Langmuir 1992,8, 2938-2946
2938
Adhesion Kinetics of Phosphatidylcholine Liposomes by Evanescent Wave Light Scattering Zheming Xia and The0 G. M. van de Ven’ Paprican and Department of Chemistry, Pulp and Paper Research Centre, McGill University, Montreal, Canada H3A 2A7 Received May 5, 1992. I n Final Form: August 10, 1992 Phosphatidylcholine liposomesof submicrometersizesubjected to a stagnationpoint flow in an impinging jet were allowed to adhere on quartz surfaces. The kinetics of the liposome adhesion was studied by measuring the intensity of the evanescent wave light scattered per unit area of the interface as a function of time. We found indirect evidence that liposomes remain intact, though possibly flattened to some extent. It was found that the initial rate of adhesion reaches a maximum value at a flow rate of 125 pLIs. The tangential component of the flow at higher flow rates is not negligible, causing an appreciablereduction in the adhesion rate. A slightly acidic environment (pH < 6.8) favors adhesion. As a result of the balance between the bending and adhesion energies, liposomes smaller than a critical radius, experimentally determined to be about 40 nm, will no longer adhere to the surface. The adhesion rate of liposomes in CaClz saline is found to be twice that in NaCl saline at salt concentrationshigher than 0.25 M, probably because Ca2+ions adsorbed on the lipid bilayer reduce the rigidity of liposomes more effectivelythan Na+ ions. Liposomes suspended in NaCl saline up to 1.5 M fail to flocculate, implying the existence of an additional repulsive force besides the electrostatic repulsion, most likely a hydration force. The rigidity of the membrane also plays a role in flocculation.
Introduction Liposomes are vesicle structures consisting of either a single lipid bilayer or several concentric lipid bilayers enclosingan aqueous compartment. Studies of liposomes are motivated by the importance of the lipid bilayer as a fundamental constituent of natural membranes. Liposome/cell interactions provide excellent models for physiologicalevents such as cell/cell recognition and adhesion. Due to their capacityof encapsulatingdesired chemicals in the compartment and incorporating proteins and other macromolecules in the lipid bilayer, and also because they are nontoxic to the human body, liposomes have been widely recognized as potential carriers for drugs. They can deliver the drug selectively to the sites in the body where the therapeutic effect is wanted, avoiding dilutions of the drug and indiscriminating destruction of vital organs and tissues by the drug before it reaches the target cells. The carriers, with or without the aid of incorporated macromolecules as targeting devices, establish the interaction with the target cells. The interaction, in turn, triggers the uptake process, either through a lysosomal mechanism by which the drug is liberated in the vicinity of the target cellsor through stable adhesion or endocytosis where the drug diffuses from intact liposomes, serving as “depots”,or even through fusion of the liposomes with the membrane of the target cells. Most of the studies on liposomes as pharmacological capsules and immunological adjuvants1 are, for the sake of simplicity, confined to in vitro experiments, although there are several successful clinical attempts to use encapsulated drugs, for instance, antitumor drugs to increase the survival time of the host, insulin2 to lower blood glucose levels in rata through oral administration: chelators injected intravenously in mice to remove plutonium from liver: and cortisol palmitate to control (1)Lewis, J.T.; Hafeman,D. G.;McConnell, H. M. Biochemistry 1980, 19, 5376.
(2)Fendler, J. H.; Romero, A. Life Sci. 1977, 20, 1109. (3) Parel, H. M.; Ryman, B. E. Biochem. SOC.Trans. 1977, 5, 1054. (4)Rahman, Y. E.; Rosenthal, M. W.; Cemy, E. A. Science 1973,180, 300.
arthritisS6 One of the most impressive and successful results of in vivo experiments was obtainede using liposome-carried antimonial drugs to trace the parasitic infection Leishmaniasis in animal models. It becomes apparent that success of the drug delivery system depends mainly on the stability of liposome suspensions either ”on the shelf“ or in the blood stream, and the intensity and specificity of the interactions of liposomes with the target cells. Therefore, most of the potential applications mentioned above will ultimately lead to the problem of coagulation and adhesion of liposomes in a variety of biological environments. Adhesion of individual liposomes has been studied by the micromanipulator technique,’ focusing on the interaction between liposomes and surfaces or cell membranes and the energy involved during the adhesion process. Research on suspensions of liposomes subjected to flow, with emphasis on the kinetics of adhesion, has been ale0 demonstrated? However, the low refractive index of the lipid bilayer confines the studies to‘giant” liposomes (1-3 Km) in order for them to be observed by a conventional microscope or by video-enhanced contrast differential interference micros~opy.~ Investigation of the adhesion kinetics of smaller liposomes (20-500 nm) proves to be a challenge. Unlike the kinetics of aggregation,measurable by a variety of techniques such as light transmissionlOJ1 and photon correlation spectroscopy,12studies on the kinetics of adhesion require the detection of submicrometer-sized liposomes at the surface/suspension interface, (5)Shaw, I. H.;Kinght, C. G.; Page-Thomas, D. P.; Phillips, N. C.; Dingle, J. T. Br. J. Exp. Pathol. 1979, 60, 142.
(6)Alving,C.R.;Steck,E.A.;Champan,W.L.;Waits,B.V.;Hendricke,
L. D.;Swartz, G. M.; Hanson, W. L. R o c . Natl. Acad. Sci. U.S.A. 1978, 75,2959. (7)Evans, E.;Metcalfe, M. BiOphy8. J. 1984,45, 715. (8)Wattenbarger, M. R.; Graves, D. J.; Lauffenburger,D. A. Biophy8. J. 1990,57,765. (9)Ninham, B. W.; Evans, D. F. Faraday Discuss. Chem. SOC.1986, 81, 1. (10)Day, E. P.; Kwok, A. Y. W.; Hark, S. K.; Ho,J. T.; Vail,W. J.; Bentz, J.; NU, S. Roc. Natl. Acad. Sci. U.S.A. 1980, 77,4026. (11)Yoshikawa, V.;Akutsu, H.; Kyogoku, Y. Biochim. Biophys. Acto 1983, 735,397. (12) Menger, F. M.; Lee, J.; Aikene, P.; Danis, S. J.Colloid Interface Sci. 1989, 129, 185.
0 1992 American Chemical Society
Adhesion Kinetics of Phosphatidylcholine Liposomes laser light
ii
interface particle evanekcent wave region
n,'
i n2
Langmuir, Vol. 8, No. 12,1992 2939
0
0,
Figure 1. Illustration of the phenomenon of total internal reflection and the resulting evanescent wave. nl and n2 are the indicesof refraction for the dense and rare media, 0 is the incident angle, and Be is the critical angle beyond which the evanescent wave occurs.
excluding the ones in the bulk of the suspension, and to observe the entire adhesion process in situ. In this study, we use a novel technique of quantifying the adhesion process for submicrometer particles by combining the evanescent wave light scattering technique with the impinging jet apparatus.13 We seek to demonstrate the kinetics of adhesion of submicrometer liposomes under well-defined hydrodynamic conditions and to examine the influences of a variety of parameters such as flow rate, size of liposomes, pH, and salt concentration on the adhesion rate.
Theoretical Section I. Evanescent Wave Light Scattering. When a ray of light traveling in an optically dense medium reaches the interface with a rarer medium at an incident angle 0 greater than a critical angle 0, given by sin 8, = n2/nl (1) where nl and n2 are the indices of refraction for the dense and rare media, respectively, the amplitude of the electric field of the light does not drop to zero as at a metallic interface. Instead, it penetrates into the rarer medium (cf. Figure 1). Since the part of the electromagnetic field transmitted into the rarer medium is no longer propagating sinusoidally but decays exponentially with distance in a direction normal to the interface, it is commonly referred to as an evanescent wave.14J5 The depth of the penetration, d,, defined as the distance within which the electromagnetic field of the wave falls to e-l (Figure 11, is given by
where XO is the wave length of the light in vacuum. Any object of a refractive index different from n2 within the range of an evanescent wave will scatter light. It has been shownl6 that for a nonadsorbing spherical particle located far enough from the surface, i.e., neglecting the multiple scattering between the particle and the surface, the scattered electric field at a distance far from the particle, in spherical coordinates (r,6,q5)with the origin at the center of the sphere, has the form (13) Albery, W. J.; Kneebone, G. R.; Foulds, A. W. J.Colloid Interjace Sei. 1986, 108, 193. (14) Bryngdahl, 0. IV. Evanescent Waves in optical imaging. In Progress in optics XI;Wolf, E., Ed.; North-Holland: Amsterdam, 1973; pp 16!+221. (16) Harrick, N. J. Internolrejlectionspectroscopy;Wiley: New York, 1967. Kerker, M. Appl. Opt. 1979, 18, 2679. (16) Chew, H.; Wang, D.-S.;
where z is the separation between the sphere and the surface, B l , m contain vector spherical harmonics and are functions of the size of the sphere, the refractive indices, and the incident evanescentwave, and Kt is the transmitted wave vector. The intensity of the scattered light is proportional to E,,2. The inevitable multiple scattering between a particle and a surface when the particle is attached to the surface complicatesthe expressionsfor the scattered electricfield. Furthermore, if the scattered light is collected in the dense medium at a distance from the interface, a correction is necessary for the reflection and refraction of scattered light at the boundary. So far, evanescent wave light scattering has been applied to particles at a small distance from surface^."-^^ No theory is available for the calculation of the scattered light of an adhered particle in the evanescent wave region. Quantitative analysis of particles or molecules on surfaces was usually achieved with the aid of fluorescence labeling.21 For a collection of particles on the surface, assuming that (i)particles are homogeneous in size, (ii) particles are evenly distributed in the illuminatedarea, (iii) the particles are far apart to avoid lateral multiple scattering, and (iv) the characteristic length of the area is much smaller than the distance between the surface and a photon sensor (placed along an axis normal to the interface through the origin of the wave vector), the amount of light detected by the sensor can be considered as a summation of an equal amount of light scattered from individual particles. Therefore, the total intensity of light is proportional to the number concentration of particles on the surface. 11. Kinetics of Adhesion. Similar to the expression for the exponential increase in the number of particles on the surface per unit area, n,, with time, t (eq 8 in ref 221, the intensity of scattered light from particles on the surface per unit area, I,, can be expressed as
I, = I J I - e-+) (4) where I, is the intensity per unit area at t = and T the characteristic time of the adhesionprocess. For short times (t 0), eq 4 reduces to
-
I, = J I t
(5)
where JI = I m / T . Thus, the initial deposition rate is constant. For spherical particles approachingthe surface, the rate constant JI equals
where Cis the amount of light scattered per particle (which depends on the size and shape of the liposomes), a the strength of the flow characterized by the geometry of the impingingjet and the Reynolds number of the flow; ho the particle concentration, D the diffusion coefficient, and Cud the ratio of the experimental adhesion rate to the theoretical one, which is the efficiency of adhesion. CYd is less than 1in the presence of an energy barrier. Since the (17) Lan, K. H.; Ostrowsky,N.; Sornette, D.Phys. Reu. Lett. 1986,67, 17. (18) Prieve, D. C.; Frej, N. A. Langmuir 1990,6, 396. (19) Bike, S. G.; Prieve, D. C. Int. J. Multiphase Flow 1990,16,727. (20) Schumacher, G. A., van de Ven, T. G. M. Langmuir 1991,7,2028. (21) Lok, B. K.; Cheng, Y.;Robertson, C. R. J. Colloid Interjace Sci. 1983,91, 87. (22) Xia, Z.; Woo, L.; van de Ven, T. G. M. Biorheology 1989,26,359.
2940 Langmuir, VoZ. 8, No. 12,1992
intensity is proportionalto the number of particles on the surface, we will refer to JI as the initial rate of adhesion. 111. Kinetics of Coagulation. Coagulation in the absenceof any repulsive force between particles is defined as fast coagulation,the rate of which wase first calculated by Smoluch~wski.~~ The change in the apparent radius aappof coagulating spheres observed by dynamic light scattering can be expressed
-
Xia and van de Ven n
as24925
(7) where k d is a constant which can be calculated from the particle size distribution, a is the particle radius at t = 0, W is the stability ratio, and T1p is the half time of fast coagulation, given by Tl12= 1/8?rDan, The equivalent radius of a doublet can be calculated from the Stokes-Einstein equation (6aqa,D, = kT), where q is the viscosity and the translational diffusion constant for a doublet, D,, is given by26
surface
1
(8) D , = (1/3)(011+ m,) whereDll= D/1.294 and D l =D/1.432. In case the doublets fuse, a, is given by a, = 2lI3a.
Experimental Section I. Preparation of Liposomes. A. Multilamellar Liposomes. A 2-mL sample of L-a-phosphatidylcholinesolutionwith a concentration of 100 g/L in hexane (Sigma Chemical Co.) was allowed to deposit overnight on the sides of a round-bottom flask by removal of the organic solvent on a rotary evaporator under nitrogen gas. The thin layer of dried lipid was hydrated with 20 mL of saline or other buffer solutions for 1 h, and was then dispersed in the aqueous phase by gentle shaking. B. Size Extrusionof Liposomes. Homogeneousdispersions of liposomes were prepared by sequential extrusion of multilamellar liposomes mentioned above. A size extrusion device was built similar to the design described in ref 27. Essentially, it contains a cylindrical compartment of a volume of 200 mL separated by a metal grid disk in the middle. Two polycarbonate membranes (Nuclepore Corp.) of desired pore size (50-400 nm) and two drain disks (NucleporeCorp.) were placed alternatively on the metal disk. A second metal disk was pressed against the membranes by a cylinder when the cap was screwed on. The gaps at both ends of the cylinder were sealed with rubber rings. Suspensions of multilamellar liposomes were pushed through the polycarbonate membranes five times under a pressure of 3000-4000 kPa from a nitrogen gas cylinder. To minimize the loss of lipid during the process, the membranes were rinsed with a saline solution which was in turn used to dilute the homogeneous liposomes to the desired concentration. The average apparent diameters of the liposomes formed, measured by photon correlation spectroscopy (PCS), were close to the pore diameter of the polycarbonatemembranes. The size was sensitiveto the pressure applied, though reproducible at the same pressure. C. Quality of Liposome Suspensions. The liposomes prepared by size extrusion were unilamellar2' and fairly monodisperse, with a standard deviation of about l o % , as estimated from the PCS measurements. No coagulation was observed for suspensions of 10l0/mLin 150 mM NaCl at 4 "C, for a period of up to 1week. D. Concentrationof Liposome Suspensions. The number concentration of a liposome suspension was estimated from the weight concentration of lipid assuming (i) there is no loss of lipids during the preparation procedures, (ii) all the liposomes are (23) Smoluchowski, M. Phys. 2.1916, 17,55. (24) Xia, Z. Ph.D. Thesis, McGill University, 1992. (25) Thomas, J. C. J . Colloid Interface Sci. 1987, 117, 187. (26) Brenner, H.Int. J. Multiphase Flow 1974, 1, 195. (27) Hope,M. J.; B d y , M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985,812,55.
b sheath fluid
sheath flutd
Figure 2. (a) Schematic representation of the experimental setup: C, video camera; Co, computer; G, gas cylinder; Go, goniometer;H, prism holder;J, impingingjet; L, laser;M, monitor, Mi, microscope; Mr, mirror; R, reservoir; Pi, pinhole; PMT, photomultiplier; Pr, prism; V, VHS recorder; W, waste. (b) Schematic graph of the double jet. unilamellar and have a uniform diameter equal to the average hydrodynamicdiameter obtained from PCS measurements,and (iii) egg phosphatidylcholinehas a molecular weight of 77lB and occupies an area of 0.75 nm2 in the lipid bilayer.29 11. Evanescent Wave Scattering/ImpingingJet Apparatus. An evanescentwave is created at a quartz/water interface by directing a laser beam of wavelength 632.8 nm (in vacuum) from a Spectra-Physics Model 127 (35 mW) helium-neon laser perpendicularly on the 80' slope of a trapezoid-shaped prism of refractive index 1.5. As shown schematically in Figure 2a, the prism was placed on top of an impinging jet, the bottom surface being the surface on which particles adhere and which serves as the illuminated area around the stagnation point of the flow created by the jet. The light scattered from particles adhered on the surface in the region of the evanescentwave was collected by a photomultiplier (PMT) mounted on a Zeiss microscope with an objective lense of lox; no eye piece was used during a measurement. The output of the PMT, as counts of photons per unit time, was sent to an IBM 286/AT computer for data processing via an analog/digital interface. Alternatively, the PMT can be replaced by a video camera, and the image of the adhered particles can be observed on a TV monitor and recorded for further analysis. It is possible to compare the outputs from both PMT and video when a slot, the same size as the CCD (7 X 9 mm2)on the video camera, is placed in front of the 16-mm-diameter PMT sensor. This modification confines the observation area to 590 X 760 pm at the solution/ prism interface as "seen" through the video camera. The control in the direction of the laser light was achieved by using a double front surface mirror assembly, the first mirror at an ahgle of 45O from the horizontal beam &d the second one adjustable by mounting it on a goniometer and a x-y-z trans(28) Maclean, H.Lecithin and allied substances: lipins; Longmans, Green and Co.: New York, 1918. (29) Rand, R. P. Annu. Rev. Biophys. Bioeng. 1981,10,277.
Langmuir, Vol. 8, No. 12,1992 2941
Adhesion Kinetics of Phosphatidylcholine Liposomes lational stage. The beam was narrowed by passing it through two pinholes (typically of 0.6- and 0.2-mm diameter) separated at a distance of about 20 cm. An optical fiiter and a shutter were installed in front of the PMT sensor so that only light with the same wavelength as that of the laser beam was allowed to pass through; consequently, experiments could be carried out in ambient light. In order to align the apparatus, the geometric center of the orifice of the impinging jet was brought to the center of a cross scaleon an eye piece of the microscope. A prism holder, adjustable translationally in x , y, z directions and rotationally with respect to the z axis, allows centering of the prism along the r axis and maintenance of the sides along the y axis perpendicular to the plane of the beam. The angle of the incident light was verified by adjusting the sets of mirrors until the reflected light from the &/prism interface coincided with the incident light, Le., till no bright spot appeared around the second pinhole. If the prism is well aligned, an oval illuminated area at the interface, viewed through the microscope, will appear to be symmetric around the center of the scaled cross hair in the eye piece; furthermore, the spots on the sides of the prisms, where the incident and internally reflected beams pass through, will be at the same distance from the edges. Prior to an experiment, the inner jet containing unfiltered suspending medium was switched on so that dust particles would be observed as tracers, indicating the location of the stagnation point and confiiing the alignment of the apparatus. The prism was then elevated by its holder and was replaced by a fresh one before a real experiment started. In order to eliminate the effect of multiple reflection at the &/prism interface, in case the laser beam was not precisely perpendicular to the slope, R thin layer of antireflection material was coated (Laser Optics Inc.) on the slope of the prism. The internally reflected light on the other slope of the trapezoidal plate was led away either by a mirror or by a beam stopper. 111. Double Impinging Jet. The double impinging jet (Figure 2b) consisted of a cone-shaped inner jet of a diameter of 0.5 mm and an angle of 30° and an outer jet with a diameter of 2 mm. Several precautions were taken when the double jet was designed. (i) The disturbance caused when the liquid enters the inner tube must vanish before reaching the exit, which requires that L/R > 0.8Re," where L (27 cm) and R (0.1 cm) are the length and the radius of the outer jet and Re is the Reynolds number of the flow (maximum value of 70). Under this condition, flow near the exit of the outer jet is a fully developed Poiseuille flow.50 Hence, the velocity, u, of the jet is given by
where r is the radial distance from the axis, R the radius, and Q the flow rate. (ii) The radius of the cross section, Ri, of the core fluid must be larger than that of the area of observation. This can be achieved by adjusting the flow rates of the core fluid, Qi, and the sheath fluid, Q, given by (10) By performing the integration of this equation, we obtain (11)
(iii)The diffusion of liposomes from the core fluid to the sheath fluid must be negligible. This occurs when tj < t,; tj and t, are given by t , = (R?)/2D tj = L/(u) (12) where D is the diffusion coefficient of liposome particles, t , the time necessary for liposomes to diffuse radially into the sheath fluid, and tj the time for liposomes to travel from the exit of the inner jet to the surface.
(30)Schlichting,H.Boundury-Layertheory; McGraw-Hill: NewYork, 1968.
5
.....
4 -
3 -
2 -
..Y
Io 1 0
b I
I
I
I
5
10
15
20
25
Time (min)
Figure 3. Intensity of scattered light recorded by a photomultiplier as a function of time. ZO is the background scattering. Arrows indicate the turning on and off of the flow of a suspension. Dots are measured values, and the solid line is the best fit to the equation Z - ZO= A(l - exp(-t/B)). On the other hand, calcium ions with a diffusion coefficient of 7.9 X 10-lomz s-l can diffuse from the sheath fluid to the core fluid; i.e., for them, t, < tj. Therefore, it is feasible to perform adhesion experimenta when the salt concentration is sufficiently high to cause coagulation, by allowing the extra salt to diffuse into the liposomesuspensions immediately before adhesion t a k a place. IV. Cleaning of the Quartz Prism. The antireflection coating on the side of the prism makes it impossibleto completely or partially immerse the prism in any acidic solutions or strong organic solvents. It was recommended by the company which providesthe coating to use acetone or methanol to treat the coated surface. Therefore, the prisms were subjected to ultrasonic waves for 20 min in a detergent solution, followed by thorough rinsing with distilled water. After a second stage ultrasonication in acetone for 10 min and thorough rinsing with distilled deionized (d-d) water, the prisms remained in the d-d water and ready for use. Immediately before placing a prism onto the impinging jet, the upper surface and the sides were sprayed dry by a can of compressed air to avoid refraction of light due to the remaining water drops. V. Method of Analysis. A typical case of scattered light intensityvs time is shown in Figure 3. The background scattering was subtracted from the total intensity, and the initial rate was obtained by finding the best fit of eq 4 to the measured values. VI. Test of the Techniquewith Latex Particles. Spherical polystyrene (PS) latex particles 3.4 pm and 100 nm in diameter (Seradyn) were used to test the technique. Adhesion of SUBpensions of 3.4-pm latex particles with a concentration of 1 X W / m L in 0.01 M KCL was carried out in a single jet at five different flow rates. The latex on the surface was recorded with both a PMT and a video camera by switching them alternatively during the 20-min run for each experiment. Figure 4a shows the increase in the number concentration on the surface with time, recorded by the video camera, whereas Figure 4b shows the increase in the light scattered by latex on the surface with time, recorded by the PMT. The initial rates obtained from both figures for the same experimental run were compared, and the ratios of the two are listed in Table I. The number of particles per unit area increases linearly with time during the 20-min experiments for all the flow rates ueed. The particles cover only less than 2% of the surface area a t the end of the experiments, slightly less than the 2-3 % of coverage reported for 3-pm latex particles when the fiial coverage is reached.31 It is not surprising that particles are still at the linear adhesion stage for such low concentrations of suspensions, 10fold lower than those used in other ~ t u d i e s . ~The ~ J ~low surface coverage was deaigned intentionally to minimize possiblemultiple scattering among the particles in the evanescent region. The adhesion rate, Jv,defined by the slope of the lines, increases with (31)Varennes, S.,van de Ven, T. G. M. PCH,PhysicoChem. Hydrodyn. 1987,9, 537. (32)DeWitt,J.A.;vandeVen,T.G.M.ColloidsSurf.,tobepublished.
2942 Langmuir, Vol. 8,No. 12, 1992
Xia and van de Ven so,
I
h
dE
? ! v
20
t
.a**
I a
a
a
a a a a
5
0
10
15
0
20
Time ( m i n )
+d
r
130 0
I
/
5
10
15
20
Time ( m i n )
Figure 4. (a) Surface density of adhered polystyrene latex particles as a function of time, recorded by a video camera. (b) Intensity, I, of scattered light from adhered polystyrene latex particles 3.4 pm in diameter as a function of time, recorded by a photomultiplier for the corresponding experimental runs for various flow rates indicated in the figure (pL/s). The particle concentration is 1 X 106/mL, and the salt concentration is 0.01 M KCl. Table I. Comparison between the Adhesion Rate from Evanescent Wave Light Scattering and That from Microscopic Image Analyzing adhesion adhesion flow rate rate J, X 1P rate J1 X 109 J1/Jv X 1P 51 72 86 89 130
35.6 86.9 108.0
115.3 172.2
3.26 7.74 8.59 9.25 15.74
20
I 30
Time (min)
b
0
000 000000 090000
10
Figure 5. Intensity of scattered light of adhered submicrometer polystyrene latex particles 180nm in diameter on a quartz surface as a function of time. Open dots indicate adhesion of a latex suspension, 9.2 X 10' particles/mL, in the absence of flow; filled dots indicate adhesion of the suspension at a flow rate of 92pL/s. The arrow indicates the turning on of the flow. The salt concentration is 0.05 M KCl.
20
.-m
Le!:
0
0.91 0.89 0.79 0.80 0.91 av: 0.86
0.06
flow rate. The reproducibility of the increase can be estimated by comparing the values for flow rates of 86 and 89 pL/s. The intensities of scattered light per unit area, or scattered light densities, in Figure 4b also increase linearly with time. The rate of the increase, denoted as adhesion rate JI for the convenience of comparison, follows the same trend with flow rate as in Figure 4a. The ratio of JI to J , can be considered as the amount of light scattered per particle on the surface, provided that particles are uniformly distributed on the surface, the distances between the particles are much greater than particle size, and no multiple scattering occurs between particles and the surface. For the five different flow rates listed in Table I, the ratio JdJv varies from 7.93 X 103 to 9.15 X 103 counts/particle with an average of 8.63 X 103 and a standard deviation of 600, less than 7 % of the mean value. The consistent values for the scattering density indicate that multiple scattering is limited for a surface coverage of 2 %. A small amount of doublets, observed to be about 1%of the population, and a slightly uneven distribution of particles on the surface, could be the cause of the error. Multiple scattering between particles and the surface, if not negligible, is included in the amount of light scattered per particle. The significance of the linear correlation between J, and JI is that the scattering density, Le., the scattering intensity per unit area, can be
considered as a measure of the actual surface flux of adhesion, at least for latex particles at the initial stage of adhesion. It was also verified that the evanescent wave light scattering technique has sufficient sensitivity to measure submicrometer particles, though the above comparison is not feasible due to the limited resolution of a conventional microscope. Figure 6 shows the scattering density as a function of time for PS latex particles 180 nm in diameter suspended in 0.01 M KC1 solution with a particle concentration of 9.2 X lOI3/m3. Particles adhere onto the surface even in the absence of flow with a very small adhesion rate. At a flow rate of 92 pL/s the scattering density initially increases substantially, with a rate of 1 X 10l2counts s-l m-2, followed by an exponential leveling off to a maximum density of 90 X 1013counts m-2. It can been seen that the initial rate is 100 times larger than that for 3-pm latex particles at comparable flow rates. Consideringa nearly 100-foldincrease in concentration compared to the suspensions in Figure 4b, the flux of particles will also increase 100 times, if the size of particles in the two suspensions is the same. However, the flux is inversely proportional toa2I9,which predicts a 7-fold increase for 180-nmparticles compared to a particle of 3.4 pm. Therefore, it requires only 0.07 of the intensity of scattered liiht per particle for a 180-nmparticle to reach the same scattering density as in the case of 3.4." particles. It is expected that the amount of light scattered per particle in the evanescent region is highly size dependent. VII. Test To Study Whether Liposomes Remain Intact after Adhering on Surface. Reports have shownthat liposomes may be broken to form lipid layers when brought in contact with surfaces, under certain chemical environmenta.89.MMultilammellar PC liposomes adsorbed on glass surfaces were demonstrated to remain intact by taking electron micrographs of the dried liposomes on the surface.gs The increase in the intensity of light scattering observed during an adhesion experiment of a suspension of liposomes described above does not necessarily prove that liposomes still remain intact once attached to the surface. However, by increasing the penetration depth of the evanescent wave to a value close to the diameter of the liposomes, the intensity of the scattered light is expected to increase if the liposomes are intact. Figure 6 shows the penetration depth and the intensity of the scattered light from adhered 280-nm liposomes for various incident angles. The liposome suspension (in 0.1 M CaC12) in the jet was replaced by a saline solution, after 20 min of adhesion at a flow rate of 76 pL/s, to eliminate the contribution of scattered light from the suspending liposomesin the evanescent wave region. Hence, the increase in the intensity at larger penetration depths suggests that the lipid material extends from (33)Horn, R. G.Biochim. Biophys. Acta 1984, 778, 224. (34)Jackeon, S.M.; Reboiras, M.; Lyle, 1. G.; Jones, M. N. Faraday Discuss. Chem. SOC.1986,81, 291. (35)Tenchov, B.G.;Petaev, D. N.; Koynova, R. D.; Vassilieff, C. S.; Meyer, H.W.; Wunderlich, J. Colloids Surf. 1989, 39,361.
Langmuir, Vol. 8, No.12,1992 2943
Adhesion Kinetics of Phosphatidylcholine Liposomes Incident angle 80.0 77.5
75.0
('1
72.5
70.0
h r
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.-E C
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'
80
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.-m
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.-C
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.-
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0
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l n
60 150
175
200
225
P e n e t r a t i o n d e p t h (nm)
Figure 6. Intensity of scattered light as a function of the penetration depth for a fiied amount of adhering liposomes 280 nm in diameter. The incident angle, 0, corresponding to the penetration depth is indicated in the upper horizontal scale.
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200
225
Figure 7. Rate of increase in the density of the scattered light, or adhesion rate, JI,of adhered liposomes of 200 nm (diameter) as a function of flow rate. The fiied circles are results from experiments using a single jet, and the open ones are from experimentsusing doublejets. The dashed l i e is the theoretical predictionbased on eq 6. The lipid concentrationof the liposome suspension is 400 mg/L. the surface to a distance close to the liposome diameter. This is strong evidence that liposomes remain intact on the surface.
Results and Discussion Effect of Flow Rate. PC liposomes 200 nm in diameter suspendedin a physiological saline solution, 150mM NaCl (pH 6.0-6.5),with a lipid concentration of 400 mg/L, were subjected to flow in an impinging jet at various flow rates, ranging from 75 to 200 pL/s. The light scattered from the liposomes, adhered on the quartz surface in an area of 0.45 mm2 around the stagnation point, was recorded continuously within the experimental time of 20 min. The initial rates of increase in the scattered light density, JI, obtained by finding the best fit of eq 4 to the experimental results, are plotted against flow rate in Figure 7. The solid circles indicate average values of two to three experimental runs. About 2&30 % error, indicated by the error bars, could come from two major sources: (i) there was a 5% deviation in flow rate for three experimental runs due to the accuracy of the low-rangepressure regulator of the N2 cylinder; (ii) the trapezoidal quartz prisms may not be exactly the same, causing slight changes in the amplitude of the electric field of the evanescent wave, and consequently in the incident light experienced by the liposomes, although the difference of 345% in the back-
Diameter (nm)
Figure 8. Adhesion rate, JI,of adhered liposomes as a function of liposome size. All the suspensions are of the same lipid concentration, 400 mg/L, and are subjectadto a flow with a flow rate of 95 pL/s. The circles are experimental results; the solid line is the best fit of the equation I, = AdZ+ Bd + C. ground scattering was eliminated after being subtracted from the total intensity (see Experimental Section). In spite of the scatter in the data, it is obvious that the adhesion rate, JI,increases with flow rate until it reaches a maximum value at around 125 pL/s, beyond which the adhesion rate decreases with the flow rate. However, theory predicts a monotonic increase of the adhesion rate with flow rate, as indicated by the dashed line in Figure 7. The values were calculated from eq 6. The unknown product adC is estimated assuming that the first experimental value (at 75 pL/s) matches the theoretical one. The decrease in JI,or rather the dependence of a d on shear rate, could be attributed to the fact that, besides the force normal to the surface experienced by the particles, the radial component of the flow near the stagnation point, not included in the theory, exerts a tangential force on the particles and forces them to travel, rolling or sliding, on the surface for a small distance before they are held to the surface. The radial flow rate (Vr)is proportional to the radial distance (r) and the normal distance (2) from the stagnation point, the proportionality constant being the strength of the flow, a. Although Vr is negligible when r is small, for the maximum r of 380 pm in our experiments, it could cause liposomes, which would adhere within the observed data if V , = 0, to adhere further down the stream, resulting in a low scattering intensity. Furthermore, this tangential force increases with the flow rate and could simply reduce the probability of adhesion when it becomes comparable to the adhesion force. A decrease in adhesion rates at high flow rates was also observed for the cases of other particles on glass surfaces, such as spherical latex particles coated with polymers,3s rod-shaped bacteriaz2 and hardened spherical erythrocytes?' although the critical flow rate at which adhesion reached its maximum rate is different for each system, probably due to the differences in particle size, shape, surface smoothness, adhesion energy, etc. The open circles in Figure 7 represent the results obtained by using double jets, an inner capillary jet of a radius of 250 pm containing liposome suspension and a concentric outer jet of a radius of 1 mm carrying saline. The values are consistent with the ones obtained from the single jet within the range of experimental error. Effect of Liposome Size. The effect of liposome size on the adhesion rate is summarized in Figure 8 for the (36) Varennes, 5.;van de Van, T. G.M. PCH, PhysicoChem. Hydrodyn. 1988,10,229.
(37) Xia, Z.; Goldsmith, H. L.; van de Ven, T. G. M. To be published.
2944 Langmuir, Vol. 8, No. 12, 1992 same lipid concentration of 400 mg/L in physiological saline and for the same flow rate of 95 f 4 pL/s. The adhesion rate increases with the diameter of liposomes varying from 90to 400 nm. The experimental results (filled dots),were fitted with a second-order polynomial (solid line). We were not able to form liposomes of less than 80 nm (diameter) by the size extrusion method, due to the difficulties in pressing large multilamellar liposome SUBpensions through two stacked polycarbonate membranes under a reasonable pressure. Extrapolation of the fit to zero adhesion rate yields the result that liposomes smaller than 78 nm in diameter do not adhere. In Figure 8, initial rates are compared for the same lipid concentration, not for the same number concentration of liposomes. Correctingfor this differenceand for the effects of particle size increases the estimate of this critical diameter by a few percent. The increase in JI with size for the same particle flux on the surface can be attributed partly to the fact that a big particle will scatter more light than a small particle of the same shape, and that a large flattened liposome probably scatters even more light by occupying a bigger area at the interface where the electric field of the evanescent wave is stronger. However, if this was the only reason for the increase in J I ,one would expect the line to pass through a point very close to the origin for which the light scattered by the liposomes falls below the detection limit of the instrument. This is obviously not the case for liposomes of 78 nm. A liposome adheres on a surface by minimizing its free energy:
AG = AGK+ AG7 + AGpv + AGw
(13) where Gw is the free energy of liposome/surface interactions, which is the product of contact area A and the surface energy y, G, is that of liposome deformation which is proportional to the rigidity of the lipid bilayer, K , A G p y is the contribution from changes in the volume of the liposomes or from the difference between the internal and external pressures of the liposome, and ACTresults from the surface tension of the lipid bilayer due to changes in the surface area of a liposome. For a given total area of lipid bilayer, and constant volume and pressure difference, the gain in free energy, through bending a liposome with rigidity K > 0, is compensatedby the loss of energy through the increase in its contact area. Small liposomes tend to have a higher bending energy, the extreme case being liposomes that remain spherical when brought in contact with a surface. Hence, the competition between the bending energy and the adhesion energy results in a critical radius, a,, given by38 a, = (2K/W)'I2 (14) where w is the adhesionpotential. Liposomes with radius a < a, no longer adhere to the surface. For a fluid bilayer, i.e., T > Tc (T,is the chain melting point for the lipid), at room temperature such aa a PC 5% and w is approximately equal lipid bilayer, K = to the free energy potential for formation of a unit area of contact, y. For the liposomelliposome interface of neutral lipids in salt water, y is given as (1-2) X lO-'3 J/m2;39i.e., a, cy 100nm. The experimental value of about 40 nm is less, but nevertheless of the right order of magnitude. Effect of pH. Figure 9 shows the adhesion rate of 175nm liposomes suspended in phosphate buffer solutions of (38)Seifeit, U.;Lipowsky, R. Phys. Reu. A 1990,42,4768. (39)Evans, E. A. Colloids Surf. 1984,10, 134.
Xia and van de Ven
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.-C
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I
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-
0 v
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6.0
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r
r
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Figure 9. Adhesion rate, JI,as a function of pH. The average diameter of the euspensions is 175 f 5 nm, and the flow rate is 95 pL/s.
various pH values. At the same lipid concentration and the same flow conditions, the adhesion rate is not sensitive to pH at pH < 6.5 or pH > 7.0, yet changes dramatically with pH in a small range around pH 6.8, favoringthe acidic suspensions. For instance, the adhesion rate is nearly 10 times higher at pH 6.0 than at pH 7.0. Since phosphatidylcholine is a zwitterionic lipid with both cationic and anionic functional groups such as [Pod]-, [N(CHs)sl+,it is possible that [PO4l-groups are associated with protons and the [N(CH3)31+groups are exposed, which facilitate the binding to the negatively charged quartz surface. The isoelectric pH reported for phosphatidylcholine covers a An alternative wide pH range varying from 3.5 to interpretation could be that the binding of protons on the lipid may change the orientation and consequently the arrangement of the zwitterionic headgroups, resulting in a higher liposome flexibility and lower resistance to adhesion. It has been suggested that pathologic tissues have a pH considerably lower than that of normal tissues. For instance, sites of primary tumors, metaetaeis, inflammation, and infection have reduced local pH environments."14 In fact, the use of pH-sensitiveliposomeswhich incorporate lipide containing pH-sensitive groups of pK,, between 4 and 5, such as N-palmitoylhomocyeteine,was attempted to provide a convenient means of enhancing the delivery of However, negatively charged liposomes are not resistant to high salt concentrations, especially in the presence of Ca2+ions. It waa demonstrated that fusion of liposomes of phosphatidylserine occurs at a threshold Ca2+concentration of 1 mM.4BTherefore, it is significant that liposomes containing PC lipide can also adhere to target cells triggered by a local acidic environment. Effect of Salt. Another important parameter that governs the stability of the liposomes is the salt concentration, especially in the presence of divalent cations such as calcium ions. The effect of NaCl and CaC12 concentrations on the kinetics of adhesion is shown in Figure 10. To prevent liposomes from coagulating prior to adhesion (40)Chapman, D. Introduction to lipide, McGraw-HiU: London, 198% p 95. (41)Kahlar, H.; Robertson,W. V.B.J. Natl. Cancerlnst. 194B,S,495. (42)Gullimo, P.M.; Feantham,F. H.; Smith,S. H.; Haggerty, A. C. J. Natl. Cancer Inst. 1966,34,857. (43)Naeslaud, J.; Swenson, K.E.Acta Obstet. Gynecol. Scand. 19S3, 32, 359. (44)Yatvin, M. B.; Kreutz, W.; Horwitz, M.;Shinitzky, M. Science 1980,210,1263. (45)Gregoriadis, G. Liposomes as drug carriers; Wiley Chichestar, New York, 1988. (46)Pap+djopodoe, D.; Vail, W. J.; Pangbom, W.A.; Ponta, G. Biochim. Biophys. Acta 1976,448,265.
Adhesion Kinetics of Phosphatidylcholine Liposomes
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r
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CaCI,( M )
when the salt concentration is sufficiently high to cause coagulation (see below), a double jet was applied with a core fluid of liposome suspension in 0.15 M of salt and a sheath fluid of saline with concentrations indicated in the figure. The flow rate at the outlet of the double jet was s = 10 8 ) . It takes 6 and 10 maintained at 85 f 3 ~ L / (tj s for Na+ and Ca2+with diffusion coefficients of 1.3 X lo4 and 7.9 X 10-lom2/s,re~pectively,4~to diffuse into the core fluid 250 pm in diameter; Le., t, I tj. The lipid content in the suspensions was 300 mg/L. The Ca2+concentration has a substantial influence on the kinetics of adhesion. The adhesion rate in the presence of CaC12 initially increases slowly with concentration (below 0.15 M),and then it starts to increase much faster. The value of JIat 0.5 M is more than 5 times that at 0.2 M. The adhesion rate of suspensions in NaCl saline follows the same trend, but reaches a plateau value of 2.5 X 10l2 m-2 min-' at a Na+ concentration of around 0.15 M, only less than half of the adhesion rate at high Ca2+concentrations. The classical DLVO theory describes the energy of interaction between a spherical particle and a plate as a competition between van der Waals attraction and electrostatic repulsion. However, the DLVO theory cannot explain the salt dependence of the adhesion rate, since for the salt concentration used, electrostatic repulsion is negligible due to screening by counterions. Besides van der Waals attraction and electrostatic repulsion, an additional repulsive force, the hydration force, which decays exponentially with distance between lipid bilayers,&should also be taken into account. It might be responsible for the low adhesion rate at higher Na+ concentrations. Studies have s h 0 w n ~ that ~ 9 ~calcium ions adsorb onto bilayers of zwitterionicphosphatidylcholine. The binding constants of Ca2+were determined to be 10-100 M-'for an egg PC membrane51 and 21 f 9 M-'for a dipalmitoylphosphatidylcholine (DPPC)membrane.b0Evidence of segregation according to the type of PC in an egg PC bilayer induced by 30 mM Ca2+was also shown.51 We may speculate that ions bound to the liposomes may also (47) Adameon, A. Physical chemistry; Academic Press: New York, 1979. (48) Afzal, S.;Tesler, W. J.; Blessing, S. K.; Collins, J. M.; Lie, L. J. J. Colloid Interface Sci. 1984, 97, 303. (49) Mclaughlin, A. C.; Grathwohl, C.; Mclaughlin, S. G. A. Biochim. Biophys. Acta 1978,513,338. (50) Ohshima, H.; Inoko, Y.; Mitaui, T. J. Colloid Interface Sci. 1982, 86, 51. (51) Lie, L. J.; Lie, W. T.; Parsegian, V. A.; Rand, R. P. Biochemistry 1981,20, 1771.
Figure 11. Intensity of scattered light from 280-nm (diameter) liposomeson a surface exposed to various CaClz concentrations. The liposomes were preadhered (flow rate 76 fiL/s; lipid concentration 400 mg/L) in a horizontal jet mounted under a hemicylindrical quartz prism.
change the rigidity of the liposome membrane. Thus, the critical radius varies with salt concentration, since both K and w in eq 16 are functions of salt concentration. A low adhesion rate occurs when only a fraction of the liposomes have radii larger than the critical value. By increasing the salt concentration, a, may be reduced to a value less than the radii of the liposomes in the suspension, resulting in a much higher adhesion rate. The change from a low rate to a high rate occurs at a higher salt concentration of Ca2+ than that of Na+. The plateau value of JI is also higher for Ca2+than for Na+. These differences indicate that the calcium solution has a lower value of K, yet a higher ratio of K to w than the sodium solution for the same salt concentration. Figure 11 illustrates how the intensity of the scattered light changes when the 280-nmliposomes already adhered on the surface were exposed to various Ca2+concentrations. This increase in intensity indicates that binding of Ca2+ ions to the liposomes on the surface at a higher Ca2+ concentration might have changed the rigidity of the liposomes, and consequentlyflattened the liposomes, thus causing a higher scattering intensity. However, we cannot rule out the possibility that fusion or phase transition induced by high Ca2+concentration was takingplace, which might change the vesicles into other forms of lipid assembly, suchas tubular microshctures. However, since the liposome concentration on the surface is very low, this is unlikely. Also flattening could occur as a result of osmotic stress. As the salt concentration increases from 0.15 to 0.4 M, the refractive index varies from 1.333 to 1.344,measured by an Abbe refractometer. This change in the refractive index will also contribute to the increase in the scattering intensity shown in Figure 11,mainly due to the increase in the penetration depth. However, the penetration depth only increases by less than 4% (from 158to 164nm)which, accordingto Figure 5, only results in about a 1076 increase in the scattering intensity. Because of the flattening of the liposomes, the rates given in Figure 10 need correction, since we assumed that the intensity of the scattered light is proportional to the number of liposomes. However, in comparing different runs at various salt concentrations, it is clear from Figure 11 that the proportionality constant depends on the salt concentration. However, the change in intensity scattered per particle increases by about 30% when the CaC12 concentration is increased from 0.15 to 0.4 M,while the
Xia and van de Ven
2946 Langmuir, Vol. 8, No. 12, 1992
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1 140 nm
01
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-0.6
I
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I
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Figure 12. Logarithmic plot of the stability ratio, W, of PC liposome suspensions vs salt concentration. a, is estimated by eq 8 (open symbols), or based on the assumption that doublets are formed by fusion of singlets (fiied symbols). The lines are the linear regression of the values.
rate increases by 700%. Hence, the correction amounts to only a few percent. Flocculation of LiposomeSuspensions. The kinetica of coagulation of liposome suspensions in various salt concentrations was examined by monitoring the change of the average hydrodynamic diameter of the liposomes with time in aphoton correlationspectroscope (PCS). This study was performed to see whether or not similarities between coagulation and adhesion could help us to better understand the adhesion behavior of PC liposomes in the presence of salt. Liposomes were initially prepared in deionized water. The suspensionswere mixed with NaCl or CaC12 solutions (1:l in volume), by gently inverting the test tube immediately before the measurements. No coagulation was observed for liposome suspensions containing NaCl up to a concentration of 1.5 M for all three sizes of liposomes studied (diameters 84,200, and 280 nm). The results of the flocculation in the presence of CaClz are presented in Figure 12, which shows the stability ratio (obtained from eq 7) for various salt concentrations. Liposomes of 42 nm start to coagulate at 0.3 M CaClz, a threshold much higher than 4 mM Ca2+,reported for liposomes composed of negatively charged lipids such as phosphatidylserine.s2 The minimum Ca2+concentration necessary to induce coagulation is lower for larger liposoma, approximatelyO.25,0.2, and 0.15 M for suspensions of 42,100, and 140nm,respectively. Whether or not fusion was taking place in the suspensions is not known. The fact that sodium ions at high ionic strength fail to induce coagulation, consistent with the low adhesion rates of liposome suspensions in Figure 10, and that the salt concentrations at which the liposomescoagulate are much higher than what is predicted by the DLVO theory, supports the hypothesis that an additional repulsive force, a hydration force, exists to stabilize the suspension. Study of the aggregation of negatively charged phosphatidylserinelO has shown that the concentration threshold (52) Laneman, J.; Haynee, D.H. Biochim. Biophys. Acta 1975,394, 335.
for aggregation induced by sodium ions is 0.55 M, higher than required in the case of calcium ions. The significant difference between Na+ and Ca2+in triggering liposome coagulationsuggests,once again, that the simple screening of the double layer of counterions does not appear to be the flocculation mechanism. Rather, the bivalency of calcium ions could be responsible. A possible explanation might be the bridging of calcium ions between the two [Pod- groups on the adjacent liposomalmembranes. If this were true, one would expect a maximum coagulation rate when the surface coverage of the calcium ions on the lipid bilayers reaches 60%. However, we do not observe a decrease in the coagulation rate with salt concentration, even at a surface coverage greater than 50%, estimated from the binding constants of Ca2+. An alternative explanation is that calcium ions reduce the rigidity of the liposomal membranes, thus causing coagulation. It should be noticed that the DLVO theory predicta no size dependence of the CCC (critical coagulation concentration). However, by extrapolating the lines in Figure 12 to log W = 0, we find that large liposomes reach the fast coagulation regime at a lower salt concentration, confirming that the flexibility of the liposome membrane is equally important in the coagulation process. The critical liposome radius, a,, for coagulation is expected to be smaller than that of adhesion, in the absence of shape fluctuations, because a liposomeinteracts with another flexible liposome rather than with a rigid substrate, as for the case of adhesion.
Concluding Remarks The initial adhesion rate of submicrometer phosphatidylcholine liposomes, represented approximately by the rate of increase in the intensity of the scattered light per unit area, JI,in the evanescent wave region, increases with flow rate as predicted by the theory, up to a maximum value at a flow rate of 125 pL/s. At higher flow rates, the tangential component of the flow, which is not included in the theory, is not negligible, causing an appreciable reduction in the adhesion rate. Adhesion is favored in suspensions with pH < 6.8. As a result of the balance between the bending and adhesion energies, liposomes smaller than a critical radius, ac, experimentally determined to be about 40 nm, w i l l not adhere to the surface. Caz+ions significantly enhance the adhesion at concentrations higher than 0.25 M, with an adhesion rate twice that found for Na+ ions, possibly because Ca2+ ions associated with the lipid bilayer reduce the rigidity of the liposomes more effectively than Na+ ions. PC liposomes flocculate when suspended in CaClz saline above 0.15 M, dependingon the liposome size, yet not in NaCl saline up to 1.5 M, indicating that an additional repulsive force, a hydration force, and the rigidity of the membrane play important roles in flocculation. Acknowledgment. The authors are indebted to Mr. A. Kluck and Mr. W. Bastian for making various parta for the experimental setup, to Mr. M. Polverari for technical assistance in the measurements with varying incident angles, and to Professors R. Cox, M. Frojmovic, and T. Dabros for valuable discussions. Registry No. NaCl, 7647-14-5;ClC12, 10043-52-4.
