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Langmuir 1996, 12, 4976-4978
Relation between the Molecular Structure of Phosphatidyl Nucleosides and the Morphology of their Supramolecular and Mesoscopic Aggregates
Table 1. Chemical Structure and Abbreviations of the Phosphatidyl Nucleosides Synthesized and Used
Silvio Bonaccio, Michaela Wessicken, Debora Berti, Peter Walde, and Pier Luigi Luisi* Institut fu¨ r Polymere, ETH-Zu¨ rich, Universita¨ tstrasse 6, CH-8092 Zu¨ rich, Switzerland Received April 2, 1996. In Final Form: June 25, 1996
Surfactants, including phospholipids, are able to aggregate into a variety of organized structures such as micelles, lamellae (closed liposomes or vesicles), and hexagonal and cubic phases. Together with this well established aggregation behavior new supramolecular association at a higher hierarchic level of structural organization has been emerging during the last few years. These so-called “mesoscopic self-assembled structures” that vary in size from about 100 nm to several micrometers can take forms as complex as helical ribbons, disks, rods, rings, or tubes.1,2 The self-assembly into mesoscopic structures is particularly fascinating, as the question of morphology thus extends from the dimensions of supramolecular chemistry into dimensions which are often typical of biology. From the chemical point of view, a very central question is the relationship betwen the molecular structure of the surfactant monomer and the morphology of the supramolecular and mesoscopic aggregates. At this stage, one can safely state that this relationship is not well understood. In the case of simpler supramolecular surfactant aggregates, one may attempt to predict the most probable structure (e.g. micelles versus vesicles) on the basis of packing geometry following the classic scheme of Israelachvili, Mitchell, and Ninham.3 As is well-known, this has however only a limited applicability. In the case of mesoscopic structures, some guidelines are emerging thanks to the pioneering studies by the groups of Kunitake,1a,2,4 Fuhrhop,1b,5,6b Helfrich,6 and Yanagawa;7 however, the relationship is also not understood. * To whom to address correspondence. (1) Examples are: (a) Helical ribbons of chiral double-chain ammonium amphiphiles: Nakashima, N.; Asakuma, S.; Kunitake, T. J. Am. Chem. Soc. 1985, 107, 509-510. (b) Helical fibers from chiral N-alkylgluconamides: Ko¨ning, J.; Boettcher, C.; Winkler, H.; Zeitler, E.; Talmon, Y.; Fuhrhop, J.-H. J. Am. Chem. Soc. 1993, 115, 693-700. (c) Helical fibers from chiral N-acyl-L-aspartic acids: Imae, T.; Takahashi, Y.; Muramatsu, H. J. Am. Chem. Soc. 1992, 114, 3414-3419. (d) Helical ribbons formed by hydrated galactosylceramides: Kulkarni, V. S.; Anderson, W. H.; Brown, R. E. Biophys. J. 1995, 69, 1976-1986. (2) The term “mesoscopic structure” is used for expressing a higher hierarchical level of supramolecular organization. (a) Kimizuka, N.; Kawasaki, T.; Hirata, K.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6360-6361. (b) Kimizuka, N.; Fujikawa, S.; Kuwahara, H.; Kunitake, T.; Marsh, A.; Lehn, J.-M. J. Chem. Soc., Chem. Commun. 1995, 21032104. (3) (a) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Biochim. Biophys. Acta 1977, 470, 185-201. (b) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1985. (4) (a) Kunitake, T.; Okahata, Y.; Shimomura, M.; Yasunami, S.; Takarabe, K. J. Am. Chem. Soc. 1981, 103, 5401-5413. (b) Kunitake, T. Angew. Chem., Int. Ed. Engl. 1992, 31, 709-726. (5) Fuhrhop, J.-H.; Ko¨ning, J. Membranes and Molecular Assemblies: The Synkinetic Approach; The Royal Society of Chemistry: Cambridge, U.K., 1994. (6) (a) Helfrich, W.; Prost, J. Phys. Rev. A 1988, 38, 3065-3068. (b) Fuhrhop, J.-H.; Helfrich, W. Chem. Rev. 1993, 93, 1565-1682. (7) (a) Yanagawa, H.; Ogawa, Y.; Furuta, H.; Tsuno, K. Chem. Lett. 1988, 269-272. (b) Yanagawa, H.; Ogawa, Y.; Furuta, H.; Tsuno, K. Chem. Lett. 1989, 403-406. (c) Yanagawa, H.; Ogawa, Y.; Furuta, H.; Tsuno, K. J. Am. Chem. Soc. 1989, 111, 4567-4570. (d) Itojima, Y.; Ogawa, Y.; Tsuno, K.; Handa, N.; Yanagawa, H. Biochemistry 1992, 31, 4757-4765.
S0743-7463(96)00315-0 CCC: $12.00
POP DPP
R1-COOH R2-COOH HO-X R1-COOH R2-COOH HO-X
Cyt
Ino
palmitic acid oleic acid cytidine palmitic acid palmitic acid cytidine
palmitic acid oleic acid inosine palmitic acid palmitic acid inosine
In order to shed some light into this area, it would be useful to perform investigations by systematic variation of the chemical structure of the surfactant monomers with small changes at a time and to study whether and to what extent, on the basis of these changes, it is possible to go from one type of aggregation to the other, and in particular from a supramolecular to a mesoscopic form. In this paper, we will follow this approach. We will consider a series of phosphatidyl nucleosides in which we will vary both the chemical structure of the nucleoside (a purine versus a pyrimidine base) and the structure of the lipidic moiety (1-palmitoyl-2-oleoyl-sn-glycero(3)phosphatidyl (POP) versus 1,2-dipalmitoyl-sn-glycero(3)phosphatidyl (DPP)). In particular, we will investigate the aggregation forms of POP-Cytidine (POP-Cyt), POPinosine (POP-Ino) and their 1:1 molar mixture and of DPPCyt, DPP-Ino, and their 1:1 molar mixture (see Table 1).8 We will show that these relatively subtle variations of the molecular structure of the surfactant monomers are sufficient to cause dramatic change in the aggregation morphology. The morphology of some phosphatidyl nucleosides has already been reported by Yanagawa et al.,7 who actually were the first to investigate the self-association of this family of lipids. Of the four compounds listed in Table 1, only DPP-Cyt has been studied by this group, and in a different perspective. Our studies with these compounds have been aimed primarily at finding the conditions under which stable unilamellar liposomes are formed,9 with the idea to translate the linear binding and recognition chemistry of polynucleotides into the spherical topology of liposomes. Since the aggregate morphology may be in principle dependent on how the sample is prepared, as well as on pH, buffer composition, storage time, and temperature,4,5,6b,10 all lipids were dispersed in the same way, using the same buffer, molar lipid concentrations, and “mechanical” treatment (extrusion). In brief, the phosphatidyl nucleosidessynthesized9 following a procedure described by Shuto et al.11 swas first dissolved in chloroform/methanol (between 1:1 and 5:1, v/v) and a thin film of the lipid was prepared in a round bottom flask (8) The stereospecific numbering (sn) for the glycerophospholipids and the numerical symbols and trivial names for the fatty acids are used according to the IUPAC-IUB recommendations: IUPAC-IUB Commission on Biochemical Nomenclature (CBN) Eur. J. Biochem. 1977, 79, 11-21. (9) Bonaccio, S.; Walde, P.; Luisi, P. L. J. Phys. Chem. 1994, 98, 6661-6663 and 10376. (10) (a) Schnur, J. M.; Ratna, B. R.; Selinger, J. V.; Singh, A.; Jyothi, G.; Easwaran, K. R. K. Science 1994, 264, 945-947. (b) Thomas, B. N.; Safinya, C. R.; Plano, R. J.; Clark, N. A. Science 1995, 267, 1635-1638. (11) (a) Shuto, S.; Ueda, S.; Imamura, S.; Fukukawa, K.; Matsuda, A.; Ueda, T. Tetrahedron Lett. 1987, 28, 199-202. (b) Shuto, S.; Imamura, S.; Fukukawa, K.; Ueda, T. Chem. Pharm. Bull. 1988, 36, 5020-5023.
© 1996 American Chemical Society
Notes
Langmuir, Vol. 12, No. 20, 1996 4977
Table 2. Morphological Characteristics of Aqueous Dispersions of Phosphatidyl Nucleosides (3 mM), Prepared in 30 mM Tris/HCl, 20 mM KCl, 0.1 mM EDTA, pH 8 Cyt
Ino
Cyt/Ino (1:1)
unilamellar lipsosomesc (diameter ∼ 40 nm)
POP Immediately after Extrusiona,b unilamellar lipsosomes (diameter ∼ 40 nm)
unilamellar lipsosomes (diameter ∼ 37 nm)
the liposomes are stable for at least 1 week
POP upon Storage at 25 °Cd the liposomes aggregate with timee
the liposomes are stable for at least 1 week
rings (diameter 50-200 nm; ring thickness 10-13 nm) and short rods with helical surface structure (left handed;h see Figure 1b
DPP Immediately after Extrusionf,g stacked disks and partially rolled, flat disks (diameter e 70 nm); see Figure 1d longitudinally opened cylinders; see Figure 1c
no change within 1 weeki
DPP upon Storage at 25 °C the cylinders slowly start to precipitate
no change within 1 week
transformation into flat disks
DPP upon Storage at 50 °C formation of flat disks of different sizes
no change within 1 week
a
The extrusion (50 nm final pore size) was performed at room temperature (for details see text). b The samples were rapidly frozen from room temperature (within 10 min after extrusion) and then analyzed by the freeze-fracture electron microscopic technique (for details see text). c The CD spectrum shows a maximum in the near UV range at ∼280 nm with an intensity slightly higher than that of cytidine monophosphate. [θ]280(25 °C) ) 12 000 deg cm-2 dmol-1. d Stability tests by dynamic light scattering measurements; see ref 9. e At 35 °C, the liposomes are stable for at least 1 week. In the presence of 10 mol % POPC the liposomes are also stable for at least 1 week at 25 °C. f The extrusion (50 nm final pore size) was performed at 50 °C (for details see text). g The samples were analyzed by electron microscopy using the negative stain technique (for details see text). h The CD spectrum shows a maximum in the near UV range at 275 nm with an intensity about four times higher than that of cytidine monophosphate. [θ]275(20 °C) ) 40 000 deg cm-2 dmol-1. i The CD spectrum did not change with time.
under vacuum rotatory evaporation. After drying at high vacuum over night, the lipids were dispersed either at room temperature (POP-lipids) or at 50-55 °C (DPP lipids) in buffer solution (30 mM Tris/HCl, 20 mM KCl, 0.1 mM EDTA, pH 8, adjusted at room temperature) by the help of a vortex apparatus. The lipid dispersion (always 3 mM lipid) was then passed repetitively through polycarbonate membranes with final pore diameters of 50 nm as described before.9,12 All lipid dispersions were analyzed by electron microscopy within one day after extrusion, applying the negative staining13 or freeze fracture technique.14 Let us consider now the results, as summarized in Table 2, and the corresponding figures; in particular, in Table 2 an idealized schematic view of some of the structures is given, whereas Figure 1 represent the actual micrographs. A first very significant effect of the molecular structure of the surfactant monomer on the morphology of the aggregates is to be seen in the fact that liposomes are obtained only with the POP lipidic moiety (see Table 2). On the contrary, with the same purine or pyrimidine headgroups, the DPP lipidic moiety does not form stable liposomes but mesoscopic structures in the form of rods or disks (see the drawings in Table 2 and Figure 1). Thus, substitution of one palmitoyl chain of DPP, with one oleoyl chain containing a single double bond, is sufficient to drastically change the morphology of the aggregates. At this point one may recognize that at our operational (12) (a) Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1979, 557, 9-23. (b) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168. (c) Nayar, R.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1989, 986, 200-206. (13) The samples were stained with 2% (wt/v) uranyl acetate. (14) Mu¨ller, M.; Meister, N.; Moor, H. Mikroskopie (Wien) 1980, 36, 129-140.
Figure 1. Transmission electron micrographs of negatively stained13 phosphatidyl nucleoside dispersions (3 mM), prepared in 30 mM Tris/HCl, 20 mM KCl, 0.1 mM EDTA, pH 8: (a and b) DPP-Cyt, before (a) and after (b) extrusion; (c) DPP-Ino, after extrusion; (d) DPP-Cyt/DPP-Ino (1:1), after extrusion. Length of the bar: 100 nm for parts a, b, and d; 60 nm for part c.
temperature of 25 °C we are in the fluid analogue state15 in the case of the POP compounds whereas for the DPP nucleosides we are well below it, namely in the crystalline analogue state. This expresses again the importance of the molecular structure of the surfactant on the morphology of the aggregates at the level of the physical properties. Let us consider now the influence of the headgroup, starting with the POP compounds. A significant difference is observed between the purine and the pyrimidine base, in the sense that whereas the POP-Cyt liposomes at room temperature are stable with respect to their size, the POPIno liposomes are not and aggregate upon storage at 25 (15) While the main phase transition temperature (Tm) of the POP lipids is below 0 °C, Tm for DPP-Cyt is around 40 °C (experimentally determined by differential scanning calorimetry: 41 °C (heating run) and ∼35 °C (cooling run)).
4978 Langmuir, Vol. 12, No. 20, 1996
°C. Interestingly, liposomes obtained with the mechanical mixture of the two lipids are stable. If we now consider the DPP analogs, we also observe a significant sensitivity to the headgroup structure also at the level of the mesoscopic forms: not only is the morphology of DPP-Cyt aggregates different from that of DPP-Ino aggregates (compare Figure 1b and c) but the latter are (in analogy perhaps to the POP-Ino liposomes) less stable upon storage at 25 °C. Again, the forms obtained from the mechanical mixture of the two DPP lipids are more stable at room temperature, even after prolonged storage, and the morphology is somewhat different from that of the two pure forms. Notice from Table 2 the transformation upon storage at 50 °C of the DPP-Cyt and DPP-Ino structures: they both tend to form flat disks, whereas no significant change is observed with the same experiment (storage at 50 °C) in the case of the lipidic mixture. The behavior of DPP-Cyt is particularly interesting, since DPP-Cyt forms helical rods and rings with a defined surface structure (Table 2, Figure 1a and b). Upon dispersion of DPP-Cyt in buffer, long, spaghetti-like rods are formed, as shown in Figure 1a. These rods are transformed into closed rings (Figure 1b) by extrusion through the polycarbonate membranes. In conclusion, it appears from this study that small changes of the monomeric surfactant are sufficient to induce large variations of the architecture of the supramolecular/mesoscopic aggregates. It is also clear that
Notes
at this stage there is no simple way to predict the direction and entity of these effccts. We believe that it is necessary to build first a good data bank system, based on the systematic variation of the structure of the monomers, a data bank system from which eventually perhaps, through a combination of molecular modeling and thermodynamic considerations, some general laws may be derived. For this, more systematic work is needed. At this stage, one may however recognize in these phenomena one important general principle: that the aggregation into ordered forms is a cooperative process, in which namely small effects at the monomeric level can be exponentially magnified by the multimeric aggregation process. One may think as an analogy that chemically similar compounds may crystallize into quite different forms orseven more challengingsas an analogy with the morphology of biological forms. In fact, for the study of the biological morphology there is practically no available experimental model system, and the study of the morphology of the simpler synthetic mesoscopic structures as a function of the chemical structure of the monomers may prove particularly useful and interesting at this regard. Acknowledgment. The authors are greatful to Dr. Ernst Wehrli of the institute of cell biology at the ETH for performing some of the electron microscopy analysis. LA960315L
