Tracking success of interaction of green-synthesized Carbopol nanoemulgel (neomycin-decorated Ag/ZnO nanocomposite) with wound-based MDR bacteria

2.1 Chemicals

Cetyl trimethyl ammonium bromide (CTAB), mercaptoethanol, proteinase K, sodium dodecyl sulfate, chloroform, isoamyl alcohol, isopropanol, and triethanol buffer were used for DNA (desoxyribonucleic acid) extraction and purification. Silver nitrate (AgNO₃, 99.9%) and ZnO salts (99.5%) were used to synthesize the NC. Tween 80, polyethylene glycol (PEG) 400, Carbopol 940 (99.99%), and triethanolamine (99.0%) were used to formulate the emulgel. Acetic acid and India ink were used to prepare the fungal stain. All these chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). Pure (HPLC-grade) neomycin (see datasheet in Suppl. Mat. File) and olive oil were purchased from the local market. All the reagents used were of analytical grade and did not require further purification.

2.2 Strains

The three microbial strains, i.e., P. aeruginosa, S. aureus, and C. albicans, were isolated from infected wounds of human patients to check their antibacterial and antifungal activity. They were kindly gifted from the National Institute of Health (Islamabad, Pakistan). These skin microbial strains were maintained on their respective growth media at 4°C and were refreshed after 2–3 months.

2.3 Isolation, purification, and genomic identification of fungal strains

Leaf discs of fresh and healthy Madagascar periwinkle and Cupressus plants were prepared. Their surfaces were rinsed with distilled water (dH₂O) and ethanol; subsequently, they were dried before being cultivated on potato dextrose agar plates. Plates were sealed with a sealing film and incubated at 26°C for 4 days. The fungi that grew on the leaf discs were sub-cultured on potato dextrose agar plates several times to obtain a pure isolate (single species); the isolates were then preserved at 4°C [29]. The fungal strains IIUI313 (the purified strain from the Madagascar periwinkle) and IIUI314 (the purified strain from the Cupressus plant), which had rapid growth and large biomass production, were selected to synthesize the NC.

The isolated fungal species were identified phenotypically based on colonial and cellular morphology [30,31], followed by molecular characterization for genotypic identification. A former CTAB method [32] with certain modifications was used to extract the genomic DNA. Nuclear ribosomal internal-transcribed spacers, i.e., forward ITS1 5′ TCCGTAGGTGAACCTGCGG-3′ (19mer) and reverse ITS4 5′-TCCTCCGCTTATTGATATGC-3′ (20mer), obtained from Macrogen (Seoul, South Korea) were amplified using the polymerase chain reaction (PCR) thermal cycler (BIOER, LifeECO, Shanghai, China) in a final reaction volume of 25 µL [32]. The PCR end-product was run on gel electrophoresis (1.5%) at 100 V for 40 min to separate DNA and confirm the amplicon size. The amplified PCR product was sequenced by Macrogen (Seoul, South Korea) using the Sanger sequence technology, and the obtained internal-transcribed spacer-related data of both strains were analyzed using NCBI’s (National Center for Biotechnology Information) BLAST for strain identification [6].

2.4 Mycosynthesis of the Ag/ZnO NC

The Ag/ZnO NC was synthesized using the method described previously [33]. It was modified by mixing cell-free culture extracts from two fungal strains, as shown in Figure 1. Briefly, the NC was prepared by co-inoculation of the fungal extracts M. guilliermondii (IIUI313) and A. welwitschiae (IIUI314). Both species were isolated as endophytes and grown separately in 250 mL of Sabouraud dextrose broth using 500 mL flasks. Flasks with spores were incubated at 25 ± 2°C under shaking (120 rpm) for 4 days in dark conditions to produce biomass. This fungal biomass was isolated from the liquid media by physical separation and washed with deionized water (DIW) to remove all unwanted substances. Fresh, clean biomass (24 g) from each strain was placed in two separated 500 mL flasks containing 250 mL of sterile DIW and incubated in a rotatory shaker under the same previously described conditions. A cell-free filtrate was extracted by filtering the suspension with Whatman filter paper # 1. Then, sterile aqueous salt solutions of 20 mL of 1 M AgNO₃ and 2 M ZnO were mixed with 50 mL of the fungal extracts (25 mL each), followed by continuous stirring (200 rpm) and heating (40°C) on a magnetic hot plate for 3–4 h. The percentage-to-weight ratio of Ag:Zn was 1:1, and the ratio of salts to extract was 4:5 v/v. The reduction of salts was visualized by the color change and the precipitate formation. A light-brown aggregate at the bottom of the flask was separated by centrifugation at 4,000 rpm, followed by a double washing with sterile dH₂O and a single washing with ethanol to remove all unreacted molecules. The particles were eventually dried, calcined at 400°C, and stored at room temperature (RT) until use.

Figure 1

Illustration of the fungal consortium-based synthesis of Ag/ZnO NC.

2.5 Preparation of NEG formulations

The synthesis of the NEG is a two-step process, which was based on the method published earlier [36]. It was slightly modified by using pure olive oil in the organic phase and drug dissolution in the aqueous phase (Table S1). Three Carbopol NEGs were prepared (Table S2) and named as follows: (i) blank Carbopol NEG, Formulation 1 (blank NEG, F1); (ii) Carbopol NEG-loaded Ag/ZnO NC, Formulation 2 (Ag/ZnO NEG. F2); and (iii) Carbopol NEG-loaded Neo-Ag/ZnO NC, Formulation 3 (Neo-Ag/ZnO NEG, F3). F3 was selected after optimization using various experimental conditions, as shown in Figure S3.

2.6 Physicochemical characterization of the mycosynthesized nanoformulations

2.7 Organoleptic properties

2.8 Biological and safety characterization of the mycosynthesized Neo-Ag/ZnO NEG

2.9 In vivo wound-healing potential

2.10 Statistical analysis

The one-way ANOVA, repeated measures (RM), one-way ANOVA, and unpaired t-test were performed using Graphpad Prism 8.0.2 (San Diego, CA, USA). Experiments were performed thrice, and the obtained values were presented as mean ± SD. p-values <0.05 were considered as statistically significant.

1. Ethical considerations: Animal experimentations were approved on 12/27/21 by the ethical review board (ERB) of the International Islamic University, Islamabad, Pakistan (Reg #470-FBAS/MSBT/F20). The experiments were conducted according to the recommendations and guidance of this ERB. All precautions have been undertaken to minimize the suffering of animals.

3.1 Identification of fungal strains

Morphological features of major fungal strains, respectively, found in M. periwinkle and Cupressus plant leaves, were examined. The colonial and cellular morphology revealed with India ink and without India ink (control) is shown in Figure 2; the findings were confirmed by molecular characterization. The IIUI313 strain found in the M. periwinkle leaves was M. guilliermondii (GenBank accession number: OP216058), and the IIUI314 strain found in the Cupressus leaves was A. welwitschiae (GenBank accession number: OP216102). A phylogenetic tree of these identified fungal strains was constructed using Mega-7 software by the neighbor-joining method with 700 bootstrap replications, as represented in Figure 3.

Figure 2

Phenotypic identification by optical microscopy of M. guilliermondii (a) and A. welwitschiae (b): ((a) colonial morphology; (b) cellular morphology without staining; (c) cellular morphology with India-ink staining. Magnification ×40).

Figure 3

Genotypic identification of M. guilliermondii (a) and A. welwitschiae (b).

3.2 Phytochemical analysis of the mycoextracts by GC-MS

Myriads of metabolic products were detected and analyzed by GC-MS in the extracts of M. guilliermondii (Figure S1, Table S3) and A. welwitschiae (Figure S2, Table S4). The main chemical compounds found in M. guilliermondii were squalene, di(1,2,5-oxadiazolo) [3,4-b:3,4-E] pyrazine, stearic acid, 10(E),12(Z)-conjugated linoleic acid, and dibutyl phthalate, whereas 1,2-benzenedicarboxylic acid, palmitic acid, phthalic acid, stearic acid, 2,2′-methylenebis[6-(1,1-dimethylethyl)-4-methyl-phenol were detected in A. welwitschiae. These metabolites can not only contribute to the identification of the fungal strain but also, like phytochemicals, might act as reducing and capping agents during the production of NC (e.g., Ag/ZnO NC). Thereby, these metabolites most likely reduced the salts Ag²⁺ to Ag⁰ and Zn²⁺ to Zn⁰ since a color shift was noticed from white to dark brown during the synthesis of Ag/ZnO NC. The selected strains produced a relatively high yield of Ag/ZnO NC (i.e., 4 g) in a relatively short time (within 3–4 h). Moreover, this metabolic diversity enhances the properties of the NPs, including stability and variations in shape. Similar to the varying reducing powers of different agents, as reported by Yoo et al. [49], the distinct bioactive compounds and enzymatic activities present in each fungal extract play a crucial role in influencing the synthesis of NPs.

3.3 Physicochemical characterizations of the mycosynthesized nanoformulations

The EE of Neo (100 mg/mL) in Ag/ZnO NC analyzed using a UV-visible spectrophotometer was calculated as 81%. The high encapsulation efficiency of the drug in the Ag/ZnO NC was probably due to the high-level solubility of Neo in the solvent (H₂O). The results were compatible with a recent study that reported the encapsulation efficiency of azithromycin to range between 50 and 87% in ZnO NPs using drug concentrations in the range of 200–500 mg [50].

The strong and broad absorbance peaks at 370 nm for ZnO, 410 nm for Ag, and 415 nm for Neo-Ag/ZnO NC are shown in Figure 4a observed during diffused reflectance spectroscopy analysis. The band gap values were calculated as 3.4 eV for Zn, 3.02 eV for Ag, and 2.95 eV for Neo-Ag/ZnO NC using the formula E g = 1 , 240 / λ abs . Diffused reflectance spectroscopy confirmed the nanoscale and absorbance range of AgNPs [51,52,53] and ZnO NPs [54,55,56]. The absorbance shifts toward a longer wavelength confirmed the successful conjugation of the Neo drug to Ag/ZnO NC [57]. The agglomeration of NPs with the antibiotic, as well as the decrease in the energy gap between the excited state and the ground state, might be the reason for this red shift in UV [58]. The synthesized particles (Ag/ZnO NC) with the aforementioned band gap values can be employed as antimicrobial agents because of the easy penetration of nano-size particles into bacterial cells [59,60].

Figure 4

(a) Diffused reflectance spectra of mycogenic nanoformulations: ((a) Ag NPs and ZnO NPs and (b) Neo-Ag/ZnO NC). (b) FTIR spectra of mycogenic nanoformulations: ((a) Ag/ZnO NC and Neo-Ag/ZnO NC and (b) blank NEG, Ag/ZnO NEG and Neo-Ag/ZnO NEG).

Figure 4b shows the FTIR spectra of Ag/ZnO NC, Neo-Ag/ZnO NC, blank NEG (pure Carbopol NEG), Ag/ZnO NEG, and Neo-Ag/ZnO NEG. The capping of NPs by functional groups present in fungal extracts was confirmed by the presence of the amide group shown in FTIR spectra. Enzymes or proteins present in the cell-free culture filtrate can bind to NPs through their C₃H₇NO₂S cysteine or free amine group and provide stability to NPs, as reported earlier [61,62]. The fingerprint region of Ag/ZnO NC and Neo-Ag/ZnO was observed below 1,500 cm⁻¹. The peak at 1,336 cm⁻¹ might be due to the bending vibration of ZnO. The small absorption signal at 1,600 cm⁻¹ was assigned to the secondary amide (−NHR) in Ag/ZnO NC. The FTIR spectrum of Ag/ZnO NC displayed a weak intensity peak due to the incorporation of Ag⁺ with Zn²⁺. When Neo was conjugated with Ag/ZnO NC, the bending vibrational bands shifted to a lower wave number, at 1,033 cm⁻¹, and an extra peak at 3,000 cm⁻¹ confirmed the conjugation of Neo with Ag/ZnO NC.

The FTIR spectrum of blank NEG (unloaded Carbopol nanocarrier) showed a strong, broad O–H stretching vibration at 3,307 cm⁻¹, and this indicated the presence of a carboxylic acid group. The adsorption band at 2,884 cm⁻¹ could be attributed to the weak U-shaped aliphatic C–H stretching. The spectral region at 1,639 cm⁻¹ showed medium C═C stretching and weak C–O–C stretching at 1,350 and 1,080 cm⁻¹ owing to the presence of alkene and alkane groups in the Carbopol NEG. The FTIR spectrum of the Carbopol NEG (Blank NEG) is characterized by the presence of carboxylic acid, alkene, and alkane groups [24,63]. An additional peak is observed at 1,458 cm⁻¹ for Ag/ZnO NEG and at 1,459 cm⁻¹ for Neo-Ag/ZnO NEG. The presence of an additional peak and a band shift toward a longer wavelength after decoration with Ag/ZnO NC and Neo-Ag/ZnO NC indicated some physical interactions between the nanomaterial, the drug, and the Carbopol NEG, but this interaction did not affect the permeation of the nanomaterial, as indicated by the in vitro release study. Our data agree with a recent study that showed EU interaction with Carbopol NEG [24].

XRD analysis revealed sharp diffraction peaks, which indicates the crystallinity nature of the Neo-Ag/ZnO NC. A similar diffraction pattern was observed in a recent study [10]. In accordance with JCPDS files (010891397 for ZnO, 030652871 for Ag), the XRD patterns of Ag/ZnO NC and Neo-Ag/ZnO NC showed lattice plane indices of 100, 002, 101, 111, 200, 102, 110, 103, 220, 112, and 201 related to the 2θ values 31.65, 35.0, 36.1, 38.0, 47.55, 56.55, 62.6, 67.3, 68.1, 69.7, and 78.1, respectively. Neo-Ag/ZnO NC showed similar peaks with a slight reduction in the peak intensity at planes 111 and 200 compared to that of Ag/ZnO NC; this revealed the amorphous nature of the Neo drug and the maintenance of the original NC structure (Figure 5a). Nevertheless, the dominant peaks associated with Ag/ZnO NC are of significantly higher magnitude, which may mask the presence of neomycin in the XRD pattern. This phenomenon underscores the challenge of detecting minor components in samples dominated by more abundant phases. Similar results were found in a study by El-Banna et al. [64], where they observed the absence of neomycin peak in the XRD spectra of neomycin-silver nanocomposite gel. The average crystalline size of pure NC (Ag/ZnO NC) was 38 nm. It was 40 nm for the drug-conjugated NC (Neo-Ag/ZnO NC) calculated from the very sharp peak obtained with diffraction analysis corresponding to the plane (101) by applying the Debye–Scherrer formula. The size calculated by the Debye–Scherrer formula was quite similar to the size of the Ag/ZnO NC reported in previous studies [54,65]. Ag–ZnO NC with a size of less than 45 nm is less toxic to the epidermal cells of the skin, as reported by Das et al. and Majhi et al. [66,67]; hence, they can be used in topical wound care as antimicrobial agents.

Figure 5

(a) XRD spectra of mycogenic nanoformulations: ((a) Ag/ZnO NC) and (b) Neo-Ag/ZnO NC)). (b) FESEM micrographs: ((a) Ag/ZnO NC at 1 µm, (b) Ag/ZnO NC at 500 nm), (c) Neo-Ag/ZnO NC at 1 µm, and (d) Neo-Ag/ZnO NC at 500 nm. (c) EDX spectra: ((a) Ag/ZnO NC) and (b) Neo-Ag/ZnO NC).

Remarkably, the FESEM images (Figure 5b) depicted a diversity of shapes (rectangular, triangular, pentagonal, and sheet-like crystalline shapes) owing to the use of two different fungal strains and the significant release of many different secondary metabolites that might act as reducing agents and ultimately impact NP properties. This observation is in line with the findings reported recently; indeed, two distinct morphologies for ZnO NPs with two different fungal strains were reported [68]. The average crystalline size of the pure NC (Ag/ZnO NC) and the drug-conjugated NC (Neo-Ag/ZnO NC) was 38 nm (range of 23–251 nm) and 40 nm (30–295 nm), respectively. The porosity increased with the increase in the PS, which is evident in the comparison between Neo-Ag/ZnO NC and pure Ag/ZnO NC. This increase in porosity was found to enhance the antimicrobial properties of Neo-Ag/ZnO NC against resistant strains, attributed to the higher surface area-to-volume ratio. This finding aligns with a study conducted by Bhosale et al., which demonstrated similar results [69]. They observed enhanced antibacterial and photocatalytic applications of ZnONPs after doping with AgNPs, attributing this enhancement to an increase in porosity and surface area as PS increased [69]. Additionally, EDX spectra revealed the purity of the synthesized NPs as no extra peak was observed (Figure 5c). These data indicated the successful conjugation and absorption of the Neo drug in the NC. The Neo-Ag/ZnO NEG with an average PS of 215 nm and a polydispersity index (PDI) of 0.019 suggests both uniform PS distribution and homogeneous dispersion of the active pharmaceutical agent. Figure S9 shows a PS of 215 nm, within the range favorable for topical applications, facilitating enhanced drug penetration and release. Additionally, the low PDI value indicates consistent PS distribution, further supporting the formulation’s suitability for efficient drug delivery in topical applications [38]

3.4 Organoleptic and stability features of the myconanoformulations

All the mycosynthesized nanoformulations (blank-NEG, Ag/ZnO NEG, and Neo-Ag/ZnO NEG) were inspected visually for organoleptic and stability features (Table 1). They were time-independent and similar in color compared to that of the freshly prepared Blank-NEG at different times: the color was creamy white for the Blank-NEG, light brownish for Ag/ZnO NEG, and greyish for Neo-Ag/ZnO NEG. The homogeneity and consistency were observed to be excellent; no differences in odor were observed, and no phase separation (sedimentation) was seen upon centrifugation for the period of 28 days. Therefore, all the synthesized formulations were considered stable.

Statistically significant differences (p < 0.05) were observed in the pH values of fresh Ag/ZnO NEG (F2) and Neo-Ag/ZnO NEG (F3) in comparison with the fresh Blank-NEG (F1) after 12 h, as analyzed by one-way RM ANOVA (Table 2); this was due to the addition of triethanolamine. Also, F2 and F3 were slightly acidic (p > 0.05) compared to F1, and this may be due to the acidic nature of the nanomaterials added to F2 and F3. No other significant changes (p > 0.05) in pH from 12 h to day 28 were found in F1, F2, or F3, which denotes an overall pH stability. Overall, the pH values recorded were within the skin pH range of 5–6 and, thus, suitable for topical application [36]. All the values are expressed as mean ± SEM.

Also, the viscosity determination is a fundamental step in evaluating emulgel formulations because it determines the permeability and bioavailability of bioactive molecules in topical DDS [36]. The unpaired t-test showed significant changes (p < 0.05) in the F1, F2, and F3 viscosities at low (6 rpm) and high (12 rpm) shear rates over the period of 28 days following non-Newtonian fluid (viscosity change with applied stress). F1 was significantly more viscous than F2 and F3 on different days (Table 3) since it was slightly acidic. The calculated spreadability value was 9.8 ± 1.20 s for F1, while it was 10 ± 1.25 s for F2 and 12.25 ± 1.21 s for F3. The spreadability of F1 was lower compared to that of F2 and F3 due to its higher viscosity; conversely, the spreadability of F3 was higher compared to that of F1 and F2 due to its lower viscosity. Herein, the rheological study showed that the synthesized nanoformulations followed a pseudoplastic behavior based on a recent study [70]. A decrease in viscosity at a high shear rate results in easier spreadability and improved pharmacodynamics after topical applications [71]. Viscosity of F1 > F2 > F3 (p < 0.05). Viscosity in F1 or F2 or F3 was overall stable (p > 0.05), as analyzed by unpaired t-test. All values are expressed as mean ± SEM.

3.5 In vitro drug release behavior, drug content, hemocompatibility, and antimicrobial activity of Neo-Ag/ZnO NEG

The most efficient nanoformulation, Neo-Ag/ZnO NEG (#F3), was assessed for its in vitro release and drug content. The in vitro study of Neo, ZnO, and Ag ions released from Neo-Ag/ZnO NEG showed a slow and sustained release for 12 h. The cumulative release (Neo, ZnO, Ag) increased to 81% after 12 h (Figure S5). The in vitro release study showed a good pharmacokinetic profile of the mycosynthesized NEG; this was due to the optimum viscosity of 1% Carbopol 940 [72] and PEG, used as the permeation enhancer [73]. The release of a drug from an emulsion is controlled by the interaction of a drug with the emulsion’s surfactant and co-surfactant; formulations with sustained release for a long time are preferred for topical delivery [36]. Any pharmaceutical dosage form must have an even distribution of pharmaceuticals to achieve a good interaction within the body, and this can be determined by finding the percentage of drug content. The drug content in Neo-Ag/ZnO NEG (F3) was as high as 98.70% obtained by RP-HPLC (Figure S6). The drug content in the F3 was found to be within the standard limit, and a relatively good LC of the Carbopol emulgel with minimum drug loss during formulation was observed [36].

Besides, Neo-Ag/ZnO NEG (test, F3) exhibited no cytotoxicity against human erythrocytes (red blood cells – RBC) since no halo was observed (Figure S7). This was also the case for Neo (standard). A negligible (p > 0.05) RBC lysis zone (1 mm) was observed with 1× PBS (negative control). F3 was negligible (1 mm) compared to that of Triton-X 100 (positive control), which was 23 mm (p < 0.0001). The antimicrobial effects of the synthesized nanoformulations (i.e., Ag/ZnO NC, Neo-Ag/ZnO NC, blank NEG, Ag/ZnO NEG, and Neo-Ag/ZnO NEG) against most of the prevalent MDR wound pathogens, such as P. aeruginosa, S. aureus, and C. albicans, were assessed by DDM (Figure 6a and Table 4) as well as by MIC (Figure 6b) and biofilm inhibition assay (Figure 6c). From DDM, B-NEG (Blank NEG) did not exhibit zones against selected strains. Prominent inhibition zones were observed for Ag/ZnO NC and the Neo-Ag/ZnO NEG, whereas Neo-Ag/ZnO NEG showed the greatest antimicrobial potential against S. aureus, with an inhibition zone of 46 mm compared with other tested strains (p < 0.05) and compared to that of other tested nanoformulations (p < 0.05). The relative efficiency could be ordered as follows: Neo-Ag/ZnO NEG > Neo-Ag/ZnO NC > Ag/ZnO NEG > Ag/ZnO NC ≫ Neo (pure and validated antibiotic – see datasheet suppl. mat. file) ⋙ B-NEG when tested against P. aeruginosa, S. aureus, and C. albicans (Figure 6a and Table 4). F3 manifested superior antimicrobial properties compared to that of the Neo ointment (used as control) and the other mycosynthesized nanoformulations against selected microbial strains; this is most likely due to the successful incorporation and release of Neo, Ag⁺, and Zn²⁺ from NEG and its small (nano) size [36]. Such a nanoformulation could easily interact with the cell wall surface of microorganisms, resulting in DNA disruption, enzyme inactivation, and inhibition of protein synthesis by ROS formation [42]. These findings are in accordance with a recent study that used NEG for the topical delivery of Neo and reported an increase in the efficacy of Neo in terms of antibacterial activity after incorporating it into a tea tree oil-based nanoemulsion [74]. The MIC of Neo-Ag/ZnO NEG was 0.002 µg/mL against MRSA, indicating that this test formulation (F3) was active at low concentrations (Figure 6b). The percentage of MRSA biofilm inhibition by Neo-Ag/ZnO NEG was 22% (Figure 6c and Figure S8). Significant differences (p < 0.0001) were observed in the antibiofilm potential between the positive control (i.e., untreated MRSA cells) and in Neo-Ag/ZnO NEG-treated MRSA cells. The small size and broad-spectrum activity of Neo, Ag, and ZnO led to an excellent biofilm inhibition potential against MRSA at MIC. Biocompatibility studies against human erythrocytes showed that Neo-Ag/ZnO NEG was safe for in vivo application in an animal model following the 3R principles (i.e., Replacement, Reduction, and Refinement) in animal research.

Figure 6

(a) Antimicrobial activity of samples against ((a) P. aeruginosa, (b) S. aureus, and (c) C. albicans). (b) MIC of Neo-Ag/ZnO NEG against MRSA: ((a) agar well diffusion plate and (b) graphical representation). (c) Antibiofilm activity of Neo-Ag/ZnO NEG against MRSA. Positive control: untreated microorganisms. Neo: neomycin; NC: ananocomposite; B-NEG: Blank NEG; NEG: nanoemulgel; Ag/ZnO: silver/zinc oxide.

3.6 In vivo wound healing

Neo-Ag/ZnO NEG had excellent wound-healing activity in mice when compared with the control (untreated wound). Indeed, the WTC achieved by Neo-Ag/ZnO NEG was found to be markedly higher (100%) compared to that of the control (55%) on day 12. Because of skin contraction and fast skin re-epithelialization, WTC was observed from day 4 onward with a concentration as low as 0.01% of 100 mg Neo-Ag/ZnO NEG. Treated mice showed a significant (p = 0.0001) increase in wound contraction either on day 4, day 8, or day 12 in comparison to the control. No itching, irritation, or inflammation was observed. Pictures, graphical representation, and tabulated data representing wound closure on various days are summarized in Figure 7a–c, respectively.

Figure 7

In vivo (in mice) wound-healing potential of Neo-Ag/ZnO NEG over a period of 12 days. (a) Pictures were taken at 4-day interval after treatment with Neo-Ag/ZnO NEG. (b) Graphical representation of the Neo-Ag/ZnO NEG effect on wound area (mm²) at indicated days. Data are presented as the mean ± SEM. N = 12 (****p = 0.0001). (c) Tabulated data. WTC: wound total closure.

3.7 Hypothetical wound infection and healing mechanism

When the skin’s integrity is compromised, whether through surgery or physical trauma, microbes from the skin surface invade the wound site, initiating colonization and releasing toxins, eventually forming a biofilm. This impedes immune cells like macrophages and neutrophils from effectively eliminating the microbes, leading to persistent inflammation and disrupting collagen synthesis [75] as shown in Figure 8a. Oral antibiotics are not recommended as microbes build tolerance against antibiotics [76]. Hence, our synthesized Neo-Ag/ZnO NEG offers a solution by gradually releasing nano-antibiotics from the Carbopol NEG at the wound site as shown Figure 8b, effectively eradicating biofilms and killing microbes due to its broad-spectrum antimicrobial properties by ROS production. Moreover, the NEG prevents microbial entry, mitigates infection, promotes collagen deposition and maintains optimal moisture levels for wound contraction. This facilitates immune cell migration to the wound site with neoangiogenesis and skin cell formation, promoting early wound healing [36]. In a recent research, Majhi et al. stated that Ag/ZnO NPs stimulate the production of antimicrobial peptides and enhance the migration and antibacterial abilities of keratinocytes, which can potentially aid in wound healing and combating bacterial infection [67]. In comparison to prior studies, our research findings align with previous evidence indicating that Ag/ZnO NPs stimulate the production of antimicrobial peptides and enhance the migratory and antibacterial capabilities of skin cells (keratinocytes) against S. aureus. This consistency reinforces the potential therapeutic utility of Ag/ZnO NPs in promoting wound healing and combating bacterial infections. However, it is noteworthy that our study contributes novel insights by specifically highlighting the effectiveness of Carbopol NEG.

Figure 8

Illustration of putative skin wound infection mechanism by microbes (a). Neo-Ag/ZnO NEG-induced wound healing mechanisms (b).