Smart Hydrogels with Antibacterial Properties Built from All Natural

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Smart Hydrogels with Antibacterial Properties Built from All Natural Building Blocks Mengyu Li, Hui Wang, Junfei Hu, Jingjing Hu, Song Zhang, Zhen Yang, Yiwen Li, and Yiyun Cheng Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.9b02547 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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Chemistry of Materials

Smart Hydrogels with Antibacterial Properties Built from All Natural Building Blocks Mengyu Li,a Hui Wang,b Junfei Hu,c Jingjing Hu,a Song Zhang,a Zhen Yang,c Yiwen Li,*c and Yiyun Cheng*a,b Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, P.R. China. a

South China Advanced Institute for Soft Matter Science and Technology, South China University of Technology, Guangzhou 510640, P.R. China. b

c College

of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China.

ABSTRACT: Smart hydrogels with multi-stimuli responsiveness have been increasingly exploited as soft matters for biomedical applications in drug delivery, tissue engineering, and bioactuators. Whilst many of the effects have been well documented, the facile fabrication of multi-responsive hydrogels with specific functionality is met with profound challenges, particularly in the redundant synthesis of macromolecular building blocks and the integration of multiple dynamic linkages to construct hydrogel networks. To address this issue, herein we report a simple and rapid fabrication of smart hydrogels by direct gelation of all naturally occurring building blocks including aminoglycosides, protocatechualdehyde, and Fe(III) via two types of dynamic chemical bonds. The resulting smart hydrogels could perform excellent dynamic features and promising multi-responsiveness to different stimuli including temperature, light, pH, redox, and electricity, and also exhibited high antibacterial activities. This study offers new opportunities in the facile preparation of smart hydrogels for a wide range of applications.

1. INTRODUCTION Smart hydrogels are three-dimensional network functional materials that could remarkably change their shape or mechanical properties in response to various physical stimuli (e.g., heat, light and magnetic field) and chemical stimuli (e.g., redox, pH, and enzyme).1-6 They have attracted great interests in the recent years, owing to fascination with promising applications in tissue engineering, controllable drug delivery, sensors and actuators.7-14 In particular, precise and fast response to complex and multiple stimuli is quite critical for new innovations in the design and fabrication of those smart matters for biomedical purposes. This is because the physiological environment is complicated, and single responsive feature sometimes cannot ensure the accurate and remarkable feedback. One common strategy towards this goal is to generate multifunctional polymers with sophisticated structures and properties by the incorporation and well control of several types of stimuli responsive functionalities into macromolecular systems, which can create multi-responsive behaviors in crosslinking gels over different length scales.15-17 The resulting polymer hydrogel systems usually are able to perform excellent self-healing and tunable mechanical and optical properties under different conditions.18-19 Another widely used strategy involves the sophisticated use of one or more

kinds of gelator(s), which include peptides, nucleotides, and many other amphiphilies, to rationally design and develop a class of low-molecular-weight hydrogels or supramolecular hydrogels.20-29 Their multi-responsiveness characters can be easily included into the gel systems via the pre-designed non-covalent interactions as well as dynamic covalent bonds. These hydrogels also demonstrated unique and promising properties in drug delivery and tissue engineering, such as specific bioresponsiveness, biomimetic behavior, good biocompatibility and biodegradability. Whilst various kinds of multistimuli responsive smart hydrogels have been well documented so far, concern has arisen that the facile fabrication of multi-responsive smart hydrogels with specific functionality is largely limited by the redundant synthesis of gel building blocks and challenges in multiple function integration. For example, the pre-design and synthesis of most polymer and selfassembling peptide gelators often involves extra synthetic, modification and purification works.30 In addition, current gelation process usually requires sophisticated physical operations like sonication, heating/cooling or protonation/deprotonation treatment of the gelators.30-31 Note that these methods might involve large pH changes or high temperature to the surrounding environment, probably hindering the in situ gelation within biological

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Figure 1. Fabrication and characterizations of the smart aminoglycoside hydrogel. (A) Fabrication of PA/Fe(III)/aminoglycoside smart hydrogel via two kinds of dynamic covalent bonds. (B) UV-Vis absorbance spectra of PA/Fe(III) chelate solution at different pH conditions. The PA/Fe(III) chelate was prepared by mixing PA with FeCl3·6H2O at molar ratio of 3:1. The green-colored curve is taken at pH 5.0, where the Fe(III)(PA)(H2O)2 is the major form; the bluecolored curve is taken at pH 6.0 where the Fe(III)(PA)2(H2O) is the dominant form; and the red-colored curve is taken at pH 10.0 where the Fe(III)(PA)3 is the dominant form in the chelate. (C) Phase diagram of PA/Fe(III)/TOB mixtures. The circulated gelation condition ([Fe(III)]=0.29 M, [TOB]=0.31 M) was used to prepare the model hydrogel sample for further investigations. (D) Time-dependent rheology measurement of the model hydrogel conducted at 2% strain and an angular frequency of 10 rad/s. (E-F) Gelation time (E), storage moduli G′ and loss moduli G″ (F) of hydrogels with different TOB concentrations ([Fe(III)]=0.29 M) measured by a rheometer. (G) Raman spectrum of the model PA/Fe(III)/TOB hydrogel.

systems consisting of living cells and bioactive molecules.32 Therefore, it remains a crucial challenge to seek for new strategies towards the ease and low cost fabrication of smart hydrogels with sophisticated stimuli-responsive properties, by using readily available molecular building blocks with minimum set-up and synthesis and physical operation. One promising solution towards this goal is looking to nature as a source of molecular libraries.33-37 The diverse structures present within naturally occurring building blocks enable the fast screen and rational design of suitable dynamic chemistries that are applicable for the ease fabrication of a variety of smart hydrogels with desirable biological functions. In this work, we report a simple assembly of smart gels by the direct gelation of three types of naturally available and inexpensive building blocks (e.g.

aminoglycosides, protocatechualdehyde, and ferri ion Fe(III) via two types of dynamic chemical bonds) (Figure 1A). In particular, aminoglycosides refer to a family of potent antibiotics that contain multiple amine groups and saccharides. They have been widely used as antibacterial drugs against both Gram-negative and Gram-positive bacteria.38-40 Very recently, many promising structural features of aminoglycosides make this class of naturally occurring antibiotics under active investigations as functional gelators for new hydrogel fabrication and other biomedical applications.41-43 For instance, multiple amine groups tethered on the saccharide ring offer ideal scenarios for the development of three-dimensional dynamic covalent networks by imine formation reaction, while the presence of saccharide unit could introduce both good hydrophilicity (for water absorption) and multiple hydrogen bonding


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interactions (for the secondary crosslinking) into the gel system.41 In addition, protocatechualdehyde (PA), a kind of naturally-occurring phenolic aldehyde, is popularly observed in green cavendish bananas, grapevine leaves and barley.44 Previously studies demonstrated that PA could exhibit pro-apoptotic and antibacterial properties.4445 Considering its interesting molecular structural characters (i.e. aldehyde and catechol group could form reversible covalent bonds spontaneously via imine formation reaction and catechol-metal ion coordination chemistry, respectively), we hypothesize that PA can be efficiently incorporated in situ by co-gelation with aminoglycosides and Fe(III) for the facile construction of all-small-molecule smart hydrogels in response to five different kinds of stimuli. 2. EXPERIMENTAL SECTION 2.1 Materials. PA and isopropyl-beta-D-thiogalactopyranoside (IPTG) were purchased from Meryer Chem. Tech. (Shanghai, China). Tobramycin (TOB), neomycin sulfate (NEO), gentamicin sulfate (GEN), paromomycin sulfate (PAR), ribostamycin sulfate (RIB) and netilmicin sulfate (NET) were obtained from Dalian Meilun Biotech. (Dalian, China). Iron chloride hexahydrate (FeCl3·6H2O), silicone oil PMX-200 and sodium borohydride (NaBH4) were purchased from Aladdin Biochem. (Shanghai, China). Potassium chloride (KCl), sodium hydroxide (NaOH) and hydrochloric acid (HCl) were obtained from Sinopharm Chem. (Shanghai, China). Sodium chloride (NaCl), disodium phosphate (Na2HPO4) and bromothymol blue (BTB) were from Macklin Biochem. (Shanghai, China). Potassium dihydrogen phosphate (KH2PO4) were purchased from J&K Chem. (Beijing, China). Yeast extract, tryptone were obtained from Oxoid (Basingstoke, UK). Agar was purchased from Sangon Biotech. (Shanghai, China). Escherichia coli (E. coli, DH5α) was obtained from American type culture collection (ATCC). 2.2 Preparation and characterizations of PA/Fe(III) chelate. PA dissolved in 80 ℃ deionized water was mixed with FeCl3·6H2O solution at a molar ratio of 3:1. The mixture was stirred for 3 h at room temperature and the pH value of the solution was adjusted to 10.0 by the addition of NaOH solution. The mixture was then concentrated to Fe(III) concentrations in the range of 0.35 M to 1.25 M for further use. The UV-Vis spectroscopy experiments were carried out by a PerkinElmer Lambda 35 UV/Vis spectrophotometer to measure the absorbance of the PA/Fe(III) chelate and other building blocks from the gel at different pH conditions. The measured spectral range is 350-950 nm with slit of 1 nm. For the Fe/C value study, the carbon content in the chelate was measured by the element analyses using a CE 440 elemental analyzer (Vario EL III, Elementar) and the Fe content in the chelate was determined by ICP-OES (Leeman Prodigy), respectively. 2.3 Preparation and characterization of PA/Fe(III)/aminoglycoside hydrogel. The PA/Fe(III)/TOB hydrogel was prepared by rapid mixing of PA/Fe(III) chelate and TOB solution with different molar concentrations at room temperature. Note that the typical gelation condition ([Fe(III)]=0.29 M, [TOB]=0.31 M) was used to prepare the model hydrogel sample for further investigations. The morphology of the model hydrogel was

characterized by Cryo-scanning electronic microscope (Cryo-SEM, S-4800, Hitachi, Japan). And the model gel was also freeze-dried and characterized by DXR Laser microcofocal Raman Microscope (USA) under 780 nm near infrared laser excitation. The other kinds of aminoglycoside hydrogels including PA/Fe(III)/NEO ([Fe(III)]=0.12 M, [NEO]=0.11 M), PA/Fe(III)/GEN ([Fe(III)]=0.26 M, [GEN]=0.21 M), PA/Fe(III)/RIB ([Fe(III)]=0.29 M, [RIB]=0.37 M), PA/Fe(III)/NET ([Fe(III)]=0.29 M, [NET]=0.29 M), and PA/Fe(III)/PAR ([Fe(III)]=0.26 M, [PAR]=0.17 M) were all prepared by the similar methods. 2.4 Temperature responsiveness of PA/Fe(III)/TOB hydrogel. The temperature-dependent rheology measurement was performed at 10 rad/s angular frequency and 2% strain using a Hybrid Rheometer (TA Instrument, USA) with 20 mm diameter parallel plate. 170 μL model hydrogel was added to glass vials and incubated in 25 ℃ or 50 ℃ water bath, respectively. The gel responsiveness was monitored every 1 min by manual shaking and washed with DI water to check its erosion performance. 2.5 Light responsiveness of PA/Fe(III)/TOB hydrogel. 170 μL model hydrogel prepared in glass bottle was irradiated by an NIR laser (800 nm, Tsunami, SpectraPhysics) at various laser power densities (0.06, 0.25, 0.32 and 0.36 W∙cm-2, respectively). The temperature of the hydrogel was monitored by a digital temperature sensor (Minggao, Beijing, China). For the photothermal behavior mechanism investigation of the gel, different building block solutions were prepared (2.5 mg/mL) and then put in a vial and irradiated with an 808 nm laser (2 W·cm-2) for 600 s. The temperatures of the testing solutions were recorded by both of an electric coupling thermo-detector and an IR camera. 2.6 Acid responsiveness of PA/Fe(III)/TOB hydrogel. 100 μL model hydrogel was incubated with 150 μL deionized water or 2 M HCl at room temperature. At scheduled time intervals, the solution in the vials was removed and the remaining hydrogels were then weighed. Three repeats were conducted for each sample. For 1H NMR analysis, PA and TOB were mixed at a molar ratio of 1:10 at room temperature. After 2 h, 2 M HCl was added and the samples were lyophilized and characterized by 1H NMR in D2O (Varian, 699.804 MHz). The sample without treating by HCl was tested as a control. 2.7 Redox responsiveness of PA/Fe(III)/TOB hydrogel. NaBH4 (10.47 M) was dissolved in 1 M NaOH solution (150 μL). Then the mixture solution was added into glass vials loaded with 100 μL model hydrogel. At scheduled time intervals, the solution was removed and the remained gels were then weighed. The model hydrogel incubated with 1 M NaOH (150 μL) was used as the control. Three repeats were performed for each sample. 2.8 Electricity responsiveness of PA/Fe(III)/TOB hydrogel. The electricity responsive assay was conducted under 12 V DC voltage. Copper electrodes (Cu-electrodes) or graphite electrodes (C-electrodes) were used to connect the 340 μL model hydrogel. Morphological changes of the hydrogels were recorded by a digital camera. 2.9 Bacterial culture. Fresh culture of E. coli was prepared by inoculating a single bacteria colony from a



Luria-Bertani (LB) plate and then suspended in 5 mL sterile LB medium at 37 °C. The number of bacteria was estimated by measuring the medium absorbance at 600 nm (OD600) using a microplate reader (Thermo Fisher). The bacteria were used when they enter the logarithmic growth period (OD600 = 0.6-0.8). 2.10 In vitro release of Fe(III) and TOB. The PA/Fe(III)/TOB model hydrogel (100 μL) was incubated with 100 uL PBS solution (2X, pH= 7.4) in the vial at room temperature. The samples were centrifuged at 0.5, 1, 2, 4, 6, 8, 10 h, respectively, and the supernatants in the vials were collected for further analysis. The volumes of collected solutions were recorded. The concentration of TOB in the supernatant was evaluated by a well-established ninhydrin assay. And the concentration of released Fe(III) from the model hydrogel was measured by ICP-OES. Three repeats were conducted for each sample. 2.11 In vitro antibacterial properties of PA/Fe(III)/aminoglycoside gels. For the in vitro antibacterial assay, the bacterial solutions (108 CFU/mL, 100 μL) were added to the aminoglycoside gel, sterile water, PA/Fe(III) chelate only, and TOB only at equal concentrations to those in the gels, respectively. Three repeats were conducted for each sample. The above 96-well plate was incubated at 37 °C for 24 h. Next, the bacterial suspension in the plate was inoculated on LB agar medium. The in vitro antibacterial ability of these materials was determined by counting the number of individual bacterial colonies. Moreover, RFP-expressing E. coli suspension (300 μL, 108 CFU/mL) was added into the plates coated with the model hydrogel. After incubation at 37 °C for 24 h, 100 μL of the bacteria suspension was extracted from the well and then inoculated on an LB agar plate. For the control group without the gel, the bacteria suspension was diluted by 104 times before inoculation. To induce RFP expression by the bacteria, IPTG was added to the bacteria suspension at a final concentration of 1 mM and then incubated for another 12 h at 37 °C. The live bacteria on the plates were observed by a fluorescent microscopy (Olympus, Japan). The antibacterial activities of PA/Fe(III)/NEO, PA/Fe(III)/GEN, PA/Fe(III)/PAR, PA/Fe(III)/RIB and PA/Fe(III)/NET hydrogels were also measured by the same method. 3. RESULTS 3.1 Hydrogel Fabrication and Characterization. Our design strategy is to use PA as the binding motif to coordinate Fe(III) at pH 10 with a defined molar ratio of 3:1, and that further act as the cross-linkers to react with aminoglycosides by imine formation reaction for the facile fabrication of dynamic hydrogels (Figure 1A). Notably, PA and Fe(III) could form a stable Fe(III)(PA)3 tri-complex molecule (named PA/Fe(III) chelate) in the aqueous solution at pH>8.5, which was supported by the observation of a strong UV absorbance peak at 450 nm (Figure 1B).46 This can be further confirmed by the experimental analysis of Fe/C weight ratio (22.3%) in the chelate, which was in agreement with the theoretical result (23.4%). TOB was employed as a model aminoglycoside compound, and the hydrogels can be successfully obtained within a wide concentration range of both TOB and PA/Fe(III) chelate

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(Figure 1C). Note that the circulated gelation condition ([Fe(III)]=0.29 M, [TOB]=0.31 M) was used to prepare the model hydrogel sample for further investigations. It was found that a black-colored model hydrogel could be rapidly formed with porous microstructures (Figure S1) within a few minutes by simply mixing of PA/Fe(III) chelate and TOB aqueous solution at room temperature ([Fe(III)]=0.29 M, [TOB]=0.31 M). As shown in Figure 1D, the gelation time of the model PA/Fe(III)/TOB hydrogel was 94 s, noted by the cross-over time point of the storage modulus (G′) and the loss modulus (G″) lines. Both of gelation time and mechanical properties (G′ and G″) of the PA/Fe(III)/TOB system could be finely tuned by changing the concentration of TOB and Fe(III) (Figure 1E-F and Figure S2), which suggested that the formed gel property was greatly influenced by the cross-linking density in the gel. Notably, the presence of multiple Fe(III)-(catecholate)3 coordination bonds in the model gel was also confirmed by the observation of characteristic peaks at 470-670 cm-1 and 1200-1500 cm-1 in the Raman spectrum (Figure 1G), which was in good agreement with a reference result.47 Moreover, further investigations have demonstrated the excellent shear-thinning, thixotropic, and self-healing properties of the PA/Fe(III)/TOB hydrogel. For example, it was found that the total viscosity of the model hydrogel ([Fe(III)]=0.29 M, [TOB]=0.31 M) gradually decreased with the increase of testing shear rate (Figure 2A), and the gel could be injected through a syringe, and then quickly recovered after withdrawing the shear force (in-set of Figure 2A). The shear-thinning property could allow us to easily fabricate this gel into various macroscopic shapes using specific molds, including tubular-, columned-, quadrate- and asteroidal-shaped ones (Figure 2B). The thixotropic property of the model hydrogel was also investigated by imposing alternating strain of 2% and 200% at a constant angular frequency of 10 rad/s as shown in Figure 2C and Figure S3. It was observed that the hydrogel could exhibit a liquid-like behavior at a high strain of 200% (G″ > G′, network disruption) and rapidly recover back to a solid gel-like behavior at the low strain of 2% (G″ < G′, G′ almost recovered to the original moduli, network reconstruction). In addition, the crack/fracture-recovery testing was further employed to confirm the self-healing property of the model PA/Fe(III)/TOB gel. As shown in Figure 2D, two pieces of hydrogels completely healed to form one gel without any cracks within 5 min, and all visible fractures in hydrogel disappeared completely in several hours. These results demonstrated the dynamic feature of the PA/Fe(III)/TOB hydrogel, which was attributed to the existing of multiple reversible interactions in the gel. 2.2 Multistimuli Responsive Behaviors of the Hydrogels. The presence of a large number of dynamic and reversible chemical linkages (i.e. imine bond and Fe(III)-(catecholate)3 coordination bond) and physical interactions (i.e. hydrogen bonding) in the gel usually allows reversible formation and disruption of hydrogel networks under various kinds of stimuli. We then measured the tunable properties of the model PA/Fe(III)/TOB gel in response to five different stimuli, including temperature, near infrared (NIR) light, redox, pH, and electric field. For instance, the hydrogen bonding interactions are usually reversible in temperature-


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Figure 2. Dynamic property characterizations of the model gel. (A) Shear-thinning of the model PA/Fe(III)/TOB hydrogel. The study was carried out at 2% strain and angular frequency swept from 0.1 to 100 rad/s. (B) Tubular-, columned-, quadrate- and asteroidal-shaped PA/Fe(III)/TOB model hydrogel using different molds. (C) Thixotropic test by continuous step strain measurement of the model hydrogel. Recovery and breaking of the hydrogel with alternating oscillation forces at 2% and 200% strains, respectively, was conducted for three recycles. The test was performed at a constant angular frequency of 10 rad/s. (D) Self-healing behavior of the model hydrogel.

Figure 3. Temperature and NIR light responsive properties of the model gel. (A) Temperature-dependent rheology of the model PA/Fe(III)/TOB hydrogel. (B) Melting temperature of the hydrogel with different TOB concentrations ([Fe(III)]=0.29 M) measured by a rheometer. (C) Temperature curve of the model hydrogel irradiated at various NIR laser power densities (0.06, 0.25, 0.32 and 0.36 W∙cm-2, respectively). (D) Schematic diagram of the model hydrogel irradiated by a NIR laser on the left. Photographs of the hydrogel irradiated at 0.36 W∙cm-2 on the right.

sensitive manners, which could further induce the hydrogel formation/dissociation at different temperature points. Figure 3A clearly showed that both of G″and G′ were decreasing with the increase of temperature, and the gel-sol phase transition temperature (G″ > G′, also called melting

temperature) was measured to be 41 oC. The gel could be completely recovered (G″ < G′) by cooling the mixture solution to temperatures below 41 oC. Note that the melting temperature elevations could be achieved when we increased the TOB and Fe(III) concentrations in the gel system,



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Figure 4. Redox, acidity and electricity responsive properties of the model gel. (A) Schematic diagram of reduction of the PA/Fe(III) chelate in the presence of NaBH4. (B) Redox-responsive behavior of the model hydrogel. (C) Schematic diagram of degradation of the hydrogel in the presence of H+. (D) Acid-responsive behavior of the model hydrogel. The insets are the optical images of PA/Fe(III)/TOB model gels under various pH conditions. (E) Conductive property (left panel) and Cuelectrode responsive behavior (right panel) of the model hydrogel. (F) Conductivity property (left panel) and C-electrode responsive behavior (right panel) of the model hydrogel.

respectively (Figure 3B and Figure S4). This indicated that higher content of the chemical cross-linking bonds within the system could significantly increase the gel stability. Additionally, due to the promising photothermal convention feature of PA/Fe(III) chelate (as evident by the UV-vis-NIR absorption and photothermal testing in Figure S5),48-49 the model hydrogel could also efficiently absorb NIR light and convert it into heat immediately (Figure 3C). As a result, NIR light can also be regarded as a kind of stimulus to control the temperature-related hydrogel disruption and reformation. This hypothesis was confirmed by the occurrence of gel-sol phase transition behaviors (recorded by both of optical and IR thermal images) under NIR irradiation at different power intensities and irradiation times (Figure 3C and 3D, Movie S1 and S2). Furthermore, the redox-responsive behavior of the model PA/Fe(III)/TOB gel was investigated using sodium borohydride (NaBH4) as a reducing agent, which was able to degrade the PA/Fe(III) chelate cross-linker via the reduction of Fe(III) to Fe(II) or Fe (Figure 4A). Such a behavior can be confirmed by weighing the residual gels after treatment with NaBH4. It was observed that the gel weight was slightly decreased when immersed in a NaOH solution, while the degradation process was greatly promoted in the presence of NaBH4 during the same period, clearly supporting its redox-responsive behavior. In addition, both of the imine bonds and Fe(III)-(catecholate)3 coordination bonds within the gel performed pH-sensitive

behaviors (Figure 4C),35, 50-53 which could be confirmed by the 1H NMR spectra for the model reactions in Figure S6. This could further lead to gel erosion in the presence of acid, as shown in Figure 4D. Notably, the pH-responsive behavior of the model gel was completely reversible (Figure S7), again suggesting the rapid dynamic nature of those two types of bonds within the gel. More interestingly, the model gel displayed a promising conductive property, allowing to light up the light-emitting diode (LED) bulb in the circuits (Figure 4E-F), like other kinds of metallogels.54-55 Note that the gel also exhibited responsiveness to electric field, which was demonstrated by the fast gel degradation behaviors on both of Cu-electrode (Figure 4E and Movie S3) and Celectrode (Figure 4F and Movie S4). In details, the electricity responsive behavior of the gel on Cu-electrode was explained by the reduction of Fe(III) to Fe(II) at cathode (Figure 4E and Movie S3), resulting in the rapid disassociation of networks and gel erosion in a few minutes; while its responsive performance on C-electrode could be attributed to the occurrence of acid at anode and the decrease of the Fe(III) at cathode (Figure 4F and Movie S4), which could both strongly destroy the crosslinking networks within the gel and finally cause gel degradation. Therefore, all those results unambiguously revealed the promising multistimuli responsive feature of the PA/Fe(III)/TOB smart gel. 2.3 Antibacterial Properties of the Smart Hydrogels.


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Figure 5. Antibacterial activity of the model gel. (A) Schematic concept of antibacterial activity of the PA/Fe(III)/TOB model hydrogel. (B) In vitro release of Fe(III) and TOB from the model hydrogel when immersed in PBS. (C) In vitro antibacterial activity of the PA/Fe(III)/TOB model hydrogel against E. coli. PA/Fe(III) chelate, TOB and bacteria only were tested as controls. (D) Optical images of LB agar media cultured with bacteria (after incubation with uncoated or coated substrates for 24 h). (E) Fluorescence images of RFP-expressing E. coli incubated on uncoated and coated substrates, respectively. The treated bacteria suspension was inoculated in LB media, and 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to induce the expression of RFP for fluorescent imaging. Red spots represent live bacteria.

Figure 6. Fabrication and antibacterial activities of the aminoglycoside hydrogels. (A-E) Time-dependent rheology of different aminoglycoside gels (PA/Fe(III)/NEO, [Fe(III)]=0.12 M, [NEO]=0.11 M; PA/Fe(III)/GEN, [Fe(III)]=0.26 M, [GEN]=0.21 M; PA/Fe(III)/RIB, [Fe(III)]=0.29 M, [RIB]=0.37 M; PA/Fe(III)/NET, [Fe(III)]=0.29 M, [NET]=0.29 M; and PA/Fe(III)/PAR, [Fe(III)]=0.26 M, [PAR]=0.17 M) carried out at 2% strain and an angular frequency of 10 rad/s. (F) In vitro antibacterial activity of the aminoglycoside hydrogels.



We further investigated the antibacterial activity of the resulting hydrogels consisting of various aminoglycoside antibiotic units (Figure 5A). Notably, the bacteria suspension could cause an erosion-based aminoglycoside release behavior of the gel to further kill the bacteria, which might be due to the slight disassociation of dynamic bonds under acidic conditions. Indeed, the PA/Fe(III)/TOB model gel showed a sustained drug release and gel degradation behavior when immersed in PBS. Fe(III) and TOB in the hydrogel were released in a synchronous manner (Figure 5B). Like many other kinds of aminoglycoside hydrogels,4041, 43 this PA/Fe(III)/TOB model gel also demonstrated promising antibacterial activities against E. coli. As shown in Figure 5C, the bacterial survival of E. coli has been significantly inhibited after incubation with the PA/Fe(III)/TOB gel, which clearly supported the excellent antibacterial efficiency of our hydrogel. This result was in good agreement with the observations from Figure 5D-5E on the antibacterial performances of the hydrogel. Particularly, the fluorescent intensity of red fluorescent protein (RFP)-expressed by live E. coli greatly decreased after bacteria incubation on the gel coated culture plate (Figure 5E). More importantly, considering the similar chemical features of different aminoglycoside antibiotics, this antibacterial PA/Fe(III)/TOB hydrogel can be easily and widely extended to many other kinds of aminoglycoside smart gels with promising antibacterial activities in a modular way. Based on the results from Figure 6, we could successfully fabricate another five types of aminoglycoside hydrogels consisting of neomycin (NEO) ([Fe(III)]=0.12 M, [NEO]=0.11 M) (Figure 6A), gentamicin (GEN) ([Fe(III)]=0.26 M, [GEN]=0.21 M) (Figure 6B), ribostamycin (RIB) ([Fe(III)]=0.29 M, [RIB]=0.37 M) (Figure 6C), netilmicin (NET) ([Fe(III)]=0.29 M, [NET]=0.29 M) (Figure 6D), and paromomycin (PAR) ([Fe(III)]=0.26 M, [PAR]=0.17 M) (Figure 6E), respectively. Similar to the PA/Fe(III)/TOB gel system, those five kinds of aminoglycoside hydrogels also unambiguously showed in vitro promising antibacterial activity against E. coli, as shown in Figure 6F. 3. CONCLUSION In summary, we reported the facile fabrication of smart gels by the direct gelation of three kinds of naturally occurring and low molecular weight building blocks such as aminoglycosides, protocatechualdehyde, and Fe(III) by two types of dynamic chemical bonds. Such unique hydrogel design provides several advantages compared with many established gel fabrication strategies, including no redundant pre-synthesis of gelators, facile gelation, and inherent antibacterial property. Note that the presence of a large number of dynamic and reversible linkages and hydrogen bonding in the gel network could introduce dynamic features i.e. thixotropic and self-healing properties and promising multi-responsiveness to different stimuli (e.g. temperature, light, redox, pH, and electricity) to the smart gel. And this class of aminoglycoside hydrogels also demonstrated excellent antibacterial activities. We believe that this work could stimulate new opportunities in the facile preparation of smart hydrogels using natural occurring building blocks with tunable properties and

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desirable biological functionalities for a wide range of applications.

ASSOCIATED CONTENT Supporting Information. Experimental details including gel characterizations and responsive behaviors. This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author * Corresponding author. E-mail: [email protected] (Y.C.), Tel: +86 021-54341001; E-mail: [email protected] (Y.L.), Tel: +86 028-85401066. Author Contributions M.L. carried out the synthetic work, stimuli-responsive experimentsand the antibacterial studies. H.W., S.Z., J.H. and Z.Y. conducted part of the experiments on gel preparation, optimization and characterization. J. H. performed part of antibacterial experiments. Y.L. and Y.C. designed and supervised the study, and wrote up the manuscript.

Notes

The authors declare no competing interests.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21725402 and 21774079), the Fok Ying Tong Education Foundation (151036), and the Shanghai Municipal Science and Technology Commission (17XD1401600).

ABBREVIATIONS PA, protocatechualdehyde; TOB, Tobramycin; NEO, neomycin; GEN, gentamicin; RIB, ribostamycin; NET, netilmicin; PAR, paromomycin.

REFERENCES (1) Qiu, Y.; Park, K. Environment-sensitive hydrogels for drug delivery. Adv. Drug Delivery Rev. 2012, 64, 49-60. (2) Roy, D.; Cambre, J. N.; Sumerlin, B. S. Future perspectives and recent advances in stimuli-responsive materials. Prog. Polym. Sci. 2010, 35, 278-301. (3) Gao, S.; Tang, G.; Hua, D.; Xiong, R.; Han, J.; Jiang, S.; Zhang, Q.; Huang, C. Stimuli-responsive bio-based polymeric systems and their applications. J. Mater. Chem. B 2019, 7, 709-729. (4) Hu, J.; Chen, Y.; Li, Y.; Zhou, Z.; Cheng, Y. A thermo-degradable hydrogel with light-tunable degradation and drug release. Biomaterials 2017, 112, 133-140. (5) Wang, C.; Wang, X.; Dong, K.; Luo, J.; Zhang, Q.; Cheng, Y. Injectable and responsively degradable hydrogel for personalized photothermal therapy. Biomaterials 2016, 104, 129-137. (6) Dai, T.; Wang, C.; Wang, Y.; Xu, W.; Hu, J.; Cheng, Y. A nanocomposite hydrogel with potent and broad-spectrum antibacterial activity. ACS Appl. Mater. Interfaces 2018, 10, 1516315173. (7) Sydney Gladman, A.; Matsumoto, E. A.; Nuzzo, R. G.; Mahadevan, L.; Lewis, J. A. Biomimetic 4D printing. Nat. Mater. 2016, 15, 413-418. (8) Annabi, N.; Tamayol, A.; Uquillas, J. A.; Akbari, M.; Bertassoni, L. E.; Cha, C.; Camci-Unal, G.; Dokmeci, M. R.; Peppas, N. A.; Khademhosseini, A. 25th anniversary article: Rational design and applications of hydrogels in regenerative medicine. Adv. Mater. 2014, 26, 85-124. (9) Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Delivery Rev. 2012, 64, 18-23.


Page 9 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 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 51 52 53 54 55 56 57 58 59 60


(10) Wang, X.; Wang, C.; Wang, X.; Wang, Y.; Zhang, Q.; Cheng, Y. A polydopamine nanoparticle-knotted poly (ethylene glycol) hydrogel for on-demand drug delivery and chemo-photothermal therapy. Chem. Mater. 2017, 29, 1370-1376. (11) Wang, C.; Wang, D.; Dai, T.; Xu, P.; Wu, P.; Zou, Y.; Yang, P.; Hu, J.; Li, Y.; Cheng, Y. Skin Pigmentation-Inspired Polydopamine Sunscreens. Adv. Funct. Mater. 2018, 28, 1802127. (12) Zheng, Z.; Hu, J.; Wang, H.; Huang, J.; Yu, Y.; Zhang, Q.; Cheng, Y. Dynamic softening or stiffening a supramolecular hydrogel by ultraviolet or near-infrared light. ACS Appl. Mater. Interfaces 2017, 9, 24511-24517. (13) Wang, X.; Wang, C.; Zhang, Q.; Cheng, Y. Near infrared lightresponsive and injectable supramolecular hydrogels for ondemand drug delivery. Chem. Commun. 2016, 52, 978-981. (14) Zou, Y.; Zhang, L.; Yang, L.; Zhu, F.; Ding, M.; Lin, F.; Wang, Z.; Li, Y. “Click” chemistry in polymeric scaffolds: Bioactive materials for tissue engineering. J. Control. Release 2018, 273, 160179. (15) Vermonden, T.; Censi, R.; Hennink, W. E. Hydrogels for protein delivery. Chem. Rev. 2012, 112, 2853-2888. (16) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 2009, 324, 59-63. (17) Van Vlierberghe, S.; Dubruel, P.; Schacht, E. Biopolymerbased hydrogels as scaffolds for tissue engineering applications: A review. Biomacromolecules 2011, 12, 1387-1408. (18) Zhang, Q.; Shi, C.-Y.; Qu, D.-H.; Long, Y.-T.; Feringa, B. L.; Tian, H. Exploring a naturally tailored small molecule for stretchable, self-healing, and adhesive supramolecular polymers. Sci. Adv. 2018, 4, eaat8192. (19) Li, C.-H.; Wang, C.; Keplinger, C.; Zuo, J.-L.; Jin, L.; Sun, Y.; Zheng, P.; Cao, Y.; Lissel, F.; Linder, C.; You, X.-Z.; Bao, Z. A highly stretchable autonomous self-healing elastomer. Nat. Chem. 2016, 8, 618-624. (20) Draper, E. R.; Adams, D. J. Low-Molecular-Weight Gels: The State of the Art. Chem 2017, 3, 390-410. (21) Onogi, S.; Shigemitsu, H.; Yoshii, T.; Tanida, T.; Ikeda, M.; Kubota, R.; Hamachi, I. In situ real-time imaging of self-sorted supramolecular nanofibres. Nat. Chem. 2016, 8, 743-752. (22) Frederix, P. W. J. M.; Scott, G. G.; Abul-Haija, Y. M.; Kalafatovic, D.; Pappas, C. G.; Javid, N.; Hunt, N. T.; Ulijn, R. V.; Tuttle, T. Exploring the sequence space for (tri-)peptide self-assembly to design and discover new hydrogels. Nat. Chem. 2015, 7, 30-37. (23) Su, H.; Koo, J. M.; Cui, H. One-component nanomedicine. J. Control. Release 2015, 219, 383-395. (24) Liu, S.; Tang, A.; Xie, M.; Zhao, Y.; Jiang, J.; Liang, G. Oligomeric hydrogels self-assembled from reduction-controlled condensation. Angew. Chem. Int. Ed. 2015, 54, 3639-3642. (25) Wang, H.; Shi, Y.; Wang, L.; Yang, Z. Recombinant proteins as cross-linkers for hydrogelations. Chem. Soc. Rev. 2013, 42, 891901. (26) Ceylan, H.; Urel, M.; Erkal, T. S.; Tekinay, A. B.; Dana, A.; Guler, M. O. Mussel inspired dynamic cross-linking of self-healing peptide nanofiber network. Adv. Funct. Mater. 2013, 23, 20812090. (27) Liu, G.; Sheng, J.; Wu, H.; Yang, C.; Yang, G.; Li, Y.; Ganguly, R.; Zhu, L.; Zhao, Y. Controlling Supramolecular Chirality of TwoComponent Hydrogels by J- and H-Aggregation of Building Blocks. J. Am. Chem. Soc. 2018, 140, 6467-6473. (28) Xiang, P.; Chen, H.; Xiang, H.; Zhao, Y. Selective Coassembly of Aromatic Amino Acids to Fabricate Hydrogels with Light Irradiation ‐ Induced Emission for Fluorescent Imprint. Adv. Mater. 2018, 30, 1705633. (29) Hu, J.; Wang, H.; Hu, Q.; Cheng, Y. G-quadruplex-based antiviral hydrogels by direct gelation of clinical drugs. Mater. Chem. Front. 2019, 3, 1323-1327. (30) Du, X.; Zhou, J.; Shi, J.; Xu, B. Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165-13307.

(31) Raeburn, J.; Cardoso, A. Z.; Adams, D. J. The importance of the self-assembly process to control mechanical properties of low molecular weight hydrogels. Chem. Soc. Rev. 2013, 42, 5143-5156. (32) Poolman, J. M.; Boekhoven, J.; Besselink, A.; Olive, A. G. L.; Van Esch, J. H.; Eelkema, R. Variable gelation time and stiffness of low-molecular-weight hydrogels through catalytic control over self-assembly. Nat. Protoc. 2014, 9, 977-988. (33) Shen, W.; Wang, Q.; Shen, Y.; Gao, X.; Li, L.; Yan, Y.; Wang, H.; Cheng, Y. Green Tea Catechin Dramatically Promotes RNAi Mediated by Low-Molecular-Weight Polymers. ACS Cent. Sci. 2018, 4, 1326–1333. (34) Liu, C.; Shen, W.; Li, B.; Li, T.; Chang, H.; Cheng, Y. Natural Polyphenols Augment Cytosolic Protein Delivery by a Functional Polymer. Chem. Mater. 2019, 31, 1956–1965. (35) Wang, C.; Sang, H.; Wang, Y.; Zhu, F.; Hu, X.; Wang, X.; Wang, X.; Li, Y.; Cheng, Y. Foe to Friend: Supramolecular Nanomedicines Consisting of Natural Polyphenols and Bortezomib. Nano Lett. 2018, 18, 7045-7051. (36) Xiang, S.; Yang, P.; Guo, H.; Zhang, S.; Zhang, X.; Zhu, F.; Li, Y. Green Tea Makes Polyphenol Nanoparticles with RadicalScavenging Activities. Macromol. Rapid Commun. 2017, 38, 1700446. (37) Cheng, X.; Li, M.; Wang, H.; Cheng, Y. All-small-molecule dynamic covalent gels with antibacterial activity by boronatetannic acid gelation. Chin. Chem. Lett. 2019, DOI: 10.1016/j.cclet.2019.07.013 (38) Levy, S. B.; Bonnie, M. Antibacterial resistance worldwide: Causes, challenges and responses. Nat. Med. 2004, 10, S122-S129. (39) Andersson, D. I.; Hughes, D. Antibiotic resistance and its cost: Is it possible to reverse resistance? Nat. Rev. Microbiol. 2010, 8, 260-271. (40) Huang, Y.; Ding, X.; Qi, Y.; Yu, B.; Xu, F.-J. Reductionresponsive multifunctional hyperbranched polyaminoglycosides with excellent antibacterial activity, biocompatibility and gene transfection capability. Biomaterials 2016, 106, 134-143. (41) Hu, J.; Quan, Y.; Lai, Y.; Zheng, Z.; Hu, Z.; Wang, X.; Dai, T.; Zhang, Q.; Cheng, Y. A smart aminoglycoside hydrogel with tunable gel degradation, on-demand drug release, and high antibacterial activity. J. Controlled Release 2017, 247, 145-152. (42) Wang, H.; Cheng, Y. All-small-molecule dynamic covalent hydrogels with multistimuli responsiveness. Mater. Chem. Front. 2019, 3, 472-475. (43) Hu, J.; Zheng, Z.; Liu, C.; Hu, Q.; Cai, X.; Xiao, J.; Cheng, Y. A pH-responsive hydrogel with potent antibacterial activity against both aerobic and anaerobic pathogens. Biomater. Sci. 2019, 7, 581584. (44) Zhou, L.; Zuo, Z.; Chow, M. S. S. Danshen: An overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J. Clin. Pharmacol. 2005, 45, 1345-1359. (45) Li, S.; Yu, Y.; Chen, J.; Guo, B.; Yang, L.; Ding, W. Evaluation of the antibacterial effects and mechanism of action of protocatechualdehyde against Ralstonia solanacearum. Molecules 2016, 21, 754. (46) Charkoudian, L. K.; Franz, K. J. Fe(III)-coordinatior properties of neuromelanin components: 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid. Inorg. Chem. 2006, 45, 3657-3664. (47) Filippidi, E.; Cristiani, T. R.; Eisenbach, C. D.; Herbert Waite, J.; Israelachvili, J. N.; Kollbe Ahn, B.; Valentine, M. T. Toughening elastomers using mussel-inspired iron-catechol complexes. Science 2017, 358, 502-505. (48) Liu, T.; Zhang, M.; Liu, W.; Zeng, X.; Song, X.; Yang, X.; Zhang, X.; Feng, J. Metal Ion/Tannic Acid Assembly as a Versatile Photothermal Platform in Engineering Multimodal Nanotheranostics for Advanced Applications. ACS Nano 2018, 12, 3917-3927. (49) Zhao, G.; Wu, H.; Feng, R.; Wang, D.; Xu, P.; Jiang, P.; Yang, K.; Wang, H.; Guo, Z.; Chen, Q. Novel Metal Polyphenol Framework for



MR Imaging-Guided Photothermal Therapy. ACS Appl. Mater. Interfaces 2018, 10, 3295-3304. (50) Holten-Andersen, N.; Harrington, M. J.; Birkedal, H.; Lee, B. P.; Messersmith, P. B.; Lee, K. Y. C.; Waite, J. H. pH-induced metalligand cross-links inspired by mussel yield self-healing polymer networks with near-covalent elastic moduli. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2651-2655. (51) Lee, B. P.; Konst, S. Novel hydrogel actuator inspired by reversible mussel adhesive protein chemistry. Adv. Mater. 2014, 26, 3415-3419. (52) Vigato, P. A.; Tamburini, S. The challenge of cyclic and acyclic schiff bases and related derivatives. Coord. Chem. Rev. 2004, 248, 1717-2128.

Page 10 of 11

(53) Kim, B. J.; Cheong, H.; Hwang, B. H.; Cha, H. J. MusselInspired Protein Nanoparticles Containing Iron(III)-DOPA Complexes for pH-Responsive Drug Delivery. Angew. Chem. Int. Ed. 2015, 54, 7318-7322. (54) Rahim, M. A.; Björnmalm, M.; Suma, T.; Faria, M.; Ju, Y.; Kempe, K.; Müllner, M.; Ejima, H.; Stickland, A. D.; Caruso, F. Metal– Phenolic Supramolecular Gelation. Angew. Chem. Int. Ed. 2016, 55, 13803-13807. (55) Darabi, M. A.; Khosrozadeh, A.; Mbeleck, R.; Liu, Y.; Chang, Q.; Jiang, J.; Cai, J.; Wang, Q.; Luo, G.; Xing, M. Skin-Inspired Multifunctional Autonomic-Intrinsic Conductive Self-Healing Hydrogels with Pressure Sensitivity, Stretchability, and 3D Printability. Adv. Mater. 2017, 29, 1700533.


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Smart Hydrogels with Antibacterial Properties Built from All Natural

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