Combating multidrug-resistant infections: Gold nanoparticles–chitosan–papain-integrated dual-action nanoplatform for enhanced antibacterial activity

2.6.1 Antibacterial activity of AuNPs, Pap, and AuNPs–CS–Pap

Antibacterial activities were tested by the MHA diffusion method [25]. MHA was prepared and kept solidifying. The prepared bacterial suspension was compared with the McFarland standard and disseminated through cotton swabs, while wells were punched out of each agar media using a sterilized cutter, and all the wells were loaded with 100 µL of Pap, and AuNPs at the concentration of 50 µg mL⁻¹, and AuNPs–CS–Pap at concentrations of 25, 50, and 100 µg mL⁻¹, and incubated at 37°C for 24 h for all bacterial samples. Inhibition zones were measured as diameters in millimeters (mm) using a ruler, and the values for each sample were recorded. Distilled deionized water was used as the negative control.

The fold increase percentage (F.I.%) was also calculated to evaluate the enhancement of antibacterial activity of the nanocomposite compared with the individual components. The F.I.% was determined using the following equation [26].

2.6.2 Evaluation of the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of AuNPs, Pap, and AuNPs–CS–Pap

All the prepared nanomaterials, AuNPs, Pap, and the AuNPs–CS–Pap, were subjected to MIC and MBC assays against some of the Gram-negative and Gram-positive, including E. coli, K. pneumoniae, S. aureus, and S. mutans. Cultures prepared in nutrient broth were incubated for 24 h at 37°C for optimal growth. The resultant cultures were diluted, adjusted to a 0.5 McFarland standard turbidity to achieve a standardized bacterial density for all the experimental wells. Subsequently, 100 µL of sterile nutrient broth (minus bacterial inoculum) was dispensed in each well of a 96-well microtiter sterile plate. An equal volume (100 µL) of nanomaterials, AuNPs, Pap, and the AuNPs–CS–Pap, prepared as serially diluted materials placed in the wells. Each treatment was performed in triplicate (n = 3) to ensure reproducibility. Afterward, 10 µL of standardized bacterial suspensions were added into the wells, excluding the negative control well, which contained only sterile broth. The plates were incubated for 24 h at 37°C, and after 24 h incubation, 20 µL of resazurin dye as a solution was added to all the wells to assess the viability of the bacterial suspension. Furthermore, the plates were incubated for an additional 4 h, keeping the conditions the same. Colorimetric changes determination provided each added nanomaterials’ MIC. The lowest concentration of the tested nanomaterials that prevented color change (from blue to pink) indicated the extent of bacterial growth inhibition and was defined as MIC. A sub-culturing of aliquots from wells with no visible growth on the fresh agar plates was used to determine the MBC, which was concluded from the values of the lowest concentrations that produced no colony [27].

2.6.3 Antibiofilm test

Cultivated bacterial isolate (concentration 1 × I0⁶) was incubated all day with nanomaterials, AuNPs, Pap, and AuNPs–CS–Pap, at 50 µg mL⁻¹ concentrations. After sample cleaning, the adherent bacteria stained with CV (0.1%) were washed twice with double-distilled water. Ethanol (95%, 0.2 mL) was added to CV-stained wells to determine the biofilm growth. Wells were shaken for 2 h, and subsequently optical density (OD) was determined at 595 nm [28].

2.6.4 Bacterial viability using acridine orange and ethidium bromide (AO/EtBr) staining

Staining assay using AO/EtBr was employed to check bacterial viability [29]. Bacterial suspension (50 µL) were treated with the materials, Pap, AuNPs, and AuNPs-Cs-Pap. Then the solutions were mixed with 50 µL of AO/EtBr (µg mL⁻¹). Control test was also conducted. All the mixtures were incubated at room temperature for 5 min. A fluorescence microscope (GENEX, USA) was used to examine the sample as a single drop of stained mixture. AO-stained viable cells showed green fluorescence. The damaged portions of the cells exhibited red color under EtBr staining.

2.6.5 In vivo toxicity assay

Mice (n = 5, 4 groups, aged 6–8 weeks, weighing about 26–30 g) were housed in a 24 ± 2°C temperature at 55 ± 10% humidity. All the animals were maintained in a 12:12 light/dark cycle with continuous access to food and water. The local Ethics Committee guidelines, National Institutes of Health (NIH) Publication No. 86-23, and ARRIVE recommendations, procedures, and guidelines were adhered to for all animal experiments. The Ethical Committee, Department of Biotechnology, University of Technology, approved the experimental protocols (Approval # BCSR9 February 9, 2025). Group I animals, as a control, received normal saline, and these animals were not under any treatment. Group II animals were administered a 50 mg kg⁻¹ dose of AuNPs. The Group III animals were administered Pap enzyme at the same dose. The animals in Group IV were administered the same dose of the nanomaterial, AuNPs–CS–Pap. Mice under treatment were intraperitoneally administered ∼100 µL of AuNPs, Pap, and AuNPs–CS–Pap, wherein the final administered volumes were adjusted in accordance with the animal’s weight differences. Doses of 50 mg kg⁻¹ for 3 days a week were injected. The negative control animals’ group was given normal saline (0.1 mL⁻¹, thrice weekly). Parametric data on weight, and the animals’ behavior were recorded prior to dosing. In the end, after the treatments, the animals were sacrificed, and the organs, including lungs, liver, kidney, spleen, and blood, were extracted from the animals. Serum was obtained from blood after centrifugation (3,000 rpm/10 min), and liver sera samples were stored (−20°C). Enzymatic-colorimetric method, aspartate aminotransferase (AST), alkaline phosphatase (ALP), and alanine aminotransferase (ALT) were tested from the liver samples. Estimation of blood urea, serum creatinine, and uric acid was also assessed. Chosen organs from treated and untreated mice were rinsed with PBS, embedded in paraffin, and fixed in 10% formalin for histological investigation. A microtome was used to slice the tissues after staining them with hematoxylin and eosin (H&E). The obtained slices were stained and processed according to the standard procedures recommended for histopathology work [30].

3.2.1 UV–Vis spectral analyses

UV–Vis spectra provided insights into the interactions and successful coating and immobilization of Pap onto the AuNPs–CS. The spectral analyses revealed distinct absorption peaks corresponding to each step of the preparation, confirming the successful formation of the final nanocomposite. For AuNPs alone, an absorption maximum (λ _max) was observed at 525 nm, which is characteristic of the size and spherical shape of the synthesized nanoparticles Figure 1 [20,24].

Figure 1

UV–Vis absorption spectra; (a) AuNPs; (b) CS; (c) AuNPs–CS; (d) Pap; and (e) AuNPs–CS–Pap.

Albert et al. [34] found a distinct absorption peak at 525 nm for AuNPs using a UV–Vis spectrophotometric analysis, which is in agreement with our findings. Sathiyaraj et al. [23] discovered that the UV absorption of AuNPs occurred in the 527 nm band, and our results are similar to the reported observations [35,36].

CS alone, Figure 1b, exhibited a strong absorbance band around 225 nm, which is attributed to n → σ* transitions of amine and hydroxyl functional groups. This observation is consistent with previously reported UV behavior of CS-based systems, confirming the presence of active functional groups capable of interacting with gold and protein molecules [37]. Modifications to the absorption intensity and overall width of the half-maxima occur upon ligand attachments to the NP surfaces [38]. Results showed that AuNPs–CS in Figure 1c absorbed UV light at a peak of 535 nm. Compared to the AuNPs, the SPR peak showed a slight redshift (shift to a longer wavelength) and a change in peak intensity. This indicated successful coating of the CS on the AuNPs, which modified their surface properties. The redshift and broadening of the peak can be attributed to an increase in the particle size and changes in the local refractive index due to the CS layer. Results also showed that the UV–Vis spectrum of free papain (Figure 1d) revealed a prominent absorbance peak at 298 nm, associated with the aromatic amino acids such as tyrosine and tryptophan in the protein’s structure. This peak is characteristic of intact enzyme conformation and confirms the optical activity of free Pap in solution, as supported by previous studies [39]. Moreover, results also showed in Figure 1e that AuNPs–CS–Pap absorbed UV radiation at a peak maxima λ _max of 555 nm. The SPR peak further exhibited redshift, and a slight reduction in intensity compared to the AuNPs–CS was observed. This additional shift was indicative of the successful immobilization of the Pap onto the CS-coated AuNPs. The reduction in peak intensity may be due to the increased aggregation or changes in the particle’s surface properties that are caused by the presence of Pap [40]. These observations suggested that the “LSPR phenomenon was to blame for the fact that the AuNPs typically showed a lower UV–Vis absorption than the product containing AuNPs, but that the AuNPs displayed LSPR when they were dispersed in a solution. This process caused radiation to absorb at certain wavelengths, resulting in a peak in the UV–Vis spectrum. The size, shape, and surrounding factors of the NPs have a role in peak intensity and absorption wavelength shift. The absorbance of products containing AuNPs varied, thereby suggesting the successful assembly of the AuNPs–CS–Pap as a result of interactions between the metal-cored AuNPs and other molecules and entities in the prepared nano-platform [41,42].

3.2.2 FTIR analysis

FTIR spectroscopy provided confirmation for the presence of characteristic functional groups for the gradual surface modification of the synthesized nanomaterial, AuNPs–CS and AuNPs–CS–Pap (Figure 2).

Figure 2

The FTIR analysis of (a) AuNPs; (b) CS; (c) AuNPs–CS; (d) Pap; and (e) AuNPs–CS–Pap.

The spectrum of AuNPs exhibited relatively weak but distinct absorption bands at 3,373 cm⁻¹ (O–H stretching), 2,928 and 2,868 cm⁻¹ (C–H stretching), 1,635 cm⁻¹ (H–O–H bending), and 1,064 cm⁻¹, which likely correspond to the citrate capping layers [20,43]. CS displayed strong and characteristic peaks at 3,382 cm⁻¹ (O–H and N–H stretchings), 2,882 cm⁻¹ (C–H stretchings), 1,653 cm⁻¹ (amide I), and 1,026–1,064 cm⁻¹ (C–O–C stretching), confirming its functional groups relevance for ionic and covalent interactions with gold surfaces [44].

The coating of CS onto the AuNPs, the resulting AuNPs–CS intermediate’s FTIR spectrum showed a broad peak at 3,304 cm⁻¹ (O–H/N–H), while other peaks at 754 and 551 cm⁻¹ were observed for Au–O and Au–N bonding, which suggested CS anchoring to the AuNP surface, while the AuNPs–CS–Pap FTIR spectrum retained key absorption peaks at ∼1,650 cm⁻¹ (amide), and shifts in absorption bands at ∼3,430 and 1,380 cm⁻¹ indicated the conjugation as compared to the free Pap bands [43]. These observations collectively suggested the structural features of the freshly prepared AuNPs–CS–Pap.

3.2.3 X-ray diffraction (XRD) analysis

XRD analysis was performed to evaluate the crystallinity of the synthesized materials, including uncoated AuNPs, free Pap, and the final AuNPs–CS–Pap formulation (Figure 3). The XRD pattern of pure AuNPs exhibited sharp and intense peaks at 2θ ≈ 38.1, 44.3, 64.6, and 77.5°, corresponding to the (111), (200), (220), and (311) planes of the face-centered cubic structure of metallic gold (JCPDS No. 04-0784), confirming the crystalline nature of the nanoparticles. In contrast, the XRD profile of free Pap displayed a broad, diffuse hump with no sharp diffraction peaks, indicating that Pap exists in an amorphous state – a characteristic commonly observed in proteinaceous materials [45].

Figure 3

XRD analysis of the different samples: the blue line represents AuNPs, the black line represents free Pap, and the red line corresponds to the AuNPs–CS–Pap nanocomposite.

Following surface modification with CS and Pap, the XRD pattern of the AuNPs–CS–Pap nanocomposite retained the main characteristic peaks of AuNPs, albeit with slightly reduced intensity and broader peak shapes. This observation suggests that the gold cores preserved their crystalline structure, while the coating with amorphous Pap and CS induced surface attenuation and minor peak broadening. Additionally, the absence of any new crystalline peaks from Pap further confirms its successful incorporation in an amorphous or molecularly dispersed form. Collectively, these results validate the structural integrity of the AuNPs post-coating and confirm the amorphous nature of the biopolymeric shell, supporting the successful formation of a hybrid nanocomposite system [46].

3.2.4 Zeta potential analyses

As shown in Figure 4, zeta potential measurements provide key insights into the surface charge characteristics and colloidal stability of the synthesized nanostructures. Figure 4a shows that AuNPs displayed a negative zeta potential of –15.6 mV, attributed to adsorbed citrate ions from the reduction process. This moderately negative surface charge indicates limited electrostatic repulsion, increasing the likelihood of NP aggregation under physiological conditions.

Figure 4

Zeta potential measurements of (a) AuNPs, (b) CS, (c) AuNPs–CS, (d) Pap, and (e) AuNPs–CS–Pap.

In contrast, Figure 4b shows that CS exhibited a strongly positive zeta potential of +38.6 mV, reflecting the abundance of protonated amino groups under physiological pH. Figure 4c demonstrates that coating AuNPs with CS led to a zeta potential shift to +36.8 mV, indicating successful surface functionalization and improved colloidal stability through enhanced electrostatic repulsion [47].

Pap in Figure 4d exhibited a positive zeta potential of +17.4 mV, which reflects the net surface charge of the enzyme in PBS at pH 5.5. This value is consistent with the protonation of amino groups in the enzyme structure, especially at slightly acidic pH conditions. The positive charge enhances the electrostatic interaction potential with negatively charged bacterial membranes and contributes to Pap’s inherent antibacterial activity. Furthermore, this surface charge plays a key role in stabilizing the enzyme when conjugated to other positively charged systems such as CS, minimizing repulsion and supporting cohesive integration into the nanocomposite. Following the immobilization of Pap, Figure 4e shows that the zeta potential of the Pap nanocomposite decreased to +21.8 mV. This reduction is likely due to the partial neutralization or shielding of surface charge by the adsorbed enzyme molecules, which possess zwitter ionic or weakly charged amino acid residues. Despite the decline in surface charge, the nanocomposite retained a sufficiently positive zeta potential to prevent aggregation and maintain suspension stability, consistent with prior studies on enzyme–NP conjugates.

The stability of suspensions is deemed satisfactory when the zeta potential exceeds ±30 mV. Consequently, both CS (38.6 mV) and AuNPs–CS (36.8 mV) exhibit exceptional stability, while Pap (17.4 mV) and AuNPs–CS–Pap (21.8 mV) demonstrate intermediate stability. AuNPs (−15.6 mV) exhibit reduced stability and may be susceptible to aggregation without adequate stabilization. The integration of CS with AuNPs markedly enhances the stability of the colloidal material, as shown by the elevated zeta potential values of the composite systems relative to the unmodified nanoparticles.

This trend has also been reported in related systems. For instance, Nascimento et al. observed a marked decrease in zeta potential following the immobilization of Pap onto CS-based membranes, attributing the shift to surface interactions that enhance dispersion stability while maintaining enzymatic activity [45]. Similarly, Benucci et al. demonstrated that Pap immobilization on CS–clay nanocomposites resulted in reduced surface charge, likely due to molecular coverage and conformational rearrangements upon binding [48].

Furthermore, the electrophoretic mobility of the AuNPs–CS–Pap nanocomposite was measured at 0.000113 cm²/V s, supporting the favorable colloidal behavior expected for polymer-stabilized gold nanostructures. The moderate positive surface charge is particularly advantageous in biomedical applications, where electrostatic interaction with negatively charged bacterial membranes enhances adhesion and internalization. In addition, such surface properties may be exploited in electro-kinetically driven systems, including bio separation or targeted delivery under applied fields.

Collectively, the observed zeta potential trends and supporting electrophoretic data confirm the structural integrity and dispersion stability of the developed AuNPs–CS–Pap nanocomposite and align with previously reported behavior in enzyme-functionalized nanosystems. Furthermore, studies have shown that zeta potentials above +20 mV favor antibacterial activity while maintaining colloidal stability [49].

3.2.5 FE-SEM with EDX analysis

The FE-SEM analysis provided detailed surface morphology of the synthesized nanoparticles. In Figure 5a(i), the uncoated AuNPs appeared as uniformly dispersed and well-defined spherical particles with smooth surfaces and minimal aggregation, consistent with effective stabilization by citrate ions, which impart a negative surface charge, promoting electrostatic repulsion. In contrast, Figure 5b(ii) shows the AuNPs–CS–Pap nanoconjugates, which exhibited a noticeable increase in particle size (mean size = 30.1 nm) compared to uncoated AuNPs (20.23 nm). The particles appeared less uniformly distributed, with signs of mild aggregation or flocculation. This morphological change is attributed to the coating layer of CS and Pap, which adds thickness and may induce partial clustering due to hydrophobic interactions among Pap amino acid residues and electrostatic bridging by CS.

Figure 5

(a) AuNPs: (i) FE-SEM image; (ii) particle size distribution (mean size = 20.23 nm); and (iii) EDX analysis of AuNPs (100% Au). (b) AuNPs–CS–Pap: (i) FE-SEM image, (ii) particle size distribution (mean size = 30.1 nm); (iii) EDX analysis showing C, O, Au, N, and S; and (iv) Elemental mapping for C, O, Au, N, and S.

Moreover, the rougher texture observed in the coated nanoparticles compared to the smooth AuNPs suggests successful surface functionalization and the presence of organic layers.

FE-SEM validated the morphological alterations seen in TEM, corroborating the assertion that bio-coating modifies both dimensions and surface characteristics.

These features confirm that the surface chemistry and charge properties were significantly altered by the bio-functional material’s coating, potentially influencing both stability and biological interactions [50]. EDX was employed to assess the elemental composition of the synthesized nanostructures and to verify the successful surface functionalization of the AuNPs. Figure 5a(iii) and b(iii) presents the EDX analysis for two samples: unmodified AuNPs and the fully functionalized nanocomposite (AuNPs–CS–Pap).

In Figure 5a(iii), the FE-SEM micrograph of pure AuNPs shows uniformly dispersed nanoparticles. The corresponding EDX spectrum reveals distinct peaks exclusively corresponding to gold (Au), with no detectable signals from other elements. The quantitative profile confirms that gold constitutes nearly 100% of the sample by weight, indicating high purity and the absence of surface functionalization. This information serves as a baseline for evaluating subsequent modifications.

In contrast, Figure 5b (iii) shows the EDX results of the AuNPs–CS–Pap nanocomposite. The spectrum exhibits the presence of carbon (C, 40%), oxygen (O, 30%), nitrogen (N, 6%), sulfur (S, 4%), and gold (Au, 20%), demonstrating significant changes in elemental composition following functionalization. The carbon and oxygen elements are attributed to the CS polymer backbone, while nitrogen originates from amino groups in both CS and Pap. Notably, the appearance of sulfur is a key indicator of Pap incorporation, as the enzyme contains sulfur-rich residues such as cysteine and methionine. These findings are consistent with earlier studies that used EDX to confirm Pap immobilization through sulfur detection [44,51].

Additionally, elemental mapping at the bottom of Figure 5b(iv) confirms the uniform spatial distribution of C, O, N, S, and Au across the sample surface. This homogeneity reflects a successful and consistent coating of the AuNPs with CS and Pap, further supporting the formation of a stable and integrated nanobiocomposite system. Similar mapping-based confirmation of organic functionalization and enzyme conjugation was previously reported in nanomaterial studies using polymer-based carriers [52].

The EDX spectra and elemental distribution data validate the effective surface engineering of AuNPs via biopolymer coating and enzyme immobilization. The transition from a pure Au signal to a complex organic–inorganic profile highlights the successful formation of a multifunctional nanoplatform, essential for achieving targeted biological activity.

3.2.6 TEM analysis

The TEM analysis Figure 6a revealed spherical and relatively well-dispersed AuNPs with a mean diameter of 27.1 nm, whereas the AuNPs–CS–Pap nanomaterial (Figure 6b) showed a slightly larger average size of 29.1 nm. This increase in particle size can be attributed to the coating layer composed of CS and Pap, which adds structural bulk and affects electron density. The variation in shape and the occasional clustering may result from molecular interactions among the NP surface, the CS polymer chains, and the hydrophobic amino acid residues of the Pap enzyme [53]. The observed increase in size in TEM correlates with the shift in UV-Vis and the widening of peaks in XRD, therefore validating the efficacy of the surface coating. Consequently, the establishment of a stable and efficient bio-nanoplatform, appropriate for prospective biomedical or enzymatic applications.

Figure 6

(a) TEM image and particle size distribution histogram with a mean size of 27.1 nm of AuNPs. (b) TEM image and particle size distribution histogram with a mean size of 29.1 nm of AuNPs–CS–Pap.

3.3.1 Antibacterial activity

Antibacterial activity of the freshly prepared nanomaterial AuNPs–CS–Pap was evaluated against the pathogenic isolates, S. aureus, S. mutans, K. pneumoniae, and E. coli. The activities were compared with the Pap enzyme and AuNPs alone. As illustrated by the agar diffusion assay (Figure 7) and summarized in the graphs (Table 1), the AuNPs–CS–Pap preparation (labeled D) exhibited the largest inhibition zone against all the tested bacterial isolates. The zones measured 27.00 ± 1.00 mm for the S. aureus, 25.00 ± 1.00 mm for the S. mutans, 23.33 ± 0.58 mm for the K. pneumoniae, and 21.00 ± 1.00 mm for the E. coli. The comparatives of the inhibition zones revealed that the bacterial inhibitions for the prepared nanomaterial, AuNPs–CS–Pap, were higher at significant levels (p < 0.0001), as compared to the zone inhibition values achieved by the Pap (B) and AuNPs (C) nanomaterials, separately [54].

Figure 7

Qualitative and quantitative assessments of antibacterial activity against S. aureus, S. mutans, K. pneumoniae, and E. coli. A – control, B – Pap (50 µg mL⁻¹), C–E – AuNPs–CS–Pap (25, 50, 100 µg mL⁻¹), F – AuNPs (50 µg mL⁻¹). Top: diffusion images; bottom: mean inhibition zones (mm). Data shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

The calculated Fractional Inhibitory Index (F.I.) confirmed the presence of a synergistic interaction between the components in the nanocomposite, with values of 59.26% for S. aureus, 53.33% for S. mutans, 55.71% for K. pneumoniae, and 52.38% for E. coli Table 1. These results confirm that the antibacterial effect of the combined formulation was greater than the sum of its individual components [26]. This enhanced activity is attributed to a multifactorial mechanism of action. The AuNPs–CS–Pap nanocomposite carries a positive surface charge at physiological pH, facilitating electrostatic attraction to the negatively charged bacterial membranes. This interaction disrupts membrane integrity, enhances permeability, and promotes the internalization of the nanocarrier and the enzyme Pap into the bacterial cytoplasm. CS contributes further by adhering to the cell surface and increasing membrane leakage [55]. Previous studies have demonstrated that CS alone exhibited moderate antibacterial activity, with inhibition zones of 9.1 ± 1.9 mm for S. aureus, 8.7 ± 2.4 mm for B. subtilis, and 9.8 ± 3.9 mm for P. aeruginosa, and 8.2 ± 2.8 mm for K. oxytoca. However, when combined with AuNPs, the antibacterial activity significantly improved, with inhibition zones increasing to 16 ± 2.1, 19 ± 1.8, 26 ± 1.8, and 22 ± 1.8 mm, respectively. This highlights the synergistic effect between CS and gold in enhancing antibacterial efficacy [56]. It is worth noting that under the experimental pH of 7.4, CS remains partially protonated and positively charged, thus maintaining its dispersion ability and functional contribution [57,58].

Simultaneously, Pap enzymatically cleaves critical bacterial proteins, weakening structural components [59]. The AuNPs act as carriers that improve delivery efficiency and dispersion of both CS and Pap, thereby enhancing the bioactivity of the system [60]. Gram-positive bacteria S. aureus and S. mutans showed greater susceptibility, consistent with their simpler cell wall structure, leading to larger inhibition zones compared to Gram-negative bacteria K. pneumoniae and E. coli, which possess an additional outer membrane rich in lipopolysaccharides that restricts NP penetration.

Overall, these findings provide compelling evidence that the antibacterial potency of AuNPs–CS–Pap results from a true synergistic effect. The nanocomposite demonstrates broad-spectrum, dose-dependent activity, supporting its potential for use as a promising antibacterial platform against both Gram-positive and Gram-negative pathogens, including antibiotic-resistant isolates.

Several other studies corroborated the current findings, where the Pap improves the actions of NPs [54]. The antibacterial activity of poly-lactic-co-glycolic acid (PLGA)-phosphatidylcholine (PC) NPs loaded with Pap showed higher activity than the PLGA-PC NPs. Oliveira et al. [61] found that the substance has anti-staphylococcal properties. Manuel Xavier et al. [62] proved that the hybrid AuNPs that they made selectively killed the S. aureus cells by increasing the quantity of ROS within their cells. Asanarong et al. [63] detected that the bacterial cellulose loaded with Pap composite was found to have antibacterial properties according to the results of the agar diffusion and cell growth inhibition assays. Under in vitro tests, the isolates of E. coli, P. aeruginosa, and S. aureus were completely inhibited. Studies on the use of AuNPs as antibacterial agents have been extensive owing to the gold’s durability, low toxicity, large specific surface area, and ease of functionalization. The NPs’ antibacterial efficacy is known to be dependent upon their size and dispersibility [64]. The strong electrostatic attractions between NPs and bacterial cell membranes cause an increase in NP accumulation on the membrane. This, in turn, may cause the membranes to undergo significant stress, NPs penetrating into the cytoplasm, resulting in cell lysis. Another potential effect on DNA replication might result from NPs’ interactions with the bacterial proteins’ thiol (–SH) groups. An increase in the generation of ROS (hydroxyl radicals and singlet oxygen) and the disruption of cellular functions due to the accumulation of NPs on the bacterial cell wall, in the cytoplasm, or in periplasmic regions are two potential mechanistic routes of antibacterial activity that have been theorized previously. The NPs also have the potential to influence bacterial junctions [65]. The antibacterial activity of the mixture of Pap and AuNPs–CS was amplified by combining the two substances. The AuNPs–CS seemed to have enhanced the stability of Pap due to the combination of their respective characteristics. Because of its increased stability, the Pap seemed to have maintained its antibacterial action for a longer time. On the other hand, the Pap seemed to enhance the antibacterial activity of AuNPs–CS in many ways. The enzymatic characteristics of Pap seemingly also aided in the breakdown of the bacterial cell wall, which in turn allowed the AuNPs–CS to penetrate the bacterial cells more effectively. Because of this enhancement, the AuNPs’ antibacterial potency seemed to be amplified [66].

3.3.2 Evaluation of the MICs and MBCs of the microorganisms

The MIC and MBC results presented in Figure 8 and Table 2 demonstrated the enhanced antibacterial efficacy of the AuNPs–CS–Pap nanocomposite compared to its components (AuNPs and Pap) when tested against both Gram-negative E. coli and K. pneumoniae and Gram-positive S. aureus and S. mutans bacterial isolates.

Figure 8

MIC assay of (a) AuNPs, (b) Pap, and (c) AuNPs–CS–Pap against four pathogenic bacterial isolates: E. coli, S. mutans, S. aureus, and K. pneumoniae. Serial dilutions of each compound (1.56–100 µg mL⁻¹).

As shown in Figure 8a bar for AuNPs exhibited moderate antibacterial activity, with MIC and MBC values of 12.5 and 25 µg mL⁻¹, respectively, which is consistent with previous findings on the antibacterial activity of CS-coated AuNPs [56]. In contrast, in Figure 8b, free Pap required higher concentrations (MIC = 25 µg mL⁻¹, MBC = 50 µg mL⁻¹) to exert a comparable effect, which aligns with earlier reports confirming its modest antibacterial potential when used alone [67].

Remarkably, the fresh AuNPs–CS–Pap nanocomposite showed in Figure 8c significantly enhanced potency, with MIC values as low as 3.12 µg mL⁻¹ for E. coli and 6.25 µg mL⁻¹ for the other tested isolate. This enhanced efficacy can be attributed to a synergistic mechanism that includes improved bioavailability and structural stability of the Pap enzyme when immobilized on AuNPs. The mucoadhesive and positively charged nature of the CS polymer fosters strong interactions with bacterial membranes, facilitating the penetration of AuNPs into biofilms and bacterial outer membranes, particularly in Gram-negative species. Notably, the CS polymer becomes entangled with the biofilms at the interaction sites, further enhancing its effectiveness. These results are in line with previous studies on CS–gold nanocomposites, which showed strong antibacterial effects at low concentrations due to the combined action of membrane disruption and NP-mediated enzyme delivery [56].

Interestingly, E. coli – a Gram-negative bacterium typically more resistant due to its outer membrane – was the most susceptible to the treatment. This observation may result from the combined effects of Pap’s proteolytic activity and the membrane-disrupting action of the positively charged nanocomposite. Similarly, S. aureus and S. mutans (Gram positive) also showed marked inhibition, supporting the broad-spectrum nature of the nanoplatform.

Overall, these findings confirm that combining AuNPs, CS, and Pap into a single multifunctional system reduces the effective antibacterial dose and enhances therapeutic potential. The results strongly suggest that this nanocomposite could serve as a safer and more potent alternative to conventional antibacterial agents, particularly in the era of antibiotic resistance.

3.3.3 Anti-biofilm activity

Effects of Pap and the prepared nanomaterials, AuNPs, and AuNPs–CS–Pap on biofilm generations were evaluated by the procedure of staining through CV (Figure 9). The staining of the experimental results with 0.1% CV exhibited bacterial efficacy of biofilm production, wherein each stained wells’ OD of staining was estimated through an ELISA microtiter plate reader at 590 nm. A homogeneous re-solubilization of stained wells was performed to avoid discrepancies in the OD measurement owing to the OD variations in the middle and sides of the well before homogenization.

Figure 9

Biofilm formation activity reduction of pathogenic bacteria. (a) Control; (b) Pap; (c) AuNPs; and (d) AuNPs–CS–Pap, against K. pneumoniae, S. aureus, S. mutans, and E. coli. Top: histological staining; bottom: inhibition rate percentages.

Pathogenic bacterial isolate, S. aureus, E. coli, S. mutans, and K. pneumoniae, growth inhibitions were observed by the Pap, AuNPs, and AuNPs–CS–Pap. AuNPs–CS–Pap performed well with a significant anti-biofilm activity level against all four bacterial isolates, when compared with the other two, the Pap and AuNPs. Pap alone was a weak inhibitor of biofilm generation against all the pathogenic isolates, wherein the film’s biomass reduction percentage ranged between 11 and 44%, and this suggested that Pap had some intrinsic biofilm-disruption properties. Thus, the Pap is not effective enough as a standalone agent for inhibiting biofilm formation, while the AuNPs–CS–Pap nanomaterial exhibited a significant multi-fold increase in the inhibition of biofilm production in all the tested isolates, wherein biofilms’ biomass reduction ranged between 70 and 90% Figure 9. The biofilm formation was inhibited by 91, 81, 92, and 71% for K. pneumonia, S. aureus, S. mutans, and E. coli, respectively. The Pap’s inhibitions were 44, 35, 43, and 11% for the same sequenced bacterial isolate as mentioned earlier for AuNPs–CS–Pap. Moreover, the bare AuNP biofilm inhibitions were estimated at 41, 53, 41, and 8%. These observations proved the point that Pap loading on AuNPs–CS enhanced the anti-biofilm formation activity of the final nanomaterial, AuNPs–CS–Pap, prepared incorporating the Pap. The better activity from AuNPs–CS–Pap can be credited to multiple factors that include the enzymatic degradation, through Pap, of the extracellular polymeric substances (EPS), thereby weakening the structures of biofilms. However, as such, the Pap possesses weak antibacterial activity; hence, it is incapable of annihilating the bacteria. Nonetheless, the Pap’s enzymatic action degrades the biofilm matrix and makes bacteria more vulnerable. However, during antibacterial bioactivity testing, the AuNPs annihilated the bacteria by producing ROS to cause oxidative damage. Therefore, the disruption of bacterial outer membrane and an increase in vulnerability of the bacterial entity, which eventually leads to cell damage and death, together with the inhibition of quorum sensing, prevented bacteria from coordinating the biofilm generation [68,69,70].

The CS enhances the membrane permeability, allowing better delivery of AuNPs and Pap into the forming layers of biofilm. The combination of these mechanisms amplifies the biofilm inhibition compared to the Pap alone. The AuNPs–CS improved the delivery and retention of Pap at the biofilm surfaces. The CS binds electrostatically to negatively charged bacterial surfaces, thereby ensuring that the NPs remain attached to the biofilm. This improved the penetration of Pap into the dense biofilm matrices, allowing it to work more efficiently [71,72,73]. Pap is an enzyme that can lose activity over time due to environmental factors (e.g., temperature, pH), while the AuNPs–CS–Pap CS coating protects and stabilizes the Pap, thereby ensuring sustained enzymatic activity within the biofilm. While Pap is limited to degrading the biofilm matrix, the AuNPs–CS target the bacterial cells owing to the presence of the AuNPs. This dual approach (degrading the matrix and killing bacteria) ensured that the biofilm was dismantled. The bacteria were killed, preventing regrowth and further biofilm formation. Biofilms are highly resistant to enzymatic treatments due to their dense structure and protective matrix [74,75,76,77]. The NP-based system AuNPs generated ROS and damaged the bacterial membranes by breaking down the biofilm’s defenses. This weakened the structure and allowed Pap to work more effectively. The combination of Pap with AuNPs–CS provided AuNPs–CS–Pap, which enhanced the anti-biofilm generation efficiency through synergistic integration of multi-factors to produce the elevated levels of the antibacterial and anti-biofilm generating activity. The degradation of biofilm matrices by Pap, the destruction of bacterial cells through ROS generation, and the disruption of cellular walls by AuNPs collectively contribute to significant biofilm destruction and enhanced antibacterial activity. The presence of CS further improves penetration capability and ensures the stability of the nanomaterials at the site of action. As a result, this combination demonstrates superior effectiveness compared to the use of Pap alone.

3.3.4 Evaluation of bacterial viability using AO/EtBr staining

The enzyme Pap, nanomaterials AuNPs, and AuNPs–CS–Pap were further validated for their antibacterial activity using the AO/EtBr staining for assessing bacterial viability (Figure 10). The images obtained from fluorescence microscopy exhibited the marked differences among the treated and untreated bacterial cells for these two isolates, i.e., E. coli and S. aureus. For the untreated control group, green fluorescence was predominant, which indicated the majority of bacterial cells as viable. The treatment with Pap, AuNPs, and AuNPs–CS–Pap showed red-colored fluorescence, which indicated severe membranes’ damage, and eventual cell death observed as intense red fluorescent entities. This highlighted AuNPs-treated samples’ enhanced antibacterial activity, as also observed from the strong and intense fluorescent observations, confirmed the AuNPs–CS–Pap activity and its enhancements with the AuNPs–CS–Pap preparation, which seemed to be generated from the synergistic actions of the components of this nanopreparation. The AuNPs produce ROS, which is prone to induce oxidative stress, and eventual bacterial cell damage and death. The AuNPs are known to interact electrostatically with the negatively charged bacterial membranes to destabilize them and result in cell lysis [78,79]. The Pap added to the antibacterial effects through enzymatically disrupting the EPS of the biofilms, degrading the membrane proteins, and thus increasing the bacterial cells’ susceptibility. CS incorporation with AuNPs facilitated delivery and improved interactions with the bacterial cells. The CS also assisted internalization of AuNPs and linked Pap enzyme inside the bacterial cells, thereby amplifying the membrane disruption and, in turn, increasing the antibacterial efficiency. The combined synergistic effects of the AuNPs–CS and Pap led to enhanced bacterial neutralizations and annihilations as compared to either the Pap or the AuNPs separately. These results of the bioactivity testings confirmed the enhanced antibacterial efficacy of the AuNPs–CS–Pap over the Pap and AuNPs separately. The combination used the unique properties of AuNPs, CS, and the Pap enzyme, thereby creating a robust nanosystem [80], which has the potential to be developed as a unique and highly potent therapeutic agent for combating present and future bacterial infections and multidrug-resistant (MDR) situations.

Figure 10

AO/EtBr dual staining: (a) non-treated bacterial cells; treated bacterial cells with (b) AuNPs; (c) Pap; and (d) AuNPs–CS–Pap, against E. coli and S. aureus. Magnification 400×.

3.3.5 In vivo toxicity

Further evaluation of the viability and antibacterial efficacy of the prepared nanomaterial, AuNPs–CS–Pap, in vivo experiments were designed Figure 11. Treatments of mice with AuNPs, Pap, and AuNPs–CS–Pap at doses of 50 mg kg⁻¹ for 14 days showed no mortality. Also, no statistically significant (P ≥ 0.05) body weight differences among the treated and control mice were observed. Nonetheless, no abnormal or offline and adverse clinical signs, or mice behavior were observed. The AuNPs, Pap, and AuNPs–CS–Pap treatments caused no apparent toxicity in the animals, and all the animals remained healthy till the end of the bioactivity experiments. Necropsy at the end also didn’t show any macroscopic organ changes in the treated groups. According to the previous reports [81,82,83], enzyme Pap and the AuNPs’ treatments for infections also show no fatality and unusual behavior, which included trouble in breathing, difficulty in moving, hunching, or other unusual interactions with the cage mates of the animals. Additionally, there were no clinical manifestations, including redness, swelling, movement difficulties, hunching, and any other abnormal activity. The control group animals showed a steady and consistent increase in body weight over the 14-day period. At the end of these in vivo experiments, the body weights of the control group animals reached approximately 29 g, demonstrating normal growth without any adverse effects, while mice treated with Pap enzyme exhibited the slowest increase in body weights when compared to other animal groups. The body weight increase was modest, reaching around 28 g by the end of the study. This suggested that Pap may have caused certain levels of mild physiological stress, potentially due to its proteolytic activity, which could have impacted the nutrient absorption and/or metabolism [84]. The mice treated with AuNPs showed a slightly higher weight gain as compared to the Pap-treated animal group, reaching over just 28 g by the end of the experiment. The observations reflected the AuNPs’ biocompatibility with minimal impact on the overall growth of the mice [85,86]. The AuNPs–CS–Pap-treated animals exhibited weight gain similar in parallel to the control group animals, thereby reaching nearly 28.5–29 g at the end of the experiment. These growth patterns were steady, thereby indicating that the combination of AuNPs, CS, and Pap did not induce any significant adverse effects on the mice. This also suggested that the CS coating might have mitigated any potential toxicity of the individual components, thereby enhancing the biocompatibility of the developing nanomaterial [87,88]. All treatments resulted in body weight gains, suggesting that none of the materials caused severe toxicity or adverse systemic effects during the period of the experiments. These results demonstrated that the AuNPs–CS–Pap was well-tolerated by the experimental animals, with no significant adverse effects on their body weight. The steady weight gains across all mice groups of animals suggested that the treatments were biocompatible. With the prepared NP material, AuNPs–CS–Pap, showing the most favorable profile, this result favored the safe applications of AuNPs–CS–Pap, and it is recommended for further research and preparation of newer potential therapeutic agents for the antibacterial drugs segment for the future.

Figure 11

Body weight changes for mice treated with AuNPs, Pap, and AuNPs–CS–Pap at the dose of 50 mg kg⁻¹ by using intraperitoneal injection. Body weights were measured every 2 days. Each point represents the mean ± SD of four mice.

For further investigations into the effects of AuNPs–CS–Pap on the relative weight, the weights of key organs, i.e., lungs, kidney, liver, and spleen, were compared before and after the treatment with AuNPs, Pap, and AuNPs–CS–Pap Figure 12. The relative organ weights are an important measure of potential toxicity and physiological changes induced by these treatments. The relative lung weights in the control group were significantly higher as compared with all the treated groups. Mice treated with AuNPs showed a slight but significant reduction (p < 0.05) in lung weight as compared to the control. Treatments with Pap and AuNPs–CS–Pap resulted in similar lung weights, with no significant differences between these groups and the control. The decrease in lung weight with AuNPs treatment might indicate minor stress or interaction with lung tissue. However, the inclusion of Pap and CS in AuNPs–CS–Pap mitigated this effect but maintained lung weights close to control levels.

Figure 12

Relative organ weights of mice treated with AuNPs, Pap, and AuNPs–CS–Pap. Data shows the relative weight (g) of lungs, kidneys, liver, and spleen. Statistical significance is indicated by asterisks (*p < 0.05, **p < 0.01, ****p < 0.001, ns = not significant).

No significant differences were observed in relative kidney weights across all groups, except for a slight reduction in the AuNPs group as compared to the controlś (p < 0.05). This indicated that none of the treatments, including AuNPs–CS–Pap, caused significant kidney toxicity or structural changes. The kidney remained unaffected in terms of relative weight, suggesting competitive levels of biocompatibility. The control group exhibited the highest relative liver weight, significantly greater than all the treated groups (p < 0.01 to p < 0.0001). Among the treated groups, the AuNPs–CS–Pap and Pap showed similar liver weights, while the AuNPs group exhibited slightly lower liver weights as compared to the Pap and AuNPs–CS–Pap. This reduction in liver weight after treatment suggested that the treatments had influenced metabolic activity and/or caused mild physiological adaptations in the liver. However, the similarity in liver weights among treated groups indicated that the combination of AuNPs, CS, and Pap (AuNPs–CS–Pap) did not exacerbate the effects observed with the individual components. The control group had a significantly higher spleen weight as compared to the AuNPs-treated mice (p < 0.01) and Pap-treated mice (p < 0.05). No significant differences were observed between the AuNPs–CS–Pap group and the control group, thereby suggesting that the combination treatment-maintained spleen weight at normal levels. The slight reduction in spleen weight in the AuNPs and Pap groups could indicate mild effects on immune or hematopoietic activity, while the AuNPs–CS–Pap treatment showed no adverse impact on spleen functions.

The analysis of relative organ weights suggested that the AuNPs–CS–Pap treatment exhibited the highest biocompatibility among the tested materials. While slight reductions in organ weights were observed with AuNPs and Pap treatments, the AuNPs–CS–Pap did not induce any significant changes [89,90,91], thereby demonstrating its potential as a safer nanomaterial for therapeutic applications. These findings highlighted the importance of CS in reducing the potential toxic effects of the AuNPs on vital organs. The organ index results were consistent across all organ types, with similar trends observed in the effects of AuNPs, Pap, and AuNPs–CS–Pap Figure 13. The consistency between relative weights and index results also underscored the reliability of the measures for assessing organ-specific effects of treatments.

Figure 13

Organ index of mice treated with AuNPs, Pap, and AuNPs–CS–Pap. Data show the lung, kidney, liver, and spleen indices. Statistical significance is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant).

The kidney function of experimental animals treated with AuNPs, Pap, and AuNPs–CS–Pap, compared to the control group, was evaluated Figure 14. The parameters assessed included urea, uric acid, and creatinine levels in the blood, which are the standard biochemical markers for kidney function [92]. The urea levels across all treatment groups, AuNPs, Pap, and AuNPs–CS–Pap, were similar to the control group, with no statistically significant differences (ns). The results indicated that none of the treatments caused significant impairments of the kidney functions related to urea excretion. Urea is a key marker of renal function, and its stability suggests that the treatments were well tolerated by the kidneys. Uric acid levels showed no significant differences between the control group and any of the treated groups, including AuNPs, Pap, and AuNPs–CS–Pap (ns). Uric acid is a byproduct of purine metabolism and is excreted by the kidneys. The absence of significant changes in uric acid levels suggested that the treatments did not impact the kidneys’ ability to process and excrete metabolic waste products. Creatinine levels were also consistent across all groups, with no significant differences observed between the control and treated groups (ns). Since creatinine is a crucial marker for assessing glomerular filtration rate and the kidneys’ proper functioning, the consistency of creatinine levels indicated that the treatments did not affect the filtration efficiency of the kidneys. The levels of urea, uric acid, and creatinine remained within normal ranges and showed no significant differences across all groups. These observations suggested that none of the treatments, AuNPs, Pap, and AuNPs–CS–Pap, caused renal toxicity or impaired any of the kidney functions.

Figure 14

In vivo kidney function tests of mice treated with AuNPs, Pap, and AuNPs–CS–Pap. Data show kidney activity in terms of urea, uric acid, and creatinine levels. Statistical significance is indicated by asterisks (ns = not significant).

The serum levels of liver enzymes in male mice following treatment with AuNPs, Pap, and AuNPs–CS–Pap are shown in Figure 15. The mean ALT levels ranged between 35 and 70 U/L, while AST concentrations ranged from 130 to 230 U/L across all groups. These values fall within the internationally recognized physiological reference ranges for healthy male mice – ALT: 41–131 U/L, AST: 55–352 U/L – as reported by Silva‐Santana et al. [93]. This information indicates that none of the tested formulations caused hepatocellular damage or liver inflammation, thereby confirming their non-hepatotoxic and biocompatible nature.

Figure 15

In-vivo liver function tests of mice treated with AuNPs, Pap, and AuNPs–CS–Pap. Data show liver enzyme activity in terms of ALT, AST, and ALP levels. Statistical significance is indicated by asterisks (p < 0.05, ns = not significant).

For ALP, enzyme levels ranged between 60 and 90 U/L. Although the Pap-treated group showed a significant increase (p < 0.05) in ALP activity compared to the control, the levels were still lower than the normal range for male mice (118–433 U/L) [93]. This evidence suggests that the elevation was mild and not indicative of pathological liver injury. Furthermore, the observed increase in ALP activity may reflect a physiological response rather than a toxicological effect, warranting further investigation into the underlying mechanisms. Future studies should focus on long-term assessments of liver function and additional biomarkers to fully understand the implications of these formulations. Importantly, ALP levels in both the AuNPs and AuNPs–CS–Pap groups were not significantly different from the control (ns), indicating a stabilizing effect of the NP formulations. Moreover, ALP levels in the AuNPs–CS–Pap group were significantly lower than those in the Pap-only group (p < 0.05), implying that encapsulation of Pap within the nanocomposite effectively mitigated the enzyme elevation induced by free Pap.

Since ALP is a key biomarker for biliary function and bone metabolism, the isolated elevation observed in the Pap group may reflect transient biliary stress. The normalization of ALP levels in the AuNPs–CS–Pap group further underscores the hepatic safety and improved biocompatibility of the nanoformulated system [80]. These findings suggest that the use of nanocomposites for enzyme delivery not only enhances therapeutic efficacy but also minimizes potential side effects associated with free enzymes. Consequently, this approach could pave the way for more effective treatments in conditions where precise enzyme regulation is crucial [94].

The histological morphology was detected for mouse organs, including the kidney, liver, spleen, and lung, using H&E staining after treatments with AuNPs, Pap, and AuNPs–CS–Pap. The results revealed that there were no pathological effects on the tissues of all tested organs (Figure 16).

Figure 16

Histological effects on organs of mice treated with AuNPs, Pap, and AuNPs–CS–Pap. Images show the histological staining of kidney, liver, spleen, and lung tissues from different treatment groups: control, AuNPs, Pap, and AuNPs–CS–Pap.