In vitro evaluation of cellulose-gelatin film with silver nanoparticles and its particle size influences for wound dressing application

1. Introduction

Cellulose, a biopolymer derived from plant cell walls, has gained significant attention as a multipurpose material due to its safe interaction with living tissues, environmental friendliness, and structural integrity [1]. Natural origin and abundant availability make it an attractive alternative to synthetic materials. Moreover, the properties of cellulose-derived materials might be improved by combining them with other biopolymers, such as gelatin [2,3]. As a natural polymer derived from collagen, it complements cellulose in medical applications by enhancing the healing properties of the composite materials [4]. Its high biocompatibility and biodegradability also make it suitable for medical use, while its excellent film-forming capabilities provide good elasticity and tensile strength, making it more durable and capable of conforming to various shapes and sizes [5-7]. By embracing these advantages, cellulose and gelatin can significantly improve structural, mechanical, and biological properties, resulting in efficient and multipurpose biofilm, especially for novel wound dressing materials.

Nevertheless, both cellulose and gelatin exhibit poor antibacterial activity [8,9]. To overcome this limitation, it is crucial to incorporate antibacterial agents into wound dressing; e.g., chitosan [10], benzalkonium chloride [11], silver sulfadiazine [12], and metallic nanoparticles [13]. The remarkable antibacterial capabilities of metallic nanoparticles have been highlighted in recent reports, largely attributed to their nanostructure and significant surface area [14,15]. Silver nanoparticles (AgNPs) have been the focus of numerous studies targeting bacteria, fungi, and viruses. However, controlling its size and tendency to aggregate is a critical issue that must be considered in AgNPs synthesis [16]. One fundamental aspect that needs to be studied is how the precursor concentration affects nanoparticle size and its antibacterial effectiveness. Besides, to our knowledge, research on AgNP size in cellulose-gelatin film for wound dressing application has never been studied.

In this study, we developed a multipurpose film with excellent mechanical properties and strong antibacterial activity. The film utilizes a sustainable biopolymer matrix derived from extracted avocado seed waste, a novel approach that promotes circular economy principles and, to our knowledge, has not been previously explored. We further enhanced the film’s properties by integrating cellulose and gelatin within a citric acid crosslinking system, which significantly improved both mechanical strength and biocompatibility. Importantly, citric acid also contributed to the film’s antibacterial function. In vitro assays were conducted against E. coli and S. aureus for 7 days (7×24 h) to evaluate the impact of AgNP size on antibacterial performance. The results demonstrated sustained antibacterial activity against both Gram-negative and Gram-positive bacteria, a level of performance that has not been reported in previous studies utilizing AgNPs within biopolymer matrices. These findings suggest that the developed film holds strong potential for biomedical applications, particularly as a wound dressing material.

2. Materials and Methods

3. Results and Discussion

The AgNPs produced from AgNO₃ precursors with different concentrations have different colors, as shown in Figure 1(a). The AgNPs-1 have a yellow color with the lowest color intensity, the intensity of yellow increases in AgNPs-3 and approaches a brownish color at AgNPs-5. Results with similar trends were also obtained from spectrophotometer analysis. The absorbance curve shown in Figure 1(b), AgNPs-1 has an absorbance peak at a wavelength of 420 nm, a higher intensity, followed by AgNPs-3 with an absorbance peak at a wavelength of 423 nm, and AgNPs-5 has an absorbance peak with the highest intensity at a wavelength of 429 nm. Furthermore, Figure 1(c) shows the size distribution of AgNPs, which increases as the concentration of AgNO₃ increases. In addition, a simple analysis was carried out using laser light directed at demineralized water and AgNPs colloids, as shown in Figure 1(d). The light beam directed at demineralized water will be forwarded so that the light is invisible. A different phenomenon occurs when the light beam is pointed at colloidal AgNPs; it dissipates and leaves a visible trace. It is the initial evidence of AgNPs colloid formation, other than the color change, known as the Tyndall Effect.

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Figure 1.

(a) Visual appearance and different colors of AgNPs colloidal resulted from different silver nitrate concentrations, (b) absorbance curve of AgNPs, (c) AgNPs size distribution and (d) simple experiment of Tyndall effect between demineralized water and AgNPs-[3]. [1] for Ag-NPs-[1] with AgNO₃ concentration of 1x10^-3 M, [2] for Ag-NPs-[3] with AgNO₃ concentration of 3x10^-3 M, [3] for Ag-NPs-[5] with AgNO₃ concentration of 5x10^-3 M.

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After AgNPs were embedded in the cellulose-gelatin film, the SEM image (Figure 2) showed the presence of brighter components on the film’s surface. These components were none other than silver nanoparticles scattered over the film surface. Furthermore, an analysis of the Ag composition using Energy-dispersive X-ray spectroscopy (EDX) shows that the Ag composition increases as the concentration of AgNO₃ used increases. In addition, it was observed that the size of Ag-NPs appeared larger as the concentration of AgNO₃ precursor used increased, which was in line with the size distribution results obtained. Not only that, Ag-NPs on the surface of the CA/S-Ag-5 film also appear to agglomerate or gather close together into one. It can occur because the higher concentration of AgNO₃ precursors will increase the number of particle nuclei formed in the nucleation process and accelerate particle growth. Without being accompanied by a sufficient stabilizer, in this case, trinatrium citrate (Na₃C₆H₅O), particle agglomeration is more prone to occur. Meanwhile, Figure 3 shows the chemical bonds in cellulose-gelatin films before and after Ag-NPs embedded in various concentrations. There is no significant difference in the resulting FTIR spectra, indicating that adding Ag-NPs to the film does not change the film’s chemical bonding or the film or the crosslinking behavior of the film.

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Figure 2.

(a-c) SEM images and Ag elemental compositions of the synthesized film with Ag-NPs in various concentrations.

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Figure 3.

FTIR spectra of the synthesized film with- and without Ag-NPs addition.

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Antibacterial properties are essential for specific functional applications, such as wound dressings. Cellulose-gelatin films with the addition of Ag-NPs show antibacterial properties that can persist up to 7 × 24 h in Gram-negative and positive bacterial media cultures such as E. coli and S. aureus. This result indicates that adding Ag-NPs has a much better antibacterial ability than without adding Ag-NPs in CG-CA/S-10 films.

Based on the inhibition zone until the 7^th day of observation on both E. coli and S. aureus bacterial cultures, cellulose-gelatin films with the addition of Ag-NPs have the best antibacterial performance on CA/S-Ag-1 film, followed by CA/S-Ag-3 and CA/S-Ag-5. It indicates that the concentration of AgNO₃ precursor, which then affects the size of Ag-NPs formed, can have different antibacterial effects on cellulose-gelatin films.

This phenomenon can be explained through the antibacterial mechanism that Ag-NPs can perform. Researchers have accepted two common antibacterial mechanisms by Ag-NPs: direct contact with microorganisms and the release of Ag⁺ ions. In the mechanism of direct contact with microorganisms, it is reported that Ag-NPs can adhere to the bacterial cell wall so that Ag-NP penetration can occur inside. It will undoubtedly cause physical changes to the bacterial membrane, such as membrane damage that leads to bacterial death [21,22]. It is also the reason why the antibacterial performance of Ag-NPs is stronger on E. coli, which is a Gram-negative bacterium, compared to S. aureus, which is a Gram-positive bacterium, because Gram-positive bacteria are reported to have cell walls with a thickness of up to 10x that of Gram-negative bacteria, so that their resistance is stronger [23]. After attachment and penetration into the bacterial cell wall, there is a relationship between the antibacterial effect and the size of Ag-NPs. Smaller nanoparticles will have a larger surface area, making contact with bacterial cells more intense. In addition, the smaller size will make it easier for Ag-NPs to penetrate the bacterial wall, and they tend to be more reactive to bacterial cell components, which makes it easier to cause death [22] [Figure 4].

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Figure 4.

Digital photos of the antibacterial activity assays of the films with the addition of Ag-NPs using E. coli and S. aureus media cultures at 1, 3, and 7$\times$24 h of observation.

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The second mechanism is the release of Ag⁺ ions by Ag-NPs. In this mechanism, one of the crucial parameters in Ag-NPs against bacteria is the surface area of the nanomaterial [24-26]. Ag-NPs can release Ag⁺ ions both inside and outside the bacterial wall. Previous studies convey that Ag-NPs release higher concentrations of Ag⁺ ions with a larger surface area, and conversely, the smallest Ag⁺ ions are released by Ag-NPs with the lowest surface area [27]. It can be explained why CA/S-Ag-1, with the smallest size distribution of Ag-NPs, has the best antibacterial performance. Nanomaterials with smaller sizes will have a larger surface area, so the ability to release Ag⁺ ions will also be higher. Furthermore, Ag⁺ ions will bind to proteins in the cell membrane and form stable bonds that cause protein deactivation. These proteins play an important role in the transportation system and cellular respiration, so disruption of the proteins will disrupt the process of cell division and reproduction, leading to bacterial death [15,28].

On the other hand, it is considered that the Ag-NPs have spherical geometry, and the release of Ag⁺ ions follows an unsteady state diffusion process, as described by Fick’s second law of diffusion, shown in Eq. (1). According to the equation, the diffusion rate is influenced by both the diffusion coefficient (δ) and the spatial gradient of concentration. For smaller values of $x$, which corresponds to the particle size (radius) or the distance from the particle’s center to its surface, the concentration gradients become steeper, resulting in a faster diffusion rate. This relationship highlights that smaller sizes of Ag-NPs led to reduced diffusion distances and exhibited accelerated release dynamics compared to larger particles. Such behavior aligns with the fundamental principles of diffusion, where reduced spatial constraints promote rapid molecular transport.

Moreover, ensuring that the material is free from toxic or hazardous substances is crucial for its application as a functional material. To confirm this, XRF analysis was conducted to detect the presence of any toxic elements in the synthesized film, as shown in Table 2. Typically, elemental impurities are categorized into class 1 and class 2. Class 1 elements, such as Pb, Cd, Hg, and As, must be absent due to their high toxicity and environmental risks, while class 2 elements, like Cu, V, and Ni, are less toxic but still need to be present in limited amounts [29,30]. XRF analysis confirmed that class I and II elements were absent or present in trace amounts below safety thresholds, indicating that the synthesized biopolymers are non-toxic and environmentally safe.

4. Conclusions

AgNPs were successfully embedded into the cellulose gelatin as antibacterial agents. AgNPs were distributed with an average size of 37.6-59.1 nm. SEM-EDX provided clear evidence for the presence of AgNPs in the cellulose-gelatin film with a mass fraction of about 0.8-0.96%. FTIR spectrum confirmed that there is no significant difference between the films with and without AgNP materials, indicating that the chemical bonding of the film isn’t affected by the presence of AgNPs. Importantly, in vitro evaluation against E. coli and S. aureus bacteria was observed up to 7 × 24 h. The results reveal that the size of AgNPs influences the antimicrobial activity of the film, with smaller sizes having a better ability. One probable explanation is that a smaller size allows for a larger surface area, resulting in better direct contact with bacteria and providing sufficient Ag⁺ ion release. Besides, AgNPs with a larger size tend to aggregate, which is unfavorable for the antibacterial performance. This study confirms that a novel cellulose-gelatin film with AgNPs was successfully produced from avocado seed waste with excellent antibacterial activity, pointing out their promising possibilities for medical use, especially in wound dressing applications.