DOTAP cationic liposomes and their interactions with DNA and drugs

Chemistry and Physics of Lipids 158 (2009) 91–101

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Spectroscopic characterization of DMPC/DOTAP cationic liposomes and their interactions with DNA and drugs Jean-Franc¸ois Labbé a , Francis Cronier a , René C.-Gaudreault b,c , Michèle Auger a,∗ a Département de Chimie, Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Centre de Recherche sur les Matériaux Avancés, Université Laval, Québec, Québec, Canada G1V 0A6 b Unité des Biotechnologies et de Bioingénierie, Centre de recherche, CHUQ, Hôpital Saint-Franc¸ois d’Assise, Université Laval, Canada G1L 3L5 c Faculté de médecine, Québec, Québec, Canada G1V 0A6

a r t i c l e

i n f o

Article history: Received 25 August 2008 Received in revised form 12 December 2008 Accepted 9 January 2009 Available online 22 January 2009 Keywords: Cationic liposomes Membranes DNA Arylchloroethylureas FTIR NMR

a b s t r a c t Gene and synthetic drug-delivery vectors have been developed and characterized to treat several genetic diseases and cancers. Our study aims at characterizing cationic liposomes containing the zwitterionic phospholipid DMPC and the cationic lipid DOTAP as well as their interactions with two types of DNA and a new class of antineoplastic agents derived from arylchloroethylureas (CEU). Results obtained using FTIR spectroscopy as well as 31 P and 2 H NMR indicate that DMPC and DOTAP form cationic liposomes in a highly disordered fluid phase at a molar ratio of 1:1. In addition, the FTIR results indicate that the presence of DNA or CEUs within the liposomes does not significantly affect the conformational order of both the DMPC and DOTAP acyl chains. Our results therefore provide a detailed characterization of complexes between cationic liposomes and both DNA and drugs and indicate that these complexes are stable and fluid assemblies. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The concept of gene therapy is based on the administration of therapeutic nucleic acids using viral or synthetic vectors. To date, the most promising and investigated non-viral approach is the use of cationic liposomes as gene carriers (Davies, 2006; Felgner et al., 1987; Rosenecker et al., 2006; Ziady and Davis, 2006). Cationic liposomes have shown promises, notably, for the treatment of cystic fibrosis in phase I clinical trials (Porteous et al., 1997; Ziady and Davis, 2006). Cationic liposomes and DNA form spontaneously supramolecular self-assemblies called lipoplexes (Felgner et al., 1997; Kreiss and Scherman, 1999), allowing a very efficient gene transfer at least in vitro. DOTMA (N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium chloride) was the prototype cationic lipid used by Felgner et al., who have shown that formulations made of DNA and cationic and zwitterionic lipids can transfer genes with a high efficiency in vitro without seric proteins (Felgner et al., 1987; Kreiss and Scherman, 1999). Lipoplexes show a wide degree of polymorphism that depends on lipid concentration, cationic liposomes/DNA ratio (R +/−)

∗ Corresponding author at: Département de Chimie, CREFSIP, CERMA, Université Laval, 1045 Avenue de la médecine, Québec, Québec, Canada G1V 0A6. Tel.: +1 418 656 3393; fax: +1 418 656 7916. E-mail address: [email protected] (M. Auger). 0009-3084/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2009.01.002

(Almofti Mohamad et al., 2003) and the composition of the medium (Litzinger and Huang, 1992; Zuidam et al., 1999). These parameters affect critically the structure of the lipoplexes and their transfection efficiency. More specifically, transfection efficiency is reported to be related to the morphology of the cationic lipid formulation, which can be modulated by the presence of non-bilayer forming neutral co-lipids such as DOPE (1,2-dioleoyl-sn-glycero3-phosphoethanolamine), well known to form inverted hexagonal phase. The use of this lipid was proposed to enhance DNA transfection in vitro (Simberg et al., 2001). On the other hand, according to thermodynamic modeling studies, lamellar complexes, in which DNA intercalates between lipid bilayers, are the most stable (Fig. 1) (Dan, 1998). In addition, this is the most studied and proposed morphology (Dan, 1997, 1998; Harries et al., 1998; Koltover et al., 1999; Lasic et al., 1997). Lamellar complexes have an ordered liquid-crystalline structure made of locally plane bilayers (Safinya et al., 2006), maintained in that morphology by using a mixture of cationic and neutral lipids leading to a bilayer structure. While cationic liposomes prepared using both cationic and zwitterionic phospholipids are exhaustively studied as gene vectors, the potential of these drug-delivery systems has not been extensively exploited (Campbell et al., 2001a). However, Campbell et al. have shown using circular dichroism that the inclusion of DOTAP (1,2-dioleoyl-3-N,N,N-trimethylammonium propane) in PC bilayers increases the stability of liposome preparations containing paclitaxel (taxol), an anticancer drug (Campbell et al.,

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used without further purification. DMPC-d4 was deuterated at both the CH2 ␣ position (closer to the phosphate group) and the CH2 ␤ position (closer to the choline head group). Calf thymus DNA was obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Plasmidic DNA pBQ6.2 containing the CFTR gene was obtained from Mrs. Johanna Rommens (Hospital for Sick Children, Toronto, ON) and purified using the protocol Quiagen maxi (Quiagen, Chatsworth, CA). 4-n-Butyl and 4-sec-butyl CEU were synthesized as described previously (Béchard et al., 1994; C.-Gaudreault et al., 1988). 2.2. Preparation of samples

Fig. 1. Schematic structure of a lamellar complex.

2001a). Moreover, it is reported that formulations for drug delivery could use unsaturated and saturated phosphatidylcholines, such as DLPC (1,2-dilauryl-sn-glycero-3-phosphocholine) and DMPC (1,2dimyristoyl-sn-glycero-3-phosphocholine) (Kitagawa et al., 2004). It has also been shown by Kitagawa and Kasamaki that the intradermal delivery of retinoic acid is achievable using DMPC or DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) vesicles and that the incorporation of the drug is enhanced and dependent on the DOTAP concentration (Kitagawa and Kasamaki, 2006). Recently, Cortesi et al. have developed cationic liposomes formulations for the ocular administration of peptides exhibiting anti-herpetic activity (Cortesi et al., 2006). To vectorize drugs using cationic liposomes, the main challenge is to reach selectively tumours and inflammation sites to achieve therapeutical concentrations of the drugs (Barenholz, 2001). Considering this, liposomes differ from other drug-delivery systems, which act in plasma or at the site of administration. As discussed above, several applications associated to drug-delivery systems involve the incorporation of large uncharged hydrophobic molecules in the hydrophobic domains of the liposome membranes (Campbell et al., 2001a). Nevertheless, drug vectorization is a promising area of research that remains rather poorly explored and studied so far. In addition, a number of issues such as (i) the efficiency of the drug encapsulation, (ii) their stability in biological media, (iii) the use of small lipid particles having a high transition temperature for an optimal biological half-life in the blood circulation, and (iv) the selectivity of tissues and organs localization (Campbell et al., 2001a) are important to be considered in their development. In this paper, we have characterized cationic liposomes made of DMPC and DOTAP, as well as their interactions with DNA and amphipathic anticancer drugs, using Fourier transform infrared (FTIR) and 31 P and 2 H solid-state NMR spectroscopy. We report results obtained with two types of DNA, namely linear calf thymus DNA and the plasmid pBQ6.2 containing the CFTR (cystic fibrosis transmembrane conductance) gene. We also report results obtained with two drugs, which are constitution isomers, 4-n-butyl and 4-sec-butyl chloroethylurea (CEU) (Béchard et al., 1994; C.-Gaudreault et al., 1994). More specifically, our aim is to compare the stability and fluidity of DMPC/DOTAP liposomes in the presence of DNA and both drugs. 2. Materials and methods 2.1. Materials DMPC ± d54 (deuterated on both acyl chains), DMPC-d4 and DOTAP were obtained from Avanti Polar Lipids (Albaster, AL) and

2.2.1. Cationic liposomes Each lipid was supplied in a chloroform solution and an appropriate volume of each solution was used according to the desired molar ratios. Chloroform was evaporated under a nitrogen stream and lyophilized overnight before storage at −25 ◦ C. Approximately, 24 h before the analysis, the lipid mixtures were hydrated with a 20 mM Hepes, 150 mM NaCl and pH 7.4 buffer solution to get lipid dispersions at 4% (w/v) for FTIR experiments and 15% (w/v) for NMR experiments. The pH of the aqueous dispersions was maintained between 5 and 7, leaving the liposomes with a residual positive charge (DOTAP is fully charged at all pH (Zuidam and Barenholz, 1997) and the phosphate polar head group of DMPC has a pKa ≤ 1 (Marsh, 1990)). The samples were submitted to three cycles of freeze (−195 ◦ C)–thaw (37.5 ◦ C)–vortex cycles immediately after hydration and stored at −25 ◦ C until the analysis, when five additional cooling–heating–vortex cycles were performed before the addition of DNA. 2.2.2. DNA Two types of DNA were used for a comparative study: linear calf thymus DNA and the plasmid pBQ6.2 containing the CFTR gene. An aqueous suspension of linear DNA was prepared at 3 ␮g/␮l by the dispersion of 3 mg of DNA in 1000 ␮l of the buffer solution. The pH of that solution was maintained upon the addition of DNA. The replication of the plasmidic DNA was made using a transformation procedure by the E. coli bacteria and purification on a column. E. coli DH5 was transformed by electroporation using the “Gene pulser II” apparatus (Bio-Rad Laboratories, La Jolla, CA) and the E. coli culture was made in the LB (Luria Broth) medium. The purified DNA was resuspended in a Tris–HCl 10 mM buffer adjusted to pH 8 and the concentration was varied from 0.8 to 2.94 ␮g/␮l depending on the efficacy of the transformation by the bacteria. Results obtained by electrophoresis elution showed that the plasmid is in the supercoiled circular conformation. 2.2.3. Cationic liposomes/DNA complexes An adequate volume of DNA suspension was added to the cationic liposomes to obtain DMPC/DOTAP/DNA molar ratios of 1:1:0.10 and 1:1:0.25. The suspension was stirred to optimize the contact between liposomes and DNA and to homogenize the dispersion. The molar ratios of liposomes/DNA were calculated according to the number of moles of DNA compared to the number of moles of cationic lipid (DOTAP). The average molecular weight of DNA is that per negatively charged nucleotide (325 g/mol). 2.2.4. Cationic liposomes/CEU mixtures The appropriate quantities of the cationic and zwitterionic lipids were co-dissolved with CEU in a chloroform/methanol solution 1:1 (v/v) to obtain samples containing 5 mg of total lipids and molar ratios of total lipids/CEU of 1:0.10 and 1:0.25. The solvent was evaporated under a nitrogen stream and the samples were placed at 40 ◦ C under reduced pressure overnight. Approximately, 24 h before the analysis, the cationic liposomes/CEU mixture was hydrated with

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the buffer solution to obtain a 5% (w/v) lipid dispersion. The samples were submitted to five freeze–thaw–vortex cycles.

where AQ is the quadrupolar constant (∼167 kHz) and S is the segmental orientational order parameter.

2.3. FTIR experiments

3. Results and discussion

The FTIR spectra were recorded with a Nicolet Magna 550 Fourier transform spectrometer equipped with a liquid nitrogencooled mercury cadmium telluride detector, a temperature controller (±1 ◦ C) and the Nicolet Instrument Corp. OMNIC 2.1 software (Madison, WI). The sample (15 ␮l) was spread between CaF2 windows (Wilmad Glass Co. Inc., Buena, NJ) using a 13 ␮m Mylar spacer. 100 interferograms were recorded with a resolution of 2 cm−1 using the Happ-Genzel apodization. The sample temperature was equilibrated for 2 min before the acquisition of each spectrum. All spectra were corrected for the contribution of water by subtracting a third degree polynomial function and the data treatment was performed with the GRAMS/386 software (Galactic Industries Corp., Salem, NH). The symmetric CH2 (DOTAP) and antisymmetric CD2 (DMPC-d54 ) stretching mode frequencies were determined by calculating the center of gravity (CG ) of the bands at 90% of their height as a function of temperature (0–45 ◦ C) (Cameron et al., 1982).

3.1. Characterization of the DMPC/DOTAP mixture

2.4. Solid-state NMR experiments The NMR spectra were acquired on a Bruker ASX-300 spectrometer (Bruker BioSpin, Milton, ON) operating at 1 H, 2 H and 31 P frequencies of 300.0, 46.1 and 121.5 MHz, respectively. 2.4.1. 31 P NMR experiments The static 31 P NMR spectra were acquired with a home-built probehead and using a Hahn echo pulse sequence (Hahn, 1950) with proton decoupling. Using 1024 data points, typically between 2000 and 12,000 scans were acquired with a 90◦ pulse length between 4 and 5 ␮s, an interpulse delay of 30 ␮s, and a recycle delay of 4 s. The sweep width was set to 50 kHz and a line broadening of 150 Hz was applied to all spectra. The chemical shifts expressed in parts per million (ppm) were referenced relative to the signal of phosphoric acid at 0 ppm. Simulations were made to calculate the lipid phase proportions using the Xedplot (Bruker BioSpin, Milton, ON) software. The chemical shift anisotropy (CSA) values were measured to a height corresponding to 10% of the total height of the spectra. For MAS experiments, a 4 mm Bruker probehead (Bruker BioSpin, Milton, ON) was used. Samples were analyzed at 25 ◦ C and at a spinning speed of 2.8 kHz. Between 2000 and 36,000 scans were acquired with 8192 data points. A line broadening of 150 Hz was applied to all spectra. The chemical shift and the full width at half-maximum (FWHM) were measured using the GRAMS/386 (Galactic Industries Corp., Salem, NH) software.

We have first characterized in the present study the DMPC/ DOTAP mixture at different molar ratios. FTIR spectroscopy was first used to assess the order of the lipid acyl chains, 31 P NMR was used to assess the lipid phases and the order and dynamics of the lipid head group and 2 H NMR was used to assess the effect of the surface charge on the DMPC head group. 3.1.1. Thermotropism of the lipid acyl achains It has been shown that the internal vibrations of the lipid acyl chains, accessible by FTIR, are very useful to characterize the transition from the well ordered and rigid gel (L␤ ) phase to the more disordered and fluid (L␣ ) phase. A shift in the wavenumber value of the CH2 or CD2 stretching mode frequencies occurs at the melting temperature, Tm , of the lipid. This shift is related to a higher conformational disorder due to the introduction of gauche conformers in the lipid acyl chains (Asher and Levin, 1977; Casal and Mantsch, 1984). We have investigated the pure lipids and the DMPC/DOTAP mixture at the following molar ratios: DMPC/DOTAP 8:1, 4:1, 2:1 and 1:1 to study the miscibility of both lipids and their reciprocal effect on the fluidity of their acyl chains and the sensitivity of DMPC to DOTAP. DMPC-d54 was used for these studies. Fig. 2A and B shows the thermotropic curves for DMPC and DOTAP where respectively the wavenumber of the antisymmetric CD2 stretching and the symmetric CH2 stretching mode frequencies were monitored as a function of temperature for each mixture. A Tm of 21 ◦ C was

2.4.2. 2 H solid-state NMR experiments The 2 H NMR spectra were acquired with a home-built probehead and using a quadrupolar echo sequence (Davis, 1983; Davis et al., 1976). Using 2048 data points, between 100,000 and 275,000 scans were acquired with a 90◦ pulse length between 4 and 5 ␮s, and interpulse delay of 60 ␮s and a recycle time of 500 ms. The sweep width was set to 250 kHz and a line broadening of 300 Hz was applied to all spectra. The spectra were transformed into 90◦ oriented spectra using the dePaking algorithm (Bloom et al., 1981; Lafleur et al., 1989; Sternin et al., 1983) to get a better resolution and to facilitate the calculation of the quadrupolar splittings (Q ) which can be related to an orientational order parameter by: Q =

3 AQ S 4

Fig. 2. Temperature dependence of the (A) CD2 antisymmetric and (B) CH2 symmetric stretching mode frequencies for (A) DMPC-d54 and (B) DOTAP in the DMPC/DOTAP 8:1, 4:1, 2:1 and 1:1 mixtures.

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Fig. 3.

31

P NMR spectra (A) static and (B) MAS of the DMPC/DOTAP 1:0, 8:1, 4:1, 2:1 and 1:1 mixtures at 25 ◦ C.

obtained for pure DMPC-d54 , which is in agreement with the value of 22 ◦ C reported by Saint-Laurent et al. (2001). Heating DMPC in the fluid phase only induces a monotonic increase of the wavenumber, showing a small increase of gauche conformers (Casal and Mantsch, 1984; Kauppinen et al., 1991). The results presented in Fig. 2A show a significant increase of the wavenumber of the CD2 antisymmetric stretching mode frequency of DMPC in the gel and fluid phases in the presence of DOTAP. This effect is observed from the molar ratio of 8:1 (11 mol%) and is proportional to the DOTAP concentration in the mixture. Moreover, the results indicate a gradual loss of cooperativity of the DMPC phase transition, which is also proportional to the DOTAP concentration. This fluidizing effect of DOTAP and loss of cooperativity have also been observed by Campbell et al. using fluorescence polarization with membranes made of DPPC and DMPC mixed with DOTAP (Campbell et al., 2001a,b). More specifically, a gradual decrease of the Tm of phosphatidylcholines has been observed in the presence of DOTAP, even at very low DOTAP concentrations (0.4–2.5 mol%) (Campbell et al., 2001a,b). The results presented in Fig. 2A indicate a linear decrease of the Tm of DMPC, from 21, 17 to 14 ◦ C, as a function of molar ratio. More specifically, the phase transition abolishes gradually, leaving a non-measurable Tm beyond a DMPC/DOTAP molar ratio of 4:1 (20 mol%) and is completely abolished at a molar ratio of 1:1 (50 mol%). Therefore, DMPC is in a homogenous fluid phase (Campbell et al., 2001b) stable in the range of temperatures investigated. The studies reported by Campbell et al. suggest that the two DOTAP acyl chains penetrates in the plane of the DMPC bilayers and that both lipids are miscible in all proportions (Campbell et al., 2001a) to form stable vesicles at physiological temperature (Kitagawa and Kasamaki, 2006). Hence, to the fluidizing effect of DOTAP would be associated an increase of the gauche/trans ratio of the DMPC acyl chains. This effect is present in both phases but is more pronounced in the gel phase. As opposed to DMPC, DOTAP is in the fluid phase in the range of temperatures investigated, stable and significantly more disordered than DMPC. The results presented in Fig. 2B indicate that DMPC induces conformational disorder in the DOTAP acyl chains but to a lesser extent than the effect of DOTAP on DMPC, suggesting a smaller sensitivity of the DOTAP acyl chains to trans/gauche isomerization than DMPC. This could be due to the fact that DOTAP is already highly fluid and disordered in this range of temperatures and molar ratios. This effect could be explained by the DOTAP molecular structure, especially due to the unsaturation of its two flexible acyl chains (Brown and Seelig, 1977). Moreover, our results indicate that the DOTAP thermotropic curves are becoming more similar to those of DMPC for a given mixture. Indeed, for the DMPC/DOTAP molar ratios of 8:1 and 4:1, we observe a

crossing of the curves with that of pure DOTAP at 17 and 14 ◦ C, respectively, which is the Tm of DMPC in these mixtures. Hence, below these temperatures, DMPC is in the gel phase and would weakly order the DOTAP acyl chains, which are in the fluid phase, without inducing the reappearance of its gel-to-fluid phase transition at Tm = −12 ◦ C, as reported by Hirsh-Lerner and Barenholz using DSC (Hirsch-Lerner and Barenholz, 1998). For higher DOTAP molar ratios, DMPC induces only disorder, which is consistent with the thermotropism results showing that the detection of the phase transition is impossible beyond a molar ratio of 4:1. 3.1.2. 31 P static solid-state NMR We have used 31 P static NMR to access the lipid organization (phases) and the polar head group dynamics of DMPC in the DMPC/DOTAP mixture. Since DOTAP is not a phospholipid, the spectra only show the contribution of DMPC in interaction with DOTAP. The static powder spectra were obtained for DMPC/DOTAP mixtures at the following molar ratios 1:0, 8:1, 4:1, 2:1 and 1:1 and are shown in Fig. 3A at 25 ◦ C. The results depicted in Fig. 3A indicate that DMPC forms multilamellar vesicles (MLVs) (Mitrakos and Macdonald, 2000) and has a spectrum typical of bilayers in the fluid phase at 25 ◦ C (Brown and Seelig, 1977). A small isotropic contribution is present as well (∼3%), indicating the formation of smaller isotropic structures (Cullis and de Kruijff, 1979). A CSA value of ∼51 ppm is measured for pure DMPC (Fig. 3A and Table 1), which is in good agreement with the value of 47 ppm at 35 ◦ C reported by Marassi and Macdonald (Macdonald et al., 1991). DOTAP also forms MLVs (Mitrakos and Macdonald, 2000) and our results indicate that the bilayer organization of DMPC is largely maintained upon the addition of DOTAP and that the CSA increases as a function of the DMPC/DOTAP molar ratio (Fig. 3A and Table 1). More specifically, the mixtures containing more DOTAP give broader spectra and reach a plateau from a DMPC/DOTAP molar ratio of 2:1, where a value of ∼68 ppm is reported (Fig. 3A and Table 1). A small isotropic Table 1 31 P NMR parameters for DMPC/DOTAP ± DNA at 25 ◦ C. Systems

DMPC DMPC/DOTAP 8:1 DMPC/DOTAP 4:1 DMPC/DOTAP 2:1 DMPC/DOTAP 1:1 DMPC/DOTAP/DNA (p) 1:1:0.25 DMPC/DOTAP/DNA (l) 1:1:0.25

31

P static NMR

31

P MAS NMR

CSA (ppm)

FWHM (ppm)

ıiso (ppm)

51 56 62 68 66 68 68

1.9 – – – 2.9 5.4 5.2

−0.93 – – – −0.83 −0.95 −0.82

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contribution is also observed (∼3–15%) but is not correlated to the DMPC/DOTAP molar ratio. This increase of the CSA was also observed by 31 P static NMR studies of the effect of the surface charge on the DMPC polar head group mixed with a cationic lipid, didodecyldimethylammonium bromide (DDAB) (Macdonald et al., 1991). Our results therefore suggest that DMPC and DOTAP are miscible and that the resulting mixture is made of stable fluid bilayers. The broadening effect on the DMPC spectra observed with the increase in the DOTAP concentration suggests that the cationic lipid induces a reorientation of the DMPC polar head group and/or a decrease of its wobbling motion around the rotation axis (Macdonald et al., 1991). At the molecular level, this suggests an electrostatic interaction between these two lipids at the level of their polar head groups. This finding could be correlated with the physical macroscopic state of the DMPC/DOTAP 1:1 mixture, which is more viscous than pure DMPC. This state could come from the fusion or aggregation of cationic liposomes. It has been proposed by Zuidam and Barenholz that the DOTAP quaternary amine head group forms a salt bridge with the phosphate group of several phospholipids (Zuidam and Barenholz, 1997). Moreover, DOTAP could also induce the formation of larger aggregates or promote the fusion between DMPC MLVs when present at 50 mol% in the mixture. It was shown that DOTAP would have the property to reduce the average size and the heterogeneity of the size distribution of PCs in general (Campbell et al., 2001b). Indeed, the average diameter of MLVs made of DMPC/DOTAP 1:1 has been estimated at ∼0.5–1.0 ␮m, by opposition to pure DOTAP, estimated at ∼0.6 ␮m (Campbell et al., 2001b). For comparison, results obtained by Campbell et al. on vesicles made of DMPC/DOTAP 1:1 in the presence of 4% of paclitaxel have shown that cationic liposomes are amenable to aggregation or fusion (Campbell et al., 2001a). All these results would lead to the spectral broadening observed in the presence of DOTAP. 3.1.3. 31 P solid-state MAS NMR We have also used MAS NMR to get information on the linewidths and isotropic chemical shifts of DMPC in the presence of DOTAP. The MAS spectra obtained for DMPC in the absence and presence of DOTAP at a molar ratio of 1:1 are shown in Fig. 3B at 25 ◦ C while the full width at half maximum (FWHM) and ıiso are given in Table 1. The results indicate that the presence of DOTAP induces a broadening of the DMPC spectrum, from 1.9 to 2.9 ppm. This effect is in agreement with the 31 P NMR static results and supports the hypothesis of a change in the aggregation state of DMPC MLVs. The results presented in Table 1 also indicate that the ıiso value shifts from −0.93 to −0.83 ppm upon the addition of 50 mol% DOTAP. This supports the presence of electrostatic interactions between the DOTAP quaternary amine head group and the DMPC phosphate head group. Indeed, the DOTAP quaternary amine bearing a positive charge would induce a deshielding of the 31 P nucleus in the DMPC phosphate head group. 3.1.4. 2 H NMR study of the lipid head group We have also used 2 H solid-state NMR to access the orientational order of the four deuterons of DMPC-d4 and therefore determine whether DMPC acts as a molecular voltmeter (Macdonald, 1997; Macdonald et al., 1991; Marassi and Macdonald, 1992; Pinheiro et al., 1994; Seelig et al., 1987) in the presence of DOTAP. More specifically, it was shown that when a positively charged molecule is added to PC membranes, the quadrupolar splitting of the outermost doublet, assigned to the two deuterons at position ␣, decreases while the quadrupolar splitting of the innermost doublet, assigned to the two deuterons at position ␤, increases (Bouchard et al., 1998; Macdonald, 1997; Macdonald et al., 1991; Marassi and Macdonald, 1992; Seelig et al., 1987), while the opposite effect is observed when

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a negatively charged molecule is added. This excludes a general increase of the flexibility and can only be explained by a conformational change of the choline head group (Seelig et al., 1987). The spectra obtained for the DMPC/DOTAP mixture in the following molar ratios: 1:0, 8:1, 4:1, 2:1 and 1:1 are shown in Fig. 4A and B at 50 ◦ C. The spectra were obtained at 50 ◦ C because of the lack of resolution for DMPC-d4 at 25 ◦ C. These spectra show an isotropic contribution of ∼5–20%, without being correlated to the DOTAP concentration. This is consistent with the values obtained using 31 P NMR for the DMPC/DOTAP 1:1 mixture (∼12% at 50 ◦ C). The two doublets are better resolved in the dePaked spectra shown in Fig. 4B. Fig. 4C shows the variation of the Q as a function of the DMPC/DOTAP molar ratio. These results indicate that the addition of DOTAP perturbs the orientational order of the choline head group of DMPC. The addition of DOTAP results in a linear decrease of Q ␣ and a linear increase of Q ␤ for the DMPC/DOTAP proportions of 8:1 to 1:1. For the 4:1 proportion, the signal due to the CD2 ␣ disappears, appearing as a strong isotropic contribution (Fig. 4A). This phenomenon could be explained by the progressive interchange of the respective positions of the signals assigned to the CD2 ␣ and ␤, leading to a change in the sign of the Q values for the proportions of 2:1 and 1:1. This effect is consistent with the results obtained by Seelig et al. with studies using DMPC-d2 labelled at position ␣, in the presence of a cationic lipid (Seelig et al., 1987). This interchange effect happens until the proportion of 1:1 is reached. These results indicate that DMPC acts as a molecular voltmeter for all proportions in DOTAP. More specifically, the presence of DOTAP would modify enough the surface charge to induce a conformational change of the DMPC choline head group, hence affecting the alignment of the P− –N+ dipole relative to the membrane surface, as proposed by the molecular voltmeter model (Macdonald et al., 1991). 3.2. DMPC/DOTAP/DNA lipoplex We have also characterized in the present study DMPC/ DOTAP/DNA lipoplexes. To do so, two types of DNA were used, namely plasmidic DNA pBQ6.2 including the CFTR gene and calf thymus linear DNA in molar ratios of 0.10 and 0.25 in DOTAP/DNA, corresponding to R+/− of 10 and 4, respectively. A DMPC/DOTAP molar ratio of 1:1 was used since this molar ratio of neutral/cationic lipid is of great relevance for non-viral gene therapy (Mok and Cullis, 1997; Zuidam and Barenholz, 1997, 1998; Zuidam et al., 1999). Concomitantly, for drug vectors, this molar ratio of 1:1 has been shown to allow an optimal stability of paclitaxel-containing LCs (Campbell et al., 2001a). So, the presence of 50 mol% of neutral colipid would result in a relevant lipid mixture for both applications, with an important charge density and reducing the cytotoxicity of the cationic lipid as well. DMPC-d54 was used for this study and we have verified that the DNA CH2 bands do not overlap with those of DOTAP (results not shown). 3.2.1. Thermotropism of DMPC/DOTAP/DNA (plasmidic) lipoplexes We have investigated the effect of plasmidic DNA on the lipid conformational order. The results shown in Fig. 5 indicate that the general lipid thermotropic behavior is maintained and there is no reappearance of the gel-to-fluid phase transition. Plasmidic DNA at a proportion of 0.10 has no significant effect (not shown) on DMPC. By opposition, the proportion of 0.25 induces a small ordering effect (Fig. 5A). Similar effects are also observed on the DOTAP acyl chains (Fig. 5B). These results suggest that DMPC and DOTAP interact with DNA and that the later induces similar effects on both lipids as a function of concentration. Indeed, zwitterionic DMPC possesses, such as DOTAP, a positive charge on the quaternary amine of its polar head group, which can interact with the negative charge of the DNA phosphate group. Hence, DNA could, in sufficient concentration, screen the lipid positive charges and favour the packing of the

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Fig. 4. 2 H NMR spectra (A) powder (B) dePaked of DMPC-d4 in the DMPC/DOTAP 1:0, 8:1, 4:1, 2:1 and 1:1 complexes at 50 ◦ C. (C) Variation of the Q ␣ and Q ␤ of DMPC-d4 in the DMPC/DOTAP 1:0, 8:1, 4:1, 2:1 and 1:1 mixtures at 50 ◦ C.

acyl chains. This could lead to the observed ordering effect. Another hypothesis could be that in the DMPC/DOTAP 1:1 mixture, all the positive charges on the DOTAP quaternary amine are neutralized by the phosphate group of DMPC and that the residual positive charge of the cationic liposomes comes only from the DMPC quaternary amine. Hence, DMPC could interact with DNA and induce its thermotropic behaviour to DOTAP, as proposed for pure lipid mixtures (Fig. 2). 3.2.2. Thermotropism of DMPC/DOTAP/DNA (linear) lipoplexes For comparison, we have also used linear DNA to investigate the DMPC/DOTAP/DNA (linear) lipoplexes 1:1:0.10 and 1:1:0.25. Results are somewhat different from those obtained using plasmidic DNA, while maintaining the general thermotropic profile, i.e. the thermal stability and the non-reappearance of the gel-to-fluid phase transition (Fig. 5). The DNA proportion of 0.10 has no significant effect (not shown) while the proportion of 0.25 induces a small disordering effect on the DMPC acyl chains (Fig. 5A). On the other hand, the DOTAP acyl chains seem to be insensitive to linear DNA (Fig. 5B). Results obtained with lipoplexes containing DMPC/DOTAP/DNA (plasmidic and linear) suggest that weak interactions are taking place between the lipid acyl chains and DNA. More specifically, DNA perturbs only weakly the conformational order of both lipid

acyl chains. This effect seems to be dependent on the type of DNA and its relative concentration in the liposome. A proportion of 0.10 did not affect the order of both lipids while a proportion of 0.25 of plasmidic DNA induces ordering in the lipid acyl chains. However, a small disordering effect is observed also in the DMPC acyl chains with linear DNA used in the proportion of 0.25. These different behaviors might be explained by the capacity of each type of DNA to screen charges on adjacent lipid polar head groups. More specifically, plasmidic DNA has a greater charge density than linear DNA (Mahato et al., 1997), which could explain the ordering effect of DNA in lipoplexes. This suggests that DNA intercalates between the lipid bilayers to interact with the lipid polar head groups and then to induce a small conformational change on the lipid acyl chains. 3.2.3. 31 P static solid-state NMR We have also used 31 P NMR to investigate the effect of DNA on the lipid head group. The spectra for the lipoplex made of DMPC/DOTAP/DNA (plasmidic and linear) 1:1:0.25 at 25 ◦ C are shown in Fig. 6A. The results obtained with a DNA proportion of 0.10 do not show significant effects and are not presented. These spectra contain only the DMPC signal since the use of the Hahn echo pulse sequence for the acquisition of the spectra makes the 31 P nucleus of DNA invisible because of its reduced mobility in comparison to that of phospholipids. The weaker signal-to-noise ratio

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From a macroscopic point of view, both DMPC/DOTAP/DNA preparations are more fluid than the DMPC/DOTAP 1:1 mixture but this increased fluidity does not seem to involve the mobility of the DMPC polar head group. This suggests that the addition of DNA induces a change in the state of aggregation of cationic liposomes and produces smaller aggregates upon dilution. These results are independent of the concentration and nature of DNA, suggesting similar interactions leading eventually to the formation of smaller aggregates. Previous 31 P NMR and FTIR studies involving DOPE/DOTAP 1:1 lipoplexes in the presence of both plasmidic and linear DNA indicated that the addition of linear DNA favours the formation of inverted-hexagonal phases, whereas plasmidic DNA favours the formation of isotropic lipid structures (Couture, 2000). In addition, Mitrakos and Macdonald (2000) investigated the DOPE/DOTAP system in the presence of different oligonucleotides. Their results demonstrated that the addition of the polyelectrolytes affects strongly the initial dominant lamellar structure of the DOPE/DOTAP system to induce a transition to inverted hexagonal and isotropic structures.

Fig. 5. Temperature dependence of the (A) CD2 antisymmetric and (B) CH2 symmetric stretching mode frequencies for (A) DMPC-d54 and (B) DOTAP in the DMPC/DOTAP/DNA (plasmidic) and (linear) 1:1:0.0.25 lipoplexes.

of the spectra obtained in the presence of plasmidic DNA is due to the dilution of the lipid dispersion after the addition of the aqueous suspension of plasmidic DNA. The results presented in Fig. 6A indicate that the incorporation of either plasmidic or linear DNA to the DMPC/DOTAP cationic liposomes does not significantly perturb the global lipid organization in MLVs. However, the spectral lineshape of the DMPC/DOTAP/DNA lipoplex is somewhat different, reflecting an orientational distribution of phospholipids and incidentally, a somewhat perturbed morphology of MLVs. The CSA values do not vary in the presence of DNA, with a constant value of 68 ppm (Table 1). This suggests that DMPC still interacts with DOTAP in the presence of DNA but that DNA does not affect the mobility of the DMPC polar head group.

Fig. 6.

31

3.2.4. 31 P MAS solid-state NMR We have also investigated the effect of plasmidic and linear DNA on the DMPC/DOTAP 31 P MAS spectra. The results presented in Fig. 6B indicate that DNA induces a broadening of the band, with linewidths of respectively of 5.4 and 5.2 ppm in the presence of plasmidic and linear DNA (Table 1). This broadening effect could be due to a decrease of the T2 values in the presence of DNA and could be explained by a faster lateral diffusion (Bloom and Sternin, 1987) of the lipids in the smaller lipid structures obtained in the presence of DNA. When DNA is added to cationic liposomes, the ıiso values remain at −0.82 ppm in the presence of linear DNA and shifts to −0.95 ppm in the presence of plasmidic DNA. Hence, the higher charge density of plasmidic DNA could be responsible for the difference observed between the two types of DNA. Fluorescence studies performed by Zuidam and Barenholz suggested that the distance between plasmidic DNA and the cationic bilayers are less than 6 Å (Zuidam and Barenholz, 1998), and thus can induce a shielding of the lipid phosphate group due to the DNA negative charges. 3.2.5. 2 H NMR We have also characterized the lipoplexes containing DMPC/DOTAP and plasmidic and linear DNA prepared at molar ratios of 1:1:0.10 and 1:1:0.25 using 2 H NMR spectroscopy to describe the interactions occurring between LCs and DNA at

P NMR spectra (A) static and (B) MAS of the DMPC/DOTAP/DNA for the plasmidic and linear 1:1:0.25 lipoplexes at 25 ◦ C.

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Fig. 7. Top: 2 H NMR (powder only) spectra of DMPC in the DMPC/DOTAP/DNA 1:1:0.25 (A) plasmidic and (B) linear lipoplexes at 50 ◦ C. Bottom: Variation of the Q ␣ and Q ␤ of DMPC-d4 in the DMPC/DOTAP/DNA 1:1:0.25 (C) plasmidic and (D) linear lipoplexes at 50 ◦ C.

the level of the DMPC choline head group. The 2 H NMR spectra obtained for DMPC-d4 /DOTAP in the presence of plasmidic and linear DNA are shown in Fig. 7A and B, respectively. Concomitantly, Fig. 7C and D are illustrating the variation of the Q as a function of the DNA molar ratio in the DMPC/DOTAP 1:1 mixture. Only the powder spectra are shown because of the weak signals obtained for the CD2 ␣ and ␤ and leading to an important loss of resolution. This makes the dePaking of the spectra almost impossible, especially for a DNA proportion of 0.25. However, it was possible to estimate the Q values for the DNA proportion of 0.25 by using the powder spectra. Results shown in Fig. 7A indicate that the addition of plasmidic and linear DNA perturbs the lipid bilayer organization. More specifically, the spectra are still representative of MLVs in the fluid phase but we notice the presence of an isotropic contribution that depends on the proportion of DNA. The proportions of isotropic contribution are ∼15 and 35% for plasmidic DNA and ∼15 and 29% for linear DNA at molar ratios of 0.10 and 0.25, respectively. This suggests that the isotropic structures are the result of an interaction between the cationic liposomes and DNA, instead of excess lipids not interacting with DNA. Therefore, DNA would not completely intercalate between the lipidic bilayers, but might participate to the formation of small isotropic structures with lipids as well. Therefore, a larger proportion of DNA would induce more isotropic structures. Results shown in Fig. 7 also suggest that the presence of plasmidic or linear DNA in the DMPC/DOTAP 1:1 mixture does not

induce the molecular voltmeter effect. Indeed, we observe a small decrease of both Q ␣ and Q ␤ only in the proportion of 0.25 in DNA, the proportion of 0.10 having no significant effect. Therefore, a small global decrease of the order of the DMPC choline head group is observed, which seems to depend on the concentration of DNA. In comparison, results obtained by 31 P NMR did not show a variation in the CSA values, suggesting no significant effect of DNA on the mobility and orientation of the DMPC phosphate head group. This suggests that the choline head group is closer to DNA, hence more sensitive to its presence, and induces a weak disordering effect. It is noteworthy that the choline head group is adjacent to the DMPC quaternary amine, which is most likely in interaction with the DNA phosphate group. The absence of voltmeter effect might be related to the inclusion of DNA between bilayers that does not modify sufficiently their surface charges. The disordering effect induced by DNA at the level of the C–D bonds would not induce a conformational change within the DMPC choline head group to change the orientation of the P− –N+ dipole. Studies using POPC (1-palmitoyl,2-oleoyl-sn-glycero-3phosphocholine) lipids (Mitrakos and Macdonald, 1996) have shown that polyelectrolytes, such as single-stranded DNA (Mitrakos and Macdonald, 1996), exhibit a propensity to induce lateral phase separation and therefore the formation of lipid domains. This conclusion was made using separately POPC-␤-d2 and POPC-␣-d2 mixed with various cationic lipids such as DOTAP (Macdonald, 1997). The authors observed two overlapping Pake patterns in the

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NMR spectra, indicating the coexistence of two distinct lipid domains. This phenomenon was not observed however in our spectra. Therefore, we cannot conclude that DNA induces such oppositelycharged-lipid-enriched domains, but only conformational disorder of the choline headgroup. These results could be due to the different lipid formulation and molar ratio of anionic double-stranded nucleic acid used in the two studies. In addition, we have used a buffer at high ionic strength, mimicking physiological conditions. Indeed, the screening charge effect could decrease electrostatic interactions to the extent that distinct lipid domains can no longer be distinguished. 3.3. DMPC/DOTAP/CEU mixture

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effect on DMPC and the Tm of pure DMPC remains at 21 ◦ C for all proportions of CEU investigated (Fig. 8A). On the other hand, the addition of 4-n-butyl CEU induces an increase of the wavenumber of the CH2 symmetric stretching mode frequency, and therefore an increased disordering effect in the pure DOTAP acyl chains, this disorder being more significant for the CEU proportion of 0.10 than 0.25, the latter having no effect (Fig. 8B). Finally, the addition of 4n-butyl CEU to the lipid mixture induces a similar increase of the wavenumber of the CH2 symmetric stretching mode of DOTAP for both proportions of CEU (Fig. 8B). These results suggest that 4-nbutyl CEU could have a preferential interaction with DOTAP so that the cationic lipid would ease the inclusion of 4-n-butyl CEU into liposomes. This greater sensitivity toward this lipid could be due to its polar head group and/or the greater fluidity of its unsaturated acyl chains.

3.3.1. DMPC/DOTAP/4-n-butyl CEU We have also investigated the stability of the DMPC/DOTAP system in the presence of two drugs to determine whether these cationic liposomes could be used as drug-delivery vectors. These drugs, namely, 4-n-butyl and 4-sec-butyl CEU, are constitution isomers and have log P values of 3.23 and 3.16 and IC50 (half maximal (50%) inhibitory concentration) values of 14 and 2 ␮M, respectively (Béchard et al., 1994; C.-Gaudreault et al., 1994). Since both drugs are neutral and have been previously shown to incorporate into lipid membranes (Saint-Laurent et al., 2001), we have only used FTIR spectroscopy to determine the effects of the two drugs on both the DMPC and DOTAP acyl chains. More specifically, we have investigated the effect on 4-n-butyl CEU on the conformational order of the pure DMPC and DOTAP acyl chains and on the DMPC/DOTAP 1:1 mixture. DMPC-d54 was used for these studies and two CEU proportions were investigated, namely 0.10 and 0.25. First, the results indicate that 4-n-butyl CEU has no significant effect on the pure DMPC acyl chains (Fig. 8A). In addition, the addition of 4-n-butyl CEU to the lipid mixture has no

3.3.2. DMPC/DOTAP/4-sec-butyl CEU For comparison, we have investigated the effect of 4-sec-butyl CEU on the conformational order of the pure DMPC and DOTAP acyl chains and on the DMPC/DOTAP 1:1 mixture. DMPC-d54 was also used for these studies and two CEU proportions were investigated, namely 0.10 and 0.25. The results presented in Fig. 9A indicate that 4-sec-butyl CEU has different effects on both the gel and fluid phases for pure DMPC. In the gel phase, there is a more significant increase of the wavenumber of the CD2 antisymmetric stretching mode frequency for the proportion of 0.25 than 0.10 of CEU. In the fluid phase, a weaker increase of the wavenumber is observed regardless of the proportion of CEU. There is also a greater loss of the DMPC phase transition cooperativity for the proportion of 0.25 than 0.10. Moreover, Tm values of 15 and 12 ◦ C have been obtained for the DMPC/4-sec-butyl CEU proportions of 0.10 and 0.25, respectively. For pure DOTAP, the results indicate that 4-sec-butyl CEU decreases the CH2 symmetric stretching mode frequency of the acyl chains, effect that is significantly more pronounced for the propor-

Fig. 8. Temperature dependence of the (A) CD2 antisymmetric and (B) CH2 symmetric stretching mode frequencies for (A) DMPC-d54 and (B) DOTAP in the DMPC/DOTAP 1:1 ± 4-n-butyl CEU 0.10 and 0.25 mixtures.

Fig. 9. Temperature dependence of the (A) CD2 antisymmetric and (B) CH2 symmetric stretching mode frequencies for (A) DMPC-d54 and (B) DOTAP in the DMPC/DOTAP 1:1 ± 4-sec-butyl CEU 0.10 and 0.25 mixtures.

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tion of 0.25 than 0.10 (Fig. 9B). For the DMPC/DOTAP 1:1 mixture, these effects vanish for both lipids while a slight increase of the DOTAP wavenumber is observed in the presence of 4-sec-butyl CEU, indicating a weak disordering effect. The results presented above suggest that 4-sec-butyl CEU acts in different ways on both lipids. First, it induces disorder in the pure DMPC acyl chains. In the fluid phase, the proportion of CEU has no effect, conversely to the gel phase, where more disorder is induced by the proportion of 0.25 of the CEU. This could be explained by the fact that the fluid phase is so disordered that the addition of CEU only weakly increases the disorder and the proportion of the CEU added to the mixture has no significant effect. By opposition, the gel phase is more ordered and the effect of the different proportions of 4-sec-butyl CEU is more pronounced. For the lipid mixture, the system is so much fluid and disordered that 4-sec-butyl CEU does not induce a disordering effect on DMPC. In contrast to DMPC, 4-sec-butyl CEU has an ordering effect on the pure DOTAP acyl chains, which is dependent on the CEU proportion. This effect is probably due to the CEU hydrophobic moiety, which could penetrate between the disordered lipid acyl chains, and hence have an ordering effect. There is significantly more ordering effect for the proportion of 0.25 than 0.10 indicating that DOTAP is sensitive to the 4-sec-butyl CEU proportion. However, for the lipid mixture, there is a loss of this ordering effect and the presence of 4-sec-butyl CEU induces a weak disordering effect. The results presented above indicate that the two CEUs, which are constitution isomers and which differ by their R substituent, have very different effects on the order of the lipid acyl chains. These effects seem related to their respective cytotoxicity (Saint-Laurent et al., 2001). Indeed, 4-sec-butyl CEU is significantly more cytotoxic than 4-n-butyl CEU and it is related to a higher conformational disordering effect on the lipid acyl chains, as shown by Saint-Laurent et al. (2001). In addition, our results indicate that the DMPC/DOTAP 1:1 mixture would be potentially interesting for the conception of synthetic vectors because it is very fluid, offers a great conformational stability toward the incorporation of CEU and a good charge density. Campbell et al. have suggested that the conformational disorder induced by DOTAP in the DMPC acyl chains could promote drug incorporation and that the fluid domain formation upon the presence of cationic lipid could favour its retention (Campbell et al., 2001a). 4. Conclusions The goal of the present study was to characterize a synthetic vector made of DMPC and DOTAP as a gene and drug carrier by making a comparative study using DNA and two anticancer drugs. Indeed, the mixture made of DMPC/DOTAP has been shown to have a good potential for the transport of paclitaxel (taxol) (Campbell et al., 2001a). However, this system had never been characterized as a gene carrier. Results obtained by FTIR as well as 31 P and 2 H NMR indicate that DOTAP and DMPC are miscible. More specifically, DOTAP induces a high degree of conformational disorder in the DMPC acyl chains and a decrease of Tm . This suggests that the mixture made of DMPC/DOTAP 1:1 is in a highly disordered homogenous fluid phase. The results obtained with DNA suggest that there is an interaction between DNA and the DMPC polar head group and that the presence of DNA leads to the formation of smaller aggregates. Our results also indicate that the presence of DNA or CEUs does not significantly affect the conformational order of both the DMPC and DOTAP acyl chains, regardless of the fact that DNA and CEUs interact with cationic liposomes according to very different mechanisms. A detailed characterization of complexes between cationic liposomes and both DNA and drugs was obtained in the present study and indicates that these complexes are stable and fluid assemblies.

Acknowledgements The authors would like to thank Pierre Audet and Serge Groleau for technical assistance, as well as Nathalie Turgeon and Hélène Deveau for the preparation of plasmidic DNA. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Institute for Health Research (grant #MOP-79334), le Fond Québécois de Recherche sur la Nature et les Technologies (FQRNT), le Centre de Recherche en Science et Ingénierie des Macromolécules (CERSIM) and le Centre de Recherche sur la Fonction, la Structure et l’Ingénierie des Protéines (CREFSIP).

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