Anthranilamide-based Short Peptides Self-Assembled Hydrogels as Antibacterial Agents

In this study, we describe the synthesis and molecular properties of anthranilamide-based short peptides which were synthesised via ring opening of isatoic anhydride in excellent yields. These short peptides were incorporated as low molecular weight gelators (LMWG), bola amphiphile, and C3-symmetric molecules to form hydrogels in low concentrations (0.07–0.30% (w/v)). The critical gel concentration (CGC), viscoelastic properties, secondary structure, and fibre morphology of these short peptides were influenced by the aromaticity of the capping group or by the presence of electronegative substituent (namely fluoro) and hydrophobic substituent (such as methyl) in the short peptides. In addition, the hydrogels showed antibacterial activity against S. aureus 38 and moderate toxicity against HEK cells in vitro.


Results and Discussions
Synthesis of anthranilamide-based short peptides. The library of anthranilamide-based short peptides was designed based on three main modifications, which include modifying the acyl group R1 (modification A), introducing substituents at the 5 position of the capping group (modification B), and incorporation of anthranilamide-based short peptides into the bola amphiphile (BA) scaffold (modification C) Fig. 2.
A capping group of a short peptide-based hydrogelator could govern the stiffness and stability of the resulting hydrogels 18,26 . Therefore, hydrogels 1-4, bearing different aromaticity on their capping groups, are envisaged to  exhibit different properties. Fluorine, a small electronegative atom, and methyl were introduced (modification B) to investigate the effect of an electron-withdrawing or electron-donating group on the subsequent scaffolds, yielding compounds 5 and 6, respectively.
Aside from conventional, linear low molecular weight gelators (LMWG), BA and C 3 -symmetric molecules comprise rather different molecular structures and are reported to have self-assembly properties [27][28][29][30][31] . Therefore, anthranilamide-based, short peptides were incorporated into BA and C 3 -symmetrical scaffolds. A Bola-amphiphile is an amphiphile which contains two hydrophilic ends that are connected via a hydrophobic spacer and which can cooperatively induce fibre formation [32][33][34] . The anthranilamide-based short peptides 7-9 were incorporated in different positions (ortho-, meta-, and para-) of benzene dicarboxamide to examine the effect of molecular structure on the properties of corresponding hydrogels. In addition, hydrogelators 10, linked via an oxalyl spacer, were also synthesised to investigate the effect of using a shorter hydrophobic spacer.
Further building on the bola-amphiphile scaffolds 7-10 which contain two hydrophilic ends connected by a hydrophobic spacer, the anthranilamide-based peptides were incorporated into the C 3 symmetric benzene-1,3,5-triscarboxamide (BTA)scaffold. BTA derivatives have been reported to form helical columnar stacks as the result of cooperative π-π stacking, which led to excellent hydrogelation 35 . Hence, the BTA-bridged anthranilamide-based short peptide 11 was expected to form a supramolecular hydrogel.
Initially, to obtain anthranilamide-based short peptide, isatoic anhydride was ring-opened using a modified known procedure 36 . Isatoic anhydride was heated under reflux with methyl l-Phenylalanyl-l-phenylalaninate hydrochloride in the presence of an inorganic base to provide compounds 12a-c as pure products after purification with column chromatography. Their structures were confirmed by 1 H NMR as characteristic singlets around δ = 6.3 ppm and δ = 8.8 ppm corresponded to aniline and amide protons, respectively. Compounds 12a-c were treated with the respective acyl or sulfonyl chloride followed by ester hydrolysis reaction to give the carboxylic acids 1-6 in excellent yield (70-83%) (Scheme 1).
Self-assembly of anthranilamide-based short peptides. Molecular self-assembly can be defined as a spontaneous process where disordered molecules or systems form more defined structures as result of intermolecular interactions 37 . In order to form a self-assembled gel, intermolecular interactions and balance between hydrophobicity and hydrophilicity of a short peptide-based gelators often play a significant role. An aromatic capping group of a short peptide-based hydrogel is usually designed in such a way that it could provide not only intermolecular interaction sources, but also a balance between hydrophobicity and hydrophilicity in the molecule. The partition coefficient, log P value, is a universal tool used to predict the hydrophobicity of a molecule. Having log P values between 2.53 and 5.25, which are considered to be ideal 38 , the anthranilamide-based hydrogelators 1-11 are expected to formed stable hydrogels. Additionally, the anthranilamide-based hydrogelator also provides a carbonyl group which might act as a hydrogen bond acceptor.
Several triggers such as physical stimuli, pH switch, solvent switch, or a combination of these, were employed to assess the self-assembly of compounds 1-11 in water. Initially, hydrogel formation of compounds 1-11, at 1% (w/v), was investigated using a combination of pH switch and temperature switch. The compounds were heated in dilute sodium hydroxide (NaOH) in order to deprotonate the carboxylic acid group at the C-terminus of the short peptides. Upon cooling the solutions to room temperature, clear or opaque hydrogels with pH ranging from 9-12 were observed for N-acetyl 1, N-benzoyl 2, N-naphthoyl 3, fluoro substituted 5 and methyl-substituted 6, as shown in Fig. S1. The other compounds (4, 7-11) remained as clear solutions even after 48 hours.
In an attempt to induce gelation in peptides which failed to form hydrogels using the previous method, glucono-δ-lactone (GdL) was added to basic solutions of 4 and 7-11. Compared to a mineral acid, GdL promotes the formation of a homogenous hydrogel due to its slow hydrolysis and fast dissolution rate in water 39,40 . Addition of 3 equivalents of GdL, to protonate the C-terminus of the peptide, resulted in formation of translucent hydrogels for BA 8-10 (Table 1, Fig. S2).
However, using this method, a white precipitate was observed from the N-naphthalene sulfonyl 4, ortho 7, and C 3 -symmetric 11. In addition, the effect of salt which can induce gelation in basic peptide solutions 41 , due to high ionic was also investigated for peptides 1-11. Addition of NaCl or CaCl 2 to high pH solutions of compounds 1-11 resulted in precipitation of these short peptides, presumably due to a salting out process.
In a final attempt to induce gelation in peptides 4, 7 and 11, a solvent switch method using dimethyl sulfoxide (DMSO), methanol, or ethanol as co-solvent was employed [42][43][44][45][46][47] . The majority of the anthranilamide-based short peptides formed a turbid solution which then clarified to various degrees, over the course of seconds to minutes, and subsequently formed clear or opaque hydrogels (Table S1, Fig. S2). However, gels were observed only for the first 5 minutes for N-naphthalene sulfonyl 4 and BA 7 (ortho-), as water and a white precipitate was observed to evolve over time when using DMSO: water at 1% w/v (Fig. S4). Peptide 11 formed a precipitate for all solvent switch conditions tested. Given that these hydrogels were designed with antibacterial applications in mind, due to the potential toxicity concerns arising from the use of organic co-solvents, further characterisation of peptides 4, 7 and 11 was not carried out.
The critical gel concentration (CGC) represents the minimum amount of the anthranilamide-based short peptide required to form a hydrogel. The CGC of anthranilamide-based short peptides were qualitatively assessed by varying the short peptide concentrations and conducting vial inversion test [48][49][50] . Hydrogels composed of anthranilamide-based short peptides exhibit relatively low CGCs ranging from 0.07-0.30% (w/v) with gelation achieved either through combinations of pH and temperature switch (Table 1). Here, gelation time represents the time required for these short peptides, at 1% (w/v), to form self-supporting hydrogels as assessed through the vial inversion test. characterisation of anthranilamide-based hydrogels. It has been reported that interaction between aromatic units, in short peptide hydrogelators, plays a prominent role for self-assembly to occur 51,52 . Therefore, the π-π stacking interaction between aromatic groups of anthranilamide-based short peptides was investigated using 1 H NMR, UV-Vis, and Circular Dichroism (CD) spectroscopy.

H NMR and UV-Vis
To investigate the role of the π-π stacking interactions, concentration dependent 1 H NMR studies were performed on N-acetyl 1, N-benzoyl 2, and N-naphthoyl 3, bearing different aromatic cap. Upon increasing the concentration of these hydrogelators, notable up-field chemical shifts ( δ = 0.1 ppm) were observed for the aryl proton from the aromatic cap which indicated their involvement in π-π stacking that initiate self-assembly process (Figs. 3a and S5) 53,54 . The peak broadening (Fig. 3a, green) indicated that the self-assembly of short peptides 1-3 started to occur at very low concentrations. However, complete transformation from solution phase into gel phase was observed at their CGC, as indicated by disappearance of the 1 H NMR features (Figs. 3a and S5, red) 55 .
To support the 1 H NMR analysis, concentration dependent UV-Vis was carried out. UV-Vis absorption of N-benzoyl 2 showed a bathochromic shift (from 203 nm − 217 nm) and enhancement of a shoulder peak ranging from 240 nm -350 nm as the concentration was increased from 0.003 mg mL −1 to 0.050 mg mL −1 (Fig. 3b). Similarly, N-acetyl 1 (bearing less aromatic cap) and N-naphthoyl 3 (bearing a more aromatic cap) also exhibited bathochromic shifts from 202 nm − 213 nm and enhancement of shoulder peaks ranging from 240-350 nm (Fig. S6). These results further support the observation that aromatic groups promote the self-assembly of anthranilamide-based short peptides to form well defined nanofibres leading to hydrogel formation. Circular Dichroism (CD) spectroscopy and ATR-FTIR Peptides often exhibit conformational motifs such as a-helix, ß-sheets, or disordered coils which can be determined using CD spectra and FTIR spectroscopy. Initially, the far UV region (240 nm -190 nm) where the main absorbing group is a peptide bond, was investigated using a CD spectrophotometer. The negative band at ~195 nm and relatively low ellipticity above 210 nm observed for N-acetyl 1 indicated formation of a random coil secondary structure 56 . On the other hand, the N-benzoyl 2 and N-naphthoyl 3 possessed ß-sheet secondary structures as indicated by the presence of positive maxima at around 190-200 nm (π → π*) and negative minima around 230 nm (n → π*) (Fig. 4a) 57 . The presence of two positive maxima, at 197 nm and 220 nm, for 2 and 3 suggested formation of dipeptide nanotubes which are rich in ß-sheet secondary structure facilitated through π-π stacking interactions from the aromatic capping group 58,59 .  www.nature.com/scientificreports www.nature.com/scientificreports/ Anthranilamide-based short peptides 5 and 6, bearing fluoro and methyl as substituents, exhibit similar CD patterns to those of N-benzoyl 2 (Fig. 4b), which suggest that introduction of a substituent at the 5-position of anthranilamide-based short peptides does not affect their secondary structure (Fig. 4c,d).
ß-sheet secondary structure was also observed from dilute solutions of both BA 8 (meta-) and 9 (para-). On the other hand, BA 10 (linked via an oxalyl linker) showed a characteristic disordered coil by appearance of a strong negative band below 200 nm and a weak positive band at ~218 nm 60 .
In addition, CD spectroscopy was also used as a tool to investigate gradual thermal denaturation of anthranilamide-based short peptides. Despite the CD signal of N-benzoyl 2 (as model compound) being gradually decreased, the overall ß-sheet feature was preserved upon increasing temperature from 25 °C to 60 °C. This result indicates that there are no significant conformational changes and demonstrates thermal stability of N-benzoyl 2 under physiological conditions (Fig. 5), which is important to note for future antimicrobial and cytotoxicity studies.
FTIR was used to further confirm the formation of either ß-sheet structure or disordered coil in anthranilamide-based hydrogel 1-10 (Figs. S7-14). The amide I region of D 2 O gels made from N-benzoyl 2, N-naphthoyl 3, and BA 8-9 exhibited peaks that correspond to ß-sheet structure (1625 cm −1 −1640 cm −1 ) 57 . Meanwhile, hydrogels made from N-acetyl 1 and BA 10 showed peaks at 1647 cm −1 and 1640 cm −1 , respectively, which support their disordered coil structure observed by CD spectroscopy 61,62 . In addition, peaks correspond to their respective secondary structure were also observed in xerogels (air-dried H 2 O gel) of these anthranilamide-based short peptides 1-10 (Figs. S7b-S14b). This suggested that secondary structures of these hydrogels were retained, regardless of whether the peptide was in a lyophilized or hydrated environment 63 .
Mechanical properties. Mechanical properties of hydrogelators prepared from anthranilamide-based short peptides were investigated using a rheometer. All hydrogels, except those composed of 6 displayed frequency-independent behavior during frequency sweep tests (FST) (Fig. S15). Hydrogels composed of 6 appeared to undergo irreversible deformation at frequencies >2 Hz, indicating that the gel network is metastable 58,59 .
The stiffness of a hydrogel can be approximated by its Gʹ value, where a higher value corresponds to a stiffer hydrogel 60 . It can be seen that changing the capping group from N-acetyl to N-benzoyl or N-naphthoyl significantly increases the stiffness of the resulting hydrogel from 3.4 kPa to 16.9 and 5.7 kPa, respectively ( Table 2). This result might be ascribed to enhanced aromatic or hydrophobic interactions due to N-benzoyl 2 and N-naphthoyl 3 capping groups.
Hydrogel 5 (with a fluoro substituent), also showed characteristics of a stable hydrogel with similar stiffness (11.4 kPa) to N-benzoyl 2, indicating that the installation of an electron-withdrawing fluoro group at this position does not affect the overall mechanical properties of the hydrogel. In contrast, hydrogel 6 bearing an electron-donating methyl substituent showed a notable decrease in strength with Gʹ = 120 Pa (Table 2). Hydrogel 6 showed frequency-dependent behaviour at frequencies above 2 Hz, where hydrogel rupture ultimately occurred. At frequencies below 1 Hz, however, frequency independent behaviour was observed.  www.nature.com/scientificreports www.nature.com/scientificreports/ For the bola amphiphile-type peptides, depending on the spacer, diverse mechanical properties were observed for hydrogel 8-10 owing to their different self-assembled structures. BA 8, which is linked at the meta-position, gives a moderately stiff hydrogel with Gʹ = 1.6 kPa. Interestingly, the structural isomer (para-9) yields a stiffer hydrogel (Gʹ = 12.9 kPa), possibly due to a more linear packing motif which could stabilize the multilayer nano-structure. In addition, BA connected through an oxalyl spacer 10 also showed characteristics of stable hydrogels with Gʹ = 10.3 kPa.
The strain sweep test (SST) was conducted to determine the linear viscoelastic region (LVER) of a hydrogel. A larger LVER suggests that the hydrogel is more resistant to an applied oscillatory strain, such as that which can be applied by cells 64 . Hydrogels composed of N-acetyl capped peptide 1 show deformation upon the application of a relatively small strain (0.2 ± 0.01%), indicating its unstable nature, potentially due to a lack of aromatic/hydrophobic interactions 65 . Aromatic -aromatic interactions are known to form more stable supramolecular hydrogels 66 . In agreement, the N-benzoyl 2 and N-naphthoyl 3 were more amenable to applied strains, with LVERs up to 5.6 ± 0.03% and 1.9 ± 0.02%, respectively (Fig. S16).
The presence of fluoro, as an electron-withdrawing substituent, in hydrogel 5 increased the LVER from 5.6% to 8.5%. In contrast, hydrogel 6, bearing a methyl substituent, exhibited a significantly shorter LVER (0.6%). This result might be accounted for by the electronic contributions from the substituent on the anthranilamide-core, which affected the overall mechanical properties of the resulting hydrogels.
Compared to BA 8 (meta-), the isomeric structure BA 9 (para-) exhibited a shorter LVER, presumably due to its more linear packing which reduces the flexibility of the resulting hydrogels. In addition, BA 10 (oxalyl-) displayed a significantly shorter LVER, potentially due to the less aromatic and shorter spacer compared to BA 8 and 9.
network morphology of the self-assembled gels. To gain some insight into the morphology of the hydrogels, xerogels of 1-3, 5-6, and 8-10 were imaged using atomic force microscopy (AFM). In general, these hydrogels possessed fibre-like structures with different diameters as shown in Fig. 6. N-acetyl bearing hydrogel 1 consisted of two fibre populations, small fibres with diameter of 60 ± 13 nm and larger fibres with diameter of 180 ± 20 nm (Fig. 6a). Straight fibres with no junction zones were observed and might explain the brittle characteristics of these hydrogels as measured by rheology. It has been reported that less aromatic hydrogelators tend to form thicker fibres than more aromatic hydrogelators 17,18 . Consistently, N-benzoyl 2 and N-naphthoyl 3 (bearing more aromatic groups) exhibit fibres with smaller diameters of 18 ± 5 nm and 12 ± 5 nm, respectively.
Introducing substituents, such as fluoro or methyl, did not significantly change the overall morphology of the fibres (Fig. 6d-e). The fluoro substituted hydrogel 5 possessed fibres with similar diameter (16 ± 0.1 nm) to those of hydrogel 2. Meanwhile, hydrogel 6 (having a methyl substituent) showed a notable decrease in diameter (9 ± 0.1 nm). This is somewhat surprising, given the significant differences observed in the mechanical properties (storage modulus and LVER) for hydrogels of 5 and 6.
In addition to exhibiting distinct mechanical properties, the isomeric peptides BA 8 (meta-) and BA 9 (para-) also showed divergent fibre morphology (Fig. 6f-g). Hydrogel 8 (meta-), showed formation of small straight fibres (15 ± 2 nm), while the hydrogel made from para-9 displayed twisted fibres with diameter of 112 ± 10 nm. The formation of thicker fibres along with extensive bundling, observed from BA 9 (para-), clarify their much higher Gʹ value compared to BA 8 (meta-) shown in rheology 67 . In addition, BA 10 (linked via an oxalyl spacer) showed formation of slightly curved fibres with diameter of 29 ± 7 nm.  N-acetyl 1, N-benzoyl 2 and N-naphthoyl  3 was assessed, in order to evaluate the effect of increased hydrophobicity on antibacterial activity. Further, the stiffest (BA 9) and softest (methyl 6) of the remaining gelators were tested to discern the relationship of hydrogel stiffness to antimicrobial activity. Staphylococcus aureus, the most common causative organism for skin and soft tissue infections, was chosen as the bacterial strain to assess the hydrogels antibacterial activity 68 . These hydrogel surfaces was challenged with bacterial inoculum of 3 × 10 4 cfu mL −1 and viability was measured after 18 hour of incubation. Interestingly, N-benzoyl 2, N-naphthoyl 3, and BA 9 (para-) exhibited significant bacteria activity with bacteria reduction of 4.4 Log 10 , 9.0 Log 10 , and 1.9 Log 10 , respectively (Fig. 7a). In contrast, the less stiff hydrogels (N-acetyl 1 and methyl 6) did not show significant bacterial reduction. This result was anticipated, as rheological properties (in particular higher Gʹ value) were reported to give rise to antibacterial function of a hydrogel by providing mechanical support to individual fibres and fibrous networks 69 . Furthermore, the observed antibacterial activity was not due to the presence of NaOH as the concentration of NaOH in all of the hydrogel tested were found to be below their MIC against S. aureus (7.5 mg/mL) 70 . This results is consistent with previous studies, where short peptide-based hydrogels without cationiccharge, were shown to exhibit antibacterial activity against Gram positive and Gram negative bacteria 71-74 . Cytotoxicity of hydrogels 2, 3, and 9. In order to successfully treat infection, antimicrobial compounds need to exhibit high toxicity towards bacteria but low toxicity towards mammalian cells. Although the diphenylalanine moiety is known to be non-toxic, the cytotoxicity of short peptide based hydrogelators are often defined by their capping group 19,[75][76][77] .
With this in mind, the cytotoxicity of lead candidates N-benzoyl 2, N-naphthoyl 3, and BA 9 (para) from the antibacterial studies were examined against HEK293T cells, as a robust mammalian cell model. As the antibacterial activity of the 2, 3 and 9 was evaluated in the gel phase, contact cytotoxicity of hydrogels at concentrations of 0.25, 0.5, and 1% (w/v) was examined. At these concentrations, which are far above their CGC, both 2 and 9 exhibited moderate cytotoxicity towards HEK cells (Fig. 7b) with no significant variation upon concentrations. Unfortunately, the N-naphthoyl hydrogel 3 showed poor cell viability against HEK cells (Fig. 7b, green), potentially owing to its increased hydrophobicity, which has previously been shown to correlate with cytotoxicity for short peptide gelators 78 . conclusions Anthranilamide-based short peptides have been successfully incorporated as LMWG, bola amphiphile (BA), and C 3 symmetric molecule via ring-opening reactions of isatoic anhydride in solution phase with excellent yield. The short peptides reported herein formed hydrogels using combinations of pH switch and heat or solvent switch as a trigger in relatively low concentrations (0.07-0.30%). The hydrogel properties (such as mechanical strength, secondary structure, and fibre morphology) can be modulated by varying hydrophobicity or introducing substituents on the capping group. Hydrogels made from N-benzoyl 2 and BA 9 showed most favorable viscoelastic properties. In addition, these hydrogels exhibit antibacterial activity against S. aureus and moderate toxicity against HEK cells. Further modification to the hydrogel scaffold is required to improve the cytotoxicity of the hydrogels for biomedical applications, such as topical antibacterial gels.

Materials and Methods
Synthesis. All chemicals and solvents used were purchased from Chemimpex, Combi Blocks, or Sigma Aldrich and were used without any further purification.
The anthranilamide short peptides were synthesised via ring opening reactions of isatoic anhydride. The detailed synthetic procedure for hydrogelators 1-11 along with their characterization data (IR, 1 H NMR, 13 C NMR, and HRMS) are given in Supplementary materials. preparation of hydrogels. To a pre-weighed compound in a glass vial, 1 M NaOH (2-8 molar equiv.) was added followed by addition of Mili-Q water. The suspension was then heated and vortexed vigorously for 15 minutes to completely dissolve the compound. In the case of N-acetyl 1, N-benzoyl 2, N-naphthoyl 3, fluoro 5 and Figure 7. (a) Antibacterial activity against S. aureus 38 using viable count method (n = 2, *p < 0.001, ns = nosignificant difference between hydrogels and negative control) of hydrogels made from N-acetyl 1, N-benzoyl 2, N-naphthoyl 3, methyl 6, and BA 9 (para-) at 1% w/v. (b) Cytotoxicity of hydrogels made from N-benzoyl 2, N-naphthoyl 3, and BA 9 (para-) against HEK293T cells.
cD Spectroscopy. The anthranilamide-based hydrogels were prepared at 0.8% (w/v) and were diluted 10 times before being transferred into a 0.5 mm path length cuvette. Meanwhile, for the concentration dependence, the hydrogel made from N-benzoyl 2 at 0.8% (w/v) was diluted to make up concentrations ranging from 0.058 mM -0.470 mM before being transferred into a 0.5 mm path length cuvette.
CD spectrum were obtained using a ChirascanPlus CD spectrometer (Applied Photophysics, UK) scanning wavelengths of 180-500 nm with a bandwidth of 1 nm, 0.6 s per point, and step of 1 nm 22 . The outcome of three experiments were then averaged and plotted into a single plot value. The high tension (HT) value of each experiment was maintained to be below 600 mV (Fig. S17).

Attenuated total reflectance fourier-transform infrared spectroscopy (ATR-FTIR). D 2 O gels of
anthranilamide-based short peptides 1-10 were pre-formed in a glass vial at 3%w/v. Heat was applied to these D 2 O gels to trigger the transformation to their solution-phase. Subsequently, two drops of each D 2 O gels was placed on the ATR crystal and allowed to stand for 5 minutes. On the other hand, xerogels were formed in-situ by applying nitrogen to two droplets of hydrogel made from anthranilamide-based short peptides 1-10 at 3% (w/v). The spectrum was recorded using a Spectrum 100 FTIR spectrometer (PerkinElmer, USA) fitted with a 1 mm diamond-ZnSe crystal from 4000-650 cm −1 with 4 cm −1 resolutions and 32 scans.
Rheology measurements. The viscoelastic properties of hydrogels made from anthranilamide-based short peptides were determined using Anton Paar MCR 302 rheometer with a 25 mm stainless steel parallel plate configuration, as previously described 22,23 . Pre-formed hydrogel was warmed, using a heat gun, and the resulting solution (560 µL of 1% (w/v)) was transferred onto the rheometer plate. The other plate was lowered to its measuring position, 1 mm gap, and the solution was allowed to stand for 2-24 h for the gel to completely form. To prevent solvent evaporation, a solvent trap using Mili-Q water and a Peltier temperature controller hood was employed. The frequency sweep test (FST) was performed at constant strain of 0.1% using frequency of 10 Hz to 0.01 Hz. Meanwhile, strain sweep test (SST) was conducted at constant frequency of 1 Hz using 0.1% to 100% strain. In addition, a temperature sweep test was examined to determine Tg values using constant frequency of 1 Hz and constant strain of 0.1% with temperature ramping from 25 °C to 90 °C. The rheology data presented are an average of three repeats.
Atomic force microscopy. A drop of these pre-gel solution was casted onto a mica substrate and the droplet was gently spread using a glass slide. Pre-gel solution of anthranilamide-based short peptides were obtained by either heating the thermo-reversible hydrogels 1-3 and 5-6, or quickly transferring solutions of 8-10 after GdL addition but prior to gelation. The samples were left to dry overnight before being imaged. Imaging was undertaken on a Bruker Multimode 8 Atomic Force Microscope in Scanasyst Air (PeakForce Tappings) mode, which is based on tapping mode AFM, but whereby the imaging parameters are constantly optimized through the force curves that are collected, preventing damage of soft samples 22 . Bruker Scanasyst-Air probes with a spring constant of 0.4-0.8 N m −1 and a tip radius of 2 nm were used in this experiment.
Antibacterial activity. Antibacterial activity of the anthranilamide-based hydrogels were performed using modification of a known method 79 . Initially, a single colony of S. aureus 38 was grown overnight in Luria-Bertani (LB) broth medium (Sigma-Aldrich) at 37 °C. The resulting bacteria culture was centrifuged and harvested. The bacteria pellet was re-suspended in the same volume of LB, twice. Optical density (OD) of the resulting culture was adjusted to 0.1 at 600 nm in LB (10 8 cfu mL −1 ) and the bacterial solution was further adjusted to 3 × 10 4 cfu mL −1 . The bacteria solution (1 mL) was carefully casted on top of the pre-formed hydrogels (1% w/v; 1 mL) in glass vials. As a negative control, bacteria solution without a hydrogel was used in the experiment. These vials were incubated at 37 °C for 18 h. Serial dilution was performed to the 100 µL of the bacteria solution (on top of the hydrogels) using phosphate-buffered saline (PBS). 20 µL of each dilution, were carefully transferred into nutrient agar plates and incubated at 37 °C for another 18 h. The following day, bacterial growth inhibitions were recorded using viable count methods. This experiment was performed twice in triplicate. Multiple sample comparison was performed using one-way ANOVA at p < 0.05. www.nature.com/scientificreports www.nature.com/scientificreports/ cytotoxicity assays. Similar to previously reported method 22 , cytotoxicity measurement was performed using an Alamar Blue colorimetric assay on HEK293T cells. HEK293T cells were passaged using standard cell culture procedures. Cells were detached with trypsin and centrifuged (1000 rpm for 3 min). After supernatant was removed, the cells were re-suspended in Dulbecco's Modified Eagle Medium (DMEM) at a concentration of 60,000 cells/mL. Cells were seeded onto hydrogels at a concentration of 6,000 cells/well. Hydrogels were prepared as described above and 100 µL cast into the wells of a 96 well plate in triplicate. After incubating overnight, 100 µL media was added to the set hydrogels and incubated overnight. The following day, excess media was aspirated and cells seeded atop the hydrogels as above. After incubation for 24 hours, 10 µL Alamar Blue was added to each well, followed by further incubation for another 4 hours. Wells containing cell-free hydrogels, no hydrogel substrate, and a negative control of 20% (v/v) DMSO were prepared as controls. BioRad Benchmark plate reader was used to measure the absorbance at 570 nm and 596 nm. Each experiment was repeated at least three times.