Mel’nikov, A. B.; Ryumtsev, E. I.; Zezin, A. B.; Kabanov, V. A. Dokl. Phys. Chem. 1996, 349, 772-775. (8) Sergeyev, V. G.; Pyshkina, O. A.; Zezin, A. B.; Kabanov, V. A. Vysokomol. Soed. 1997, 39A, 17-21. (9) Sergeyev, V. G.; Pyshkina, O. A.; Gallyamov, M. O.; Yaminsky, I. V.; Zezin, A. B.; Kabanov, V. A. Prog. Colloid Polym. Sci. 1997, 106, 1198-203. (10) Kabanov, V. A.; Zezin, A. B.; Sergeyev, V. G.; Pyshkina, O. A.; Yaminsky, I. V. In Abstracts of Eighth International Symposium on Recent Advances in Drug Delivery Systems; Salt Lake City, UT, 1997; pp 250-251. (11) Sergeyev, V. G.; Mel’nikov, S. M. 1996, unpublished data.
10.1021/la981255h CCC: $18.00 © 1999 American Chemical Society Published on Web 02/17/1999
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polyelectrolytes with oppositely charged amphiphilic molecules leads to the neutralization of polyelectrolyte chains and formation of hydrophobic domains in the resulting complexes. In this connection, the dissolution of complexes between synthetic polyelectrolytes and oppositely charged surfactants in organic liquids was also reported recently.12-14 Besides the importance of previous reports, some questions concerning the DNA-lipid complex dissolution in organic solvents are still open: (i) Is it possible to dissolve a large DNA molecule with the order of several tens kilobase pairs (kbps), complexed with a cationic lipid, in a low-polar solvent? (ii) Will the fluorescently labeled DNA molecules remain visible in organic solvent? (iii) What is the actual conformation of a complex between large single DNA and a cationic lipid in a low-polar medium? In relation to question iii, it was shown that DNA-lipid complex has a compact structure in organic solvents,6 but this conclusion was made for the short DNA fragments (0.3-2.0 kbps). In the present study we examined the possibility of the transfer of giant linear double-stranded DNA molecules, complexed with cationic lipids, to low-polar organic solvents. The effects of the chemical structures of lipids and organic solvents, as well as the effect of temperature, on the possibility of DNA-lipid complex solubilization in a low-polar medium were considered. The fluorescence microscopy technique, which we successfully used for the studies of DNA-lipid interaction in aqueous solutions,15-19 was applied for the direct visualization of the dynamics of DNA-lipid complexes in a low-polar hydrophobic medium. 2. Experimental Section 2.1. Materials. Coliphage T4 DNA (Mr ) 1.1 × 108 Da, ca. 167 kilobase pairs) was supplied by Sigma. Synthetic cationic lipids cetyltrimethylammonium bromide (CTAB) and didodecyldimethylammonium bromide (DDAB) were obtained from Sigma; CTAB was recrystallized twice from acetone, and DDAB was used as received. The DNA concentration was determined spectrophotometrically, considering the molar extinction coefficient of DNA bases to be equal to 6600 M-1 cm-1;20 the ratio of the absorbance of a DNA stock solution at 260 nm to that at 280 nm was found to be 1.9. The fluorescent dye, 4′,6-diamidino2-phenylindole (DAPI), and the antioxidant, 2-mercaptoethanol (ME), were from Sigma. Organic solvents, chlorobenzene, toluene, chloroform, and cyclohexane, were of analytical grade (Merck). Bidistilled water was purified with a Millipore filter (pore size 22 µm). 2.2. Preparation of DNA-Lipid Complexes. Dry DNAlipid complexes were prepared as follows: A stock DNA solution was diluted with the 0.5 × TBE buffer (45 mM Tris, 45 mM (12) Bakeev, K. N.; Shu, Y. M.; MacKnight, W. J.; Zezin, A. B.; Kabanov, V. A. Macromolecules 1994, 27, 300-302. (13) Kabanov, A. V.; Sergeev, V. G.; Foster, M. S.; Kasaikin, V. A.; Levashov, A. V.; Kabanov, V. A. Macromolecules 1995, 28, 3657-3663. (14) Bakeev, K. N.; Shu, Y. M.; Zezin, A. B.; Kabanov, V. A.; Lezov, A. V.; Mel’nikov, A. B.; Kolomiets, I. P.; Rjumtsev, E. I.; MacKnight, W. J. Macromolecules 1996, 29, 1320-1325. (15) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 2401-2408. (16) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 9951-9956. (17) Mel’nikov, S. M.; Sergeyev, V. G.; Mel’nikova, Yu. S.; Yoshikawa, K. J. Chem. Soc., Faraday Trans. 1997, 93, 283-288. (18) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K.; Takahashi, H.; Hatta, I. J. Chem. Phys. 1997, 107, 6917-6924. (19) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. In Recent Research Developments in Chemical Sciences; Pandalai, S. G., Ed.; Transworld Research Network: Trivandrum, India, 1997; Vol. 1; pp 69-113. (20) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Plainview, NY, 1989.
Mel’nikov and Lindman borate, 1 mM EDTA, pH ) 8.0), containing DAPI. Final concentrations were as follows: DNA in nucleotide units, 0.6 mM; DAPI, 0.1 mM. Lipid solutions were gently added to the DNA solution in an equimolar ratio. The formed precipitate was separated, washed with buffer solution, and freeze-dried overnight at 25 °C to completely remove water from the precipitate. The resulting dry complexes were placed into the organic solvents for 24 h before the observation. DNA-lipid complexes in solution were prepared as follows: DNA molecules were diluted with the 0.5 × TBE buffer (pH ) 8.0) containing 4% (v/v) ME, a free-radical scavenger, and a fluorescent dye. The resulting solution was gently mixed with the aqueous lipid solutions and was then kept for 2 h. The final concentrations were as follows: DNA in nucleotide units, 0.5 µM; DAPI, 0.5 µM; CTAB, 0.1 mM; DDAB, 0.1 mM. In these conditions, the binding number of DAPI per one DNA base pair in an aqueous buffer solution is estimated to be equal to 0.05 and the persistence length of DNA chain is expected to remain nearly the same as in the absence of DAPI,21,22 and all DNA molecules were found to be in a compacted globular state. The resulting solutions were mixed with the organic solvents, gently vortexed, and left for 24 h before the observation. 2.3. Fluorescence Microscopy. A fluorescence microscopy study was performed as follows: The samples were illuminated with a UV-mercury lamp; the fluorescence images of single DNA molecules were observed using a Zeiss Axioplan microscope, equipped with a 100 × oil-immersed objective lens, and digitized on a personal computer through a high-sensitive SIT video camera and an image processor, Argus-20 (Hamamatsu Photonics, Japan). The apparent long-axis length of the DNA molecules, L, was defined as the longest distance in the outline of the fluorescence image of single DNA. Images of the dynamic motion of single DNA-lipid complexes in organic solvents were taped with a conventional S-VHS video recorder. The observations were carried out at 25 °C. Special care was taken to clean the microscope glasses (No. 0, Chance Propper, England) thoroughly before the observation to prevent DNA degradation, as well as precipitation to the glass surface.19
3. Results and Discussion It has been shown in previous studies that the transfer of DNA-lipid complexes into organic solvents strongly depends on the water content in the sample.19 Therefore, we have studied the solubilization of both freeze-dried desiccated DNA-lipid complexes and the aqueous buffer solution of DNA-lipid globules in low-polar organic liquids. First, dry DNA-lipid complexes were introduced into the hydrophobic solvents at 25 °C. Fluorescence microscopy observations of T4 DNA-DDAB complexes showed that in the case of chlorobenzene and toluene the transfer of complexes into the low-polar organic medium occurs, whereas for the chloroform and cyclohexane no fluorescent particles were detected in the sample solution. At the same time, T4 DNA-CTAB complexes were found to be insoluble in all studied organic solvents at 25 °C. An increase of the temperature to 65 °C showed that under those conditions all four organic solvents solubilize DNA-DDAB complexes, whereas only chlorobenzene was found to be effective for the solubilization of DNA-CTAB complex in the organic phase. The DNA-lipid complexes in organic solvents were observed as compact particles of a nearly spherical shape (Figure 1), with L values of complexes within a 0.6-1.0 µm interval. However, due to the blurring effect in observation, the actual size of the particles of this size, visualized with FM, usually is significantly less than the apparent L values. Later on, we will present (21) Matsuzawa, Y.; Yoshikawa, K. Nucleosides Nucleotides 1994, 13, 1415-1423. (22) Matsuzawa, Y.; Minagawa, K.; Yoshikawa, K.; Doi, M. Nucleic Acids Symp. Ser. 1991, 25, 131-132.
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Figure 1. Fluorescence microscopy images of T4 DNA-lipid complexes in organic solvents at 25 °C: (A) T4 DNA-DDAB complex, solubilized in toluene at 25 °C; (B) T4 DNA-CTAB complex, solubilized in chlorobenzene at 65 °C; (a) black-and-white video frames of the fluorescence images (scale bar 10 µm); (b) three-dimensional fluorescence intensity distribution of the (a) photographs. Table 1. Solubility of T4 DNA-Lipid Complexes in Low-Polar Organic Solvents at Various Temperatures
a
solvent
DDAB-DNA (dry)
chlorobenzene toluene chloroform cyclohexane
+ + -
chlorobenzene toluene chloroform cyclohexane
+ + + +
DDAB-DNA (aq soln)
CTAB-DNA (dry)
CTAB-DNA (aq soln)
(a) At 25 °C -
-
-
(b) At 65 °C -
+ -
-
“+”, complex is soluble, “-”, complex is insoluble.
calculations of the actual DNA-lipid complex size in the organic phase. Next, the possibility of T4 DNA-lipid complex transfer into low-polar organic solvents from the aqueous solutions was examined. The aqueous buffer solutions of DNACTAB and DNA-DDAB complexes in a globular state were mixed with organic solvents, as described above, and observed at 25 °C with fluorescence microscopy. We found that, independently of the solvent temperature, neither DNA-DDAB nor DNA-CTAB complexes exhibit a transfer into the hydrophobic organic solvents. The results of fluorescence microscopy observations are summarized in Table 1. Here we have to mention that since fluorescently labeled DNA-lipid complexes were successfully observed in all organic solvents used for this study, it is natural to expect that the invisibility of complexes at some experimental conditions is not related to the weakening of the fluorescence dye binding to T4
DNA molecules in the low-polar medium but suggests the insolubility of DNA-lipid complexes in organic liquids. The analysis of the experimental observations presented in Table 1 leads us to some important conclusions. First, there is a great difference between the dissolution properties of dry and wet DNA-lipid complexes. For instance, dry DNA-DDAB complexes were detected in all of the studied organic solvents, whereas no transfer of DNA-DDAB globules from aqueous solutions was found. This effect can be explained by taking into account the fact that DNA molecules are strongly associated with water molecules.23 The hydrophilic headgroups of cationic lipids are also largely hydrated, even after the formation of the complex with oppositely charged polyelectrolytes.24 The interaction of DNA with positively charged amphiphilic molecules leads to the neutralization of charges (23) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman & Co.: San Francisco, CA, 1988.
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along the DNA chain and compaction of elongated DNA. However, the resulting DNA-lipid compex particles still contain a notable amount of water, which is strongly bound to both DNA nucleotides and polar headgroups of cationic lipid. The hydration of DNA-lipid globules brings about their incompatibility with the hydrophobic solvents. Only complete removal of water from the complexes ensures the dissolution of hydrophobic lipid-covered DNA in lowpolar liquids. In this connection, it was reported that the complex between highly polymerized (1-3 kbps) DNA and cationic lipid in aqueous solution showed no transfer into the hydrophobic phase upon mixing with chloroform but was concentrated at the water-chloroform interface.6 Second, chlorobenzene and toluene are found to be more effective for the solubilization of DNA-lipid complexes. This is natural, since these solvents are known to be better solubilizing agents for the saturated polyalkenes, such as polyethylene and polypropylene.25 The chemical structure of hydrophobic alkyl substitutes of the studied cationic lipids is quite similar to the short-chained polyalkenes. Thus, chlorobenzene and toluene act effectively as solvents for the hydrophobized DNA chains, covered with the lipid molecules. This observation may be meaningful for the increase of the efficiency of the DNA-lipid complexes delivery through the biological membranes. The chemical structure of lipid should provide its high affinity to the constituents of the biomembrane, and this will facilitate the transport of DNA-lipid complex into the patient’s cell. Also, it is of value to mention that polar substituted aromatic hydrocarbons are known to be solubilized in the headgroup region of CTAB micelles, whereas nonpolar hydrocarbons are concentrated in the lipophilic part of the micelles.26 The difference in the solubilization mechanisms may explain the solubility of DNA-CTAB complex in chlorobenzene, comparing to other studied hydrophobic solvents. Third, complexes of T4 DNA with DDAB are shown to be easier solubilized by organic liquids than those with CTAB. This can be explained by the higher hydrophobicity of DDAB molecules. Whereas CTAB is well soluble in water and forms nearly spherical micelles, the monomer solubility of DDAB in water is very low. DDAB does not form micelles in aqueous solutions but organizes into vesicles and then displays a lamellar phase upon a further increase of lipid concentration. Moreover, DDAB itself is moderately soluble in hydrophobic organic solvents, and its solubility is enhanced in the presence of water.27 Thus, DDAB-DNA complexes have a higher affinity for hydrophobic solvents. A similar trend was noticed by Sergeyev et al., who have found that, at the experimental conditions corresponding to the solubilization of complexes between low-molecular DNA and distearyldimethylammonium bromide in chloroform, DNA-CTAB complexes remain insoluble.9 Recently we showed with the X-ray scattering technique that the aggregate structure of DNA-CTAB and DNA-DDAB complexes is quite different. DNA-CTAB complexes are characterized by a hexagonal packing of the surfactant rodlike micelles, whereas lamellar lipid structures were observed for DNA-DDAB complexes.18 This fact also supports our interpretation, since lamellar packing of a (24) Jo¨nsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solution; John Wiley & Sons: New York, 1998. (25) Polymer Handbook, 2nd ed.; Brandrup, J., Immergut, E. H., Eds.; J. Wiley & Sons: New York, 1975. (26) Eriksson, J. C.; Gillberg, G. Acta Chem. Scand. 1966, 20, 20192027. (27) Evans, F. D.; Wennerstro¨m, H. The Colloidal Domain: Where Physics, Chemistry, Biology and Technology Meet; VCH Publishers: New York, 1994.
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Figure 2. Mean square displacements of the center of mass of T4 DNA-lipid complexes in low-polar organic liquids: (A) DNA-lipid complexes, dissolved at 25 °C (], DNA-DDAB in chlorobenzene; [, DNA-DDAB in toluene); (B) DNA-lipid complexes, dissolved at 65 °C (O, DNA-DDAB in chloroform; b, DNA-DDAB in cyclohexane; 9, DNA-CTAB in chlorobenzene).
lipid provides better protection of the highly charged DNA macromolecule from the hydrophobic solvent. We also supposed that the increase of temperature will facilitate the transfer of complexes between giant linear DNA and cationic lipids into low-polar media. The experimental results in Table 1 supported our expectations, showing that at a higher temperature hydrophobic solvents are more effective solubilizing agents for the DNA-lipid complexes. It is also known that some organic solvents, which are inert to the polyalkenes at room temperature, dissolve them when the temperature becomes higher than 60-70 °C.25 Next, we address the question on the actual conformational state and dimensions of T4 DNA-lipid complexes in organic solvents. Direct observations of DNA-lipid complexes in hydrophobic solvents showed that the DNA chain has a compact globular conformation (Figure 1). However, in our previous studies we found that the actual size of the DNA globule is always much smaller than its apparent long-axis length, L.15 This difference comes from the significant blurring effect in FM observation. Since the size of globular DNA is of the same order as the wavelength of the fluorescent light,19 the actual DNA dimensions in a globular state could not be evaluated from the direct measurement of the DNA apparent length. Recently we demonstrated that the actual size of DNA globules, i.e., the hydrodynamic radius, may be calculated from the translational diffusion coefficients for the individual DNA molecules.15 In the present study we also analyzed videotapes with the recorded Brownian motion of DNA-lipid complexes in organic solvents and evaluated the hydrodynamic radii of DNA-lipid complexes. The translational diffusion coefficient of DNA-lipid complexes, D, was evaluated from the time t dependence of the mean square displacement (MSD) of the center of mass of DNA macromolecules in a two-dimensional area of observation, as is exemplified in Figure 2. During the observation, a minor spontaneous convective flow in the sample solution was recognized, probably due to the illumination effect. As the bulk flow rate was almost
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Table 2. Translational Diffusion Coefficients and Hydrodynamic Gyration Radii of DNA-Lipid Complexes in Hydrophobic Organic Solventsa complex
solvent
solubilization temp, °C
ηS, mPa‚s
D, µm2/s
ξH, µm
DDAB-DNA DDAB-DNA DDAB-DNA DDAB-DNA CTAB-DNA
chlorobenzene toluene chloroform cyclohexane chlorobenzene
25 25 65 65 65
0.74 0.55 0.54 0.90 0.74
1.86 2.35 2.21 1.43 2.08
0.16 0.17 0.18 0.17 0.14
a Independently of the dissolution temperature of a complex, fluorescence microscopy observations were performed at 25 °C for all samples. Viscosity values are given according to the ref 30.
constant for the observation area, one can eliminate the effect of the convection from the two-dimensional diffusional movement, on the basis of the following equation:28
〈(RG(t) - RG(0))2〉 ) 4Dt + At2
(1)
Here RG(t) ) (Rx, Ry) is a coordinate of a center of the DNA globule at a given time t and A is a numerical constant as a scale of a convective flow magnitude. From the leastsquares fitting to the second-order polynomial of t for the various time-intervals observed, we have evaluated the actual diffusion constant D. The hydrodynamic gyration radius ξH was calculated from D according to the StokesEinstein equation:29
ξH )
kBT 6πηSD
(2)
Here T is the temperature, kB is the Boltzmann constant, and ηS is the viscosity of the organic solvent. In Table 2 we present the hydrodynamic radii of DNA-lipid globules in organic solvents, calculated according to eqs 1 and 2 at T ) 298 K. The obtained values of ξH for the DNA-lipid complexes in hydrophobic solvents are quite similar to those reported previously for the DNA-lipid complexes in aqueous solution.15,19 This observation presumes that the solubilization of complexes between giant linear DNA and cationic lipids in low-polar organic solvents does not induce the unfolding transition in large DNA. Despite the solubility of large DNA, covered with the hydrophobic lipid molecules, in organic solvents, the internal stiffness of the DNA chain hinders the unfolding of compacted DNAlipid complexes in a low-polar medium. Supporting our observation, it was reported that complexes between short DNA fragments and cationic surfactants are densely packed in chloroform and cyclohexane.10 Another important conclusion deals with the actual structure of DNA-lipid complexes in hydrophobic solvents. It is known that for the DNA-lipid globules in aqueous solutions an aggregation process might take place. As a result, the DNA-lipid complex in water might consist of several individual globules, which leads to an increase of the hydrodynamic gyration radius of DNA.15 If one compares the obtained values of the linear dimensions of DNA (Table 2) with the recent data attained with the use of fluorescence microscopy,15,31 scanning tunneling microscopy,32,33 and atomic force microscopy,9 it becomes (28) Matsumoto, M.; Sakaguchi, T.; Kimura, H.; Doi, M.; Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K. J. Polym. Sci., Part B: Polym. Phys. 1992, 30, 779-783. (29) Doi, M.; Edwards, S. F. The Theory of Polymer Dynamics; Clarendon: Oxford, U.K., 1986. (30) CRC Handbook of Chemistry and Physics, 55th ed.; Weast, R. C., Ed.; CRC Press: Boca Raton, FL, 1974. (31) Yoshikawa, K.; Matsuzawa, Y. Physica D 1995, 84, 220-227.
evident that globular DNA-lipid complexes in low-polar organic solvents consist of a single DNA macromolecule. Finally, we would like to make some remarks on the calculation of the actual size of DNA-lipid complexes. The above calculation of ξH is based on the assumption that DNA-lipid complexes are nearly spherical particles with a relatively high spatial density. However, recently it was shown that the DNA-lipid complexes, formed from short DNA fragments, have a toroidal shape.9 Since up to date there were no data on giant linear DNA, we found acceptable to use a conventional way of calculations. If it is toroidal, due to the change in the friction coefficients, there would be a difference in the ξH values. Considering DNA toroid as an oblate ellipsoid, one can calculate the difference between its friction coefficient according to the Stokes equation and the friction coefficient of a sphere of the same volume. For the DNA-lipid complex it was found with the use of the atomic force microscopy technique that the ratio between the height of the toroid and its diameter is equal to 0.1.9 Taking into account this value, the difference in friction coefficients and, consequently, the diffusion coefficients between a DNA toroid and a sphere of the same volume is equal to 1.46.34 This will have an effect on the numerical values, obtained in our study, but would not change the qualitative situation and would not affect the comparison with the previously published data on ξH of DNA-lipid complexes,15,19 since they were also obtained assuming nearly spherical particles. 4. Conclusions The results obtained in the present study clearly show that large bacteriophage T4 DNA molecules may be transferred into low-polar organic solvents after DNA complexation with positively charged cationic lipids. DNA molecules, which were stained with a conventional fluorescence dye, DAPI, in hydrophobic solvents, were successfully visualized with a fluorescence microscopy technique. Depending on the structure of the cationic lipid, i.e., its hydrophobicity, the T4 DNA-lipid complexes showed a marked difference in their solubilization in a low-polar medium. It was demonstrated that DNA-DDAB complexes are much better solubilized in organic solvents than the DNA-CTAB complexes; thus, the lipophilicity of the amphiphilic molecules, complexed with DNA, is a key factor, which determines the successful transfer of DNA molecules into a low-polar organic solvent. It was also found that the solubilization of DNA-lipid complexes in organic solvents directly depends on the ability of the solvent to solubilize the hydrocarbons, corresponding to the hydrophobic groups of lipid molecules. (32) Kidoaki, S.; Yoshikawa, K. Biophys. J. 1996, 71, 932-939. (33) Noguchi, H.; Saito, S.; Kidoaki, S.; Yoshikawa, K. Chem. Phys. Lett. 1996, 261, 527-533. (34) Marshall, A. G. Biophysical Chemistry: Principles, Techniques and Applications; John Wiley & Sons: New York, 1978.
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The aromatic compounds, chlorobenzene and toluene, which are known to be effective solvents for polyalkenes, were shown to be good solvents for the DNA-lipid complexes. On the other hand, chloroform and cyclohexane were found to be less effective solubilizing agents. The preferential solubilization of substituted aromatic compounds in the polar headgroup regions of surfactant micelles also contributed to the higher solubility of DNACTAB complex in the presence of chlorobenzene. An increase of temperature led to the enhancement of the solubility of DNA-lipid complexes. At last, from the measurements of the hydrodynamic radii of DNA-lipid complexes in low-polar solvents it was shown that, despite solubilization of complex, DNA molecules do not unfold but remain in a compact globular
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state. The size of DNA-lipid globules presupposes that they are formed by single T4 DNA chains. Acknowledgment. The Crafoord Foundation (Lund, Sweden) is gratefully acknowledged for the financial support in the updating of the microscope imaging system. Thanks are due to Dr. B. A° kerman and Prof. B. Norde´n (Chalmers University of Technology, Go¨teborg, Sweden) for the kind permission to use their image analysis equipment and to Dr. V. G. Sergeyev (Moscow State University) for the valuable discussions. This work was supported in part by grants from the Swedish Natural Sciences Research Council (NFR), the Swedish Research Council for Engineering Sciences (TFR), and the Swedish Royal Academy of Sciences. LA981255H
