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Ricin Antitoxins Based on Lyotropic Mesophases Containing Galactose Amphiphiles P. G. Hartley,*,† M. R. Alderton,‡ R. M. Dawson,‡ and D. Wells† CSIRO Molecular & Health Technologies, Private Bag 10, Clayton South, Victoria 3169, Australia, and Defence Science and Technology Organisation, PO Box 4331, Melbourne, Victoria 3001, Australia. Received July 14, 2006; Revised Manuscript Received November 14, 2006
Lyotropic mesophases of the inverse hexagonal or cubic type are nanostructured materials that result from the self-assembly of amphiphilic surfactant molecules in water. The extremely large area of the surfactant-water interface inherent within these structures makes them attractive media for sorbent or encapsulant systems. Here, we report on the development of a new class of polyvalent materials that are based on the incorporation of bioactive ligands within lyotropic mesophases. In particular, we have studied the potential for these materials to behave as polyvalent antitoxins by incorporating synthetic galactose amphiphiles, which mimic the natural cell surface ligand for the protein toxin ricin. The study demonstrates that cubic morphology lyotropic mesophases containing galactose amphiphiles exhibit high specificity ricin uptake, with favorably high dissociation constants and high capacities. We suggest that lyotropic mesophase polyvalent ligands are thus promising materials for the incorporation of a broad range of cell surface recognition moieties and hence may have wide applicability as materials capable of partaking in biological recognition processes.
INTRODUCTION Polyvalent ligand-receptor interactions are employed in many biological recognition processes (1). There has been recent interest in designing synthetic polyvalent inhibitors for the therapeutic inactivation of biological entities, including viruses, bacteria, and toxins. In such systems, molecular architectures are employed to act as scaffolds for the presentation of high densities of bioactive ligands. These polyvalent ligands act to saturate receptor sites on the target, thereby blocking its ability to bind to cellular recognition sites, reducing uptake and cytotoxicity. Of particular interest have been polyvalent ligand systems based on synthetic branched polymer architectures such as dendrimers. Such systems are currently under commercial development for antiviral and microbiocidal applications (2). Pathogenic protein toxins are produced by a number of microbial species such as Clostridium botulinim, Vibrio cholera, and Shigella species (3). Numerous plant protein toxins with pathogenic activity have also been identified (4). Within the latter family, the toxin ricin, derived from the castor bean plant Ricinus communis, has unprecedented toxicity and has been utilized as the active agent within immunotoxins (5) and as a potent poison or biowarfare agent (6, 7). Both plant and bacterial toxins act by binding with high specificity to cell surface moieties. This recognition process then triggers cellular uptake Via several possible routes, resulting in the transport of the cytotoxic component of the toxin to its site of action. In the case of ricin, the cell binding component (ricin B chain) specifically attaches to multiple cell surface galactose moieties and facilitates internalization of the cytotoxic A chain within the cell, which acts to disrupt ribosomal function (5). The spontaneous self-assembly of certain lipid and synthetic surfactant molecular architectures results in the formation of * To whom correspondence should be addressed. Tel: +61 3 9545 2595. Fax: +61 3 9545 8106. E-mail:
[email protected]. † CSIRO Molecular & Health Technologies. ‡ Defence Science and Technology Organisation.
extended three-dimensional hydrated structures in solution (812). Under certain circumstances, these structures exist in equilibrium with an excess of solution and are thus stable against dilution. In particular, the structures of the lyotropic mesophases of glyceryl monooleate (monoolein) and 3,7,11,15,-tetramethyl1,2,3-hexadecanetriol (phytantriol) have been extensively studied, and proposed for use in drug delivery formulations (1317). These materials also possess attractive properties with respect to the development of polyvalent ligand systems. Most notably, they exhibit an extraordinarily large surfactant-water interface, which is potentially available for incorporation of biological or chemical functionality. Additionally, the formulation of lyotropic mesophase colloidal dispersions has been demonstrated in several systems (10, 11, 15, 18, 19). These low viscosity dispersions are readily amenable to in ViVo administration. In an earlier paper, we studied the ricin binding activity of a number of dendrimer systems derivatized with pendent galactose functional groups (20). We also introduced a new class of polyvalent ligand, in the form of the biofunctionalized lyotropic mesophase, which compared favorably with the dendrimer systems with regard to ricin binding. In the current study, we have developed a variety of novel synthetic surfactant systems, which form dilutable lyotropic mesophases and which incorporate high concentrations of biologically active ligands for ricin based on galactose surfactants. We discuss the role of surfactant chemical and anomeric structure in determining the efficacy of these materials for ricin binding and uptake. The broad aim of this work is to demonstrate unequivocally the potential for employing lyotropic mesophase systems as a new class of polyvalent ligands.
EXPERIMENTAL PROCEDURES Surfactant Synthesis and Formulation. Synthesis and chemical characterization information for the novel galactose surfactants discussed in this paper are available in the Supporting Information. In particular, synthesis was directed toward surfactants that possessed alkylation of galactose at the C1 position.
10.1021/bc060216b CCC: $37.00 © 2007 American Chemical Society Published on Web 12/23/2006
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Figure 1. Structures of surfactant amphiphiles used in this study. Galactose-6-oleate existed as a solid (melting point 90-98 °C). Galactose-6phytanoate and all galactosides (oleyl R and β and phytanyl β) existed as viscous mesomeric liquids at room temperature.
In this way, free galactose functionality in the C3-C6 positions is retained, and it is these positions that are known to be important in galactose binding to ricin (21). C6 alkylations were also performed for comparative purposes. The various surfactants employed in this study are shown in Figure 1. Mixed surfactant formulations were prepared by dissolving the appropriate proportions of each compound in dichloromethane to a total surfactant concentration of ∼40-100 mg/mL. Phytantriol was obtained from Sigma-Aldrich Pty. Ltd. Castle Hill, NSW. All water used in these experiments was Milli-Q grade. Surfactant Lyotropic Phase Behavior. Water Penetration BehaVior. Small samples of each surfactant were sandwiched beneath a coverslip on a microscope slide and stabilized at room temperature prior to addition of water at the edge of the coverslip. Capillary action drew the water between the two glass surfaces to surround the surfactant island. The optical behavior of the surfactant-water interface was observed during water penetration using an Olympus IMT-2 microscope equipped with crossed polarizing lenses. The sample was then heated at 2 °C/ min or less in a Mettler FP82HT hot stage controlled by a FP90 central processor and changes in the optical textures at the surfactant-water interface were monitored and correlated with phase structures according to established correlations (22). Water penetration scans were usually performed in the temperature range 22-99 °C. Images were captured Via a video camera, and observations were noted concerning viscosity of any observed mesophase forms. Further detailed confirmation of the structure of selected surfactant mesophases was obtained using small-angle X-ray scattering (SAXS). Samples of surfactant in excess water solution were loaded into a small sample cell consisting of two very thin mica windows separated by a 1 mm stainless steel spacer ring. A Bruker “nanostar” SAXS camera equipped with a position sensitive 2-D “Hi-Star” detector using point collimated copper KR radiation was employed. Experiments were performed under vacuum or under a helium atmosphere to minimize background scattering. Sample transmissions were estimated using a glassy carbon standard, which avoided the need to remove the instrument beam stop and thus protect the detector. Data acquisition was performed at a sample-to-detector distance of 23 cm, which allowed analysis of intensity behavior over the wavevector range 0.0498 Å-1 e q e 0.950 Å-1. The scattering profiles of the empty sample geometries were subtracted from the final scattering profile of the sample (perpixel subtraction) after first correcting for the intensity of the X-ray beam.
Ricin Binding Efficiency. Ricin binding to lyotropic mesophases was facilitated by the addition of 10 µL aliquots of ∼49 mg/mL surfactant solutions in dichloromethane or dichloromethane-methanol to glass test tubes and allowing the solvent to evaporate. The final mass of surfactant deposited in the tubes was in the range 0.43-1.28 mg. Ricin solutions, of known concentrations between 6.25 and 200 ng/mL in blocking buffer (130 µL, 0.25% bovine serum albumin in 0.05%Tween/PBS, pH 7.4), were added to the tubes, and they were placed on a rocker platform and gently oscillated for 24 h at room temperature. Aliquots (100 µL) of the supernatants from these tubes were then assayed for ricin, as described below. Aliquots (100 µL) of ricin control solutions (known ricin concentrations) were also analyzed in each experiment. Ricin concentrations in control and test solutions were quantified using an enzyme-linked immunosorbent based microtiter plate binding assay described elsewhere (23). Briefly, Nunc microtiter test plates were prepared by treating overnight with 0.1 mL per well of 2 µg/mL of the ricin binding protein asialofetuin in 0.1 M carbonate/bicarbonate buffer, pH 9.6. After washing with PBS-Tween and incubating 1 h with blocking buffer, the plates were incubated sequentially for 1 h each with ricin-containing control and test solutions, followed by rabbit anti-ricin antibody (1:15 000 dilution), and goat antirabbit IgG alkaline phosphatase conjugate (1:4000 dilution), each in blocking buffer, with washing of the plate with PBS-Tween 3 times after each incubation. An additional wash with distilled water followed the incubation with antibody-enzyme conjugate. The substrate for alkaline phosphatase (p-nitrophenyl phosphate in Tris buffer) was then added, and the rate of increase of absorbance at 405 nm was recorded for either 17.5 min (at 2.5 min intervals) or 55 min (at 5 min intervals) with a TitertekPlus MS212 plate reader. All incubations were at room temperature (approximately 20 °C). The known ricin concentration control solutions were used to construct a calibration curve for solution ricin concentration versus absorbance change per minute (Abs/min) for each ELISA assay. Calibration curves were recorded to accompany each ricin binding experiment, in order to reduce errors resulting from solution preparation and handling. The calibration curve data was conveniently fit in the ricin concentration limit 0-100 ng/ mL using a logarithmic relationship, Abs/min ) A + B ln[ricin]. The A and B factors in this relationship were obtained from the linear plot of Abs/min vs ln[ricin] for control solutions. These factors were then used to derive a residual ricin concentration (final [ricin]) in test solutions (i.e., ricin solutions that were
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exposed to lyotropic mesophase samples). In this way, the relationship between initial [ricin] and final [ricin] following exposure to lyotropic mesophase samples was quantified. The ricin affinity constants (Ka) and dissociation constants (Kd ) 1/Ka) for the different mesophase formulations were ascertained from the ELISA assay data by assuming 1:1 binding and using the initial and final equilibrium solution ricin concentrations ([ricin]in and [ricin]eq) with the initial concentration of galactose surfactant monomers within the mesophase ([active]in), in the relationship: Ka ) [bound ricin]eq/([unbound active]eq[ricin]eq), where [bound ricin]eq ) [ricin]in - [ricin]eq and [unbound active]eq ) [active]in - [bound ricin].
RESULTS A number of surfactant amphiphiles possessing a galactose headgroup that might be capable of self-assembling into lyotropic mesophases of the cubic or inverse hexagonal type were synthesized (Figure 1). The approach was based on employing hydrophobic moieties (phytanyl and oleyl groups) found in surfactants that exhibit these mesophase behaviors (9, 14). Once produced, we found that addition of water to these materials at room temperature resulted in the formation of myelinic lamellar phases, as determined by polarizing light microscopy. Since we were seeking to produce materials that existed in the dilutable cubic or inverse hexagonal mesophase phase forms, a second approach was adopted that relied on incorporation of the galactose surfactants within binary surfactant mixtures. Secondary “matrix surfactants” were selected for these mixtures on the basis of their known propensity to support mesophase formation. The phase behavior properties of these matrix surfactants are described in detail elsewhere (10, 14). To summarize, oleyl glycerate (OG) shows a viscous anisotropic phase (inverse hexagonal, HII) in equilibrium with water above 22 °C, which melts into a mobile isotropic phase at 66-73 °C (inverse micellar, LII). Phytanyl glycerate (PG) also forms an HII phase spontaneously in equilibrium with water, which melts at a slightly lower temperature, 39.5 °C. Phytantriol exhibits a cubic phase in equilibrium with water over a broad temperature range with an inverse hexagonal phase appearing at higher temperatures (14). In our measurements, the cubic-inverse hexagonal phase transition occurred at 54.5 °C, which contrasts with a literature value of 44 °C (14). From discussions with other researchers, we understand that these elevated phase transition temperatures for different phytantriol samples are not an uncommon observation. Addition of water at room temperature to mixtures of monoalkyl galactose surfactants with matrix surfactants yielded a number of systems, which exhibited lyotropic mesophase behavior of the desired type (cubic and inverse hexagonal, see Figure 2 and Table 1). We found that the mesophase behavior of these mixtures could be manipulated by adjusting the ratios of the component surfactants. The aim of this approach was to obtain mixtures in which a high concentration of galactose surfactant could be incorporated within a dilutable mesophase material while retaining the mesophase structure. As such, the compositions (Figure 2 and Table 1) represent the upper limit of galactose surfactant concentrations that could be added while retaining the desired mesophase structure, as determined by the polarizing microscopy-water penetration technique. We found that high concentrations of the galactose surfactants (20-50%) could be incorporated within these mixtures. This is an important observation, since it suggests that a high surfactant-water interfacial concentration of ligand (galactose)
Figure 2. Lyotropic phase behavior as viewed through cross polarizers. The optical textures are used to assign mesophase forms. The viscosity of both the isotropic and anisotropic phases is clear from the “rough” nature of the interface of the phases with water and is a hallmark of dilutable mesophases of the inverse type. Panel A shows water penetration behavior of 40% oleyl R-1-galactoside/60% oleyl glycerate initial penetration at room temperature (25 °C). Panel B shows the water penetration behavior of 20% oleyl R-1-galactoside/80% oleyl glycerate at room temperature for 30 min (23 °C).
should be available for binding to the protein toxin ricin within the mesophase structure, which was the aim of this work. Broadly speaking, the phase behavior of the mixed surfactant systems appears to lie between the behavior of the two isolated components. This can be rationalized by considering the curvature of the surfactant-water interface, according to the model suggested by Israelachvili (24). Isolated galactosyl surfactants preferentially form lamellar phases (of minimal curvature), while the matrix surfactants discussed above preferentially form structures of greater (inverse) curvature, the cubic and inverse hexagonal phases. For mixtures comprising >20% galactose surfactants in oleyl glycerate, addition of the lamellar-preferring galactoside reduces the preference for the negatively curved inverse hexagonal phase, yielding a cubic phase in equilibrium with water. We postulate that the low concentration of galactoside in the 20% oleyl R-1-galactoside/oleyl glycerate mixture is insufficient to significantly alter the phase behavior from the pure oleyl glycerate system; hence the inverse hexagonal phase is retained. We note the absence of phase behavior data for one of the surfactant mixtures prepared, namely, 40% oleyl β-1-galactoside/ 60% oleyl glycerate. This surfactant was prepared in quantities sufficient only for ricin binding analysis (see later). The phase behavior for this mixture is expected to be qualitatively similar to 40% oleyl β-1-galactoside/60% oleyl glycerate, which showed a cubic phase in equilibrium with water at all temperatures studied. When phytanyl glycerate was employed as the matrix surfactant, the effect of adding the galactoside surfactants was less marked, and the inverse hexagonal phase was retained in
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Table 1. Summary of Compositions and Lyotropic Phase Behavior Observed through Cross Polarizers Using Water Penetration Analysis sample 40% galactose-6-oleate/60% oleyl glycerate
40% galactose-6-phytanoate/60% phytanyl glycerate
20% oleyl R-1-galactoside/80% oleyl glycerate
40% oleyl R-1-galactoside/60% oleyl glycerate
50% phytanyl β-1-galactoside/50% phytanyl glycerate
30% oleyl R-1-galactoside/70% phytantriol
40% phytanyl β-1-galactoside/60% phytantriol
optical textures observed
temperature range
likely phases present
100% water viscous isotropic viscous isotropic isotropic anisotropic isotropic 100% surfactant
RT to 65 °C RT to >100 °C 24-92 °C RT to 92 °C 24-100 °C
cubic (CII) cubic (CII) cubic (CII) lamellar (LR) inverse micellar (LII)
100% water anisotropic 100% surfactant
RT to 99 °C
inverse hexagonal (HII)
100% water anisotropic mobile isotropic 100% surfactant
RT to 88 °C >81 °C
inverse hexagonal inverse micellar (LII)
100% water viscous isotropic anisotropic 100% surfactant
>RT RT to 42°
cubic (CII) lamellar (LR)
100% water anisotropic 100% surfactant
RT to 100 °C
inverse hexagonal (HII)
100% water viscous isotropic second viscous isotropic anisotropic mobile isotropic 100% surfactant
40-78 °C RT to 78 °C RT to 68 °C >68 °C
cubic (CII) cubic (CII) lamellar (LR) inverse micellar (LII)
100% water viscous isotropic second viscous isotropic anisotropic 100% surfactant
RT to 100 °C RT to 100 °C RT to 78 °C
cubic (CII) cubic (CII) lamellar (LR)
most cases. This suggests that the phytanyl-chained surfactants exhibit more robust inverse phases, probably due to the large volume occupied by the hydrophobe, and the disorder that it confers within self-assembly structures. The addition of galactosides to the less negatively curved matrix surfactant phytantriol (cubic phase), results in a slight reduction in the curvature relative to the pure phytantriol system. This manifests itself in the elimination of the high temperature inverse hexagonal phase of phytantriol, as was observed in phytantriol mixtures comprising 30% oleyl R-1-galactoside or 40% phytanyl-β-1-galactoside. These mixtures also showed the presence of a lamellar phase at lower water contents, which is also a feature of the pure phytantriol-water phase diagram. In order to study the structural properties of the mesophase systems in more detail, small-angle X-ray scattering (SAXS) was employed on a selection of the samples, which were prepared in equilibrium with excess water, and analyzed at room temperature (Figure 3). As expected from the polarizing microscopy optical texture observations, the 50% phytanyl glycerate/50% phytanyl β-1-galactoside in equilibrium with excess water at room temperature (∼22 °C), exhibited Bragg reflections with scattering vector (q) spacings consistent with the (inverse) hexagonal phase (1:x3:x4). SAXS analysis of the 40% phytanyl β-1-galactoside/60% phytantriol, 30% oleyl R-1-galactoside/70% phytantriol and 40% oleyl R-1-galactoside/ 60% oleyl glycerate mixtures in equilibrium with excess water revealed Pn3m cubic symmetry (Bragg peak spacings x2:x3: x4:x6:x8:x9), (25) again consistent with the polarizing microscopic observation of viscous isotropic phases in these systems. SAXS analysis also allows the quantification of structural parameters associated with lyotropic mesophases (25). The lattice parameter, a, is readily derived from the q spacings of the Bragg reflections (Figure 3). The straight line relationship
again reinforces the assignment of the Pn3m space group for the cubic phases (Figure 3B-D). The lattice parameter for the inverse hexagonal phase (Figure 3A) was obtained from the scattering vector position of the initial [10] reflection Via the relationship a ) q(x3/4π). Lattice parameters supply information regarding the centerto-center distances of repeating structural units within the mesophases. Larger lattice parameters indicate larger center to center distances, implying a more expanded mesophase structure (26). For the inverse hexagonal phase, this can readily be envisaged as the distance between the centers of adjacent aqueous channels. For inverse cubic phases, it corresponds to the cubic unit cell dimension. Considering the similarity between the various surfactant molecular dimensions involved in this study, changes in the lattice parameter within the cubic phases can reasonably be assumed to correlate with the diameter of aqueous channels within the structure. The addition of galactoside surfactants dramatically increases the lattice parameter from the previously described value of 64-66 Å for pure phytantriol (14) to values for the mixtures of 90.9-93.5 Å (Figure 3). Expansion of the mesophase structure is to be expected for the addition of the relatively bulky sugar headgroups possessed by the galactoside surfactants, causing reduced negative interfacial curvature, as discussed earlier. The phytantriolderived mesophases do possess somewhat narrower aqueous channel diameters than the corresponding oleyl glycerate mixture, with the largest lattice spacing encountered for 40% oleyl R-1-galactoside/60% oleyl glycerate (a ) 121.2 Å). The inverse hexagonal phase structure based on phytanyl glycerate possesses the most compressed (narrowest) unit cell (a ) 60 Å). These are important observations with regard to understanding the optimal mesophase structures for protein uptake within mesophase polyvalent ligands, as will be discussed later.
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Figure 3. SAXS data for different surfactant formulations recorded at room temperature (22 °C): (A) 50% phytanyl β-1-galactoside/50% phytanyl glycerate; (B) 40% phytanyl β-1-galactoside/60% phytantriol; (C) 30% oleyl R-1-galactoside/70% phytantriol; (D) 40% oleyl R-1-galactoside/60% oleyl glycerate; (E) plots used to derive lattice parameters from SAXS data for cubic phase mixtures; h, j, and k refer to the Miller indices for Pn3m symmetry assigned to each peak in the scattering data.
The ricin binding exhibited by surfactant mixtures possessing inverse hexagonal mesophase morphology was examined using the enzyme-linked immunosorbent binding assay (ELISA) described in the methods (Figure 4). This assay measures the residual ricin in solution following exposure of known ricin concentration solutions to small samples of mesophase formulations. A control measurement for a pure phytantriol mesophase sample shows that the galactoside-free mesophase has little or no affinity for the ricin in solution, since final [ricin] approximates initial [ricin] over the initial ricin concentration range studied. A similar poor affinity for ricin is demonstrated for mesophase samples containing galactose-6-phytanoate. Measurable reduction in [ricin] is however observed for mesophases containing galactosides possessing alkyl groups attached at the C1 carbon of the sugar (phytanyl β-1-galactoside and oleyl R-1galactoside). Referring to Table 2, we find that the maximum proportion of ricin removed from solution for these inverse hexagonal materials is 39.6%, (for the 20% oleyl R-1-galactoside/oleyl glycerate formulation). The linear nature of the initial versus final ricin concentration relationship indicates that an equilibrium between bound and unbound ricin exists in these systems. The lack of an observable plateau in the binding relationship also suggests that these systems are not saturated at the ricin concentrations that we have investigated. The equilibrium nature of the ricin binding to the mesophase systems
Figure 4. Ricin binding assay data for inverse hexagonal phase structures (with phytantriol control): (b) phytantriol (control); (O) 40% galactose-6-phytanoate/phytanyl glycerate; (0) 50% phytanyl β-1galactoside/phytanyl glycerate; (9) 20% oleyl R-1-galactoside/oleyl glycerate.
permits calculation of the affinity constant for ricin binding, as described in the methods section (Table 2).
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Ricin Antitoxins Table 2. Efficiency of Removal of Ricin from Solution by Mesophase Formulations with Calculated Affinity and Dissociation Constants composition
% ricin removed
Ka (M-1)
phytantriol (control) 8.6 8.3 Inverse Hexagonal Mesophase Mixtures 40% galactose-6-phytanoate/ 12.2 43.7 phytanyl glycerate 50% phytanyl β-1-galactoside/ 22.4 70.6 phytanyl glycerate 20% oleyl R-1-galactoside/ 39.6 374.5 oleyl glycerate Inverse Cubic Mesophase Mixtures 40% galactose-6-oleate/ 40.5 200.7 oleyl glycerate 40% oleyl R-1-galactoside/ 80.3 1164.2 60% oleyl glycerate 40% oleyl β-1-galactoside/ 85.1 1631.2 oleyl glycerate 40% phytanyl β-1-galactoside/ 90.3 2844.6 60% phytantriol 30% oleyl R-1-galactoside/ 91.3 3996.2 phytantriol
Kd (mM) 120 23 14 2.7
5.0 0.86 0.610 0.35 0.25
The inverse cubic mesophase formulations showed considerably different ricin binding behavior (Figure 5). When incorporated within a cubic mesophase, galactose-6-oleate is able to bind a significant quantity of ricin from solution (40.5%), which is in contrast to the analogous galactose-6-phytanoate, when contained within an inverse hexagonal mesophase (12.2%, Figure 4). However, it is again the C1-substituted galactose surfactants that show the most dramatically increased potency for binding ricin when contained within inverse cubic mesophase structures (Figure 5 and Table 2). These materials are capable of binding 80-91.3% of ricin in solution over the concentration range studied. The optimal ricin binding activity (91.3%) was observed for a 30% oleyl R-1-galactoside/phytantriol mixture. The most unequivocal observation from this data is the superior ability of cubic phase materials to absorb ricin. A direct comparison between different mesophase forms possessing identical ligands may be made by comparing the 40-50% phytanyl β-1-galactoside containing inverse hexagonal and inverse cubic mesophases. Here, despite decreasing the galactoside concentration by 10% in moving from inverse hexagonal to inverse cubic morphologies, a 40-fold decrease in ricin dissociation constant (Kd) is observed (14 mM for 50:50 phytanyl β-1-galactoside/phytanyl glycerate versus 0.35 mM for 40:60 phytanyl β-1-galactoside/phytantriol). A similar comparison can be made between oleyl R-1-galactoside/phytantriol formulations. Twenty percent oleyl R-1-galactoside (inverse hexagonal phase) yields Kd ) 2.7 mM, while 30% oleyl R-1galactoside (cubic phase) gives Kd ) 0.25 mM.
DISCUSSION The high affinity for ricin of the cubic morphology galactoside mixtures may be explained through analysis of the SAXS data. The typical lattice parameters for the cubic mesophases are on the order of 9.1-12.1 nm (Figure 3) versus 6 nm for the inverse hexagonal mesophase of 50% phytanyl glycerate/50% phytanyl β-1-galactoside. A significantly increased pore diameter within the cubic phase for the phytantriol-based systems is therefore observed. We propose that this allows greater diffusion of the ricin protein (whose longest dimension is on the order of 12 nm according to crystallographic data (21)) within the mesophase aqueous channels and hence greater opportunity to bind to the ligands immobilized therein. This further suggests that the mesophases incorporate a molecular size exclusion capabil-
Figure 5. Ricin binding asay data: inverse cubic structures (with phytantriol control): (A) b, phytantriol (control); 0, 40% galactose6-oleate/oleyl glycerate (cubic phase 24-100 °C); 9, 40% phytanyl β-1-galactoside/60% phytantriol (cubic phase 24-100 °C); (B) b, phytantriol (control); ], 40% oleyl R-1-galactoside/60% oleyl glycerate; [, 40% oleyl β-1-galactoside/oleyl glycerate; O, 30% oleyl R-1galactoside/phytantriol.
ity, which may have implications for other delivery and uptake applications (17). Further work is currently underway in our laboratory to probe this phenomenon. In the current work, the role of the anomeric form of the galactose ligand on ricin binding was also probed. The data indicates that the anomeric conformation of the galactoside has little influence on mesophase ricin binding, since little difference in ricin binding was observed between oleyl R-1 and oleyl β-1 surfactant containing mesophases. This observation contrasts with earlier evaluation of methyl-substituted galactose inhibitors of ricin, which indicated that the β anomer of the methylsubstituted sugar exhibited significantly greater binding to ricin (27). Comparing binding data between the C6 and C1 alkylated galactose surfactants reveals that alkylation at the C1 position is optimal for ricin binding. This is not surprising, given that the major protein-sugar interactions for ricin appear to occur through the C3, C4, and C6 OH groups (21). An indicative lower limit to the ricin binding capacity of the mesophases investigated here is obtained by assuming 100% binding of a 130 µL aliquot of 100 ng/mL ricin by approximately 1 mg of mesophase. This yields a capacity of ∼13 µg of ricin per gram of mesophase. It is important to reiterate
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that this represents a lowest limit to binding capacity. Work is currently underway to define the upper limits of mesophase binding capacity for ricin; however, it is interesting to note that the lethal dose (LD50) of ricin for humans is on the order of 0.1-1 µg/kg (28). We note that the kinetics of ricin binding have not been explored in the current study, since the measurements were performed after a 24 h incubation period. This is clearly a key consideration for the development of either an effective antitoxin or sequestration material. Preliminary measurements suggest that equilibrium ricin binding is attained within 5-10 h of incubation with ricin solutions. The kinetics of binding will be critically dependent on the diffusion of ricin within the mesophase, which in turn will depend on the surface area of the mesophase in contact with ricin binding solution. Given that these materials can be readily prepared in microparticulate dispersion form (10), we would anticipate that dramatic improvements in binding kinetics should be achievable. Further work is underway to prepare these materials in dispersion form and elucidate the ricin binding kinetics in such systems. In summary, we have prepared a number of dilutable (i.e., insoluble) mesophase materials, incorporating the biological ligand galactose, which is known to specifically bind the B-chain of the protein toxin ricin. The aim of this work was to demonstrate that lyotropic mesophases can be effective in the selective separation of biological solutes, by virtue of their internally mobile structure and high interfacial area with respect to water. The self-assembly behavior exhibited by these systems has been characterized by polarizing microscopy and smallangle X-ray scattering. Specific ricin uptake characteristics have been measured by means of a modified ELISA assay. Mesophase materials composed of C1 alkylated galactose surfactants that formed cubic phase structures were found to be highly effective at the specific extraction of ricin from solution. This efficacy was not found for inverse hexagonal mesophase structures. This is explained on the basis of the structural parameters of the different mesophase forms. We postulate that the more open cubic mesophase structure permits in-diffusion and uptake of the toxin more readily. Ricin assay data shows that in all cases, binding occurs Via an equilibrium that can be manipulated through changes in mesophase structural form or ligand chemistry. In these studies, 0.49 mg samples of mesophase materials were capable of removing in excess of 90% of ricin from a 100 ng/mL ricin solution, with a corresponding dissociation constant of 0.25 mM, which compares favorably with literature values for dissociation constants measured for the active component of the natural cell surface receptor, galactose (0.15-0.6 mM) (23). While this binding efficiency is at the lower level of that required to compete with cell surface receptors for ricin binding in ViVo, these data show that synthetic mesophase materials represent promising systems for ricin sequestration. Optimization of these systems by, for example, improving accessibility for ricin binding by incorporating spacer groups between surfactant alkyl chains and galactose moieties can readily be envisaged. In forthcoming publications, we demonstrate the incorporation of biological lipid ligands into mesophase materials and their increased effectiveness in the extraction of ricin from solution. Work is now underway to determine the efficacy of these materials in cytotoxicity inhibition assays and to form dispersions from them. The ease with which mesophase materials can be prepared further offers an economic route to their development. The fact that self-assembled mesophase forms mimic to some extent the surface properties of the plasma membrane of the cell offers the possibility of exploring many cell surface recognition processes using this approach.
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ACKNOWLEDGMENT We thank Dr. Jamie Booth and Prof. Suresh Bhargava of RMIT, Melbourne, for assistance with SAXS measurements. We also thank Dr. Peter Gray of DSTO for valuable discussions. Supporting Information Available: Details of galactose surfactant synthesis and characterization. This material is available free of charge Via the Internet at http://pubs.acs.org/BC.
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