Green silver nanoparticle-embedded chitosan-alginate hydrogel: A novel antibacterial approach for potential wound healing

Green synthesis of silver nanoparticles using matcha green tea

For the preparation of the matcha green tea extract as illustrated in Figure 1, one g of dried matcha green tea powder (Camellia sinensis) was added to 50 mL of distilled water and stirred at 70°C for 5 minutes. Then, the mixture was allowed to cool to ambient temperature, and the extract was obtained using vacuum filtration. The green synthesis of Ag-NPs was carried out as follows: 50 mL of the matcha green tea extract was transferred to an Erlenmeyer flask and placed in an ice bath with continuous 200 r/min stirring. The extract was swirled and cooled to room temperature. An aqueous solution of 1 mM silver nitrate (AgNO3) was prepared. This solution was then added dropwise (1 drop/sec) into the cooled extract under moderate stirring. ³⁵ The Ag-NPs solution was centrifuged at 10000 r/min for 20 minutes to separate the nanoparticles from silver ions and other particles. All free silver cations were removed in the supernatant fluid while the bluish pellet within the tube containing the Ag-NPs was retrieved and rinsed several times with distilled water to eliminate remaining free silver ions.OPEN IN VIEWER

Silver nanoparticles characterization

Synthesis of hydrogel

1 g chitosan was added to 70 mL of 1% (v/v) acetic acid solution and was stirred overnight at room temperature until a clear solution was obtained. Separately, 1 g of sodium alginate was added to 70 mL of distilled water and stirred for 5 hours at room temperature. Both chitosan and alginate solutions were added together and mixed for an extra 1 hour to produce chitosan-alginate blend. This blend has been divided into two equal parts. In the first part, 0.1 g NMB and 1 g of APS were added to the polymer blend solution, and the resulting hydrogel was named as Chitosan-Alginate (Ch-Al). In the second part, 10 mL Ag-NPs, 0.1 g of NMB, and 1 g of APS were added to the polymer blend solution, and the resulting hydrogel was named Ch-Al-AgNPs. The two samples were ultrasonicated for 30 min to remove air bubbles and then poured into a pottery dish. The resulting samples were completely dried at room temperature. After that, the samples were taken out and soaked in deionized water (change the water every 6 h/ 6 time for the removal of excess reagents and byproducts.

Characterization of hydrogel

Water absorption

Swelling studies were conducted by measuring the weight of the hydrogel discs after immersing them in distilled water for 24 hours at room temperature. Water absorption (WA) is generally used to describe the swelling behavior of the prepared hydrogels. The dry hydrogel was soaked in distilled water (DW). The swollen samples were allowed to drain using a plastic strainer for 10 minutes. Then, the swollen hydrogel was weighed after soaking.^36,37 WA is given by Equation (1) as follows:

W A ( g / g ) = W s − W d W d

(1)

where Ws and W_d are the weights of the swollen and dry samples, respectively. WA was calculated as grams of absorbed water per gram of dry hydrogel (g/g).

Mechanical properties

The mechanical properties of the prepared hydrogel films, tensile strength (TS) and percentage elongation at break (EAB), were determined using a texture analyzer (XT plus, Stable Micro Systems, Godalming, England) following the standard ASTM D882 method. For the present test, film strips (60 mm length and 7 mm width) were used. The equations (2) and (3) presented below were used to determine the values of the TS and EAB for the film samples.

T S = F A

(2)

F is the maximum force; A is the cross-sectional area of the film.

E A B % = L f − L i L i * 100

(3)

where, L_f is the final length of the film; L_i is the initial length of the film.

Minimum inhibitory concentration determination

The prepared MGTAg-NPs was evaluated for their antimicrobial properties where the materials were analyzed using both quantitative (Green synthesized Silver Nanoparticles loaded on Hydrogel and attached culture growth) and qualitative (growth inhibition zone evaluation) techniques against all tested strains (P. aeruginosa, E. coli, S. aureus, Candida albicans and Aspergillus niger). The developed materials’ 1 cm × 1 cm inhibition zone fragments were sterilized using UV exposure for 30 minutes in order to assess the growth. Each sample’s sterile components were aseptically added to nutrient agar plates that had already been inoculated with 0.5 McFarland (1.5 × 10^8 colony forming units/ml) microbial suspensions made from the strains of microorganisms that had been tested. Using a sterile swab, the inoculation of microbial strains was carried out in accordance with the disc-diffusion antibiogram assay. To promote microbial growth, the inoculated Petri dishes containing the materials under test were incubated for 24 hours at 37°C. Development of growth following incubation. Growth inhibition was assessed after incubation. Following the period of incubation, the growth inhibition zone appeared.

Antimicrobial assessment

The antibacterial performance of the green synthesis of AgNPs incorporated hydrogel have been analyzed against specific selected microbial species by microbial growth inhibition tests. Cultures in nutrient broth with the hydrogel samples were made with respect to the microbial growth in attached (biofilm) as well as a desorbed population (planktonic). Sterile hydrogel pieces were grown on nutrient broth with microbial suspensions at a turbidity equivalent to 0.5 McFarland standard and incubated at 37°C for 24 hours for biofilm development. Microbial growth of the hydrogel samples was determined by absorbance values obtained on a spectrophotometer at 650 nm after incubation.

In this study, the minimum inhibitory concentration (MIC) was assessed by disc diffusion and well diffusion methods. Unfortunately, the data regarding growth analysis are quite tricky. Due to that, an appropriate form of agar, BHI Agar and Tryptic Soy agar for bacteria, SD agar for fungi, was incubated with microbial suspensions. Pesticidal discs or wells were prepared by impregnating various concentrations of the Ch-Al and Ch-Al-AgNPs hydrogels, including 300 and 400 μg/mL for fungi, and 1.0 and 2.0 mg/mL for bacteria. At 24-h incubation at 37°C the optical density of the microbial cultures determined the MIC representing the minimum inhibitory concentration of the compounds most effective against bacterial species.

For Bacterial and Fungal strains, SEM has been used for assessing the morphological changes of treated bacterial or fungal cultures treated by (MGEAg-NPs). Cut agar was used to prepare the strains, which were then fixed for at least 3 hours in 2.5% glutaraldehyde (100 mm phosphate buffer solution, pH 7.2) and 1 hour in 1% osmium tetroxide. A series of graduated ethanol (30, 50, 60, 70, 80, 90, 95, and 100%; each level applied twice for 15 min each time) and ethanol: isoamyl acetate (3:1, 1:1, 1:3, and 100% isoamyl acetate twice for 30 min) were used to dehydrate the agar blocks. The agar blocks were coated with a gold coater for 5 minutes after being dried with a critical-point drier using liquid CO₂.

Biofilm assessment and statistical analysis

Using sterile Eppendorf tubes filled with 1 mL of sterile saline water, the Ch-Al and Ch-Al-AgNPs hydrogel previously prepared were placed in order to evaluate the microbial cells’ capacity to attach and form biofilms following a 24-h incubation period. Colony forming units (CFU)/mL values were determined by dilution and inoculation of the resulting suspensions on agar plates after the washed coatings with attached cells were again persuasively vortexed to separate the attached cells. With the Student’s T test, statistical significance was examined. Significant statistical values were defined as p values less than 0.05. The experimental data were analyzed using appropriate statistical methods, Version 19 of SPSS was utilized to perform the statistical analysis.

UV-Vis spectroscopy

The UV-visible absorption spectrum of synthesized AgNPs displayed a characteristic surface plasmon resonance (SPR) peak at 405 nm (Figure 2(a))), indicative of the formation of spherical or quasi-spherical AgNPs. This absorption peak aligns with the reported wavelength range of 390–420 nm for AgNPs and confirms the successful synthesis of AgNPs using matcha green tea extract as a reducing agent. It is basically a phenomenon where the free electrons in certain metallic nanoparticles as Ag and Au particles oscillate in a particular direction due to the incidence of a specific wavelength of the E. M radiation.^22,38OPEN IN VIEWER

As per light reflection phenomenon of SPR, either light is absorbed or scattered hence resulting in the appearance of the absorption peak in the UV-Visible spectrum. The reflection coefficient increases and the SPR peak shifts depending on the size and form of nanoparticles, their chemical composition, and the dielectric constant of the environment. Among all the synthesized AgNPs, spherical NPs have the SPR peak at a wavelength of 390–420 nm due to the choice of the method and the condition used. From the spectral characteristic given above, the maximum SPR is seen at 405 nm which confirms the formation of spherical or quasi-spherical AgNPs while using the green tea extract as a reducer. This spectral characteristic demonstrates the plasmonic property of the developed AgNPs.^22,38

Lack of peaks or humps in the UV-Vis spectrum also supports the lack of structural anisotropy or aggregation of nanoparticles. This essentially means that the green synthesis approach using matcha green tea extract lead to the formation of highly dispersed and uniform AgNPs. Matcha green tea extract probably by polyphenolic compounds may have exerted a vital influence on the decrease and stabilization of AgNPs by avoiding their aggregation and the preserve of spherical structured nanoparticles. This biocompatible and green reducing agent also reduces the usage of highly toxic chemical reductants in the entire synthesis process. The SPR peak shape of the synthesized AgNPs matches the recorded UV-Vis spectrum which is equal to their plasmonic capability for further uses in biosensing, SERS, or photocatalysis.^23,29

Transmission electron microscopy

Transmission Electron Microscopy (TEM) analysis (Figure 2(b))) revealed well-dispersed AgNPs with sizes ranging from 20 to 40 nm. The image shows distinct nanoparticles dispersed,the TEM image confirms the successful synthesis of AgNPs and provides qualitative information about their presence, distribution, and general size range.

The dark spots or particles dispersed on the gray background represent individual or small clusters of AgNPs. The uniform distribution of the nanoparticles suggests their potential for consistent antimicrobial activity. These homogeneous distribution and absence of cluster formation similar to what is seen in the TEM images are suggestive of probable high overall antibacterial properties and higher biocompatibility of the synthesized AgNPs. Distribution of nanoparticles is also favorable for interacting with bacterial or fungal cells to ensure the maximum potential of the antimicrobial nanoparticles. Apart from the routinely used bright field TEM, HRTEM analysis was performed to determine the crystal structure and atomic planes of the AgNPs. From the observed HRTEM images however, clear lattice fringes were observed indicating that the synthesized AgNPs have a crystalline character as shown in the images below.^5,22

Scanning electron microscope

SEM analysis (Figure 3(a)–(d))) revealed the SEM of chitosan, alginate, and the two prepared hydrogel film samples. Chitosan, which appears as irregular, crystalline-like particles or chunks. ³⁹ Whereas alginate has a more amorphous and rougher surface morphology. ⁴⁰ Meanwhile, Ch-Al exhibits a fibrous, interconnected network structure. The fibers or strands appear to be randomly oriented, forming a porous and open matrix. Ch-Al-AgNPs also displays a fibrous network structure but with some differences. The inclusion of AgNPs resulted in a more compact matrix with visible AgNPs clusters. These morphological changes indicate that the hydrogel’s structure was influenced by the presence of AgNPs.OPEN IN VIEWER

The results thoroughly characterize the nature of hydrogels, the used Transmission Electron Microscopy to visualize the presence of AgNPs within the Ch-Al matrix. Furthermore, Scanning Electron Microscopy has allowed the study of surface morphology and interference of nanoparticles with microbial cell walls. This feature makes Ch-Al-AgNPs suitable for use as a wound dressing because it provides an appropriate space for cell growth and migration as well as absorption of tissue exudates.^4,8

Energy dispersive X-ray spectrometer for Ch-Al-AgNPs hydrogel

EDX spectra (Figure 3(e)) confirmed the elemental composition of the Ch-Al-AgNPs hydrogel. The presence of Ag was detected, at a low weight percentage (0.28%), verifying the successful embedding of AgNPs within the hydrogel. ²² Several peaks corresponding to different elements present in the sample. The prominent peaks are labeled as C K (carbon), O K (oxygen), Ag M (silver M-shell), and Ag Lα, Ag Lβ, and Ag Lβ2 (silver L-shell emission lines).

The weight percentage of carbon is (60.48%), and the atomic percentage is (67.22%). This high carbon content is likely due to the presence of chitosan and alginate polymers, which are primarily composed of carbon and oxygen. While the weight percentage of oxygen is (39.24%), and the atomic percentage is (32.74%). The presence of oxygen is expected due to hydroxyl, carboxyl, and other functional groups in the chitosan and alginate polymers. Finally, the weight percentage of silver is (0.28%), and the atomic percentage is (0.03%). The relatively low but detectable amount of silver confirms the presence of AgNPs in the hydrogel formulation. ²²

FTIR spectroscopy

The FTIR spectra (Figure 3(f)) confirmed the incorporation of AgNPs within the Ch-Al matrix. Key peaks, such as the broad peak around 3400 cm⁻¹ (O-H and N-H stretching) and peaks in the 1650–1500 cm⁻¹ range (amide bands), which is due to stretching vibrations of the hydroxyl groups. The peaks around 1600 cm⁻¹ and 1420 cm⁻¹ may be attributed to the asymmetric and symmetric stretching vibrations of the carboxylate groups, respectively. ⁴¹ The peak at 1030 cm⁻¹ may be associated with C-O-C stretching vibrations of the pyranose ring. Furthermore, the spectrum of Ch-Al shows the main characteristic peaks of both used polymers (chitosan and alginate), which confirms the presence of both polymers in the sample Ch-Al. The peak at 3400 cm⁻¹ is due to the overlapping O-H and N-H stretching vibrations from both polymers. The peaks around 1600 cm⁻¹ and 1420 cm⁻¹ may be attributed to the carboxylate groups of alginates, while the peaks in the 1650–1500 cm⁻¹ region are related to the amide I and amide II bands of chitosan. ²³ Finally, the spectrum of Ch-Al-AgNPs is similar to that of Ch-Al, exhibiting the characteristic peaks of both chitosan and alginate. However, there are a few differences in both the peak intensity and the peak shape of certain peaks, which may be attributed to the presence of AgNPs and their potential interactions with the polymer’s matrix. ²⁹

Water absorption

Water absorption tests (Figure 3(g)) demonstrated that the Ch-Al hydrogel exhibited a higher swelling ratio (35 g/g) compared to the Ch-Al-AgNPs hydrogel (30 g/g). The presence of AgNPs within the hydrogel matrix reduced the swelling capacity, likely due to steric hindrance and interactions between the nanoparticles and the polymer chains.^4,41 This lower swelling ratio may offer advantages for wound healing by preventing excessive absorption of wound exudates. ⁴

Figure 3(g) illustrates the swelling behavior of the two hydrogel samples over 24 hrs. It can be noted that, for initial swelling rate, both hydrogel samples exhibit a rapid initial swelling rate during the first 5 hours. This initial speed swelling is due to the hydrophilic nature of the polymeric chains. The swelling behavior analysis revealed that the pristine chitosan-alginate (Ch-Al) hydrogel exhibited a higher maximum swelling ratio of approximately 35 g/g, whereas the incorporation of green-synthesized silver nanoparticles (AgNPs) into the hydrogel matrix (Ch-Al-AgNPs) resulted in a slightly lower maximum swelling ratio of around 30 g/g. Swelling equilibrium as after reaching their respective maximum swelling ratios, both samples appear to reach an equilibrium state, where the swelling ratio remains relatively constant over the remaining time period. ⁴²

The lower swelling ratio observed for the Ch-Al-AgNPs can be attributed to the presence of Ag NPs within the hydrogel matrix. The incorporation of Ag NPs can lead to interactions with the polymeric chains, potentially altering the hydrophilicity and water absorption capacity of the hydrogel. Additionally, Ag NPs may create steric hindrance or obstruction within the hydrogel matrix, limiting the accessibility of water molecules to the polymer chains and reducing the overall swelling capacity.^4,32

Furthermore, the lower swelling ratio of Ch-Al-AgNPs may be advantageous in certain wound healing applications, as it can provide controlled swelling and prevent excessive absorption of wound exudate. This controlled swelling can help maintain structural integrity and minimize potential disruption of the wound bed. Also, the presence of Ag NPs imparts antimicrobial properties to the hydrogel, aiding in combating wounds.

This swelling behavior of hydrogels is one important aspect that affects the function of delivering therapeutic agents. A high swelling ratio may enhance the diffusion of drugs through the hydrogel, thereby improving the drug bioavailability at an applied site. In the current study, a moderate swelling ratio recorded for the Chitosan-Alginate hydrogel is considered an optimum value for controlled release of the drug with the aim to provide long-lasting antimicrobial activity while reducing the risk of early drug release. The high swelling capacity reduction of the Ch-Al-AgNPs hydrogel with respect to the pristine Ch-Al hydrogel points to its useful modification in relation to wound-healing applications. This controlled swelling behavior will not allow excessive absorption of wound exudate, hence maintaining structural integrity for longer periods of time.

Mechanical properties

Mechanical testing illustrated in Table 1 revealed that the Ch-Al hydrogel had a higher tensile strength (25.4 MPa) and elongation at break (42.7%) compared to the Ch-Al-AgNPs hydrogel (18.2 MPa and 35.9%, respectively). The addition of AgNPs slightly compromised the mechanical strength and flexibility of the hydrogel, although the properties remain within the acceptable ranges for wound dressing applications.^22,35

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Table 1.

Mechanical properties of the prepared hydrogel films.

Sample	Tensile strength (MPa)	Percentage elongation at break (%)
Ch-Al	25.4	42.7
Ch-Al-AgNPs	18.2	35.9

As illustrated in Table 1 sample Ch-Al exhibits a higher tensile strength of (25.4 MPa) compared to Ch-Al-AgNPs , which has a tensile strength of (18.2 MPa). Sample Ch-Al also displays a higher percentage elongation at break of (42.7%), while Sample Ch-Al-AgNPs has a lower value of (35.9%).

The mechanical properties of edible hydrogel films are crucial for their intended applications, such as wound healing, food packaging, or drug delivery. ⁴ The tensile strength and percentage elongation at break provide valuable information about the films’ mechanical behavior and performance. Sample Ch-Al demonstrates superior mechanical properties compared to Ch-Al-AgNPs, with a higher tensile strength and greater elongation at break. This suggests that Sample Ch-Al can withstand higher tensile forces before failure and has better stretchability or flexibility compared to sample Ch-Al-AgNPs.

The differences in mechanical properties between the two samples can be attributed to various factors, including the composition of the films, the types and proportions of polymers, plasticizers, and other additives used, as well as the processing conditions employed during film preparation. When compared to literature values for similar edible hydrogel films, the tensile strength of Sample Ch-Al (25.4 MPa) is within the range reported for chitosan-based films (15–30 MPa),^22,35 while Sample Ch-Al-AgNPs (18.2 MPa) falls slightly below this range. The percentage elongation at break for both samples (42.7% for Ch-Al and 35.9% for Ch-Al-AgNPs ) is comparable to the values reported for edible films containing polysaccharides and plasticizers (30%–50%). ⁴³

The addition of AgNPs has slightly compromised the mechanical properties of the Ch-Al-AgNPs hydrogel; however, these properties have remained within acceptable limits for eventual clinical use. This reduced tensile strength and flexibility may be a compromise with the excellent antimicrobial activity, which is, in fact, much needed to prevent infection of open wounds.

Drug delivery and swelling rate are directly impacted by the swelling ratio; therefore the mild swelling (30 g/g) of the Ch-Al-AgNPs hydrogel allows for controlled drug release , to maintain antibacterial activity and avoid early drug loss. Morover, AgNPs lower the bacterial growth , which may promote neovascularization by reducing inflammation and infection. Therefore, a balanced wound environment that promotes tissue regeneration is produced. The Ch-Al-AgNPs hydrogel’s product swelling ratio helps preserve structural integrity and prevent over-absorption. This regulated swelling provides a consistent healing environment and is matched with the requirements for wound dressings.