Antimicrobial gel with silver vanadate and silver nanoparticles: antifungal and physicochemical evaluation

Abstract

Aim: To develop a β-AgVO₃ gel and evaluate its physicochemical stability and antifungal activity against Candida albicans.

Materials & methods: The gel was prepared from the minimum inhibitory concentration (MIC) of β-AgVO₃. The physicochemical stability was evaluated by centrifugation, accelerated stability (AS), storage (St), pH, syringability, viscosity and spreadability tests and antifungal activity by the agar diffusion.

Results: The MIC was 62.5 μg/ml. After centrifugation, AS and St gels showed physicochemical stability. Lower viscosity and higher spreadability were observed for the higher β-AgVO₃ concentration and the minimum force for extrusion was similar for all groups. Antifungal effect was observed only for the β-AgVO₃ gel with 20xMIC.

Conclusion: The β-AgVO₃ gel showed physicochemical stability and antifungal activity.

Plain Language Summary

We used silver and vanadium to make a gel that can kill fungi in the mouth. We looked at the color of the gel, it's smell and also checked how well it lasted. The gel turned yellow and had no smell and did not spoil for at least 2 months. When we tested the gel against a type of fungus, it worked as well as another medicine called chlorhexidine, which is sold in pharmacies. But when we compared it with another medicine called nystatin, our gel was not as effective in killing the fungus.

Plain language summary

Article highlights.

• β-AgVO₃ has broad-spectrum antimicrobial activity.

Methods

• Gels with β-AgVO₃ were prepared, and their physicochemical properties were evaluated by AS, St, pH, syringability, viscosity and spreadability tests.

• The antifungal activity of the gels was evaluated by agar diffusion test.

Results

• Gels obtained with β-AgVO₃ have adequate physicochemical characteristics to keep the product viable for an estimated period of 2 months.

• β-AgVO₃ gels have a compatible pH for intraoral application.

• β-AgVO₃ gels show antifungal efficacy against Candida albicans.

Conclusion

• The gel containing 20 x β-AgVO₃ MIC showed similar efficacy to 0.12% chlorhexidine, but less than nystatin.

1. Introduction

Fungal infections in the oral cavity by Candida spp. are reported from birth (~37% of newborns) to old age, and among the most common species, approximately 80% of the strains isolated from patients are Candida albicans [1,2]. This species lives commensally in the oral cavity, and when the patient's health is imbalanced due to poor nutrition, environmental factors, immunosuppression and lack of hygiene, C. albicans can cause local oral infections such as oral candidiasis (pseudomembranous, erythematous, hyperplastic, atrophic), angular cheilitis and denture stomatitis, as well as systemic infections (oropharyngeal infections and sepsis) [1–7]. The various forms of these fungal infections reduce patients' quality of life by causing pain, burning, tenderness, bleeding, taste changes and halitosis [4,6,7].

In addition, C. albicans has yeast-to-hyphae dimorphism, exhibits high adhesion to dental materials, biofilm formation and co-aggregation affinity with Streptococcus spp. and Staphylococcus spp., which increase its virulence and induce more severe local and systemic infections [1–7]. These infections are treated with topical (nystatin and miconazole) and systemic (fluconazole) antifungals [4,8,9], however, these drugs are poorly accepted by patients due to their unpleasant taste, induction of burning, erythema, nausea, vomiting and drug interactions with hepatotoxic and nephrotoxic effects [4,8,9], impaired cognition, memory loss [10–12] and decreased therapeutic action due to fungal resistance [1,2,13–15].

A promising alternative for the control and treatment of oral infections by C. albicans are antimicrobial gels based on silver nanoparticles (AgNPs) [16], which also have a spectrum of activity against Gram-positive and -negative bacteria [16–24] resistant or not to conventional antimicrobials [25,26], and its use at low concentrations (0.005%) does not induce cytotoxic effects against human keratinocytes and fibroblasts [17,27]. However, pure AgNPs have a tendency to agglomerate due to their synthesis method, where Ag⁺ ions attract each other due to electrostatic and van der Waals forces [28,29], which reduces their antimicrobial efficacy and induces toxicity in tissues where they are applied [30].

In order to prevent AgNPs agglomeration and optimize their antimicrobial activity, they can be intercalated on the surface of silver vanadate nanowires to form the nanostructured silver vanadate decorated with silver nanoparticles (β-AgVO₃) with high stability and a synergistic antimicrobial activity mechanism of silver and vanadium [5,31–35]. The β-AgVO₃ has been incorporated into dental materials such as acrylic resin [5,31,32,36–38], irreversible hydrocolloid [39], denture liners [40], ceramics [29,41,42] and endodontic [33,43–46] and orthodontics cements [47] and showed broad-spectrum antimicrobial action.

Gels are used in dentistry to facilitate the delivery of medicines and have advantages including low cost, ease of obtaining, versatility of application, adherence to oral structures, resistance to removal by speech and saliva and controlled and sustained drug release, making gels an important alternative method for controlling pathogenic microorganisms and treating infections [3,16,17]. Factors that affect their use and performance in antimicrobial efficacy include inadequate application, inefficient exposure time, storage and formulation composition, which in turn can make the gel unusable due to its high viscosity that does not allow for spreading in tissues, the release of the antimicrobial agent and an inadequate pH [3,16–24].

The development of gels loaded with β-AgVO₃ shows promise as an alternative for the control, prevention and treatment of C. albicans infections that are resistant or not to conventional drugs due to the synergistic antimicrobial action of silver and vanadium [37,38,44,46], the antibiofilm effect of AgNPs [48], low toxicity against gingival fibroblasts [45] and their prolonged topical action [3]. However, after the development of new gels, they need to be evaluated to ensure their constant physicochemical stability, such as viscosity, pH and other characteristics that certify that the product can be safely applied in the oral cavity [49–51]. The aim of this study was to develop an antimicrobial gel with β-AgVO₃ and evaluate its physicochemical characterization and antifungal activity against C. albicans. In this article, we highlight the innovative use of β-AgVO₃ added to gels to be explored as a possible oral antifungal agent. Conventional drugs cause side effects and drug interactions, and some clinical strains of C. albicans are resistant to these drugs. In addition, β-AgVO₃ disperses AgNPs on the surface of its nanowires and optimizes the antifungal effect at a lower application concentration. This makes this material promising for oral fungal control, with a potential reduction in the cytotoxic effects of silver.

2. Materials & methods

2.1. Solvents & reagents

To synthesize β-AgVO₃, silver nitrate (AgNO₃, 99.8%, Merck KGaA, Germany) and ammonium metavanadate (NH₄VO₃, 99%, Merck KGaA, Germany) were used. To obtain the antimicrobial gel, Carbopol (CP) 940 (Lubrizol, Brazil), glycerin (Buckminster Química, Brazil) and triethanolamine (TEA) (Smart Química, Brazil) were used.

2.2. Experimental

2.2.1. Synthesis of β-AgVO₃

The nanomaterial was synthesized as described in the literature [35] by solubilizing 0.9736 g of NH₄VO₃ in 200 ml of distilled water at 65°C under magnetic stirring (752A, Fisatom, Brazil) for 10 min and 1.3569 g of AgNO₃ under the same conditions. A precipitation reaction was then performed by adding the AgNO₃ solution drop by drop to the NH₄VO₃ solution and stirring for 30 min. After mixing the solutions described above, the β-AgVO₃ solution was formed, and the precipitate obtained was vacuum filtered, washed with distilled water and absolute ethanol and vacuum dried for 10 h to obtain the powder.

2.2.2. Characterization of β-AgVO₃

The characterization was performed by Transmission Electron Microscopy (TEM) (JSM-6610LV, JEOL, Japan) and Fourier Transform Infrared Spectroscopy (FTIR) (FTIR-IRTracer-100, Shimadzu, Japan), where the samples were diluted and processed as tablets using analytical grade potassium bromide (KBr), and subsequently Fourier transform infrared spectra were collected using transmittance in the 4000 – 400 cm^-1 region.

2.3. Preparation of gels

2.3.1. Minimum inhibitory concentration of β-AgVO₃ against C. albicans

The minimum inhibitory concentration (MIC) was determined by the Clinical and Laboratory Standards Institute (CLSI) serial dilution method in duplicate. The C. albicans strain (ATCC 10231) was obtained by recent culture on Sabouraud Dextrose (SD) agar (Kasvi, Brazil), inoculated in SD broth (Kasvi, Brazil) and cultured in a microbiological oven (B2DG, DeLeo, Brazil) at 37°C until exponential growth. The culture was then centrifuged at 6,000 rpm for 5 min, the supernatant was discarded and the pellet was washed twice in phosphate-buffered saline (PBS) containing NaCl, KCl, Na₂HPO₄ and KH₂PO₄. The C. albicans inoculum was standardized by counting the cells in a Neubauer chamber (HBG, Germany) coupled to a light microscope (Axio Observer A1, Carl Zeiss, Germany) due to the genus variable morphology.

A 96-well plate (Kasvi, Brazil) was prepared by inoculating 100 μl of culture medium at a concentration of 1 × 10⁶ CFU/ml. A solution of β-AgVO₃ powder in sterilized distilled water was prepared at a concentration of 1000 μg/ml, and 100 μl was added to the first well (500 μg/ml) and homogenized. Successive dilutions were made by transferring 100 μl of the suspension to the next well so that the concentration of the β-AgVO₃ solution was reduced by half with each dilution. Ten concentrations of β-AgVO₃ were obtained, as well as a positive control (inoculated culture medium and PBS) and a negative control (culture medium and β-AgVO₃ solution). The plate was incubated at 37°C and after 48 h, the microbial growth was assessed by the turbidity of the sample with the naked eye. An aliquot of the plate wells was seeded into a Petri dish to confirm the MIC.

2.3.2. Obtaining the gels

Gels were prepared by mixing 0.5% CP 940 in 88% sterilized distilled water at 70°C under magnetic stirring at 50°C. After homogenization, the antimicrobials were manually mixed with 10% glycerin, loaded onto the gel, and added 0.75% triethanolamine (TEA). The antimicrobials used were β-AgVO₃ (1, 2, 4, 5, 8, 20 x MIC against C. albicans), nystatin (Sigma-Aldrich, USA) and chlorhexidine (Sigma-Aldrich). The following groups were used: no antimicrobial added to gel (G0); 0.0062% β-AgVO₃ added to gel (G1); 0.012% (G2); 0.025% (G4); 0.03% (G5); 0.05% (G8); and 0.12% (G20) for the physicochemical characterization and microbiological tests; and nystatin 100,000 IU (GN) and 0.12% chlorhexidine (GC) for the microbiological test.

2.4. Physicochemical characterization

2.4.1. Centrifugation, accelerated stability & storage

To evaluate the physicochemical stability of the formulations, we used a modification of the guidelines proposed by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) [52]. The aim of this protocol is to initially study the physicochemical behavior of the gel and, if necessary, modify the formulation until we find the right proportions of each component to obtain an effective antimicrobial gel. One centrifugation cycle at 3000 rpm for 30 min (Centrifuge 5430/5430 R, Eppendorf SE, Germany) was performed on 5 g of each group (n = 3).

For AS, 5 g of each group (n = 5) were subjected to six heating and cooling cycles, where one cycle corresponded to exposing the samples to temperatures of 37.5°C for 24 h and 4°C for 24 h. The 5 g from each group (n = 5) were stored for 2 months in polypropylene tubes (JProlab, Brazil) at room temperature with ambient light.

The pH and color characteristics, odor and macroscopic changes of the gels were visually evaluated by a single operator before and after the AS test and during 0, 2, 4, 6 and 8 weeks of St. For this, a pHmeter (PHTEK Ultrabasic; Denver Instrument Company, CO, USA) calibrated with buffer solutions with pH values of 4.0 and 7.0 was used. The appearance, color and odor of the samples were always analyzed in the same location and under the same light source and were classified as unchanged, slightly modified, modified and intensely modified.

2.4.2. Viscosity

The viscosity of the gels (n = 6) was assessed using a rotational viscometer (Brookfield DV-III, Brookfield Engineering Laboratories, USA) calibrated at each sample change using spindle no. 25 at 8 rpm at a temperature of 31° ± 0.3°C.

2.4.3. Spreadability

The spreading diameter of the gels (n = 10) was measured by placing 1 g of each gel between two glass plates and applying a 125 g weight to the upper plate for 1 min. After the weight was applied, the plates were photographed using a professional camera with a 4.8–144 mm lens (ILCE7M3K/B, Sony, Tokyo, Japan). The photographs were exported to ImageJ software (National Institutes of Health, MD, USA) to measure spreadability.

2.4.4. Syringability

Syringability (n = 10) was evaluated using a universal testing machine (DL 2000, EMIC, Brazil) equipped with a 5 Kg/F load cell and a special device for attaching the syringes. Disposable 3 ml plastic syringes (DESCARPAK, Brazil) with an inner diameter of 1.3 cm and a height of 10 cm were used without a needle attached. The gels were transferred into the syringes without the formation of air bubbles at a fixed height of 15 mm. The plunger of the syringes was moved in a compression module at a speed of 1 mm/s, covering a distance of 10 mm and the minimum force to start the plunger movement and allow the gels to extrude was recorded.

2.5. Agar diffusion test against C. albicans

C. albicans strain was obtained, cultivated and standardized as described above in Section 2.3.1. The SD agar culture medium was sterilized and prepared with an inoculum concentration of approximately 1 × 10⁶ CFU/ml. A base layer of 12 ml containing sterilized culture medium was added to Petri dishes (90 mm²), followed by a layer of 8 ml of the inoculated medium. After the culture medium had solidified, 5 mm-diameter wells were drilled perpendicularly and 0.5 mg of the gels (n = 10) were added to the wells. Petri dishes were incubated at 37°C for 24 h to form the inhibition halo and then photographed using a professional camera with a 4.8–144 mm lens. The photographs were exported to ImageJ software, where the diameter of the inhibition halo was measured.

2.6. Statistical analysis

SPSS Statistics 20.0 software (IBM, USA) was used. The Shapiro-Wilk test was applied to verify the normality of the data distribution and the Levene test to verify homoscedasticity. The Kruskal-Wallis and posthoc Dunn tests were used to compare pH between groups, and the Friedman and posthoc Nemenyi tests were used to compare pH at different times in the same group in the AS and St. The one-way ANOVA and posthoc Tukey tests with Bonferroni adjustment were used to compare the groups in the viscosity, spreadability, syringability and agar diffusion tests. A significance level of 5% was used.

3. Results

3.1. Synthesis & characterization of β-AgVO₃

The TEM showed β-AgVO₃ crystals with acicular geometry (needle-shaped), an average diameter of 100 nm, and a length of micrometric order similar to the pattern observed in previous studies (Figure 1) [29,31,32,34–36,38,39,43,44,46].

Figure 1.

Transmission electron microscopy of nanostructured silver vanadate decorated with silver nanoparticles (β-AgVO₃) powder (× 200,000 magnification).

FTIR showed bands located between 400 and 1000 cm^-1 (attributed to the bonds between the oxygen and vanadium atoms), a band at 501 cm^-1 (associated with the V-O vibrational mode), bands in the range of 940 to 775 cm^-1 (indicative of polymeric vanadate groups) and a band located at 1633 cm^-1 (corresponding to the vibration of the -OH bond in the water molecule) [32,38]. Furthermore, there was an intensification of the band around 849 cm^-1, associated with the V-O stretching vibration and the Ag-O-V bond vibration mode (Figure 2). To confirm the formation of β-AgVO₃, the band at 1409 cm^-1 (attributed to symmetrical N-H deformation) present in the spectrum of the NH₄VO₃ precursor disappeared in the spectrum of β-AgVO₃ [32,34,35,38].

Figure 2.

FTIR spectra of ammonium metavanadate and β-AgVO₃.

3.2. Minimum inhibitory concentration of β-AgVO₃ against C. albicans

The minimum inhibitory concentration (MIC) of β-AgVO₃ against C. albicans was 62.5 μg/ml. Inhibition of the fungus was also observed at concentrations of 500, 250 and 125 μg/ml.

3.3. Evaluation of physicochemical characteristics

3.3.1. Centrifugation, accelerated stability & storage

The gels were prepared and showed a yellow color with color intensity proportional to the percentage of β-AgVO₃ added, with the exception of G0, which showed a transparent color. All the gels were odorless, viscous and homogeneous, with dispersed β-AgVO₃ particles.

After centrifugation, a visual observation was made showing the maintenance of the organoleptic characteristics, viscosity and dispersion of the β-AgVO₃ particles, indicating physicochemical stability. After AS, G0 showed physicochemical stability with maintenance of color, appearance and odor. G1, G2, G4, G5, G8 and G20 maintained their odorless characteristics and viscosity; however, there was an intense color change proportional to the percentage of β-AgVO₃ added. The color change was visually characterized as green for G1, G2, G4 and G5 and dark brown for G8 and G20. At the end of the St, G0 showed physicochemical stability with maintenance of color, appearance and odor. G1, G2, G4, G5, G8 and G20 maintained their viscosity and odorless characteristics; however, their color was intensely modified.

Before the AS cycles, all the groups had a similar pH (p = 0.299). After the cycles, there was a decrease in the pH of G2 and G5 (p = 0.043). In addition, after the cycles, there was a decrease in the pH of G1 (p = 0.012), G2 (p = 0.000) and G3 (p = 0.006) compared with G0 and a lower pH for G2 compared with G20 (p = 0.015) (Figure 3).

Figure 3.

pH values of the gels before and after accelerated stability (AS). Kruskal-Wallis and Posthoc Dunn - equal capital letters represent the statistical difference before and after AS. Friedman and posthoc Nemenyi - equal lowercase letters represent statistical difference for the same time (before or after).

Before gel storage, G2 had a lower pH than G20 (p = 0.045). In the second week, G20 had a higher pH than G1 (p = 0.000) and G2 (p = 0.019) and G8 had a higher pH than G1 (p = 0.036). At the fourth week, G20 had a higher pH than G1 (p = 0.000) and G0 (p = 0.005). In the sixth and eighth weeks, G20 had a higher pH than G1 (p = 0.000). At the end of storage, there was a decrease in pH for G0 (p = 0.027), G2 (p = 0.014), G4 (p = 0.001), G5 (p = 0.014), G8 (p = 0.001) and G20 (p = 0.001). During the storage test, a decrease in pH was observed between the second and eighth week for G4 (p = 0.027), G8 (p = 0.027) and G20 (p = 0.0) (Table 1).

Table 1.

Hydrogen potential (pH) of gels with nanostructured silver vanadate decorated with silver nanoparticles (β-AgVO₃) throughout the storage in weeks.

Groups	T0	T2	T4	T6	T8
G0	7.21 (7.03–7.38)a	6.61 (6.43–6.80)	6.42 (6.19–6.66)A	6.57 (6.28–6.74)	6.58 (6.14–6.72)a
G1	7.20 (7.01–7.36)	6.30 (6.18–6.39)B.C	6.17 (6.14–6.24)B	6.27 (6.20–6.33)A	6.20 (5.84–6.38)A
G2	6.90 (6.83–6.98)A.a	6.46 (6.23–6.75)A.b	6.70 (6.57–6.84)	6.63 (6.49–6.78)	6.59 (6.24–6.73)a.b
G4	6.98 (6.92–7.06)	6.78 (6.53–7.03)	6.74 (6.49–6.86)	6.61 (6.51–6.72)	6.45 (6.34–6.56)
G5	7.10 (7.02–7.26)	6.60 (6.54–6.65)	6.72 (6.53–6.89)	6.61 (6.41–6.71)	6.53 (6.36–6.70)
G8	6.81 (6.72–7.13)a	6.79 (6.72–6.83)C.b	6.69 (6.68–6.72)	6.59 (6.53–6.70)	6.53 (6.49–6.58)a.b
G20	7.26 (7.21–7.35)A.a	7.19 (7.13–7.21)A.B.b	7.05 (7.00–7.08)A.B	6.93 (6.89–7.22)A	6.80 (6.72–6.84)A.a.b

Median CI. Kruskal-Wallis and posthoc Dunn (p < 0.05). ^A.B.C.D Equal capital letters represent statistical difference between rows in the same column. Wilcoxon (p < 0.05). ^a.b.c.d Equal lowercase letters represent statistical difference between the rows of the two columns.

3.3.2. Viscosity

G0, G1 and G4 had higher viscosity compared with the other groups (p < 0.05). G2 showed similar viscosity to G0 and G4 (p > 0.05). Lower viscosity values were observed for G8 and G20 compared with the other groups (p < 0.05) (Figure 4).

Figure 4.

Viscosity values (cPs) of the gels containing β-AgVO₃. The one-way ANOVA and posthoc Tukey with Bonferroni adjustment - equal capital letters represent the statistical difference between groups.

3.3.3. Spreadability

A higher spreadability diameter was observed for G8 and G20 compared with G0 (p = 0.003), G1 (p = 0.010), G2 (p = 0.000) and G5 (p = 0.000). G4 had a higher spreadability diameter compared with G2 (p = 0.001) (Figure 5).

Figure 5.

Diameter of spreadability (mm) of gels containing β-AgVO₃. The one-way ANOVA and posthoc Tukey with Bonferroni adjustment - equal capital letters represent the statistical difference between groups.

3.3.4. Syringability

The minimum force (gF) to extrude the gels from the syringes was similar for all groups (p = 0.379) (Figure 6). The gels were extruded continuously and showed no phase separation of the chemical components.

Figure 6.

Minimum force values (gF) to start the extrusion of gels containing β-AgVO₃. The one-way ANOVA and posthoc Tukey with Bonferroni adjustment - equal capital letters represent statistical similarity.

3.4. Agar diffusion test against C. albicans

Of the gels containing β-AgVO₃, only G20 showed antimicrobial activity by forming an inhibition halo against C. albicans, with a similar effect to GC (p = 0.121). GN showed a higher inhibition halo compared with G20 and GC (p < 0.05) (Figure 7).

Figure 7.

Inhibition halo values (mm) of gels containing β-AgVO₃ against Candida albicans. The one-way ANOVA and posthoc Tukey with Bonferroni adjustment - equal capital letters represent the statistical difference between groups.

4. Discussion

β-AgVO₃ is an antimicrobial semiconductor with the synergistic activity of Ag and V [5,29–33,36–47,53–57]. Its oxidation and release of Ag+, V4+ and V5+ ions form ROS that cause the death of microorganisms by altering their plasma membrane, proteins, microbial DNA, nutrition and metabolism [29,36,38,39,43,44]. To ensure that the gel proposed in this study had properties such as physicochemical stability and viscosity that would allow it to form long-lasting antifungal thin films, CP 940 was used as a thickener [3–5,58–60].

The centrifugation, AS and St analyses were applied to evaluate the physicochemical stability of the β-AgVO₃ gels and the results showed that it was maintained even with different ratios of the nanomaterial, suggesting its resistance to degradation and safety for oral use [58,59]. Our results corroborate those of other authors who also reported the stability of formulations containing CP and AgNPs after centrifugation [19], AS [18,19,22] and St for 2 [19] to 4 months at 40°C [16,22], showing that gels modified with β-AgVO₃ are promising for the effective delivery of the antimicrobial without compromising its therapeutic properties over time.

The neutral pH of the studied formulations was observed immediately after preparation and after AS and St for 8 weeks, it showed values from 6.2 to 7.6. TEA was used to neutralize the pH of the CP gels, which also helps to maintain their viscosity [58,60–63]. This is due to the acid-base reaction between the trihydroxyethylamine groups of TEA and the carboxyl groups of CP, resulting in the formation of a quaternary ammonium salt that increases the pH and viscosity of the gel [58,60–63]. Other authors who have studied the incorporation of antimicrobials into gels have shown that the addition of TEA ensures the shelf life of the product and allows for safe oral administration [58,60–64].

Bustamante-Torres et al. [58] showed that the decrease in pH during St is due to the continuous release of H+ ions by CP into the aqueous medium, which can be controlled by adding a higher amount of TEA [65]. Similarly, in our study, a decrease in the pH of β-AgVO₃ gels was observed; however, it presented values compatible for application in the oral cavity [66,67], which is consistent with that observed in other formulations of CP, AgNPs, and TEA freshly prepared [16,18,19,22,24], after AS [19] and St [16,19,22]. In addition, the pH values obtained in this study allow them to be balanced with the pHs of the oral cavity and saliva, which may prevent mucosal inflammation, exacerbation of lesions, demineralization of dental tissues and microbial proliferation [66,67].

The results of organoleptic analysis showed an intense color change in the β-AgVO₃ gels due to their oxidation by interaction with oxygen and ultraviolet (UV) light as a result of non-airtight storage during the study period [34,35,68,69]. Sunita et al. reported that CP and AgNPs gels maintained their initial color for 4 months when stored airtight in a light-free environment [16]. Xu et al. [68] and Shahzadi et al. [69] reported that the formation of oxides and sulfides of Ag from its oxidation can interfere with its therapeutic effect by reducing the amount of Ag+; however, antioxidants can be added to the formulation to prevent this.

The addition of AgNPs to gels affects their viscosity, and a similar behavior occurred with our product, since the addition of β-AgVO₃ at a lower concentration increased its viscosity by acting as a charge particle, while a higher concentration made it more fluid due to the binding of Ag to the -COOH of the CP, breaking its polymeric bonds [16,18,59]. Despite this reduction, β-AgVO₃ gels are still characterized as having a high viscosity (26538 - 48207 cP), which corroborates the literature [16,18,19,22–24] and these values allow them to adhere to tissues and release the antimicrobial efficiently [70–74].

This study showed a decrease in gel rheology with a higher concentration of β-AgVO₃ (from 43860.50 ± 7256.70 to 26538.17 ± 1566.38 cP), providing higher spreading, uniformity and prolonged contact in the tissues, which contributes to its effective activity and confirms that reported by Silva et al. [59]. The syringability test evaluated the extrusion behavior of the gel from a syringe under compression forces ranging from 520.83 ± 91.08 to 615.97 ± 87.03 gF, as well as the homogeneity of the extrusion, which contributes to dosing precision, waste prevention and under- and overdosing [70–73]. In addition, we observed that changing the viscosity of the groups did not affect the force required for their extrusion, which in turn was considered low due to the use of syringes without an attached needle, as described in the literature [70–73].

In this study, 0.12% chlorhexidine was used to compare the effect of β-AgVO₃ because it is a fungistatic agent against C. albicans, can be applied in the oral cavity, and remains effective for 12 h [75,76]. The β-AgVO₃ gel showed antifungal results similar to those of chlorhexidine, suggesting its use as an alternative for this purpose. The superiority of the β-AgVO₃ gel is believed to be due to the fact that the nanomaterial showed low toxicity against VERO cells from the epithelium of African monkeys in the study by Alvim et al. [77]. Higher efficacy was observed for nystatin, but gels with higher concentrations of β-AgVO₃ could be studied, as fungi are sensitive to the ROS generated by AgNPs [4,6,8].

The evaluated formulations showed suitable properties for use in the oral cavity, such as physicochemical stability and neutral pH after centrifugation, AS and St, viscosity, spreadability and syringability, which allow easy administration, spreading and efficient release of the antimicrobial agent, confirming findings in the literature [16,18,19,22,24,70–74]. In addition, evaluation of its cytotoxic potential as well as clinical studies could determine the safety, feasibility and dosage of its use as an antifungal.

5. Conclusion

It was concluded that the freshly prepared β-AgVO₃ gels showed physicochemical characteristics such as a yellowish color proportional to the concentration of nanomaterial added, pH compatible for oral application, high viscosity and the ability to spread and extrude from the inside of syringes. After 8 weeks of AS and St, the gels showed a change in color and a decrease in pH. The addition of higher concentrations of β-AgVO₃ reduced the viscosity of the gels and increased their spreadability. The 0.12% β-AgVO₃ gel showed an antifungal effect similar to chlorhexidine 0.12%, and the highest antifungal effect was observed for 100,000 IU nystatin.

It is suggested that the physicochemical stability of these gels be evaluated for periods of 3–12 months in accordance with international ICH guidelines to determine their shelf life as well as to verify the maintenance of the product's antifungal activity after this period. In addition, the evaluation of the antimicrobial activity of these gels against multi-species biofilms and their biocompatibility will allow us to understand their efficacy in the treatment of infections and ensure their safety for application to oral tissues.

Funding Statement

This work was supported by the National Council for Scientific and Technological Development (CNPq) (processes 131495/2022-4 and 314132/2021-0).

Financial disclosure

This work was supported by the National Council for Scientific and Technological Development (CNPq) (processes 131495/2022-4 and 314132/2021-0). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.