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A shear-induced network of aligned wormlike micelles in a sugar-based molecular gel. From gelation to biocompatibility assays

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Abstract

A new low molecular weight hydrogelator with a saccharide (lactobionic) polar head linked by azide-alkyne click chemistry was prepared in three steps. It was obtained in high purity without chromatography, by phase separation and ultrafiltration of the aqueous gel. Gelation was not obtained reproducibly by conventional heating-cooling cycles and instead was obtained by shearing the aqueous solutions, from 2 wt% to 0.25 wt%. This method of preparation favored the formation of a quite unusual network of interconnected large but thin 2D-sheets (7 nm-thick) formed by the association side-by-side of long and aligned 7 nm diameter wormlike micelles. It was responsible for the reproducible gelation at the macroscopic scale. A second network made of helical fibres with a 10–13 nm diameter, more or less intertwined was also formed but was scarcely able to sustain a macroscopic gel on its own. The gels were analysed by TEM (Transmission Electronic Microscopy), cryo-TEM and SAXS (Small Angle X-ray Scattering). Molecular modelling was also used to highlight the possible conformations the hydrogelator can take. The gels displayed a weak and reversible transition near 20 °C, close to room temperature, ascribed to the wormlike micelles 2D-sheets network. Heating over 30 °C led to the loss of the gel macroscopic integrity, but gel fragments were still observed in suspension. A second transition near 50 °C, ascribed to the network of helical fibres, finally dissolved completely these fragments. The gels showed thixotropic behaviour, recovering slowly their initial elastic modulus, in few hours, after injection through a needle. Stable gels were tested as scaffold for neural cell line culture, showing a reduced biocompatibility. This new gelator is a clear illustration of how controlling the pathway was critical for gel formation and how a new kind of self-assembly was obtained by shearing.

Introduction

Low molecular weight (LMW) hydrogelators provide an alternative family of gelling agents compared with polymers, leading to soft materials with possible applications in the field of wet materials, switchable gels, controlled release or uptake, cell culture. LMW gelators belong to different structural families (peptides, cholesteryl, nucleobase or sugar amphiphiles, etc…) and the now quite large amount of work done on these self-assembling molecules has been well reported in several reviews [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. We are more especially interested in sugar-derived molecular gelators (see the recent review on this family of gelators and the following references for the most recent works [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50]). Sugar gelators provide generally a neutral hydrophilic polar head, with a low sensitivity to temperature changes on the contrary to PEG amphiphiles. In the context of biological applications, carbohydrate derived hydrogels will interact differently with biomolecules or cells compared with PEG or peptide derived gelators, notably by mimicking to some extent the saccharidic components of the glycocalix, composed of glycoproteins and glycolipids [22].

From a practical point of view, simple, rapid and cheap syntheses are essential when considering sugar-based LMW hydrogelators applications. In former results, a family of hydrogelators based on a disaccharide head has been described. It has been shown that the presence of a triazole linker enhanced gelation, but the synthetic pathway consisted of six steps [23], [24], [25], [26]. Other amphiphilic molecules with close structures (namely, a sugar head, triazole linkers, and a fatty chain) have been described as well, including hydrogelators [27], [28], [29], organogelators [30] and micelles [31], [32], [33], all of them involving also protection-deprotection multistep synthesis and purification by chromatography. In this work, a new gelator inspired from these structures has been prepared with a simpler synthetic route with only three steps starting from lactobionic acid as the polar head (Scheme 1). Another important aspect in the field of LMW hydrogelators is to control in a precise manner the supramolecular structure sustaining the gel. In the case of very flexible molecules many conformations are possible. It can give rise to polymorphism. Polymorphism is the main cause for the lack of reproducible gelation [34], [35], [36], [37]. Accordingly, the importance of controlling the conditions of the self-assembly in order to reach a reproducible final state, with the related macroscopic properties, has been well pointed out in several papers. But it still remains underestimated, quite unexplored and not well controlled [38], [39], [40], [41], [42]. Compound 3 is a typical example of such a situation. Its gelation behaviour appeared more complex than expected and gave the opportunity to explore the effect of different pathways for the gel preparation. The self-assembled structures have been elucidated by electronic microscopy and Small Angle X-ray Scattering (SAXS). The rheology and thermal transitions of the gel have also been studied. Finally, the results related to the use of these gels for cell culture are briefly discussed.

Section snippets

Preparation of the gelator

An easy access to the sugar based gelator 3 is represented in Scheme 1. The gelator was obtained in three synthetic steps and was purified without chromatography. The fatty chain 1 was purified by recrystallization (yield 60%) while the product 2 was obtained nearly pure by extraction and was used without further purification in the next step. After the azide-alkyne “click chemistry” step, leading to the compound 3, the unreacted and sparingly soluble azide was discarded by centrifugation. The

Conclusions

This new sugar-based gelator, despite an easy synthetic access, displayed quite a complex gelation behaviour in pure water due to polymorphism. The presence of two amides bonds, but separated from each other by a rigid spacer (triazole), forces the molecule to bend and favours the polymorphism. This observation may help the design of gelators. Without shearing, the gelation was capricious. Starting from 2 wt% solutions and applying a strong shear enabled to get reproducible gelation. The

Acknowledgements

We acknowledge the following people for their technical assistance: TEM: I. Fourquaux, D. Goudounèche (CMEAB); V. Sartor, S. Gineste (IMRCP); NMR: P. Lavedan, M. Vedrenne (ICT). We acknowledge the European Union for funding (AFM, DSC) (FEDER 35477: “Nano-objets pour la biotechnologie”) and The French National Research Agency (ANR) for financial support (ANR Neuraxe). We thank the Integrated Screening Platform of Toulouse (PICT, IBiSA) for providing access to HPLC equipment. LB and EF kindly

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