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Langmuir 1991, 7, 16-18
Self-Assembling Properties of 1,2-Diacyl-sn-glycero-3-phosphohydroxyethanol: A Headgroup-Modified Diacetylenic Phospholipid Michael Markowitz and Alok Singh' Center for BiolMolecular Science and Engineering, Code 6090, Naval Research Laboratory, Washington, D.C. 20375-5000 Received October 29, 1990 An acidic diacetylenic phospholipid, 1,2-bis(tricosa-lO,l2-diynoyl)-sn-glycero-3-phosphohydroxyethanol, has been prepared by exchanging choline functionality of corresponding phosphocholine with ethylene glycol by means of phospholipase D transphosphatidylation. The morphologies formed by this compound as aqueous dispersions in both the presence and absence of metal ions at pH 5.6 were directly observed by transmission electron micrograph. In the presence of mono- and bivalent cations, cylindrical microstructures were observed, whereas in the absence of cations, no definable microstructures were detected. Scanning electron microscopicanalysis of the Ni-plated cylindricalmicrostructures showed that they were hollow. Monolayer studies of the phospholipid in the presence and absence of metal ions indicate that differences in headgrouppacking and acyl chain alignmentbetween the charged and neutralized lipid are responsible for the observed morphologies. Phosphatidylcholines with acyl chains containing diacetylenic moieties have generated a great deal of interest because of their ability to produce tubules along with other self-assembled microstructures and to stabilize those microstructures through polymerization.lS2 The influence of the structure of bilayer-forming surfactants on the formation of tubular microstructures has been reported.*5 In addition, the protein-induced formation of tubular myelin structures from phospholipids has been thoroughly studied.6 Ample reports exist in literature concerning the effect of surface charge on the self-assembly behavior of surfactant^.^ In particular, the formation of cochleate cylinders by phosphatidylserine in the presence of Ca2+ has been well characterized.8 Recently, we initiated a study on the relationship between headgroup structure and dispersion medium on the bilayer properties of diacetylenic phospholipid^.^ The results of those studies prompted us to proceed further and to investigate the factors that influence the types of microstructures formed from this class of phospholipids in greater detail. For the present study, we were interested in examining the role that surface charge plays in determining the morphology of bilayer microstructures formed from acidic diacetylenic phospholipids. Therefore, a compound has been prepared in which the choline functionality of 1,2-bis(tricosa-10,12-diynoyl)(1) Yager, P.; Schoen, P. E. Mol. Cryst. Liq. Cryst. 1988,106,371-381. (2) Biotechnological Applications of Lipid Microstructures; Gaber, B., Schnur, J. M., Chapman, D., Eds.; Plenum Press: New York, 1988; pp 305-320. Schnur, J. M.; Price,R.; Schoen, P.; Yager, P.;Calvert, J.;Singh, A. Thin Solid Films 1987, 152, 181-206. (3) Singh, A.; Schoen, P. E.; Schnur, J. M. J.Chem. Soc., Chem. Commun. 1988, 1222-1223. Georger, J. H.; Singh, A.; Price, R. R.; Schnur, J. M.; Yager, P.; Schoen, P. E. J. Am. Chem. SOC.1987,109,6169-6175. Yager, P.; Schoen, P.; Davies, C. A.; Price, R.; Singh, A. Biophys. J.1985, 48,899-906. Yager, P.; Price, R. R.; Schnur, J. M.; Schoen, P. E.; Singh, A.; Rhodes, D. Chem. Phys. Lipids 1988, 46, 171-179. (4) Helfrich, W. J. Chem. Phys. 1986, 85, 1085-1087. (5) de Gennes, P. G. C. R. Seances Acad. Sci. 1987,304, 259-263. (6) Boggs, J. M.; Moscarello, M. A. Biochim. Eiophys. Acta 1978,515, 1-21. (7) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: New York. 1985.
(8)Papahadjopoulos, D.; Vail, W. J.; Jacobson, K.; Poste, G. Biochim. Biophys. Acta 1975, 394,483-491. (9) Singh, A,; Marchywka, S. Polym. Mater. Sci. Eng. 1989, 61,675678.
-sn-glycero-3-phosphocholine~~ (DC,8,9PC)has been replaced by hydroxyethanol ( 1).11J2 Thus, the charge and CH,-
O-C-(CHz),-C=-C-C~C-(CH2)o-CH,
II
I
0
CH -0-c
I
CHz -0-P
- ( c H ~ ) ~ - c G C -C G C -(CHz),-CH,
II O7
II
-O-CH~-CHz-OH
0 1
headgroup structure of DC8,9PC have been altered, which in turn should affect the headgroup hydration ofthe lipid.'3 The effect that the modulation of the charge by monovalent and divalent cations had on microstructure morphology was investigated. The morphologies of the bilayer assemblies formed from 1 in aqueous media (pH 5.6)in the presence and absence of monovalent and divalent cations were observed by transmission electron microscopy (Figure l ) . 1 4 In the (10) Chapman, D. Biocompatible surfaces, US.Patent 4,348,329, Sept 7, 1982. (11) Eibl, H.; Kovatchev, S. Methods Enzymol. 1981, 72, 632. (12) The lipid was characterized by IR and NMR spectra and purity
was routinely checked by TLC using two solvent systems: (A) CHCls: MeOH:H20 (65:25:4 (v/v/v)) and (B)CHCla:MeOHNH3 (25% in water) (65303 (v/v/v)). Compound 1 was revealed at R10.56 as compared with R, 0.38 for DCa,9PC in solvent A and in solvent B Rf values were 0.69 and 0.17, respectively. In addition, the spot corresponding to compound 1 tested negative for choline with Dragendorfs reagent. To ensure the absence of any ion, the lipid was treated with an ion exchange resin (Biorad AG 50W-X8).Fast atom bombardment mass spectral analysis established that the treated compound was ion-free (13) Cevc, G. Biochemistry 1987,26,6305-6310. (14)Transmission electron micrographs were recorded on a Zeiss EM10 transmission electron microscope. All samples were unstained. The samples were prepared by evaporating a CHC13solution of each compound along the walls of a test tube. After keeping the samples under vacuum overnight, an aliquot of one of the Following buffers was added to each sample so that the final concentration of phospholipid was 2.38 m M (1) 0.2 M aqueous acetate buffer (pH 5.6); (2) 0.2 M aqueous acetate buffer (pH 5.6) containing either 1mM CuCl2 or 1mM CaCl2; (3) 0.1 M aqueous acetate buffer (pH 5.6) containing either 0.1 M NaCl or 0.1 M CsCl. The samples were then hydrated above their phase transition temperatures for 1 h, vortexed to disperse the phospholipid, and then allowed to cool to room temperature gradually.
0743-7463/91/2407-0016$02.50/0 0 1991 American Chemical Society
Letters
Langmuir, Vol. 7, No. 1, 1991 17
u
-
Figure 1. Transmission electron micrographs of microstructures of 1 prepared in (a) 0.1 M acetate buffer (0.1 M CsC1, pH 5.6), (b) 0.1 M acetate buffer (0.1 M NaCl, pH 5.6), (c) 0.2 M acetate buffer (1 mM CaC12, pH 5.6), and (d) 0.2 M acetate buffer (1 mM CuC12, pH 5.6). Bar represents 0.1 pm.
absence of metal ions or in the presence of a low concentration of Na+ (7.5 mM), 1 did not form any recognizable microstructures. However, in the presence of divalent cations (Ca2+,1.5 mM; Cu2+,1 mM) or a high concentration of monovalent cations (Na+, Cs+, 0.1 M), vesicular and varyingyields of cylindrical microstructures were obtained. The appearance of the diagonal striations on the cylinders formed in the presence of Na+, Ca2+,or Cu2+indicated that they were tightly wrapped, while the cylinder formed in the presence of Cs+ appeared featureless. The diameters of the cylindrical microstructures ranged from 0.05 to 0.6 pm and they appeared to be hollow. The results indicate that neither the formation nor the diameter of the cylindrical bilayer assemblies is metal ion specific under these conditions. In order to determine whether these bilayer assemblies were hollow or had a solid core, the cylindrical microstructures formed in the presence of Cs+ and of Cu2+were plated with colloidal Ni and then further examined by scanning electron microscopy.15J6 Figure 2 illustrates the end-on views of the Ni-coated cylinders, which confirmed their hollow
nature. The minimum diameters of the metal-coated tubules were greater than had been observed by TEM for the nonplated tubules. This could be attributed to the change in the pH that occurred during the Ni coating process. The neutralization of the negative charge of the headgroup by the binding of the metal ions would enable the acyl chains of 1 to pack in a gel state in which the diacetylenic moieties were aligned in a manner similar to that found for DC,,9PC, whereas the acyl chains of the charged lipid would be more likely to pack in a liquidcrystalline state in which the diacetylenic moieties would not be stacked. This explanation is supported by monolayer studies (pH 5.6,21 "C) of the packing of 1 in the absence and presence of monovalent and divalent cations (Figure 3).17 The transition exhibited by the monolayer in the absence of (15) Schnur, J. M.; Yager, P.; Price, R.; Calvert, J. M.; Schoen, P. E.; Georger, J. H. Metal clad lipid microstructures. U.S.Patent 4,911,981, March 27,1990. (16) Scanning electron micrographs of the Ni-coated microstructures were recorded on an ISI-DS130 scanning electron microscope.
18 Langmuir, Vol. 7, No. 1, 1991
Letters
Figure 2. Scanning electron micrographs of Ni-coated microstructures of 1 prepared in (a) 0.2 M acetate buffer (1mM CuC12, pH 5.6) and (b) 0.1 M acetate buffer (0.1 M CsCl, pH 5.6). Bar represents 0.1 pm. 45,
1
0 20
40
60
80 100 120 AREA (k?/Molecule)
140
160
Figure 3. Monolayers of 1 on subphases of 0.2 M acetate buffer (pH 5.6) (-), 0.2 M acetate buffer (1mM CuC12; pH 5.6) (- - -), and 0.1 M acetate buffer (0.1 M CsCl, pH 5.6) The monolayers were spread from CHC13 solutions of the phospholipids and compressed at a rate of 50 cm2/min up to their collapse pressures. (e
0).
metal ions (6 mN/m), characteristic of diacetylenic phospholipids with acyl chains of similar length,18is much less pronounced when the monolayer is formed in the presence (17) Force-area isotherms were recorded on a Wilhelmy plate film balance (NIMA) with an automatic data collection unit. The monolayers were spread from CHCl3 solutions of the phospholipids (1 X 10-3 M) onto a subphase of one of the following buffer solutions: 0.2 M aqueous acetate buffer (pH 5.6); 0.2 M aqueous acetate buffer (pH 5.6) containing 1mM CuCl,; 0.1 M aqueous acetate buffer (pH 5.6) containing 0.1 M CsCl. A 5X mol portion of 1 was used in each experiment. The monolayers were compressed to their collapse pressures a t a rate of 50 cm2/min. (18) Johnston, D. J.; McLean, L. R.; Whittam, M. A.; Clark, A. D.; Chapman, D. Biochemistry 1983,22, 3194-3202.
of the metal ions. In the absence of metal ions, 1 formed a liquid-expanded monolayer, whereas in the presence of metal ions (Cs+,0.1 M, Cu2+,1mM), the monolayer became more condensed. Only at high mechanical pressure did the charged monolayer obtain the same packing, and then it collapsed quickly. One possible explanation for the ability of this compound as opposed to phosphatidylserine to form tubules in the presence of metal ions is that the neutralization of the negatively charged headgroup by the metal ions in the aqueous medium would enable 1 to behave in a similar fashion to the known tubule forming phospholipid DC8.9PC. However, the size of the headgroup of 1 is small relative to that of phosphatidyl serine and it is possible that by altering the structure of the headgroup of 1 the packing and alignment of the diacetylenic chains could be drastically altered and a variety of microstructures could be obtained. We intend to pursue this possibility by preparing acidic diacetylenic phospholipids with a variety of hydroxyalkanol headgroups and examining the microstructures formed from their aqueous dispersions at different pH values and ionic strengths.
Acknowledgment. We wish to thank Dr. Joel Schnur and Dr. R. Shashidhar for helpful discussions, Dr. Jim Callaghan for the mass spectral analysis, Dr. Tom Fare for use of the NIMA film balance, Ms Susan Marchywka for aid in the synthesis of the phospholipids, Mr. Ronald Price for the electron microscopy, and Dr. Subhash Baral for the Ni plating of the tubules. This research was partially supported by the Office of Naval Research. Dr. Michael Markowitz is a recipient of an Office of Naval Technology postdoctoral fellowship.
