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Preparation and biological evaluation of self-assembled cubic phases for thepolyvalent inhibition of cholera toxin

Scott J. Fraser,ab Rachel Rose,b Meghan K. Hattarki,b Patrick G. Hartley,b Olan Dolezal,b

Raymond M. Dawson,c Frances Separovica and Anastasios Polyzos*bd

Received 11th March 2011, Accepted 15th April 2011

DOI: 10.1039/c1sm05428g

The inverse cubic phase derived from the self-assembly of surfactants in water offers a unique three-

dimensional platform for protein binding. Colloidally stable, sub-micron dispersions of the inverse

bicontinuous cubic phase (cubosomes) impart an unusually large interfacial area for presentation of

small molecules to selectively bind proteins of interest. Cubosomes of the phytantriol/water systemwere

prepared and the receptor for cholera toxin (CT), monosialoganglioside GM1 (GM1), was integrated

within the cubic phase. Our results show that GM1-functionalised cubosomes display a strong

inhibitory response against CT with a high specificity for the toxin. Surface plasmon resonance (SPR)

studies demonstrate that CT and cholera toxin B subunit (CTB) both specifically bind and form a very

stable complex with GM1–phytantriol cubosomes, demonstrated by an absence of binding to control

proteins (mouse IgG, lysozyme and ricin). The inhibitory activity of the GM1–phytantriol cubosomes

against CT was evaluated by a modified enzyme linked immunosorbent assay (ELISA). Using this

method we have determined a nanomolar inhibitory activity (e.g., IC50 ¼ 2.31 nM against 10 ng ml�1

CT) for these particles and a dissociation constant of the GM1–CT complex (KD) of 1.75 nM,

highlighting the remarkable inhibitory activity of the self-assembled cubic phase systems.

1. Introduction

Polyvalent interactions are ubiquitous biological processes that

involve the recognition of a target protein by multiple receptors

on a cell surface.1 Many cell–pathogen interactions are charac-

terized by a polyvalent binding between a protein and multiple

carbohydrate moieties.2 A very well characterized polyvalent

interaction involves the cell-surface recognition of cholera toxin

(CT) by ganglioside GM1, a pentasaccharide-containing glyco-

lipid found on the host epithelial cell surface.3 CT is a hetero-

hexameric protein that is secreted by the Gram-negative

bacterium Vibrio cholerae,4,5 which is responsible for the diar-

rheal disease cholera. The toxin belongs to the AB5 class of toxins

and consists of a catalytic A subunit that is surrounded by an

axisymmetric arrangement of five identical B subunits (Fig. 1).6

An attractive approach to the generation of cholera therapeutics

involves the development of receptor-binding antagonists that

interfere with B-subunit recognition of GM1. Polyvalent antag-

onists that mimic the CT–GM1 interaction have proven

aSchool of Chemistry, Bio21 Institute, The University of Melbourne,Melbourne, VIC, 3010, AustraliabCSIRO Material Science and Engineering, Bayview Avenue, ClaytonSouth, VIC, 3169, Australia. E-mail: tash.polyzos@csiro.aucDSTO Melbourne, Defence Science and Technology Organisation, POBox 4331, Melbourne, VIC, 3001, AustraliadDepartment of Chemistry, University of Cambridge, Lensfield Road,Cambridge, CB2 1EW, UK

This journal is ª The Royal Society of Chemistry 2011

a successful strategy toward the establishment of anti-infective

drugs for cholera. Several groups have made advances in devel-

opment of polyvalent CT inhibitors, which include galactose-

terminated macromolecules,7 glycopolymers,8 glycodendrons,9

and glycodendrimers.10 The conformational rigidity and surface

topology of these classes of inhibitors have often led to moderate

affinities, and the synthetic complexity associated with such

approaches is often significant. An alternative approach is to

employ self-assembled scaffolds in which mobile galactose

moieties are available to ‘pattern match’ with CT binding sites.

Amphiphile based systems, such as liposomes, have recently been

reported in such applications.11–13 However, polyvalent scaffolds

Fig. 1 The structure of cholera toxin (left) and the pentameric

arrangement of the B-subunit (right).6

Soft Matter, 2011, 7, 6125–6134 | 6125

Fig. 3 The structure of phytantriol.

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based on extended 3-dimensional self-assembled architectures

remain largely unexplored.

The inverse bicontinuous cubic phase (QII) is a 3-dimensional

structure of cubic symmetry that is formed by the self-assembly

of particular amphiphiles. The QII phase represents a family of

closely related structures, where the underlying crystal lattice can

be described by the gyroid (G), diamond (D) and primitive (P)

minimal surfaces, which correspond to the Ia3d (G), Pn3m (D)

and Im3m (P) crystallographic space groups, respectively.14–16

The 3-dimensional structure affords a self-assembled scaffold

with a remarkably high surface area and extensive porosity.17

These properties, coupled with the liquid crystalline nature of the

phase, result in a nanoporous material that is not susceptible to

osmotic or mechanical rupture, in contrast to the properties of

liposomes or micelles.18 The thermodynamic stability of the QII

phase affords a structure that co-exists in equilibrium with excess

water over a broad temperature range.19,20 We recently demon-

strated that the Pn3m (D) cubic phase of the binary surfactant

mixture of ‘‘phytantriol’’ (3,7,11,15-tetramethyl-1,2,3-hex-

adecanetriol) and GM1 binds AB-type toxins including CT21,22

(Fig. 2). The material, however, has limited clinical application in

this context owing to a high viscosity and pronounced rigidity.

The high viscosity of these liquid crystalline materials can be

ameliorated by dispersion in water. The dispersion of the bulk

cubic phases can be achieved via a variety of methods including

mechanical or ultrasonic treatment and usually requires the

addition of a dispersant to provide colloidal stability. This results

in the formation of a stable dispersion of sub-micron sized

particles that retain the internal structure of the parent bulk

cubic phase.23 Dispersions of these cubic phase particles (cubo-

somes) have a viscosity approximately equal to water, which is

desirable for enteric administration.24 Furthermore, the large

Fig. 2 A representation of the Pn3m (diamond) cubic crystallographic structu

curved blue/red bilayer structure represents the bilayer composed of phytan

separate, non-intersecting, regions of continuous water channels within the c

6126 | Soft Matter, 2011, 7, 6125–6134

surface area may lead to favourable mass transport of the toxin

within the internal cubic phase structure and improved binding

kinetics compared to the bulk cubic phase material.

In this paper, we report the preparation of cubosomes

comprised of binary mixtures of GM1 and phytantriol. To

demonstrate the effectiveness of cubic phases as scaffolds for the

polyvalent presentation of small molecules, for this study we

chose GM1 as the polysaccharide to bind CT, although any

galactose-containing lipid could be used, and phytantriol as the

amphiphile to form the inverse cubic phase scaffold. Phytantriol

(Fig. 3) is a non-ionic surfactant, which readily forms cubic

phases at ambient temperatures (20–40 �C) when the water

content exceeds 15%.25 The surfactant is comprised of an iso-

prenoid hydrophobic tail, which confers a large volume relative

to the headgroup, a structural requirement for inverse cubic

phase formation, according to the critical packing param-

eter,18,26,27 CPP ¼ v/(lca0), where lc is the effective length of an

amphiphilic chain, a0 is the effective amphiphilic headgroup

area, and v is the average volume occupied by the amphiphilic

hydrophobic chain. Furthermore, the attachment of the triol

head-group to the hydrophobic tail is through a C–C bond rather

than an ester linkage as found in related lipid systems, which

confers a greater chemical stability.

We examine the binding of CT by these materials using surface

plasmon resonance (SPR) and enzyme linked immunosorbent

assay (ELISA). SPR is a sensitive, non-labeled technique that

re formed by minimal surfaces in bicontinuous amphiphilic systems. The

triol (left) and GM1 (right). The blue and red surfaces represent the two

ubic structure.

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measures the adsorption of biomolecules to a gold or silver

surface.28 The binding interaction of ligand and receptor mole-

cules can be monitored in real time and the binding kinetics

readily measured. The utilization of SPR for the measurement of

the binding interaction between CT and surface immobilised

cubic phases is further enhanced by previous work that has

quantified the binding of CT to surface immobilized GM1.29,30 We

also extend previous work31 using ELISA to examine the inter-

action of cubic phases with proteins and we anticipate that in

addition to SPR, this will provide reliable, quantitative infor-

mation about the inhibitory activity of cubosomes against CT.

2. Experimental section

2.1 Materials

Monosialoganglioside (GM1) (95%, Sigma Aldrich, St Louis,

MO), bovine serum albumin (BSA) (>96%, Sigma Aldrich, St

Louis, MO), 3,7,11,15-tetramethyl-1,2,3-hexadecane-triol (phy-

tantriol) (96%, DSM Nutritional Products, Parsippany, NJ),

Pluronic F127 (Aldrich, St Louis, MO), dichloromethane (HPLC

grade) and methanol (HPLC grade) were used without further

purification.Milli-Qwaterwas usedwhere indicated (R>18.2MU

cm�1). Cholera toxin (CT) fromVibrio cholerae (Inaba 569B, azide

free) and cholera toxin B subunit (CTB) (choleragenoid, sodium

salt) were obtained from List Biological Laboratories, Campbell,

CA. Purified polyclonal goat anti-cholera toxin was a product of

Biogenesis, Poole, UK. Rabbit anti-goat alkaline phosphatase

conjugate and p-nitrophenyl phosphate tablets were purchased

from Sigma-Aldrich, St Louis, MO. HBS-E (10 mM HEPES,

150 mM NaCl, 3 mM EDTA, pH 7.4) instrument running buffer

was freshly prepared as required and de-gassed before use.

2.2 Cubosome preparation and characterization

The required quantity of GM1 was suspended in phytantriol (1 g)

and dissolved in a solution of 10 ml of 20% methanol/dichloro-

methane. The mixture was thoroughly dissolved until an opti-

cally transparent solution resulted. The solvent was then

removed using rotary evaporation to afford a clear viscous oil.

Cubosomes were prepared by adaptation of the method of

Gustafsson and co-workers.32 The surfactant/lipid mixture (1 g)

was placed in a small glass beaker and Pluronic F127 (0.07 g) was

added. The mixture was heated in an 80 �C oven for 3 min. The

heated mixture was then slowly transferred into heated water

(80 �C) (29 ml) under continuous shear in a high shear mixture

(Polytron PT2100, Kinematica, Inc., Bohemia, NY). The

mixture was then cooled to room temperature whilst still under

shear for 5 minutes. The resulting dispersion was then processed

by 7 passes through an Avestin C5 high-pressure homogenizer

(Avestin Inc., Ottawa, Canada) at �25 000 psi pressure/65 �Cyielding a dispersion of cubic phase particles of typical particle

size 100–150 nm. The dispersions were added to water re-circu-

lated within the instrument until a final concentration of

approximately 40 mg ml�1 was achieved.

2.3 Particle size measurements

Particle size distributions were measured using a Coulter LS-230

particle sizer (Beckman-Coulter, Inc., Miami, FL). Data were

This journal is ª The Royal Society of Chemistry 2011

collected over 240 s. For the particle size calculations, a standard

model was employed based on homogeneous oil spheres with

a sample density of 1.0 g ml�1.

2.4 Cryo-transmission electron microscopy (cryo-TEM)

Samples were prepared for imaging by cryo-TEM according to

the method of Adrian et al.33 A laboratory-built vitrification

system was used, allowing the humidity to be maintained at

approximately 90% during sample plunging and vitrification. A

4–5 ml aliquot of sample was applied to a 200 mesh copper TEM

grid coated with lacy carbon film (ProSciTech, Thuringowa,

Australia) and allowed to settle for 30 s. The grid was manually

blotted for 10–15 s, and the resulting thin film then vitrified by

plunging into liquid ethane. Grids were stored in liquid nitrogen

before transferring into a Gatan 626-DH Cryo-holder (Gatan

Inc., Pleasanton, CA). Imaging was carried out using a FEI

Tecnai 12 TEM (FEI Co., Hillsboro, OR), operating at 120 kV,

with the sample at a temperature of �180 �C. Images were

recorded using a MegaView III CCD camera equipped with

AnalySis imaging software (Olympus Soft Imaging Solutions

GmbH, M€unster, Germany), using standard low-dose proce-

dures to minimize radiation damage.

2.5 Synchrotron small angle X-ray scattering (SAXS)

measurements

The SAXS beamline at the Australian Synchrotron (Melbourne,

Australia) was used to perform SAXS experiments on cubosome

samples. Briefly, the synchrotron X-ray beam was tuned to

a wavelength of 0.8266 �A with a typical flux of 1013 photon per s.

The 2-D diffraction patterns were recorded on a Bruker 6000

CCD detector (Bruker Optik, Ettlingen, Germany) with the

detector offset to access a greater q-range, allowing for detection

of small and wide-angle X-ray scattering. Silver behenate

(a ¼ 58.38 �A) was used as the low-angle X-ray diffraction cali-

brant for all measurements. Samples were loaded into 1.5 mm

glass capillaries and analyzed from 20–40 �C at 2.5 �C incre-

ments, with an equilibration time of 15 min for each temperature.

A custom designed Peltier driven cell was used to control the

temperature (�0.1 �C). Diffraction images were analyzed using

the IDL-based AXcess software package, developed at Imperial

College, London, UK.34 The measured X-ray spacings are

accurate to within �0.1 �A.

2.6 CT binding by surface plasmon resonance (SPR)

All SPR binding experiments were performed at 25 �C using an

L1 sensor chip35 docked in a Biacore T100 instrument (GE

Healthcare, Uppsala, Sweden)36 with 1� HEPES buffered saline

containing EDTA (1� HBS-E; 10 mM HEPES, 150 mM NaCl,

3 mM EDTA, pH 7.4) as the instrument running buffer. After

docking, the L1 chip was cleaned by twice injecting 20 mM

CHAPS for 30 s at a flow rate of 100 ml min�1. This was followed

by an instrument priming routine by running through with

1� HBS-E buffer for 60 min. All cubosomes used for SPR

experiments (prepared as described above) were diluted 1/100

into 1� HBS-E buffer and filtered through a 0.22 mm syringe

filter prior to injection.

Soft Matter, 2011, 7, 6125–6134 | 6127

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Cholera toxin (CT) binding to GM1–phytantriol cubosomes. The

GM1–phytantriol cubosomes were assayed for binding using

a capture assay approach whereby each binding cycle consisted

of three consecutive steps: (1) capture of cubosomes onto an

L1 chip surface, (2) binding of CT to captured GM1–phytan-

triol cubosomes, and (3) regeneration of the L1 chip surface.

Accordingly, cubosome preparations were injected for 120 s in

flow cell 2 of an L1 chip at a flow-rate of 5 ml min�1, typically

resulting in a capture of �2500 RU of cubosomes (SPR

Response Units; 1 pg protein mm�2 z 1 RU37). Cholera toxin,

cholera toxin B subunit (CTB) and appropriate negative

control proteins (mouse IgG, lysozyme and ricin) were diluted

in HBS-E and injected sequentially across flow cells 1 and 2

for 60 s at 5 ml min�1. The CT/GM1–phytantriol cubosome

complex was allowed to dissociate for 1 min. Captured GM1–

phytantriol cubosomes were completely removed from the L1

chip surface by a 15 s injection of 20 mM CHAPS at 30 ml

min�1. In order to ascertain the kinetics of binding, in

a separate experiment, serial dilutions (two-fold) of CT and

CTB in HBS-E buffer were injected over captured GM1–phy-

tantriol cubosomes and the association and dissociation phases

were monitored for 60 s and 600 s respectively. A control

measurement of the instrument running buffer (‘‘zero-buffer’’

blank) solution was also included for double referencing

purposes.38

SPR data processing and analysis. All SPR sensorgrams were

processed using Scrubber software (version 2c; Biologic Soft-

ware, Canberra, Australia; www.biologic.com.au). Sensorgrams

were first zeroed on the y-axis and then x-aligned at the initial

injection. Bulk refractive index changes were removed by sub-

tracting the reference flow cell (Fc 1) responses. The average

response of all blank injections was subtracted from all analyte

injections and blank sensorgrams to remove systematic artifacts

in the experimental and reference flow cells. Scrubber analysis

software was used to determine association/dissociation rate

parameters (ka and kd) from the processed datasets by globally

fitting to a 1 : 1 bimolecular binding model. This model assumes

simple pseudo first order bimolecular association, A + B $ AB

with a supposition that no interaction occurs between separate

receptor molecules. Consequently, the rate parameters (ka and

kd) of this interaction were derived in accordance with SPR

kinetic theory previously adopted for cholera toxin interacting

GM1.30 Thus, the dissociation rate constant (kd) was derived from

the equation:

Rt ¼ Rt0e�kd(t�t0) (1)

where Rt is the response at a time t and Rt0is the response at

a time t ¼ 0. The association rate constant (ka) was derived from

the equation:

Rt ¼ kaCRmaxð1� e�kaCþkdtÞkaC þ kd

(2)

where Rmax is the maximum binding response (proportional

to the amount of immobilized ligand) and C is the

concentration of injected analyte. The equilibrium dissociation

constant (KD) for the binding reaction was calculated from the

quotient kd/ka.

6128 | Soft Matter, 2011, 7, 6125–6134

2.7 CT binding by modified enzyme-linked immunosorbent

assay (ELISA)

The binding of CT to the receptor (GM1) was determined as

described previously.31 In brief, a solution of GM1 in phos-

phate-buffered saline (PBS) was used to coat wells of a Nunc-

Immuno Maxisorp 96-well plate (Invitro Technologies, Mel-

bourne, Australia) (100 ml per well) overnight at room

temperature. After this and subsequent incubations, the plate

was washed 3 times with PBS. The wells of the plate were

initially incubated for one hour with 360 ml blocking buffer

(0.25% bovine serum albumin in PBS-0.05% Tween 20) to

block non-specific binding sites, and then with 100 ml CT

(1–100 ng ml�1), 100 ml goat anti-CT antibody and 100 ml

rabbit anti-goat alkaline phosphatase conjugate, each in the

blocking buffer. These incubations were at room temperature

for one hour. Finally, the plate received an additional wash

with distilled water, followed by the addition of 100 ml alkaline

phosphatase substrate (p-nitrophenyl phosphate) in Tris buffer.

The rate of change of absorbance at 405 nm was measured over

10 minutes using a Bio-Tek Synergy HT plate reader (Bio-Tek

US, Winooski, VT). Blank wells (without GM1 or GM1 without

CT) gave very low readings (�0.001 abs per min). Inhibition of

the binding of CT to immobilised GM1 was determined by

incubating GM1–phytantriol cubosomes or free GM1 (no phy-

tantriol or Pluronic F127) with CT (total volume 130 ml) for

one hour in disposable glass test tubes with gentle rocking on

a rocking platform mixer (Ratek Instruments, Melbourne,

Australia) during the incubation of the 96-well plate with

360 ml blocking buffer. A 100 ml aliquot from each test tube

was then added to wells of the washed plate, after which the

assay proceeded as normal. Both GM1–phytantriol cubosomes

concentration and CT concentration were varied, as was the

initial molar concentration of GM1 in GM1–phytantriol cubo-

somes. The inhibitory potency of free GM1 was determined

against 10 ng ml�1 cholera toxin only.

Data analysis was achieved by constructing calibration curves

for the assay absorbance per minute (abs per min) versus the

known CT concentrations ([CT]). Data were fit using the Hanes–

Woolf linearized version of the Langmuir equation, which has

been shown to be the most reliable of the linear methods.39 This

equation is:

[CT]/(abs per min) ¼ 1/k1k2 + [CT]/k2 (3)

where k1 and k2 are rate constants. A plot of [CT]/(abs per min)

vs. [CT] should therefore be linear, and this was found to be the

case (see Fig. 4 for results of a typical experiment). The amount

of CT that was not bound to GM1–phytantriol cubosomes (‘‘free

CT’’) was determined by reference to net rates of change of

absorbance in control wells (CT only). A 1 : 1 stoichiometry for

the binding of CT to GM1 cubosomes was assumed, despite the

possibility of polyvalent binding by the AB5-type toxin, to enable

an estimate of the potency of the cubosomes as inhibitors of CT–

receptor binding to be determined. Accordingly:

CT + G $ CT$G (4)

where CT ¼ cholera toxin, G ¼ GM1 cubosome and CT$G is the

toxin–receptor complex. Therefore,

This journal is ª The Royal Society of Chemistry 2011

Fig. 4 Calibration plot for cholera toxin control wells of ELISA assay

(no inhibitor of binding to immobilised GM1 present). Rate ¼ rate of

hydrolysis of p-nitrophenol ¼ rate of change of absorbance at 405 nm

(abs per min), and [CT] ¼ concentration of cholera toxin (ng ml�1).

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KD ¼ [CT]$[G]/[CT$G] (5)

where KD is the dissociation constant of the toxin–receptor

complex. The concentration of GM1 cubosome for which free CT

is reduced by 50% in the assay is designated as IC50. Since 50% of

the initial CT (defined as [CT]init) is free and 50% is bound to GM1

cubosome at the IC50, the ratio [CT]/[CT$G] ¼ 1 by definition

under these conditions. The value of [G] at the IC50 is the initial [G]

minus the amount ofG (GM1 cubosome) in the bound form,which

is the same as the amount of bound CT ¼ [CT]init/2. Therefore,

KD ¼ IC50 � [CT]init/2 (6)

3. Results and discussion

3.1 Preparation and characterization of GM1–phytantriol

cubosomes

The ganglioside-containing cubosomes were prepared by high-

pressure homogenization as described in Section 2.2. The

dispersed materials were stable for many months without visible

coalescence occurring during this period. Fig. 5 shows a Cryo-

genic Transmission Electron Micrograph (Cryo-TEM) image of

the cubosomes. The internal cubic periodicity of the cubosomes

is clearly observable within the particles. The average particle

diameter was 150 nm as determined by dynamic light scattering,

supporting the observed particle sizes in the cryo-TEM images.

The Synchrotron-SAXS diffraction patterns for the cubosome

samples in water and phosphate buffered saline (PBS), are shown

in Fig. 5 (25 �C).The Bragg reflections gave reciprocal spacings in the ratio 1 :

O2,O3,O4,O6,O8, indicating that the particles comprise a cubic

phase of Pn3m (Q224) space group in excess water at room

temperature (Table 1).40 The allowed Bragg reflections for

a Pn3m space group correspond to the Miller indices, hkl ¼ 110,

111, 200, 211, 221. From the scattering vector positions (qhkl) of

the Bragg reflection maxima, the lattice spacing (a) of the cubic

phase can be determined using relationship (7):

qhkl ¼ (2p(h2 + k2 + l2)1/2)/a (7)

This journal is ª The Royal Society of Chemistry 2011

The lattice parameter for a Pn3m (Q224) space group for the

0.5% (w/w) GM1-containing cubosomes (Fig. 5(ii)) was found to

be 71.3 �A. Pure phytantriol cubosomes (Fig. 5(i)) also showed

a Pn3m (Q224) space group with a lattice parameter of 70.2 �A in

the presence of excess water and at 25 �C, which is consistent with

other studies of this system,25,41 suggesting that the 0.5% w/w

GM1 does not significantly influence the Q224 cubic phase of

phytantriol. X-Ray scattering in a higher electrolyte concentra-

tion (PBS) showed a slight increase in lattice parameter to

73.05 �A (Fig. 5(iii)).

3.2 Binding studies

CT binding by surface plasmon resonance. Surface Plasmon

Resonance (SPR) was used to measure the binding between

immobilized cubosomes and CT or the pentameric subunit, CTB.

Binding experiments were conducted using a Biacore L1 chip

comprising a standard carboxymethyl dextran surface that has

been modified with lipophilic anchor molecules.35 Shown in

Fig. 6a is a typical sensorgram observed during the (1) capture of

GM1–phytantriol (containing 0.5% w/w of GM1) cubosomes on

the sensor chip from a 0.10 mg ml�1 cubosomes solution, fol-

lowed by washing with 1� HBS-E and (2) injection of serially

diluted CTB. The injection of the GM1–phytantriol cubosomes

was accompanied by a large response of approximately 3000 RU

corresponding to an efficient capture of the cubosomes on the L1

chip surface. The shallow curve observed after washing is

indicative of a slow dissociation of GM1–phytantriol cubosomes

but the small change in RU is consistent with minimal loss of

mass. The injection of CTB across the immobilized GM1–phy-

tantriol cubosomes resulted in a response of approximately 150

RU, followed by a concomitant slow dissociation step. Regen-

eration of the chip surface (removal of the cubosomes) was

readily achieved after washing with CHAPS buffer, whereupon

the SPR response signal returned to the starting baseline. A

closer inspection of the CTB cubosome interaction (Fig. 6b)

revealed that all of the sensorgrams exhibited exponential

binding behavior, whereby a plateau in the time-absorbance

curve was observed. The rate of binding to the cubosomes and

overall response, as indicated by the slope of the association

curve, increased with increasing concentration of CTB.

Control experiments were undertaken to examine the speci-

ficity of the GM1–phytantriol cubosomes. Firstly, non-specific

binding was assessed through the capture of cubosomes

comprised of phytantriol and Pluronic F127, followed by expo-

sure to 180 nM CTB. Fig. 6b clearly shows a negligible baseline

increase (#5 RU) following injection of CTB supporting the

absence of significant non-specific binding. Secondly, the binding

of control proteins to the cubosomes was evaluated by flowing

three control proteins, lysozyme, ricin and mouse IgG. As shown

in Fig. 6c, there was no detectable response for all three proteins

at concentrations up to 20 nM, highlighting the retention of GM1

specificity within the cubic phase materials. It should be noted

that ricin is also carbohydrate (galactose) specific, demonstrating

the specificity of the GM1-functionalised cubosomes for CT and

CTB. Moreover, the lysozyme (pI ¼ 10.7) is expected to be

positively charged at the experimental pH and this suggests that

non-specific electrostatic interactions with the negatively charged

Soft Matter, 2011, 7, 6125–6134 | 6129

Fig. 5 Cryogenic transmission electron micrograph (cryo-TEM) and synchrotron small angle X-ray diffractograms at 25 �C of: (i) phytantriol

cubosomes in water, (ii) GM1–phytantriol cubosomes (0.5% w/w) in water, and (iii) GM1–phytantriol cubosomes (0.5% w/w) in PBS buffer.

Table 1 Synchrotron SAXS data for different preparations of cubo-somes measured at 25 �C

Cubosome preparation SolventLatticespacing, a (�A) Space group

Phytantriol mqH2O 70.2 � 0.6 Pn3m (Q224)PBS 71.1 � 0.6 Pn3m (Q224)

Phytantriol/GM1(0.5% w/w) mqH2O 71.3 � 0.2 Pn3m (Q224)PBS 73.1 � 0.7 Pn3m (Q224)

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GM1 head-group are not a major determinant factor of protein

binding in this system.

Next we attempted to utilize an SPR assay to estimate the

affinity (KD) for the interaction of CT and CTB with the cubo-

somes. Triplicate binding data of serially diluted CTB and CT

interacting with the GM1–phytantriol cubosomes are shown in

Fig. 6d and e, respectively. Binding data were globally fit to the

simple 1 : 1 binding model described in Section 2.6. A 1 : 1 model

was used to simplify the data fitting, although CT (and CTB) is

a pentameric protein and, therefore, may bind up to five GM1

molecules.42 Similar studies35 have successfully used a global 1 : 1

binding model for analysis of CT interaction with GM1

embedded within liposomes; however, this model did not fit our

data well (orange lines in Fig. 6d and e; c2 > 100 RU2). Derived

kinetic binding values for CT binding to GM1–phytantriol

cubosomes were ka ¼ 2.75� 1.0� 105 M�1 s�1, kd ¼ 4.5� 0.90�10�5 s�1 giving a KD of 0.18 � 0.07 nM. However, c2 of statistical

parameter of 189 RU2 indicated a significant deviation of fitted

model from experimental data. Further inspection of these SPR

sensorgrams showed two unusual SPR binding properties; firstly,

binding results did not conform to the SPR supposition that

larger analytes (by mass) invoke a larger SPR response. In our

case, the binding responses for CT (Mr ¼ 87 kDa) and CTB

(Mr ¼ 58 kDa), when injected at the same concentration, were

quite similar, despite a similar quantity of GM1-cubosomes

available at the chip surface for each run (compare Fig. 6d and e).

Secondly, the binding of serially diluted concentrations of CT

and CTB did not conform to classic SPR sensorgrams. Typically,

at a higher concentration, Rmax (saturation of immobilized

ligand/cubosomes) is attained and then, at lower analyte (CT)

6130 | Soft Matter, 2011, 7, 6125–6134

concentrations, a gradual and uniform decrease from saturation

is normally observed. Instead, at concentrations of less than

40 nM binding responses were lower than expected, eventually

leading to a complete loss of signal at concentrations that would

otherwise be expected to show at least minimal binding. Inter-

estingly, binding signal for CTB injections could be detected at

$10 nM and for CT at$20 nM. This observation was, yet again,

contrary to the SPR detection principles, which suggests that the

larger CT molecule should be detectable at lower concentrations.

It is clear that GM1-cubosomes present challenges for

measuring binding kinetics. Similar difficulties have also been

reported with structures even less complex such as liposomes43

and model membranes.28,44 In this case, consideration must also

be given to the overall particle size of the GM1–phytantriol

cubosomes bound to the SPR sensor surface and the diffusion

kinetics (mass transport) of the analytes (CT and CTB) within the

cubosome interior. The optimal maximal distance for measuring

binding using the Biacore chips is approximately 150 nm from

the gold surface. Given that the L1 chip comprises a carboxy-

methyl dextran (CMD) film of approximately 100 nm thickness,

then the addition of GM1-functionalized cubosomes (average size

of 100–150 nm) to the CMD layer results in a significant

proportion of the GM1 to be located beyond the optimal Biacore

chip sensing distance (assuming a uniform distribution of GM1

throughout the phytantriol bilayer of the cubosome). Conse-

quently the diffusion of CT and CTB to a distance within the

optimal sensor range is likely to influence the measurements,

particularly at a lower toxin concentration. We further note here

that the mean diameter of lipid vesicles utilized in Cooper et al.35

was 25 nm and may therefore explain why a good fit of binding

data was obtained in those studies. Compromised diffusion

theory is also consistent with elucidating the observed CTB/CT

SPR paradox whereby the binding of the smaller CTB was better

detected than the larger CT molecule. We postulate a greater

diffusion of CTB within the GM1 cubosome owing to its smaller

size.

Interestingly, when the CT binding data for the two highest

concentrations of CT (80 and 40 nM) were fit globally to a simple

1 : 1 binding model, an acceptable fit was obtained (Fig. 6f).

From this fit, derived kinetic values were ka ¼ 5.2 � 2.1 �

This journal is ª The Royal Society of Chemistry 2011

Fig. 6 SPR analysis of cholera toxin (CT/CTB) binding to captured

GM1–phytantriol cubosomes (0.5% w/w of GM1 in cubosomes) on the L1

sensor chip surface. (a) Overlay of two typical binding cycles consisting of

three injection steps: (1) cubosome capture, (2) CT binding, and (3)

regeneration of the L1 chip surface with 20 mM CHAPS. Arrows mark

the beginning and end of each injection respectively. (b) Processed and

double referenced binding data showing interaction between CTB

(injected at 20, 60, and 180 nM) and captured cubosomes comprised of

phytantriol (Phyt) (blue line) or GM1–phytantriol (Phyt-GM1) (black

lines). (c) Processed and double referenced binding data showing specific

interaction between Phyt-GM1 (black lines) and CT and no interaction

with mouse IgG (blue line), lysozyme (red line) and ricin (orange line). (d)

Serial two-fold dilutions (80, 40, 20, 10, 5 and 0 nM) of CTB (d) and CT

(e) binding to captured Phyt-GM1 cubosomes (black lines). (f) The same

data as in (e) showing data and fit only for 80 nM and 40 nM binding

curves. Triplicate binding data are shown. Data were globally fit to the

1 : 1 binding algorithm described in the Experimental section (orange

lines).

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105 M�1 s�1, kd ¼ 5.2 � 2.0 � 10�5 s�1 giving a KD of 0.10 �0.03 nM (c2 ¼ 9.6 RU2). This result further conforms with our

hypothesis of limited analyte (CT) penetration within cubo-

somes. Thus, when lower CT concentrations are injected, insuf-

ficient analyte penetration to the chip surface compromises

affinity estimates, in particular ka, which is concentration

dependent. Irrespective of our data fitting approach our SPR

affinity estimates in the order of 0.1–0.2 nM compared well with

values reported by other groups who utilized similar SPR

methodology and binding model fitting for analysis of CT

interactions with GM1 presented on a liposome surface

(KD values of 0.005 nM and 0.26 nM).30,35 It should, however, be

noted here that in all these studies (including ours) issues asso-

ciated with the polyvalent nature of cholera toxin significantly

influences the overall accuracy of affinity estimates and is likely

to be a significant factor responsible for widely ranging estimates

reported in the literature.45,46 In addition, our experimental

observations demonstrate the complexity associated with

immobilizing large cubosomes particles with an extended 3D

internal structure at the biosensor surface. Consequently cubo-

somes pose a significant challenge for SPR technology.

CT binding by modified ELISA. An enzyme-linked immuno-

sorbent assay (ELISA) was used to assess the inhibitory activity

of the GM1 modified cubosomes against CT. Preparations con-

taining 0.1–2.0% w/w GM1 in 0.5 mg ml�1 phytantriol cubosomes

were used as inhibitors and values of the IC50 (the concentration

of cubosomes required to bind 50% of CT in solution) were

determined.

For each CT concentration, the amount of free CT was plotted

against the final concentration of GM1. In most cases, the rela-

tionship was not linear and the data were fit to a second-order

polynomial equation to enable determination of the IC50 for

GM1. A typical plot is shown in Fig. 7 (10 ng ml�1 CT (0.12 nM)

with a dilution of 0.5 mol% GM1 cubosome). The data points

represent the means of three experiments, with the standard error

of the mean (s.e.m.) varying from 0.4 to 11.2%. The IC50 of

2.31 nM is substantially less than those from the non-soluble

inverse cubic phases where we reported 0.10 mM and 0.27 mM

versus 100 ng ml�1 CT for 10% GM1 (w/w) and 20% GM1 (w/w) in

phytantriol inverse cubic phases respectively.21 Accordingly,

cubosomes display 40–120-fold greater potency than the non-

soluble cubic phases, although the latter were evaluated at

a higher CT concentration. Of particular note is the improve-

ment in IC50, despite a lower loading of GM1 (0.5% (w/w)) in the

cubosome preparations. We reason that such an increase in

potency is due to the larger surface area available for binding

between CT andGM1 in the cubosomes. The IC50 values reported

here are also lower than those of other soluble polyvalent

inhibitors such as pentavalent and decavalent inhibitor systems,

which generally have IC50 values in the micromolar range.10,47–49

The dissociation constantKD was calculated from the IC50 (see

Section 2.7). Both IC50 and (to a lesser extent) KD were found to

vary significantly with the concentration of CT. The KD depen-

dency on [CT] suggests that the interaction of CT with the GM1

cubosomes is not in accordance with the simple model repre-

sented in eqn (4) above (Section 2.7). This may be attributed

primarily to the diffusion of the protein throughout the porous

interior of the cubosome. CT binding of the GM1 polysaccharides

Soft Matter, 2011, 7, 6125–6134 | 6131

Fig. 7 Concentration of free cholera toxin [CT] (toxin that is free to bind

to immobilised GM1) after incubation of the toxin with GM1 cubosomes

(dilution of 0.5 mol% GM1 cubosomes).

Fig. 8 Variation of a function of the dissociation constant, KD, for GM1

in cubosomes vs. the initial CT concentration. [CT] ¼ the initial

concentration of cholera toxin, and KD ¼ the dissociation constant of the

GM1–CT complex.

Fig. 9 Inhibition of binding of 10 ng ml�1 CT to immobilised GM1 by

free GM1 and by dilute 1% GM1 cubosomes on the same 96-well plate.

Free [CT] ¼ the concentration of cholera toxin free to bind to immobi-

lised GM1 after incubation with GM1 alone or with GM1 cubosomes.

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that decorate the external surface of the cubosome presumably

proceeds first and when these binding sites are exhausted,

binding to the remaining GM1 polysaccharides requires the

transport of CT to the internal structure, which may be diffusion

limited by the tortuosity of the aqueous channels. Furthermore

at high concentrations of CT, the possibility of steric crowding in

the aqueous channels by water, GM1, F127, and GM1–CT

complexes, cannot be discounted, thus limiting transport of CT

into the internal cubosome structure and thereby increasing the

KD (decreasing affinity).45

The variation of KD with [CT] was consistent with the Lang-

muir equation (Section 2.7), as illustrated by the linear plot in

Fig. 8 (compare Fig. 4). The intercept of this graph is significantly

different from zero at the 95% confidence level. The reciprocal of

the slope of Fig. 8 is the theoretical maximum value of KD

(KD(max)) at infinitely high [CT] and was found to be 3.37 nM

for diluted 0.5% w/w GM1. Corresponding values for diluted 0.1,

0.2, 1.0 and 2.0% w/w GM1 cubosomes were determined by the

same process to be 1.56, 0.60, 2.76 and 1.89 nM, respectively.

These values were determined using pooled values of [CT] not

bound to GM1 cubosomes from 2–5 experiments. The s.e.m. of

each mean value was #13% in all but 4 cases, and the maximum

s.e.m. was 18.7%. The overall mean of the five values of KD(max)

above (from the five concentrations of GM1 in cubosomes) is

1.75 � 0.23 nM (s.e.m. of 13.0%). The IC50 for GM1 alone

(i.e., not incorporated into cubosomes and in the absence of

phytantriol and Pluronic F127) as an inhibitor of binding

with10 ng ml�1 CT under the same experimental conditions as for

the cubosomes, and in some cases on the same 96-well plate, was

found to be 12.8 � 0.63 nM (n ¼ 4). An example of one such

control experiment (GM1 alone, and dilute 1%GM1 cubosomes as

inhibitors of binding of 10 ng ml�1 CT, on the same plate) is

illustrated in Fig. 9.

We previously reported an IC50 of 80 nM for GM1 alone versus

100 ng ml�1 CT;31 these results are consistent with the depen-

dence of the IC50 on CT concentration (above). The mean IC50 of

the GM1–phytantriol cubosomes versus 10 ng ml�1 CT was found

to be 1.18� 0.17 nM; they are, therefore, approximately 11 times

6132 | Soft Matter, 2011, 7, 6125–6134

more effective as inhibitors of cholera toxin binding than free

GM1 and 60 times as effective as the corresponding bulk GM1–

phytantriol mesophases.21 Furthermore, the cubosomes have an

approximate 10-fold greater inhibitory activity than macromo-

lecular conformationally restricted GM1 analogues50,51 and

2-dimensional self-assembled systems such as supported lipid

bilayers.30,52 However, we note that establishment of the CT/GM1

binding equilibrium may be limited by the diffusion of CT to the

cubosome interior and binding measurements may underesti-

mate KD if equilibrium has not been established. Further work is

continuing to calculate the rate of diffusion of CT within the

GM1–phytantriol cubic phase to determine if this is indeed the

case. The Kd values from the ELISA experiments reported above

are an order of magnitude higher than that calculated from the

SPR data, although the difference is lower when compared with

a literature Kd value of 0.26 nM.35 However, the two methods of

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assaying inhibitory potency are very different, and others have

shown that a direct comparison between SPR and ELISA is

difficult to quantify.53

4. Conclusions

In this paper, we have demonstrated that homogeneous cubic

phase particles (cubosomes) are a promising new class of scaffold

for the polyvalent presentation of an inhibitor against CT.

Structural characterization using SAXS and cryo-TEM revealed

that the Pn3m (Q224) cubic phase of phytantriol tolerates the GM1

glycolipid without disruption of the cubic structure. Surface

plasmon resonance (SPR) analysis has shown that immobilized

GM1–phytantriol cubosomes specifically bind CT and, owing to

avidity effects, form a very stable complex with the toxin. We

have estimated an approximate overall KD value for this system

to be in the subnanomolar range. However the use of SPR to

calculate the overall KD for large, complex self-assembled

systems is limited by the small detection range and ineffective

modeling of a 1 : 5 (GM1 : CT) interaction. An improved esti-

mate of binding data, and therefore inhibitory activity, was

facilitated by our modified enzyme linked immunosorbent assay

(ELISA). This method established a nanomolar IC50 value for

the cubosomes, and in conjunction with the SPR data, further

demonstrates the remarkable inhibitory activity of these inhib-

itor systems. A key feature of the cubosome CT inhibitors is that

their design does not require consideration of precise structural

information of the toxin. Rather, the lateral mobility of the

glycolipid receptors within the mesophase bilayer allows for

precise pattern matching of receptors and the protein ligand. The

reorganization of ligands with toxin recognition is a unique

mechanism that is unavailable to conformationally restricted

polyvalent inhibitors. Along with the relative ease of preparation

and low cost of these materials, the encouraging biological data

support our novel approach for the use of cubosome systems as

a new class of polyvalent inhibitors in the development of anti-

toxin prophylactics and therapeutics for cholera, and related

infectious diseases.

Acknowledgements

The authors are grateful to the Office of the Chief Executive

(CSIRO) for PhD Studentship funding (SJF) and the Australian

Synchrotron for SAXS beamline allocation. The views expressed

herein are those of the authors and are not necessarily those of

the owner or operator of the Australian Synchrotron. We thank

beamline scientists, Nigel Kirby and Stephen Mudie, for their

assistance during data collection. The contributions of Lynne

Waddington (CSIRO) for providing Cryo-TEM images and

Dr Xavier Mulet (CSIRO) for his assistance in the preparation of

graphical material are greatly appreciated.

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