1988
Langmuir 1991, 7, 1988-1990
Notes Aggregation of Aqueous Suspensions of Phospholipid Tubules
O=P-0 I
Min-Hua Lu,t Jerome B. Land03 J. Adin Mann, Jr.,l,t Rolfe G. Petachek,’ and Charles Rosenblatt’JJ Case Western Reserve University, Cleveland, Ohio 44106
Received March 18, 1991. In Final Form: April 25,1991
0
I
0 CH2
II I
CH3(CH2)gCEC-C=C(CH2)8COCH
O II 1
CHg(CH2)gC=C-CGC(CHp) &OCH 2
Introduction Figure 1. Schematic representation of the lipid DC8,oPC. Several years ago the diacetylenic lecithin 1,2-bis(l0,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DCagPC) a result of the tubule’s large anisometry, the log jams tend (Figure 1) was found to form microstructures in water to be quite dilute. Moreover, the interactions appear to consisting of bilayers which wrap around a hollow ~ o r e . l - ~ be weak, since gentle stirring is often sufficient to redisThese so-called “tubules” are straight, tens of micrometers perse the tubules. From an experimental standpoint, log in length, and 0.30-0.75 pm in diameter. The thicknesses jams are often viewed as a nuisance. To perform light of the walls range between 1 and 15 bilayers5 and are scattering or optical birefringence measurements on a generallyuniform over an entire tubule or over large regions suspension of tubules, for example, it was necessary to of the tubule. Since the tubules can be easily polymeragitate the samples from time to time in order to redisized, one can imagine a variety of applications. In order perse them.k8 In this work our goal is to understand the to exploit these structures, of course, one must be able to mechanism for binding and the circumstances under which manipulate the tubules when dispersed in solution, as well the tubules can be prevented from binding. as understand their interactions both with each other and wi h appropriate confining surfaces. To that end we are Experimental Procedure and Results c!ll rently involved in an ongoing program in which the The phospholipid DC8,sPC was obtained from Avanti tubule is treated as a structured colloid. In this vein we Polar Lipids and used without further purification. Ten have been able to investigate the orientability of tubules milligrams of lipid was added to a 10-mLsolution consisting in both electric and magnetic field~5*~*’ the effects of of absolute ethanol and water in a ratio 70:30 by volume. suspending tubules in a ferrofluid,8 and the interactions The mixture was heated to -60 “C, removed from the of tubules with an acrylic s ~ r f a c e .In ~ this latter work we oven, and allowed to cool. After approximately 1 h the calculated the separation-dependent partition function mixture had become extremely cloudy. Examination of of the tubule-surface system. Owing to the trade-off the material with optical microscopy showed the presence between energetic and entropic terms which arise from of needlelike structures several tens of micrometers in geometric effects, we showed that a hump of order kBT length, the signature of tubules. The suspension was then exish in the Helmholtz free energy near the acrylic surface. centrifuged and the water-ethanol supernatant was withHere k g is Boltzmann’s constant and T is the temperadrawn. Pure water was then added, the tubules redisture. In effect the hump serves as a weak barrier against persed, the suspension centrifuged, and the supernatant tubule binding, stabilizing the suspension for relatively again withdrawn. This process was repeated 4 times in long periods of time. order to remove the ethanol from solution. The final step In this paper we report on observations regarding involved resuspension of tubules to a concentration of 1 tubule-tubule aggregation. Ever since the discovery of mg/mL in a mixture of H20 and DzO, such that the water these structures it has been known that tubules in aqueous density was 1.093. By means of centrifuging tubules in suspension tend to stick together on close approach, water as a function of [HzO]:[DzO], we had earlier found forming macroscopic structures which are often referred this to be the mean tubule density. This density, in fact, to as “log jams”. Optical microscopy of these structures is completely consistent with the calculations based on indicates that the tubules are rarely close-packed and, as the X-ray results of Lando and Sudiwala.lo Thus, in this way we avoided sedimentation during our observations of * To whom correspondence should be addressed. + Department of Physics. tubule aggregation. Finally, using optical microscopy we f Department of Macromolecular Science. determined the average tubule length, L, to be approxi8 Department of Chemical Engineering. mately 30 pm. (1)Yager, P.; Schoen, P. E. Mol. Cryst. Li9. Cryst. 1984, 106, 371. Approximately 3 mL of the suspension (-0.1 mg/mL) (2) Yager, P.; Schoen, P. E.; Davies, C.; Price, R.; Singh, A. Biophys. J. 1985,48, 899. was placed in a vial and allowed to equilibrate at (3) Singh, A.; Schnur, J. Polym. Prepr. (Am. Chem. SOC.,Diu.Polym. approximately 27 “C for at least 24 h. After the initial Chem.) 1985,26, 184. randomization of orientations (approximately 5 min), it (4) Georger, J. H.; Singh, A.; Price, R. R.; Schnur, J. M.; Yager, P.; Schoen, P. E. J. Am. Chem. SOC.1987,109,6169. was found that the sample remained uniformly turbid, ( 5 ) Rosenblatt, C.; Yager, P.; Schoen, P. E. Biophys. J. 1987,52,295. indicating the absence of significant log jams. Within 10 (6) Li, Z.; Rosenblatt, C.; Yager, P.; Schoen, P. E. Biophys. J. 1988, min we observed the onset of a nonuniform turbidity; 54. - -,-289. - -. within 1-2 h we observed significant nonuniformities, (7)W+s, D.M.; Li, Z.; Rosenblatt, C.; Yager, P.; Schoen, P. E. Mol. Cryst. Ltq. Cryst. 1989, 167, 1. a structure best described as a “dust ball” began to where (8)Lu, M.-H.; Rosenblatt, C. Appl. Phys. Lett. 1990, 56, 590. Lu, M.-H.; Petschek, R. G.; Rosenblatt, C. J. Colloid Interface Sci. (9) 1991,142, 121. (10)Lando, J. B.; Sudiwala, R. V. Chem. Mater. 1990,2, 594. 0743-7463/91/2407-1988$02.50/0
0 1991 American Chemical Society
Notes
materialize in the middle region of the vial. Over time, 24 h, this structure became more dense, depleting tubules from the surrounding regions. The structure never occupied less than approximately 5% of the total volume, indicating that the tubules were not close packed. In addition, we found that gentle aggitation by slow rotation of the vial was sufficient to destroy the “dust ball”, whereupon the aggregation process recommenced. We note that upon cooling the sample to room temperature (- 23 “C) the process was similar, but on a time scale a factor of -2 slower. In this narrow temperature range no significant structural changes are expected to occur in the lipid. Although the chain melting temperature T , is a function of ethanol concentration,ll even if we had been unable to extract any ethanol, T , would still have been in the neighborhood of 35 “C; this is well above the temperature region we studied. We thus need to examine sources other than changes in structure to explain the temperature dependence of the aggregation. Van der Waals interactions are expected, of course, to be the primary mechanism for attraction. Balancing the van der Waals interactions are repulsive forces, which inhibit aggregation. Superficially the most likely repulsive interactions would be the short-ranged hydration forces, which is a catch-all term for mechanisms such as ordering of the water and undulation forces.12-14 Thus, if the potential well arising from the sum of the hydration and van der Waals forces were sufficiently deep, aggregation would occur. On the other hand, for sufficiently large repulsive interactions, the well would be shallower than kBT and no binding would occur. It turns out that the magnitudes of the hydration forces increase with increasing temperature.12J3 Since the van der Waals force varies only weakly with temperature, the temperature variation of the hydration forces would result in a decrease in the depth of the potential well and, thus, a decrease in the tubules’ propensity to bind at higher temperatures. This result is contrary to our observations, indicating that hydration forces play at most a minor role in preventing aggregation. The diacetylenic lecithin DC8,gPC is a zwitterion, having transferred an electron from the nitrogen to the phosphate in the head group. Since the lipid can have a nonzero net charge as a function of pH (with counterions comprising a diffuse Guoy-Chapman layer), we investigated the possibility that the repulsive forces are Coulombic in nature. For separations less than the Debye screening length K - ~ ,the tubule-tubule interaction involves a tradeoff between van der Waals attraction (largely unaffected by screening) and the classical double layer (DLVO) repulsion.l5 Any temperature-dependent aggregation or dispersion would arise from the temperature dependence of the tubule’s net charge, in effect the association constants for the ionized species and the lipid. To this end we prepared a number of samples ranging in pH between 1.3 and 11.1using HC1 as the acid and KOH as the base. All samples were observed at five different temperatures: 4, 15,20, 25, and 34 “C. At all temperatures except 34 O C it was found that samples of pH = 2.6 and 3.0 remained dispersed. At the highest temperature both (11) Safinya, C. R. Private communication. (12) Israelachvili, J. Acc. Chem. Res. 1987,20, 415. (13)Guldbrand,L.;Jbnason, B.;Wennerstrbm, H. J . Colloid Interface Sci. 1982,89,532. (14) Tanford, C.The Hydrophobic Effect: Formation of Micelles and Biological membranes; Wiley: New York, 1980. (15) Verwey, E.J. W.; Overbeek,J. Th. G. Theory of the Stability of Lyophobic Colloids; Elsevier: New York, 1948.
Langmuir, Vol. 7, No. 9, 1991 1989
of these samples began to exhibit weak aggregation, although not to the same extent as the other samples. Samples from the next highest pH value (pH = 4.2) through pH = 11.1 were found to aggregate at all temperatures, although formation of comparably sized “dust balls” at the lowest temperatures occurred on much slower time scales (days) than at the highest temperatures (hours). The difference in time scales is far more than can be explained simply on the basis of a temperature-dependent diffusion constant for the tubules or on the basis of the temperature dependence of the van der Waals interactions. The one sample at the lowest pH (pH = 1.3) was also found to aggregate a t all temperatures, with comparable thermal behavior. In addition to the above observations, we added different salts to two samples at pH = 3.0. A sample was prepared containing 0.008 M of the monovalent salt NaC1, corresponding to an ionic strength of p = 4.8 X 10l8 e2/cm3, where e is the elementary charge. We prepared a second sample containing 0.004 M of Al&O4)3, corresponding to p = 3.6 X 10’9 e2/cm3. In the absence of the electrolyte the pH = 3.0 sample did not, of course, aggregate. The monovalent salt, however, caused aggregation of the tubules, and the trivalent salt with much higher ionic strength resulted in very rapid formation of the “dust balls”.
Discussion These observations as a function of pH and ionic strength can be explained by the following model. The tubuletubule potential V ( x ) has a deep well for small tubule separations (typically tens of angstroms) arising from van der Waals attractions; at larger separations the longer ranged Coulombic repulsions (for low ionic strength) contribute a positive hump to V ( x ) . For all values of pH greater than approximately 3.5 and less than the isoelectric point, the lipid molecule carries a net positive charge arising from the association of an H+ ion. The net charge, however, is sufficiently small so that the height of the Coulombic barrier is comparable to or smaller than kBT. Thermal motion drives the tubules over this Coulombic barrier, resulting in aggregation. Note that even if shortranged hydration forces were present, only the equilibrium separation would be affected, leaving the result qualitatively unchanged. For pH values between 2.6 and 3.0 the net lipid charge is sufficiently large that the barrier is substantially larger than kBT, thus preventing aggregation and stabilizing the suspension. (In our earlier calculation9 for the interactions of tubules near an acrylic surface, it was necessary to introduce a short ranged hydration repulsive force. Nevertheless, stabilization against binding to the surface comes about there primarily because of special geometric consideration^.^ It turns out that Coulombic forces play essentially no role in that calculation, as would be expected in the mid-pH region.) Finally, as the acidity is further increased, the tubules again aggregate. Here the ionic strength is of order 10lg e2/cm3, corresponding to a reduced Debye screening length K - ~in the neighborhood of 30 A. For separations larger than K-1 the repulsive Coulombic interactions are screened; at separations smaller than K - ~the potential barrier is either small or absent and only a deep potential well dominated by van der Waals attractive interactions remains, resulting in tubule aggregation. We note as an aside that the precise pH range for tubule stability may depend slightly on the isotopic concentration of water, although the qualitative results would remain unchanged. In light of this result at very low pH we performed a simple calculation for the attractive van der Waals force
1990 Langmuir, Vol. 7,No. 9, 1991
Notes
between two infinite parallel sheets a t various separations d; the Hamaker constant was taken to be 2 X 10-13 erg.g For each separation we overlooked the discrete nature of the charges and determined the continuous surface charge density p on each sheet needed to balance the attractive force. For d of order 15 A we found p 0.15 e/nm2, and ford of order 25 A, p 0.06 e/nm2. This would correspond to a minimum of approximately 0.1 to 0.3 charges per head grouplo needed to balance the van der Waals attractions a t these short distances; even more would be necessary when screening is taken into account. These charge densities are quite reasonable, and this rough calculation indicates that as the pH and thus the screening distance decrease, it would be difficult for the lipids to carry sufficient net charge to overcome the attractive forces at separations d < K - ~ . To support this picture we note that the addition of a monovalent salt to the nonaggregating pH = 3.0 sample resulted in a reduced screening length of order 40 A, and ultimately aggregation; for the trivalent salt K - ~ 12 A, resulting in rapid aggregation, the onset of which occurs on time scales of order 10 min. As an aside, we note that the low pH result might instead have come about if the acid were to hydrolyze the lipid head group. A neutral lipid would result, and aggregation would occur owing to the lack of a long range repulsive term in the potential. To test this hypothesis we titrated a sample from pH = 7 to pH = 1.3, noting that at pH = 2.75 no aggregation was observed. Finally, at pH = 1.3, we centrifuged the aggregated tubules (which had aslightly higher density than the water), drew off the supernatant, and resuspended the tubules in water such that the final pH was again 2.75. If the lipid had been hydrolyzed, the tubules would have aggregated, even at this pH. No aggregation was observed, however, giving further credence to our suggestion that aggregation in fact occurs at very
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low pH due to a small Debye screening length rather than the lipid hydrolyzing. For a sufficiently weak Coulombic barrier, the onset of aggregation is determined largely by tubule diffusion. For tubules of length L = 30 pm and havin a typical diameter of 0.5 pm and wall thickness of 200 , we calculate the volume V per tubule to be approximately lo4 cms for a concentration of 0.1 mg/mL. Thus, the averageseparation 20 pm. The diffusion constant D for an d [=W3] ellipsoid of these dimensions is D 7 X 10-lo cm2/s.18J7 In a random walk process we find that 7 = d2/4D, where 7 is a characteristic time for diffusion (over half the tubule separation) which results in tubule contact. For the experimental parameters T is of order 10 min, consistent with the observed onset of aggregation. Moreover, since each tubule is expected to approach another tubule approximately 3 to 6 times per hour, a diffusion-based model cannot serve as an alternate to that proposed above for tubule stability in the pH range 2.6-3.0. In conclusion, we have found that tubules do not aggregate in a range 2.6 < pH < 3.0. The process of aggregation involvesa sensitive balance between dispersive and Coulombic repulsive forces and, at low pH, the screening length as well.
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Acknowledgment. We wish to thank Steven Walsh and Paul Yager for useful discussions. This work was supported by the National Science Foundation under Grants ECS-8822228 (M.-H.L. and C.R.) and DMR8901854 (R.G.P.). J.A.M. was supported by grants from the National Science Foundation and Office of Naval Research. (16) Perrin, F. J . Phys. Radium 1934,5, 497; 1936, 7, 1. (17) Perrin, F. J. Chem. Phys. 1942, 10, 415.
