Hybrid gum tragacanth/sodium alginate hydrogel reinforced with silver nanotriangles for bacterial biofilm inhibition

Abstract Biomaterial associated bacterial infections are indomitable to treatment due to the rise in antibiotic resistant strains, thereby triggering the need for new antibacterial agents. Herein, composite bactericidal hydrogels were formulated by incorporating silver nanotriangles (AgNTs) inside a hybrid polymer network of Gum Tragacanth/Sodium Alginate (GT/SA) hydrogels. Physico-chemical examination revealed robust mechanical strength, appreciable porosity and desirable in vitro enzymatic biodegradation of composite hydrogels. The antibacterial activity of AgNT-hydrogel was tested against planktonic and biofilm-forming Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. For all the strains, AgNT-hydrogel showed a dose-dependent decrease in bacterial growth. The addition of AgNT-hydrogels (40-80 mg ml−1) caused 87% inhibition of planktonic biomass and up to 74% reduction in biofilm formation. Overall, this study proposes a promising approach for designing antibacterial composite hydrogels to mitigate various forms of bacterial infection. GRAPHICAL ABSTRACT


Introduction
Bacterial colonization and biofilm formation on abiotic or biotic surfaces has turned out to be the fundamental survival mechanism by bacteria to endure conventional antibacterial treatments (Khatoon et al. 2018;Muhammad et al. 2020). It could be developed on various implanted materials, causing biomaterial associated infections (BAIs), which have become a health menace with millions of cases reported annually in the United States alone, and remarkably higher in developing nations (Zander and Becker 2018). Through the transition of bacteria from planktonic to more persistent and pathogenic biofilm form, they could escape the host immune system which is otherwise active against planktonic bacteria . Moreover, the increasing rate of bacterial resistance to prevailing antibiotic treatments, accompanied by the absence of new antibiotics is the foremost reason for escalating biofilms (Haidari et al. 2021;Targhi et al. 2021). Also, extracellular polymeric substances (EPS) provide structural strength to the biofilm and prevent the accessibility of antibacterial agents (Bi et al. 2021). So, biofilm control is of utmost importance to reduce the virulence of bacteria, and hence the management of BAIs by multidrug-resistant (MDR) bacteria. In a nutshell, the end of the golden period for antibiotics that could not avoid such deeprooted infections has highlighted the necessity for alternative antimicrobial materials and strategies that could effectively contribute against these complex clinical issues. In the quest for new therapies, Agbased nanomaterials have been extensively studied for creating antibacterial surfaces along with a proven potential to circumvent bacterial antibiotic resistance Urnukhsaikhan et al. 2021;Tripathi and Goshisht 2022). In addition, anisotropic AgNTs with sharp edges and corners exhibit superior antibacterial effects than spherical Ag nanoparticles (Agostino et al. 2017;Goyal et al. 2017;Djafari et al. 2019;Malekzadeh et al. 2019;Parit et al. 2020;Al-Zahrani et al. 2022). The highly effective contact area of AgNTs helps them to strongly bind the cell membrane producing a change in permeability, as well as adversely inhibiting the bacterial metabolism by the release of silver ions (Urnukhsaikhan et al. 2021;Takeda et al. 2022). Furthermore, the use of antibacterial hydrogels has emerged to be a well-established strategy to combat biofilm infections by MDR bacteria (Gonz alez-S anchez et al. 2015;Fasiku et al. 2021;Tarawneh et al. 2022;Bhattacharjee et al. 2022). Such hydrogels act as reservoirs for antibacterial agents, primarily silver nanoparticles which could be released in a sustained manner to the implant site, thereby minimalizing cytotoxic effects (Dai et al. 2018;. Over the past few decades, natural polymers such as chitosan (De Mori et al. 2019;Fasiku et al. 2021), alginate (Karnik et al. 2016;Pawar et al. 2018;Porter et al. 2021), hyaluronic acid (Boot et al. 2020;Liao et al. 2020), gelatin (Suleman et al. 2021), among others, have been extensively reported for hydrogel synthesis with biofilm inhibitory potential. Amongst these, sodium alginate (SA), an anionic polysaccharide has been preferably used, wherein carboxyl groups in its structure interact with the divalent metal cations like Ca 2þ to form a stable gel network (Cikrikci et al. 2018;Abasalizadeh et al. 2020;Gir on-Hern andez et al. 2021). However, SA hydrogels cross-linked with Ca 2þ ions exhibit poor mechanical properties, low enzymatic degradation, and therefore uncontrolled payload release. Previous studies suggested that SA can be blended with other polymers to develop hydrogels to control these limitations (Matai et al. 2019;Apoorva et al. 2020;Kumar et al. 2020). Gum tragacanth (GT) is a biodegradable, branched anionic polysaccharide with D-galacturonic acid, Dgalactose, D-xylose, L-arabinose, and L-fucose units. Hydrogels derived from GT are known to be stable over a wide pH range, and the presence of abundant hydroxyl and carboxyl groups of GT provides sites for ionic interactions leading to the stable gel network (Verma et al. 2019;Nagaraja et al. 2021). Recently, few studies have reported the fabrication of GT and SA microstructured gel-based biomaterials for wound healing and drug delivery applications (Kulanthaivel et al. 2017;Cikrikci et al. 2018;. Inspired by this, this study intends to combine the properties of SA with GT and investigate their suitability as an anti-biofilm material. The blending of polysaccharides GT and SA to form an inter-crosslinked structure can essentially augment the mechanical strength, allow tunable porosity and hence the release of antibacterial agents. However, the use of toxic and complex cross-linking mechanisms complicates their applicability. Therefore, there is still a need to develop a simple method to design biodegradable and eco-friendly antibacterial hydrogel suitable for biofilm inhibition. Therefore, silver nanotriangles (AgNTs) composite hydrogels comprising GT and SA via simple ionic cross-linking with calcium ions were synthesised. To the best of the authors' knowledge, this is the first report illustrating the incorporation of AgNTs in the natural hydrogel platform as a potential anti-biofilm agent. In the ensuing work, the AgNTs composite hydrogel provides the following advantages: (1) In situ hydrogel synthesis approach makes it amenable to biomaterial coating applications; (2) The use of natural polymers, namely GT and SA for hydrogel formulation renders inherent biocompatibility and biodegradability, thereby bypassing the toxicity issues; (3) Sustained release of anisotropic AgNTs from the hydrogel polymer networks reduces the systemic toxicity and enhances the overall antibacterial efficacy of AgNTs. Following, these hydrogels were assessed for their ability to reduce the planktonic as well as biofilm growth by Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Concentrations of AgNT-hydrogel needed for biofilm inhibition were higher than the MIC for planktonic bacteria.

Synthesis of citrate stabilized silver nanotriangles (AgNTs)
The silver nanotriangles were synthesized using a previously reported seed-mediated process with slight modifications (Aherne et al. 2008). Initially, an aqueous solution was prepared by mixing TSC (2.5 mM, 5 ml), PSSS (500 mg L À1 , 0.25 ml) and fresh NaBH 4 (10 mM, 0.3 ml). To this solution, AgNO 3 (0.5 mM, 5 ml) was added dropwise with continuous stirring at 500 rpm under 25 C. The transparent solution turned bright yellow which indicated the formation of Ag seeds.
For AgNTs synthesis, AA (10 mM, 75 ml), and seed solution (200 ml) were sequentially added to DI water (5 ml). Following, the dropwise addition of AgNO 3 (0.5 mM, 3 ml) turned the solution gradually blue-coloured, indicating the formation of AgNTs. The synthesized AgNTs were further stabilized with the addition of TSC (25 mM, 0.5 ml) under constant stirring at 500 rpm for 15 min. Finally, the obtained AgNT solution was stored at room temperature for further use.

Preparation of AgNT-hydrogels
GT and SA hydrogels were prepared using a previously reported ionotropic gelation method with few modifications (Apoorva et al. 2020). In brief, the constituent polymers, namely GT (3 w/v %) and SA (2 w/v %) were added to 10 ml of DI water and magnetically stirred until both polymers were homogeneously dissolved. This polymer mixture was then transferred to a glass casting mould (dimensions-50 Â 16 mm 2 ). Next, the ionic cross-linking was carried out by pouring CaCl 2 (10 w/v %) over the polymer mixture. After 15 min incubation, the excess CaCl 2 solution was discarded and the resulting hydrogel (40 Â 6 mm 2 ) was washed twice with DI water to remove non-crosslinked chains, water molecules or other chemicals.
Similarly, for the synthesis of AgNT-hydrogels, an aqueous solution of as-prepared AgNTs was added to GT and SA mixture before cross-linking with CaCl 2 ( Table 1). The prepared AgNT hydrogels were further utilized for physico-chemical characterization and antibacterial assessment.

Characterization
The optical absorption spectra of the samples were recorded using a UV-visible spectrophotometer (LabIndia, UV 3200) in the wavelength range of 200-800 nm. Size and morphological analysis of the samples was done using a High Resolution-Transmission Electron Microscope (HR-TEM, JEOL JEM 2100 plus). The particle size and charge distribution studies were performed by recording hydrodynamic diameter (H.D.) and zeta potential (f) using a Zetasizer (Malvern, ZS90). X-Ray Diffraction (XRD) pattern of the samples was obtained by Bruker D8 Advance X-Ray diffractometer with monochromatic Cu-Ka radiation (k ¼ 1.5406 Å) in the 2h range of 5-80 and a scan rate of 1 min À1 . The elemental and internal structure of the samples were examined using a Field Emission-Scanning Electron Microscope (FE-SEM, Hitachi SU8010) working at an operating voltage of 5 eV coupled with an Energy-Dispersive X-ray spectrometer (EDX). For FE-SEM analysis, the hydrogel samples were soaked in DI water, lyophilized and exposed to gold ion sputtering before recording images. Fourier transform infrared spectroscopy (FTIR) was carried out with a Nicolet iS10 spectrometer in the range of 400 to 4000 cm À1 to ascertain the chemical composition of the samples using standard potassium bromide (KBr) pellet procedure.

Wettability
The wetting behaviour of the hydrogels was analyzed at 25 C using a goniometer (Drop Shape Analyzer DSA-100E) . The sessile drop method was employed to measure direct water contact angle by dropping 4 ml of DI water onto the hydrogel surface, followed by a digital image capturing and data analysis by the contact angle measuring apparatus.

Swelling behaviour
The water retention potential of the hydrogels was recorded by a standard gravimetric method, as reported previously (Kumar et al. 2020). Hydrogels were weighted (W o ) and separately soaked in two different buffer solutions i.e. phosphate buffer saline (PBS) (pH 7.4, 30 ml) and phosphate buffer (PB) (pH 5.5, 30 ml), at 25 C. The weight (W t ) of the swollen hydrogels was recorded at pre-defined intervals (1, 3, 5, 7, 12, 24 h) after carefully wiping the water droplets on the surface with tissue paper. The percentage swelling was then calculated using the following equation (eq 1):

Porosity
To estimate the porous behaviour of hydrogels, porosity measurements were carried out using the liquid displacement method (Nie et al. 2020). Typically, the lyophilized hydrogels of known weight were dipped in a pre-determined volume of DI water (V 1 ). After 24 h, the level of water with submerged hydrogel (V 2 ), and after hydrogel removal (V 3 ) were measured. The porosity was calculated using the following equation (eq 2): In vitro enzyme degradation assay The degradation tendency of the hydrogels was assessed via in vitro degradation assay utilizing lysozyme as the primary model enzyme (Osi et al. 2021). Pre-weighed hydrogels were first immersed in PBS (pH 7.4) for 24 h at 37 C to achieve swelling equilibrium. Afterwards, the swollen hydrogels were soaked in lysozyme solution (2 mg ml À1 , 3 ml) prepared in PBS buffer, and incubated at 37 C. At fixed time intervals (1, 3, 5, 7 and 14 days), the hydrogel samples were taken out and washed with DI water. Subsequently, the water droplets on the samples were carefully removed with delicate paper wipers and then weighed. Moreover, the lysozyme solution was changed after every three days to maintain uniform behaviour. In vitro degradation of the hydrogels was estimated by calculating the residual mass (%) using the following equation (eq 3): where, W o and W t denote the weight of the hydrogel before immersion in lysozyme and at different time intervals, respectively.

Rheology
The mechanical and shear thinning properties of hydrogel were analyzed with a rotational rheometer (Bohlin Rheometer CVO 100, Malvern Instruments Ltd.) at 25 C. The measurements were taken using cone plate geometry having a diameter of 40 mm and a 4 angle. The disc shaped hydrogels (10 Â 6 mm 2 ) were cut and positioned on the lower plate and the gap was fixed at 150 mm. The frequency sweep test was performed with an angular frequency varying from 0.6 -439 rad sec À1 at a constant strain of 1%. Storage or elastic (G') and loss moduli (G'') were examined and recorded. Further, the shear thinning properties of hydrogel were assessed with shear viscometry tests over the shear rate of 0.1-1000s À1 .

Bacterial cultures
All the bacterial strains (E. coli, P. aeruginosa and S. aureus) were revived by inoculating a single colony from a nutrient agar plate (stored at 4 C) and cultured into NB medium at 37 C for 12 h. The optical density (OD) measurements of the culture medium were recorded at 600 nm using UV-visible spectroscopy and standardized to 0.8-1.0, which corresponds to a bacterial concentration of $10 8 to 10 9 CFU ml-1. Unless mentioned, for evaluating antibacterial performance, the standardized culture medium was further diluted 1:100 times, which is equivalent to 1% v/ v. All the experiments were performed in triplicates.
In vitro antibacterial assay for planktonic bacteria Different AgNT-hydrogel samples (40 mg ml À1 ) were sterilized under UV light for 15 min and immersed in 5 ml bacterial suspension (1% v/v). The samples were kept in a shaking incubator at 37 C for 12 h. For comparison, the untreated bacteria medium and nutrient medium containing hydrogel served as positive and negative controls, respectively. After 12 h incubation, the OD of samples was recorded at 600 nm by spectrophotometry. The AgNT-hydrogel formulation which showed the highest bacterial inhibition was selected to evaluate the MIC of different bacterial species. The broth dilution assay was conducted to determine the minimum inhibitory concentration (MIC) of AgNT-hydrogel against E. coli, P. aeruginosa and S. aureus. The cells (10 6 CFU ml À1 ) were incubated with different concentrations of UV-sterilized AgNThydrogels (10, 20, 40, 60, and 80 mg ml À1 ) and kept in a shaking incubator (150 rpm) at 37 C for 12 h. After the completion of incubation, the bacterial growth inhibition was examined by measuring OD 600 of the culture medium. The minimum hydrogel concentration tested and found capable of inhibiting the visible bacterial growth was identified as the MIC for that particular bacteria. The inhibition efficiency of AgNT-hydrogels against planktonic cells was calculated using the following equation (eq 4): where OD positive control, OD negative control and OD sample are the optical density (recorded at 600 nm) of untreated bacteria, AgNT-hydrogel containing nutrient medium, and bacterial treated with AgNT-hydrogel, respectively.

Spread plate antibacterial assay
The bacterial cells (1% (v/v)) were cultured in the presence of AgNT-hydrogel (respective MIC values) at 37 C for 12 h. An aliquot from the treated culture was diluted 1:100 fold and spread onto nutrient agar plates. The plates were then incubated for 24 h at 37 C. The reduction in the number of bacterial colonies on plates incubated with treated bacteria as compared to the untreated bacteria was observed visually, and digital images were acquired.

Time inhibition assay
AgNT-hydrogels (at MIC values) were added to the culture tubes containing bacterial cells (1% (v/v)) and kept in a shaking incubator at 37 C. Positive and negative controls were included in the study. After the designated incubation times (1, 3, 5, 7, 9, and 12 h), each culture tube was taken from the incubator and the growth inhibition of the treated cells was estimated by recording OD at 600 nm. The killing curves were plotted as log 10 (CFU ml À1 ) as a function of time.

Reusability assay
The bacterial cultures ((1% (v/v)) were exposed to AgNT-hydrogels and kept in a shaking incubator at 37 C for 12 h. After incubation, OD 600 of the medium was measured at 600 nm and growth inhibition was calculated. This corresponded to cycle 1. To reuse the hydrogels, they were carefully removed from the bacterial culture and washed with PBS buffer to remove adhered bacteria. The hydrogels were sterilized under UV light and added to fresh bacterial cultures ((1% (v/v)), incubated at 37 C for 12 h under shaking conditions and OD 600 was measured. This was referred to as cycle 2 and the mentioned process was repeated for consecutive cycles.

Morphology analysis of bacterial cells by FE-SEM
The morphological changes in E. coli cells after interaction with the AgNT-hydrogel were visualized by FE-SEM (Hitachi SU8010) at 5 KeV. For analysis, the cell samples were prepared according to the previously reported method . In brief, bacterial cells in the logarithmic phase ($10 8 CFU ml À1 ) were incubated with AgNT-hydrogel for 6 h. The cells were then centrifuged at 5000 g for 10 min to obtain pellets, rinsed thrice with PBS (pH 7.4) and fixed with 2% (v/v) glutaraldehyde for 2 h. Post chemical fixation, the cells were rinsed three times with PBS, followed by incubation for 15 min. Then, the cells were dehydrated with gradient ethanol concentration (10, 30, 50, 70, 90 and 100%, 15 min each). The dehydrated cells were air-dried on a clean glass coverslip, sputter coated with a thin gold layer by gold sputtering and imaged under FE-SEM.

In vitro biofilm formation assay
The potential of AgNT-hydrogel to impede bacterial biofilm formation was evaluated by the Crystal Violet staining method (Targhi et al. 2021). In brief, overnight cultured bacterial suspensions were diluted in the ratio of 1:100 in TSB medium for S. aureus and E. coli, and LB medium for P. aeruginosa). From the above dilution, 100 ml of bacterial suspension was added to each well in a 96-well plate (Tarsons, India), along with AgNT-hydrogel at different concentrations (40, 60, and 80 mg ml À1 ). For comparison, positive and negative controls were included in the assay. The plate was then incubated at 37 C for 24 h, under static conditions to ensure undisturbed cell attachment to the plate surface. After incubation, the growth media were discarded, and the wells along with hydrogels were rinsed thrice with PBS (pH 7.4) to separate the remaining planktonic bacteria. The biofilm cells were fixed with 100 ml methanol for 10 min. The supernatant was removed and subsequently air-dried. The biofilms were stained with 200 ml CV (0.1 v/v %) solution at ambient temperature for 20 min. The wells were again washed three times with PBS to remove excess stain, and air-dried. The CV-bound with biofilm cells were then solubilized with 33% acetic acid and incubated for 15 min. The biofilm cells were thereafter detached thoroughly by vigorous pipetting and absorbance was read at 590 nm using a spectrophotometer and the biofilm biomass was quantified.

Statistical analysis
The experimental data were reported as mean ± standard deviation (SD) and performed in triplicate (n ¼ 3). The graphs were plotted in OriginPro 8.5. The error bars for all the graphs represent the SD.

Synthesis and characterization of AgNTs
Silver nanotriangles (AgNTs) were synthesized by a seed-based method using environmentally benign reagents and under mild conditions (Scheme 1(A)). During the synthesis of AgNTs, a strong reducing agent i.e. sodium borohydride mediated seed production, whereas ascorbic acid (weak reducing agent) ensured a slow and controlled reduction rate during the growth step. The method involved the use of TSC as a stabilizer for seed production and growth steps (Zhang et al. 2011). Low concentration of citrate during the seed production step has been reported for facilitating anisotropic growth to form AgNTs (Bakshi et al. 2008;Gao et al. 2013;Khan et al. 2017). Moreover, PSSS was added at the stage of seed production which prevented the crystal growth along Ag f100g directions (corresponding to spherical Ag nanoparticles) and promoted the growth along Ag f111g crystal planes, thereby resulting in the formation of monodisperse AgNTs (Aherne et al. 2008). Altogether, the interplay of chemical selection and their concentrations play a considerable role in the production of high-quality monodispersed AgNTs. The formation of citrate-capped AgNTs was visualized by a colour change -from yellow (seed solution) to dark blue, owing to the shift in surface plasmon resonance for metallic Ag nanoparticles (Figure 1a, inset). UV-Vis absorption spectra of the blue-coloured solution showed three different absorption peaks representing discrete modes of plasmon excitation, thereby indicating the anisotropic shape of Ag nanoparticles, as reported previously Vo et al. 2019). The resonance band at 330 nm appeared as a result of the out-of-plane quadrupole resonance. Strong in-plane quadrupole and dipole plasmon resonance bands at 405 and 650 nm, respectively, were characteristics of triangular structured Ag nanoparticles. The AgNTs were further characterized by TEM (Figure 1b). Anisotropic, triangular-shaped nanoparticles with truncated structures and sharp edges were revealed. The average size of AgNTs was 82 nm with a broad distribution of edge length (15-150 nm). Further, the differences in edge length were responsible for the origin of a wide resonant band (at 650 nm) in the absorption spectra of AgNTs. The DLS measurements further Scheme 1. Schematic illustration of synthesis of (a) AgNTs and (b) AgNT-hydrogels prepared by ionic cross-linked method.
ascertained the hydrodynamic diameter of AgNTs to be $63 nm ( Figure S1), corroborating with the TEM analysis. The crystallinity of AgNTs was ascertained by XRD and four diffraction peaks at 38. 25 , 44.31 , 64.70 , and 77.65 corresponding to (111), (200), (220), and (311) planes of Ag were observed ( Figure  S2). A sharp and highest intensity diffraction peak at 38.5 pertaining to f111g lattices planes of the facecentred cubic structure indicates the fabrication of triangular shaped silver nanoparticles (Van Dong et al. 2012).
Next, the zeta potential (f) measurements of AgNTs were carried out to understand their colloidal stability at different pH values ( Figure S3). When dispersed in DI water (pH 6.5), AgNTs exhibited a f value of À14 ± 0.32 mV under the citrate ions adsorbed on the surface, which prevents the aggregation of particles under physiological conditions. Under highly acidic conditions (pH 3), f was found to be À5.05 ± 0.35 mV which implies low stability of AgNTs. A high number of protons are available at low pH, which interacts with the negatively charged citrate ions, thereby resulting in reduced efficiency for AgNTs stabilization (El et al. 2010;Fernando and Zhou 2019). As pH increased, the electrostatic repulsions interactions between citrate stabilized AgNTs and maintained the stability of the particles. As a result, a corresponding increase in f values at pH 5 (f ¼ À10.02 ± 0.45 mV) and 7 (f ¼ À18.8 ± 2.43 mV) was observed. Interestingly, in alkaline conditions (pH 8 to 12), f values reached up to À26.4 ± 0.53 mV, possibly due to the presence of hydroxyl ions which contribute to the enhanced stability of AgNTs. Overall, the synthesized AgNTs were negatively charged and changes in proton and hydroxyl ion concentration influenced their electrostatic stability.

Synthesis and characterization of AgNTs-hydrogels
During the formation of AgNT-hydrogels, numerous hydroxyl and carboxyl groups of GT/SA were ionically cross-linked by Ca 2þ , thus leading to a stable gel network (Scheme 1 (B)). Benefitting from the hydrophilic nature of AgNTs, their addition before the gelation process ensured their incorporation inside hydrogel networks due to multiple electrostatic interactions and hydrogen bonding between surface functional groups of AgNTs and GT/SA (Varaprasad et al. 2010;Jing et al. 2022). Specifically, AgNTs interacted with GT and SA through the hydroxyl and carboxyl groups present in the polymeric chains. This further enhanced the cross-linking and improved the overall mechanical strength of the composite hydrogels. These hydrogels could be formed in 15 min, as evidenced by the bottle inversion method (Figure 2a). Hydrogel formulations (denoted as TAA 1-TAA 4) were prepared accordingly with different AgNTs concentrations and evaluated for antibacterial characteristics (vide infra).
The morphology and internal network of lyophilized blank (without) and AgNT-hydrogels were examined by FE-SEM. The microscopic analysis of blank hydrogel revealed a planar surface with an interconnected and porous network (Figure 2b). A change in the morphology of hydrogels was observed after the incorporation of AgNTs. FE-SEM images of AgNT-hydrogels showed crystal-like particle morphology at different ratios (Figure 2c and d). No distinguished difference was found for the hydrogels with different AgNT contents. The mechanism behind this change in hydrogel microstructure is not clear and may require further investigation. In addition, an increase in the concentration of AgNTs caused a concomitant decrease in hydrogel porosity. Interestingly, the pores in the network could serve as the interaction sites for the trapped bacteria and AgNTs. EDAX analysis confirmed the presence of C (carbon), O (oxygen) and Ag (silver) elements that constituted the TAA 4 hydrogel matrix (Figure 2e). The elemental composition of AgNT-hydrogels revealed that the weight percentages of silver in the formulations were about 0.1% to 0.41%. Apart from this, the peaks of Ca (Calcium) and Na (Sodium) in the spectrum were attributed to the cross-linking agent (CaCl 2 ) and sodium alginate, respectively. Further, this nullified the possibility of AgNTs leaching out from the polymer network during Ca 2þ crosslinking. The probable chemical interactions occurring between AgNTs, and the polymer network of the hydrogels were revealed by FTIR spectra (Figures 2f  and S4). The usual band in the range of 3270-3870 cm À1 is characteristic of O-H stretching (Zhong et al. 2018). The FTIR spectra of TAA and TAA 4 revealed a vibration band in the range of 1020-1060 cm À1 corresponding to C-OH stretching in GT and SA. These bands are characteristic of the polysaccharide groups consisting of galacturonic and guluronic units (Cikrikci et al. 2018;Apoorva et al. 2020). The characteristic band in the range 1610-1640 cm À1 was ascribed to asymmetric and symmetric stretching vibrations of carboxyl groups of GT, SA as well as composite hydrogels respectively (Zhong et al. 2018). Interestingly, a sharp and high-intensity band at 1738 cm À1 associated with C ¼ O stretching in the spectrum of GT disappeared after hydrogel formation (Moghaddam et al. 2019;Garg et al. 2020). This might be attributed to the fact that most of the functional groups are involved in bond formation, thereby reducing the intensity of the band. Additionally, citrate capped AgNTs showed bands at 1638 and 1402 cm À1 attributed to the asymmetric and symmetric stretching vibrations of carboxyl groups. Moreover, the oxygen atoms of OH and COOH groups get associated with AgNT clusters. This leads to the presence of a large number of hydroxyl and carboxyl groups in close proximity inside AgNThydrogels, and the possible hydrogen bonding between them caused the broadening of the peaks at $3400 cm À1 and $1030 cm À1 (Rao et al. 2010).
To analyze the mechanical and physico-chemical characteristics of the hydrogels, their swelling behaviour was demonstrated by mimicking the physiological conditions at 37 C using PBS (pH 7.4), and PB buffer (pH 5.5). Clearly, both the hydrogels (TAA and TAA 4) showed rapid swelling in the first 6 h, followed by attaining equilibrium. Noteworthy, the hydrogels showed pH-dependent swelling behaviour as shown in Figure 3a. The swelling of TAA and TAA 4 hydrogels increased from $57% to 100% and $47% to 84%, respectively, as the pH increased from 5.5 to 7.4. In general, hydrogels tend to swell owing to the presence of hydrophilic groups (mainly COOH and OH groups) in their polymeric network that interacts with water molecules, thereby resulting in swelling (Ahmed 2015). For instance, at pH 7.4, a large number of carboxyl groups (-COO -) of alginate (pKa, 3.49) and GT (pKa, 3) were protonated, leading to strong electrostatic repulsion between the negatively charged polymer chains, and consequently increased swelling. Contrarily, in the acidic environment (pH 5.5), due to the decreased electrostatic repulsion between the less protonated carboxyl groups, the hydrogel network tends to swell less. The addition of AgNTs caused a reduction in the swelling ratio of hydrogels at pH 7.4 as well as 5.5. The crosslinking density of TAA 4 was denser than that of TAA, thereby resulting in a stable internal structure with smaller pores, and hence less swelling (Deen and Chua 2015;Zhang et al. 2017). Next, the water contact angle of hydrogels was determined to ascertain their wettability and hydrophilicity (Figure 3b). The contact angle for blank TAA and TAA 4 hydrogels obtained was 28.85 ± 3.64 to 34.85 ± 5.26 , respectively, indicating the overall hydrophilic nature (contact angle < 90 ). Moreover, the increase in contact angle of TAA 4 hydrogels is consistent with the presence of AgNTs in their matrix, wherein the reduction in the free hydrophilic groups led to a slight decrease in hydrophilicity. Besides, the porosity of the hydrogels was estimated by the liquid displacement method (Figure 3c). With the addition of AgNTs, the porosity of hydrogel decreased from 91% to 67%. Such an outcome can be explained by the increased cross-linking density in TAA 4 which restricts the free space available for water uptake, in corroboration with swelling studies. Considering the application of AgNT-hydrogels in the biomedical field, it becomes imperative to evaluate their degradation behaviour. The degradation profiles of TAA and TAA 4 hydrogels were determined at simulated physiological conditions (PBS, pH 7.4) by examining the weight loss in the presence of lysozyme at 37 C (Figure 3d). The residual mass ratios for TAA and TAA 4 were more than 65% in 5 days. The hydrogels were susceptible to loss of structural integrity upon lysozyme exposure and showed complete biodegradation within 14 days. These results certify the biodegradability and long-lasting shelf life of the hybrid hydrogels. The mechanical properties and viscoelastic nature of AgNT-hydrogels were verified by rheological experiments. To this end, an oscillatory frequency sweep test was carried out at a constant strain of 0.1% to determine the storage (G 0 ) and loss (G 00 ) moduli of AgNT-hydrogels as a function of frequency ( Figure 4a). As expected, the hydrogels showed viscous nature at low frequencies with G 0 smaller than G 00 . With an increase in frequency, the G 00 values became considerably smaller than G 0 which signified a shift towards elastic behaviour. This trend in the change of loss and storage moduli values denotes the viscoelastic nature and stable structure of the hydrogels. As shown in Figure 4b, the storage modulus (G 0 ) of TAA and TAA 4 hydrogels at a fixed angular frequency of 271 rad sec À1 was 900 Pa and 260 Pa, respectively. After the incorporation of AgNTs in TAA 4, the G' values showed a significant increase compared to TAA, implying the augmented inter-particle interactions and enhanced cross-linking density (Matai et al. 2019;Cheng et al. 2021;Ferrag et al. 2021). Furthermore, as demonstrated by shear rate tests (Figure 4c), both the hydrogels depicted shear thinning behaviour associated with non-newtonian fluids. As the shear rate increased, a concomitant decrease in the viscosity was observed. Similar to storage/elastic moduli experimental outcomes, TAA 4 hydrogels showed slightly higher viscosity which may be attributed to the increase in molecular weight upon the incorporation of AgNTs into the networks. Therefore, the above findings endorse the selection of AgNT hydrogel formulation with optimum morphological and structural characteristics for anti-biofouling applications.

Antibacterial assessment
Hydrogels endowed with antibacterial properties could decrease the risk of infection in biomedical implants. To this end, the antibacterial performance of AgNT-hydrogels was evaluated against three different bacterial strains namely, S. aureus, E. coli, and P. aeruginosa. All these bacterial organisms were selected for the study since they are capable of developing multi-drug resistance and account for most implant associated infections. Firstly, the bacterial inhibition potential of TAA hydrogels was elucidated by the broth dilution method (Figures 5a and S5). With the increase in the concentration of AgNTs in the hydrogel, a corresponding increase in growth inhibition was observed. This clearly illustrated that the antibacterial performance of hydrogels primarily relied on the AgNTs, which could inhibit bacterial growth by contact killing as well as ion release. For all hydrogel formulations, bacterial inhibitory activity (highest to lowest) followed the order: E. coli > P. aeruginosa > S. aureus, similar to as reported previously . Because the released Ag þ ions attack the cell wall of bacteria and cause protein denaturation, the structural difference in the cell wall of different bacteria might be responsible for the variation in inhibitory effect. The highly negative-charged lipopolysaccharide present in E. coli (Gram-negative) displayed higher electrostatic interaction with the positive-charged Ag þ ions than weakly negativecharged S. aureus (gram-positive), thereby enhancing penetration and cell lysis (Song et al. 2018). The literature reports that tuning the morphology of Ag nanoparticles to a triangular shape could strengthen its antimicrobial performance owing to the high anisotropy of sharp edges and tips (Acharya et al. 2018;Mohsen et al. 2020). Likewise, different nanomaterials viz. zinc oxide nanoparticles (Sharma et al. 2022), Au NPs (Lagha et al. 2021), and iron oxide NPs (Velsankar et al. 2022) have been documented to confer enhanced antimicrobial efficiency owing to their anisotropic structures. Song and his group compared the antimicrobial efficiency of different shapes of nanosilver namely triangular nanoplates, spheres and rods, synthesized via solution phase routes (Pal et al. 2007). Amongst these, triangular nanoplates with the highest reactivity and interaction with E. coli cells exhibited the highest inhibitory potential. Interestingly, previous studies have indicated that nanoparticles with a large number of f111g lattice planes exhibit higher bacterial inhibition potential in contrast to the spherical nanoparticles having less f111g planes (Djafari et al. 2019;Truong et al. 2022). The minimum inhibitory concentration (MIC) of TAA 4 hydrogel was evaluated quantitatively by the broth dilution method. TAA 4 hydrogels in the concentration range of 10-80 mg ml À1 were incubated with various bacterial strains for 12 h. Figure 5b shows the concentration-dependent bacterial inhibition, wherein the hydrogels at a concentration of 80 mg ml À1 exhibited the highest inhibition of E. coli ($87%), P. aeruginosa ($73%) and S. aureus ($54%). Below 10 mg ml À1 , turbidity owing to the bacterial growth was visible. Further, TAA 4 hydrogel at a MIC value of 40 mg ml À1 could inhibit $77% of the E. coli biomass. However, owing to the lower inhibitory potential of TAA 4 against S. aureus and P. aeruginosa, the corresponding MIC value was found to be > 40 mg ml À1 . Correspondingly, the MIC value for S. aureus and P. aeruginosa was determined to be 60 mg ml À1 .
Next, the bactericidal effects of TAA 4 hydrogel (40 mg ml À1 for E. coli, and 60 mg ml À1 for P. aeruginosa and S. aureus) were also studied by colony formation on agar plates for 24 h (Figure 5c). As a consequence of TAA 4 exposure, the number of E. coli colonies decreased greatly when compared to the untreated plates. A similar trend was documented for S. aureus and P. aeruginosa bacterial colonies as well ( Figure S6). However, the reduction in colonies is less prominent than E. coli, thereby revealing a relatively lower bactericidal potential of TAA 4 hydrogels against P. aeruginosa, and lowest for S. aureus.
Further, a time-dependent inhibition assay of TAA 4 was performed. Figure 6a-c shows a gradual reduction in the cell density of treated bacteria as compared to the untreated counterparts as the contact time increased. The results revealed a significant reduction in bacterial cell number from 3 h treatment onwards. It was observed that E. coli, S. aureus, and P. aeruginosa cells treated with TAA 4 hydrogels showed 0.95, 0.57, and 0.61 log units of decrease in their growth compared to the untreated cells under similar conditions after 12 h. Thus, it was ascertained that TAA 4 exhibits bacteriostatic potential against the bacteria tested.
The FE-SEM analysis of E. coli and S. aureus was performed to discern the changes in cellular morphology and cell membrane following interaction with TAA 4 hydrogel (Figure 7a-d). The untreated E. coli showed normal surface characteristics with a smooth, intact rod-shaped structure. Contrastingly, TAA 4 hydrogel treatment caused a significant reduction in the cell count and compromised membrane integrity with subsequent leakage of cytoplasmic components (indicated by red arrow). S. aureus (control) cells exhibited intact grape-shaped morphology, while their treated counterparts were found in clusters with distorted morphology due to the bactericidal effects of TAA 4 hydrogel. The changes in the cellular morphology of treated cells are consistent with the previous research findings on the antibacterial effects of AgNTs (Truong et al. 2022). The mechanism behind the bactericidal properties of AgNTs can be associated with the death of microbial cells due to damage to the microbial enzymes or cell membrane. Probably, anisotropic shaped AgNTs released from the hydrogel altered the cell membrane integrity. The sharp edges and vertices of AgNTs enabled easy penetration and puncturing of the membrane (Acharya et al. 2018). Owing to membrane disruption, the extracellular materials which are otherwise protected by cellular membrane were released. Moreover, other mechanisms implicate the release of Ag þ ions from nanoparticle suspension for intracellular ROS production, interference with DNA replication and respiration pathway as well as enzyme deactivation (Sadeghi et al. 2012;Hu et al. 2016). Subsequently, the reusability of TAA 4 hydrogel for examining bacterial inhibition was studied for three consecutive cycles. Figure S6 shows the percentage growth inhibition post-treatment of bacterial suspension with TAA 4 for 12 h. The TAA 4 hydrogel was able to inhibit E. coli growth for three consecutive cycles with $45% inhibition during the third cycle. Similarly, for P. aeruginosa, after three consecutive cycles $40% reduction in growth was observed after treatment with TAA 4. A similar trend was observed for S. aureus, wherein TAA 4 was able to retain bactericidal potential and could inhibit $38% of bacterial cells even in the third cycle. Ionically cross-linked hydrogels, such as AgNT hydrogels in the present case, have good stability and are suitable for antibacterial activity. However, these types of hydrogels tend to disintegrate upon repeated use. Therefore, AgNT-hydrogels were intact and able to retain an antibacterial effect up to three experimental bactericidal cycles.

Anti-biofilm assay
In contrast to planktonic bacteria, the eradication and disruption of the bacterial biofilm are considered a challenging task due to antibiotic resistance and the presence of EPS matrix which hinders the accessibility of antibacterial agents. In the past few years, plenty of studies have demonstrated the efficacy of spherical silver nanoparticles for biofilm inhibition as they could bypass the problem of resistance (Hetta et al. 2021;Martinez-Gutierrez et al. 2013;Siddique et al. 2020). However, the performance of triangular shaped Ag nanoparticles for biofilm inhibition is still less explored, which is expected to exhibit an enhanced reduction in biofilm growth. In this context, the biofilm inhibitory potential of AgNTs incorporated in hydrogels was quantified by the crystal violet assay.  After incubation at stationary conditions for 24 h, the control groups (untreated bacteria) showed the formation of thick biomass of biofilm as implicated by the dark blue colour of crystal violet and measured the absorbance at 590 nm (OD 590 ) (Figure 8). In sharp contrast, treatment with TAA 4 hydrogel at increasing concentrations resulted in a concomitant reduction in the intensity of the blue colour ( Figure  8d-f). The quantitative assessment showed that for E. coli, $74% reduction in biofilm formation was achieved at a TAA 4 concentration of 80 mg ml À1 . Similarly, in the case of S. aureus and P. aeruginosa, TAA 4 at the same concentration exhibited biofilm reduction efficiency of $63% and 67%, respectively. A higher TAA 4 concentration was required to inhibit an equivalent level of bacteria in the biofilm form in comparison to those in the planktonic state. These observations confirm that the biofilms were effectively inhibited upon treatment with (40-80 mg ml À1 ) of AgNT-hydrogels for 24 h and thus certify that AgNTs released from the hydrogels potentially penetrated and disintegrated the biofilms. Similar results were reported by Zhang et al. (2020) which showed concentration-dependent biofilm reduction in P. aeruginosa by Ag nanoparticles. In general, bacterial flagellum plays a crucial role in bacterial attachment to the surface, pathogenicity, and hence biofilm formation. That study established that Ag nanoparticles down-regulated the production of proteins associated with the flagellum motility of P. aeruginosa, thereby affecting its activity. The impairment of such flagellar movement using Ag nanoparticles seems to affect the biofilm formation significantly (Shah et al. 2019;Alavi et al. 2019). Additionally, Ag nanoparticles may interact and reduce the synthesis of exopolysaccharides, proteins and extracellular DNA involved in bacterial attachment and biofilm formation (Ansari et al. 2015;Joshi et al. 2020). These are capable of down-regulating genes for quorum sensing (QS), thereby reducing the secretion of virulence factors and biofilm formation (Singh et al. 2015;Qais et al. 2021). Overall, AgNTs can plausibly follow the various mechanisms proposed above for biofilm inhibition.

Conclusions
In this study, a facile methodology was utilized to synthesize ionically cross-linked GT/SA hydrogel for encapsulating AgNTs. The incorporation of AgNTs in the hydrogels increased its mechanical performance and maintained appreciable hydrophilicity, porosity and swelling characteristics.
AgNT-hydrogels demonstrated enhanced antibacterial effects against planktonic and biofilm-forming bacterial pathogens (S. aureus, E. coli, and P. aeruginosa). However, the concentration of AgNT-hydrogel needed to effectively retard and eliminate the biofilm growth was higher than the MIC values obtained for planktonic bacterial cultures. The antibacterial potential of hydrogels accounted for the sustained release of AgNTs triggering the mechanical disruption of bacterial cell membrane first, resulting in leakage of cellular components. The consequent intracellular release of Ag þ ions induced metabolic imbalance, leading to the inhibition of bacterial cell growth and cell lysis. Additionally, these novel hydrogel formulations could easily penetrate and disrupt the biofilm matrix, which is a clinically relevant outcome for controlling biomaterial associated infections.