S 1

Supporting information

Shape and release control of a peptide decorated vesicle through pH sensitive orthogonal supramolecular interactions

Frank Versluis,a Itsuro Tomatsu,

a Seda Kehr,

b Carlo Fregonese,

b Armand W.J.W. Tepper,

a

Marc C. A. Stuart,c Bart Jan Ravoo*,

b Roman I. Koning

d and Alexander Kros*

a

aLeiden Institute of Chemistry, University Leiden, P.O. Box 9502, 2300 RA, Leiden, the Netherlands. bOrganisch-Chemisches Institut and CeNTech, Westfälische Wilhelms-Universität Münster, Corrensstrasse

40, 48149 Münster, Germany. cBiophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology

Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. dMolecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, P.O. Box 9600 2300 RC, Leiden,

the Netherlands.

Experimental Section

Preparation of Ad-(leu-glu)4 (2): Octapeptide 2 was synthesized using a peptide synthesizer by

conventional Fmoc solid phase peptide synthesis on a Sieber amide resin at a 250 µmol scale

with a loading of 0.64 mmol/g.1 To modify the peptide with adamantane, the final coupling was

performed with 1-adamantanecarboxylic acid in the same manner as the amino acid residues.

The desired peptide was obtained by treating the crude product with TFA/water (9:1, v/v), to

cleave it from the resin and to remove the t-butyl groups on the glutamic acids, yield: 181 mg (63

%). The obtained crude compound was purified with RP-HPLC, with two different eluents; 1 %

TFA in water and water/CH3CN mixture with a gradient of 8:2 → 0:10, yield: 70 mg (24 %).

Preparation of the cyclodextrin-vesicles2: Amphiphilic β-cyclodextrin 1 was synthesized as

described.2 To prepare unilamellar bilayer vesicles, 1 was dissolved in CHCl3 and the solvent

was evaporated with a rotary evaporator and a vacuum oven for one hour. The obtained thin film

of 1 was hydrated with phosphate buffered saline and was vortexed extensively at room

temperature (PBS, pH 7.4) to provide a 1 mM turbid aqueous solution of 1. After sonication

under heating for 30 minutes, a clear solution was obtained. The size of the vesicles was set

around 100 nm by extrusion through a polycarbonate membrane. The average diameter of the

particles was estimated to be 77 nm with dynamic light scattering.

S 2

ββββ-Sheet formation on the surface of the CD vesicle: After the appropriate amount of peptide 2

(typically 0.5 mM solution in PBS) was added to vesicles composed of 1 (typically 1.0 mM

solution in PBS), the pH of the mixture was set to 5.0 with a minimum amount of a 0.1 M H3PO4

or 1 M HCl.

Circular dichroism (CD) spectroscopy: CD spectra were measured with a J-815

Spectropolarimeter (JASCO) at room temperature. A 0.1 cm quartz cuvette was used. The CD

spectra were obtained after averaging of six scans and subtraction of the background. The Molar

ellipicity, [θ] (deg cm2 dmol

-1), was calculated from the observed ellipticity, θobs (mdeg), by

applying the following equation: [θ] = θobs·M/(10·l·c), where M is the mean residue molecular

weight, l is the path length of the cuvette and c is the concentration of the peptide.

Dynamic Light Scattering (DLS): DLS data were obtained using a Zetasizer Nano ZS

(Malvern Instruments Ltd). The observed intensity autocorrelation function, g(2)

(t), was

measured experimentally, which is related to the normalized autocorrelation function, g(1)

(t), by

the Siegert relation, g(2)

(t) = 1 + β·[g(1)

(t)]2, where β is a constant parameter for an optical

system. To obtain the average relaxation time (τ), the first cumulant method was employed to

analysis of g(1)

(t) according to the equation, g(1)

(t) = exp(-t/τ). The apparent translational

diffusion coefficient, D, is calculated from D = Γ�/q2, where Γ is the relaxation rate, Γ = 1/τ, and

q represents the magnitude of scattering vector expressed as q = (4πn/λ )·sin(θ/2), where θ is the

scattering angle, 173°, n is the refractive index of the solution and λ is the wavelength (633 nm).

The apparent hydrodynamic radius, r, is given by the Einstein-Stokes equation r = kBT/(6πηD),

where kB is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity.

Cryo-TEM: Glow-discharged carbon-coated lacey Formvar grids (300 mesh, Ted Pella) or

Quantifoil R2/2 grids (Quantifoil, Jena Germany) were loaded with 3 µL sample. Grids were

blotted and plunged into liquid ethane using a fully automated home-built vitrification device

operating at 100 % humidity and 22 ºC. Electron microscopy images were recorded on a FEI

Tecnai 12 electron microscope at an accelerating voltage of 120 keV and equipped with a 4k x

4k Eagle camera (FEI, Eindhoven, The Netherlands). Images were recorded at liquid nitrogen

temperature using a Gatan 626 cryo-holder (Gatan Inc., Pleasanton, U.S.A.) under low-dose

conditions. As we observed absorptions of the material onto the Quantifoil grids (Figure 2a), we

used lacey Formvar grids (Figure 2b and 2c).

S 3

Fluorescence Spectroscopy: Fluorescence measurements were performed using a FS920

fluorometer (Edinburgh Instruments Ltd.) with a DTMS-300X excitation monochrometer and a

peltier-controlled thermostatic cell. All spectra were obtained at 25 °C using a quartz cuvette

with a 1 cm path length. Each spectrum was measured with the step size of 1.0 nm, and a

sampling time of 0.1 s, and single scan. Excitation and emission slits were 1 nm. The excitation

wavelength was 282 nm. For the fluorescence experiment we used 10 µM of tetrasodium 1,3,6,8-

pyrenetetrasulfonate and 100 mM of NaI. To destroy the assembly, we used a minimum amount

of 20 wt% Triton X-100 solution. The experiment was performed as follows: tetrasodium

1,3,6,8-pyrenetetrasulfonate (Py) was added during the preparation of the vesicular system

composed of 1 and 2. Next, NaI was added to quench all non-encapsulated dyes, the observed

signal was due only to encapsulated dyes. The pH was then lowered to 5.0 with 1 M HCl and the

fluorescence intensity was measured for 1 hour. Triton X-100 was then added and the

fluorescence intensity was measured again.

Isothermal Titration Calorimetry (ITC): ITC titrations were performed with a Nano-ITC III

(Calorimetry Sciences Corporation, USA). The reference cell was filled with Milli-Q grade

water. All solutions were degassed for 10 min before use. A β-CD solution (5.24 mM) was

titrated in 35 injections of 7 µL to a peptide solution (0.51 mM). Both β-CD and peptide were

dissolved in a buffer of 0.137 mM NaCl, 3.35 mM KCl, 8.03 mM Na2HPO4 and 1.84 mM

KH2PO4 in Milli-Q grade water. Titration parameters were set as follow: equilibration time 300

s, injection interval 1080 s, stirring rate 150 rpm, temperature 23 °C. The experiment was

repeated three times. Raw data are corrected for the heat of dilution, which was measured by

performing a titration of β-CD (5.24 mM) into buffer using identical experimental settings.

Experimental data are analysed with NanoAnalyze software.

References

1. a) W. C. Chan, P. D. White, Fmoc Solid-phase Peptide Synthesis: A Practical

Approach; Oxford University Press: Oxford, 2000. b) G. B. Fields, R. L. Noble, Int.

J. Pept. Protein Res. 1990, 35, 161-214.

2. a) B. J. Ravoo, R. Darcy, Angew. Chem. Int. Ed. 2000, 39, 4324 – 4326. b) P. Falvey,

C.-W. Lim, R. Darcy, T. Revermann, U. Karst, M. Giesbers, A. T. M. Marcelis, A.

Lazar, A. W. Coleman, D. N. Reinhoudt, B. J. Ravoo, Chem. Eur. J. 2005, 11, 1171 –

1180.

S 4

Interaction of ββββ-CD and Ad-(leu-glu)4

The interaction of β-CD and octapeptide 2 was investigated using isothermal titration calorimetry

(Figure S-1). The binding constant (Ka) of the complex is 3.5 × 104 M

-1 and the stoichiometry is

close to 1:1. These findings imply that the presence of the peptide moiety did not alter the

inclusion of adamantane in the cyclodextrin cavity.

This titration could not be performed with the CD vesicles, since the concentration required for

ITC (5 mM CDV and 0.5 mM peptide) lead to inhomogeneous solutions which gave rise to

significant additional heat effects during the titration. However, we have investigated the

interaction of various adamantane derivatives with CDV with capillary electrophoresis and ITC

(see ref. 6) and invariably found Ka ~ 10000 M-1

, thus we assume that also the octapeptide binds

to CDV in the same way as it binds to β-CD.

Figure S-1. Isothermal titration calorimetry (ITC) for a Ad-(leu-glu)4 solution titrated with a β-

CD solution.

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Time evolution of the transition from random coil to β-sheet upon changing pH

The CD spectrum shows that the transition from random coil to β-sheet is faster than one minute

(the experimental limit of our setup) upon changing the pH and it does not show a development

in time.

0 10 20 30 40 50 60-2.5x10

4

-2.0x104

-1.5x104

-1.0x104

-5.0x103

0.0

5.0x103

[θ]

/ deg cm2 dmol

−1

time /min

Figure S-2. Time dependency of the CD signal of 2 after changing pH from 7.4 to 5.0;

monitored at 217 nm.

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Optimum concentration of the peptide 2

CD spectra at different concentration of 2 in the presence of the same amount of the CDV were

measured. In Figure S-3 normalized cd signals at 215 nm are plotted against concentration of

Ad-(leu-glu)4 at pH 5.0. It is clearly seen that 40 to 50 mol % of Ad-(leu-glu)4 gives strongest

signal; β-sheet formation occurs efficiently in about 2:1 mixture of 1 and 2, not 1:1. This

observation indicates that CDV preserve their bilayered structure after deformation into a fiber

like structure. Only a half of cyclodextrins, which faces to the exterior, is available for the

complex formation with the adamantane terminus of the peptides.

0 10 20 30 40 50 60 70 80 90 100 110-2.5x10

4

-2.0x104

-1.5x104

-1.0x104

-5.0x103

[θ]

/ deg cm2 dmol

−1

concentration of Ad-(leu-glu)4 / mol%

Figure S-3. Concentration dependency of the cd signals of 2 at 215 nm at pH 5.0 in the presence

of the CDV.

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Cryo-TEM images taken at different concentration of the peptide 2

The peptide concentration dependency of the morphological change on the complex was

investigated using cryo-TEM. At a ratio of 5:1 mainly deformed vesicles are observed. At a 2:1

ratio fibers and some deformed vesicles are predominantly present. At a 1:1 ratio many fibers are

observed with very few deformed vesicles.

(a) (c) (b)

Figure S-4. Cryo TEM images of a mixture of 1 and 2 at pH 5.0 at: a) 5:1 ratio; b) 2:1 ratio; c)

1:1 ratio. Scale bars represent 100 nm. Images were taken 1 hour after acidification.

Adsorption of the vesicles on the grid for Cryo-TEM

Figure S-5. Contrast optimized image of the Quantifoil R2/2 grid (from the same picture with

Figure 2a) showing the absorption of the vesicles onto the grid.

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DLS measurements at different pH

The structural change of the CDVs was investigated with dynamic light scattering (DLS). Figure

S-5 shows the autocorrelation functions and the average relaxation time (τ) calculated from the

autocorrelation functions measured at pH 7.4 and 5.0 alternatingly. At pH 7.4, τ is ca. 0.22 ms,

which is close to the value for the CDVs and corresponds to a spherical object with a diameter of

76 nm. On the other hand, τ is ca. 0.32 ms at pH 5.0, corresponding to an object having a mean

effective hydrodynamic diameter (Deff) of 110 nm which can be a fiber in dimension of 8 nm ×

450 nm. (Deff = L/ln(L/d), where L and d are the length and the diameter of fiber, respectively.)

Figure S-6. (a) Autocorrelation functions from DLS measurements performed on a mixture of 1

(1 mM) and 2 (0.5 mM) at pH 7.4 and 5.0, and a solution of 1 (1 mM) at pH 7.4. (b) Relaxation

time (τ) obtained from the mixture of 1 and 2 upon switching pH between 7.4 (○) and 5.0 (●).

at pH 7.4 at pH 5.0

delay time (µsec)10-1 100 101 102 103 104

0

0.2

0.4

0.6

1.0

auto

corr

ela

tio

n f

un

ctio

n

0.8

0.22 msec 0.32 msec

without peptide

0.22 msec

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1,3,6,8-pyrenetetrasulfonate (Py) release

The release data shown in Figure S-6 was calculated by taking the ratio of the fluorescence

intensities at 383 nm after NaI was added, after the pH was lowered to 5.0 and after Triton X-100

was added (figure S-4). Since the estimated interior volume of the fiber was significantly smaller

than that of the spherical vesicle, we assume all the change is due to the diffusion of Pys from

inside to outside of the complex.

Figure S-7. Fluorescence spectra of a mixture of 1 and 2 at a concentration of 1mM and 0.5 mM

respectively, containing the fluorophore Py (10 µM) and quencher NaI (100 mM). At pH 7.4, the

observed signal is due to encapsulated dyes (dashed line). Upon lowering the pH to 5.0, the

intensity drops significantly, indicating that the dye is released (solid line). Upon adding Triton

X100, the complex is dissolved and the signal is completely quenched (short dashed line).

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Estimation of the volume difference in a vesicle and a fiber

By assuming that the surface area of the complex remains constant while the shape of the

complex changes from a vesicle to a fiber, the length of the tubes (lt) can be calculated from

radius of the spherical vesicles (rs) and radius of the tubes (rt). Both rs and rt were obtained from

the cryo-TEM images and showed to be 35 nm and 4 nm, respectively.

The surface area of the vesicle (Ss) is ~1.5 × 104 nm

2 calculated from Ss = 4⋅π⋅rs

2.

If one vesicle transforms into one fiber then the surface area of the tube (St) is equal to Ss,

St = 2⋅π⋅rt⋅lt = 1.5 × 104 nm

2, and therefore the length of the tube (lt ) ~6.1 × 10

2 nm.

This value is in good agreement with the cryo-TEM data.

Using these values the volumes of sphere and tube (Vs and Vt) can be calculated as Vs =

(4/3)⋅π⋅rs3

= 1.8 × 105 nm

3 and Vt = π⋅rt

2⋅lt = 3.1 × 10

4 nm

3.

As a result the interior volume of a vesicle is ~6 larger compared to the interior volume of a fiber

(ratio of the two (Vt/ Vs) is ~ 0.17).

By applying this simple geometrical calculation, we concluded that the internal volume of a fiber

is much smaller compared to the volume of a vesicle. Therefore it is reasonable to assume that

the dye is expelled from the aggregate upon the vesicle-to-fiber transition into the surrounding

medium where it will be quenched by the iodine ions.

It is noteworthy that the difference of the inner volumes will be even larger than this estimation

due to the volume occupied by the membrane.