Physicochemical Investigation of a Lipid with a New Core Structure

Max Planck Institute of Colloids and Interfaces, Research Campus Golm, Am Muehlenberg 1, D-14476 Potsdam, Germany, and Institute of Pharmacy, Martin ...
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Langmuir 2007, 23, 3919-3926

3919

Physicochemical Investigation of a Lipid with a New Core Structure for Gene Transfection: 2-Amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan-1-ol Maria N. Antipina,*,† Ingo Schulze,‡ Bodo Dobner,‡ Andreas Langner,‡ and Gerald Brezesinski*,† Max Planck Institute of Colloids and Interfaces, Research Campus Golm, Am Muehlenberg 1, D-14476 Potsdam, Germany, and Institute of Pharmacy, Martin Luther UniVersity, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle/Saale, Germany ReceiVed September 27, 2006. In Final Form: December 20, 2006 Cationic liposomes/DNA complexes can be used as nonviral vectors for direct delivery of DNA-based biopharmaceuticals to damaged cells and tissues. In order to obtain more effective and safer liposome-based gene transfection systems, the new cationic lipid 2-amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan-1-ol (AHHP) was synthesized. In this paper we report on the synthesis of AHHP and investigations of its physical-chemical properties. Langmuir monolayers of AHHP were studied at the air/buffer interface by film balance measurements, grazing incidence X-ray diffraction (GIXD), and infrared reflection absorption spectroscopy (IRRAS). Structure and thermotropic phase behavior of AHHP in aqueous dispersion were examined by small-angle and wide-angle X-ray scattering (SAXS/WAXS) and differential scanning calorimetry (DSC). The results show clear differences in structure and phase behavior of AHHP, both in the monolayer system and in aqueous dispersions, in dependence on the subphase pH due to protonation or deprotonation of the primary amine in the lipid head group. Thermodynamic data derived from π-A isotherms provide information about the critical temperature (Tc), which is in rough agreement with the temperature of the lipid phase transition from gel to fluid state (Tm) found by X-ray and calorimetry studies of AHHP aqueous dispersions. The packing properties of the molecules in mono- and bilayer systems are very similar. DNA couples to the monolayer of the new lipid at low as well as at high pH but in different amounts. The DNA coupling leads to an alignment of adsorbed DNA strands indicated by the appearance of a Bragg peak. The distance between aligned DNA strands does not change much with increasing monolayer pressure.

1. Introduction Fabrication of intelligent nanosized systems for direct delivery of DNA-based biopharmaceuticals to the appropriate damaged cells and tissues is an important goal of gene therapy, which is one of the most interesting and promising methods in the treatment of inherited and chronic diseases, including cancer, AIDS, neurological disorders such as Parkinson’s disease and Alzheimer’s disease, and cardiovascular disorders.1,2 At present two types of DNA delivery systems are available. The first type of DNA carriers based on viruses is currently used in more than 70% of human clinical gene therapy trials worldwide.3 In spite of high transfection efficiencies in a variety of human tissues, such disadvantages as the toxicity of the viruses and the potential for generating a strong immune response often make the viral delivery systems dangerous for clinical application. Chemical nonviral carriers make up another type of vector-assisted delivery system. Nonviral transfection of genetic material avoids problems with immunogenicity and achieves now more importance. Commonly used nonviral vectors for DNA transfection, according to the nature of the synthetic material, are DNA/polymer complexes and liposomal delivery systems, where DNA is entrapped in and/or complexed with liposomes.4-6 Polymeric transfection systems often demonstrate high transfection efficacy.7 * Corresponding authors: (M.N.A.) tel: +49 331 567 9232, fax: +49 331 567 920, e-mail: [email protected]; (G.B.) tel: +49 331 567 9234, fax: +49 331 567 9202, e-mail: [email protected]. † Max Planck Institute of Colloids and Interfaces. ‡ Martin Luther University. (1) Stull, R. A.; Szoka, F. C. Pharm. Res. 1995, 12, 465-483. (2) Patil, S. D.; Burgess, D. J. AAPS Newsmag. 2003, 6, 27. (3) Walther, W.; Stein, U. Drugs 2000, 60, 249-271. (4) Merdan, T.; Kopecek, J.; Kissel, T. AdV. Drug DeliVery ReV. 2002, 54, 715-758.

In spite of this, problems of quality control and inherent pharmacological properties of some polymers (such as hypocholesterolemia induced by chitosans) make polymeric delivery systems unfavorable for human use.8,9 Complexation of DNA with cationic liposomes seems to be a more versatile tool for the fabrication of gene delivery nonviral vector systems. Liposomes are generally nonimmunogenic because of the lack of proteinaceous components. They are able to improve the biological stability of DNA and to protect gene material from nucleases. By using mixtures of different lipids (cationic/zwitterionic) for liposome preparation, it is easy to adjust the size and surface charge. Immobilization of different short peptides/proteins or sugar molecules on cationic liposomes/DNA complexes is one of the possibilities to obtain gene delivery systems able to specifically target the membranes of the appropriate cells.10 Introduction of pH-sensitive lipids into liposomes can provide in situ release of DNA therapeutics by changing the complex structure and morphology. Although it has been already shown that nonviral systems constructed of a wide class of cationic lipids were able to implement gene transfection, an intelligent multifunctional gene delivery system is still a dream of contemporary gene therapy. In fact, liposome-mediated transport of genetic material into the (5) Fattal, E.; Delattre, J.; Dubernet, C.; Couvreur, P. S.T.P. Pharma Sci. 1999, 9, 383-390. (6) Pedroso de Lima, M. C.; Simoes, S.; Pires, P.; Faneca, H.; Duzgunes, N. AdV. Drug DeliVery ReV. 2001, 47, 277-294. (7) Kamiya, H.; Tsuchiya, H.; Yamazaki, J.; Harashima, H. AdV. Drug DeliVery ReV. 2001, 52, 153-164. (8) Merdan, T.; Kopecek, J.; Kissel, T. AdV. Drug DeliVery ReV. 2002, 54, 715-758. (9) LeHoux, J. G.; Grondin, F. Endocrinology 1993, 132, 1078-1084. (10) Fattal, E.; Dubernet, C.; Couvreur, P. S.T.P. Pharma Sci. 2001, 11, 3144.

10.1021/la062840i CCC: $37.00 © 2007 American Chemical Society Published on Web 02/20/2007

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target cells suffers from low efficacy and toxicity of lipids in use. Therefore, the success of nonviral gene therapy depends in a crucial manner on novel efficient lipids. For that purpose we have investigated the suitability of 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) as backbone for a new lipophilic core molecule in which the amino group is an anchor for certain basic residues. The third hydroxy group is free or suitable for further derivatization to more complex compounds. The central compound, 2-amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan-1-ol (AHHP), was tested in an biological assay using NIH5T3 and Hep G2 cells and the galactosidase gene as marker for a successful transfection (manuscript in preparation). Under these conditions a transfection was found already with this rather simple compound. Further elongation with basic residues at the amino function will be performed with basic amino acids. Extensive physicochemical investigations of this new promising substance were carried out. Thermodynamical properties of AHHP were studied in two-dimensional Langmuir monolayers and compared with those obtained in volume systems. Coupling of DNA with the monolayer of the new cationic lipid under different pH conditions was also investigated in the present work.

Antipina et al.

2.1. Materials. For all measurements and sample preparations, Milli-Q Millipore water with a specific resistance of 18.2 MΩ‚cm was used. The synthesis of AHHP is described below. Doublestranded deoxyribonucleic acid (DNA) used in the monolayer experiments was purchased from Sigma (Taufkirchen, Germany) and is a highly polymerized natural product originating from calf thymus. The sample was used without further purification. To minimize the danger of DNA denaturation, all experiments have been performed at 20 °C and only fresh solutions of DNA containing 1 mM of NaCl were used. Sodium chloride for the DNA solution was heated to 600 °C to reduce the content of potential organic impurities. Monolayer and bulk-phase experiments were performed either in citric buffer, pH 4, containing 50 mM C6H8O7 and 0.2 M NaOH, or in 10 mM Tris buffer, pH 8, containing 50 mM NaCl. 2.2. Synthesis. Commercially available and cheap Tris was transformed into a compound containing two lipophilic residues. Therefore, a blocking group strategy was necessary to obtain the above-mentioned structure. The amino and two hydroxy groups were blocked with an excess of benzaldehyde to produce a heterobicyclic system according to Pierce et al.11 The resulting free hydroxy function was blocked by alkylation with benzyl chloride to the corresponding benzyl ether derivative. The following reductive ring opening reaction with NaBH4/TFA yields the 2-benzyloxymethyl-2-dibenzylaminopropane-1,3-diol. This selectively blocked compound was alkylated with 1-bromohexadecane to the corresponding bishexadecyl ether under alkaline conditions in toluene. After purification of the crude product by column chromatography, the three benzyl groups were removed by hydrogenation with palladium hydroxide under a pressure of 20 bar. 2.2.1. 1-Aza-2,8-dibenzyl-5-benzyloxymethyl-3,7-dioxabicyclo[3.3.0]octane (2). 1-Aza-2,8-dibenzyl-5-hydroxymethyl-3,7-dioxabicyclo[3.3.0]octane (1) (10 mmol, 3 g) was dissolved in 50 mL of tetrahydrofuran (THF).11 Then 12 mmol (1.35 g) of potassium tertbutylate was added at room temperature. After addition of 15 mmol (2.56 g) of benzyl chloride, the mixture was heated under reflux for 2 h. After cooling, 50 mL of chloroform and 50 mL of water were added to the mixture. The organic layer was separated, washed two times with water, and then washed with brine. The solution was dried over sodium sulfate, the solvent was evaporated, and the residue was recrystallized from heptane. Yield 82% white substance, C25H25NO3, 387.48 g‚mol-1, fp 80-82 °C. ESI-MS m/e 388 (M+ + H), calcd C 77.49, H 6.50, N 3.61; found C 77.84, H 6.43, N 4.07. 1H NMR (CDCl3) δ 3.59-3.64 (m, 4H, 2[-OCH2C-]), 3.74-3.78 (m,

2H, [-CCH2OCH2-]), 4.60-4.61 (m, 2H, [-CCH2OCH2-]), 5.235.25 (m, 2H, [C6H5CHNCHC6H5]), 7.04-7.40 (m, 15H, 3[C6H5]). 2.2.2. 2-Benzyloxymethyl-2-dibenzylaminopropane-1,3-diol (3). Trifluoroacetic acid (10 mmol, 1.14 g) was dropped into a suspension of 10 mmol (0.38 g) of sodium borohydride in 5 mL of THF. Into this mixture was dropped 2 mmol (0.77 g) of compound 2 with stirring under cooling by an ice bath. After being stirred for 1 h at room temperature, the mixture was again cooled down and 10% sodium hydroxide solution was poured slowly into it. The mixture was then evaporated and extracted three times with 25 mL of ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to dryness under vacuum. The crude product was purified by column chromatography with chloroform/ether and gradient technique. Yield 87%, white substance, C25H29NO3, 391.5 g‚mol-1, fp 65-67 °C. ESI-MS m/e 392 (M+ + H), calcd C 76.7, H 7.47, N 3.56; found C 76.21, H 7.13, N 3.29. 1H NMR (CDCl3) δ 2.66 (s, 2H, 2[OH]), 3.77-3.98 (m, 10H, [-OCH2C-], [HOCH2CCH2OH], [C6H5CHNCH2C6H5]), 4.60-4.61 (m, 2H, [C6H5CH2O]), 7.10-7.43 (m, 15H, 3[C6H5-]). 2.2.3. 2-Benzyloxymethyl-2-dibenzylaminopropane-1,3-bis(hexadecyl ether) (4). Compound 3 (25 mmol, 10 g) and 100 mmol (5.6 g) of powdered potassium hydroxide in 200 mL of xylene were heated under reflux by use of a water separator. After 45 min, 75 mmol (23 g) of 1-bromohexadecane was added. The mixture was stirred for 24 h under reflux. The cooled reaction mixture was filtered and then diluted with 200 mL of diethyl ether. The clear solution was washed with water, with 1 N hydrochloric acid, with 5% sodium hydrogen carbonate solution, and again with water. After the solution was dried over sodium sulfate, the solvent was distilled in vacuum and the residue was purified by flash chromatography. Yield 68%, white substance, C57H93NO3, 840.37 g‚mol-1, fp 30-31 °C. ESIMS m/e 841 (M+ + H), calcd C 81.47, H 11.15, N 1.67; found C 81.38, H 11.21, N 1.51. 1H NMR (CDCl3) δ 0.87 (t, 6H, 2[CH3-), 1.19-1.53 (m, 56H, 2[CH3(CH2)14CH2O-]), 3.29 (t, 4H, 2[CH3(CH2)14CH2O-]), 3.55-3.99 (m, 10H, [C6H5CH2NCH2C6H5], [-OCH2CCH2O-], [OCH2CN-]), 4.39 (m, 2H, [C6H5CH2O-]), 7.03-7.32 (m, 15H, 3[C6H5-]). 2.2.4. 2-Amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan1-ol (5). Compound 4 (1.2 mmol, 1 g) was dissolved in 30 mL of ethyl acetate. After addition of 50 mg of Pd(OH)2, the slurry was hydrogenated for 48 h at 60 °C and 40 bar. The catalyst was filtered off and washed with ethyl acetate. The solution was filtered once more and evaporated to dryness. The residue was purified by column chromatography with chloroform/methanol and gradient technique. Yield 48%, white solid, C36H75NO3, 569.98 g‚mol-1, fp 76-77 °C.ESI-MS m/e 571 (M+ + H), calcd C 75.86, H 13.25, N 2.46; found C 75.57, H 13.11, N 2.41. 1H NMR (CDCl3) δ 0.86 (t, 6H, 2[CH3-]), 1.23-1.55 (m, 56H, 2[CH3(CH2)14CH2O-]), 3.30-3.41 (m, 8H, 2[CH3(CH2)14CH2O-], [-OCH2CCH2O-]), 3.48 (s, 2H, [HOCH2-]). The chemical structure of newly synthesized cationic lipid is shown in Figure 1 (inset). 2.3. Monolayer Experiments. For monolayer experiments, 1 mM stock solutions of AHHP were prepared in chloroform (Merck, Germany; purity >99.8%). The corresponding solution was placed on the aqueous subphase with a microsyringe and allowed to relax for 5 min before compression to facilitate the evaporation of the solvent. 2.3.1. Film Balance Measurements. The pressure-area (π-A) isotherms were measured on a computer-interfaced Langmuir trough (R&K, Potsdam, Germany) during the monolayer compression. The setup is equipped with a Wilhelmy balance for surface tension measurements and a temperature control system. The subphase temperature was maintained constant with an accuracy of (0.1 °C. The films were compressed at a rate of approximately 2 Å2 molecule-1 min-1. 2.3.2. Grazing Incidence X-ray Diffraction. Measurements were performed on the liquid surface diffractometer (undulator beamline BW1) at HASYLAB, DESY, Hamburg, Germany.12-15 The Lang-

(11) Pierce, J. S.; Lunsford, C. D.; Raiford, R. W.; Rush, J. L.; Riley, D. W. J. Am. Chem. Soc. 1951, 73, 2595-2596.

(12) Als-Nielsen, J.; Jaquemain, D.; Kjaer, K.; Lahav, M.; Levellier, F.; Leiserowitz, L. Phys. Rep. 1994, 246, 251-313.

2. Experimental Section

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Figure 1. Pressure/area isotherms of AHHP monolayer at different temperatures. Left: citric buffer, pH 4; T ) 20 °C (a), 25.8 °C (b), 28.1 °C (c), 30.6 °C (d), 33.7 °C (e), 38.2 °C (f), or 39.8 °C (g). Right: Tris buffer, pH 8; T ) 20 °C (a), 40.4 °C (b), 45.4 °C (c), 47.7 °C (d), or 50.9 °C (e). (Inset) Chemical structure of 2-amino3-hexadecyloxy-2-(hexadecyloxymethyl)propan-1-ol. muir trough is located in a thermostated, tightly closed and Heflashed container. A monochromatic beam (λ ) 1.304 Å) from a beryllium (0 0 2) crystal strikes the water surface at an angle of 0.11°, equal to 85% of the critical angle for total external reflection at this X-ray wavelength. A linear position-sensitive detector (PSD) (OED-100-M, Braun, Garching, Germany) with a vertical acceptance 0 < Qz < 1.27 Å-1 was used for recording the diffracted intensity as a function of both the vertical [Qz ≈ (2π/λ) sin Rf] and the horizontal [Qxy ≈ (4π/λ) sin Θ] scattering vector components. Rf is the vertical and 2Θ is the horizontal scattering angle. The horizontal resolution of 0.008 Å-1 was determined by a Soller collimator mounted in front of the PSD. The accumulated position-resolved counts were corrected for polarization, effective area, and Lorentz factor. Model peaks taken as a Lorentzian in the in-plane direction and a Gaussian in the out-of-plane direction were least-squares-fitted to the measured intensities. Bragg rod profiles normal to the surface can be obtained by integration of the scattered signal over Qxy intervals. From the peak positions of the horizontal (in-plane) diffraction data, the lattice spacing d can be determined as d(hk) )

2π Qxyhk

(1)

where Qxy is the maximum of the Lorentz curve. From this, the lattice parameters a, b, and γ can be calculated, giving the unit cell area Axy. For the hydrocarbon chains of the lipid, this is the projection of the cross-sectional area A0 ) Axy cos t onto the horizontal plane. The tilt angle t is the angle between the normal of the water surface and the symmetry axis of the hydrocarbon chain. An undistorted hexagonal lattice exhibits a single diffraction peak. Two Bragg peaks are detected when the monolayer forms an orthorhombic (distorted hexagonal) lattice. In this case, the chains may be tilted in a symmetry direction either toward the nearest (NN) or the next-nearest (NNN) neighbors. For an orthorhombic chain lattice with NN tilt (Qzn ) 0), the tilt angle can be calculated by16-17 tan t )

Qzd (Qxyd)2 - (Qxyn/2)2

(2)

2.3.3. Infrared Reflection Absorption Spectroscopy. IRRA spectra were recorded on an IFS 66 Fourier transform infrared (FTIR) spectrometer (Bruker, Germany) equipped with a liquid-nitrogen(13) Jacquemain, D.; Leveiller, F.; Weinbach, S.; Lahav, M.; Leiserowitz, L.; Kjaer, K.; Als-Nielsen, J. J. Am. Chem. Soc. 1991, 113, 7684-7691. (14) Kaganer, V. M.; Mo¨hwald, H.; Dutta, P. ReV. Mod. Phys. 1999, 71, 779819. (15) Kaganer, V. M.; Peterson, I. R.; Kenn, R. M.; Shih, M. C.; Durbin, M.; Dutta, P. J. Chem. Phys. 1995, 102, 9412-9417. (16) Scalas, E.; Brezesinski, G.; Mo¨hwald, H.; Kaganer, V. M.; Bouwman, W. G.; Kjaer, K. Thin Solid Films 1996, 284/285, 56-61. (17) Kaganer, V. M.; Brezesinski, G.; Howes, P. B.; Kjaer, K.; Mo¨hwald, H. Phys. ReV. E. 1999, 59, 2141-2152.

cooled mercury cadmium telluride (MCT) detector. The IR beam was conducted out of the spectrometer and focused onto the water surface of the Langmuir trough. The angle of incidence was either 40° or 62° with respect to the surface normal, and the IR beam was polarized by a BaF2 polarizer in the plane of incidence (p) and perpendicular to this plane (s). Measurements were done by switching between two troughs at regular intervals (every 10 min) by use of a trough shuttle system controlled by the acquisition computer. One trough contained the monolayer system under investigation (sample), whereas the other (reference) was filled with pure water. The spectra from the reference trough were subtracted from the sample spectra in order to eliminate the water vapor signal. To maintain a constant water vapor content, the setup was placed in a hermetically sealed container. Spectra were recorded with a spectral resolution of 8 cm-1; 200 scans were collected for s-polarized light and 400-800 scans for p-polarized light. 2.4. Experiments with Aqueous Lipid Dispersions. For bulkphase experiments, a suitable quantity of the new cationic lipid was dispersed in the corresponding buffers (80 wt %). The samples were hydrated overnight at 4 °C, afterward heated above 60 °C, and kept in the sonication bath for 4 h. Each hour the samples were also vortexed for 5 min. The resulting dispersions were divided into two parts. One part was used for SAXS/WAXS measurements. The other part was diluted to 1.5 mg‚mL-1 lipid concentration and then used for DSC experiments. The samples were equilibrated at 4 °C for 7-10 days before measurements. 2.4.1. Small-Angle and Wide-Angle X-ray Scattering. Timeresolved SAXS/WAXS experiments were performed at the soft condensed matter beamline A2 at HASYLAB, DESY, Hamburg, Germany, at a fixed wavelength of 0.15 nm.18 The diffracted intensities in the small- (SAXS) and wide-angle (WAXS) regions were simultaneously recorded by two linear delay-line detectors connected in series.19 The data acquisition system has been described elsewhere.20 The diffraction patterns were recorded during heating and cooling scans at a rate of 1 K‚min-1. To minimize the X-ray dose on the sample, a fast shutter was kept closed when no data were acquired. In the experiments, an alternating sequence of 5 s exposure time and 55 s waiting time was applied. The reciprocal spacing s ) 1/d were calibrated with the diffraction pattern of dry rat-tail collagen with a long spacing of 65 nm (SAXS) and p-bromobenzoic acid (WAXS). Samples were prepared as described above and transferred into capillaries (1 mm diameter) (Hilgenberg, Malsfeld, Germany). The flame-sealed capillaries were placed in the temperature-controlled sample holder. 2.4.2. Calorimetry. Differential scanning calorimetry (DSC) measurements were performed on the microcalorimetry system (MCS DSC, MicroCal Inc., Northampton, MA) as described elsewhere.21 The heating rate for DSC scans was 60 °C‚h-1. Transition temperatures obtained with slower scanning rates are within (0.1 °C in accordance with those obtained at 60 °C‚h-1. The cells were kept under 2 bar of nitrogen pressure. Tm was determined as the maximum of Cp curves. The values of the transition enthalpies were obtained by numerical integration of the baseline-corrected DSC curves. The estimated error of the transition enthalpy for lipid aqueous dispersions is about 10%. The exact control of the total amount of lipid transferred inside the DSC cell gives the main contribution to the resulting error.

3. Results and Discussion 3.1. Thermodynamics of AHHP Monolayers. The cationic group of AHHP is represented by the primary amine, which can be protonated and therefore positively charged or deprotonated/ uncharged depending on the environmental pH value. All experiments were performed in buffer solutions with pH 4 or pH 8 as examples of mostly charged and mostly uncharged states (18) Rapp, G. Acta Phys. Pol. A. 1992, 82, 103-120. (19) Gabriel, A. ReV. Sci. Instrum. 1977, 48, 1303-1305. (20) Rapp, G.; Gabriel, A.; Dosiere, M.; Koch, M. H. J. Nucl. Instrum. Methods Phys. Res., Sect. A. 1995, 357, 178-182. (21) Johnson, C. M.; Cooper, A.; Stockley, P. G. Biochemistry 1992, 31, 97179724.

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Figure 2. (Left) Surface pressure (πt) of main phase transition (LE-LC) of AHHP monolayer on citric buffer, pH 4 (2), and on Tris buffer, pH 8 (b), versus temperature. (Right) Transition enthalpy (|∆H|) as a function of temperature for 2-amino-3-hexadecyloxy2-(hexadecyloxymethyl)propan-1-ol monolayer on citric buffer, pH 4 (2), and on Tris buffer, pH 8 (b). ∆H was calculated by use of a modified Clausius-Clapeyron equation.

of the lipid hydrophilic head groups, respectively. The doublechain cationic lipid AHHP forms stable monolayers at the air/ buffer interface. The surface pressure π was measured as a function of the molecular area A (π-A isotherms) at different temperatures between 20 and 50 °C. Figure 1 shows the temperature-dependent behavior of the AHHP monolayer on subphases with pH 4 and pH 8. The monolayer is fully condensed at 20 °C on both buffer subphases. At higher subphase temperatures, the monolayer shows the first-order transition from the liquid-expanded (LE) to a condensed (LC) phase during compression. The plateau region corresponding to the coexistence of LE and LC phases shifts to higher surface pressures with increasing temperature for both systems. The transition pressure πt is a linear function of temperature (Figure 2, left) with slight deviation from linear behavior close to T0, the temperature below which no LE phase exists. Linear extrapolations to π ) 0 yield T0 ) 25.7 °C and T0 ) 43.8 °C for the AHHP monolayer on subphases with pH 4 and pH 8, respectively. Additional information can be obtained by studying the dependence of the transition enthalpy ∆H of the LE/LC phase transition on the subphase temperature. For two-dimensional systems a modified Clausius-Clapeyron equation can be used:

dπ ∆H ) dT T(AE - AC)

(3)

where T is the subphase temperature, AE is the molecular area in the LE phase at the plateau onset, AC is the molecular area in the LC phase extrapolated to the transition pressure, and dπ/dT is the temperature coefficient of the transition pressure. |∆H| as a function of temperature is plotted in Figure 2 (right). Linear extrapolation toward |∆H| ) 0 yields the critical temperature Tc, above which no LC phase exists. Tc values are found to be approximately 53 °C for the monolayer at pH 4 and approximately 60 °C for the monolayer on the subphase with pH 8, where a mostly deprotonated state is expected. For comparison, the Tc value for 1,2-dipalmitoylphosphatidylcholine (DPPC) monolayer, which exhibits the LE/LC phase transition even at 20 °C, amounts to 41.5 °C on a pure water subphase.22 The phase transition enthalpy values of the AHHP monolayer on pH 4 and pH 8 buffer subphases can be directly compared at the same reduced temperature θ, determined as θ ) T/Tc. At θ ) 0.76, |∆H| is found to be 35.8 kJ‚mol-1 and 65.9 kJ‚mol-1 on a subphase with pH 4 and pH 8, respectively. The temperature coefficient dπ/dT amounts to 1.25 and 1.34 on subphases with pH 4 and pH (22) Blume, A. Biochim. Biophys. Acta 1979, 557, 32-44.

Figure 3. Pressure/area isotherms of AHHP monolayer at 20 °C: on citric buffer, pH 4 (a); on Tris buffer, pH 8 (b); on citric buffer, pH 4, in the presence of DNA (c); and on citric buffer, pH 8, in the presence of DNA (d).

8, respectively. For example, the temperature coefficient of DPPC is 1.38.23 Figure 3 presents pH-dependent changes in the pressure/area isotherms of the AHHP monolayer in the absence and presence of DNA. In spite of the fact that the monolayer of AHHP is condensed at 20 °C on the pure buffer subphases, electrostatic repulsion between charged head groups leads to an expansion of the monolayer (shift to larger molecular areas) at pH 4 in comparison with the mostly uncharged state at pH 8 (Figure 3, curves a and b). In the presence of DNA, the monolayer on the pH 4 subphase is much more expanded at surface pressures below 10 mN‚m-1 (DNA adsorbs to and penetrates into the monolayer) compared to that on the pure buffer subphase (Figure 3, curves a and c). In this case, the liquidlike compressibility of the film could be an indication of a disordered state. Indeed, no ordered lipid phase can be observed by GIXD at low-pressure values (see below). A typical condensed π-A isotherm was observed when the lipid film was formed on the subphase with pH 8 in the presence of DNA. Under these conditions, the monolayer is only slightly expanded at low surface pressures in comparison with the film on the pure buffer subphase ((Figure 3, curves b and d). 3.2. Lattice Structure Analysis by GIXD. In order to better understand the influence of subphase pH and presence of DNA on organized 2D structures, the monolayer of AHHP was examined by grazing incidence X-ray diffraction. The observed maxima of the in-plane (Qxy) and the out-of-plane (Qz) scattering vector components as well as tilt angles of the aliphatic chains and cross-sectional areas are presented in Table 1. Figure 4 shows selected contour plots of the corrected X-ray intensities as a function of Qxy and Qz on pure buffer subphases at various surface pressures. In accordance with the pressure/area measurements, at a lateral pressure just above the transition from the gaseous into a condensed phase, the monolayer exhibits two low-order diffraction peaks, the degenerate (1, (1) reflection above the horizon and the nondegenerate (0, 2) reflection at zero Qz, at both investigated pH values. Such an intensity profile is characteristic for a centered rectangular chain lattice with the chains tilted in the direction toward nearest neighbors (NN) along the short axis of the orthorhombic in-plane unit cell (L2 phase).14 On increasing pressure, the 2-fold degenerated peak moves to smaller Qz values, indicating a decrease of the tilt angle of the aliphatic chains. The type of the chain lattice on pH 4 buffer subphase does not change with increasing surface pressure, (23) Yu, Z.-W.; Jin, J.; Gao, Y. Langmuir 2002, 18, 4530-4531.

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Table 1. Bragg Peak (Qxy) and Bragg Rod (Qz) Maxima, Cross-Sectional Area A0, Tilt Angle t, and Distortion of the 2-Amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan-1-ol Langmuir Monolayer Qxy(1) Qz(1) Qxy(2) Qz(2) π (mN/m) (Å-1) (Å-1) (Å-1) (Å-1) A0 (Å2) t (deg) distortion 0 0 0 0 0 0

Citric Buffer, pH 4 1.323 0.756 20.3 1.33 0.735 20.4 1.354 0.687 20.3 1.377 0.64 20.3 1.406 0.56 20.4 1.452 0.42 20.3

0.7 4.8 10 19.7 29.5 39.6

1.454 1.451 1.456 1.456 1.46 1.478

34.4 33.4 31.0 28.7 25.0 18.6

0.13 0.12 0.1 0.08 0.05 0.02

9.7 19.6 29.3 39.5

Citric Buffer, pH 4, in Presence of DNA 1.448 0 1.323 0.745 20.4 33.9 1.451 0 1.362 0.697 20.2 31.2 1.457 0 1.399 0.603 20.3 26.8 1.466 0 1.432 0.516 20.2 22.8

0.12 0.09 0.05 0.03

0.8 5 10.1 19.8 29.7 39.5

1.466 1.469 1.473 1.482 1.493 1.514

23.5 22.1 20.4 16.6 11.4 0

0.04 0.03 0.02 0.01 0.004 0

1.4 4.8 10 19.9 29.5 39.6

Tris Buffer, pH 8, in Presence of DNA 1.465 0 1.424 0.573 20 25.1 1.465 0 1.431 0.551 20 24.1 1.469 0 1.443 0.514 20 22.4 1.479 0 1.464 0.419 20 18.3 1.496 0 1.487 0.25 20.1 11 1.511 0 20 0

0.04 0.03 0.02 0.01 0.008 0

0 0 0 0 0 0

Tris Buffer, pH 8 1.427 0.533 20.2 1.438 0.502 20.1 1.449 0.463 20.1 1.471 0.379 20.1 1.489 0.26 20.1 20.1

whereas the monolayer on pH 8 (mostly deprotonated state of the lipid head groups) undergoes a phase transition into a nontilted phase. Only one Bragg peak at zero Qz can be seen in the diffraction pattern at higher pressure values indicates the chains to be upright and packed in a hexagonal lattice (LS phase). A detailed analysis of the GIXD data indicates that the aliphatic chains in the monolayer are more strongly tilted at pH 4 than

Figure 4. Contour plots of corrected X-ray intensities as a function of in-plane (Qxy) and out-of-plane (Qz) scattering vector components of AHHP monolayer at 20 °C on citric buffer, pH 4, at π ) 10 mN‚m-1 (a), on citric buffer, pH 4, at π ) 40 mN‚m-1 (b); on Tris buffer, pH 8, at π ) 10 mN‚m-1 (c); and on Tris buffer, pH 8, at π ) 40 mN‚m-1 (d).

Figure 5. Tilt of the aliphatic chains t vs monolayer surface pressure π. The subphase is citric buffer, pH 4 (2); Tris buffer, pH 8 (b); citric buffer, pH 4, in the presence of DNA (4); or Tris buffer, pH 8, in the presence of DNA (O).

at pH 8 (Figure 5). At lower pressures, DNA can penetrate into the AHHP monolayer at pH 4. As expected from the pressure/ area isotherms, the presence of DNA in the lipid monolayer leads to its fluidization at low surface pressures. Indeed, no lateral order of lipid chains can be detected below 9 mN‚m-1. DNA/ lipid interactions lead also to an increase of the tilt angle of the aliphatic chains. At pH 8, no changes in the phase behavior of the monolayer due to interactions with DNA are observed. The unit cell of the chain lattice transforms from orthorhombic into hexagonal at surface pressures above 30 mN‚m-1 in the absence and presence of DNA. At low surface pressures, the tilt angle of the aliphatic chains is slightly larger on the DNA-containing subphase compared with the pure buffer subphase, indicating a weak lipid/DNA interaction. Since the molecular area is only slightly increased, a much smaller amount of DNA is penetrated into the lipid layer compared to the situation on pH 4 subphases. At higher lateral pressures, the DNA is obviously squeezed out since the tilt angle is the same on pH 8 subphases with and without DNA. π-A isotherms and GIXD data show clearly that DNA adsorbs on the AHHP monolayer even in solution with pH 8, at which a deprotonated state of the lipid head groups is expected. As the pKa of the new compound is unknown, we have to assume that a small amount of the AHHP molecules is protonated even at pH 8, which produces a certain charge density and provides favorable conditions for DNA adsorption. Since the lipid/DNA interactions are weak under these conditions, the adsorbed DNA does not influence the structure and properties of the AHHP monolayer on the pH 8 subphase. The results obtained in this study are contrary to the condensation effect observed for the triple-chain lipid methyltrioctadecylammonium bromide (TODAB) on pure water.24 This shows that the final state of a cationic Langmuir monolayer while interacting with DNA depends strongly on the surface charge density and also on the van der Waals interactions between the lipid chains. In the case of long-chain molecules and low surface charge density, van der Waals interactions can partly or fully prevent the penetration of DNA into the monolayer, as observed for AHHP on pH 8 buffer solution. If we assume a linear relationship between pressure and molecular area (Axy ) B - kπ) and a constant cross-sectional area A0, then plotting of 1/cos t as a function of π allows us to determine the tilting phase transition pressure πt by extrapolation toward 1/cos t ) 1 (Figure 6). The observed tilting pressures are (24) Symietz, C.; Schneider, M.; Brezesinski, G.; Mo¨hwald, H. Macromolecules 2004, 37, 3865-3875.

3924 Langmuir, Vol. 23, No. 7, 2007

Figure 6. Plots of 1/cos t versus surface pressure for the AHHP monolayer. The subphase is citric buffer, pH 4 (2); Tris buffer, pH 8 (b); citric buffer, pH 4, in the presence of DNA (4); or Tris buffer, pH 8, in the presence of DNA (O).

Figure 7. Lattice distortion of the AHHP monolayer versus sin2 t. The subphase is citric buffer, pH 4 (2); Tris buffer, pH 8 (b); citric buffer, pH 4, in the presence of DNA (4); or Tris buffer, pH 8, in the presence of DNA (O).

55 mN‚m-1 on pH 4 and 37 mN‚m-1 on pH 8 buffer subphase. At pH 4, interactions of DNA with the cationic lipid monolayer shift πt to 60 mN‚m-1. At pH 8, the phase transition in the nontilted state occurs at a surface pressure that is only 2 mN‚m-1 higher than in the absence of DNA. The lattice distortion d versus sin2 t of the AHHP monolayer on various subphases is shown in Figure 7. Landau theory predicts that the tilt contribution to the distortion is proportional to sin2 t.14 Then, by plotting distortion measured along an isotherm as a function of sin2 t and extrapolating to zero tilt, one can separate the contribution of the tilt from other contributions to the distortion. Figure 7 clearly demonstrates that in the deprotonated state (pH 8) the only reason for the monolayer lattice distortion is the tilt of the aliphatic chains independent of the presence of DNA. In contrast, the zero-tilt-angle intercepts, d0, are nonzero when the lipid head groups are highly protonated (pH 4). The absolute values of d0 are 0.036 and 0.059 for the AHHP monolayer on the pure buffer subphase at pH 4 and on the buffer/DNA solution, respectively. One reason for a distorted lattice with upright chains could be the high surface charge density, which prevents the hexagonal packing of lipid molecules on subphases with pH 4. If DNA adsorbs to a Langmuir monolayer and forms a onedimensional periodicity, a diffraction peak ascribable to DNA lateral ordering appears at small Qxy values.24 Assuming that DNA adsorbs to the lipid monolayer as aligned cylinders, it is possible to calculate the lattice spacing (dDNA) as a distance between two neighboring DNA helixes. dDNA as a function of surface pressure π is shown in Figure 8. It can be seen, that the lattice spacing of DNA is on average 6 Å larger when the lipid monolayer is formed on the buffer/DNA solution at pH 8

Antipina et al.

Figure 8. Molecular model of DNA alignment at the monolayer of AHHP. Lattice spacing between the DNA chains, dDNA, is plotted as a function of the surface pressure π. The monolayer is formed on citric buffer, pH 4, in the presence of DNA (4) or on Tris buffer, pH 8, in the presence of DNA (O).

compared with pH 4. The lateral ordering of DNA must be a result of two counteracting processes: The electrostatic attraction of oppositely charged lipid head groups and DNA and the repulsion of like-charged DNA molecules. Thus, because of the smaller amount of positively charged molecules in the monolayer on the subphase at pH 8, DNA cannot compensate effectively its negative charge and, therefore, forms a more loosely packed structure. From Figure 8 one can conclude that the DNA spacing is independent of the surface pressure at pH 4, although the monolayer surface charge density increases by roughly 10% during compression. In contrast, changes in charge density in the monolayer as well as in the DNA adsorption layer (slight decrease of dDNA on compression) are directly linked on the pH 8 subphase. A possible explanation of this result could be that in the case of low pH the monolayer surface charge density is always much higher than the density of the accessible charges in the DNA adsorption layer. Hence the changes of the surface pressure do not influence the DNA packing mode. The resulting average lattice spacing of DNA (approximately 30 Å) exceeds the diameter of the DNA double helix (20 Å) because not all DNA charges at the DNA cylinders are screened by electrostatic interactions with the lipid head groups. This behavior is different compared with a bilayer system where the DNA cylinders are in contact with the lipid head groups on both sides.25 In the case of high pH, only a small part of the monolayer is charged and attracts the DNA. Therefore, the distances between DNA strands are larger. Attracted DNA leads possibly to an increase in the protonation state of the monolayer. Therefore, the lipid monolayer and the accessible charges on the DNA adsorption layer could have very similar charge densities. Hence, an increase in monolayer charge density by compression yields a decrease of dDNA. The unexpected finding of GIXD measurements was that at pH 4 the diffraction peak indicating the ordered DNA structure does not appear at surface pressures below 9 mN‚m-1. Obviously, DNA adsorbed at the liquid-expanded monolayer (see IRRAS results) does not form a one-dimensional periodicity. 3.3. IRRAS Monolayer Experiments. Conclusions about the phase state of the Langmuir monolayer can be drawn from the methylene stretching vibrations, which are sensitive to the conformational order and packing of the hydrocarbon chains. The liquid-expanded state of the monolayer is characterized by the predominance of gauche conformations. Thus, the position (25) Ra¨dler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810-814.

Lipid with New Core Structure for Gene Transfection

Langmuir, Vol. 23, No. 7, 2007 3925

Figure 9. IRRA spectra of AHHP on buffer subphases containing DNA (π ) 40 mN‚m-1) at pH 4 (a) or at pH 8 (b).

of the asymmetric (νas) methylene stretching vibration band is approximately 2924 cm-1. Due to the all-trans conformation of the hydrocarbon chains in the condensed state of the monolayer, this band is shifted to lower values of approximately 2919 cm-1. In agreement with isotherms and GIXD measurements, IRRAS shows that the AHHP monolayer is fully condensed at pH 8: around zero surface pressure νas amounts to 2919 cm-1 and shifts to 2918 cm-1 at high surface pressure. Nearly the same behavior was observed when the subphase with pH 4 was used. In this case, the position of this band changes from 2920 cm-1 at low surface pressure to 2919 cm-1 at high pressure. DNA binding does not influence the asymmetric methylene stretching vibration band in the almost deprotonated state of the monolayer at pH 8. In contrast, on the subphase with pH 4 the monolayer undergoes a phase transition from the liquid-expanded to the condensed state. At zero pressure, the band position of 2922 cm-1 indicates that the interactions with adsorbed DNA lead to a fluidization of the lipid monolayer. This is in good agreement with the observed isotherm, which shows a lift-off area of approximately 150 Å2 and a plateau at approximately 5 mN‚m-1 as well as with the GIXD experiments, which show no ordered structures at low lateral pressures (see Table 1). However, this LE phase is better ordered compared to LE phases of pure lipid monolayers with band positions around 2925 cm-1. Only above 9 mN‚m-1 is the AHHP layer fully condensed. If DNA couples to the monolayer, intensive absorption bands are expected in the sugar-phosphate backbone vibration range, 1250-950 cm-1. One of them, 1052 cm-1, is attributed to the complex mixed O5-C4-C5-O4 vibrations and the others (1223 and 1085 cm-1) are attributed to PO2- asymmetric (νas) and symmetric (νs) stretching vibrations.26-28 The absorption band at approximately 1712 cm-1 indicates that DNA molecules are regular double helixes and is attributed to CdO stretching vibrations of stacked guanine/thymidine base pairs. If the double helix is destroyed, a new band at 1692 cm-1 due to carboxylic group vibrations of unstacked bases appears.26 The IRRA spectra of AHHP on the pure buffer subphases have no absorption bands in the 1250-950 cm-1 region. In the presence of DNA in the subphase, the absorption bands of νs and νasPO2- and mixed O5-C4-C5-O4 vibrations appear at both studied subphase pH values (Figure 9). The absorption band due to CdO stretching vibrations of stacked guanine/thymidine base pairs is observed at 1719 cm-1. The intensity of the absorption bands due to the (26) Tsuboi, M. Appl. Spectrosc. ReV. 1969, 3, 45-90. (27) Sukhorukov, B. I.; Semyenov, M. A.; Maleev, Y. V.; Shabarchina, L. I. Biophysics 1979, 24, 611-619. (28) Sukhorukov, G. B.; Monterl, M. M.; Petrov, A. I.; Shabarchina, L. I.; Sukhorukov, B. I. Biosens. Bioelectron. 1996, 11, 913-922.

Figure 10. Thermotropic phase behavior of aqueous dispersion of AHHP. (a) Lamellar repeat spacing d vs temperature for the system at pH 4 (O) and pH 8 (2); (b1, b2) selected WAXS spectra; (c) DSC curves.

DNA coupling to the AHHP monolayer is clearly higher at pH 4 compared with pH 8, indicating that the monolayer can bind more DNA molecules when the lipid head groups are in the protonated state. This is in agreement with the higher packing density of aligned DNA observed via GIXD. Comparing the intensity of νasCH2 stretching vibrations of the AHHP monolayer on the subphase containing DNA with that on the pure buffer subphase can give information about the amount of DNA penetrated into the lipid monolayer. For example, at 5 mN‚m-1 approximately 56% of the surface is occupied by penetrated DNA molecules on the pH 4 subphase, in contrast with only 17% when the monolayer is in the mostly deprotonated state (pH 8). Compression of the layer leads to a complete squeezing out of DNA at pH 8. At pH 4, a small amount of penetrated DNA (less than 10%) was still detected at 40 mN‚m-1. 3.4. Thermotropic Phase Behavior in Aqueous Dispersions. The thermotropic phase behavior of AHHP in aqueous dispersions was studied by time-resolved small- and wide-angle X-ray diffraction measurements and DSC technique (Figure 10). In both citric (pH 4) and Tris (pH 8) buffers, the system is in a lamellar state in the whole investigated temperature range (from 30 up to 70 °C). Below 56-57 °C the lamellar spacing d decreases slightly with increasing temperature but does not depend on the pH value (Figure 10a). The influence of the pH value on the packing properties of AHHP becomes apparent only in the type of the chain lattice. Two Bragg peaks in the WAXS region indicate that at pH 4 the system is in the tilted lamellar gel phase (Lβ′) with an orthorhombic chain lattice (Figure 10b1). In pH 8 solution, AHHP forms a nontilted Lβ gel phase, as only one Bragg peak can be seen in the WAXS spectra (Figure 10b2), indicating hexagonal packing of nontilted chains. The corresponding values of the cross-sectional area for the aqueous dispersions at pH 4 and pH 8 are 20.3 and 20.1 Å,2 respectively. WAXS analysis shows that the bulk systems form the same type of chain lattice with similar parameters as the Langmuir monolayers: orthorhombic lattice with tilted chains at all pressures at pH 4 and a hexagonal lattice with nontilted molecules above 37 mN‚m-1 at pH 8. The fact that the lamellar spacing d is the same in the tilted and nontilted gel phases can be a result of different interlayer

3926 Langmuir, Vol. 23, No. 7, 2007

water thicknesses. At pH 4, the water layer between the bilayers could be thicker due to electrostatic repulsion between the charged lipid layers. Above 57 °C, the halo in the WAXS spectra indicates molten chains of lipid molecules in the AHHP aqueous dispersion at both investigated pH values (Figure 10b1,b2). The phase transition leads into the lamellar LR phase. Decrease of the lamellar spacing d above the phase transition is due to formation of gauche conformations in the lipid chains in the LR phase (Figure 10a). DSC measurements show clear phase transitions with Tm ) 56.3 and 57 °C for AHHP in pH 4 and pH 8 dispersions, respectively. Thus, the calorimetric results are in good agreement with the X-ray measurements. The phase transition enthalpy amounts to 21 kJ‚mol-1 in the solution with pH 4 and 29 kJ‚mol-1 in pH 8 buffer solution. If the bilayer can be considered as two weakly coupled monolayers, the temperature of the lipid “main” phase transition from gel to fluid state (Tm) in aqueous dispersions is the bilayer equivalent of the monolayer critical temperature Tc. Similar Tc and Tm values have been observed, for instance, for DPPC.29-30 In the case of AHHP, there is only a rough agreement of these transition temperatures ((3 °C).

4. Conclusion The cationic lipid 2-amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan-1-ol (AHHP) was synthesized and investigated in two- and three-dimensional systems at two different pH values. The new compound can be used in pH-sensitive liposomemediated transfection of genetic material into cells and tissues. Indeed, AHHP was tested in a biological assay using NIH5T3 (29) Kim, J. T.; Mattai, J.; Shipley, G. G. Biochemistry 1987, 26, 6592-6598. (30) Meyer, H. W.; Semmler, K.; Rettig, W.; Pohle, W.; Ulrich, A. S.; Grage, S.; Selle, C.; Quinn, P. J. Chem. Phys. Lipids 2000, 105, 149-166.

Antipina et al.

and Hep G2 cells and the galactosidase gene as a marker for successful transfection. The physical-chemical properties of AHHP were investigated depending on pH conditions and hence on the protonation state of the lipid head group. Coupling of DNA with the new lipid was studied with a Langmuir monolayer as a model system. It was shown that the structure and phase behavior of AHHP depend on the subphase pH both in the monolayer system and in aqueous dispersions. Monolayer critical temperature (Tc) is proved to be in rough agreement with the temperature of the lipid phase transition from gel to fluid state (Tm) determined by X-ray and calorimetry studies of AHHP aqueous dispersions. Molecules in mono- and bilayer systems have very similar packing properties. The DNA coupling to the AHHP monolayer was detected by the appearance of a Bragg peak arising from ordered adsorbed DNA strands with a one-dimensional periodicity and in IRRA spectra by intensive absorption bands in the sugar-phosphate backbone vibration range, 1250-950 cm-1, and at 1712 cm-1. The results presented demonstrate DNA binding to the AHHP monolayer at low as well as at high pH. However, the AHHP monolayer couples a much smaller amount of DNA at pH 8 than at pH 4. Acknowledgment. This work was supported by the Max Planck Society and the Alexander von Humboldt Foundation (M.N.A.). The help of Kristian Kjaer and Dmitri Novikov at beamline BW1 and of Sergio S. Funari at beamline A2 with setting up the X-ray experiments is gratefully acknowledged. We thank HASYLAB at DESY, Hamburg, Germany, for beam time and for providing excellent facilities and support. LA062840I