2872 Langmuir 1995,11, 2872-2880

Study of the Surface and Biological Activity of a Trivalent Cage Surfactant

Marilyn E. Karaman,? Richard M. Pashley," and Nicholas K. Bolonkin

Department of Chemistry, The Faculties, and Department of Applied Mathematics, RSPhyS & E, The Australian National University, Canberra, A.C.T. 0200, Australia

Received September 6, 1994. In Final Form: March 2, 1995@

We have studied a novel cobalt cage surfactant which has significant in vivo biological activity on parasitic nematodes. This activity was investigated by the confocal microscopy of sheep red blood cells, allowing the mode of action to be studied at a cellular level. Surface force measurements were carried out using colloid probe atomic force microscopy on adsorbed bilayers of this surfactant, which were found to be mechanically weak. The surface properties of the surfactant were measured and correlated with its biological activity. The theory of membrane curvature was also used to describe the surfactants biological activity.

Introduction The surfactant used in this investigation, shown in

Figure 1 and known for convenience as CS12 (i.e., a cage surfactant with a C-12 tail), belongs to a class ofcompounds known as sarcophagines. These are large macrobicyclic ligands consisting of an organic cage containing nitrogen atoms which chelate metal ions tightly into the center of the cage making them chemically stable in their unreactive crypt.l This compound was synthesized by a condensation reaction involving cobalt(II1) ethylenediamine complex ion as a template and formaldehyde and nitromethane as the capping reagents. A wide range of related molecules can be synthesized by either varying the parent diamine or installing various functional groups at the apical positions of the caps. Because of their selectivity for metal ions, they are interesting molecules that have potential biological and environmental applications. The cage metal ions show considerable potential diagnostic applications in nuclear magnetic resonance and radionuclide imaging. These compounds may also be used in the removal ofheavy metal pollutants from waste water. The metal ion can be selectively encapsulated into the vacant crypt, thus making the ion unreactive. Due to the selectivity for metal ions they encapsulate, they have the ability to extract minute amounts ofheavy metals from solution in the ppb range.'

These compounds may also find use in the treatment of specific diseases involving the accumulation of heavy metals such as Wilson's disease, a congenital deficiency condition in which the inflicted children lack the mech- anism required for the excretion of copper, and thus, accumulated copper builds up in the child's liver and brain and results in liver failure. Laboratory tests using the sarcophagines on mice liver cells have shown effective copper removal from the cells.' Possible treatment using the sarcophagines is based upon the premise that once a metal ion is captured inside the cage, it is stable and is eliminated readily in urine, rendering it nontoxic. It is also known not to interfere with the Na+/K+ balance or Ca2+ metabo1ism.l

These compounds also show biological activity in nematodes (parasitic round and tapeworms) by their ability to insert into and destabilize the organisms' outer membrane, making them an effective nemacidal agent. The sarcophagine cage without the paraffin tail shows no

+ Department of Applied Mathematics. @ Abstract published in Advance ACS Abstracts, July 1, 1995. (1) Sargeson, A. M. Chem. Aust. 1991, 58, 176-8.

0743-746319512411-2872$09.00/0

biological activity demonstrating that it is the amphiphilic nature of the molecule which allows membrane insertion and disruption. Previous biological studies on cage surfactants have shown that their biological activity is dependent on charge, concentration, the number and length of the surfactant tails, and the presence of cosurfactants.2

Wanless et al.3 investigated a cobalt-encapsulated cage surfactant having a methyl substituted sarcophagine head-group and a branched hydrocarbon tail containing two amine groups (known as CS3). The two acidic amine groups were found to possess different dissociation characteristics, believed to be due to the effect of the high head-group charge. The average degree of dissociation was found to be 0.73 giving an effective +4.27 charge on the cation. The head-group area a0 was estimated to be 154 A2, which gives a critical packing parameter of 0.23, which supported observations that this surfactant formed micellar aggregates. Because of the complicated (pH- dependent) nature of cage surfactants such as CS3, we chose here to study the simpler, single chained (CS12) cage surfactant.

Since the membrane activity of this class of compound depends on the amphiphilic nature of the molecule, we have characterized the surface activity of CS12. The cmc was determined by two methods: conductivity and surface tension. These provided not just a measure of the cmc but also an estimate of the degree of ionization of the surfactant (from conductivities) and the head group area a0 (from surface tension values). The latter can be used to predict aggregate structure, which depends upon a critical packing parameter, Vla$,, based on simple geo- metrical argument^.^-^ This parameter is simply obtained from the volume of the hydrocarbon tail, V, the extended hydrocarbon chain length, l , , and the optimal head group area, ao. The critical packing parameter provides a useful predictive guide to the molecular architecture of the aggregates formed for a particular ~ u r f a c t a n t . ~ Thus, if V/aoZ, < 113 spherical micelles are expected, in the range 113 =- VIaoZ, < 112, polydispersed cylindrical micelles form

(2) Behm, C. Personal communication. (3) Wanless, E. J., Pashley, R. M. Colloids Surf. 1991,56,201-215. (4) Tartar, H. V. J . Phys. Chem. 1955, 59, 1195. ( 5 ) Tanford, C. J . Phys. Chem. 1972, 76,21, 3020. (6) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J . Chem. Soc.,

(7) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Biochim. Faraday Trans. 2 1976, 72, 1525.

Biophys. Acta 1977, 470, 185.

0 1995 American Chemical Society

Study of a Trivalent Cage Surfactant

CI - X n

Langmuir, Vol. 11, No. 8, 1995 2873

Figure 1. (A) Schematic diagram of the CS12 molecule and (B) space-filling model of CS12.

and in the range l/2- 1 vesicles, oblate micelles or lamellar phases form. For values > 1 inverted phases are observed.

In an attempt to understand some of the biological effects of this surfactant at the cellular level, a confocal microscopy study was carried out using sheep erythrocytes in isotonic 150 mM NaCl to study morphological changes induced by the surfactant-membrane interaction. In addition, a colloid probe7 was used in an atomic force microscope as a substrate for the adsorption of surfactant bilayers, and the interaction forces between bilayers were studied. The results obtained enabled us to build a picture of monolayer/ bilayer formation and surfactant-packing characteristics, which can be related to the interaction forces involved in the membrane disruption process.

Materials and Methods The water used in this study was produced from tap water

which was fed into a Memtec Krystal Kleen unit using a three- stage purification process. The first stage involved a prefilter treatment to remove suspended solids. Next, the water was passed through a reverse osmosis membrane, where dissolved electrolytes were removed, and finally through an activated charcoal stage, where dissolved gases such as chlorine were removed. The permeate was then distilled and collected in a positive pressure, dust-free laminar flow cabinet. The surface tension of the water was measured using the rod-in-free-surface method (RIFS).8

Glassware used for critical experiments was thoroughly cleaned by first rinsing in 10% NaOH; it was then washed in a warm teepol solution and rinsed in tap water and AR grade BDH ethanol to remove any surface active material. It was then finally rinsed in the water purified as explained above.

The C 12 cage surfactant used was synthesized in The &search School of Chemistry at the Australian National University and given to us by Professor Alan Sargeson. It was obtained by reacting the cobalt diamser with tetradecanal in the presence of triethylamine and methylcyanide and then reducing the double bond with sodium borohydride in methanol. It was then purified by eluting down a silica column in methanol and then recrystal- lized from a solution of ethanol and hydrochloric acid. It was then analyzed using both NMR and total elemental analysis and

(8) Padday, J. F.; Pitt, A. R.; Pashley, R. M. Faraday Trans. 1 1975, 71, 1919.

found to be greater than 99% pure and was, therefore, used without any further purification.

The erythrocytes used in the confocal investigation were obtained from an adult sheep from the John Curtin School of Medical Research at the Australian National University.

Solution conductivities were measured at 25 "C using a radiometer CDM 80 conductivity meter.

All surface force measurements were obtained using a Digital Instruments Nanoscope I1 atomic force microscope, which was adapted to allow measurement of the forces acting between the stylus or stylus attachment and a planar s~bstrate .~ The force between the surfaces was detected by the deflection of the cantilever attached to the stylus using a laser reflectance system; the displacement of one surface was varied using a piezoelectric crystal. Knowledge of the cantilever spring constant and the deflection of the spring with the piezoelectric displacement allowed us to determine the separation distance between surfaces relative to the constant compliance position, where both surfaces were in intimate contact.

Silica probes were prepared by attaching 5 pm silica glass spheres (obtained from Polysciences) using Epicote 1009 resin to commercial cantilevers (produced by Digital Instruments) using a micromanipulator. The silica flat was prepared by oxidation of a silicon wafer glued with araldite to a magnetic stainless steel stub. Both the silicon flat and silica probe were treated with a water plasma at 30 mTorr Ar, 36 mTorr H20, and 10 W for 30 s.l0

The confocal microscope consisted of a Nikon microscope coupled with laser enhancement, computer, storage facility, and a video recorder. Both static and dynamic experiments were carried out to directly observe the effect of CS12 surfactant on sheep erythrocytes. This technique allowed the morphology of the cell as a function of concentration to be observed as well as the concentration at which cell lysis occurred in.isotonic media (150 mM NaCl). In the static experiments 10 pL of sheep red blood cells (RBC) was added to 90pL of 150 mM saline in epidorf tubes and 10-50 pL of 5 x M CS12 was added, then mixed using a vortex mixer. A small sample was then transferred to a concave microscope slide covered with a coverslip and sealed with nail varnish; the slide was then viewed in the confocal microscope and the cell morphology noted as a function of time

(9) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991,353,

(10) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Langmuir 1992,8 239-41.

(7), 1833.

Langmuir, Vol. 11, No. 8, 1995 Karaman et al.

I 0 0 1 2 3 1

conoentratlon (mM)

Figure 2. Electrical conductivity of a range of aqueous CE solutions. The critical micelle concentration (cmc) is den0 by the dashed line and occurs at approximately 1.8 mM. The degree of micelle ionization (a) was estimated from these results to be 0.66 from a comparison of the slopes above and below the cmc value.

and concentration. In the dynamic experiments the microscope slide and coverslip were sealed with the nail varnish except at two faces. The RBC in saline were introduced through one of these faces followed by addition of 1 x M CS12. This allowed observation of surfactant diffision across the slide and the behavior of cells distant from the point of administration.

Results and Analysis Surface Tension and Conductivity. The surface

adsorption and solution properties of the cage surfactant were studied by surface tension and conductivity mea- surements over a wide concentration range in aqueous solutions. Conductivity measurements provide estimates of the critical micelle concentration (cmc) and the degree of micelle ionization (a). If conductivity is plotted as a function of CS12 concentration, the change in slope before and after the cmc allows the degree of ionization of the CS12 aggregate to be estimated, assuming that the solution conductivity is entirely due to the free C1- present in solution and that the conductivity of the CS123+ ions and even larger micelles is negligible due to the large viscous drag forces associated with the size ofthe species.l' The ratio in slopes above and below the cmc gives the effective degree of ionization of the micelle.

For this highly charged surfactant the change in slope was small but distinct (cf. Figure 2). By use of linear regression analysis, the cmc was estimated to be ap- proximately 2 mM and the degree of ionization 0.66. "he latter result indicates that only one C1- ion is bound per CS12 surfactant molecule in the micellar aggregate. As will be shown below, a mass action description of the micelle formation process predicts a weak transition in the counterion ((21-1 concentration at the cmc for multi- valent surfactants such as CS12.

The surface tension values obtained using the rod-in- free-surface technique are given in Figure 3. The critical aggregation concentration was determined in both water and 150 mM NaCl and was found to occur a t approximately

(11) Pashley, R. M. Personal Communication.

I I I I

'Ctt""1 25 0.001 0 01 0.1 I 10

CS 12 Concentration (mM) Figure 3. Surface tension as a function of CS12 concentration in water at room temperature and in aqueous 150 mM NaCl solution at 37 "C, denoted by filled triangles and circles, respectively. The cmc in water and electrolyte solution were found to occur at about 2 and 0.2 mM, respectively. From the maximum slope and the Gibbs adsorption isotherm, the head- group area in both cases was estimated at 85 k, which would give a critical packing parameter (V/a$,) of 0.25, corresponding to a micellar aggregation state.

2 and 0.2 mM, respectively. From the maximum slope and the Gibbs adsorption isotherm, the head-group areas in 150 mM NaCl at 37 "C and in water at room temperature were both found to be approximately 85 A2, which would give a critical parameter Wad, of 0.25, corresponding to a micellar aggregation state.

Langmuir Trough. Another direct measure of head group area was attempted using the Langmuir trough on a 10-1 M NaCl subphase, however, because of the high solubility ofthe surfactant and the nature ofthe aggregate formed (i.e. micellar), very rapid monolayer desorption was evident, and no head group area could be estimated. By comparison, when soluble vesicle forming surfactants are deposited onto a Langmuir trough, the monolayers do not readily desorb because of the unfavorable phase transition (monolayer to bilayer to vesicles).12 These results also support the view that CS12 aggregates must be micellar, as predicted by the critical packing parameter for this surfactant.

Static and Dynamic Confocal Microscopy. Both static and dynamic confocal microscopy experiments were carried out to observe directly the effect of CS12 surfactant a t the cellular level with sheep erythrocytes. The mor- phology of the cells as a function of concentration was observed and the concentration at which cell lysis occurred in isotonic media (150 mM NaC1) determined. In static experiments with no added surfactant the cells appeared unstressed and spherical and showed distinct external membrane features. As the CS12 concentration was increased to 0.046 mM, the distinct boundary associated with the membrane disappeared and the cells appeared to be stressed and tended to clump together indicating changes in surface charge, probably by the incorporation of the positively charged surfactant a t sublytic concentra- tions. At 0.083-0.100 mM CS12 the cells appeared swollen and stressed, exhibiting clumpiness, but cellular membranes still remained intact. When the concentration was increased to 0.106 mM, the cells appeared to be

(12) Karaman, M. E.; Ninham, B. W.; Pashley, R. M. J. Phys. Chem. 1994,98, 11512-18.

Study of a Trivalent Cage Surfactant Langmuir, Vol. 11, No. 8, 1995 2875

L

Figure 4. Photographs taken from video-recorded, dynamic confocal microscopy images of sheep red blood cells in the presence of 1 mM CS12 with time. (A) shows a number of healthy intact erythrocytes, whereas (B) and (C) show fewer and fewer intact erythrocytes and the presence of ghost cells (i.e., erythrocyte membranes without hemoglobin). (D) shows only a single intact erythrocyte and coagulated ghost cell debris.

stressed with extensive clumping evident and some ghost cells were observed due to cell lysis causing intercellular material exclusion leaving only an empty cell membrane. At 0.115-0.167 mM only strongly clumped ghost cell membranes and extensive cellular debris were evident.

In the dynamic confocal experiments (Figure 4) 10 pL of RBC in 90 pL of 150 mM saline was transferred to a microscope slide and sealed on only two faces. Then either the CS12 or hexadecyltrimethylammonium bromide (CTAB) was added to one face, and the diffusion of the surfactant across the microscope slide was observed. In these experiments it was found that close to the open faces the RBC appeared stressed undergoing dehydration, evident by their change in shape, which one would expect because of the increase in extracellular NaCl concentration close to the open faces, due to water evaporation. The osmotically affected cells were shown to revert back to an unstressed resting state (of about 5 pm in diameter and spherical) by the addition of saline a t the open edges. A droplet of 1.5 mM CS12 was added close to the open edge of the slide, and it was noted that as the cage surfactant diffused across the slide, the resting unstressed RBC became stressed and transformed into ghost cells. These tended to clump together and showed a darker node in the membrane, possibly where surfactant insertion and membrane rearrangement took place. The procedure was repeated for comparison using CTAB, another single chained cationic surfactant, a t a similar concentration. This comparative study using quaternary ammonium surfactants showed no membrane-disrupting properties a t similar concentrations. However, single chained sur- factants usually cause membrane disruption a t concen- trations near their cmc.13

(13) Ashman, R. B.; Blanden, R. V.; Ninham, B. W.; Evans, D. F. Immunol. Today 1986, 7 (91,278-283.

Table 1. Parameters Used to Fit the SilicdSilica Measured Force Curves in Water and Sodium Chloride

Solutions concentration Debye surface surface

(mol L-l) length (nm) potential (mV) charge (C m-2) water 50 -90 -2.1 10-3 1 x 10-3 11 -50 -3.8 x 10-3 1 x 10-2 3 -25 -6.1 10-3 1 x 10-1 1 -23 -1.8 x

We initially attributed the activity of the CS12 sur- factant a t this concentration (approximately 1/10 cmc in water) to its high charge and large head-group in addition to its cationic nature electrostatically interacting strongly with the anionic membrane. But the critical micelle concentration in 150 mM NaCl was reduced to 0.2 mM due to head-group screening effects. Some biological activity was found to occur as low as 0.046 mM, but cell lysis only occurred above 0.106 mM in saline. Therefore, it appears that the high head-group charge had little effect on the biological activity, and that it behaved in a similar manner to monovalent surfactant species in that cell lysis occurred close to the cmc. This may be due to the large head-group size and the shape of the molecule causing packing difficulties into the cellular membrane. With this in mind, it would be interesting to study a double chained multivalent cationic surfactant to elucidate the effect of surfactant packing on biological activity.

Atomic Force Probe Microscopy. In an attempt to understand the bilayer forces involved in membrane disruption, forces were measured using colloid probe atomic force microscopy between surfactant bilayers adsorbed onto silica substrates. The force curves for silica in water and in and 10-1 M NaCl are shown in Figure 5. The observed Debye lengths (K-~), surface potentials, and surface charges, obtained by fitting theoretical curves for 1:l electrolytes using the method of Chan et al.,14 are summarized in Table 1 and were found to be in agreebent with those of Ducker et al.'

Addition of 0.125 mM CS12 removed the repulsive double-layer forces between the silica surfaces in water and only a van der Waals (vdw) jump into contact was observed (see Figure 6). This is in good agreement with the predicted vdw jump for SiO2/H2O/SiO2 taking a Hamaker constant of 0.85 x J. Thus, a t this concentration a submonolayer must have adsorbed and neutralized the negative charge but without creating a hydrophobic attraction. l5 As the concentration was increased to 0.75 mM (Figure 7) a t a line rate of 240 ms, the forces became repulsive again, indicating that the silica surfaces were then positively charged, the repulsive force extending out to approximately 22 nm. The observed forces fell between an envelope embraced by the two extremes of double-layer force a t a constant potential/ constant charge range of +do mV and 3.9 x C m-2, with an observed K - ~ of 450A, which is in good agreement with that expected of 45.6 A. A short-range van der Waals attraction was also observed; again, the adsorption process was not accompanied by a hydrophobic attraction a t this concentration. The constant potential and constant charge theoretical curves were obtained using a numerical solution to the nonlinear Poisson-Boltzmann equation for 3: 1 and 1: 1 electrolyte mixtures. l6 The 3: 1 electrolyte was of the type M3+x3l-, as used in these experiments. Thus, for a positively charged surface (as shown in Figure

(14) Chan, D. Y. C.; Pashley, R. M.; White, L. R. J. Colloid Interface

(15) Pashley, R. M.; McGuiggan, P. M.; Ninham, B. W.; Evans, D. F.

(16) Pashley, R. M. J. Colloid Interface Sci. 1984, 102, 23-28.

Sci. 1980, 77, 283-285.

Science 1985,229, 1088-1089.

2876 Langmuir, Vol. 11, No. 8, 1995

AFM INVESTIGATION - SilicalSilice

10000

- v-

E 5 1000 - s

I: ; - -

Karaman et al.

0 1 0 2 0 30 4 0 5 0 60 7 0 a 0 90 1 0 0

Distance (nm)

Figure 5. Forces measured between silica surfaces in water and in and lo-' M NaC1, which are denoted by open triangles, filled circles, open circles, and open squares, respectively. The observed force was scaled with the radius of the particle as a function of separation distance (D) . These forces were fitted with theoretical curves for 1:l electrolytes using the method of Chan et a1.I0 The fitting parameters obtained are summarized in Table 1.

Figure 6. Forces measured between silica surfaces in the presence of 0.125 mM CS12. The open circles are the experimentally determined results, and the solid line is the theoretical vdw force, assuming the complete absence of any double-layer repulsion. AHamaker constant (A) of0.85 x J was used in the nonretarded equation, FIR = -Af6D2.

7) the multivalent cation is the co-ion and the monovalent anion is the counterion.

At 1.25 mM CS12 (Figure 8) at a line rate of 1000 ms the force was repulsive extending out to about 18 nm with

i 10 20 30 DISTANCE (nm)

Figure 7. Forces between silica surfaces in the presence of 0.75 mM CS12. The upper solid line is the constant charge (3.9 x C m-2) theoretical curve, and the lower solid line is the constant potential (30 mV) curve. Line rate was 240 ms.

a fairly long-range jump into a soft barrier from ap- proximately 11 nm to about 3-4 nm. The forces beyond 11 nm were fitted with a +50 mV potential, a charge of 8.7 x C m-2 and K - ~ of 35 A, which is in good agreement with the expected K - ~ of 35 A. At this concentration, however, the adsorbed surfactant layers must have produced a hydrophobic surface, which gave the strong attraction observed. Similar forces were also observed at 2.25 mM CS12 (Figure 9) at a line rate of 1000 ms. The experimental data in this case were fitted with a constant potential of +40 mV and a constant charge of 9 x C m-*, with a Debye length of 26 A, in close agreement with that expected for this electrolyte.

Study of a Trivalent Cage Surfactant Langmuir, Vol. 11, No. 8, 1995 2877

DISTANCE (nm)

Figure 8. Forces between silica surfaces in the presence of 1.25 mM CS12. The upper solid line is the constant charge case (8.7 x C m-2), and the lower solid line is the constant potential (50 mV) curve. Line rate was 1000 ms. A strong attractive force was also observed.

DISTANCE (nm)

Figure 9. Forces between silica surfaces in the presence of 2.25 mM CS12. The upper solidlineis the constant charge (9.1 x C m-2), and the lower solid line is the constant potential (40 mV) boundary condition. Line rate was 1000 ms. Again, a strong attractive force was also present.

At 3 mM CS12 (at a line rate of240 ms) it appeared that a bilayer adsorbed giving a strongly repulsive double- layer force with no attractive jump (see Figure 10). The observed forces were fitted with a +50 mV potential and a constant charge corresponding to 7.8 x C m-2. The observed K - ~ was found to be 39 A, which is substantially longer than the calculated (3:l) value of 23 A. However, in a further complication these forces were also found to be strongly dependent on the rate a t which repeat measurements were taken (see Figure 11). It appears

4000 45000

. I O 0 10 20 30 40 50 60 70 DISTANCE (nm)

Figure 10. Forces between silica surfaces in the presence of 3.0 mM CS12. A constant charge of (7.8 x C m-2) and constant potential (50 mV) were used to fit these results. Line rate was 240 ms.

6010 I

5 0 1 0 h 401 0

1

E

I 3 3010

2010

1010

10

0 5 1 0 1 5 20 25 3 0 3 5 4 0

DISTANCE (nm)

Figure 11. Forces between silica surfaces in the presence of 3.0 mM CS12 at different approach rates. The open squares, filled squares, and the filled diamonds are for approach rates (line rates) of 80, 240, and 1000 ms, respectively. that a t rates of 240 ms and above the inward force curves were totally repulsive. However, as the rate was decreased to 1000 ms, the long-range initially repulsive interaction was followed, by a strongly attractive jump into a soft barrier. These slower rate measurements were very similar to those observed at 1.25 mM (compare Figure 8), where the Debye length was also similar. This comparison suggests that for rapid rates the adsorbed bilayer remains

2878 Langmuir, Vol. 11, No. 8, 1995 Karaman et al.

4000

3000

2000

1000

- r

E

9 0 - U t

-1000

-2000

.3000

.4000

DISTANCE (nm)

Figure 12. Observed forces between silica surfaces in the presence of 3.0 mM CS12 as the surfaces are withdrawn at different line rates. The triangles, squares, and the circles denote line rates of 80, 240, and 1000 ms, respectively.

intact, but at slower rates it can be displaced to expose a hydrophobic underlayer.

Outward runs from contact were also analyzed at this concentration (see Figure 12), and it was found that as the rates of approach were decreased, the adhesion increased, as illustrated below,

line rate (ms) 80 240 1000 adhesive energy 6.2 x 7.2 x 8.0 x

(J m 3

where the calculated adhesive energies were obtained using the equation E = FI2nR. These results support the suggestion that the adsorbed layer can be weakened and displaced by slow approach rates.

At 4.00 mM CS12 (Figure 13) at a line rate of 1000 ms, the repulsive force extended further but an attractive jump was still observed, indicating the presence of a hydrophobic force at this slow rate. The data were fitted with a +70 mV potential with a Debye length K - ~ of 50 A, whereas the expected K - ~ was 19.8 A.

At 8.03 mM CS12 (Figure 14) at a line rate of a 1000 ms the repulsive force became still stronger, and an attractive jump stronger than expected for vdw forces was again observed. The data were fitted with a f 7 0 mV potential and a charge of 9.8 x C m-2, and the observed K - ~ was 50 A, compared with a 3:l value of 14 A. The fitting parameters used for surfactant force curves are summarized in Table 2, and the calculated, simple 3:l electrolyte Debye lengths are compared with those ob- served in Figure 15. The results in Figure 15 indicate that the Debye length deviation from simple 3: 1 electrolyte occurs sharply at the cmc.

In an attempt to explain the complicated variation in the observed Debye lengths we have used the mass action model to describe the micelle aggregation process and hence obtain estimates of counterion and co-ion concen-

10000 , I I I i

-2000 .IO u 10 20 Distance (nm) 30

Figure 13. Forces between silica surfaces in the presence of 4.0 mM CS12. The upper solid line is for constant charge (9.8 x C m-2), and the lower solid line is for constant potential (70 mV). Again, at this slow line rate (1000 ms) the adsorbed bilayer can apparently be displaced exposing a hydrophobic surface and a correspondingly strong attraction.

.IO 10 20 30 40 50 60 DISTANCE (nm)

Figure 14. Forces between silica surfaces in the presence of 8.03 mM CS12. The upper solid line is for constant charge (9.8 x 10-3 C m-2), and the lower solid line is for constant potential (70 mV). Line rate was 1000 ms.

trations. The mass action equations and definitions of the cmc value are similar to those used in earlier work.3 The results obtained are shown in Figure 16 and clearly demonstrate that the trivalent monomer will substantially fall in concentration above the cmc for highly charged

Study of a Trivalent Cage Surfactant Langmuir, Vol. 11, No. 8, 1995 2879

Table 2. Summary of the Double-Layer Fitting Parameters Used to Model the Forces Observed in a Range of CS12 Solutions

[CS121 surface charge surface potential K-1 K - 1 K - ~ a micelle K - ~ a counterion (mM) (x C m-2) w o (mv) observed (A) 3:l (A) exclusion (A) only (A) 0.125 (112) 111.8 d a d a 0.750 3.9 30 45 45.6 d a d a 1.250 8.7 50 35 35.3 d a d a 2.250 9.1 40 26 26.3 30.0 75.9 3.000 7.8 50 39 22.8 31.5 69.6 4.000 9.8 70 50 19.8 33.1 62.9 8.030 9.8 70 50 13.9 34.8 46.9

a Denotes value estimated from the mass action aggregation model.

1 2 0

1 0 0

80

d 5

2 6 0 C

0

h

40

20

0

1.0QE.04 1.00E.03 1.00E-02

CSlZ Concanlratlon (M)

Figure 15. Theoretical (using a simple 3:l electrolyte model) and measured Debye lengths as a function of CS12 concentra- tion.

surfactants. Assuming that micelles are essentially excluded from between the interacting surfaces, as observed earlier,17 the Debye length will then increase above the cmc, as observed. Two extreme cases are calculated in Table 2: one where only free counterions contribute to the Debye length and the other which includes the free monomers. A comparison with the observed Debye lengths suggests that the real case is between these two with some exclusion of the trivalent co-ions. As mentioned earlier, this model predicts a weak transition in C1- concentration at the cmc (see Figure 16), and hence, only a slight slope-change in conductivity would be expected.

Microelectrophoresis. In order to obtain further details about CS12 adsorption on silica, a microelectro- phoresis study was carried out to measure the electrostatic potentials as a function of concentration and to find the point a t which a bilayer was adsorbed. The results are shown in Figure 17. The observed zeta potential for the silica sol used in 1 mM NaCl was -41.8 mV, which became increasingly more positive with the addition of CS12. The isoelectric point was found to occur a t a CS12 concentration

E Total aumotanl concentrallon n / mM

Figure 16. Calculated concentrations of monomers (nl), micelles (nm), and counterions (nz) as a function of the total surfactant concentration (n), using a mass action aggregation model (see text).

of 4.2 x M, and a bilayer adsorption step was evident a t about the cmc value at a zeta potential of about +28 mV.

Discussion and Conclusions Critical micelle concentration determinations using

different methods oRen give up to a 50% difference in values of the cmc. This has been attributed to differences in the degree of polydispersity in the micelles, the existence of impurities, and variations in the extrapolation proce- dures used to locate the cmc.18

The change in the conductivity slopes was found to be very slight for CS12. This behavior can be explained by the mass action model using the measured value for the degree of ionization (a = 0.661, since it shows that the concentration of the trivalent monomers decreases rapidly above the cmc, whereas the concentration of counterions increases fairly uniformly across the entire concentration range, thereby making an accurate cmc estimate difficult (see Figure 16).

I t is interesting to note from the surface tension study that the addition of 150 mM monovalent electrolyte had

~~ ~ ~~ ~~ ~

(17) Pashley, R. M.; McGuiggan, P. M.; Horn, R. G.; Ninham, B. W. J . Colloid Interface Sci. 1988, 126, 569-573.

~ ~ ~~ ~

(18) Kresheck, G. C. In Water-A Comprehensive Treatise; Franks, F., Ed.; Plenum: New York, 1975; Vol. 4, pp 95-167.

2880 Langmuir, Vol. 11, No. 8, 1995

.20 - 1

Karaman et al.

-40 1 I I J 0.00001 0.0001 0.001 0.01

CS12 Concentration / M

Figure 17. Zeta potentials of a colloidal silica dispersion as a function of CS12 concentration in 1 mM NaCl at 25 "C.

little or no effect on the head-group area of the CS12 surfactant. These results suggest that even in water this area is determined by the molecular size and geometry of the large diamser head-group rather than the electrostatic repulsion between adjacent head-groups.

The agreement in head-group area is even more remarkable because of the factor of 4 difference in the Gibbs adsorption isotherm for the two cases of water and with added electrolyte. This factor arises because of the change in bulk C1- concentration in water but not with excess electrolyte. The corresponding difference in slopes is clearly evident in Figure 3, where each corresponds to approximately the same head group area.

In the confocal microscopy investigation some biological activity (i.e,, loss ofmembrane external features) €or CS12 was found to occur a t concentrations as low as 0.046 mM, which from the microelectrophoresis investigation cor- responds to the isoelectric point for adsorption of this surfactant onto silica. This concentration is substantially below the cmc in saline (of 0.2 mM). At 0.106 mM the cells appeared stressed, extensive clumping was evident, and some swollen ghost cells were produced (i.e., cell membranes with hemoglobin and intercellular material excluded) indicative of CS 12 incorporation and preferential partitioning to the only negatively charged phospholipid (phosphatidylserine) in the inner bilayer membrane of

the erythrocyte cell. At concentrations below 0.1 mM the erythrocytes appear to be able to tolerate CS12 incorpo- ration, but above this critical uptake concentration the CS12 is apparently electrostatically attracted to the inner bilayer cell membrane where it packs into the bilayer, changing membrane curvature and thus bringing about membrane rearrangement and ultimately membrane disruption. The favorable electrostatic attraction of CS12 to negatively charged membranes is probably responsible for its biological action slightly below the cmc value.

I t appears likely that the biological activity takes place in a three-step process. Firstly, the high charge on the CS12 head-group may be responsible for delivering the amphiphile close to the negatively charged cellular membrane, but it is ultimately the hydrophobic interaction that allows surfactant uptake and incorporation which occurs close to the cmc. Once the amphiphile is taken up, the charged head-group would be electrostatically at- tracted to the oppositely charged phospholipids present in the cell membrane. The surfactant critical packing parameter (VladJ dictates how well the surfactant ag- gregate (cf. micelles) will pack into the relatively low curvature bilayer membranes of the cell. This uptake, incorporation, and membrane rearrangement must locally change the membrane curvature and ultimately result in membrane disruption.

The atomic force microscopy investigation reported here gave some insight into the fluidity and bilayer packing characteristics of the CS12 molecule. We were also able to measure interaction forces acting between bilayers and obtain the concentrations at which submonolayer, sub- bilayer, and bilayer adsorption occurred. Rate dependence effects linked to the mechanical weakness of the adsorbed bilayer were easily demonstrated using this technique. We also observed the effects of micellar exclusion which highlighted the onset of aggregationvia a sharp transition in Debye length. This transition occurred at a concentra- tion close to that determined by several other more conventional methods.

Acknowledgment. We thank Professor Barry Nin- ham, Department of Applied Maths (RSPhysSE, ANU) for his many useful discussions and for use of the atomic force microscopy facility, Professor Alan Sargeson and Mr. Glen Walker (RSC, ANU) for supplying the CS12 surfactant and for helpful discussions, Professor Brian Gunning (RSBS, ANU) and Ms. Ailsa Rowlinson (JCSMR, ANU) for use of the confocal microscope facilities, Mr. Roger Heady and Ms. Sally Stowe (EMU, RSBS) for use of the electron microscope facility, and Ms. Cathy Woodhams (JCSMR, ANU) for supplying sheep blood cells.

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