(a) DLS profile of the nanoscale micelles of TPC. (b) AFM morphological image of the micelles of TPC deposited on mica, showing that these nanoscale spheres stack together. (c) Higher magnification of (b). TPC dissolved in water at 5.0 mM at pH 3.0. The scales are marked in each panel. 

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... of heat-set gels resulting from a difference in the interaction between two component chiral gelators. 12 They also found that chiral calix[4]arene bearing L -2,3-dibenzoyltartaric acid groups at the lower rim can enantioselectively form gel only with one enantiomer of chiral amines. 12 Among the family of gels, the growth of low molecular weight hydrogels (LMWH) has advanced many fields ranging from cosmetic products to pharmaceuticals to gene delivery, due to their characteristics of biocompatibility and biodegradation. In addition, the development of an ever-increasing spectrum of functional hydrogelators continues to broaden the versatility of hydrogel applications. LMWH now play a critical role in many tissue engineering scaffolds, biosensor and biological micro- electro-mechanical systems, and drug carriers. 13 However, to the best of our knowledge, almost no studies have concerned calix- arene-based hydrogels except one recent example that Mocerino and coworkers reported the first calixarene based hydrogelator, a proline-functionalised calix[4]arene, 14 where its gelation was controlled by specific anions. 15 This work is an interesting topic not only in the field of supramolecular chemistry but also in biomedical-engineering as water-soluble calixarenes are gener- ally regarded as biologically friendly. 1,16 In this work, we constructed a series of supramolecular binary hydrogels based on tetra-proline modified calix[4]arene (TPC) and basic amino acids (arginine, histidine and lysine), as shown in Scheme 1. Binary gel systems offer a fine alternative candidate for tailoring the properties by changing one of the two components. 17 Especially, the use of bio-safe natural products such as amino acids as one component is more advantageous over synthetic gelators. Natural amino acids are benign candidates to prompt gelation of LMWH for not only are they biocompatible 18 but that they are also hydrotropes. 19 In addition, despite hardly understanding complicated supramolecular interactions in TPC/ amino acid systems, studying the influence of amino acid structures on the properties of the resulting hydrogels can also serve as a model to discover or design other new functional binary gel- phase materials. Twenty natural amino acids were all used to examine the gelation behavior of TPC; it was found that only three basic amino acids (arginine, histidine and lysine) can induce gelation of TPC successfully (Table 1), see Fig. 1 for a representative case of TPC/ arginine LMWH. The hydrogelation conditions were then opti- mized, at ratios of arginine/TPC ranging from 1/1 to 10/1 (mol/ mol), TPC concentrations of 1.0 mM to 100 mM, and the pH varying from 7.0 to 1.0. It was found that 5.0 mM of TPC with four times as much arginine yields a stable transparent hydrogel when the pH was decreased to 3.0 (Fig. 1). The same optimal conditions were also obtained in the cases of lysine and histidine. In fact, TPC can form LMWH upon the addition of a great excess of HCl (concentration up to 1.0 M), which somewhat resembles TPC gelation triggered by anions. 15 The gelation mechanism of TPC with basic amino acids was hypothesized as shown in Scheme 2. Firstly, TPCs themselves form micellar aggregates when the concentration is over the CMC (critical micelle concentration). Secondly, basic amino acids non-covalently interact with the TPC micelles (electrostatic, hydrogen-bonding, host–guest and van de Waals interactions can occur between TPC and amino acids), forming joint micelles, leading to the formation of a hydrogel matrix. As reported by Mocerino and coworkers, TPC is a kind of amphi- phile that can form micellar aggregates with a CMC of 5.6 mM in neutral conditions. 15 From another viewpoint, TPCs can be regarded as kinds of hydrophobic groups modified by amino acids or peptides that have a tendency to amphiphilically aggregate in various pH conditions. 20 X-Ray crystallography measurements shows that TPC maintains the preferable pinched- cone conformation (left in Scheme 2). Such a truncated cone structure is favourable for the formation of micellar aggregates. The CMC of TPC at pH 3.0 was measured by circular dichroism (CD) spectroscopy, giving value of 1.2 mM (Fig. S4†). The TPC concentration employed in gelation is over 4 times higher than its CMC, which ensures the formation of micellar aggregates before gelation. Dynamic laser scattering (DLS) and zeta potential measurements were further performed to identify the TPC micellar size and surface charged distribution, giving results of an average diameter of 124 nm (Fig. 2a) and an average zeta potential of –2.32 mV (Fig. S5†). DLS analysis further shows that the majority of the particles exhibit a mean diameter of 190 nm, whereas smaller particles with a mean diameter of 42 nm also exist. Nanoscaled spheres were also observed from atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images. As shown in Fig. 2, S6 and S7,† the diameters were measured as in the range of 40–200 nm, which is in agreement with the DLS data. It can be seen that it forms nanoscaled particles with negative charges in free TPC solution (5.0 mM, pH 3.0). At pH 3.0, these three basic amino acids obviously present positive charges according to their p K a values. 18 As a consequence, arginine, lysine and histidine can interact with TPC micelles well where Coulombic forces play a large role along with other possible interactions, while the other amino acids can not. Based on this assumed mechanism, TPC concentration, component ratio and pH are three key factors controlling the formation of supramolecular binary hydrogels. AFM has been performed to study the morphological features of wet gels. 15 In a previous report, TPC is an electrolyte activated hydrogelator and the density of the resulting hydrogels depends on the particular salts used. 15 As we know, basic amino acids can exist in an anionic or cationic form, and have more complicated structures than inorganic electrolytes. Hence, we hypothesized that all three LMWHs may display different structures of networks although they have similar components and were prepared under exactly the same conditions. As shown in Fig. 3a–c, the TPC/amino acid LMWHs possess frameworks of nanofibers, and the fiber shapes differ when the basic amino acid was changed. In Fig. 3a and S8†, the fibers of the TPC/arginie LMWH are composed of many long and branched fibers of 1.0–10.0 m m long. For the TPC/histidine LMWH, the fibers become shorter and twisted with lengths of 1.0–3.0 m m (Fig. 3b and S9†). Fig. 3c shows a denser network with stacks of rodlike nanofibers in the TPC/ lysine LMWH. Therefore, distinguishable microstructures are observed in LMWHs, indicating that basic amino acids with side chain variations can indeed induce significant morphological changes in binary hydrogels. Similar phenomena were also observed in SEM and TEM studies. Specifically, the SEM and TEM images reveal three dramatically different types of xerogels: fibrous, porous and lamellar. The TPC/arginine xerogel shows a fibrous framework (Fig. 3d and 3g). Fig. 3e and 3h show a porous material with stacks of sheets for the xerogel based on TPC/histidine. A lamellar structure with less thickness exists for the TPC/lysine xerogel (Fig. 3f and 3i). Here, it is important to note that the structures obtained from AFM experiments are different from the structures obtained from SEM and TEM. This is due to the fact that the method of sample preparation is different for different microscopic experiments. In comparison with AFM experiments, SEM and TEM studies require complete drying of the LMWHs, and the preparation of xerogels may result in arti- facts for gel systems that inherently exist in their solvated states. 13 Thermal stability and thermoreversibility of gels are of interest with respect to their various applications, such as drug delivery. 13,21 We have studied the thermal stability of gels based on TPC/basic amino acids in an aqueous medium at acidic pH 3.0 across a range of concentrations (Fig. S11†). The gel-sol transition temperature, T gel , increases along with increasing the concentration of TPC. As can be seen, when the concentration of TPC is below 5.0 mM, all the mixtures remain as solutions, in contrast, at high concentrations, TPC can easily form gels with basic amino acids; for instance, even at 50 C, TPC still exhibits a powerful gel capability at 20.0 mM. Furthermore, a high concentration of basic amino acids can also be employed to encourage the system to form stable gels. Therefore, basic amino acids play a crucial role in impacting and stabilizing the hydrogels. Accordingly, these results clearly demonstrated that the concentrations of TPC and basic amino acids have profound effects on the thermal stability of the LMWHs discussed here. To further investigate the thermoreversibility of the LMWHs, we performed fluorescence spectroscopic studies using 8-anilino- 1-naphthalenesulfonic acid sodium salt (ANS) as a probe, which is sensitive to a hydrophobic environment. 22 LMWHs containing 10.0 mM TPC were taken as representative examples. The emission intensity of LMWH-loaded ANS was measured in the temperature range of 25–70 C. The fluorescence decreases gradually with elevating temperature (Fig. 4 and S12†), which indicates that ANS is exposed to a more hydrophilic environment as the gel phase turns into the sol phase. While in the cooling cycle, the emission intensity increases when temperature decreased from 70 to 25 C, and the curves almost coincide with those in the heating cycle in shape (Fig. 4a, 4b and 4c). Moreover, obvious inflection points are observed in the plots of emission intensity versus temperature (Fig. 4a, 4b and 4c). The inflection points, T gel , appear at 48, 45 and 46 C for LMWHs of TPC/ arginine, TPC/histidine and TPC/lysine respectively, for samples being heated. Similar values of ...