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* Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, University of Helsinki, Finland and CNR, ICCOM—Sezione di Roma c/o Dipartimento di Chimica, Universita degli Studi di Roma "La Sapienza," P. le A. Moro 00185, Rome, Italy
Correspondence: Address reprint requests to Dr. Paavo K. J. Kinnunen, Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine / Biochemistry, Biomedicum, P.O. Box 63 (Haartmaninkatu 8), FIN-00014 University of Helsinki, Finland. Tel.: 358-9-191 25400; Fax: 358-9-191 25444; E-mail: paavo.kinnunen{at}helsinki.fi.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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0.50 conveyed the enhanced green fluorescent protein coding plasmid effectively into cells. The condensation of DNA by liposomes with XSR-1 > 0.50 was indicated by static light scattering and ethidium bromide intercalation assay, whereas differential scanning calorimetry and fluorescence anisotropy of diphenylhexatriene revealed stoichiometry dependent reorganization in the headgroup region of the liposome bilayer, in alignment with our previous Langmuir-balance study. Surface charge density and the organization of positive charges appear to determine the mode of interaction of DNA with (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide/1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes, only resulting in DNA condensation when XSR-1 > 0.50. Condensation of DNA in turn seems to be required for efficient transfection. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). Importantly, such complexes are nonimmunogenic, nonpathogenic, biodegradable, and easy to prepare. They have also been shown to pass through the blood-brain barrier (Shi and Pardridge, 2000) and have reached clinical trials (Caplen et al., 1995; Alton et al., 1999). Yet, despite intensive efforts the mechanism(s) of liposome mediated transfection (lipofection) and therefore also the characteristics of an ideal transfection agent remain incompletely understood.
Several physical or chemical properties of different cationic lipids have been proposed to be responsible for their ability to transfect cells. The number of positive charges of the cationic lipid (Wheeler et al., 1996), the linkage between the hydrophobic and cationic portion of the molecule (Leventis and Silvius, 1990), and the structure of the hydrophobic moieties (Solodin et al., 1995) have been demonstrated to influence the measured transfection efficiencies. Also properties of the lipoplexes formed have been extensively studied to establish proper composition required for efficient and reproducible transfection. Presence of inverted hexagonal HII-phase–forming dioleoylphosphatidylethanolamine (DOPE) (Farhood et al., 1995; Koltover et al., 1998; Mok and Cullis, 1997), diacylglycerol (DAG) (Paukku et al., 1997), or other nonlamellar phase promoting compounds (Pedroso de Lima et al., 1999) in the lipoplex has been shown to enhance transfection efficiency. However, this requirement is challenged by some previous studies (Hui et al., 1996; Simões et al., 1999), as well as by the findings presented here. The morphologies of cationic lipid-DNA complexes have been visualized by various electron microscopic techniques (e.g., Sternberg et al., 1994; Huebner et al., 1999; Gustafsson et al., 1995) and atomic force microscopy (Wheeler et al., 1996; Kawaura et al., 1998). These studies suggest several structures which could be responsible for transfection, e.g., "spaghetti-and-meatballs" (Sternberg et al., 1994), dense multilamellar complexes (Huebner et al., 1999), or complexes with a particular diameter (Kawaura et al., 1998). However, Xu et al. (1999) recently reported that the morphology of complex as visualized by electron microscope does not have significant impact on the transfection efficiency.
Understanding the nature of cationic lipid-DNA interaction could have broader biological significance because of the natural cationic lipid sphingosine. The latter is ubiquitously present in mammalian cells and forms complexes with DNA (Kinnunen et al., 1993; Kõiv et al., 1994), revealing "beads-on-a-string" morphology as well as larger aggregates. It is, in principle, possible that sphingosine could be involved in the regulation of gene expression and signal transduction. To this end, the well established lipid second messenger, phosphaditic acid, is able to reverse DNA-sphingosine complex formation (Kinnunen et al., 1993).
Gemini surfactants, composed of two conventional surfactant molecules connected by a spacer, have acquired a lot of attention in colloid and surface chemistry (Menger and Keiper, 2000). Recently also the use of gemini surfactants as transfection vectors has been investigated (e.g., Fielden etal., 2001). Our preliminary experiments revealed the cationic gemini surfactant (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide (SR-1; chemical structure illustrated in Fig. 1) to be an efficient transfection vehicle. Moreover, there was no requirement for the presence of nonbilayer "helper" lipids such as DOPE. In our Langmuir-balance study we found that the properties of mixed monolayers of SR-1/POPC were strongly dependent on the content of SR-1 in the films (Säily et al., 2001). These results led us to explore the impact of XSR-1 on the interaction of this cationic gemini surfactant with DNA and the efficiency of the resulting complexes in cellular transfection. Surface charge density of mixed SR-1/phosphatidylcholine liposomes is demonstrated to represent an important determinant for the transfection efficiency and condensation of DNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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) and its purity was verified by NMR. The purity of other lipids was checked by thin-layer chromatography on silicic acid coated plates (Merck, Darmstadt, Germany) using chloroform/methanol/water (65:25:4, by vol.) as a solvent system. Examination of the plates after iodine staining or, when appropriate, by fluorescence illumination revealed no impurities. Concentrations of phospholipids and SR-1 were determined gravimetrically using a high precision electrobalance (Cahn, Cerritos, CA). DNA concentrations (expressed in mM basepairs) were determined by absorbance at 260 nm (
= 6600 l/mol x cm). The 260/280 ratio of the calf thymus DNA was 
1.9. Freshly deionized filtered water (Milli RO/Milli-Q, Millipore Inc., Jaffrey, NH) was used in all experiments.
Preparation of liposomes
Multilamellar liposomes (MLVs) were prepared by mixing appropriate amounts of the lipid stock solutions in dry chloroform to obtain the desired compositions. Thereafter the solvent was removed by evaporation under a stream of nitrogen. For removal of residual amounts of solvent the samples were further maintained under high vacuum for at least 2 h. The resulting dry lipid films were then hydrated with 5 mM Hepes, 0.1 mM EDTA, pH 7.4 and thereafter incubated for 30 min at 60°C, i.e., above the temperatures of the transition endotherms of the lipid components and their mixtures (Fig. 6 A). To obtain large unilamellar vesicles (LUVs) the hydrated lipid dispersions were vortexed vigorously and then extruded with a LiposoFast small volume homogenizer (Avestin, Ottawa, Canada) by subjecting to 19 passes through polycarbonate filter (100-nm pore size, Nucleopore, Pleasanton, CA). LUVs with diameters of 100–115 nm are produced by this method (MacDonald et al., 1991; Wiedmer et al., 2001). Unless otherwise stated the liposomes were kept on an ice water bath for at least 12 h before their use. Importantly, to avoid interference with fluorescence spectroscopy resulting from light scattering due to varying lipid concentrations XSR-1 was varied in these measurements (as well as in DSC experiments) by altering both the amount of SR-1 and DMPC while keeping total lipid concentration constant. In the transfection experiments the total cationic charge (i.e., the amount of SR-1) was maintained constant and XSR-1 was varied by altering the amount of the DMPC in the liposomes.
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Transfection efficiencies were measured using COS-1 cells, derived from simian kidney cell line CV-1 transformed with a mutant simian virus 40 (Gluzman, 1981). Cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum (DMEM-10) on 24-well plastic cell culture plates in an incubator with an atmosphere of 5% CO2 in air. Three different kinds of liposome-DNA complexes were made. Accordingly, either MLVs or LUVs, as indicated, were first formed and the plasmid was then added to these liposome solutions. Preformed MLVs and LUVs were prepared as described above. Complexes of DNA with these preformed liposomes were then formed by adding the indicated amounts of pEGFP-N1 plasmid at 23°C in 0.6 ml of serum free Dulbecco's modified Eagle's medium (DMEM-SF), followed by an incubation for 15 min at 23°C. Alternatively, the indicated amount of plasmid was included in the DMEM-SF buffer used to hydrate (at T > Tm of the lipid constituents) the dry lipids to yield MLVs. Before transfection the total volume of the suspensions containing liposome-plasmid complexes was adjusted to 0.9 ml with DMEM-SF. DMEM-10 was removed from the wells of just confluent 24-well cell culture plates and the liposome-plasmid complexes were added to three separate wells, 0.3 ml to each. After a 6-h incubation at 37°C the transfection mixture was replaced by DMEM-10. Transfection efficiency was determined after 65 h of incubation at 37°C by measuring the fluorescence intensity of EGFP using SPECTRAFluor Plus fluorescence reader (Tecan AG, Hombrechtikon, Switzerland) with emission and excitation wavelengths set at 485 and 510 nm, respectively. The background measured for nontransfected cells serving as controls was subtracted from the values recorded for the transfected cells.
Cytotoxicity was assessed by Trypan Blue (Invitrogen, San Diego, CA) exclusion. Cells were cultured on 24-well culture plates in DMEM-10 and subsequently incubated at 37°C for 6 h with liposome-DNA complexes with the indicated compositions, whereafter the transfection mixture was replaced by DMEM-10 and the incubation continued for further 24 h. Subsequently, cells were detached from the wells by trypsinization and resuspended in one ml of DMEM-SF. An aliquot of 50 µl of the resulting cell suspension was mixed with 50 µl of 0.4% Trypan Blue and incubated at ambient temperature (23°C) for 5 min. The amount of nonviable cells was determined by counting the stained cells with a hemocytometer.
Light scattering
Static light scattering due to the formation of complexes by the cationic liposomes and DNA was measured with a spectrofluorometer with both excitation and emission monochromators set at 500 nm. Two ml of 50-µM liposome solution of the indicated composition was placed into a magnetically stirred four-window quartz cuvette thermostated at 37°C. LUVs and DNA were mixed at the stated molar ratios. Scattering intensities were measured 5 min after the addition of DNA to the indicated concentrations and remained constant after this period.
Ethidium bromide intercalation assay
Fluorescence emission spectra of ethidium bromide in the 520–700-nm region were recorded with excitation at 500 nm. In brief, 1.8 ml of 16 µM EtBr was first applied into a magnetically stirred four-window quartz cuvette thermostated at 37°C whereafter calf thymus DNA was added to yield a final concentration of 34 µM (expressed in basepairs). Subsequently, LUVs of the given compositions were included to achieve the indicated molar ratios. Emission spectra were measured 5 min after the addition of liposomes.
Differential scanning calorimetry
MLVs were vortexed and maintained on an ice bath for at least 12 h XSR before loading into the calorimeter cuvette (final concentration 1 mM). When indicated DNA was present in the hydration buffer and theresultingcationic lipid-DNA complexes were incubated on an ice water bath as described above. A VP-DSC microcalorimeter (Microcal, Northampton, MA) was operated at a heating rate of 0.5° per min. The instrument was interfaced to a 486 PC, and the data were analyzed using the routines of the software provided by the instrument manufacturer.
Fluorescence spectroscopy using DPH
Diphenylhexatriene (DPH) was included in LUVs to yield a 1:500 molar ratio of probe to lipid (XDPH = 0.002). Fluorescence measurements were carried out with a Perkin-Elmer LS 50B spectrofluorometer interfaced to a Pentium PC. The excitation and emission wavelengths were set at 360 and450 nm, respectively. Two ml of 50 µM SR-1/DMPC solution was applied into a magnetically stirred four-window quartz cuvette placed in the sample compartment thermostated at 37°C by a circulating water bath. When indicated DNA was added to the LUVs to yield the desired molar ratios of the cationic lipid and DNA (expressed in basepairs). Anisotropy values were measured 5 min after the addition of DNA to the given concentrations and were found to remain constant after this period.
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RESULTS |
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0.50. The expression levels achieved by preformed SR-1/DMPC MLVs (at XSR-1 0.50) were comparable or higher than those obtained with the commercial cationic liposome vector LipofectinTM (data not shown). Yet, inasmuch as our goal was to study the effect of surface electrostatics to the transfection efficiency rather than to compare the efficiency of SR-1 to the commercial lipofection systems it must be emphasized that the transfection experiments were conducted using conditions optimized for SR-1/DMPC liposomes only.
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; Ross and Hui, 1999; Zuidam et al., 1999) complexes formed by adding DNA to preformed cationic MLVs were more efficient in transfection than those formed by adding the plasmid to LUVs with similar composition. The third kind of lipoplexes were made by adding the plasmid into buffer, which was then used to hydrate the dried lipid residue. Adding the plasmid in the hydration buffer resulted in lower transfection efficiencies than measured for preformed MLVs (Fig. 2 C). However, the dependence of transfection efficiency on XSR-1 was evident also for these complexes. Further, these lipoplexes demonstrated sensitivity toward CL/DNA ratio in contrast to the other formulations employed in our study.
The above dependence of the transfection efficiency of the lipoplexes on XSR-1 could be due to different cytotoxities of the lipoplexes. To investigate this possibility we determined the cytotoxicity of SR-1/DMPC MLVs complexed with the plasmid (CL/DNA = 1) by Trypan Blue exclusion. However, the percentage of nonviable cells as a function of XSR-1 revealed only minor differences (Fig. 3). We may thus conclude that differences in transfection efficiencies are not due to cytotoxicity.
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). Thereafter, with XSR-1approaching 1.00 gradual decrement in scattering was measured. Liposomes with XSR-1 = 0.75 and 0.88 showed a weak tendency for precipitation, whereas no evidence for this was obtained at XSR-1 < 0.75. Precipitation due to DNA was evident for neat SR-1 liposomes, similarly to that observed for HADAB, acationic lipid studied previously in our laboratory (Subramaniam et al., 2000). With XSR-1 from 0.50 to 0.88 maximal effect on scattering required only 
2.5 µM DNA, corresponding to CL/DNA stoichiometries from 5.0 to 8.8.
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10-fold higher when intercalated between the bases of DNA, compared to its fluorescence in water (Lakowicz, 1999). During condensation of DNA varying amounts of EtBr are forced to dissociate from DNA and partition into the aqueous phase, resulting in a decrease in fluorescence emission intensity (Cain et al., 1978; Bhattacharya and Mandal, 1998; Geall etal., 1999). We employed this method to verify condensation of DNA by SR-1/DMPC LUVs, suggested by static light scattering. The normalized emission intensity of DNA bound EtBr at 590 nm as a function of lipid concentration is illustrated in Fig. 5 and reveals two different patterns depending on XSR-1. In brief, liposomes with XSR-1 0.50 caused only minor decrement in EtBr emission (15%), which could result from augmented scattering due to liposomes. In contrast, liposomes with XSR-1 > 0.50 caused a much more pronounced attenuation in emission (maximally by 47%), thus suggesting condensation of DNA by the added liposomes. Interestingly, the effect was not dependent on the total amount of cationic charge present in as much as the liposomes with XSR-1 = 0.75 and 1.00 show similar behavior despite significant difference in the amount of the added cationic surfactant.
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14°C and the main transition at Tm 24°C. At XSR-1 = 0.05 the value for Tp increased to 16.2°C and that for Tm to 26.0°C, in keeping with the saturated acyl chains of the cationic surfactant. When XSR-1 was increased to 0.13 the pretransition disappeared. Increasing XSR-1 elevated Tm further until a maximum of 41.8°C was reached at XSR-1 = 0.38 and 0.50. At XSR-1 = 0.63 the main transition decreased to 40.2°C, with two smaller overlapping endotherms at 31.8 and 42.7°C. MLVs with XSR-1 = 0.75 revealed a broad transition with two separated peaks at 29.7 and 35.8°C. At XSR-1 = 0.88 a local minimum in Tm at 28.9°C was evident. Neat SR-1 revealed two endotherms at 32 and 39°C, with a total enthalpy of 73 kJ/mol. Their nature remains uncertain at this stage. Moreover, the endotherms for SR-1 were not fully reproducible, similarly to recent observations on other cationic amphiphiles (Zantl et al., 1999). Accordingly, although the beginning and the end of endotherm, as well as the total enthalpy remained almost the same in every measurement, the shape and the exact peak temperatures varied, the latter within 4°C. Yet, despite this variation the composed tentative phase diagrams (Fig. 7 A) can be considered to be representative.
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Tentative phase diagrams constructed from the above DSC data recorded for binary SR-1/DMPC liposomes both without DNA and with 0.05 mM DNA were compiled in Fig. 7, A and B, respectively. The only pronounced change due to DNA was the decrement in the solidus line at XSR-1 = 0.13. However, the shape of the endotherm at XSR-1 = 0.13 made it difficult to determine the exact onset of the transition, and therefore this point in the phase diagram is somewhat uncertain. The slight increment in the liquidus line of the phase diagram between XSR-1 = 0.05 and 0.38 was also evident. This effect disappeared at XSR-1 > 0.50.
Fluorescence spectroscopy
To observe possible effects of SR-1 and DNA on acyl chain order DPH was included into liposomes with different contents of SR-1 while recording the emission anisotropy r (Lakowicz et al., 1979a, b) both with and without DNA (Fig. 8). Values for r increased with increasing XSR-1 up to 0.63. Above this SR-1 mole fraction anisotropy decreased significantly, yielding values similar to those measured for liposomes with XSR-1 < 0.25. The anisotropy measurements were done at 37°C, under the main transition temperatures of SR-1/DMPC liposomes with 0.25 XSR-1 0.63 (Fig. 6 A). In keeping with the DSC measurements these liposomes showed the highest anisotropy values.
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0.05 (Fig. 8). When XSR-1 was increased to 0.13, the effect of DNA became evident, with increased anisotropy at XSR-1 from 0.13 to 0.50. The effect of DNA saturated at 5 µM, with no additional influence at higher concentrations. In keeping with the small changes observed by DSC, minor changes in r were caused by DNA at XSR-1 0.63. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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0.50 (Fig. 2 A). Importantly, this difference is explained neither by variation in cytotoxicity (Fig. 3) nor by changes in the total cationic charge present in the liposomes, as the latter was maintained constant in the transfection experiments. Interestingly, biophysical parameters of both liposomes and DNA in the lipoplexes also demonstrated similar dependence on the lipid stoichiometry.
Increased static light scattering (Fig. 4) suggests condensation of DNA by the cationic liposomes, which has been previously shown to be required for efficient lipofection (Bloomfield, 1996; Tang and Szoka, 1998). Compared to the condensation of DNA by polycations, condensation caused by cationic amphiphiles is less thoroughly studied (Bloomfield, 1996). Moreover, the latter system is also inherently more complex, involving the association of a charged polymer with a colloid, pseudo-two-dimensional cationic surface. In our measurements with calf thymus DNA a maximum in light scattering was evident at XSR-1 0.63. Yet, it should be emphasized that aggregation could also contribute, thus making strict relationship between the observed scattering and DNA condensation somewhat uncertain. To investigate the possibility of DNA condensation further we measured fluorescence intensity of the DNA intercalating dye EtBr with SR-1/DMPC liposomes (Fig. 5). The quenching of EtBr emission evident at XSR-1 > 0.50 provides strong support for the condensation of DNA being the major reason for the observed increase in scattering. Furthermore, our preliminary transmission electron microscopy studies (data not shown) revealed that the size of the complexes did not significantly change when XSR-1 was increased from 0.25 to 0.75. Thus condensation and not aggregation is likely to represent the reason for the observed augmented scattering.
A slightly lower content of SR-1 is required to produce high transfection efficiency than to induce condensation of DNA as measured by light scattering and EtBr intercalation assay (XSR-1 0.50 and XSR-1 > 0.50, respectively). This could be due to use of the circular 4700 bp plasmid DNA in the transfection experiments, whereas longer calf thymus DNA was utilized in the other measurements. The length of DNA could influence complex morphology and possibly also condensation when comparing very short DNA fragments (with persistence length of DNA being 500 Å) to much longer molecules (Bloomfield, 1996; Bloomfield, 1998). Accordingly, this difference in XSR-1 could relate to the difference in the lengths of the two types of DNA. More specifically, the larger decrement in the conformational entropy of the longer DNA upon its coil-globule transition due to complex formation requires higher positive charge density in the liposomes (Bloomfield, 1998). Yet, this difference in the DNAs used is unlikely to have a major qualitative impact on our data so as to undermine a meaningful comparison.
Binary SR-1/DMPC liposomes have maxima in Tm with XSR-1 between 0.38 and 0.50 (Fig. 6 A), and these values for Tm exceed the main endotherm temperatures of the neat constituents. In addition, the enthalpy peaks for the mixtures are rather narrow thus resembling transitions of liposomes composed of a single lipid rather than a mixture. A plausible explanation for this counterintuitive impact of XSR-1 on Tm of DMPC is provided by an electrostatically driven reorganization in the headgroup region of the bilayer. Accordingly, the addition of cationic SR-1 into zwitterionic phosphatidylcholine bilayer is anticipated to cause a reorientation of the P--N+ dipole of the PC headgroup (Fig. 9 B). More specifically, although the orientation of PC headgroup dipole in the neat phosphatidylcholine liposomes has been shown by NMR to be almost parallel to the plane of the bilayer (Seelig et al., 1987), the presence of cationic amphiphiles in the bilayer causes the P--N+ dipole to reorient nearly perpendicular to the surface because of the Coulombic repulsion (Scherer and Seelig, 1989). This results in a reduced average area occupied by the PC headgroup. Reduction in mean molecular areas occupied by POPC molecules in the presence of SR-1 was shown in our previous Langmuir-balance study (Säily et al., 2001). Reorientation of the hydrated phosphocholine moiety can be expected to result in reduced repulsive interactions at the headgroup level. As a consequence chain–chain interactions within the hydrocarbon phase must be augmented so as to balance the forces in the membrane lateral pressure profile. The augmented packing of the acyl chains is described by reduction in the free volume of the bilayer in keeping with the elevated values for Tm and increased fluorescence anisotropy for DPH (Figs. 7 and 8). The above is in accordance with recent molecular dynamic simulations (Bandyopadhyay et al., 1999) and Langmuir-balance studies (Zantl et al., 1999; Säily et al., 2001) on cationic lipid-phosphatidylcholine mixtures. At XSR-1 > 0.50 Coulombic repulsion between the cationic headgroups of SR-1 is progressively enhanced, resulting in a lateral expansion of the bilayer, increase in membrane free volume, and thus decrement both in Tm and fluorescence anisotropy of DPH (Figs. 7 and 8, respectively).
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suggested that the cationic charge of the P--N+ dipole interacts directly with DNA. Accordingly, DNA could induce additional change in the orientation of P--N+ dipole due to Coulombic attraction (Fig. 9, A–C). In keeping with this notion the addition of DNA to the liposomes with 0.13 XSR-1 0.50 caused a significant increase in r (Fig. 8) thus suggesting that DNA induces tighter packing of the acyl chains. This mechanism would provide an explanation for the observed elevation in liquidus line in the phase diagram. Importantly, the above mechanism explains also why liposomes with 0.13 XSR-1 0.50 (and not liposomes with XSR-1 > 0.50 which bear more cationic net charge) demonstrate most pronounced changes due to the addition of DNA. In alignment with the above, compression isotherms for SR-1/POPC monolayers revealed film condensation in the presence of DNA (Säily et al., 2001).
The nature of the moiety bearing the cationic charge has been shown to be important in the condensation of DNA by cationic liposomes (Geall et al., 1999). Accordingly, the dependence of DNA condensation on the XSR-1 could be explained by electrostatically driven molecular reorientations in the surface of liposomes suggested by DSC, fluorescence anisotropy of DPH, and monolayer (Säily et al., 2001) experiments. In the liposomes with XSR-1 > 0.50 the cationic charges of SR-1 are screened by the phosphates of the P--N+ dipoles thus causing association of DNA to liposomes to be mediated by the cationic charge of the tertiary ammonium group of the choline moiety (Fig. 9 C). However, the phosphocholine headgroup is strongly hydrated and because of its three methyl groups the cationic charge of the latter is anticipated to be incapable of as strong interaction with the phosphate residues of DNA as the sterically less shielded charges of SR-1. Interestingly, this raises the possibility that the enhanced transfection by the cationic liposomes containing PE may not relate only to the promotion of the formation of the inverted hexagonal phase HII by this lipid but also to a more efficient Coulombic interaction of the weakly hydrated -N+H3 moiety of the PE headgroup with DNA. It seems feasible that in addition to the direct Coulombic interaction between SR-1 and DNA being required, also the cationic charge density is critical. The latter could be related to the lack of condensation of DNA by the divalent Mg2+, in contrast to spermidine3+ and spermine4+, for instance. The necessity for an interaction of SR-1 with DNA may also reflect the importance of the removal of hydration layers from the contacting molecular surfaces (Leikin et al., 1993). To this end, our preliminary transmission electron microscopy studies on negatively stained complexes of CT-DNA and SR-1/DMPC LUVs with XSR-1 = 0.25 and 0.75 revealed very different morphologies. Accordingly, at XSR-1 = 0.25 spaghetti-and-meatballs–like structure (Sternberg et al., 1994) was evident, whereas at XSR-1 = 0.75 more dense and irregular particles were observed (data not shown).
To conclude, our data demonstrate that the cationic charge density of liposomes is an important determinant of transfection efficiency and suggest that this is related to condensation of DNA. This observation further emphasizes the importance of compactly packed and well protected plasmid DNA for efficient lipofection. Surface electrostatics of the liposomes thus appear to have a more crucial role in the condensation of DNA and lipofection than previously anticipated (e.g., Wagner et al., 2000; Harries et al., 1998; Gelbart et al., 2000) involving complex rearrangements in the headgroup region of the bilayer. Experimental and theoretical efforts to elucidate more completely the role of electrostatics of liposomal surfaces in lipofection are in progress in our laboratory.
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ACKNOWLEDGEMENTS |
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S.J.R. is supported by the M.D./Ph.D. program of the University of Helsinki. The Helsinki Biophysics and Biomembrane Group is supported by grants from TEKES and the Finnish State Medical Research Council.
Submitted on March 26, 2002; accepted for publication September 11, 2002.
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), 1.0 (•), 2.0 (
), and 3.0 (
). See Materials and Methods for further details.
), and 15.0 µM (+). Temperature was maintained at 37°C. Buffer was 5 mM Hepes, 0.1 mM EDTA, pH 7.4 and total concentration of lipid was 50 µM.
