1Organic Chemistry Institute and CeNTech, Westfälische Wilhelms-Universität Münster, 2Laboratory of Macromolecular and Organic Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, 3Laboratory of Materials and Interface Chemistry and Soft Matter Research Unit, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology
Besenius, P., de Feijter, I., Sommerdijk, N. A. J. M., Bomans, P. H. H., Palmans, A. R. A. Controlling the Size, Shape and Stability of Supramolecular Polymers in Water. J. Vis. Exp. (66), e3975, doi:10.3791/3975 (2012).
For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5.
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained.
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by π-π stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length > 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.
Scheme 1. Self-assembly of BTA-based discotics in citrate buffer into spherical aggregates showing diameters of about 5 nm, at millimolar concentrations of building block. Increasing the ionic strength by the addition of NaCl results in the formation of elongated rods with a diameter of around 3 nm and length > 25 nm. Click here to view larger figure.
1. Preparing a BTA-Gd(III)DTPA Solutions for CD Spectroscopy and Measurement of Temperature-dependent CD Spectra as a Function of NaCl Concentration
2. Fitting the T-dependent CD Data to a Model for T-dependent Self-assembly
Above equation contains (next to the variable temperature, T, and the degree of aggregation, Φn) three parameters; i.e. the enthalpy of elongation he, the elongation temperature Te (the temperature at which the self-assembly starts) and the parameter ΦSAT, which is introduced to ensure that Φn/ΦSAT does not exceed unity, which follows from the constraint that the degree of aggregation cannot exceed unity.
Fitting renders the enthalpy of elongation he (J/mol) and the elongation temperature Te (K) that characterize the self-assembly of the molecules for a given concentration. When fitting, one restraint should be obeyed which is that only the degree of aggregation at temperatures below Te should be fitted, since equation 2.1 is only valid in the elongation regime.
Next, the experimentally found degree of aggregation in the nucleation regime can be fitted, using the following equation:
Above equation contains (next to the variables T and Φn) four parameters of which already three were determined with equation 2.1; i.e. the enthalpy of elongation he, the elongation temperature Te and the parameter ΦSAT. The only unknown parameter is the Ka value -describing the cooperativity of the nucleation phase- which is found by fitting the experimentally found degree of aggregation for temperatures above Te.
3. Preparing BTA-Gd(III)DTPA Solutions for Transmission Electron Microscopy and Visualization of Supramolecular Polymers via Cryogenic TEM
4. 1H-DOSY NMR Measurements of Spherical Self-assembled BTA-Gd(III)DTPA at Low Ionic Strength
5. Representative Results
1H-DOSY NMR and SAXS measurements on BTA-M(III)-DTPA: spherical objects in citrate buffer
The ionic character of the peripheral Gd(III) complexes introduces frustration in the one-dimensional growth of the discotic monomers whose core is designed to polymerize into elongated rod-like aggregates. The balance between attractive and repulsive interactions controls the size and the shape of the aggregates (Scheme 2).
A powerful technique to determine the size and the shape of particles in solution is synchrotron source small angle X-ray scattering (SAXS). BTA-Gd(III)-DTPA was dissolved in a citrate buffer solution and the SAXS profiles were recorded and fitted in the region 0.01 < q < 0.1 Å-1. A slope approaching zero in the low-q region (q < 0.06 Å-1) indicates a lack of shape anisotropy in the aggregate, suggesting the presence of spherical objects (Figure 1). The data measured at different concentrations were fitted using a homogeneous monodisperse spherical form factor leading to a calculated radius, R, of 3.2 nm. The calculated geometric radius of monomeric discotic BTA-Gd(III)-DTPA is 3.0 nm, which suggests the presence of aggregates with an aspect ratio close to 1.
Figure 1. SAXS profiles for BTA-Gd(III)-DTPA in citrate buffer (100 mM, pH 6) at 0.5 and 1.0 mM (top). DOSY NMR of BTA-Y(III)-DTPA in 50 mM d6-succinate buffer at 1.0 mM (bottom). Click here to view larger figure.
In order to provide further evidence for the spherical shape and nanometer-size of the self-assembled objects, we performed 1H diffusion-ordered NMR spectroscopy (1H-DOSY NMR) (Figure 1). DOSY-NMR allows determination of the diffusion coefficients of aggregates, from which the hydrodynamic radius (RH) can be calculated. Since Gd(III) is highly paramagnetic and 1H signals would thereby be broadened significantly, we changed Gd(III) for diamagnetic Y(III). The diffusion coefficients of the aggregated diamagnetic discotic amphiphile in a deuterated succinate buffer (50 mM, pH 6, c = 1 mM) was determined to be 0.69x10-10 m2s-1. Via the Stokes-Einstein relation, we calculate a hydrodynamic radius RH of 2.9 nm for the discrete objects of spherical size (Table 1). This size is in excellent agreement with value obtained from SAXS data for BTA-Gd(III)-DTPA.
a from DOSY; b from SAXS
Table 1. Results of SAXS and DOSY measurements for BTA-M(III)-DTPA.
Cryo-TEM on BTA-Gd(III)-DTPA: from spherical objects to elongated nanorods
Further evidence for successful control over one-dimensional stack length was obtained from cryo-TEM micrographs. Due to the vitrification of the aqueous solutions cryogenic TEM preserves the structural morphology of the self-assembled aggregates and avoids drying affects related to conventional TEM sample preparation. Figure 2 (left) shows that BTA-Gd(III)-DTPA produces the expected spherical objects with diameters close to 6 nm at a 1 mM concentration, which confirms the results from SAXS and DOSY measurements. According to these findings, we have been able to obtain self-assembled discrete objects that can be considered the supramolecular equivalent of dendritic macromolecules10.
Figure 2. Cryo-TEM images for BTA-Gd(III)-DTPA (left) 1 mM vitrified at 298 K in citrate buffer (100 mM, pH 6), scale bar represents 50 nm; (right) 1 mM vitrified at 298 K in citrate buffer (100 mM, pH 6) and an overall NaCl concentration of 5 M, scale bar represents 50 nm.
So far we have only worked in buffered solutions of low ionic strength. However, if electrostatic repulsive forces of the peripheral charged M(III)-DTPA complexes on BTA-Gd(III)-DTPA are at the origin of the frustrated one-dimensional growth, we expected that increasing the ionic strength of the buffered environment, using an inert 1:1 salt with highly hydrated counterions, should reduce the electrostatic interactions and hence a different type of self-assembled object should be formed. In citrate buffer comprising 5 M NaCl this effect was indeed observed (Figure 2, right). The formation of high aspect ratio rod-like supramolecular polymers is clearly observed in cryo-TEM micrographs at high ionic strength. Electrostatic screening is the most likely explanation for this finding. The shape changes from a spherical aggregate of around 6 nm in diameter to elongated rods with a diameter of 6 nm and length of up to several hundred nanometers.
CD measurements of BTA-Gd(III)-DTPA: switching on cooperative self-assembly by increasing the ionic strength
Circular dichroism (CD) spectroscopy measures the difference in absorption between left-handed and right-handed circularly polarized light. When a helical object has a preferred helical sense, left- and right-handed circularly polarized light will be absorbed to different extents, hence giving rise to a CD-effect. Since the intermolecular hydrogen bonds formed between consecutive BTA-Gd(III)-DTPA within the aggregates, are lined up in a helical fashion and the stereogenic centre at the L-phenylalanine moiety favors one helical sense over the other, we expect a clear CD spectrum from BTA-Gd(III)-DTPA based aggregates11,12. In addition, temperature-dependent CD spectroscopy is a powerful tool to assess the self-assembly mechanism of BTA-Gd(III)-DTPA polymerisation and allows to derive conclusions on the stability of the formed aggregates13.
As an example, the room temperature CD spectra of BTA-Gd(III)-DTPA (8x10-3 mM or 4x10-3 mM in a 100 mM citrate buffer) with increasing salt concentration (0 M NaCl to 1.0 M NaCl) are given in Figure 3A. Although a significantly lower concentration is applied for the CD measurements, the clear Cotton effect indicates the presence of intact aggregates, even at micromolar concentrations. The shape of the CD spectrum changes upon increasing the salt concentration, which is a good indication for reduced interactions at the periphery of the stacks and better packing of the discotics. In addition, the CD cooling curves of the same solutions (363 - 283 K, measured at λ = 269 or 278 nm) show distinct differences in shape (Figure 3B). The apparent Te -the temperature at which aggregation starts- shifts to higher temperatures at higher salt concentration and an increasingly cooperative mechanism, characterized by a more abrupt increase in the CD-effect, becomes apparent. Whereas the cooling curve at 0 M NaCl is best described by an isodesmic self-assembly process, the cooling curve at 1.0 M NaCl is typical for a cooperative self-assembly process 14. In the former case, all association constants are assumed to be equal while in the latter case self-assembly occurs in at least two distinct stages. In the first step, a "nucleus" needs to be formed which is energetically highly unfavorable. After cooling below a critical polymerization temperature, elongation and exponential growth into supramolecular polymers of high molecular weight follows. Quantifying the thermodynamic parameters of the self-assembly of BTA-Gd(III)-DTPA at 0 and 1 M NaCl using a cooperative model clearly reveals the decrease in Ka, which is the dimensionless activation constant8. Lower values for Ka indicate a higher degree of cooperativity in the self-assembly process, which is expressed in the formation of highly elongated supramolecular polymers as observed in cryo-TEM.
|8 x10-3 mM||0 M||5 10-2|
|4 x10-3 mM||1 M||1 10-4|
Table 2. Degree of cooperativity expressed by Ka in the temperature-dependent self-assembly of BTA-Gd(III)-DTPA as a function of the NaCl concentration (CNaCl).
Figure 3. BTA-Gd(III)-DTPA in a 100 mM citrate buffer (c = 8 x10-3 mM at low ionic strength and 4 x10-3 mM at high ionic strength) A] CD spectra recorded at 293 K as a function of the ionic strength, cNaCl = 0 M - 1.0 M, the molar ellipticity Δε is calculated as follows: Δε = CD-effect/(cxl) in which c is the concentration of BTA in mol L-1 and l is the optical path length in cm; B] Corresponding CD cooling curves measured at λ = 269 nm for 0 M NaCl and 278 nm for 1 M NaCl solutions expressed as the degree of aggregation Φn as a function of the NaCl concentration cNaCl = 0 M - 1.0 M, Φn is calculated by dividing the measured CD-effect by the maximal CD-effect.
The self-assembling discotic amphiphiles discussed in this contribution containing the Gd(III)-DTPA complex are currently under investigation as novel magnetic resonance imaging (MRI) agents that combine high contrast with tunable excretion times.15 Hence, the details of their self-assembling behavior and their stability in different conditions are of critical importance. The combination of spectroscopic (CD and NMR), scattering (SAXS) and microscopy (cryo-TEM) techniques allows visualization of the formed structures and the quantification of their thermodynamic parameters. This combination of techniques is generally applicable for self-assembling molecules as long as a preferential helical sense in the studied system allows a difference in the absorption of left- and right-handed circularly polarized light.
No conflicts of interest declared.
The authors gratefully acknowledge Marko Nieuwenhuizen for assistance with the DOSY-NMR.
|CD spectroscopy||Jasco||Jasco J-815 spectropolarimeter|
|NMR||Varian||Varian Unity Inova 500 spectrometer||5-mm ID-PFG probe of Varian|
|SAXS||Dutch-Belgian beamline (BM26B) at the European Synchotron Radiation Facility (ESRF) in Grenoble, France|