One-Dimensional Thermotropic Dilatation Area of Lipid Headgroups

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One-Dimensional Thermotropic Dilatation Area of Lipid Headgroups within Lamellar Lipid/DNA Complexes Giulio Caracciolo,*,† Daniela Pozzi,† Heinz Amenitsch,‡ and Ruggero Caminiti† Dipartimento di Chimica, UniVersita` degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy, and Institute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Schmiedelstrasse 6, A-8042 Graz, Austria ReceiVed December 20, 2005. In Final Form: March 5, 2006 Using simultaneous synchrotron small- and wide-angle X-ray diffraction (SWAXD), we investigated the thermotropic behavior of a cationic lipid mixture of DOTAP-DOPC (1,2-dioleoyl-3-trimethylammonium-propane-dioleoylphosphatidylcholine) liposomes complexed with calf thymus DNA. The DOTAP-DOPC/DNA complex reacts to temperature change by a bilayer compression normal to its surface and an expansion of the DNA in the plane of the rod lattice. By applying two independent recently developed models, we show here for the first time that the thermotropic dilatation area of lipid headgroups within the complexes is not isotropic but occurs parallel to the 1D DNA lattice (i.e., along the direction perpendicular to the DNA axis). Our results shed light on the role of spatial dimensionality in the DNA packing density within lamellar lipoplexes and provide experimental evidence that the interaction between DNA molecules confined between lipid bilayers can be regarded as a 1D problem.

Introduction During the past decades, a wide variety of nonviral strategies for gene delivery have been proposed and studied.1 Among these, cationic liposomes (CLs), binary mixtures of cationic and neutral lipids, were found to interact easily with DNA, leading to the formation of stable CL-DNA complexes (lipoplexes). Nowadays, lipoplexes are employed in clinical trials worldwide and represent the most consistent alternative to viruses, with the main concern in applicability remaining the low transfection efficiency (TE). When combined with DNA, CLs form self-assemblies with distinct lamellar (LCR phase) or inverted hexagonal (HCII phase) nanostructures.2-4 First, it was suggested that HCII complexes transfected more efficiently than LCR complexes because of different mechanisms of DNA release into the cell.3 More recently, experimental evidence was provided that the most efficient complexes are assembled in the LCR phase,5,6 and Safinya’s group identified the membrane charge density, σΜ, as a key universal parameter that controls transfection behavior in lamellar lipoplexes.6 As a result, redesigned LCR complexes competed with the high TE of HCII complexes.7 During the past few years, many experimental and theoretical studies8,9 have investigated the relationship between the physicalchemical properties of lipoplexes and the experimental conditions of their formation. A deeper comprehension of the exact role of physical and chemical parameters in the inner structure of * Corresponding author. E-mail: [email protected]. † Universita ` degli Studi di Roma “La Sapienza”. ‡ Austrian Academy of Sciences. (1) Lasic, D. D. Liposomes in Gene DeliVery; CRC Press: Boca Raton, FL, 1997. (2) Salditt, T.; Koltover, I.; Ra¨dler, J. O.; Safinya, C. R. Science 1997, 275, 810. (3) Koltover, I.; Salditt, T.; Ra¨dler, J. O.; Safinya, C. R. Science 1998, 281, 78. (4) Safinya, C. R. Curr. Opin. Struct. Biol. 2001, 11, 440. (5) Caracciolo, G.; Pozzi, D.; Caminiti, R.; Congiu Castellano, A. Eur. Phys. J. E 2003, 351, 222. (6) Lin, A. J.; Slack, N. L.; Ahmad, A.; George, C. X.; Samuel, C. E.; Safinya, C. R. Biophys. J. 2003, 84, 3307. (7) Ahmad, A.; Evans, H. M.; Ewart, K.; George, C. X.; Samuel, C. E.; Safinya, C. R. J. Gene Med. 2005, 7, 739. (8) Simoes, S.; Slepushkin, V.; Gaspar, R.; Pedroso de Lima, M. C.; Duzgunes, N. Gene Ther. 1998, 5, 955. (9) May, S.; Ben Shaul, A. Curr. Med. Chem. 2004, 11, 1241.

lipoplexes should help to control and manipulate the formation of specific phases.4 The last step in devising more efficient lipoplexes will be understanding the relationship between the structural properties and function of the complexes in order to direct the self-assembly of lipoplexes toward the desired structures.4 Very recently, many different liposomal formulations with temperature sensitivity have been reported.10 As a result, hyperthermia and liposomal drug delivery started to be used together in an attempt to exploit their mutual interactions against cancer.11,12 Although this combination therapy seems to hold great promise toward improving current TE, the molecular mechanisms by which it does remain unclear. It has been proposed that varying temperature modifies the permeability of the plasma membrane by changing the fluidity of the lipid bilayer of both the plasma membrane and the lipoplexes. Here we report a small- and wide-angle X-ray diffraction (SWAXD) study on the effect of temperature on the structural properties of lamellar lipoplexes composed of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), the neutral lipid dioleoylphosphatidylcholine (DOPC), and calf thymus DNA. The thermotropic phase behavior of lipoplexes made of saturated lipids has been the subject of two previous investigations.13,14 Nevertheless, for transfection applications, saturated lipids are not usually employed because of transition temperatures close to human physiological temperatures. To our knowledge, no study has been devoted to the temperature dependence of the structural properties of lipoplexes made of unsaturated lipids. Materials and Methods I. Sample Preparation. Dioleoyl trimethylammonium propane (DOTAP) (MW(DOTAP) ) 698.6) and neutral lipid dioleoyl (10) Kong, G.; Anyarambhatla, G.; Petros, W.; Braun, P. R. D.; Colvin, O. M.; Needham, D.; Dewhirst, M. W. Cancer Res. 2000, 60, 6950. (11) Mushiake, H.; Aoe, M.; Kazuhiro, W.; Andou, A.; Shimizu, N. Acta Med. Okayama 2002, 56, 35. (12) Okita, A.; Mushiake, H.; Tsukuda, K.; Aoe, M.; Murakami, M.; Andou, A.; Shimizu, N. Oncol. Rep. 2004, 11, 1313. (13) Zantl, R.; Artzner, F.; Rapp, G.; Ra¨dler, J. O. Europhys. Lett. 1998, 45, 90. (14) Zantl, R.; Baicu, L.; Artzner, F.; Sprenger, I.; Rapp, G.; Ra¨dler, J. O. J. Phys. Chem. B 1999, 103, 10300.

10.1021/la0534423 CCC: $33.50 © 2006 American Chemical Society Published on Web 03/29/2006

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phosphatidylcholine (DOPC) (MW(DOPC) ) 786) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. According to standard procedures,15 DOTAP and DOPC were dissolved in chloroform so that the weight ratio of neutral lipids in the bilayer is ΦW ) (weight of neutral lipid)/(weight of total lipid) ) 0.54, which corresponds to a molar ratio of neutral lipids in the bilayer of ΦM ) (neutral lipid)/(total lipid) ) 0.5 (mol/ mol). The solvent was evaporated under a stream of nitrogen and then under vacuum for at least 12 h. Multilamellar vesicles (MLVs) were formed by hydrating the dry lipid film in Tris-HCl buffer solution (10-2 M, pH 7.4) to reach the desired final concentration of 100 mg/mL. Calf thymus Na-DNA solution (10 mg/mL) was sonicated, inducing DNA fragmentation with a length distribution between 500 and 1000 base pairs (bp), which was determined by gel electrophoresis. Isoelectric DOTAP-DOPC/DNA lipoplexes were prepared by adding appropriate amounts of DNA solution to the mixed lipid dispersions. At the isoelectric point, the number of cationic lipid headgroups exactly matches the number of negatively charged phosphate groups on the DNA backbone. II. Synchrotron SWAXD Experiments. All SWAXD measurements were performed at the Austrian SAXD station of the synchrotron light source ELETTRA (Trieste, Italy).16,17 SAXD patterns were recorded with a gas detector based on the delay line principle covering a q range (q ) 4π sin θ/λ) of between 0.05 and 1.6 Å-1. The angular calibration of the detector was performed with silver behenate powder (d spacing ) 58.38 Å) for the SAXD regime and with p-bromobenzoic acid for the WAXD regime. The unoriented samples were sealed in 1.5-mm-diameter glass X-ray capillaries. SAXD experiments were carried out from 5 to 65 °C by increasing the temperature in 15 °C steps. The sample was held at each temperature for 5 min before the measurement was started so that the system can be considered to be in thermal equilibrium. The exposure time for each sample at each temperature was 100 s. No evidence of sample degradation due to radiation damage was observed in any of the samples at this exposure. The data have been normalized for the primary beam intensity and detector efficiency, and the background has been subtracted. Temperature was controlled in the vicinity of the capillary to within (0.1 °C (Anton Paar, Graz, Austria). III. Data Analysis. Because of the bilayer nature of lipid membranes, the electron density profile (EDP) is centrosymmetric; therefore, the electron density profile, ∆F, along the normal to the bilayer, z, can be calculated as a Fourier sum of cosine terms ∆F )

F(z) - 〈F〉 [〈F2(z)〉 - 〈F〉2]1/2

N

)

( ) z

∑ F cos 2πld l

l)1

(1)

where F(z) is the electron density, 〈F〉 is its average value, N is the highest order of the fundamental reflection observed in the XRD pattern, Fl is the form factor for the (00l) reflection, and d is the lamellar periodicity along the normal to the lipid bilayer consisting of one lipid bilayer and one water layer. Form factors Fl were calculated from the integrated intensity Il ) Fl2/Cl under the lth diffraction peak, where Cl ) ql2 is the Lorentz polarization correction factor for unoriented samples.18,19 The previous equation determines the form factors except for the phase factor, which must be (1 for symmetric bilayers. The usual phase problem was solved as previously proposed,20,21 paying additional attention to observe a maximum in (15) Caracciolo, G.; Caminiti, R.; Pozzi, D.; Friello, M.; Boffi, F.; Congiu Castellano A. Chem. Phys. Lett. 2002, 351, 222. (16) Amenitsch, H.; Rappolt, M.; Kriechbaum, M.; Mio, H.; Laggner, P.; Bernstorff, S. J. Synchrotron Radiat. 1998, 5, 506. (17) Bernstorff, S.; Amenitsch, H.; Laggner, P. J. Synchrotron Radiat. 1998, 5, 1215. (18) Zhang, R.; Suter, R. M.; Nagle, J. F. Phys. ReV. E 1994, 50, 5047 and references therein. (19) Zhang, R.; Tristram-Nagle, S.; Sun, W.; Headrick, R. L.; Irving, T. C.; Suter, R. M.; Nagle, J. F. Biophys. J. 1996, 70, 349. (20) Luzzati, V.; Mariani, P.; Delacroix, H. Makromol. Chem., Macromol. Symp. 1998, 15, 1. (21) Francescangeli, O.; Rinaldi, D.; Laus, M.; Galli, G.; Gallot, B. J. Phys. II 1996, 6, 77.

Figure 1. Synchrotron SWAXD scans for isoelectric DOTAPDOPC/DNA lipoplexes as a function of temperature (T ) 5, 20, 35, 50, and 65 °C from the bottom to the top). Each scan is shifted by multiplicative constants for better comparison. Solid and dashed lines are guides to the eye, allowing one to follow the shift of the second-order BP of the lamellar lipid bilayer-DNA monolayer structure and the movement of the DNA peak, respectively.

Figure 2. Schematics of the LCR phase. DNA rods are intercalated between cationic lipid membranes in the liquid-crystalline phase composed of cationic (grey) and neutral helper (white) lipids. The lamellar spacing, d, is the sum of the lipid bilayer thickness, dB, plus the thickness of the interbilayer water layer, dW. The DNA spacing, indicated as dDNA, is the distance between adjacent DNA rods. electron density in the middle of the water region due to the DNA molecules. The sign combination used to calculate the electron density profile is (- - + -) relative to structures factors F1, F2, F4, and F5, whereas F3 is equal to zero as a result of the systematic absence of the third-order Bragg peak (BP) in the SAXD pattern (Figure 1).

Results and Discussion Figure 1 shows the SWAXD patterns of charge-neutral DOTAP-DOPC/DNA lipoplexes as a function of temperature. The series of temperature-dependent WAXD scans of Figure 1 (1 < q < 1.55 Å-1) reveals that the DOTAP-DOPC lipid membranes are in the liquid-crystalline LR phase. The equally spaced BPs in the SAXD regime (0.05 < q < 0.55 Å-1) at q00n are caused by the alternating lipid bilayer-DNA monolayer LCR structure (Figure 2) with lamellar periodicity of d ) 2π/q001. The broad middle peak positioned at qDNA, between the first two BPs of the LCR structure, is attributed to the in-plane packing of the intercalated DNA strands with spacing dDNA ) 2π/qDNA.2-4 Figure 3 shows the EDPs calculated from the lamellar BPs of Figure 1 for three different temperatures (T ) 5, 35, and 65 °C).

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Figure 5. Thermal expansion coefficient of the bilayer thickness. Figure 3. Electron density profiles of DOTAP-DOPC/DNA lipoplexes as a function of temperature: 5 °C (- -), 35 °C (‚‚) and 65 °C (-). The distance from the center of the lipid bilayer is indicated as z.

Starting at 5 °C and on increasing temperature, the lamellar repeat distance d decreases from 64.6 to 60.3 Å. Figure 4 also shows that the reduction of the lamellar periodicity is due to the simultaneous thinning of both the membrane and the water region. At 5 °C, the lipid bilayer has a thickness of dB ) 37.4 Å that compares well with the results from Koltover and co-workers who found dB ) 38.2 Å for DOTAP-DOPC mixed bilayers.23 On increasing temperature, dB decreases almost linearly below 35 °C but then starts to display asymptotic behavior. The membrane gets thinner with increasing temperature because of the disordering of hydrocarbon chains accompanied by a decrease in the effective chain length. Rising temperature induces the continuous formation of trans-gauche rotamers within acyl chains.25 The asymptotic behavior observed above 35 °C is a well-known saturation effect in hydrocarbon chain melting, and it is due to the finite length of the acyl chains in that only a limited number of gauche isomers can be generated.26 This effect is due to both of the lipids because the dioleoyl hydrocarbon chains are identical for DOTAP and DOPC. The reduction in dB was first described by Luzzati and co-workers,22 who showed that the mechanism of thermal contraction of acyl chains is similar to that of rubberlike polymers.27,28 The value of the thermal expansion coefficient of the lipid bilayer dB

RB )

Figure 4. Temperature dependence of the lamellar spacing, d, the bilayer thickness, dB, the water layer thickness, dW, and the DNA rod lattice, dDNA, of isoelectric DOTAP-DOPC/DNA lipoplexes. The solid lines are linear fits to the data in the temperature range between 5 and 35 °C. As is evident, the bilayer thickness and the DNA-DNA spacing show asymptotic behavior for T > 35 °C.

According to general definitions,22 the bilayer thickness, dB, is defined as the distance between headgroup peaks in the EDP whereas the thickness of the interbilayer water region is the difference between the lamellar periodicity and the bilayer thickness, dW ) d - dB. The EDPs of Figure 3 show the usual lipid bilayer density plus high-density regions at the outer edges of the profile due to the DNA rod lattices intercalated between opposing bilayers. Figure 4 shows the temperature dependence of the retrieved structural parameters for DOTAP-DOPC/DNA lipoplexes. At 5 °C, DOTAP-DOPC/DNA lipoplexes exhibit a lamellar periodicity of d ) 64.6 Å that is in close agreement with those previously reported.23,24 (22) Luzzati, V.; Husson, F. J. Cell Biol. 1962, 12, 207.

∆dB 1 dB ∆T

(2)

is given as a function of temperature in Figure 5. The value of RΒ becomes more negative as the temperature increases, starting from (-1.060 ( 0.009) × 10-3 K-1 (T ) 5 °C) up to (-1.132 ( 0.007) × 10-3 K-1 (T ) 65 °C), indicating an increased bilayer elasticity that is expected for the above-stated reasons.29 Figure 4 also shows the temperature dependence of dW. Within lamellar lipoplexes, the water region is usually assumed to be nearly constant, dW ≈ 25 ( 1.5 Å, corresponding to the diameter of a double-stranded B-DNA molecule (∼20 Å) surrounded by a thin hydration shell.23 At 5 °C, dW ) 27 Å and decreases as a function of increasing temperature probably because of the growing attraction between adjacent membranes.25 Here, we emphasize that the structural changes induced by temperature were fully reversible and no hysteresis effects in the SWAXD patterns were observed after adequate equilibration times (t ≈ 1000 s). (23) Koltover, I.; Salditt, T.; Safinya, C. R. Biophys. J. 1999, 77, 915. (24) Caracciolo, G.; Pozzi, D.; Caminiti, R.; Amenitsch, H. Appl. Phys. Lett. 2005, 87, 133901. (25) Rappolt, M.; Laggner, P.; Pabst, G. Recent Res. DeV. Biophys. 2004, 3, 363. (26) Seelig, A.; Seelig, J. Biochemistry 1974, 13, 4839. (27) Treloar L. R. G. The Physics of Rubber Elasticity; University Press: Oxford, U.K., 1949. (28) Luzzati, V.; Mustacchi, H.; Skoulios, A. E.; Husson, F. Acta Crystallogr. 1960, 13, 660. (29) Pabst, G.; Rappolt, M.; Amenitsch, H.; Bernstoff, S.; Laggner, P. Langmuir 2000, 16, 8994.

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Table 1. Comparison between Experimental and Theoretical DNA Spacingsa T(°C)

(dDNA)expt

(dDNA)calcd

5 20 35 50 65

40.8 41.9 43.0 43.6 44.5

40.8 41.6 42.3 42.9 43.6

a Theoretical values were calculated according to eq 4 using the following values: ΦW ) 0.54, A ) 194 Å2, FD ) 1.75 g/cm3, and FL ) 1.07 g/cm3.

As shown in Figure 4, the DNA-DNA correlation length, dDNA, exhibits a lateral expansion, moving from 40.8 to 44.5 Å. First, the thermotropic changes in the DNA lattice can be interpreted in terms of the reduction of the bilayer thickness dB. Upon the formation of isoelectric lipoplexes, all lipid and DNA counterions are released, and the total number of cationic lipid headgroups is equal to the number of anionic phosphates on the DNA backbone. Therefore, the average charge densities of the membrane and DNA are exactly matched.23 The available area of cationic lipid membranes is entirely occupied by DNA molecules, and the DNA-DNA interhelical distance is predictable from a simple volume fraction model. Assuming a smectic arrangement of DNA chains, Koltover et al.23 provided the average spacing dDNA between DNA molecules in isoelectric complexes

ADFD MW(DOTAP) 1 dBFL MW(bp) (1 - ΦW)

dDNA ) 2

(3)

where AD is the cross-sectional area of a DNA molecule, FD and FL are the densities of DNA and the lipid, and MW(bp) ) 649 is the molecular weight of a DNA base pair. At a first sight, the temperature-induced changes in DNA spacing could be entirely ascribed to membrane thinning. Table 1 lists the DNA spacings obtained by inserting the values of dB for each temperature into eq 3. As is evident, the calculated values show very good agreement with the experimental values. The good agreement between experimental and calculated DNA spacings suggests that the thermotropic changes in the in-plane DNA rod lattices are ruled by geometric packing arguments. Nevertheless, at higher temperatures, the experimental DNA spacings show a slight deviation from the simple linear increase of dDNA with 1/dB predicted by eq 3. For the lamellar phases, the bilayer thickness and the interfacial area per lipid, A, are the most relevant structural parameters.30 The value of A is the result of interplay between attractive and repulsive forces. The main attractive components are the van der Waals forces between acyl chains and headgroup dipolar interactions whereas the main repulsive ones include steric interactions, hydration forces, and entropic contributions due to hydrophobic chain ordering. The equilibrium interfacial area per lipid molecule is therefore given by the balance of these forces that minimize the interfacial free energy. Rising temperature induces an increase in interfacial area per each lipid molecule, leading to a lateral bilayer expansion. This is the result of increased molecular motions such that the lipids require more lateral space.25,29 Thus, the continuous reduction in DNA packing density (i.e., the continuous enlargement of DNA-DNA spacing) between 5 and 65 °C could be interpreted in terms of the lateral expansion of the DOTAP-DOPC membranes. To quantify this effect, information on the temperature dependence of the lateral area (30) Petrache, H. I.; Dodd, S. W.; Brown, M. F. Biophys. J. 2000, 79, 3172.

is needed. Unfortunately, to the best of our knowledge, no experimental or theoretical studies have been performed to obtain the interfacial area of mixed DOTAP-DOPC bilayers. During the past few decades, different methods for the determination of the area have been proposed on the basis of volumetric measurements of lipid molecules.31,32 Within lamellar lipoplexes, A is a key structural parameter because it modulates the surface charge density of lipid membranes that, in turn, controls the DNA packing density.6 As a matter of fact, we tried to retrieve structural information on the temperature dependence of A by arguments concerning DNA packing density within DOTAP-DOPC/DNA lipoplexes. Some of us have recently proposed a theoretical model33 describing DNA-DNA electrostatic interactions within lamellar lipoplexes. In the model, the interaction force between adjacent DNA strands is given by

F)

[

(Ze)2 2ldDNA(1 - ΦM) -1 A 2πl2

][ 2

]

((dDNA)2 + ξp2)1/2 -1 dDNA (4)

where (Ze) is the charge of the cationic lipid headgroup, A is the average lipid headgroup area, ΦM is the molar ratio of the neutral lipid in the bilayer, ξp is the DNA persistence length (ξp ≈ 500 Å),  is the dielectric constant of water ( ≈ 80), and l ) 1.7 Å is the distance between two phosphate entities along the DNA axis. As is evident, the interaction force depends on the lipid composition (ΦΜ), on the DNA spacing (dDNA), and on the average interfacial area per lipid molecule (A). For different lipid compositions (i.e., for different ΦΜ values), the electrostatic force F exhibited deep minima at single values of dDNA that were interpreted as equilibrium distances, (dDNA)eq, of the system.33 Excellent agreement between the equilibrium distances predicted by the model and those experimentally observed in isoelectric lipoplexes, (dDNA)iso, was found, showing that charge neutrality is the most stable condition of lamellar lipoplexes. As a result, the DNA packing density was well predicted by the model, and the existence of a master curve matching the experimental observations was established.33 Here the model has been applied to estimate the average value of the lateral area A. By force minimization with respect to dDNA, one finds a relation between A and dDNA

A ) 2ldDNA(1 - ΦM)

(5)

From eq 5, we calculated the values of the lateral area per lipid molecule that minimize the electrostatic force of eq 4 at dDNA values identical to those retrieved from the series of temperaturedependent SAXD patterns of Figure 1. Figure 6 shows the DNADNA electrostatic forces, calculated for ΦM ) 0.5, exhibiting deep minima at DNA spacings equal to the (dDNA)iso of Figure 4. This procedure allowed us to reconstruct the temperature dependence of the average area per lipid molecule A (Figure 7). As expected, A increases monotonically with temperature because lipid molecules require more lateral space in the membrane plane. Using eq 4 relies on the basic assumption that the area of cationic lipid headgroups, AC, is similar to that of neutral lipid headgroups, AN (AC ≈ AN ) A). At the molecular level, both DOPC and DOTAP contain choline groups whereas DOPC contains a (31) Luzzati, V. In Biological Membranes; Academic Press: New York, 1968. (32) Nagle, J. F.; Tristram-Nagle, S. Biochim. Biophys. Acta 2000, 1469, 159. (33) Caracciolo, G.; Caminiti, R. Chem. Phys. Lett. 2004, 400, 314.

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Figure 8. Schematic illustration of the electrostatic model. Mixing DNA and cationic liposomes (a) results in the formation of locally ordered 1D arrays of DNA chains (blue rods with the helix axis parallel to the z axis) intercalated between charged membrane bilayers (b). The electrostatic attraction between cationic lipids and DNA charged molecules induces polarization of the positive charge carried by the lipid headgroups along the DNA helix axis. These distributions of charge are schematized as parallel lines (c) and, finally, as a set of parallel lines of charge in the xz plane (d). The distance between DNA chains is indicated as dDNA. Figure 6. DNA-DNA electrostatic forces calculated according to eq 4 with different values of the interfacial area, A: 67.9 Å2 (b); 69.7 Å2 (3); 71.6 Å2 (9); 72.6 Å2 ()); 74.1 Å2 (2). The forces exhibit deep minima at the same dDNA values as calculated from the SAXD patterns of Figure 1.

Figure 7. Temperature dependence of the average interfacial area A per lipid molecule of DOTAP-DOPC membranes.

phosphate entity in the headgroup. For both DOTAP and DOPC, Safinya and co-workers usually use the same value (A ) 70 Å2) to estimate the surface charge density of DOTAP-DOPC lipid membranes.6,23 Thus, this approximation is not a severe restriction in the present study, and it is realistic to compare our findings with those obtained for pure DOPC bilayers throughout the literature because, to our knowledge, no structural study has been devoted to the determination of the area per lipid molecule for pure DOTAP membranes. It is therefore gratifying to note that our estimate of A at 35 °C (A ) 71.6 Å2) is in good agreement with the value obtained for DOPC bilayers from molecular dynamics simulations by Chiu et al.34 (A ) 71 ( 1 Å2 at 30 °C). It is also noteworthy that our estimate is, within experimental error, in full agreement with the experimental fully hydrated area/DOPC molecule reported by Tristram-Nagle et al. (A ) 72.2 ( 1.1 Å2 at 30 °C).35 From the temperature dependence of both the average interfacial area and the DNA spacing, we calculated the thermal expansion coefficients27

RA )

∆A 1 A ∆T

(6)

∆dDNA 1 dDNA ∆T

(7)

and

RDNA )

(34) Chiu, S. W.; Jakobsson, E.; Subramaniam, S. Biophys. J. 1999, 76, 1929. (35) Tristram-Nagle, S.; Petrache, H.; Nagle, J. F. Biophys. J. 1998, 75, 917.

Table 2. Thermal Expansion Coefficients of Lipid Headgroups Area, rA, and DNA-DNA Spacing, rDNA, Calculated According to Equations 6 and 7, Respectively T (°C)

RA (10-3 K-1)

RDNA (10-3 K-1)

5 20 35 50 65

1.509 1.470 1.432 1.412 1.383

1.512 1.472 1.440 1.417 1.390

Table 2 lists both of the calculated thermal expansion coefficients as a function of temperature. Interestingly, the thermal expansion coefficients are almost the same. This finding suggests that the dilatation of the lipid headgroup area is not isotropic but occurs in the plane of the membrane along one direction perpendicular to the DNA axis (Figure 2). Because of the strong electrostatic interaction between cationic lipid headgroups and phosphate groups on the DNA backbone, the dilatation along the DNA axis would be forbidden. In addition, RA (Table 2) is much lower than that reported by Heimburg36 for pure fluid DPPC membranes (RA ≈ 4.2 × 10-3 K-1 at T ) 50 °C) and that reported by Pabst37 et al. for pure fluid POPC membranes (RA ≈ 3.945 × 10-3 K-1 at T ) 35 °C). Conversely, the thermal thickness expansion coefficient, RB (Figure 5), is comparable with the values reported by Heimburg (RB ≈ -1.6 × 10-3 K-1 at T ) 45 °C) and by Pabst et al. (RB ≈ -1.905 × 10-3 K-1 at T ) 35 °C). Our structural findings are plausible but do not represent definitive proof for these conclusions because the values of A calculated from eq 5 could suffer from the intrinsic approximations of the electrostatic model. In more detail, eq 4 is derived by considering that, upon lipoplex formation, the Coulombic attraction between DNA and cationic lipids induces the polarization of the positive charge carried by the lipids along the DNA axis (Figure 8). Cationic lipids are pushed out from between the DNA positions and migrate in the plane of the membrane to match the negative charge carried by the DNA. Such lipid demixing is due to the natural tendency of cationic lipids to replace DNA counterions acting like 2D condensed “counter lipids”.38-40 The basic assumption of the model is that treating DNA molecules as lines of charge with counter lipids merely modifies the charge distribution along the DNA axis, leading the (36) Heimburg, T. Biochim. Biophys. Acta 1998, 1415, 147. (37) Pabst, G.; Rappolt, M.; Amenitsch, H.; Laggner, P. Phys. ReV. E 2000, 62, 4000. (38) Harries, D.; May, S.; Gelbart, W. M.; Ben-Shaul, A. Biophys. J. 1998, 75, 159. (39) May, S.; Ben-Shaul, A. Curr. Med. Chem. 2004, 11, 1241. (40) May, S. J. Phys. Condens. Matter 2005, 17, 833.

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Table 3. Structural Parameters of DOTAP-DOPC Membranes Calculated from the Electron Density Profilea T (°C)

F˜ r

dH (Å)

dC (Å)

A (Å2)

RA (10-3 K-1)

5 20 35 50 65

1.6 1.53 1.56 1.52 1.5

4.88 4.68 4.6 4.55 4.19

13.93 13.9 13.6 13.55 13.48

68.1 69.3 70.8 71.3 74.1

1.468 1.443 1.412 1.403 1.350

a

Equation 8. Table 4. Interfacial Area Per Lipid Moleculea and Corresponding Thermal Expansion Coefficients, aA,b as a Function of Temperature

T (°C) A (Å2) (eq 5) RA (10-3 K-1) A (Å2) (eq 8) RA (10-3 K-1) 5 20 35 50 65

67.9 69.7 71.6 72.6 74.1

1.509 1.470 1.432 1.412 1.383

68.1 69.3 70.8 71.3 74.1

1.468 1.443 1.412 1.403 1.350

a Calculated from the electrostatic model, eq 5, and from electron density profiles, eq 8. b Calculated from eq 6.

DNA-DNA interaction to become a simple and analytically tractable 1D problem. Thus, before claiming that embedded DNA molecules really force the dilatation area of lipid molecules to occur only along the DNA-DNA direction, supporting evidence should be provided unambiguously. Recently, Pabst and co-workers have calculated the area per lipid on the basis of an integration of the EDP.37 The developed full q refinement model has the advantage of deriving A without further information on the specific volume of the lipid. Applying the method, the Laggner group could investigate in detail the thermal behavior of phosphatidylcholine and phosphatidylethanolamine membranes.25 To determine the area per lipid, the formalism of Lemmich et al.41 was applied ,which yields

(

)

F˜ rneC neH 1 A) dH FCH2(F˜ r - 1) dC

(8)

where FCH2 is the methylene electron density (0.317 e/Å3), F˜ r ) (FH - FCH2)/(FC - FCH2) is the ratio between the electron density of the headgroup (FH) and that of the hydrocarbon tails (FC) both relative to the methylene electron density, neC is the number of hydrocarbon electrons, neH is the number of headgroup electrons, dC is the hydrocarbon chain length, and dH is the headgroup size. This method could lead to an underestimation of the area per lipid as discussed by Rappolt et al.25, but the relative changes should be the same. All of the structural parameters were retrieved from the EDPs and are listed in Table 3. It is remarkable that, within errors, both independent methods result in the same values for the interfacial area and for the linear expansion coefficients (Table 4). We underline that the second method used for the calculation of A does not use the approximations of the discussed electrostatic model. Indeed, it is based on the exclusive use of the EDPs that are, in turn, calculated by the experimental SAXD patterns. Thus, the most convincing explanation is that charge polarization along the DNA axis really modifies the structural properties of lipid bilayers in that it forces bilayer expansion to occur along the direction perpendicular to the DNA axis. At the molecular (41) Lemmich, J.; Mortensen, K.; Ipsen, J. H.; Hønger, T.; Bauer, R.; Mouritsen, O. G. Phys. ReV. E 1996, 53, 5169.

Figure 9. DNA spacing of isoelectric DOTAP-DOPC/DNA lipoplexes as a function of surface charge density, σM, as determined according to eq 5.

level, DNA acts as a grid with lipid molecules constrained to follow the dilatation of the 1D DNA lattice. This finding demonstrates quantitatively that the lateral expansion of the lipid bilayer within DOTAP-DOPC/DNA lipoplexes is governed by the lateral expansion of the DNA inplane rod lattice and that the increase in DNA spacing is a direct and quantitative measure of the area dilatation of the lipid bilayer itself. The excellent agreement between the interfacial area values calculated by the electrostatic model and by the EDPs confirms that the complex molecular architecture of the unit cell of the lipoplex can be successfully simplified as proposed33 (Figure 8) and that the DNA-DNA interaction within lamellar lipoplexes can be regarded as a 1D problem. Furthermore, it has recently been demonstrated that the surface charge density, σΜ, is the constraint regulating DNA packing density within lamellar lipoplexes and depends on both ΦW and A according to the following relation23

σM(T) )

(1 - ΦW) A(T)

(9)

We inserted into eq 9 the values of A displayed in Figure 7, thus evaluating the effect of lateral area expansion on the surface charge density of lipid membranes. Thus, we wish to account for the variation in the DNA rod lattice in a way that depends on the renormalization of the surface charge density of DOTAPDOPC membranes, which in turn depends on the temperatureinduced lateral expansion of lipid headgroup area A. In the present study, dDNA varies as a function of σΜ as displayed in Figure 9. The present results are in close agreement with the findings of Martin-Herranz et al., who recently found that the condensation of DNA by different lipid systems forming lamellar lipoplexes follows a universal charge-condensation curve.42

Conclusions Using simultaneous synchrotron SWAXD, we have investigated the thermotropic behavior of DOTAP-DOPC/DNA lipoplexes. As a function of increasing temperature, the lipid bilayer thickness and the thickness of the interbilayer water region were found to decrease whereas the DNA spacing increased. Using two independent models, we have calculated the temperature dependence of the lipid headgroup area A. We have shown that the thermotropic dilatation area of lipid headgroups within DOTAP-DOPC/DNA lipoplexes has only one degree of (42) Martin-Herranz, A.; Ahmad, A.; Evans, H. M.; Ewert, K.; Schulze, U.; Safinya, C. R. Biophys. J. 2004, 86, 1160. (43) Caracciolo, G.; Pozzi, D.; Amenitsch, H.; Caminiti, R. Langmuir 2005, 21, 11582.

Dilatation Area of Lipid Headgroups

freedom in that it occurs only along the direction perpendicular to the DNA axis. As a consequence, the variation in DNA spacing is a quantitative measure of the dilatation area of the lipid headgroups. An understanding of the molecular packing of DNA

Langmuir, Vol. 22, No. 9, 2006 4273

within lamellar lipoplexes may be valuable for the future development of biomolecular templates for DNA storage.43 LA0534423