JAC Advance Access published online on October 10, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm382
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Role of the ammonium group in the diffusion of quaternary ammonium compounds in Streptococcus mutans biofilms
1 Department of Chemistry, Université de Montréal, CP 6128, Succ. Centre-Ville, Montréal, Québec, Canada H3C 3J7 2 Department of Stomatology, Faculty of Dentistry, Université de Montréal, CP 6128, Centre-Ville, Montréal, Québec, Canada H3C 3J7
* Corresponding author. Tel: +1-514-343-5936; Fax: +1-514-343-7586; E-mail: michel.lafleur{at}umontreal.ca
Received 4 June 2007; returned 22 June 2007; revised 21 August 2007; accepted 10 September 2007
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Objectives: Cetylpyridinium chloride (CPC), a quaternary ammonium compound, was shown to interact irreversibly with Streptococcus mutans biofilms, leading to a slow diffusion compared with poly(ethylene glycol) (PEG) molecules of similar size. The objective of this work is to determine if the retardation of CPC diffusion and its strong binding to biofilms is caused by interactions between the ammonium group of CPC and the exopolysaccharide (EPS) matrix.
Methods: First, we characterized the diffusion of two analogues of CPC in S. mutans biofilms: dodecylpyridinium chloride (DPC), carrying a shorter alkyl chain than CPC, and tetramethylene bispyridinium chloride (TMBPC), a compound carrying two positively charged ammonium groups. Second, we cultured biofilms with different densities of EPS and examined the impact of this density on the transport properties of CPC. The diffusion of these compounds was probed using infrared spectroscopy with attenuated total reflectance sampling.
Results: The diffusion of CPC, DPC and TMBPC in S. mutans biofilm is slower than that of PEG10k. In addition, TMBPC and DPC, as PEG10k, could be readily washed out from the biofilms while CPC association was practically irreversible. The penetration of CPC through the EPS matrix was found to be not significantly affected by the increased EPS density, whereas the penetration of PEG with a molar mass of 10k was considerably reduced.
Conclusions: These results suggest that the interactions between the quaternary ammonium groups and the EPS matrices are not the prime contribution of the strong CPC binding, and the alkyl chain length plays a role in this association, likely through hydrophobic interactions.
Key Words: bacteria , S. mutans , FTIR spectroscopy , diffusion , PEG
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Introduction |
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Biofilms are complex microbial growth forms that involve attachment to surface, communication between individuals (quorum sensing) and resistance towards attack.1–6 One of the most distinctive features of biofilms is the presence of a thick matrix composed mainly of exopolysaccharides (EPS), but also proteins, lipids, ions and nucleic acids, forming a charged and highly hydrated gel. This EPS matrix is thought to be a key element for many biofilm characteristics including biofilm spatial heterogeneities, growth and conversion rates, and recalcitrance towards antimicrobial agents. The biofilm structure plays a crucial role in solute transport as solutes are free to circulate in water channels while permeation may be restricted in thick EPS hydrogels and dense bacterial microcolonies. Biofilms exhibit high levels of resistance to antibiotics, disinfectants and detergents.2,7,8 Mechanisms of resistance may include cell dormancy caused by a nutrient depletion deep into biofilms, expression of specific (such as porins, ß-lactamases, etc.) or aspecific (multidrug efflux pumps, thicker cell wall) resistance mechanisms. EPS matrices have also been proposed to contribute to recalcitrance by limiting the diffusion of antibiotic solutes in biofilms, either by size exclusion or by interaction/reaction with the solutes.2,8 For example, a restricted accessibility of bacteria in biofilms based on size exclusion has been demonstrated for molecules in the range 200–10 000 Da9 and 3–900 kDa.10
Quaternary ammonium compounds (QACs) are a class of broad-spectrum bactericides used as antiseptics and preservation agents.3,7,11 They include a hydrophilic quaternary ammonium group and a hydrophobic alkyl chain. Their lethal mechanism is associated with their interaction with bacteria plasma membranes. The leakage of metabolites, such as K+ and inorganic phosphate, the lysis of the cells and the disappearance of membrane enzymes are among the reported impacts of QACs on bacteria.3,7,11 As bacterial membranes are typically negatively charged, it is believed that attractive electrostatic interactions play a prime role in the association of QACs and bacteria membranes.
It has been shown that cetylpyridinium chloride (CPC), a QAC currently used in mouth wash, for example, interacts strongly with Streptococcus mutans biofilms as it diffuses slowly in biofilms, appears to bioaccumulate and practically cannot be washed out from biofilms.9 It is proposed that attractive interactions between the ammonium group of CPC and the negatively charged species of biofilms are responsible for this affinity. Interactions between cationic antibiotics and negatively charged components of the biofilms have already been proposed.12–17
In order to investigate the influence of the ammonium functional group on solute diffusion in biofilms, we performed two series of experiments. First, we determined the diffusion of CPC in S. mutans biofilms with different EPS density. By adjusting the biofilm culture parameters, the quantity of biomass and the density of the EPS matrix can be controlled,18 and the effect of such variations on the penetration and the accumulation of solutes in the biofilms was examined. Second, we investigated the role of the positively charged ammonium group of the QAC molecules by comparing the transport properties in the biofilms of a series of molecules bearing ammonium groups: CPC, dodecylpyridinium chloride (DPC), a CPC analogue with a shorter alkyl chain, and 1,1' tetramethylene bispyridinium chloride (TMBPC), a soluble QAC molecule carrying two pyridinium moieties and a very short hydrophobic segment (see Figure 1 for their chemical structure). CPC and DPC are two amphiphilic molecules that can form micelles; the CPC critical micellar concentration (cmc) is 0.8–0.08 mM19,20 whereas that of DPC is 6 mM.19 We have also examined the transport properties of poly(ethylene glycol) with a molar mass of 10 000 (PEG10k) as a control compound. This polymer has a hydrodynamic radius similar to that of the CPC micelles and it is known to have very limited interactions with biomolecules.12 The biofilms were cultured on a horizontal attenuated total reflection (ATR) crystal and ATR-Fourier-transform infrared spectroscopy (ATR-FTIR) was used to characterize the solute diffusion.9,21–24 This setting allows the measurement of the solute penetration in real time, in situ, in fully hydrated conditions, without requiring fixing the biofilms and labelling of solutes or probes.
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Chemicals
S. mutans NCTC 10449 was used to form biofilms. CPC, DPC, TMBPC, PEG10k and deuterium oxide were purchased from Sigma (Oakville, Canada). Trypticase Yeast Extract (TYE) medium was prepared with trypticase peptone (17 g/L) (BBL Becton Dickson and Co., Cockeysville, MD, USA), NaCl (5 g/L) (Fisher Scientific, Fair Lawn, NJ, USA) and Na2HPO4 (0.7 g/L) (A&C American Chemical Ltd, Montréal, Canada).
S. mutans biofilms were obtained as described previously.9,25 Briefly S. mutans was precultured overnight in TYE with 0.50% sucrose. The resulting suspension was passed, for inoculation, in a horizontal ATR flow cell (Harrick Scientific Corporation, Ossining, NY, USA) thermostated at 36 ± 1°C, at 0.5 mL/min, using a peristaltic pump (Masterflex, Cole Palmer, Montreal, Canada). After a 5 h adhesion phase, fresh sucrose-containing TYE broth was circulated through the flow cell for 15 h, to allow the growth of the biofilms. The sucrose concentrations used during this phase were 0.10%, 0.25%, 0.50%, 1.00% and 2.00% (w/v). The diffusion experiments were performed after the growth period, according to an established approach.23,26 The diffusion of CPC, DPC, PEG10k and TMBPC in biofilms grown in different conditions was examined. After obtaining an established biofilm on the zinc selenide (ZnSe) crystal, the diffusion kinetics of a couple of solutes was measured successively, on the same biofilm. First, the diffusion of PEG10k was measured because this solute does not influence the biofilm viability.9 After washing it, CPC, DPC or TMBPC was then introduced. Each solute was introduced in the system at a flow rate of 10 mL/min during 20 s, to minimize the time period required for the condition changes. After this period, the flow was brought back to 0.5 mL/min for the rest of the diffusion experiment. The solute concentrations [expressed in % (w/v)] in the diffusion solutions, prepared with TYE broth, were 2% for CPC and DPC, 3% for PEG10k and 1% for TMBPC. These concentrations are, at least, one order of magnitude above the reported cmc of CPC and DPC.
Spectra were acquired on a BioRad-FTS25 IR spectrometer equipped with a KBr beam splitter and a DTGS detector, with a nominal spectral resolution of 2 cm–1. Background and steady-state spectra were obtained from 128 co-added scans. During the diffusion experiments, 20 scans were co-added to provide each spectrum. The ATR flow cell was equipped with a horizontal ZnSe internal reflection element (50 x 10 x 2 mm3) with an incidence angle of 45° and a refractive index of 2.4. The depth of penetration of the evanescent wave was estimated, assuming a biofilm refractive index close to that of water (
= 1.33), to vary between 1 and 4 µm in the mid-IR domain.
The diffusion of the solutes in the biofilms was probed using their increasing contribution in the IR spectra, caused by their diffusion through the biofilms, towards the ZnSe crystal. The contribution of the solutes was highlighted by spectral difference, using the biofilm spectrum recorded prior to the diffusion. Different spectral regions were integrated in order to follow the penetration of the different solutes: the CH stretching region (2830–3000 cm–1) for CPC and DPC, the CO stretching band (1020–1160 cm–1) for PEG10k (as used previously9) and the C=C and C=N in-plane vibrations of the pyridinium group (1475–1500 cm–1) for TMBPC.
In order to describe the evolution of the solute concentration at the base of the biofilms, as measured by ATR-IR spectroscopy, the equation associated with the solute diffusion through a slab of uniform thickness was adapted from ref. 27, to express the variables in spectroscopic terms:
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is the area of the solute band at infinite time and B is the rate of penetration of the solute. This simplified model assumes that the ATR crystal was inert, and the biofilms were homogeneous layers, with a constant thickness, whose transport properties could be described by a single mutual-diffusion coefficient. Despite the fact that these assumptions are rather crude, they were sufficiently respected so the model could reproduce well the main features of the penetration profiles of the different analytes studied here. The values of B and I were obtained by the least-square fit of the variation of I(t) as a function of time, using Eqn 1. From B, the mutual-diffusion coefficient, Dbiofilm, was obtained using:
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The mean thickness of the biofilms was determined as described previously.9,28 The average value was obtained, at the end of the experiments, from 30 positions measured at random positions in the biofilms. This allowed the calculation of a global average thickness encompassing both columns and the base. The thickness averaged over all the biofilms grown under given conditions (Figure 4b) was used for the determination of Dbiofilm. Dbiofilm is indeed dependent on the thickness value used in the calculations (actually proportional to l2) and the identification of a single value of l can be challenged. However, the model using this approach provides reasonable fits for all the experiments and it allows expression of the diffusion in terms of a customary parameter.
The penetration of a solute in biofilms, P, was estimated from the ratio
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The reported values correspond to the average ± sample standard deviation for, at least, n = 3.
NMR self-diffusion measurements
The measurements were performed using a AV-400 Bruker spectrometer operating at 400.13 MHz and equipped with a diffusion probe Bruker DIFF 60. The self-diffusion coefficients for DPC and TMBPC, dissolved in D2O, were measured, at 23°C, along the z-axis, using the stimulated echo pulse sequence.29 The gradient's pulse length was 1 ms, and the delay between the two gradients' pulses was 100 ms. Typically 64 scans were co-added. The gradients were calibrated to provide the known diffusion coefficient of water in a H2O/D2O mixture at various temperatures.30 The measurements were performed as a function of the solute concentration and extrapolated to a 0 concentration. The concentrations for DPC were between 75 and 200 mM to ensure that the micellar form dominated.
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Diffusion of QAC molecules
The diffusion kinetics of CPC, DPC and TMBPC were determined in biofilms grown in milieu containing 0.5% sucrose; the resulting penetration profiles are presented in Figure 2 and the calculated diffusion parameters are summarized in Table 1. During these kinetic experiments, the CPC concentration at the base of the biofilms increased steadily during nearly 2 h after a lag phase of 
10 min and reached a plateau at an integrated intensity corresponding to a penetration of 70% (i.e. the area of the band characteristic of CPC corresponded to 0.7 of that measured from the spectrum of the CPC-containing milieu in the absence of biofilm). The curves, fitted using Eqn 1, provided a diffusion coefficient of 3.1 ± 0.4 x 10–12 m2/s, a value in good agreement with 1.9 ± 0.9 x 10–12 m2/s previously reported.9 The penetration rate and diffusion coefficient of DPC were 9.7 ± 4.4 x 10–4 s–1 and 1.2 ± 1.0 x 10–11 m2/s, respectively. The penetration of DPC is in the same order of magnitude of that of CPC; the DPC concentration at the base of the biofilm reached 76% of that in the solution flowing above the biofilm. The DPC diffused faster than CPC, reaching a plateau in <1 h. The diffusion of TMBPC in biofilms was considerably faster than that of CPC and DPC. There was no lag phase in the diffusion of TMBPC, indicating that a detectable amount of the solute reached the base of the biofilm in <1 min. A plateau concentration was reached in 10 min. The diffusion coefficient of TMBPC was calculated to be 2.56 ± 0.23 x 10–11 m2/s in 0.50% sucrose biofilms, nearly 10 times greater than that of CPC. Its penetration was only 49.6 ± 1.6%. As a control, the diffusion of PEG10k in the 0.5% sucrose biofilms was examined. PEG10k concentration increased steadily to reach a plateau after 15 min. The resulting diffusion coefficient was 5.4 ± 1.3 x 10–11 m2/s, a value in agreement with a previous report (2.7 x 10–11 m2/s).9 Contrary to CPC, DPC and TMBPC could be eliminated from biofilm by washing with fresh TYE sucrose broth. TMBPC could be totally eliminated from biofilms in around 15 min, whereas 20% of DPC were still present after 3 h of washing (Table 1).
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Effect of EPS density on the diffusion
IR spectra of 20-h-old S. mutans biofilms, cultured on the ZnSe crystal with different sucrose concentrations, are presented in Figure 3, after subtracting the contribution of the aqueous milieu. It was possible to estimate the density of the biofilm exopolysaccharide matrix by measuring the surface of C–O stretching bands, between 900 and 1200 cm–1, mainly representative of the polysaccharides31,32 (Figure 4). A 3-fold increase of the sugar band area was observed when the biofilms were grown with a sucrose concentration increasing from 0.1% to 2.0%. Assuming that the volume of the biofilm sampled by the evanescent wave is constant, this result indicated that EPS quantity increased up to 3-fold at the base of the biofilm, assuming that the nature of the produced EPS remained the same. It has been previously shown that the base of similar biofilms grown with 0.5% sucrose was mainly occupied by water (98% expressed as volume percent), leaving only 2% for the mean biomass content,9 a value consistent with other studies.33,34 The biomass content was then estimated to vary from 0.7% to 2.5%, when the sucrose content of the medium was increased from 0.5% to 2%. The area of the Amide II band whose maximum is at 1540 cm–1, arising mainly from the peptidic bonds of proteins, displayed very limited variations for the investigated growth conditions. Moreover, the main band of DNA, the antisymmetric phosphate stretching observed between 1200 and 1270 cm–1, displayed a much more limited increase. These observations strongly support that the substantial area increase of the bands between 900 and 1200 cm–1 could be mainly attributed to an enhanced production of EPS. The surface of the sugar band increased rapidly with increasing sucrose concentration and reached a plateau for concentrations 
0.5% (Figure 4a). The presence of this plateau indicates that the EPS concentration in the volume sampled by the evanescent wave (1–4 µm immediately above the ATR crystal, over the whole crystal) reached a maximum. Biofilm thickness also increased with sucrose concentration from 61 ± 21 µm for 0.1% sucrose to 230 ± 23 µm for 2% sucrose biofilms (Figure 4b). This increased average thickness indicates a faster growth of the biofilms when the milieu contained a larger sucrose content, in agreement with previous results.18 It is also interesting to note that the non-linear relationship of the polysaccharide content and the biofilm thickness with the sucrose concentration is reminiscent of the S. mutans biofilm strength previously reported.18 That paper showed an increase of the biomass content when the sucrose concentration was increased in the growth medium and it was suggested that this increase originates mainly from an increase of the EPS matrix density. The data presented here provide experimental support to this hypothesis. Macroscopically our biofilms showed a structure made of a coalescence of protruding microcolonies on a thick layer completely covering the ATR crystal. Under the microscope, S. mutans biofilms consisted of columns and mushroom-like structures over a heterogeneous layer of variable thickness. No significant differences could be observed for the biofilms grown in the presence of different sucrose concentration. It should be pointed out that a previous study examining similar biofilms reported a clear difference in the microscopic-scale architecture, using confocal laser scanning microscopy, between the biofilms grown with 0.1% and 1% sucrose.18 Therefore, even though our non-stained biofilms appeared similar, differences are expected at a smaller scale.
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The penetration of PEG10k and CPC were determined in biofilms grown in the presence of various sucrose concentrations, leading to different EPS densities. Our study reveals that an increase in EPS density has a significant effect on the penetration of PEG10k in the biofilm (Figure 5). The penetration of PEG10k was 64 ± 9% for biofilms grown in the presence of 0.10% sucrose and decreased to 50 ± 16% when the medium contained 0.25% and 0.50% sucrose and to 32% for biofilms grown with 1% sucrose. No or little PEG10k penetration could be measured with biofilms grown with 2% sucrose. In fact, for several experiments with biofilms grown in these conditions (4 out of 6), no PEG10k signal could be detected, even after 2 h of diffusion, suggesting that the PEG10k could not reach the base of the biofilm. These variations are considered representative of changes in PEG10k concentration considering that the sampling depth should be practically constant. The sampling depth depends on the refractive index of the milieu above the crystal but this is only weakly influenced by the change in EPS concentration [e.g. it changes only by 2.5% when the sucrose concentration changes from 5% to 25% (w/w)].35
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The increase in EPS density had a different effect on the CPC penetration in the biofilms. When the EPS density was low, for the biofilms grown with 0.10% sucrose, CPC at the base of the biofilm was detected at concentrations above its concentration in the flowing medium, demonstrating that CPC bioaccumulates at the base of the biofilms. For a sucrose concentration in the growth medium between 0.25% and 2%, the CPC penetration was lower and rather constant at
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Discussion |
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This study was performed to investigate the role of the positively charged ammonium group of QACs on their transport properties in biofilms. In order to assess the possibility of specific interactions between QACs and biofilm matrices, we examined the impact of the EPS density on the transport properties of CPC and of the molecular details of QACs on their diffusion in biofilms. First, the diffusion of CPC in biofilms is slow compared with solutes of similar size (ref. 9 and the present results). Second, the increased EPS density triggered by increased sucrose concentration in culture medium should have considerable impact on the diffusion of CPC in the biofilms if specific interactions exist between the detergent and some components of the biofilms. However, the penetration of CPC in biofilms was practically independent of the EPS matrix density. Conversely, the penetration of PEG10k was considerably reduced for dense biofilms. It has also been shown that a significant fraction of biofilms is inaccessible to solutes and this fraction was dependent on the size of solute. The present study reports that the penetration restriction is also dependent on the density of the EPS matrix. Steric exclusion is likely to be the origin of the decreased penetration of PEG10k at the base of the biofilms observed for increased EPS density. CPC micelles and PEG10k have equivalent size so both species should experience analogous steric exclusion from the EPS matrix. A different behaviour is however observed for CPC. There is a significant decrease of penetration when the growth medium contains 0.25% relative to 0.1% and it is fairly constant for sucrose content varying between 0.25% and 2%. This indicates that at least one additional factor influences the diffusion of CPC in biofilms. It is noted that for biofilms grown in the presence of 0.1% sucrose, the concentration of CPC in the region sampled by the evanescent wave is even greater than that for the flowing medium, indicating the bioaccumulation of the antibacterial detergent in these biofilms. This phenomenon demonstrates the existence of attractive interactions between CPC and the biofilms. When the biofilm matrix becomes denser, the balance between two phenomena with opposed effects is likely the origin of the relatively constant overall accumulation of CPC in biofilms. On the one hand, there should be a more restricted access of CPC throughout the biofilms, due to steric interactions, on the basis of the PEG10k behaviour. On the other hand, the increased EPS density can promote CPC association by providing more binding sites (but in a more limited accessible proportion of the biofilm). Therefore, this data set indicates a strong interaction between CPC and biofilm components.
In order to examine the role played by the quaternary ammonium group in this association, two other molecules containing pyridinium groups were examined. The diffusion of DPC and TMBPC is faster than that of CPC, their Dbiofilm being 4–8 times larger than that of CPC. This difference includes a contribution associated with the size difference. In order to normalize this contribution and, consequently, highlight the influence of the structure, the relative diffusion coefficients, Drelative, are also included in Table 1. These values are obtained by dividing Dbiofilm by the self-diffusion coefficient in water, Dwater, at infinite dilution. The latter were obtained from the literature or measured by NMR spectroscopy. The diffusion coefficient of PEG10k in biofilm corresponds to 70% of its diffusion in water, at infinite dilution. Conversely, the three investigated QACs show a Drelative 10 times smaller. These results could suggest that the presence of a quaternary ammonium functional group reduces the diffusion of a solute in biofilms. However, the small size of TMBPC (the hydrodynamic radius, RH, is estimated at 4.5 nm) and, to a certain extent, of DPC micelles (RH 
14 nm) could also contribute to this low Drelative. It was shown that PEG 200, a molecule with a size similar to TMBPC, also displays a low Drelative (Drelative = 0.041).9 This was interpreted by the diffusion of the small molecular species in parts of the biofilms that are very dense and where the analyte diffusion is slow. The similar penetration for TMBPC and PEG 200 supports this interpretation. Therefore, the contribution of the pyridinium group on the diffusion of TMBPC appears to be limited. The pyridinium functional group does not appear to impede drastically the diffusion. In addition it does not appear to be sufficient to lead to accumulation in biofilms and to the quasi-irreversible adsorption of the molecules to biofilms, as illustrated by the fact that TMBPC could be totally eliminated from the biofilms. DPC displays a penetration similar to that of CPC but the association with the biofilms is more reversible as the large majority of DPC can be washed from the biofilms. Therefore, we conclude that the hydrophobic interactions involving the alkyl chain of the detergent likely play a considerable role in the association of CPC to biofilms.
The present study reveals that ammonium groups cannot be considered as strong binding anchors to biofilms. As a consequence, the graft of ammonium groups to molecules/particles may not warrant efficiency for the targeting towards biofilms. A combination of the ammonium group and a long alkyl chain appears to be required for strong association to biofilms. In fact, it has been shown that the bactericidal activity of QACs depends on their alkyl chain length. Chains between 10 and 18 carbon atom long were a molecular requirement to display antibiotic activity.7,11,17 Bacterial membranes are proposed as potential association sites for QAC association. However, on the basis of the present findings, the putative attractive interactions between the positively charged ammonium groups and the negatively charged lipids of bacterial membranes cannot be considered as the prime driving force for the association. This finding is consistent with the limited increase of affinity found for CPC when anionic phosphatidylglycerol was introduced in neutral phosphatidylcholine bilayers.36 There are other anionic sites in biofilms such as anionic groups of EPS, and of the cell wall components. The location of the CPC association sites and the various energetic contributions (including at least steric exclusion, hydrophobic and electrostatic interactions) responsible for its slow diffusion but high penetration must be investigated in detail.
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NSERC (Canada) provided funding. This work was also funded by FQRNT (Québec) through its financial support to the Center for Self-Assembled Chemical Systems (CSACS).
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None to declare.
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We thank NSERC (Canada) and FQRNT (Québec) for funding.
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