Deoxycholic acid and l -Phenylalanine enrich their hydrogel properties when combined in a zwitterionic derivative

Journal of Colloid and Interface Science 554 (2019) 453–462

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Regular Article

Deoxycholic acid and L-Phenylalanine enrich their hydrogel properties when combined in a zwitterionic derivative Leana Travaglini a,1, Maria Chiara di Gregorio a,⇑,1, Emilia Severoni a, Andrea D’Annibale a, Simona Sennato b,c, Franco Tardani b, Mauro Giustini a, Marta Gubitosi a, Alessandra Del Giudice a, Luciano Galantini a,⇑ a b c

Department of Chemistry, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy CNR-ISC UOS Sapienza, Sapienza University of Rome, P. le A. Moro 5, 00185 Roma, Italy Department of Physics, Sapienza University of Rome, P. le A. Moro 5, 00185 Roma, Italy

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

Article history: Received 24 April 2019 Revised 5 July 2019 Accepted 7 July 2019 Available online 8 July 2019 Keywords: Hydrogels Phenylalanine Bile salts Zwitterionic gels Metallogels

a b s t r a c t Hypothesis: Sodium Deoxycholate (NaDC) and Phenylalanine (Phe) are important biological hydrogelators. NaDC hydrogels form by lowering the pH or by increasing the ionic strength. Phe gels form from saturated solution by thermal induction and slow kinetics. The resulting gels hold great potential in medicine and biology as drug carriers and models for fundamental self-assembly in pathological conditions. Based on this background it was hypothesized that a Phe substituted NaDC could provide a molecule with expanded gelling ability, merging those of the precursors. Experiments: We coupled both building blocks in a zwitterionic derivative bearing a Phe residue at the C3 carbon of NaDC. The specific zwitterionic structure, the concurrent use of Ca2+ ions for the carboxyl group coordination and the pH control generate conditions for the formation of hydrogels. The hydrogels were analyzed by combining UV and circular dichroism spectroscopies, rheology, small angle X-ray scattering and atomic force microscopy. Findings: Hydrogel appearance occurs in conditions that are uncovered in the case of the pure Phe and NaDC: self-standing gels form instantaneously at room temperature, in the 10–12 pH range and down to concentration of 0.17 wt%. Both thixotropic and shake resistant gels can form depending on the derivative concentration. The gels show an uncommon thermal stability in the scanned range of 20–60 °C. The reported system concurrently enriches the hydrogelation properties of two relevant building blocks. We anticipate some potential applications of such gels in materials science where coordination of metal ions can be exploited for templating inorganic nanostructures. Ó 2019 Elsevier Inc. All rights reserved.

⇑ Corresponding authors at: Department of Organic Chemistry, Weizmann Institute of Science, 234 Herzl Street, 7610001 Rehovot, Israel (M.C. di Gregorio). 1

E-mail addresses: [email protected] (M.C. di Gregorio), [email protected] (L. Galantini). These authors contributed equally to this work.

https://doi.org/10.1016/j.jcis.2019.07.019 0021-9797/Ó 2019 Elsevier Inc. All rights reserved.

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1. Introduction Supramolecular hydrogels are 3D networks showing ability to entrap water and remarkable viscoelastic properties. The noncovalent character of the stabilizing interactions imparts higher structure responsiveness, reversibility, and adaptability than the polymeric gels. Owing to these properties, hydrogels turn out to be versatile materials for cosmetic formulation, tissue growth, drug delivery, catalysis and food industry [1]. Within this frame, hydrogels formed by biomolecules are of particular interest due to their higher tendency to address requirements of biocompatibility in applications occurring in biological contexts [2,3]. Bile salts (BSs) are bio-surfactants produced in the liver and stored in the gallbladder. They play a critical role in the emulsification of fats and fat-soluble vitamins for their absorption in the intestine. Moreover, they are responsible for further physiological functions such as elimination of excess cholesterol and regulation of enzymes and ion channel activities [4]. Some of the BSs form hydrogels [5,6]. The gel ability is induced by the presence of several hydrophilic (hydroxyl and carboxylate groups) and hydrophobic moieties (steroidal nucleus) that, being heterogeneously located in the molecular structure, enable for a complex and multidirectional network of hydrophobic and hydrogen bond interactions [6]. The bile salt sodium deoxycholate (NaDC) forms gels upon protonation of the carboxylic groups in water (by lowering the pH) or upon addition of charge screening salts. The resulting gels have been largely characterized in the past decades and, more recently, tested as novel drug carriers for administration of several active molecules [7–9]. Due to their thixotropic properties, NaDC gels have been shown to allow for improved performances compared to conventionally used polymeric matrices when applied on nasal and buccal membranes [10]. Gelling properties can be tuned or introduced in not naturally gelling BSs by varying ad hoc the hydrophobic/hydrophilic balance of the system [6,11–16] or by introducing additional stabilizing forces. This aim has been achieved both by the use of suitable organic additives [7] and the design of new BS derivatives [5,6,17]. Interesting gelling strategies are those that rely on the introduction of electrostatic attractions or on the use of metal cations as coordinative elements between the BS molecule carboxylic heads [5,6]. On one hand, by the first strategy, mixtures of oppositely charged BA derivatives have allowed for enhanced gel efficiency [18] and charge [19,20] and morphological [20] modulation. On the other hand, by the latter approach, a broad series of metal cholate or metal lithocholate gels have been prepared [5,21]. However much fewer examples of metal deoxycholate gels have been reported [22–24]. Metal-BS gels find many applications in material chemistry as templates and precursors of metallic, semiconductive nanostructures and metal-organic frameworks [5,8,21,25–27]. L-Phenylalanine is one of the building blocks of life. It is an essential amino acid in humans and its introduction through the diet is crucial for peptide synthesis and for the biosynthesis of tyrosine. Phe self-assembly has been extensively investigated because it is related to the formation of toxic amyloid fibers in phenylketonuria disease [28,29]. Phe has importance also in drug formulation. Indeed, Phe aids in melatonin production and it is efficient in the treatment of skin diseases as vitiligo [30] and melasma [31]. In this context, gels are often desirable for topical administration. This issue has been addressed so far by using polymeric matrices as Phe vehicles [32,33]. This is likely due to the poor knowledge about the gelling ability of pure Phe. Indeed, despite the large literature on Phe self-assembly in solid [34,35] and amyloidal [36,37] phases, hydrogel formation has been only recently observed and analyzed. In 2002, Myerson reported a mixed

crystal- hydrogel phase forming in mixtures of Phe and aspartame [38]. Thakur in 2014 [39] and Lloyd in 2018 [40] described for the first time the conditions for pure Phe hydrogel preparation. Gels turned out to form by heating supersaturated Phe water solutions (concentration range 212–605 mM) followed by slow cooling process. The process of the gel formation is quite slow, requiring from 1 to 24 h. The gels are opaque and are formed by a matrix of fully crystalline fibers, with no indication of amorphous or semicrystalline phases. As previously described for BSs, it has been shown that specific derivatizations can vary the self-assembly properties of Phe, giving rise to more efficient gelling systems than the precursor [41–44]. Such gels have been largely explored over the past decade and used not only in bio-applications, such as drug delivery and extracellular matrix for tissue engineering, but also for trapping dyes, heavy metals or pollutants, and sensing explosives [45]. In the light of the broad interest in NaDC and Phe hydrogels, in this work we couple both building blocks in a new derivative. Phe was linked to the steroidal nucleus of DC
anion at the C3 position by substituting the original OH group (b-L-PheDC, Scheme 1, left). Such a derivatization favors an increase of the hydrophobic interactions of the steroidal region. Furthermore, Ca2+ ions were introduced in the sample for the coordination of the carboxylic heads, allowing thus for an efficient involvement in the assembly of the flexible chains as well. The overall strategy enabled an unprecedented result: the formation of hydrogels that are based concurrently on Phe and NaDC. Indeed, results reported so far demonstrate that, in general, addition of aminoacidic solutions breaks the gel matrix of NaDC [46] and previously reported Phe conjugated deoxycholic acid derivatives were not gelling [47]. The self-assembly investigation was conducted by microscopic and spectroscopic techniques that revealed the formation of gels with kinetics, and under pH and concentration conditions, that are instead not efficient for the gelation of the single precursors. 2. Materials and methods 2.1. Synthesis of the b-L-PheDC Scheme 2 reports the synthetic route for b-L-PheDC preparation. Briefly, BOC protected L-phenyl alanine and 3(b)-amino-12(a)-hy droxy-5b-cholan-24-oate 1 were condensed according to a previously reported literature procedure [48,49] i to obtain the ester precursor of the final compound 2. Subsequently, BOC removal ii and hydrolysis of the ester moiety on the side chain iii afforded the desired product. Details of the synthetic procedure, reagents and compound characterization were reported in the Supplementary Material. 2.2. Sample preparation b-L-PheDC does not solubilize either in pure bi-distilled H2O or in acidic conditions, whereas transparent solutions form at high pHs (10.0-12.0). The pH of the analyzed samples was adjusted by addition of titrated solutions of NaOH or HCl. For the hydrogel formation, stock solutions of b-L-PheDC (2.0  10
2 or 6.0  10
3 M) in NaOH solution (pH 12.0) and of CaCl2 (1.0  10
2 or 3.0  10
3 M) in bi-distilled H2O were prepared. Subsequently 0.5 mL of the b-L-PheDC solution and 0.5 mL of the CaCl2 solution were withdrawn and mixed in a third vial. Homogeneous hydrogels immediately formed after vortex shaking the sample for few seconds. The final b-L-PheDC/CaCl2 molar ratio and surfactant concentration in the hydrogels were 2:1 and 1.0  10
2 or 3.0  10
3 M, respectively.

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Scheme 1. Schematic representation of the structures formed by the self-assembly of b-L-PheDC (molecular structure on the left side) at different experimental conditions: (a) at pH 12, in the presence of CaCl2 (b-L-PheDC: CaCl2 = 2:1) at pHs (b) 12 and (c) 10. Red panels on the right side report the molecular packing hypothesized for the aggregates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

In order to assess the role of CaCl2, CaCl2 solutions at concentration above or below the optimum gelling concentration were added to b-L-PheDC solutions at pH 12, following the procedure previously described for the gelling samples. The analyzed samples had a final b-L-PheDC concentration of 1.0  10
2 M and b-L-PheDC/CaCl2 molar ratio of 20:1, 10:1, 5:1 and 1:1. In order to assess the role of the pH, HCl was added to the b-LPheDC/CaCl2 2:1 sample at pH 12. For the estimation of minimum PheDC concentration for gel formation, gel at different PheDC/CaCl2 molar ratios were diluted by progressive additions of water in aliquots till the gel breaking. The samples were stirred after each addition and then let to rest for 40 min to allow the gel to reform. Each water added aliquots provided increments of the sample of 10–25% in weight. Gel was considered to be formed when the solution did not change the shape after vial tilt of 45°.

2.3. Surface tension A computerized Lauda instrument was used for the measurement of the surface tension c, based on the ring detachment method. Alkaline samples (pH 12) at increasing concentration of b-L-PheDC were prepared and measured. The temperature of each measurement was set at 20.0 ± 0.1 °C by a Peltier system. Subsequently, the measured c values were reported as a function of the sample concentration in a log scale. The critical aggregation concentration (cac) was obtained determining the point where the slope of the initial linear trend drastically changes. 2.4. UV–vis absorption and circular dichroism (CD) A JASCO instrument, model 715, was used for collecting UV–vis and CD spectra. To determine only the contribution of the b-L-

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Scheme 2. (i) Boc–Phe-OH, N-(3-Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), dry CH2Cl2, r.t., N2, 16 h, 54%; (ii) TFA/ CH2Cl2, r.t., 4 h, 82%; (iii) LiOH, H2O, MeOH, r.t.,16 h, 90%.

PheDC derivative, the curves of the solvent were subtracted to the samples. All the data were normalized for b-L-PheDC concentration and path lengths, by plotting the UV and CD spectra in molar extinction coefficient e and molar ellipticity [h], respectively. The spectra span the 190–300 nm wavelength k range. Quartz cuvettes with 0.1 or 0.5 mm path length were used. The spectral resolution was 1 nm. The plotted spectra are the average of four consecutive scans. 2.5. Small angle X-ray scattering (SAXS) The SAXS measurement on the pure b-L-PheDC sample at pH 12 was performed at the MAX II SAXS beamline I911-4 at MAXIV Laboratory in Lund, Sweden [50]. The solution was injected into a thermostated quartz capillary and equilibrated for ca 20 min before measurement. The wavelength used was k = 0.091 nm, the SAXS patterns were recorded on a 165 mm diameter MarCCD detector at a sample-detector distance of 1963 mm and were processed using the Fit2Dsoftware. The scattered intensities were recorded in the 0.1–4.5 nm
1 scattering vector q range (q = 4psin(h)/k, where 2h is the scattering angle) and corrected for the contribution of both the solvent and the capillary. The SAXS measurements on the samples formed in the presence of CaCl2 were instead performed using a Xeuss 3.0 HR P300K QXoom system (Xenocs SA, Sassenage, France), equipped with a two-dimensional Pilatus3 R 300 K detector (Dectris Ltd., Baden, Switzerland) and a micro-focus Genix 3D X-ray source (k = 0.1542 nm). The SAXS patterns were processed using the FoxTrot software developed at SOLEIL. The scattered intensities were recorded in the scattering vector q ranges of 0.06–6.0 nm
1 (for samples at pH 12) and 0.16–4.7 nm
1 (for the sample at pH 10). The data thus obtained were further analyzed by SasView [51]. The SAXS profile of the pure b-L-PheDC sample at pH 12 was fitted by using the form factor of an ellipsoid. The global pair distance distribution function p(r) was inferred for both the pure b-LPheDC and the b-L-PheDC/CaCl2 samples by the indirect Fourier transform (IFT) of the I(q) profile [52]. The maximum particle distance Dmax, the radius of gyration Rg and the scattered intensity extrapolated at zero angle I(0) were also estimated from the p(r). The cross-section pair distance distribution functions of infinitely long objects pcs(r) were obtained for the b-L-PheDC/CaCl2 samples

by the IFT [53] of the I(q)q profiles. A model-based fit with the form factor expression for a flexible cylinder was also perfomed [54]. The micelle aggregation number nagg value was estimated for the pure b-L-PheDC aggregates according to an approach previously described [8], by using the value of the scattered intensity in absolute units I(0) = 0.0091 ± 0.0001 cm
1, proper volumes for PheDC
, Na+ and Cl
ions [55] and an ionization degree a of 0.7. The a value is reported to be 0.7–0.8 for sodium deoxycholate [18] and for an analogous derivative [16]. Since the analyzed concentration is remarkably higher than the critical aggregation concentration (cac), [17] the surfactant was assumed to be totally involved in the micelle formation. 2.6. Atomic force microscopy (AFM) AFM measurements were performed using a DIMENSION ICON (Bruker AXS, Germany) equipped with a Nanoscope V controller. Images were collected in air in tapping mode by using high resolution rotated-tapping mode etched silicon probes (RTESP). These probes have nominal tip radius of 8 nm and resonant frequency around 300 kHz. The samples for measurements were prepared by depositing aliquots of 10–20 lL of each sample onto the freshly cleaved mica surface and after 5 min the surface was gently washed with 200 lL of Milli-Q water. The mica surface with the adsorbed sample was then flushed with a stream of nitrogen for drying and analyzed after 30 min. Images were analyzed using Gwiddion 2.45 free software. Images have been corrected by elimination of background and levelling defects. 2.7. Oscillatory rheology measurement A stress-controlled TA AR-1000 unit was used, where the rotor position, driven by an air flow, is controlled by a proper electronic apparatus. Cone–plate geometry was employed. To ensure reproducibility of the measurements, a volume of 0.5 mL was always used. Pre-shear procedures were applied to avoid possible errors due to different sample mechanical histories. The temperature of the plate was controlled by a Peltier unit, operating in the range between
10 and 99.9 °C with an accuracy of 0.02 °C. A cap was

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used to prevent solvent evaporation from the sample during the measurements. Stress-sweep experiments were performed to define the linear viscoelastic regime at a constant frequency of 1 Hz. Frequencysweep procedures were performed in a 0.01–40.0 Hz frequency range at a fixed applied stress and temperature.

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to those observed in samples at concentrations below the cac. These data suggest weak interactions between the b-L-Phe moieties within the micelles.

3. Results and discussion 3.1. From b-L-PheDC micelles to Ca2+ induced gels b-L-PheDC cannot be solubilized in water at neutral and acidic pHs, however it completely dissolves at high pHs (10.0–12.0). Although the pKa of the derivative can be affected by the selfassembly [56,57], based on the pKa of the precursors (deoxycholic acid HDC pKa 6.6, Phe NH2 9.3) we suppose that solubilization at these pHs occurs because of the carboxylic and ammine group deprotonation, meaning their state in the form of COO
and NH2 respectively (see Scheme 1). At pH 12.0, surface tension measurements show a cac of 0.9 mM for b-L-PheDC, lower than the value measured for the precursor HDC (2.9  10
3 M, Fig. S1). The p(r) profile indicates the presence of prolate ellipsoidal micelles at concentration above the cac (1.0  10
2 M) (Fig. 1a, bottom). The SAXS curve is well fitted by considering an ellipsoid with semi-major and semi-minor axis values of 2.38 ± 0.01 and 1.18 ± 0.01 nm, respectively (Fig. 1a, top). The aggregation number is 12.8 ± 0.5, in agreement with the values reported in the literature for NaDC micelles of similar dimensions [58]. UV absorption and CD spectroscopy provided insights on the molecular packing. The UV absorption spectrum exhibits a broad band between 200 and 230 nm (Fig. 1b), ascribable to the La and the n-p* transitions of the L-Phe aromatic ring [19] and amide bond, respectively. The corresponding CD spectrum shows in the same wavelength range a positive band centered at 220 nm. The spectra do not vary upon a temperature increase and are similar

Fig. 2. CD and UV spectra of b-L-PheDC 1.0  10
2 M at different b-L-PheDC/CaCl2 molar ratios at pH = 12.0 and at 20 °C.

Fig. 1. (a) SAXS profile (top) and inferred p(r) function (bottom) of b-L-PheDC 1.0  10
2 M at pH 12.0 and 20 °C; the fit obtained by using the form factor of an ellipsoid is reported in the top panel as red curve. (b) CD and UV spectra of b-L-PheDC 1.0  10
2 M at pH 12 as a function of temperature. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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The b-L-PheDC/CaCl2 molar ratio was varied from 20:1 to 1:1, while the b-L-PheDC concentration (1.0  10
2 M) and the pH (12.0) were kept constant: a marked change in the self-assembly was observed upon increasing the CaCl2 fraction. The first addition of CaCl2 (ratio 20:1) led to the appearance of two positive CD peaks at 200 and 215 nm. They were roughly 14- and 6-fold, respectively, more intense than the one observed in the pure b-L-PheDC. The intensity of such peaks continued to slightly increase at b-LPheDC/CaCl2 molar ratio 10:1 and 5:1 while the spectral shape remained unchanged. Once the b-L-PheDC/CaCl2 molar ratio 2:1 was reached, both UV and CD spectra drastically changed. A remarkable increase of the molar extinction coefficient took place. Concurrently in the CD spectrum (i) the peak at 200 nm dropped

down, (ii) a broad positive band (210–241 nm) with maxima at 225 and 230 nm developed and (iii) a deep negative peak appeared at 195 nm (Fig. 2). Visually, a sol-gel transition occurred (vide infra, Fig. 4a). Such critical b-L-PheDC/CaCl2 molar ratio corresponds to an equivalent amount of positive Ca+2 and negative carboxylate charges. Therefore, these results suggested that Ca+2 ions stoichiometrically coordinate the carboxylic groups, significantly influencing the aggregation. We could demonstrate that at higher PheDC concentration gel can form also at lower fractions of CaCl2, meaning higher PheDC/CaCl2 molar ratios. The minimum PheDC concentration for gel formation is lower the larger the CaCl2 fraction (lower PheDC/CaCl2 ratio). Indeed, at pH 12, we could estimate that gel can form down to PheDC concentration of 0.90 wt%

Fig. 3. (a) SAXS profile (left) and inferred pCS(r) function (right) for a b-L-PheDC 1.0  10
2 M/CaCl2 2  10
3 M solution at pH 12 and 20 °C; the fit obtained by IFT (red line) and reference slopes are also shown in the left panel; (b) AFM topography and (c) phase images of the structures formed in the b-L-PheDC 1.0  10
2 M/CaCl2 2  10
3 M solution at pH 12; the micrograph in panel c corresponds to the region marked by a white square in panel b; white and cyan arrows in panel c point to helicoidally folded and not well formed structures, respectively; (d) SAXS profile (left) and inferred pCS(r) function (right) for the b-L-PheDC 1.0  10
2M/CaCl2 5  10
3 M gel at pH 12 and 20 °C; the fit obtained by IFT (red line) and reference slopes are also shown in the left panel; (e, f, g) AFM topography and (h) phase images of the structures formed in the b-L-PheDC 1.0  10
2 M/CaCl2 5  10
3 M gel at pH 12 and 20 °C; the micrographs in panels g and h correspond to the region marked by a black square in panel f; (i) height profiles of the structures labelled as 1 and 2 in panel g. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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(1.5  10
2 M), 0.71 wt% (1.2  10
2 M) and 0.31 wt% (6.0  10
3 M) at PheDC/CaCl2 molar ratios of 4.0, 2.7 and 2.0, respectively. AFM images and SAXS data were collected on the sample b-LPheDC/CaCl2 at molar ratios 5:1 and 2:1 (Fig. 3). The SAXS profiles upon addition of CaCl2 remarkably changed compared to the one observed for the micelles. The Log-Log SAXS data plot showed a slope close to
1 at q > 0.14 nm1 both in the case of the b-LPheDC/CaCl2 5:1 solution and the b-L-PheDC/CaCl2 2:1 gel (Fig. 3a, d left). This indicates that Ca2+ promotes the formation of elongated aggregates. Such aggregates turned out to be flexible since the curves bend at lower q values with a slope of about
2, a value typical of wormlike chains. Consistently, the lowest q regions of the curves can be fitted considering flexible cylinders [54] having a Kuhn segment of about 28.1 nm and 24.9 nm for the b-LPheDC/CaCl2 5:1 solution and the b-L-PheDC/CaCl2 2:1 gel, respectively (Fig. S2). The p(r) inferred for q > 0.14 nm
1 provided a function with a maximum at small distances related to the cross section and a linearly decaying profile, characteristic of rodlike shapes (Fig. S3). The cross-action pair distance distribution functions pcs(r) showed instead a structured profile with a maximum at 2 nm and a shoulder around 5 nm (Fig. 3a, d right), suggesting a non-homogeneous distribution of the electron density in the aggregate cross-sections. AFM images of the b-L-PheDC/CaCl2 5:1 sample revealed the presence of elongated and interconnected aggregates having both not well structured (Fig. 3b, c cyan arrows) and helicoidally folded (Fig. 3b, c white arrow) area. These latter regions became dominant in the gel at b-L-PheDC/CaCl2 2:1 M ratio (Fig. 3e–h). Such morphology has been already observed in other BS derivatives [15,59] and metal-cholate gels [25,26]. The ribbons presented a pitch of about 50 nm as evidenced by the periodic oscillations in the longitudinal AFM height profile (Fig. 3i left). Considering the maxima of the height profiles, an average diameter of 4.18 ± 0.36 nm can be inferred (Fig. 3i right). This value is remarkably lower than the pcs(r) inferred Dmax (about 9 nm), indicating that dehydration and flattening of the structure occurred upon sample deposition on the AFM support.

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3.2. Zwitterionic gels: effect of pH and b-L-PheDC concentration on the gel properties We have reported that the introduction of electrostatic interactions is a strategy to improve the efficiency and mechanical properties of the gels [18]. This issue was previously addressed by preparing mixtures of oppositely charged surfactants. We envisioned that in the b-L-PheDC system a similar result could be obtained without introducing further positively charged molecules but simply inducing a zwitterionic state in b-L-PheDC through pH lowering. Therefore, it was expected that the Ca2+ ions would not be actively involved in the gel structuring at pH values pKa of the NH2 group. However, since the fully zwitterionic state did not allow b-L-PheDC solubilization, we decreased the pH in the above described Ca2+-b-L-PheDC gel by concurrently exploiting the Ca2+ mediated assembly as molecular arrangement preconditioning for the formation of the zwitterionic gels. Consistently with this hypothesis, we observed that the b-L-PheDC/CaCl2 hydrogel at 1:2 molar ratio continued to be selfstanding when the pH was lowered down to 10 (Fig. 4a top). In all the cases the gels were not thixotropic. Finally, precipitation occurred by further lowering the pH. The dynamic rheology measurements showed improved mechanical properties of the gels upon lowering the pH. Oscillatory experiments in stress sweep mode at a fixed frequency (1.0 Hz) exhibited a widening of the linear viscoelastic domain (LVD) as the pH was decreased; the LVD regions were comprised between 0.11 and 5.0 Pa, 0.11–8.0 Pa and 0.11–20.0 Pa for the gels at pH 12.0, 11.0 and 10.0 respectively. Moreover, in Fig. 4b, the moduli G0 and G00 in frequency sweep measurements at a constant applied stress (r = 0.3 Pa), are reported. G0 remained larger than G00 at all frequencies for all the samples as expected for soft solids. A comparison among the G0 data of the three samples pointed out that the elastic component of the gels increased moving in the series pH 12 < 11 < 10 (Fig. 4b). Remarkably, CD and rheological measurements showed that the gels were significantly resistant to temperature increases (up to 60 °C), indicating, at all the pH values, a remarkable contribution of hydrophobic interactions to the molecular packing and network stabilization (Fig. 4c).

Fig. 4. (a) gel appearance (top) and CD/UV spectra of b-L-PheDC 1.0  10
2 M/CaCl2 5  10
3 M samples at different pH values and at 20 °C; (b) elastic G0 (filled circles) and viscous G00 (empty circles) moduli as a function of an ascending stress (left) and frequency (right) ramp, at 1 Hz and 0.3 Pa respectively, related to the b-L-PheDC 1.0  10
2 M/ CaCl2 5  10
3 M gels at pH 12 (top), 11 (center) and 10 (bottom) and 20 °C; (c) CD/UV spectra (top) and elastic G0 (filled circles)/viscous G00 (empty circles) moduli (bottom) as a function of increasing temperatures related to the b-L-PheDC 1.0  10
2 M/CaCl2 5  10
3 M gels at pH 12 (left) and 10 (right).

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The SAXS profile still showed the presence of elongated structures (Fig. 5a). However, a more pronounced asymmetry of the pcs(r) with a Dmax of 15 nm is compatible with a nearly rectangular rather than circular section of the aggregates (Fig. 5b). Accordingly, flat ribbons, having a much lower height (2.62 ± 0.16 nm) compared to the one estimated for the gel at pH 12 (Fig. 3i), were imaged in the diluted gel at pH 10 by AFM (Fig. 5c and d). These data indicate that the initial folded ribbons unwound into flat tape when the pH was lowered to 10.

Rheological data showed that at this pH the gel was kept down to a b-L-PheDC concentration of about 3.0  10
3 M (b-LPheDC = 0.17 wt%) although a decrease in the viscoelastic properties of the zwitterionic gel as a consequence of dilution could be detected: the LVD spans in the 0.0001 and 1.0 Pa range and the G0 was lower by about a factor 103 compared to the more concentrated pH analogue (Fig. 6a). On the other hand, the weaker interaction favored the appearance of thixotropic properties.

Fig. 5. (a) SAXS profile and (b) inferred pCS(r) function for b-L-PheDC 1.0  10
2 M/CaCl2 5  10
3 M gel at pH 10; the fit obtained by IFT (red line) and reference slopes are also shown in panel a; (c) AFM height image of the structures formed in the b-L-PheDC 3.0  10
3 M/CaCl2 1.5  10
3 M gel at pH 10; (e) AFM topography and (f) phase images of the region marked by a white square in panel c; (d) height profiles of the structures labelled as 1 and 2 in panel e. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. (a) elastic G0 (filled circles) and viscous G00 (empty circles) moduli as a function of an ascending stress (top) and frequency (bottom) ramp, at 1 Hz and 0.01 Pa respectively, related to the b-L-PheDC 3  10
3 M/CaCl2 1.5  10
3 M gel at pH 10 and 20 °C; (b) CD and UV spectra of b-L-PheDC 3  10
3 M/CaCl2 1.5  10
3 M at different pH values and at 20 °C; the inset shows the gel appearance of the sample at pH 10.

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The gel formation was not observed for the fully Ca2+ mediated structures (pH 12) at the same diluted surfactant concentration. An intermediate condition of viscous solution was observed at pH 11. Minor variations in the CD spectra were detected in the dilutes samples (Fig. 6b). 3.3. Evolution of the aggregates at the molecular scale Based on these results, a model was proposed to explain the Ca2+ induced gelation of b-L-PheDC, which is summarized in the Scheme 1. We envisioned that, at pH 12, the self-assembly of b-LPheDC molecules was limited to small micelles in the absence of Ca2+ ions, due to marked repulsions among the carboxylate groups. When Ca2+ was added, self-assembly into ribbons formed by arrays of molecules alternately oriented up and down occurred. In the interior of the ribbons the molecules were mainly stabilized by hydrophobic interactions among the rigid steroidal body of contiguous molecules and perhaps by hydrogen bonds involving their hydroxyl group. On the surface, depending on the conditions, different stabilizing contributions could be identified. At high pH, stabilization is provided by carboxylic groups fully crosslinked by Ca2+ ions and by hydrogen bonds and p -p interactions occurring between adjacent Phe residues. When the pH is lowered, -NH2 groups become partially protonated into -NH+3, thus competing with Ca2+ for the interaction with the carboxylic groups and further strengthening the intermolecular interactions. The variation of the interaction framework induced by the pH lowering, implies small changes in the reciprocal orientation of the molecules, reasonably accounting for the unwinding of the ribbon from helical to flat. 4. Conclusions Phe and HDC were covalently coupled in a novel derivative to mutually improve the gelling properties of the two building blocks. The derivative design aimed at (i) generating an extended hydrophobic region while keeping good solubility and (ii) placing at the opposite sides of the molecule two residues that could switch from negative charge/neutral state respectively to zwitterionic state within few pH units (10–12). This allowed for a variegated self-assembly behavior in water, guided by multiple typologies of interactions, namely p – p stacking, hydrophobic interactions and pH dependent metal-coordination and electrostatic interactions. Under pH and concentration control, hydrogels were obtained showing (i) rapid kinetic of formation, (ii) diverse morphology and rheological properties, (iii) remarkable temperature resistance. These properties are not afforded by the two single precursors under similar experimental conditions. Indeed, NaDC is able to form gels by lowering the pH down to the values where protonation of the DC
carboxylic group starts to occur. However, DC
is known to precipitate when coordinated with Ca2+ [60,61]. By contrast, the substituted derivative is observed to give rise to a stable and strong Ca2+ binding induced gel, which is reinforced by lowering the pH close to the protonation of phenylalanine NH2 group. At this pH conditions, the gel is observed to form down to derivative concentration of 0.17 wt%. Such gelling ability is remarkably larger than that of metal promoted gels of natural bile salts [21,25,62–64]. The reported calcium promoted gelation let us to envision a general ability of the derivative to provide gels at high swelling degree by exploiting metal coordination. This ability with different metals will be investigated in future works. Another interesting finding was the formation of hydrogels comprised of a remarkable fraction of low molecular weight molecules in zwitterionic state. Zwitterionic polymeric gels are largely used in nano-application [65–67]. However, it is challenging to

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obtain analogous low molecular weight hydrogels due to the poor solubility of the zwitterionic molecules. We avoided the molecule precipitation by ‘‘pre-templating” the molecular packing through metal-coordination and concurrently aiding the assembly with hydrophobic interactions. The resulting zwitterionic supramolecular gels turned out to exhibit improved rheological properties compared to the metallo-gels due to the neutralization of the system and the arising of electrostatic interactions. Acknowledgments The authors thank Prof. Victor Hugo Soto Tellini and Eduardo Valerio for supervising the organic synthesis and for the IR spectroscopy measurements, respectively. The authors also thank Sapienza University of Rome and Universidad de Costa Rica for financing the agreement of cultural and scientific cooperation. This work benefited from the use of the SasView application. We acknowledge the SAXSLab Sapienza facility at Sapienza University of Rome for the SAXS measurements.

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