Multilamellar Liposomes Formed by Phosphatidyl Nucleosides: An

The investigation is conducted mostly via the HR-MAS technique and the general observation is that the resolution achieved in this way is superior to ...
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Langmuir 2004, 20, 1144-1151

Multilamellar Liposomes Formed by Phosphatidyl Nucleosides: An NMR-HR-MAS Characterization Oscar Cruciani,§ Luisa Mannina,*,†,§ Anatolij P. Sobolev,§ Annalaura Segre,§ and Pierluigi Luisi‡ Istituto di Metodologie Chimiche, C.N.R., Area della Ricerca di Roma, C.P. 10, I-00016 Monterotondo Stazione, Rome, Italy, Facolta` di Agraria, Dipartimento S.T.A.A.M., Universita` degli Studi del Molise, via De Sanctis, I-86100 Campobasso, Italy, and Dipartimento di Biologia, Universita` “Roma Tre” , Viale Marconi 446, I-00146 Rome, Italy Received September 26, 2003. In Final Form: December 5, 2003 We present an NMR investigation of multilamellar vesicles (MLVs) obtained from phosphatidyl nucleosides, 5′-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)cytidine (1), 5′-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)inosine (2), and their mixtures. Because of the lower stability of liposomes obtained from 2, studies have been preferentially performed in this case with mixed liposomes 2/POPC (4:1). The investigation is conducted mostly via the HR-MAS technique and the general observation is that the resolution achieved in this way is superior to that obtained in the past with small unilamellar vesicles (SUVs). A full assignment is now possible, which includes the spectral region of the ribose ring and part of the glycerol moiety. Also in the case of MLVs, both for 1 and 2, a stacking between the aromatic bases of the same liposome layer seems to be ruled out, although in both cases the nucleobases appear to be exposed to the aqueous phase. The splitting of both aromatic H-5cyt and H-6cyt is ascribed to the presence of two aggregate populations that may correspond to the two syn and anti conformations observed for cytidine monophosphate in aqueous solution. On the basis of NOESY cross-peaks, it is not always possible to discriminate between inter- and intramolecular interactions; however, the distances found for 1 appear to be compatible with the intramolecular contacts in the anti conformation of the cytidine and also with intermolecular interactions between neighboring molecules of 1. We also find that the glycerol moiety does not seem to interact with the cytidine; however, part of the ribose ring seems to be close to the glycerol moiety. More generally, the interaction of one base with the sugar moiety of a neighboring base, previously observed for SUVs, still appears to be true for MLVs. Studies have been performed also for mixed liposomes obtained from the mixture of 1 and 2, where it is observed that the HR-MAS spectra of the corresponding MLVs are not simply the sum of the spectra of the two isolated components. In particular, there is the presence of a NOESY cross-peak between the aromatic protons H-6cyt and H-2ino, and this permits us to rule out large patchwork domains containing only one nucleoside components in the mixed liposomes. Finally, a study is performed on the time evolution of the system obtained by mixing the previously prepared liposomes of 1 and 2. No interaction is obtained in this case, i.e., the spectra are constitutive, which is consistent with the general picture of liposomes as kinetic traps that are not fusing with each other.

Introduction Over the last 10 years we have been investigating liposomes obtained from phosphatidyl nucleosides, in particular 5′-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)cytidine (1) and 5′-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)inosine (2), which can be obtained enzymatically from the corresponding phosphatidylcholines and the nucleosides. These liposomes are interesting since, in principle, they display the information chemistry of nucleic acids combined with the compartment features of vesicles. The main driving idea for these studies was to investigate whether and to what extent the chemical and binding properties of nucleic acids can be expressed also on the spherical structure of the liposomes. A modest recognition between “complementary” liposomes was previously obtained,1 and also observed on the configuration of a Langmuir-Blodgett film made with phosphatidyl nucleosides.2 * To whom correspondence should be addressed. E-mail: [email protected]. † Universita ` degli Studi del Molise. ‡ Universita ` “Roma Tre”. § C.N.R., Area della Ricerca di Roma. (1) Berti, D.; Baglioni, P.; Bonaccio, S.; Borsacchi-Bo, G.; Luisi, P. L. J. Phys. Chem. B 1998, 102, 303. (2) Berti, D.; Franchi, L.; Baglioni, P.; Luisi, P. L. Langmuir 1997, 13, 3438.

However, in our previous NMR studies3 on these systems we suggested that the presence of the interaction of one given base with the sugar moiety of a neighboring base could explain why the bases are not available for complementary base pairing. Moreover, in the case of liposomes formed using complementary phosphatidyl nucleosides, no evidence of the existence of mixed liposomes was found. An analogous NMR study was also carried4 on micelles formed by hexadecylphosphoryl nucleosides and, in particular, hexadecylphosphoryladenosine (C16-AMP) and hexadecylphosphoryluridine (C16-UMP). In these supramolecular aggregates the polar heads of the two surfactants lie almost perpendicular to the micellar radius. It is important to observe that in the case of mixed micelles a significant interaction between the two complementary aromatic bases is present. Therefore, in the case of micelles formed using complementary hexadecylphosphoryl nucleosides, the existence of mixed surfaces formed by both bases has been proved. Note that in small liposomes formed by the mixture 1/2 a patchwork structure has been evoked since no experimental evidence of mixed surfaces was ever found.3 Hence (3) Bonaccio, S.; Capitani, D.; Segre, A. L.; Walde, P.; Luisi, P. L. Langmuir 1997, 13, 1952. (4) Zandomeneghi, G.; Luisi, P. L.; Mannina, L.; Segre, A. L. Helv. Chim. Acta 2001, 84, 3710.

10.1021/la035804h CCC: $27.50 © 2004 American Chemical Society Published on Web 01/23/2004

HR-MAS on Liposomes from Phosphatidyl Nucleosides

it seems possible that the curvature of the system may play a major role in determining the surface distribution of the complementary bases. In this paper we continue this investigation with special regards to multilamellar liposomes. In our previous NMR studies3 performed on liposomes, i.e., on small unilamellar vesicles (SUVs), from 1 and 2 the lack of resolution, due to the intrinsic anisotropy, was the main obstacle to the structural characterization of these systems and to the study of binding availability for the bases of the phosphatidyl nucleosides in liposomes. In the present work, we decided to study a system characterized by a very low curvature i.e., multilamellar vesicles (MLVs) obtained from the same compounds. Because of their low curvature, MLVs constitute a more reliable membrane model than SUVs, that are characterized by a high curvature and by a marked packing asymmetry of the inner and outer monolayer.5-7 In MLVs the use of magic angle spinning (HR-MAS) allows a real improvement of the resolution.8,9 It is important to underline that before the introduction of the HR-MAS method, due the to intrinsic anisotropy of the multilamellar systems, MLVs spectra were extremely broad and were not informative. In the HR-MAS technique the sample is placed in a rotor spinning around an axis, which is oriented at the so-called magic angle of 54.7° with respect to the magnetic field B0.10 Spinning the sample at a sufficient rate allows an averaging of the spin dipolar interactions and susceptibility distortions. In particular in the case of membranes the averaging of the spin like dipolar interactions by the use of magic angle spinning (MAS) is possible due to the presence of an internal fast axial rotation9,11 that reduces the intramolecular dipole-dipole interactions. Therefore, when an internal motion already reduces these dipolar couplings, the spectral broadening present under static conditions can be reduced by the use of MAS. The result is an NMR spectrum with resolution approaching that of liquid samples. Interesting applications of the HR-MAS technique in study of interactions have been reported.12,13 However this method does not work if the dipolar broadening is too severe (stiff systems) or if the vesicles present a motion that interferes with the coherent perturbation applied by the MAS as in the case of SUVs.14,16 The present paper reports the clarification, obtained by the use of the HR-MAS technique, about some aspects of the conformation of the phosphatidyl nucleosides and about the interaction of the bases with the sugar and glycerol moieties and the lipidic bilayer on a multilamellar structure. In particular, the present study will deal with MLVs obtained from the two phosphatidyl nucleosides indicated above as 1 and 2. Moreover we investigated the possibility of obtaining mixed MLVs either producing liposomes directly from the mixture 1/2 or starting with separate MLVs of 1 and MLVs of 2, which are then mixed together. To investigate intramolecular interactions between groups within the same lipid molecule and intermolecular interactions between neighboring lipid molecules, we will utilize here 1D- and 2D-1H-HR-MAS. Materials and Methods Materials. 1-palmitoyl-2-oleoyl-sn-glycero-3-phospatidylcholine (POPC) was purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. 1 and 2 have been synthesized enzymatically from POPC, and corresponding nucleosides by the help of phospholipase D as described before.16 Cytidine and inosine were obtained from Fluka (Switzerland). Phospholipase D from Streptomices sp. AA 586

Langmuir, Vol. 20, No. 4, 2004 1145 was purchased from Genzyme Diagnostics (West Malling, Kent, U.K.). D2O (99.9% atom D) was purchased by Fluka. All the other reagents of the highest-purity grade were from Fluka, Sigma, or Merck. Sample Preparation. Liposome preparation has been described extensively elsewhere.15,16 Multilamellar liposomes from 1, from the mixture 2/POPC (4:1, mol ratio), and from the mixture 1/2 (1:1 mol ratio) have been prepared by dispersing a dry film of the lipids, deposited from a CHCl3 solution on a glass wall, in an appropriate buffer solution (20 mM deuterated sodium phosphate buffer, 20 mM KCl, 0.2 mM EDTA, pD ) 8). The final lipid concentrations were: 18.1 mg/mL (20 mM) for MLVs of 1; 18.6 mg/mL (20 mM) in 2 and 3.8 mg/mL (5 mM) in POPC for MLVs of 2/POPC (4:1); 7.7 mg/mL (8.5 mM) in 1 and 7.9 mg/mL (8.5 mM) in 2 for MLVs of 1/2 (1:1). A further sample was prepared by mixing an equal volume of the multilamellar dispersion of 1 and of the multilamellar dispersion of the mixture 2/POPC. Methods. HR-MAS NMR. All HR-MAS experiments were performed on a Bruker AVANCE 600 spectrometer operating at 600.13 MHz, using a Bruker HR-MAS probe with an external lock. The samples were loaded in 4 mm ZrO2 cylindrical rotors with spherical inserts (internal volume of 12 or 45 µL) and spun at different spinning rates in the range from 3 to 12 kHz. All NMR spectra (1D and 2D) have been referenced to the palmitoyl and oleoyl ω-CH3 set at 0.90 ppm from TMS (tetramethylsilane), as previously indicated for similar compounds.19,20 To optimize the experimental conditions (temperature, spinning speed, and NMR acquisition parameters), a set of experiments on three different samples of POPC liposomes was performed as extensively shown elsewhere.8 The 2D experiments have been performed in the experimental conditions (temperature and spinning speed) to ensure a sufficient resolution and while maintaining the integrity of the rotor inserts during long time experiments like NOESY (10 h). All 1H HR-MAS NMR experiments on MLVs samples have been performed at the temperature and the spinning speed later on indicated for each sample accumulating 128 scans with 32K data (time domain) and using a spectral width of 6.0 kHz. A standard pulse sequence with water suppression by a presaturation of the HDO resonance during the relaxation delay was used. A 4-6 µs 90° excitation pulse was used with a relaxation delay of 3s. Prior to Fourier transformation, the data were zero filled to 32K points and apodized using an exponential line broadening of 1 Hz.21 All the 2D COSY experiments22 have been performed, using a spectral width of 6 kHz in both dimensions, 256 increments, 20 scans, 2K data points, and a relaxation delay of 3 s. All the 2D-1H-NOESY experiments22 have been performed in TPPI phase-sensitive mode, using 6 kHz spectral width in (5) Schuh, J. R.; Mu¨ller, L.; Chan, S. I. Biochim. Biophys. Acta 1982, 687, 219. (6) Longmuir, K. J.; Dahlquist, F. W. Proc. Natl Acad. Sci. USA 1976, 73, 2716. (7) Tauskela, J. S.; Thompson, M. Biochim. Biophys. Acta 1992, 1104, 137. (8) Cruciani, O.; Mannina, L.; Sobolev, A. P.; Cametti, C.; Segre, A. L. Colloids Surf. A: Physicochem. Eng. Asp., in press. (9) Oldfield, E.; Bowers, J. L.; Forbes, J. Biochemistry 1987, 26, 6919. (10) Mehring, M. Principles of High-resolution NMR in solids; Springer-Verlag: Heidelberg, Germany, 1983. (11) Davis, J. H.; Auger, M.; Hodges, R. S. Biophys. J. 1995, 69, 1917. (12) Handel, H.; Elke, G.; Gottschall, K.; Klaus, A. Angew. Chem. 2003, 42, 438. (13) Winter, W. T Polym. Prepr. 2003, 44, 299. (14) Maricq, M. M.; Waugh, J. S. J. Chem. Phys. 1979, 70, 3300. (15) Long, J. R.; Sun, B. Q.; Bowen, A.; Griffin, R. G. J. Am. Chem. Soc., 1994, 116, 11950. (16) Bonaccio, S.; Walde, P.; Luisi, P. L. J. Phys. Chem. 1994, 98, 6661. (17) Szoka, F.; Papahadjopoulos, D. Annu. Rev. Biophys. Bioeng. 1980, 9, 467. (18) Vemuri, S.; Rhodes, C. T. Pharm. Acta Helv. 1995, 70, 95. (19) Halladay, H. N.; Stark, R.E.; Ali, S.; Bittman, R. Biophys. J. 1990, 58, 1449. (20) Volke, F.; Pampel, A. Biophys. J. 1995, 68, 1960. (21) Ernst, R. R.; Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions, Clarendon Press: Oxford, England, 1987. (22) Braun, S.; Kalinowski, H. O.; Berger, S. 150 and More Basic NMR Experiments. A practical course. Wiley-VCH: New York, 1998.

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both dimensions, 512 increments in F1, 32 scans, 1K data points in F2, a relaxation delay of 3 s, and a mixing time of 15, 20, 25, or 100 ms as indicated later on for each sample. MLVs of 1. 1D experiments at different temperatures (298, 300, 305, 318 K) and at different spinning speeds (3, 4, 5, 6, 8, 10, and 12 kHz) have been performed. The following 2D experiments have been performed at 318 K and with a spinning speed of 5 kHz: COSY; NOESY with a mixing time of 15 ms; NOESY with a mixing time of 25 ms. MLVs of 2/POPC. 1D experiments at different temperatures (300, 318 K) and at different spinning speeds (3, 4, 5, 6, 8, 10, and 12 kHz) have been performed. The following 2D experiments have been performed at 300 K and with a spinning speed of 8 kHz: COSY; NOESY with a mixing time of 15 ms; NOESY with a mixing time of 25 ms. MLVs of the 1/2 Mixture. 1D experiments at 300 K and at different spinning speeds (4, 5, 8, and 12 kHz) have been performed. The following 2D experiments have been performed at 300 K and with a spinning speed of 8 kHz: NOESY with a mixing time of 15 ms; NOESY with a mixing time of 20 ms; NOESY with a mixing time of 25 ms. MLVs of 1 + MLVs of 2 /POPC. 1D experiments at 300 K and at different spinning speeds (3, 8, 10, and 12 kHz) have been performed. The following 2D experiments have been performed at 300 K and with a spinning speed of 10 kHz: NOESY with a mixing time of 25 ms; NOESY with a mixing time of 100 ms. A fundamental parameter in any 2D-1H NOESY experiment is the mixing time. As previously pointed out,3,23,24 due to the presence of strong spin diffusion, long mixing times produce spurious cross-peaks, which in turn may lead to a wrong interpretation. In micelles and other supramolecular lipidic systems, significant spin diffusion may be present even at relatively short mixing times. To ensure correct 2D-1H-NOESY experiments on liposomes, the mixing time must be chosen in such a way to rule out the possibility of spin diffusion effects. The criterion used was the absence of any residual cross-peak between the resonance due to the terminal methyl group of the fatty acid chains at 0.90 ppm and both the resonances due to the methylene protons of the glyceride moiety near 4.5 ppm and to the fatty acid olefinic protons at ∼5.3 ppm. Only with a mixing time as short as 15-25 ms were no spurious cross-peaks found. It must be clear that, due to this very short mixing time, only strong cross-peaks, corresponding to rather short interatomic distances, can be observed, while interatomic distances of the order of 4.5 Å or more cannot be detected. However, in the case of the sample obtained by mixing MLVs of 1 with MLVs of 2/POPC, to verify the eventual presence of NOE contacts between distant protons, a mixing time of 100 ms was also used.

Results and Discussion MLVs of 1. In the given experimental conditions, a resolution sufficient to perform a full assignment was obtained (see Table 1 and Figure 2). It is worth of noticing that, in the case of solution NMR experiments on SUVs from 13, a full spectral assignment was not possible. The full assignment obtained by 2D experiments is given in Figure 2 and Table 1, encompassing the spectral region (3.8-4.6 ppm) relative to the ribose ring and part of the glycerol moiety. In particular, the 2D COSY experiment (data not shown) allows us to assign the broad signal at 1.3 ppm to different CH2 groups of fatty chains. The chemical shifts observed for the aromatic protons in the case of MLVs of 1 agree with those previously found for cytidine monophosphate in aqueous solutions.3 Therefore, also in the case of MLVs, a possible stacking between the aromatic bases of the same layer must be ruled out, as seen previously in the case of SUVs.3 In the presence of stacking, the magnetic anisotropy of the aromatic ring would cause a strong upfield shift on the (23) Keepers, J. W.; James, T. L. J. Magn. Reson. 1984, 57, 404. (24) Olejniczak, E. T.; Gampe, R. T.; Fesik, S. W. J. Magn. Reson. 1986, 67, 28.

Cruciani et al. Table 1. Assignment of 600 MHz 1H-HR-MAS Spectrum, Performed with a Spinning Rate of 5 kHz and at 318 K, of MLVs Obtained from 1 (20 mM in 20 mM Deuterated Sodium Phosphate Buffer, 20 mM KCl, 0.2 mM EDTA, pD ) 8)a 1H

resonance 6acyt 6bcyt 5acyt 5bcyt 1′cyt 2′cyt 3′cyt 4′cyt 5′cyt 5′′cyt A B C C′ W P P′ Q Q′ F J T′′ T′ T S

((0.02 ppm) 8.05 7.98 6.21 6.15 5.95 4.31 4.30 4.17 4.08 3.97 4.10 5.32 4.52 4.26 5.32 2.41 2.36 2.31 2.26 2.02 1.61 1.35 1.32 1.31 0.90

NOESY contacts 5acyt, 1′cyt, 2′cyt, 4′cyt, 5′cyt 5bcyt, 1′cyt, 2′cyt, 4′cyt, 5′cyt 6acyt, 1′cyt, 2′cyt, 4′cyt 6bcyt, 1′cyt, 2′cyt, 4′cyt 6acyt, 6bcyt, 5acyt, 5bcyt, 2′cyt, 5′cyt, 5′′cyt 6acyt, 6bcyt, 5acyt, 5bcyt, 1′cyt B 6acyt, 6bcyt, 5acyt, 5bcyt, B 1′cyt, 5′′cyt, B 1′cyt, 5′cyt, B B C, C′, A, 3′cyt, 4′cyt, 5′cyt, 5′′cyt B, C′ B, C F, T′′ F, T, J, P′ F, T, J, P F, T, J F, T, J W, T, T′′, J, P, P′, Q, Q′ F, T, T′, P, P′, Q, Q′ F, W J S, J, F, P, P′, Q, Q′ T

a In the third column the contacts registered in the 2D-1H NOESY experiments are also reported.

aromatic 1H in the neighboring ring and vice versa.25 In this case this upfield shift is not present. As is apparent from Figure 2 (1D spectrum) and Figure 3 (2D-1H-NOESY map, an expansion of the aromatic and sugar spectral region) the NMR signals of both aromatic protons H-5cyt and H-6cyt are split. The same splitting is partially present in the resonance due to the anomeric H-1′cyt while all the other signals are not split. Moreover in the COSY spectra (not shown) the absence of H-6acytH-6bcyt cross-peaks and H-5acyt-H-5bcyt suggests that these a and b resonances are from different molecules. In the 2D-1H-NOESY maps, a cross-peak between the signals 6acyt and 5acyt is evident as well as one between the signals 6bcyt and 5bcyt. We interpret this splitting and the cross-peaks previously mentioned as due to the presence of two different populations, a (lower field) and b (higher field), which can be evaluated in the ratio 5:2. It is then possible that the two conformations syn and anti observed for cytidine monophosphate in aqueous solution3 and supposed in the case of SUVs from compound 1 are both present also when the same nucleoside is involved in a multilamellar structure. Because of the poor resolution obtainable in the static NMR experiments, this important evidence was not previously observed. In the 2D-1H-NOESY spectra cross-peaks between the signals 6acyt and 6bcyt and between the signals 5acyt and 5bcyt are present. These NOESY cross-peaks are due to a slow interconversion between the two forms syn and anti. Therefore, due to the presence of this interconversion, all signals due to the two populations present the same NOESY cross-peaks. Therefore, specific contacts for each conformer population cannot be obtained. As reported in Table 1, the resonances due to aromatic protons H-5cyt and H-6cyt and those due to some of the sugar ring show observable NOESY cross-peaks. In par(25) Waugh, J. S.; Fessenden, R. W. J. Am. Chem. Soc. 1957, 79, 846.

HR-MAS on Liposomes from Phosphatidyl Nucleosides

Figure 1. Molecular structures of POPC and of the compounds 1 and 2.

ticular, the resonance due to the aromatic proton H-5cyt shows NOESY cross-peaks with that due to sugar protons H-1′cyt, H-2′cyt, and H-4′cyt (contact less evident). The resonance due to the aromatic proton H-6cyt gives NOESY cross-peaks with that due to protons H-1′cyt, H-2′cyt, H-4′cyt, and H-5′cyt. It is important to underline that it is not always possible to distinguish between intra- and intermolecular interaction, using the information (cross-peaks and integrals) obtained from the 2D-1H-NOESY maps. This fact hampers a clear model of the 1 conformation in the vesicular aggregate which, however, is consistent with a model such as the one suggested in our previous paper.4 The distances, calculated by the integration of the NOESY cross-peaks and using as internal reference the distance (2.45 Å) between the aromatic protons H-5cyt and H-6cyt are only indicative: in fact, due to the presence of the spin diffusion, mixing times extremely short have been used and no build-up curve has been built. In particular, the distances values obtained between the aromatic protons H-5cyt and H-6cyt, are only partially useful. In fact, the values obtained for the contacts H-6cyt-H-1′cyt and H-6cyt-H-2′cyt seem compatible with intramolecular interactions in the anti conformation but also with intermolecular interactions between neighboring molecules of 1. On the other hand, the distances calculated for the contacts H-5cyt-H-1′cyt, H-5cyt-H-2′cyt and H-6cyt-H-4′cyt are consistent only with intermolecular interactions. It is also evident from the 2D-1H-NOESY experiments that no cross-peaks between the resonances due to the aromatic protons and either the resonance due to the glycerol moiety or to the fatty acid chains are present.

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Figure 2. 600 MHz 1H-HR-MAS spectrum, performed with a spinning rate of 5 kHz and at 318 K, of MLVs obtained from 1 (20 mM in 20 mM deuterated sodium phosphate buffer, 20 mM KCl, 0.2 mM EDTA, pD ) 8).

Therefore, the glycerol moiety does not seem to interact with the cytidine; this observation is also confirmed by the chemical shift values of the glycerol backbone protons. In fact in the case of MLVs from 1, these protons have practically the same chemical shifts previously observed in liposomes of POPC,8,26 and result unaffected by the presence of the nucleoside. The resonance due to the proton B of glycerol backbone, besides the obvious cross-peaks with the resonances due to the other protons (A, C, and C′) of the same molecular fragment, shows cross-peaks with the resonances due to protons H-5′cyt, H-5′′cyt, and H-3′cyt of the sugar moiety. No cross-peaks between resonances of the glycerol moiety and those of the fatty acid chains are present. Therefore, part of the ribose ring seems to be close to the glycerol moiety, whereas it is surely far away from the alkyl chains. Probably the ribose tends to bend toward the glycerol moiety so as to minimize the contact between its hydrophobic parts and the aqueous phase. The aromatic base seems to be located away both from the glycerol and from the alkyl chains and does not seem reclined back toward the bilayer but exposed toward the aqueous phase. However the information obtained from the 2D-1HNOESY maps about the contacts between the aromatic protons H-5cyt and H-6cyt and protons of the sugar moiety indicate that the interaction of one base with the sugar moiety of a neighboring base, proposed in the previous (26) Dorovska-Taran, V.; Wick, R.; Walde, P. Anal. Biochem. 1996, 240, 37.

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Cruciani et al. Table 2. Assignment of 600 MHz 1H-HR-MAS Spectrum, Performed with a Spinning Rate of 8 KHz and at 300 K, of MLVs Obtained from the Mixture 2/POPC (20 mM in 2 and 5 mM in POPC in 20 mM Deuterated Sodium Phosphate Buffer, 20 mM KCl, 0.2 mM EDTA, pD ) 8)a 1H

Figure 3. 2D-1H-NOESY spectrum, performed with a spinning rate of 5 kHz at 318 K and with a mixing time of 15 ms, of the MLVs obtained from 1: expansion of the spectral region of aromatic and sugar protons.

model,3 seems still true. Therefore, the headgroup of 1 is not suitable for complementary recognition and binding. MLVs of 2/POPC. Liposomes of 2 are not stable and tend to aggregate;16,27 Then, we chose to characterize stable MLVs obtained from a mixture 2/POPC (4:1). We report the full assignment of the HR-MAS spectrum performed at 300 K and with a spinning speed of 8 kHz in Table 2 and in Figure 4. The values of the chemicals shift indicate that also in this case a stacking between adjacent aromatic bases is absent. The splitting of the NMR signals relative to the aromatic, H-2ino and H-8ino, and anomeric, H-1′ino, protons is present also in this case, but less evident than in the case of MLVs of 1 and observable only as an elongation of diagonal peaks in the 2D spectra. The resonance due to the aromatic proton H-8ino yields observable NOESY cross-peaks with the resonance due to the other aromatic proton H-2ino and with the resonances due to all the ribose protons (see Table 2). The cross-peak between the resonances of the aromatic protons H-8ino and H-2ino is clearer in the 2D-1H-NOESY map obtained using a mixing time of 25 ms. Moreover a contact, more intense than the contact H-8ino-H-1′ino, is present between the aromatic proton H-2ino and the anomeric one H-1′ino. The resonance due to the anomeric proton H-1′ino shows other two NOESY cross-peaks with the resonance due to the ribose proton H-4′ino and with a resonance at 4.50 ppm. Because of the superimposition at 4.50 ppm of the ribose proton H-3′ino resonance and of the glycerol c resonance, an unequivocal interpretation of the NOESY cross-peak in question is not possible. However the lack of further NOESY contacts between the anomeric protons H-1′ino and the other protons of the glycerol moiety suggests (27) Bonaccio, S.; Wessicken, M.; Berti, D.; Walde, P.; Luisi, P. L. Langmuir 1996, 12, 4976.

resonance

((0.02 ppm)

8ino 2ino 1′ino 2′ino 3′ino 4′ino 5′ino 5′′ino A B C C′ W R M N P P′ Q Q′ F J T (≡T′, T′′) S

8.41 8.13 6.06 4.63 4.50 4.31 3.95 3.89 4.10 5.33 4.50 4.26 5.33 3.29 4.37 3.75 2.41 2.38 2.29 2.24 2.03 1.60 1.30 0.90

NOESY contacts 2ino,1′ino, 2′ino, 4′ino, 5′ino, 5′′ino, 4.50 ppm 1′ino, 8ino 8ino, 2ino, 2′ino, 4′ino, 4.50 ppm 8ino , 1′ino, 3′ino 2′ino 8ino, 1′ino, 5′ino, 5′′ino 8ino, 4′ino, 5′′ino, B 8ino, 4′ino, 5′ino, B B, C C′, C, A, 5′ino, 5′′ino C′, A, B C, B F, T N M F, T, J, P′ F, T, J, P F, T, J F, T, J W, T, J, P, P′, Q, Q′ F, T, P, P′, Q, Q′ S, J, F, W, P, P′, Q, Q′ T

a In the third column the contacts registered in the 2D-1H NOESY experiments are also reported.

that this cross-peak might be due to a contact between the anomeric proton H-1′ino and the proton H-3′ino. The chemical shifts values and the information obtained from the 2D-1H-NOESY maps indicate for the polar head the same situation reported in the case of MLVs of 1; therefore, again, the nucleobase is not turned back toward the bilayer but is exposed toward the aqueous phase. In the case of MLVs of 1 a distinction between intraand intermolecular contacts does not seem possible; while, for MLVs of 2/POPC, the existence of intermolecular contacts is confirmed by the presence of a NOE crosspeak between the resonances due to the protons H-8ino and H-2ino. This cross-peak can be due only to an intermolecular interaction; in fact in the same molecule these protons are too far apart (∼6.5 Å) to allow observable NOE effects. Moreover the contact H1′ino-H-2ino can only be due to intermolecular interaction. MLVs of the Mixture 1/2. It is important to underline that the HR-MAS spectra of the MLVs obtained from the mixture 1/2 are not simply the sum of the spectra registered for MLVs from a single phosphatidyl nucleoside. This evidence is not trivial, because in the case of unilamellar vesicles from the same compounds3 the spectra obtained for the vesicles formed by the 1:1 mixture of 1/2 was exactly the sum of the two separate spectra. The comparison between the 1D HR-MAS spectra of MLVs obtained from the 1:1 mixture of 1/2 (see Figure 6) and the HR-MAS spectra of MLVs from the single phosphatidyl nucleoside (carried under the same experimental conditions) emphasizes a chemical shift change for the aromatic and the anomeric protons of cytidine and inosine. In particular, on respect to the cases where only one phosphatidyl nucleoside was present, we observe an upfield shift for the resonances due to cytidine and a downfield shift for those due to inosine. This shift is quite evident for the resonances due to aromatic and anomeric protons; all the other resonance signals are unaffected.

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Figure 5. 2D-1H-NOESY map, performed with a spinning rate of 8 kHz at 300 K and with a mixing time of 15 ms, of the MLVs obtained from the mixture 2/POPC: expansion of the spectral region of aromatic and sugar protons.

Figure 4. 600 MHz 1H-HR-MAS spectrum, performed with a spinning rate of 8 kHz and at 300 K, of MLVs obtained from the mixture 2/POPC (20 mM in 2 and 5 mM in POPC in 20 mM deuterated sodium phosphate buffer, 20 mM KCl, 0.2 mM EDTA, pD ) 8).

It is important to underline that the above shifts are present for all the used spinning speeds and therefore are not an artifact dependent by the experimental conditions. In Table 4, we report the values of the shifts for the aromatic signals registered at different spinning speeds. A small splitting of aromatic and anomeric signals, as previously pointed out for MLVs derived from a single phosphatidyl nucleoside, is present also in this case and with the same pattern previously seen. In the case of MLVs from the mixture 1/2, in the 2D-1H-NOESY maps (see Figure 7) no cross-peaks are present between resonances due to the aromatic protons and those due to the glycerol moiety (see Figure 7). Therefore, it is possible to suppose that the two polar heads keep the outside orientation previously reported. With respect to the NOESY experiments performed on MLVs obtained from single phosphatidyl nucleosides, some of the cross-peaks due to the resonances of the riboses protons are absent, possibly due to the lack of resolution in this spectral region. The presence of a cross-peak between the aromatic proton 6cyt of cytidine and the aromatic 2ino of inosine constitute the main information on the whole 2D map. This cross-peak is present at any mixing time used for acquisition (15, 20, and 25 ms) and for both populations (a, b) of the aromatic proton 6cyt. Furthermore, the NOESY contact between protons 8ino and 2ino, present in the case of MLVs from 2/POPC, is totally absent in the NOESY experiment performed with

mixing time of 15 ms and is very low in the other two cases (20 and 25 ms). The chemical shift variation of resonances due to aromatic and anomeric protons is possibly due to an interaction between inosine and cytidine. Since the resonances of the two nucleobases give a NOESY crosspeak between the resonances due to the protons H-6cyt and H-2ino, they are also sensitive to a different magnetic anisotropy and therefore are shifted. All these experimental data rule out the formation of large patchwork domains containing only one phosphatidyl nucleoside and enforce a model in which the two phosphatidyl nucleosides are alternated all over the bilayer. It is possible to draw some conclusion about the interaction between inosine and cytidine when these are involved in MLVs. The values of the chemical shifts allow the exclusion of a stacking interaction. In fact in the presence of stacking, the experimental shifts should be larger. The observed contact between the proton H-2ino and the proton H-6cyt suggests an arrangement of the neighboring nucleobases that is compatible neither with a classic Watson-Crick base-pairing nor with a basepairing other than Watson-Crick (Hoogsteen, reversed Hoogsteen, and reversed Watson-Crick).28 It is worth noticing that the observed interaction H-2ino-H-6cyt (see Figure 8) indicates that the molecule side utilized by inosine in the base pairing is always engaged with the side of cytidine not used for the base pairing. MLVs of 1 + MLVs of 2. The existence of an interaction between cytidine and inosine observed in the sample of MLVs directly obtained from the mixture 1/2 suggested that one can verify the possibility that this interaction could be a sufficient drawing force to obtain MLVs of 1/2 starting by MLVs from 1 and MLVs from 2 mixed together. (28) Landolt, H.; Bo¨rnstein, R. Numerical Data and Functional Relationships in Science and Technology, New Series, Group VII, Vol. 1a: Nucleic Acids. Springer-Verlag: Heidelberg, Germany, 1989.

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Cruciani et al. Table 3. Assignment of 600 MHz 1H-HR-MAS Spectrum (Performed with a Spinning Rate of 8 kHz and at 300 K) of MLVs Obtained from the Mixture 1/2a resonance

1H

(ppm) ((0.02 ppm)

8ino 2ino 6acyt 6bcyt 5cyt 1ino 1′cyt 2′cyt 3′ino 4′cyt 5′ino 5"cyt 5"ino A B C C′ W P P′ Q Q′ F J T, T′, T′′ S

NOESY contacts

8.45 8.19 8.01 7.93 6.14 6.09 5.94 4.31 4.53 4.18 3.97 3.97 3.90 4.11 5.33 4.53 4.27 5.35 2.42 2.36 2.31 2.27 2.03 1.61 1.30 0.90

1′ino, 2ino, 4.53 ppm 1′ino, 8ino, 6acyt, 6bcyt 1′cyt, 5cyt, 2′cyt, 2ino 1′cyt, 5cyt, 2′cyt, 2ino 1′cyt, 6acyt, 6bcyt, 2′cyt 8ino, 2ino, 4.35 ppm 2′cyt, 6acyt, 6bcyt, 5cyt 1′cyt, 6acyt, 6bcyt, 5cyt

B, C A, C′, C A, C′, B B, C F T, J T, J T, J T, J T, W T, P, P′, Q, Q′ S, F, J, P, P′, Q, Q′ T

a In the third column the contacts registered in the 2D 1H NOESY experiments are also reported.

Table 4. Values (in (2 Hz) of the Differences Registered between the Resonances Due to the Aromatic Protons H-8ino, H-2ino, H-6cyt and H-5cyt in the Case of MLVs Obtained from the Mixture 1/2 and in the Case of MLVs Obtained from 1 and MLVs Obtained from 2/POPCa

Figure 6. Comparison between 1H-HR-MAS spectra registered with a spinning speed of 12 kHz at 300 K for MLVs obtained from the mixture 1/2, MLVs obtained from 1 and MLVs obtained from the mixture 2/POPC (expansion of the aromatic and anomeric protons spectral region).

To verify a possible evolution of the system, the sample obtained by mixing MLVs of 1 with MLVs of 2/POPC has been monitored over 2 weeks. After the mixing, the sample was analyzed every hour for the first 2 days and later on every 8 h. It is worth of noticing that the spectra did not show any change during the time interval considered and that the 1 H HR-MAS spectra appear always as the simple sum of the single spectra, i.e., those obtained from a single phosphatidyl nucleoside performed under the same experimental conditions. This evidence points out that the system is stable and does not evolve; therefore, we do not observe any interaction between cytidine and inosine. In the two 2D-1H-NOESY experiments performed respectively with a mixing time of 25 and 100 ms, the same cross-peaks seen in the case of MLVs from 1 and in the case of MLVs from 2 are present; no cross-peaks between signals due to the cytidine and signals due to the inosine are present and also the contact H-6cyt-H-2ino, seen in the case of MLVs obtained from the mixture 1/2, is totally absent. Concluding Remarks The most general observation arising from this study is the high-resolution achieved by HR-MAS also in the case of MLVs obtained from 1 and 2. The present paper can therefore be taken as a clear example of the great potential offered by the HR-MAS techniques. The fact that we are able to study the fine conformational details of

spinning speed, kHz

∆ν8ino

∆ν2ino

∆ν6acyt

∆ν6bcyt

5.0 8.0 12.0 av.

26 24 24 25

36 36 33 35

36 39 38

43 47 45

∆ν5acyt 46 44 45

a

All the experiments have been performed at 300 K and with the spinning speeds indicated in column 1.

groups hanging from the bilayer may suggest that the NMR HR-MAS technique can be useful for studying the structure and conformation of drugs and other guest molecules bound to the lipidic bilayer. In the case of MLVs formed by 1 and 2, indeed this technique allowed one to reach a detailed description of the structural features of the cytidine and inosine bases in phosphatidyl nucleosides that are involved in multilamellar liposomes. The present study confirms that in liposomes formed by the phosphatidyl nucleosides 1 and 2, an interaction between one given base and the sugar moiety of a neighboring base is present and that the aromatic bases are exposed toward the aqueous phase. It is evident from the 2D-1H-NOESY experiments that no cross-peaks between the resonances due to the aromatic protons and either the resonances due to the glycerol moiety or to the fatty acid chains are present. We can say, however, that part of the ribose ring is close to the glycerol moiety, whereas it is far away from the alkyl chains. As already mentioned, probably the ribose tends to bent toward the glycerol moiety so as to minimize the contact between its hydrophobic parts and the aqueous phase. In the cases of the considered multilamellar liposomes, the interaction between adjacent aromatic bases is clearly shown by the above commented NOESY contacts. The existence of this intermolecular interaction is evident in

HR-MAS on Liposomes from Phosphatidyl Nucleosides

Langmuir, Vol. 20, No. 4, 2004 1151

Figure 7. Left side: expansion of the spectral region of aromatic and sugar protons of a 2D-1H-NOESY spectrum (T ) 300 K, spinning speed ) 8 kHz, mixing time of 15 ms) of the MLVs obtained from the mixture 1/2. Right side: expansion of the aromatic region of the 2D map.

Figure 8. (a) Schematization of the contact between the proton 2ino and the proton 6cyt registered in the case of MLVs obtained from the mixture 1/2. (b) Schematization of a classical WatsonCrick bases pairing.

the case of MLVs obtained from 2/POPC and in the case of MLVs obtained from the mixture 1/2. It is important to underline that in the case of SUVs obtained from the same compounds this interaction was excluded.3 Mixed vesicles were obtained mixing the two phosphatidyl nucleosides at the beginning of the preparation of the lipidic film. The experimental data (chemical shifts variation and NOESY contacts) indicate that in these mixed vesicles the two phosphatidyl nucleosides are alternated all over the bilayer. It is worth noticing that in contrast to the case of SUVs obtained from the same compounds, in the mixed MLVs, no large patchwork domains have been found. In the mixed MLVs, some kind of interaction between inosine and cytidine is present, but this is not a stacking interaction or a classic H-bonded interaction between the two complementary bases. The precise nature of this interaction is not easy to rationalize. The fact that no interaction is observed by mixing liposomes obtained separately from 1 and 2 is unfortunate

(one might have expected a Watson-Crick interaction among “complementary” liposomes)sbut it is not completely unexpected on the basis of common knowledge about the physical behavior of phospholipids vesicles. In fact, it is known that liposomes are not chemical equilibrium systems, actually they can be more properly seen as kinetic traps, i.e., structures that are stabilized and separated by each other owing to a high activation energy that protect them from transforming into other energetically similar structures.29 In fact, it is very difficult if not impossible to cause liposomes of different sizes to simply fuse with each other.30 Thus, “complementary” liposomes of phosphatidyl nucleotides may have no tendency to fuse with each other to form mixed liposomes. However, as we have seen, stable mixed liposomes can be obtained starting from the mixture of monomers. Several other phosphatidyl nucleosides, bearing for example adenine and uracil bases, were also prepared. The corresponding MLVs are presently under investigation, and the results will be reported at a later time. Abbreviations HR-MAS: high-resolution magic angle spinning MAS: magic angle spinning POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospatidylcholine SUVs: small unilamellar vesicles MLVs: multilamellar vesicles 1: 5′-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)cytidine 2: 5′-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)inosine

Acknowledgment. We thank Dr. Giovanni Ughetto for helpful comments and discussions. LA035804H (29) Luisi, P. L. J. Chem. Educ. 2001, 78, 380. (30) Zhilang, C.; Luisi, P. L. J. Phys. Chem., in press.