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

https://doi.org/10.1016/j.jcis.2019.07.019Get rights and content

Abstract

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.

Introduction

Supramolecular hydrogels are 3D networks showing ability to entrap water and remarkable viscoelastic properties. The non-covalent 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], [8], [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], [12], [13], [14], [15], [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], [23], [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], [26], [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], [42], [43], [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 (β-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.

Section snippets

Synthesis of the β-l-PheDC

Scheme 2 reports the synthetic route for β-l-PheDC preparation. Briefly, BOC protected l-phenyl alanine and 3(β)-amino-12(α)-hydroxy-5β-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

From β-l-PheDC micelles to Ca2+ induced gels

β-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 self-assembly [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

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

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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