ACTA FACULTATIS PHARMACEUTICAE UNIVERSITATIS COMENIANAE Tomus LV 2008

POLYMORPHIC PHASE BEHAVIOR OF DNA – DOPE – GEMINI SURFACTANT AGGREGATES: A SMALL ANGLE X-RAY DIFFRACTION 1

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Pullmannová, P. – 1Uhríková, D. – 2Funari, S.S. – 3Lacko, I. – 3 Devínsky, F. – 1Balgavý, P.

Department of Physical Chemistry of Drugs, 3Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University, Bratislava 2 HASYLAB DESY, Hamburg

Polymorphic phase behavior of DNA – dioleoylphosphatidylethanolamine (DOPE) – alkane-α,ω-diyl-bis(dodecyldimethylammonium bromides) (CnGS12, spacer n = 2 and 12) aggregates was examined in the range 10 – 80 °C. At molar ratio DOPE : CnGS12 = 0.15, aggregates form a condensed lamellar phase with DNA strands packed irregularly (Lαc). The increase in temperature induces Lαc → condensed inverted hexagonal phase (HIIc) transition. The phase transition temperatures ~ 46 °C and ~ 90 °C for DNA + DOPE + CnGS12, n = 2 and 12 aggregates were determined at the equal volume fractions of both phases. Key words: gemini surfactants – DNA – dioleylphosphatidylethanolamine – small angle X-ray diffraction

INTRODUCTION Lipoplexes formed due to the DNA interaction with cationic liposomes has been found as potential gene delivery systems [1]. Cationic liposomes are prepared either from cationic surfactants (cationic lipids) or as a mixture of cationic surfactants and neutral phospholipids. Cationic surfactants are designed to interact with the phosphate groups of polynucleotides, thus reducing the negative surface charge of aggregates. This results in minimization of the electrostatic repulsion at the surface of biological membranes and facilitation of DNA entrance into cells. The best levels of transfection in vivo were obtained at isoelectric point of a mixture, when used ratio of positive/negative charges is around 1 [2]. Cationic surfactants for gene delivery may

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cause toxic effect in vivo and in vitro that is still an obstacle to the application of non-viral vectors to gene therapy [3]. The inclusion of neutral phospholipids (helper lipid) in cationic liposomes in many (but not all) cases improves lipoplex efficiency and decreases their toxicity. Helper lipids affect lipoplex electrostatics as well as the way the lipid self-assembles (micellar, lamellar, hexagonal, etc.), the level of hydration, and DNA secondary and tertiary structure ([4] and references therein). For in vitro transfection a favorable helper lipid is 1,2-di-oleoyl-sn-glycero-3-phosphatidyl-ethanol-amine (DOPE), which enables better matching of charge density of the lipid surface to DNA helices, facilitates counterion release from the lipid surface by DNA [5], and decreases lipid hydration [6]. Gemini surfactants CnGSm with two alkyl chains (m is the number of carbons in the alkyl chain) and two quaternary ammonium groups connected by a chain referred to as a spacer (n is the number of carbons in the spacer) have been intensively investigated as DNA delivery vectors [7-10]. Of almost 250 compounds of some 20 different structural types tested in [11], a majority showed very good transfection activity in vitro. Zana and Benrraou [12] reported that the interaction of a given polyanion with gemini surfactants is stronger than with the corresponding alkyltrimethylammonium bromide or two-chain surfactants dodecyldimethyl(alkyl)-ammonium bromide. In spite of many tests of DNA-CnGSm for transfection activity [11], the lipoplexes prepared as a mixture of neutral phospholipids and CnGSm have been examined rarely. It is worth to mention that cationic liposomes prepared as a mixture of gemini surfactants C3GS16 (polymethylene spacer) with saturated dipalmitoylphosphatidylcholine (DPPC) and DOPE were successfully used in vivo as a topical delivery DNA vectors across the intact skin at scleroderma in mice [13]. Surface and colloidal properties of lipoplexes determine their transfection efficiency. Three types of organized DNA – phospholipid – cationic surfactant microstructures were identified: i) spaghetti-like structures in which DNA is covered by a cylindrical lipid bilayer [14], ii) condensed columnar inverted hexagonal phase (HIIc) with linear DNA molecules surrounded by lipid monolayers forming inverted cylindrical micelles arranged on a hexagonal lattice [15], iii) condensed lamellar phase (Lc) with ordered DNA monolayers intercalated between lipid bilayers [16]. Recent data indicate that lamellar complexes (Lc) have transfection efficiencies as high as those formed by hexagonal phase (HIIc) [17]. In our previous work [18] we have found that calf thymus DNA at interaction with dilauroylphosphatidylcholine liposomes (DLPC) in the presence of butanediyl-1,4-bis-(dimethyllauroylammonium bromide) (C4GS12) at molar ratio DLPC : C4GS12 : DNA = 2 : 1: 1.6 (mol/mol/base) forms a condensed lamellar phase Lαc (so called sandwich structure). With increasing proportion of C4GS12 in aggregates prepared from unilamellar DLPC liposomes, we have observed the formation of microscopic domains enriched by surfactant molecules [19]. The condensed lamellar phase Lαc was also observed in aggregates with egg yolk phosphatidylcholine (EYPC) prepared at isoelectric point in large range of molar ratios 2 ≤ EYPC : GnGS12 ≤ 9 for CnGS12, n = 2 and 10 (polymethylene spacer). We have found that the distance between adjacent DNA strands increases linearly in the range 3.6 ≤ dDNA ≤ 6.2 nm in aggregates EYPC : C2GS12 ≤ 9 mol/mol. Two different ways of DNA ordering were observed

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in aggregates DNA + EYPC + C10GS12 in dependence on the EYPC : C10GS12 molar ratio [20]. The aim of this work is the study temperature induced polymorphic phase behavior of the aggregates prepared due to DNA interaction with DOPE and CnGS12 (n = 2 and 12). The observed Lαc → HIIc phase transition is discussed using a simplified approach based on a preferred shape of the lipid molecule and a spontaneous curvature of adopted phases [21-23].

MATERIALS AND METHODS Highly polymerized salmon sperm DNA (sodium salt) (Applichem, Darmstadt, Germany) at concentration 3 mg/ml was dissolved in 0.1 mol/l NaCl, pH ≈ 6.1. The precise value of concentration was determined spectrophotometrically, by measuring the absorbance Aλ at wavelength λ = 260 nm. The purity of DNA was checked by measuring the absorbance Aλ at λ = 260, 230 and 280 nm. We have obtained the values of A260/A230 = 2.23 and A260/A280 = 1.76. The NaCl of analytical purity was obtained from Lachema, Brno, Czech Republic. The aqueous solution of 0.1 mol/l NaCl was prepared with redistilled water, pH ≈ 6.1. Alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, n = 2 and 12) were prepared as described in [24] and purified by manifold crystallization from a mixture of acetone and methanol. Dioleylphosphatidylethanolamine (DOPE) was purchased from Avanti Polar Lipids, Alabaster, USA. The samples were prepared at isoelectric point DNA : CnCS12 = 2 : 1 base/mol, at the molar ratios CnCS12 : DOPE = 0.15 : 1. The required amounts of DOPE and alkylammonium salt (~ 5 mg per sample) were mixed in organic solvent. The solvent was evaporated under a stream of gaseous nitrogen and its residue removed by an oil vacuum pump. The dry mixture was hydrated by NaCl and DNA solutions. The sample was vortexed for a short time and the sediment created in the sample few minutes after preparation was placed between two Kapton foil (Dupont, France) windows of a sample holder for X-ray diffraction. Small- (SAXD) and wide-angle (WAXD) synchrotron radiation diffraction experiments were performed at the soft condensed matter beamline A2 at HASYLAB at the Deutsches Elektronen Synchrotron (DESY) in Hamburg (Germany), using a monochromatic radiation of wavelength λ = 0.15 nm. The evacuated double-focusing camera was equipped with two linear delay line readout detectors. The sample was equilibrated at selected temperature (20 °C) for 5 min before exposure to radiation. Temperature scans were performed at a scan rate 1 °C/min and the diffractograms were recorded for 10 s every minute. The raw data were normalized against the incident beam intensity using the signal intensity measured in the ionisation chamber. The SAXD detector was calibrated using rat tail collagen [25] and the WAXD detector by tripalmitin [26,27]. Each diffraction peak of SAXD region was fitted with a Lorentzian above a linear background. The WAXD pattern of all measured samples exhibited one wide diffuse scattering in the range s ~ 1.8 – 3.2 nm-1, characteristic for liquid-like

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carbon chains of phospholipid and CnGSm molecules. We do not show WAXD pattern in the Figure 1.

RESULTS AND DISCUSION Figure 1A shows SAX diffractograms of the fully hydrated DOPE at 20 and 60 °C. Three reflections at reciprocal spacing ratios 1 : √3 : 2 are a typical diffraction pattern of a hexagonal phase, two-dimensional periodic structure. DOPE has a relatively small and weakly hydrated headgroup and attractive headgroup-headgroup interactions. According to the Israelachvili’s concept of an effective lipid molecular shape [21], double-chained lipids with small head-group area have an inverted truncated coneshaped molecules and form inverted spherical or cylindrical micelles. The micelles can aggregate into ordered phases. DOPE has a strong tendency to adopt the inverse hexagonal phase HII. An inverted hexagonal phase is favored for molecules when the critical packing parameter VA0/lc > 1, where V is the molecular volume, A0 is the equilibrium area/molecule at the lipid – water interface, and lc is the critical chain length, which cannot be greather than the length of the fully exteded, all-trans chain. The lattice parameter a = 7.67 ± 0.01 nm at 20 °C was determined by a = 2/√3s1, where s1 is the position of the first order diffraction peak’s maximum. With increasing temperature the lipid keeps its organization. We observed the decrease in a (a = 6.92 ± 0.01 nm at 60 °C) what corresponds well with our previous data [28]. Instead, decreasing temperature retards the motion of phospholipid’s acyl chains, and the change in molecular shape may result in a change in long range order of the phase. Fully hydrated DOPE shows the inverted hexagonal → lamellar phase transition (HII → Lα) at 5 – 10 °C, depending on the ionic strength of the solution, also history and handling of sample may play a role [29,30].

The aggregates DNA – DOPE – CnGS12 Figures 1B, C show diffractograms of the DNA + DOPE + CnGS12, n = 2 and 12 aggregates at 20 °C and 60 °C. At 20 °C, we observed two reflections at the spacing 1/s1 = 2/s2 = d, characteristic for a lamellar phase. Lamellar phase is one-dimensional periodic structure with the repeat distance d. We found the repeat distances d = 6.97 ± 0.01 nm and d = 6.63 ± 0.01 nm in aggregates DNA + DOPE + CnGS12 for n = 2 and 12, respectively. As follows from the diffractograms, the aggregates show completely different long range organization in comparison to the pure DOPE. One can suppose following mechanism of the interaction: The polar groups of CnGS12 interact with the lipid head groups and due to hydrophobic interaction, its alkyl chains intercalate into the hydrophobic core of the lipid bilayer. The orientation of alkyl chains of the CnGS12 is parallel to the lipid alkyl chains. The monounsaturated oleic acid contains 18 carbons in hydrocarbon chain. Incorporation of CnGS12 into the lipid bilayer causes two effects: a creation of a free volume localized under the shorter surfactant alkyl chains and a lateral expansion of the bilayer [31]. The free volume can be eliminated by the increased frequency of trans-gauche isomerisation of hydrocarbon chains or by their

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bending. The lipid bilayer becomes positively charged due to incorporation of cationic surfactants and due to interaction with DNA polyanion, the DNA + DOPE + CnGS12 aggregates are formed. The lamellar phase is favored when the critical packing parameter VA0/lc ~ 1. L (1) L (1) ↓H (1,0)

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Fig. 1. The SAXD patterns of DOPE (A), DNA – DOPE – C2GS (B) and DNA – DOPE – C12CS12 (C), (h,k) are Miller indices.

As it follows from the Figure 1, already a small portion of CnGS12 n = 2 and 12 (CnGS12 : DOPE = 0.15 mol/mol) stabilizes the lamellar phase of DOPE to higher temperatures (in comparison to pure DOPE). However, the electrostatic interaction of DNA with the positively charged CnGS12 + DOPE bilayers may play a role too. It was documented that DNA improves the organization of the lipid bilayer stacking [18,32]. The obtained repeat distances d = 6.97 and 6.63 nm for DNA + DOPE + CnGS12, n = 2 and 12, respectively, correspond well with d = 6.90 and 6.70 nm when the aggregates DNA + EYPC + CnGS12, n = 2 and 10 were prepared with phosphatidylcholine [20]. Koltover [15] has found the repeat distance d = 6.35 nm in Lαc phase of DOTAP + DOPE mixture at weight fraction of DOPE Φ DOPE = 0.41 and a regular

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packing DNA strands with the periodicity dDNA = 5.81 nm. Diffractograms in Figure 1 do not show any reflection related to DNA organization between the lipid’s lamellae, what indicates an irregular packing of DNA strands. The DNA packing can be affected by the cation surface charge density of liposomes and by the ionic strength of the solution. The molar ratio CnGS12 : DOPE = 0.15 in our aggregates represents rather low surface charge density that was chosen to avoid a demixing of CnGS12 + DOPE mixture into a two phase system as we observed in DNA + DLPC + C4GS12 aggregates [19]. The 0.1 mol/l NaCl represents ionic strength of solution close to physiological conditions. The increasing ionic strength affects the electrostatic interaction between DNA and cationic liposomes as documented by the DNA condensation followed by fluorescence spectroscopy [33,34], the high ionic strength completely shields the interaction. Thus, probably both the low surface charge density as well as the ionic strength of solution are responsible for the absence of DNA related reflection in the diffractograms in Figure 1. In spite of many X-ray diffraction experiments on DNA-cationic liposome aggregates prepared either in pure water or at very low ionic strength (see e.g. [15-17,35,36]), structural changes induced by physiologically relevant ionic strength of solution are documented rarely. Boukhnikachvili et al. [32] have studied DNA complexes with polycationic lipid dioctadecylamidoglycylspermine (DOGS) prepared in a 0.15 mol/l NaCl solution. In the DOGS + DNA complexes they found the lamellar repeat distance d ~ 6.45 nm and no diffraction peak corresponding to DNA sub-lattice in DOGS + DNA complexes. Temperature induced changes in the structure of aggregates were examined up to 60 °C, resp. 80 °C. Figure 1 (B and C) shows SAXD patterns of DNA + DOPE + CnGS12, n = 2 and 12 at 60 °C. The diffractograms identified the coexistence of a lamellar and a hexagonal phase in both aggregates, hovewer the volume fraction of the hexagonal phase is different and depends on the length of the spacer n. The repeat distance of lamellar phase d and the lattice parameter a were determined by analysis as described above. At 60 °C, we found the repeat distance d = 6.20 ± 0.01 nm for the lamellar phase and the lattice parameter a = 6.93 ± 0.01 nm of the hexagonal phase in DNA + DOPE + C2GS12 aggregates. Temperature dependences of d and a are shown in Figure 2. With increasing temperature, we observed the decrease in the repeat distance d, and at ~ 33 °C one may recognize the onset of a hexagonal phase in DNA + DOPE + C2GS12 aggregates (Figure 2). The following increase in temperature induces Lαc → HIIc phase transition. Figure 3 displays temperature induced changes in intensities of the first order reflections of both phases. We determined t ~ 46 °C (the cross point) as the temperature of the Lαc → HIIc phase transition when volume fractions of both phases are equal. After heating the sample up to 60 °C, the presence of the lamellar phase is still apparent as show Figure 1 and Figure 3. Temperature behaviour of DNA + DOPE + C12GS12 aggregates is similar. At 60 °C, we found the repeat distance d = 6.07 ± 0.01 nm for the lamellar phase and the lattice parameter a = 6.76 ± 0.01 nm of the hexagonal phase in DNA + DOPE + C12GS12 aggregates. Temperature dependences of d and a are given in Figure 2. The aggregates prepared with C12GS12 show the onset of a hexagonal phase at higher temperature ~ 51 °C.

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7.2

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Fig. 2. Temperature dependences of the lattice parameters (d, a) of DNA – DOPE – CnCS12, n = 2 and 12 aggregates

Temperature induced changes in the first order reflection intensities are shown in Figure 3. Aggregates were heated up to 80 °C, but as follows from the Figure 3, the lamellar phase was dominant in the whole temperature range. To extrapolate the dependences in the figure, one can guess the Lαc → HIIc phase transition temperature of ~ 90 °C. Our experimental results indicate the stabilization of lamellar phase in the DOPE + CnGS12 mixtures against Lαc → HIIc phase transition. Several theoretical approaches to the molecular basis of the nonlamellar phase behavior of lipids have appeared in the literature (for review see [37]). We try to explain the observed polymorphic behavior of the aggregates using the approach based on a preferred shape of the lipid molecule and a spontaneous curvature of adopted phases [21-23]. Gruner [22] has suggested that the phase behaviour is largely the result of a competition between the tendency for certain lipid monolayers to curl and the hydrocarbon packing strains that result. The tendency to curl is quantitatively given by the intrisic radius of curvature, R0, which minimizes the bending energy of a lipid monolayer.

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DNA-DOPE-C12GS12 40000

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Fig. 3. Temperature dependences of the first order reflection intensities of Lαc and H IIc phases of DNA + DOPE + CnGS12, n = 2 and 12 aggregates

Taking simplified model coming out from the „preferred shape“ of molecule (i.e. the shape of the average volume it would occupy in a monolayer) one can assume: Lipids for which the preferred shape is of uniform cross section along their length (cylinder) would have zero spontaneous curvature and should form stable bilayers. Molecules for which the preferred shape is characterized by headgroup areas that exceed the crosssectional areas of their hydrocarbon chains (truncated cone) would have negative R0 values and should form stable micelles or type I phases (normal, oil-in-water, for nomenclature see [23]). And those lipids for which their preferred shapes are characterized by headgroup areas that are smaller than the cross-sectional areas of their hydrocarbon chains (inverted truncated cone) would have small positive R0 values and should be predisponed to form stable inverted (type II) phases (inverse, water-in-oil). Molecules CnGS12 in dependence on the length of the spacer, in excess of water and above their critical micellar concentrations, associate into miscellaneous structures as a spherical, rodlike or disc-like micelles, and also vesicles were observed [38-41]. Increase in the length of spacer is accompanied by increase in its hydrophobicity, which

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remains suppressed probably due to steric factors till the length of spacer acquires n ~ 5. Above this value the hydrophobic nature of the linking chain starts manifesting itself [42]. At n ≥ 10 the spacer becomes too hydrophobic to remain in contact with water and moves into hydrophobic interior (or to the air side of the interface) adopting the folded conformation [43]. The length of spacer may in this way affect the radius of curvature of micelle to the larger R0 value, however not the sign of curvature (negative). The simultaneous presence in a bilayer of a lipid with a small positive radius of curvature R0 (DOPE) with another lipid (or surfactants) with a larger R0 or R0 with the opposite sign (CnGS12) leads to the phenomenon known as frustration [44], that is at the critical edge of bilayer stability. The balance between lamellar and non-lamellar (such as HII) phases is governed by several factors as the mixture composition, the thermodynamics of the interaction of the membrane surface with the aqueous phase, temperature, etc. (for review see [45]). We observed Lαc → HIIc phase transition induced by temperature. The increased temperature increases the surface area per lipid in the bilayer. For lipids having small weakly hydrated headgroups, and having attractive headgroup-headgroup interactions, this leads to energetically disadvantageous contact between the aqueous and hydrophobic regions and to a weakening of any residual headgroup-headgroup bonding, resulting in the bilayers destabilization in favor of the HII phase. Our results also demonstrate that small changes in the design of molecule (the length of spacer) may significantly affect the stability of the phase. The mechanism by which interconversions between lamellar and inverted nonlamellar phases of lipids take place is poorly understood and several models has been developed resulting from 31P NMR or time resolved X-ray diffraction studies [46]. Essential requirements for a DNA transfection vector are that it should bind DNA sufficiently strongly and rapidly, readily penetrate the target cell and perhaps its nucleus, and eventually release DNA from the lipoplex inside the target cell at the right time and in the correct place [2]. The endocytosis with a several key steps was proposed as a mechanism of the lipoplex passage through the cell membrane [47]. The releasing of the lipoplex from the endosome is a strategic point at which the phase transition Lαc → HIIc in structural organization of the lipoplex can play an important role. Our experiments documents the ability of gemini surfactants to condensate DNA into the aggregates with organized structure and to induce lipoplex Lαc → HIIc phase transition as a function of composition of cationic liposome, temperature and the design of gemini surfactant molecule. Acknowledgement: This work was supported by the European Community – Research Infrastructure Action under the FP6 „Structuring the European Research Area“ Programme (through the Integrated Infrastructure Initiative „Integrating Activity on Synchrotron and Free Electron Laser Science“): RII3-CT-2004-506008 (IA-SFS), HASYLAB project II-20052037 EC; by the JINR project 07-4-1031-99/2008 and by the VEGA grant 1/3029/06 to DU.

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Doc. RNDr. Daniela Uhríková, CSc. Faculty of Pharmacy Comenius University Odbojárov 10 832 32 Bratislava Slovak Republic [email protected]

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POLYMORFNÉ SPRÁVANIE AFREGÁTOV DNA – DOPE – GEMINI SURFAKTANTY: RTG DIFRAKCIA POD MALÝMI UHLAMI 1

Pullmannová, P. – 1Uhríková, D. – 2Funari, S.S. – 3Lacko, I. – 3 Devínsky, F. – 1Balgavý, P.

1

Katedra fyzikálnej chémie liečiv, 3Katedra chemickej teórie liečiv, Farmaceutická fakulta, Univerzita Komenského, Bratislava 2 HASYLAB DESY, Hamburg

Metódou rtg difrakcie pod malými uhlami sme vyšetrovali polymorfné správanie agregátov DNA – dioleoylfosfatidyletanolamín (DOPE) – alkán-α,ω-diylbis(dodecyldimetylamóniumbromidy) (CsGS12, s = 2 a 12 je dĺžka spojovacieho reťazca) v teplotnom rozsahu 10 – 80 °C. Pri mólovom pomere DOPE : CsGS12 = 0,15 C tvoria agregáty pri 20 °C kondenzovanú lamelárnu fázu s nepravidelne usporiadnými vláknami DNA (Lαc). S rastúcou teplotou sme pozorovali fázový prechod Lαc → kondenzovaná hexagonálna fáza (HIIc). Teploty fázového prechodu ~ 46 °C a ~ 90 °C pre agregáty DNA + DOPE + CsGS12, s = 2 a 12, boli stanovené pri rovnakom objemovom podiele oboch fáz.

Acta Facult. Pharm. Univ. Comenianae 55, 2008, p. 170-182.

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