Dynamic multivalent recognition of cyclodextrin vesicles

Abstract

Cyclodextrin bilayer vesicles have dynamic membranes that recognize guest molecules through efficient multivalent host–guest interaction reminiscent of multivalent binding of a ligand with receptors in a biological membrane.

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Receptor clustering is a powerful tool that cells and bacteria use to tune affinity and select competing ligands on their membrane surface.¹ In chemical terms, this phenomenon is an example of dynamic molecular recognition through multivalent interaction.² Here we describe an artificial membrane with embedded receptor molecules that recognizes and binds a suitable ligand via multivalent interaction with a small cluster of receptors. Previously, we have reported the preparation of amphiphilic cyclodextrins (CDs) and the corresponding CD bilayer vesicles, which have the ability to recognize and bind specific guests.³ In this communication we report that a vesicle membrane composed of β-CD host molecules 1b has specific, multivalent interactions with a dye-labeled, divalent guest 3. Fluorescence resonance energy transfer (FRET)⁴ was used to monitor the complexation of fluorescent guest molecules to the CD vesicles. In vesicles composed of a minority of β-CD host 1b in a majority of “inert” α-CD 1a, clustering of β-CD host 1b leads to efficient multivalent interaction. We use a quantitative model to interpret the clustering of receptors as an increased effective concentration of receptor molecules at the membrane surface.

Fig. 1 shows a schematic illustration of a bilayer vesicle composed of amphiphilic CDs and the structure of amphiphilic CDs 1a and 1b. Fig. 1 also shows N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled cholesterol (NBD-Chol) 2, which is well known as a hydrophobic membrane probe,⁵ and N-(lissamine-rhodamine B)-labeled divalent adamantyl guest (LRB-Ad₂) 3.⁶ NBD and LRB dyes are well known as a FRET donor–acceptor pair in studies of biological and model membranes.⁴

Fig. 1 Bilayer vesicle composed of amphiphilic CDs and molecular structure of amphiphilic CDs 1a and 1b, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled cholesterol (NBD-Chol) 2 and N-(lissamine-rhodamine B)-labeled divalent adamantane guest (LRB-Ad₂) 3.

Using conventional extrusion methods, we prepared unilamellar CD vesicles (diameter ca. 160 nm) containing 1 mol% NBD-Chol 2. The formation of bilayer vesicles from amphiphilic CDs 1a and 1b in water was observed by transmission electron microscopy and dynamic light scattering consistent with previous reports.³

Fig. 2 shows the steady-state fluorescence changes upon adding guest LRB-Ad₂3 (dissolved in 5 mM phosphate buffer, pH 7.5, 0.5% DMSO) to the host vesicle solution (10 µM CD 1b in 5 mM phosphate buffer and pH 7.5) containing 1 mol% NBD-Chol 2. The fluorescence intensity of donor 2 (λ_ex = 450 nm, λ_em = 530 nm) decreased while the intensity of acceptor 3 (λ_em = 585 nm) increased upon addition of 3. The emission of 2 was nearly completely quenched upon addition of < 1 µM guest 3, indicating that efficient FRET occurred as a result of the interaction between guest 3 and β-CD host 1b, which brings acceptor 3 in close proximity to donor 2. The Förster distance (R₀) for NBD and LRB ranges from 50 to 70 Å.⁴ According to the dimensions of the CD bilayer provided by X-ray diffraction data and Langmuir Blodgett isotherms,³ acceptor guest molecule 3 and donor membrane probe 2 will approach within the Förster distance upon complexation of 3 at the surface of vesicles of CD host 1b containing 2. Since the concentration of acceptor 3 was very low ([3]_max = 0.9 µM), inner filter effects and collisional quenching will be negligible. Note also that in all experiments the concentration of guest 3 was much lower than host 1b.

Fig. 2 Fluorescence emission spectra (λ_ex = 450 nm) of vesicles of β-CD 1b (10 µM) containing 1 mol% NBD-Chol 2 (0.1 µM) upon adding divalent guest LRB-Ad₂3. [3] = 0–0.9 µM. All measurements were carried out in 5 mM phosphate buffer at pH = 7.5 and T = 25 °C.

To visualize the interaction between guest 3 and vesicles of host 1b, confocal microscopy images of CD vesicles (10 µM) containing 1 mol% NBD-Chol 2 (0.1 µM) in the absence and presence of guest 3 were collected on a Carl Zeiss LSM510 microscope (Fig. 3). Fluorescence images were obtained by recording the red emission (above 560 nm) and the green emission (between 500 nm and 550 nm), while exciting at 458 nm. Hydrophobic probe 2 is exclusively localized in the CD vesicles (see ESI for additional evidence). In the absence of guest 3, the vesicles are observed in both emission channels as a result of the broad emission spectrum of NBD-Chol 2. However, when 1 µM guest 3 was added to the NBD-Chol 2 containing vesicle solution (10 µM), fluorescent vesicles were observed only in the red emission channel, indicating that the green fluorescence of 2 embedded in the vesicles was quenched via a FRET mechanism induced by binding guest 3 to β-CD 1b on the surface of the vesicles. The intense red fluorescence observed results from acceptor 3.

Fig. 3 Confocal microscopy images of vesicles of β-CD 1b. Scale: 207 × 207 µm for each window. (a) Vesicles of β-CD 1b containing NBD-Chol 2. (b) LRB-Ad₂3 bound to vesicles of β-CD 1b containing NBD-Chol 2. Top: λ_em = 500–550 nm. Bottom: λ_em > 560 nm. See text for details.

In a negative control experiment, LRB without a divalent adamantane anchor was added to vesicles containing donor 2. Emission spectra showed that there was no significant FRET between 2 in the β-CD vesicle and LRB in bulk solution (see ESI). LRB-Ad₂3 was also added to vesicles of α-CD 1a and to non-CD vesicles³ composed of n-dodecyl triethyleneglycol (C₁₂EO₃, 90%) and n-tetradecyl triethyleneglycol (C₁₄EO₃, 10%), both containing NBD-Chol 2 (0.1 µM). Neither the vesicles of α-CD 1a (which is too small to fit adamantane) nor the non-CD vesicles (which do not contain host molecules) show any change of fluorescence of NBD-Chol 2 upon addition of divalent guest 3. The Stern–Volmer plot (Fig. 4) demonstrates that the recognition of guest 3 by the vesicles is specifically mediated by the host–guest complexation of adamantane guest 3 and CD host 1b at the vesicle surface.

Fig. 4 NBD-Chol 2 fluorescence intensity (F₀/F) versus guest concentration: (∇) Vesicles of β-CD 1b and LRB-Ad₂3. Negative controls: (▼) Vesicles of β-CD 1b and LRB; (●) Vesicles of C₁₂EO₃/C₁₄EO₃ and LRB-Ad₂3; (○) Vesicles of α-CD 1a and LRB-Ad₂3. [NBD-Chol 2] = 0.1 µM and [1a] = [1b] = 10 µM. [C₁₂EO₃/C₁₄EO₃] = 100 µM. All measurements were carried out in 5 mM phosphate buffer at pH = 7.5 and T = 25 °C.

The slope of the Stern–Volmer plot can be employed to estimate the apparent association constant K_a = 1.5 × 10⁷ M⁻¹ between host 1b and divalent guest 3.‡ The magnitude of this association constant is diagnostic for a divalent interaction of one guest molecule with two host molecules. The efficient divalent interaction of guest 3 with host 1b is characterised by the equilibrium constant K_a2 = C_eff × K_a1² where K_a1 is the monovalent association constant and C_eff is the effective concentration of host molecules 1b at the surface of the CD vesicle.⁷ C_eff reflects the high number of β-CD hosts on the membrane surface accessible to the second adamantyl group, after the first adamantyl group of guest 3 binds to the vesicle. C_eff was calculated from a straightforward geometrical model taking into account the experimental molecular surface area of the CD host molecule 1b³ and the distance between the two adamantyl groups at guest 3⁷ (see ESI). According to this model, C_eff = 0.17 M, which is consistent with the value of C_eff of CD host molecules at the surface of densely packed CD monolayers on gold and glass.^6,7 Assuming that the divalent interaction K_a2 is the result of two equal and independent monovalent interactions K_a1, it follows that K_a1 = (K_a2/C_eff)^0.5 = 9.4 × 10³ M⁻¹, which is consistent with the experimental value for the monovalent interaction of 1b with adamantane carboxylate (K_a1 = 7.0 × 10³ M⁻¹).³ We conclude that the recognition of guest 3 by host 1b is amplified by the formation of a divalent host–guest complex at the membrane surface.§

Furthermore, we measured the apparent association constant K_a of guest 3 with CD vesicles composed of mixtures of “good” host CD 1b and “inert” CD 1a (Table 1). CDs 1a and 1b differ in ring size only and can be readily mixed in all proportions. Since the alkyl chains of 1a and 1b are relatively short (C₁₂), the bilayer membranes are in a liquid crystalline-like L_α phase with rapid lateral diffusion of the cyclodextrins on the vesicle surface. We therefore assume that the vesicle surface is a dynamic, homogeneous, two-dimensional solution with fractions of 1a and 1b directly proportional to their ratio in the mixture and a random distribution of 1a and 1b across the vesicle surface.

Table 1 Apparent association constant (K_a) and effective concentration (C_eff) of CD experienced by guest LRB-Ad₂3 binding to vesicles of β-CD 1b mixed with α-CD 1a

Vesicle composition^a	100	70	50	30	10
a Percentage of β-CD 1b in mixed vesicles with α-CD 1a. b Effective concentration Ceff derived from Ka. Error ±10%. See text for details. c Calculated effective concentration Ceff for a statistical mixture of β-CD 1b and α-CD 1a. Error ±10%. See ESI for details.
K a (M^−1)	1.5 × 10^7	1.2 × 10^7	1.2 × 10^7	1.1 × 10^7	3.5 × 10^6
Ceff (M)^b	0.17	0.14	0.14	0.12	0.04
Ceff (M)^c	0.17	0.11	0.08	0.04	0.002

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Although K_a decreases with the mole percentage of 1b, K_a is still > 1 × 10⁶ M⁻¹ at a low percentage of 1b, indicative of divalent rather than monovalent interaction. Table 1 lists the value of C_eff calculated from K_a = K_a2 and K_a1 = 9.4 × 10³ M⁻¹ according to C_eff = K_a2/(K_a1)². Finally, Table 1 lists the values of C_eff calculated from the experimental molecular surface area of CDs 1a and 1b³ and the distance between the two adamantane groups at guest 3 (see ESI). The values of C_eff as a function of the percentage of 1b in the CD vesicles are also shown in Fig. 5. It can be seen from Fig. 5 that C_eff as calculated from the apparent binding constant K_a deviates strongly and positively from C_eff as calculated for a statistical mixture of 1a and 1b. In other words, the effective concentration C_eff of host 1b in a mixture with inert 1a experienced by a suitable divalent guest molecule is much higher than expected for a random, statistical mixture of 1a and 1b. A plausible and appealing explanation for the high C_eff of 1b is that divalent guest 3 induces clustering of 1b in an excess of 1a.⁸ The small entropy cost of clustering will be easily offset by the large free energy gain of forming a divalent inclusion complex. However, we can not rule out (neither a priori nor by experiment) that some cluster formation occurs in the mixed CD vesicles even in the absence of divalent guest 3.

Fig. 5 Experimental (○) and calculated (●) effective concentration (C_eff) of β-CD 1b experienced by guest 3versus percentage of β-CD 1b in vesicles composed of mixtures of CDs 1a and 1b. See text for details.

In conclusion, we have demonstrated dynamic multivalent molecular recognition in an artificial membrane, reminiscent of the way that cells and bacteria select ligands on their membrane surface.

We thank Alart Mulder for the synthesis of 3 and Jurriaan Huskens for helpful discussions.

Notes and references

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6. S. Onclin, A. Mulder, J. Huskens, B. J. Ravoo and D. N. Reinhoudt, Langmuir, 2004, 20, 5460–5466; A. Mulder, S. Onclin, M. Péter, J. P. Hoogenboom, H. Beijleveld, J. ter Maat, M. F. García-Parajó, B. J. Ravoo, J. Huskens, N. F. van Hulst and D. N. Reinhoudt, Small, 2005, 1, 242–253.
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Footnotes

† Electronic supplementary information (ESI) available: control experiments and calculation of the effective concentration C_eff of CD in mixed vesicles. See DOI: 10.1039/b510540d

‡ The efficient divalent interaction of LRB-Ad₂3 with the CD vesicles is in marked contrast to inclusion interaction of polyanionic guest polymers with the CD vesicles.³ These guest polymers bind to the vesicles in a brush- or mushroom-type conformation with a K_a = 2 × 10⁶ M⁻¹ at most. It is likely that the oligo(ethylene glycol) residues protruding from the surface of the vesicles prevent optimal multivalent interaction with the guest polymers. This type of steric repulsion is well known for colloids and surfaces decorated with poly(ethylene glycol). Also there is a degree of electrostatic repulsion between the polyanion and the vesicles, which have a negative surface potential.³

§ The apparent association constant K_a is determined from the equation: F₀/F = 1 + K_a[3], where F and F₀ are the fluorescence intensity of donor NBD-Chol 2 in the presence and the absence of acceptor LRB-Ad₂3 and [3] is the concentration of 3.

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