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(
)-Menthol based thixotropic hydrogel and its application as a universal antibacterial carrier† Yi Li,ac Feng Zhou,b Ying Wen,a Keyin Liu,a Liming Chen,a Yueyuan Mao,a Shiping Yang*c and Tao Yi*a A kind of novel hydrogelator based on (
)-menthol, a traditional cooling compound, tailed by an amino acid derivate through an alkyl chain, has been designed and synthesized. The hydrogelator containing an L-lysine can form a stable hydrogel with thixotropic character in a large pH range. An interesting feature is that the viscoelastic character of the hydrogel can be enhanced by mechanical force. The mechanism of the selfassembly process was investigated by means of IR, SEM, AFM and X-ray diffraction. The formation of three dimensional multiporous networks through acid base interactions and strong double hydrogen bonding between amino acids is proposed to be the driving force for the construction of the stable hydrogel. As a result, the hydrogelator can further gelate aqueous solutions of some confirmed antibacterial agents such as Zn2+ and a series of water soluble organic antibiotic medicines like lincomycin, amoxicillin, etc., in such a unique way that the concentration of the antibacterial agents loaded into the hydrogel can be tuned to a large extent. The antimicrobial susceptibility of the hydrogels loaded with Zn2+ or lincomycin

Received 30th November 2013 Accepted 21st January 2014

is much more effective than that of the corresponding aqueous solution tested by the Oxford cup method. Furthermore, the hydrogelator is completely innoxious to living cells by measurement of MTT

DOI: 10.1039/c3sm52999a

assay. Thus, the hydrogel can be developed as a universal carrier for antibacterial agents and may also be

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widely used in the fields of cell culture, tissue engineering, or drug delivery systems.

1. Introduction Low molecular-weight gels (LMWGs),1–10 as a kind of new functional material, have received increasing attention for their potential application in drug delivery,11–14 biological tissue engineering materials,15–19 sensing devices,20–25 etc. Intriguingly, supramolecular hydrogels, resulting from self-assembly including noncovalent interactions such as hydrogen bonding, p–p stacking, hydrophobic and van der Waals forces between small molecules in water, are inherently biocompatible and biodegradable, and the gentle physical and chemical properties of which represent considerable potential in the elds of biomedicine applications and drug delivery.26–30 Nowadays, the prevalence of infection is very high in our daily life and poses a great threat to public health. Therefore, antibiotics are widely used to inhibit bacterial growth or even kill bacteria. However, some antibiotics have been associated

a

Department of Chemistry, Fudan University, 220 Handan Road, Shanghai 200433, P. R. China. E-mail: [email protected]; Fax: +86-21-55664621

b

Department of Clinical Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, P. R. China

c College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234, P. R. China

† Electronic supplementary information (ESI) available: Tg value, temperature dependent IR spectra, additional gel images and Oxford cup experiment. See DOI: 10.1039/c3sm52999a

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with a range of adverse effects and high doses of antibiotics may result in preferential growth of resistant bacteria.31 Scientists have tried to develop some new methods for controlling of drug dosing in terms of quantity, location and so on to reduce the utilization of antibiotics with side-effects.32–34 Some biomaterials comprising different antibacterial agents including organic and inorganic agents such as metal oxide nanoparticles,35 anti-inammatory organic agents,36 etc., were also developed. For example, antibacterial surface coatings37 and antibacterial bandages for wound dressing38 have been obtained through mixing ZnO nanoparticles with a polymer hydrogel. However, compared with polymer hydrogels, LMWGs have the advantages of rapid response to the environment and thermo reversibility. Moreover, compared to the method that diffuses a drug into a preformed gel, precise concentrations of therapeutics can be encapsulated directly within the gel network during the self-assembly process.14,39 However, the gelation process is much more complicated in LMWG systems due to the competition of multiple nondirectional intermolecular interactions between gelators and gelator/solvent. In fact, foreseeing a hydrogelator design with a specic application, especially in the biological eld, is becoming even more tentative.40 Amino acids or peptides with low biotoxicity and good biocompatibility have been recognized as very useful building blocks for creating self-assembled nanostructures. There are

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some examples of amino acid or peptide-based functionalized building blocks for generating hydrogels.41 For example, B. Xu, et al. proved that D-amino acids could boost selective inhibition for the management of local inammation and confer a supramolecular hydrogel of a nonsteroidal anti-inammatory drug (NSAID).42 A lysine-containing short peptide-based bhairpin hydrogel with inherent antibacterial activity was designed and synthesized by Salick, D. A. et al.43 Hydrogels based on self-complementary oligopeptides for cell culture and cytokine release were constructed by S. Zhang, et al.44 Amphiphilic 3,4,5-trihydroxybenzoic derivatives containing (S)-2-aminopentanedioic acid which could trap a large quantity of the water-soluble drug tetracycline hydrochloride within a stable gel in aqueous ethanol were developed by our group.45 Very recently, A. Banerjee et al. reported a single amino acid based pyrene conjugated thixotropic hydrogel.46 However, a LMW hydrogel that can quantitively load antibiotics is still very rare due to the easy collapse of the gel state when interacting with medical reagents. Comparatively strong three dimensional gel networks and low toxicity are necessary for the design of LMW hydrogels for drug carrier applications. Menthol, which is a cyclic terpene alcohol found in leaves of the genus Mentha, is used in a wide range of products, such as confectionary, candy, toothpastes, vaporubs and aromatherapy inhalations.47 Moreover, menthol can be easily achieved with low cost. However, menthol has never been used for the design of a hydrogel. In this work, menthol methyl ester derivatives were introduced into a gel system as a new kind of gelator. Here, four simple new molecules based on (
)-menthol were designed and synthesized by linking with amino acid or carboxylic acid derivatives (compounds 1–4 in Scheme 1). Among them, 2 behaves as a good hydrogelator, which can gelate pure water and plenty of antibacterial agents including inorganic complexes such as zinc acetate or common organic antibiotics such as lincomycin hydrochloride, etc., to give a thixotropic hydrogel. As a result, a new kind of universal hydrogel drug carrier which could widely load different antibacterial materials was developed.

2. 2.1

Materials and methods Materials

All of the starting materials were obtained from commercial suppliers and used as received. (1R)-(
)-menthyl chloroformate

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(95%) was provided by Alfa Aesar. L-Lysine methyl ester$2HCl was obtained from GL Biochem (Shanghai) Ltd. Lincomycin hydrochloride (97%) was purchased from Accela Chem Bio Co. Ltd. Other chemicals were supplied from Sinopharm Chemical Reagent Co., Ltd. (Shanghai). Column chromatography was carried out on silica gel (200–300 mesh). 2.2

HR-MS was measured on a LTQ-Orbitrap mass spectrometer (Thermo Fisher San Jose, CA). 1H NMR (400 MHz) and 13C NMR (100 MHz) were tested on a Mercuryplus-Varian instrument. Proton chemical shis are recorded in parts per million downeld from tetramethylsilane (TMS). Melting points were determined on a hot-plate melting point apparatus XT4-100A. SEM images were obtained from a FE-SEM S-4800 (Hitachi) instrument. Samples were prepared by spinning the samples on a mica sheet and coating with Au. AFM results were obtained from Veeco Multimode Nanoscope (Bruker) with tapping mode. XRD diagrams were tested on a D8 ADVANCE and DAVINCI.DESIGN (Bruker). IR spectra were collected by FT-IR 360 (Nicolet Company). Rheology experiments were operated on an ARES Rheometer with the frequency ranging from 0.1 to 100 rad s
1, and strain ranging from 0.001% to 100%. 2.3

Structures of compounds 1–4.

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Gelation test

The gelation test of the four compounds 1–4 was carried out with various solvents using a test tube inversion method.1 A weighed sample was put into a septum-capped vial (1.5 mL) with 200–300 mL solvent. The tube was heated to 50–85  C, cooled to room temperature and le for a period of time. The hydrogels loaded with antibacterial agents were prepared by a similar method with a weighed sample of 2 placed into the septum-capped vial containing a solution of different antibacterial agents in different concentrations, heated until soluble and cooled to room temperature. If no ow was observed when inverting the vial, it was considered to be a gel (G). While if part of the solvent was gelated but a ow was still found, it was noted as a partial gel (PG). The critical gelation concentration (CGC) of the two gelators was determined by measuring the minimum amount of gelator for the formation of a stable gel at room temperature. The gel–sol transition temperature (Tg) of the thermally reversible gel was measured by the following method: a sealed vial containing the gel was immersed upside-down in a thermostated water bath. The temperature of the bath was raised at a rate of approximately 2  C min
1. Tg was dened as the temperature at which the gel began to collapse. 2.4

Scheme 1

Characterization

Cell culture and cytotoxicity assay

A human cervical carcinoma cell line (HeLa cells) was provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). Cells were cultured in RPMI 1640 supplemented with 10% FBS (Fetal Bovine Serum) at 37  C in a humidied atmosphere with 5% CO2, and routinely passaged by trypsinization when nearly conuent. In vitro cytotoxicity was measured by performing a methyl thiazolyl tetrazolium (MTT) assay on HeLa cells. Cells were

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seeded into a 96-well cell culture plate at 5  104 per well, under 100% humidity, and were cultured at 37  C and 5% CO2 for 24 h; different concentrations of compound 2 (100, 500 and 1000 mmol L
1, diluted in ethanol/RPMI 1640) were then added to the wells. The cells were subsequently incubated for 24 h at 37  C under 5% CO2. Thereaer, MTT (10 mL; 5 mg mL
1) was added to each well and the plate was incubated for an additional 4 h at 37  C under 5% CO2. The medium was discarded and 100 mL DMSO per well was added. The plates were shaken at ambient temperature for 3 min, and the optical density at 570 nm was recorded. The optical density OD570 value (Abs.) of each well, with background subtraction at 690 nm, was measured by means of a Tecan Innite M200 monochromatorbased multifunction microplate reader. The following formula was used to calculate the inhibition of cell growth: Cell viability (%) ¼ (mean of Abs. value of treatment group/mean Abs. value of control)  100%. 2.5

Antimicrobial susceptibility tests

The Escherichia coli strains (DH5a, Gram-negative strains) and Staphylococcus epidermidis strains (ATCC12228, Gram-positive strains), which are major infection-causing bacteria, were used to determine the antibacterial effect. Fresh bacterial samples were mixed with new liquid nutrient medium in Eppendorf tubes. Aer the strains were cultivated for 24 hours, the medium (3 mL) was mixed with fresh nutrient agar (100 mL, 42  C). The nutrient agar was then poured into petri dishes with four separated Oxford cups (OD ¼ 8 mm). Aer the agar coagulated, the Oxford cups were removed and 100 mL fresh nutrient agar medium was added to seal the bottom of the well so that the sample did not diffuse along the surface of the bottom of the dish. About 140 mL hydrogel was added into the well (just to ll level) and the dishes were cultivated at 37  C for 12–14 h. Also the Zn2+ or lincomycin aqueous solution was added analogously. The antibacterial effect was evaluated by the inhibition zone. The metal ion (Zn2+) and organic drugs (lincomycin) were mixed in the hydrogel as separate groups to evaluate the antimicrobial effect in the hydrogels.

3. 3.1

Results and discussion Molecular design and synthesis

All of our synthesized compounds contain the (
)-menthol group, which gives the best cooling compound with a clean desirable minty odor and intense cooling property of all eight optical isomeric compounds of menthol proved by previous research.47 The (
)-menthol group is hydrophobic, and the amide linkage or amino acid group can accelerate the formation of hydrogen bonds, which may contribute to the gelation process. Besides, both (
)-menthol and amino acid are harmless, biodegradable and biocompatible, which are suitable properties for usage in biomedicine applications. Here, compounds 1–4 were easily obtained by reaction of (1R)(
)-menthyl chloroformate with amino acids (the synthesis details were provided in ESI†). Even though the chemical

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structures of the four compounds are similar, all with the menthol methyl ester group linking with L-lysine (1 and 2) or carboxylic derivates (3 and 4), only 1 and 2 have the capability for gelating organic solvents or water due to the existence of amino acids.

3.2

Gelation capability of the compounds

According to the method for preparing gels described in the experimental section, we tested the gelation ability of compounds 1–4 in various organic solvents, water and alcohol aqueous solutions. The result is shown in Table 1. Interestingly, none of the four compounds could gelate any organic solvent in a heating-cooling treatment. 4 has a nice solubility in water and protic solvent, while 3 is insoluble in water but soluble in most of the organic solvents at room temperature. However, 2 can form a stable hydrogel in pure water and 1 can gelate aqueous ethanol solution with a certain ratio. The detail gelation properties of 1 and 2 are as follows. 1 was insoluble in pure water even if heated. However, aer a certain amount of aqueous ethanol solution (with a volume ratio of water–ethanol at 4 : 1) was put into a septum-capped vial (1.5 mL) containing a weighed sample of 1, and heated to 50–85  C, all the solid was dissolved. A stable gel formed within 30 min aer the sample vial was cooled to room temperature. The critical gelation concentration (CGC) was 1.25% w/v at room temperature. 1 could also immobilize dimethyl sulfoxide (DMSO) and get a stable organogel by means of ultrasound with a CGC of 2.5% w/v. All of the gels were thermally reversible. Scanning electron microscopy (SEM) images showed that 1 gelated from ethanol aqueous solution generated a loosened cotton-like morphology, in which thin bres of about 20 nm in diameter entangled into a bigger bundle of 100 nm in width and the bundles further cross-linked with each other to form a three-dimensional network (Fig. 1a and b). 2 was a good hydrogelator, which could form a translucent gel in water in a very stable state with a relatively low CGC

Table 1

Gelation ability of 1–4a

Solvent/compound

1

2

3

4

Water Methanol Ethanol n-Butanol Acetonitrile Ethyl acetate Acetone Chloroform Tetrahydrofuran n-Hexane Aqueous ethanol DMSO

I S S I I I I S I I CG (1.25, 4 : 1)b OG (2.5)c

OG (0.83) S S S I I I S S I G S

I S S S S S S S S S P S

S S S S I I I S S I S S

a

G: gel; CG: clear gel; OG: opaque gel; P: precipitation; S: solution; I: insoluble. b Volume ratio of water–ethanol (4 : 1). c Obtained aer sonication (0.37 W cm
2, 40 kHz) for 30 seconds at room temperature. The critical gelation concentrations of the gelators are given in parentheses [% w/v].

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(0.83%), and the gel could be maintained even for several months. Because of the existence of the terminal carboxylic acid group which is sensitive to the acidic environment, the pH of the solution for the preparation of the hydrogel was checked. The result showed that the hydrogel could be formed in a large pH range from 1 to 12. The gel was thermal reversible and thixotropic, and was changed to a clear sol by vigorous mechanical damage (Fig. 2a–c). Upon resting, the gel could be regenerated rapidly, showing self-healing character. The gel–sol phase transition temperature (Tg) of the gel was increased with the increase of the gelator's concentration from 58  C for 2.0% to 92  C for 4.8% (Fig. S1 in ESI†). The good stability and thixotropic character of hydrogel 2 have a close relationship with its self-assembly network. SEM images of xerogel 2 showed a dense three-dimensional network composed of thin bres with unit size of about 15 nm in diameter (Fig. 1c and d). The thin bres entangled to form a close-knit porous network. The meshes that appeared are so small (20–50 nm) that the solvent contained in them could not ow away easily, which elucidated the stability of hydrogel 2. A similar multiporous network was also reported by us in a selfhealing organogel containing peptide and cholesterol groups.49 Atomic force microscopy (AFM) for the diluted gel showed a dense three-dimensional network composed of thin bres, in accordance with the result of SEM. When the hydrogel was broken by shaking, the bres were slightly aggregated to bundles from the AFM image (Fig. S2 in the ESI†). The mechanical properties of hydrogel 2 were studied by rheology experiments. The strength of the hydrogel depends on

Fig. 1 SEM images of the xerogels: (a and b) 1 in the mixed solvent of H2O–ethanol (4 : 1, v/v, C2 ¼ 4.0% w/v); (c and d) 2 in water (4.8% w/v); hydrogel 2 (4.3% w/v) loaded with (e) lincomycin (0.001 mol L
1) and (f) Zn2+ (0.008 mol L
1).

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(a–c) The self-repairing phenomenon of hydrogel 2 (a, original gel; b, after mechanical damage and c, following 5 min standing at room temperature, C1 ¼ 1.9% w/v). (d) The pictures of the hydrogels with different antibiotics, from left to right, 2, 2 + Zn2+, 2 + lincomycin, 2 + streptomycin sulphate, 2 + amoxicillin, 2 + penicillin sodium (800 000 IU), 2 + cefoxitin sodium, 2 + vancomycin hydrochloride, 2 + kanamycin, 2 + ampicillin, respectively. The concentrations of 2 for the hydrogel are 1.9% for pure 2, 3.25% for 2 + Zn2+ and 2 + lincomycin, 2.3% for the others. The concentrations are 0.001 M for lincomycin and 0.01 M for the others. Fig. 2

the concentration. As shown in Fig. 3a, the linear viscoelastic region (LVR) of hydrogels of 2 in concentrations of 3.5% and 5% was determined by strain amplitudes ranging from 0.01% to 100% at 6.28 rad s
1. The storage modulus (G0 ) and the loss modulus (G00 ) remained nearly constant up to approximately 0.3% strain (yielding point) in both gels. In the LVR, both G0 and G00 for more highly concentrated gel (5.0%) are much larger than that of less concentrated gel (3.5%). In the meantime, G0 (around 2500 Pa for 3.5% gel and 15 000 Pa for 5% gel) was much higher than G00 (around 680 Pa and 4200 Pa for 3.5% and 5% gel, respectively). We noticed that both of the values of G0

Fig. 3 The rheological data for hydrogel 2 with concentrations of 3.5% and 5.0% at 25  C: (a) stain sweep of the gel at a frequency of 6.28 rad s
1; (b) frequency sweep of the gel at a strain of 0.1%; (c) the stain sweeps and (d) frequency sweep of the original gel (5.0%) and the gel after six cycles of self-repairing (additional legends: G0 , solid mark, G00 , open mark, |h*| (absolute complex viscosity), open mark with inner line).

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and G00 of hydrogel 2 are much larger than previously reported self-healing organogels.48,49 This shows the good gel state of 2 when a small strain is imposed. In the shearing process, the gels were disrupted gradually accompanied with a steep decrease in the values of both moduli but reversal of the viscoelastic value (G00 > G0 ). The transformation points are about 7% of the strain for both gels, independent of the concentration. Therefore, we choose the shear strain of 0.1% in the LVR, to ensure linear viscoelastic behaviour from angular frequency of 0.1 to 100 rad s
1 (Fig. 3b). The dynamic frequency sweep rheometry data showed a kind of classic shear viscosity curve, which indicated the existence of the yielding stress. The slightly increasing value of G0 and almost no change in G00 with increasing angular frequency indicated that the energy storage process occurred without energy dissipation, which also conrmed that the gel had a good elastic character. The thixotropic character and self-healing behaviour of the hydrogel inspired us to study the rheological changes via the self-assembly and repairing process. Specically, when the hydrogel (5.0%) was shaken by hand, it was changed to a sollike owing state. When the sol was rested for 5 minutes, the strength of G0 and G00 , as well as the gel–sol transition point, for the rst repaired gel, were reversed to almost the same value as that of the original gel (Fig. 3c and d). However, aer the repaired gel was repeatedly broken and rested, the strength of G0 and G00 were obviously enhanced compared to the original and rst repaired one, but the gel–sol transition point decreased from 7% to 3%. Moreover, the reversible switching of the rheology by alternating the thixotropic and self-healing processes could be repeated efficiently for several cycles aer the second repairing. This indicated that mechanical force can enhance the viscoelastic character of the gel and this selfhealing gel had an excellent fatigue resistance.

3.3

Driving force of the gelation

The comparison of the solubility and gelation properties of compounds 1–4 demonstrates that the terminal amino acid in 1 and 2 plays a key role on hydrogel formation. The balance of the hydrophilicity and hydrophobicity is another important factor for formation of the hydrogel. The infrared spectra would provide some important evidence for intermolecular interaction in the gelation, especially the contribution of hydrogen bonds between amides and amino acids in our compounds. Considering that all those compounds have the same menthol methyl ester head but are different in the tails, the vibration bands concerning hydrogen bonds were analyzed and shown in Fig. 4. Compound 3 has a very nice solubility in all of the organic solvents due to weak intermolecular interactions between the tailed ester, which was proved by IR spectrum. The N–H vibration band at 3390 cm
1 and the strong C]O band at 1739 cm
1 (ester) and 1690 cm
1 (carbamate) in 3 indicated that there was no strong hydrogen bond between molecules compared to our previous work.50 The multi-band between 3500 and 3000 cm
1 (OH vibration) and single band at 1695 cm
1 (hydrogen bonded C]O vibration) in the IR spectrum of 4 showed a characteristic vibrational pattern for the dimer carboxylic acid (Fig. 4a).51

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Fig. 4 IR spectra of (a) powder of 1, 3 and 4; (b) powder, hydrogel of 2, 2 + Zn2+ and 2 + lincomycin (4.3% of 2, the Czn2+ is 0.01 mol L
1 and Clincomycin is 0.001 mol L
1).

When the terminal group is replaced by an esteried amino acid, the hydrogen bonds in 1 are much more stronger than those in 3 with N–H stretching bands shied from 3390 cm
1 in 3 to 3373 cm
1 in 1, and the C]O stretching band from 1739 cm
1 in 3 to 1692 cm
1 in 1. These hydrogen bonds may be the main driving force for the gelation character of 1. The C]O stretching band of powder of 2 from the prepared organic solvent was positioned at 1723 and 1692 cm
1, which indicated a partly deprotonated or hydrogen bonded carboxylic acid (Fig. 4b). However, those bands in the xerogel from water shied to 1690 cm
1, suggesting a completely deprotonated carboxylic acid. Besides, broad and multi bands between 2400 and 3200 cm
1 in 2 strongly suggest the formation of a zwitterion between the amine and carboxylic acid. The comparison of the IR spectra of the xerogel and powder indicated the reorganization of the molecular packing in hydrogel 2. To further understand the aggregation properties in the hydrogel of 2, temperature-dependent IR spectra of the xerogel 2 from water in the temperature range of 25–205  C were measured (Fig. S3†). The stretching vibrations of N–H at 3369 cm
1 shi to 3379 cm
1 and the amino II band at 1527 cm
1 shis to 1510 cm
1 with a temperature increase from 25 to 205  C. The C]O stretching vibrations of 2 at 1691 cm
1 at 25  C shi to 1697 cm
1 and a new peak at 1723 cm
1 related to a free type of carboxylic acid appears with the increase of temperature. The above result indicates that the intermolecular interaction between the amino acid groups of 2 is the main driving force for gelation and was further conrmed by XRD data. The powder X-ray diffraction (XRD) data of xerogels of 1 and 2 were measured as shown in Fig. 5. 1 gave peaks at 29.55, 16.35 ˚ (Fig. 5a and b), with a ratio of 1 : 1/O3 : 1/O6, and 11.59 A indicating a hexagonal column structure with the cell param˚ The intensity peak at 9.63 A ˚ may belong to eter of a ¼ 34.12 A. the c direction, which was slightly larger than the size of a menthol group. The XRD signal of xerogel 2 gave peaks at 29.1, ˚ with a ratio of 1 : 1/3 : 1/4, suggesting a layered 9.51 and 7.37 A, ˚ is close to twice structure (Fig. 5c and d). The distance of 29.1 A that of the molecule 2 in linear dimensions, which indicates a dimer structure generated by acid–base interactions or intermolecular hydrogen bonding of the two terminal amino acids of 2. We inferred that both electrostatic and hydrogen bonding interactions existed in the formation of dimer of 2 at the location of the terminal amino acids. And then the dimers accumulated lengthways due to the hydrogen bonding interactions

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Fig. 5 Powder XRD profile of the xerogel: (a and b) gel 1 (4% w/v) and (c and d) hydrogel 2 (4.3% w/v).

between C]O and N–H. Finally, the one dimensional bers cross-linked into three dimensional networks by hydrophobic interactions of the menthol groups. The simulative diagram of the self-assembly process of molecules of 2 was shown in Fig. 6. The result indicates that the acid–base interactions and strength of the hydrogen bonds between molecules are the key for the formation of the hydrogel in the comparison of the IR spectra and chemical structure of the four compounds. Notably, 2 could also primely gelate aqueous solutions of zinc acetate or some commonly used organic antibiotics such as lincomycin. Zinc ions have antimicrobial action conrmed by some researchers before,35,52 and lincomycin is a well-known traditional antibiotic in the eld of medicine and is used widely in antimicrobial pharmaceuticals. Here, the properties and antibiotic effects of hydrogel 2 (4.3% w/v) loaded with zinc acetate and lincomycin was studied in detail. The gelation concentration of zinc acetate aqueous solution was in the range

Fig. 6 The simulative diagram of the self-assembly process of molecules of 2.

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of 0–0.04 mol L
1, while the concentration of lincomycin aqueous solution could exceed 0.1 mol L
1. Also the hydrogel was thixotropic and self-repairing (Fig. S4 in ESI†). The thixotropic property is important in that it allows formulations of the hydrogel to be injected using a ne needle in a unique injectable hydrogel based drug delivery system. The morphology of hydrogel 2 loaded with lincomycin (0.001 mol L
1) was similar with that of hydrogel 2, which suggests that 2 may gelate an aqueous solution of lincomycin without specic bonding interactions (Fig. 1e). The densely arranged bers in the hydrogel of 2 + Zn2+ (0.008 mol L
1 of zinc acetate) look much more spindly and straight than that of just 2 (Fig. 1f). IR spectra of xerogels of 2 + Zn(II) and 2 + lincomycin show almost no change compared with that of hydrogel 2, which indicated that the skeleton of the self-assembly of 2 was retained in the two component gels (Fig. 4b). 2 thus acts as a universal hydrogel which can gelate aqueous solutions of a large range of antibiotics with tuneable concentrations. 3.4

Cytotoxicity of compound 2

To verify the biocompatibility of the gelator 2, MTT assay was used to evaluate the cytotoxicity of it on HeLa cells. Aer 24 h incubation of HeLa cells with 2 at different concentrations, a cytotoxicity diagram was obtained. From the results depicted in Fig. 7, compound 2 could ensure that more than 98% (p > 0.05) of the HeLa cells survived whether at 100 mM, 500 mM or even 1000 mM. So, the gelator 2 was highly biocompatibility and could be used in biomaterials. 3.5 Antimicrobial susceptibility tests of the hydrogel 2 loaded with Zn(CH3COO)2 or lincomycin hydrochloride Zinc is an essential mineral in cellular metabolism. Zinc ion concentrations of 10
5 to 10
7 M are required for optimal bacterial growth of most microbes in vitro. However, it is claimed that high concentrations of zinc ion show antibacterial properties.52,53 Recently, ZnO nanoparticles as an antibacterial agent have received much attention for their good antibacterial effect, comparably modest cost and their established use in health care products.37,38 However, many related factors such as obtaining suitable size ZnO nanoparticles, stability, and the concentration in the growth medium has

Fig. 7 MTT assay on HeLa cells treated by 2.

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limited the better application of ZnO nanoparticles in the eld of antibacterial materials. Here, 2 could primely gelate the aqueous solution of zinc acetate with the gelation concentration of zinc acetate aqueous solution adjustable in a large range of 0– 0.04 mol L
1. The Escherichia coli strains (DH5a) and Staphylococcus epidermidis strains (ATCC12228) were used to test the antibacterial effect. Antimicrobial susceptibility tests were made by using the Oxford cup method. The detailed biological experiments were described in the experimental section. Each test group assay was repeated three times to average the results. Eight increasing gradient concentrations of Zn(CH3COO)2 aqueous solution, including 5.0  10
3, 6.0  10
3, 7.0  10
3, 8.0  10
3, 9.0  10
3, 1.0  10
2, 2.0  10
2 and 3.0  10
2 M were prepared to form hydrogels of 2 (4.3% w/v of 2). The photographs of the bacteriostatic circles on Escherichia coli obtained aer 12 h of incubation in different tests were shown in Fig. S5 of ESI.† The results were depicted in Fig. 8a, in which the X-axis represented log10 of CZn2+ (mol L
1) and the Y-axis represented the bacteriostatic circle diameter (the 8 mm outer diameter of the Oxford cup was included). Both the hydrogels loaded with Zn2+ and the Zn2+ aqueous solution had obvious antimicrobial effects on Escherichia coli. With the increasing of the concentration of Zn2+, the bacteriostatic circle diameters were gradually increased demonstrating more and more effective antimicrobial activity. Besides, according to the statistically standard values of deviation, the hydrogel loaded with Zn2+ showed an even bigger antimicrobial circle than that of Zn2+ aqueous solution, which means that the antimicrobial effect of the hydrogel is better than the solution. The antimicrobial effect of the hydrogel 2 only (without Zn2+) was also tested as a control and showed almost no bacteriostatic circles, indicating that the hydrogel 2 itself has no obvious antimicrobial effect. Moreover, the comparison of the antimicrobial effect of Zn2+ with different bacteria indicates that Zn2+ and its hydrogel had an inhibition effect on the proliferation of Gram-negative strains of Escherichia coli, however, no signicant antibacterial effect towards Staphylococcus epidermidis (ATCC12228). Lincomycin is a broad-spectrum antibiotic and shows high in vitro and in vivo activity against Gram-positive bacteria.54 Lincomycin hydrochloride gel is a kind of non-prescription drug widely used for various skin infections caused by mild burn wounds and insect bites.55 As a useful drug carrier, hydrogel 2 could be also used for loading this antibiotic as a special medicinal hydrogel. Antimicrobial susceptibility tests were carried out in order to test its antimicrobial effect. A series of concentrations of lincomycin hydrochloride aqueous solutions of 1.0  10
4, 2.0  10
4, 4.0  10
4, 6.0  10
4, 8.0  10
4, 1.0  10
3, 2.0  10
3, 4.0  10
3, 6.0  10
3, 8.0  10
3 and 1.0  10
2 M were used for preparing hydrogels. Photographs of the bacteriostatic circles on Staphylococcus epidermidis were obtained aer 14 h of incubation (Fig. S6†). We found that the hydrogel loaded with lincomycin had an inhibition effect on the proliferation of Staphylococcus epidermidis but no signicant antibacterial effect towards Escherichia coli, which is complementary to the Zn2+. As depicted in Fig. 8b, a similar antimicrobial effect trend with the hydrogel loaded with Zn2+ was obtained even though the antimicrobial effect was

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Soft Matter

The changes on the bacteriostatic circle diameter with addition of (a) Zn2+ in different concentrations from 0.005 to 0.03 M and (b) lincomycin hydrochloride in different concentrations from 0.0001 to 0.01 M in hydrogel of 2 and aqueous solution.

Fig. 8

obviously higher. Also, the antimicrobial effect of the hydrogel was higher than the lincomycin solution. It could be concluded that by introducing the antibiotic into the hydrogel system, a more effective antibacterial effect could be obtained, which would decrease the high concentration induced side effects on organisms to some extent. Following this topic, in order to broaden the application of our hydrogel material, the gelation capability of 2 with other antibiotic solutions was tested. The result showed that 2 could gelate almost all the antibiotic aqueous solutions (0.01 mol L
1), including amoxicillin, penicillin sodium (800 000 IU), cefoxitin sodium, vancomycin hydrochloride, ampicillin, kanamycin, streptomycin sulphate, etc. (Fig. 2f). We propose that the formation of stable three dimensional multiporous networks through acid base interactions and strong double hydrogen bonding between molecules of 2 are the main reasons for the maintenance of the gel state with the existence of different antibiotics. So, the present hydrogel material can be used as a universal carrier for loading with many kinds of antibacterial agents.

4. Conclusions In conclusion, four novel (
)-menthol derivatives were designed and synthesized, in which two compounds linked with

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esteried amino acid (1) and amino acid (2) could form stable gels in aqueous ethanol solution and pure water, respectively. The hydrogel of 2 shows thixotropic character, which is important for biological and industrial applications. Moreover, the viscoelastic character of the hydrogel can be enhanced by mechanical force. To our knowledge, this is the rst example of gelling compounds based on (
)-menthol. The gelator is completely non toxic to living cells. Furthermore, this hydrogelator 2 can also gelate aqueous solutions of some conrmed antibacterial agents such as Zn2+ and a large range of water soluble organic antibiotic medicines like lincomycin, amoxicillin, etc. in such a unique way that the concentration of the antibacterial agents loaded into the hydrogel can be accurately tuned to a large extent, which may be more simple and effective than other mixed or embedded methods. The antimicrobial susceptibility of the hydrogels loaded with Zn2+ or lincomycin is much more effective than that of the aqueous solution tested by the Oxford cup method. Besides, those harmless and environmentally friendly hydrogels may be widely used in drug delivery systems, biological tissue engineering materials, food and cosmetic industries, and so on. Further works are under progress.

Acknowledgements This work was nancially supported by National Basic Research Program of China (2013CB733700), the Chinese National Funds for Distinguished Young Scientists (21125104), National Natural Science Foundation of China (91022021, 51373039), Specialized Research Fund for the Doctoral Program of Higher Education (20120071130008), Program Innovative Research Team in University (IRT1117), Program of Shanghai Subject Chief Scientist (12XD1405900), and Shanghai Leading Academic Discipline Project (B108).

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