ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10015-2311
World Journal of Dentistry
Volume 14 | Issue 10 | Year 2023

Efficacy of Chemically and Biologically Synthesized Zinc Oxide Nanoparticles Incorporated in Soft Denture Liner against Candida albicans: A Comparative In Vitro Study

Laura APM Albuquerque¹, Sunila Sangappa², Asha Srinivasan³, Ann Archer⁴, Raghavendra Rao⁵

^1,2Department of Prosthodontics and Crown & Bridge, JSS Dental College & Hospital, JSS Academy of Higher Education & Research (Deemed to be University), Mysuru, Karnataka, India

³Department of Nanoscience, Faculty of Life Sciences, JSS Academy of Higher Education & Research (Deemed to be University), Mysuru, Karnataka, India

⁴Department of Microbiology, Faculty of Life Sciences, JSS Academy of Higher Education & Research (Deemed to be University), Mysuru, Karnataka, India

⁵Department of Microbiology, JSS Medical College, JSS Academy of Higher Education & Research (Deemed to be University), Mysuru, Karnataka, India

Corresponding Author: Sunila Sangappa, Department of Prosthodontics and Crown & Bridge, JSS Dental College & Hospital, JSS Academy of Higher Education & Research (Deemed to be University), Mysuru, Karnataka, India, Phone: +91 9591613824, e-mail: drsunilasangappa@gmail.com

Received on: 03 September 2023; Accepted on: 01 October 2023; Published on: 06 November 2023

ABSTRACT

Aim: The present study aimed to comparatively evaluate the efficacy of varying concentrations of chemically synthesized zinc oxide nanoparticles (CSZnO-NPs) and biologically synthesized ZnO-NPs (BSZnO-NPs) incorporated separately in soft denture liners against Candida albicans (C. albicans).

Materials and methods: The sample size for this experimental comparative in vitro study was 120. ZnO-NPs were biologically synthesized from Azadirachta indica (A. indica) (neem) leaves and characterized by ultraviolet visible (UV-vis), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray powder diffraction (XRD). CSZnO-NPs were procured from Sigma Aldrich. CSZnO-NPs and BSZnO-NPs were then incorporated separately into soft liner in concentrations of 0 (control), 3, 5, 7, 9, and 11 wt% for the purpose of comparison. The disk diffusion method was used to evaluate antifungal activity and the zones of inhibition (ZOI) were measured. Data was compiled and analyzed using the Statistical Package for the Social Sciences (SPSS) software version 20. Inferential statistics used were analysis of variance (ANOVA) and independent t-test. A p-value of <0.05 was considered significant.

Results: Characterized BSZnO-NPs subjected to SEM showed particles of <100 nm in size with spherical morphology, UV-vis spectroscopy showed the absorption peak at a wavelength of 220 nm, XRD showed a good crystalline structure formation, and FTIR showed the presence of functional groups. For antifungal evaluation, the mean ZOI were measured, and an ANOVA test was performed, which showed a significant difference (p < 0.0001) between the concentrations of CSZnO-NPs and BSZnO-NPs incorporated in soft liners, respectively. Least significant difference (LSD) post hoc analysis showed a significant difference (p < 0.001) between the various concentrations, with the most significant difference seen at 7 wt% of CSZnO-NPs and 9 wt% of BSZnO-NPs. On performing an independent t-test, it was inferred that 3, 5, 7, and 11 wt% incorporation of CSZnO-NPs showed significantly higher ZOI against C. albicans as compared to the respective concentrations of BSZnO-NPs. At 9 wt%, there was no significant difference between the two groups.

Conclusion: In conclusion, the optimal antifungal concentration of CSZnO-NPs and BSZnO-NPs is 7 and 9 wt%, respectively, when incorporated into the soft liner. Overall, CSZnO-NPs proved to be more efficacious than BSZnO-NPs against C. albicans.

Clinical significance: Incorporation of optimal concentrations of CSZnO-NPs or BSZnO-NPs into soft denture liner is efficacious in combating the incidence of denture stomatitis (DS). Valuing the advantages of green synthesis, BSZnO-NPs can also be recognized for their antifungal efficacy.

How to cite this article: Albuquerque LA, Sangappa S, Srinivasan A, et al. Efficacy of Chemically and Biologically Synthesized Zinc Oxide Nanoparticles Incorporated in Soft Denture Liner against Candida albicans: A Comparative In Vitro Study. World J Dent 2023;14(10):851–859.

Source of support: Nil

Conflict of interest: None

Keywords: Azadirachta indica (Neem), Candida albicans, Denture stomatitis, Soft liner, Zinc oxide nanoparticles

INTRODUCTION

Edentulism is the absence of one or more natural teeth, mainly caused by dental caries and periodontal disease. This condition undermines the quality of life of an individual as it results in difficulties in mastication, articulation of speech, and poor facial esthetics.¹ Due to the above challenges, completely edentulous people seek artificial replacement of missing teeth. One of the most commonly used treatment options for such patients is a removable denture owing to its relatively low cost, which gives adequate aesthetics and restores function. Candida-associated denture stomatitis (DS) is a common occurrence in denture wearers. An international study stated that 66.7% of denture wearers exhibited growth of oral Candida albicans (C. albicans) as compared to those without dentures,² while a regional study in Karnataka revealed that 48% of denture wearers exhibited growth of C. albicans.³ This is due to a combination of factors ranging from yeast entrapment on the denture base and denture relining material to several systemic influences.⁴ Additionally, intraorally, denture usage creates an environment characterized by an anaerobic environment and acidic pH suitable for C. albicans growth facilitated by reduced salivary flow under the intimately adapted denture base.⁵ A predominant systemic influence is diabetes mellitus. These diabetics suffer from oral candidiasis more frequently than healthy individuals, and if they are denture wearers, the risk increases.⁶ Globally, in 2019, 9.3% of the adult population was estimated to be living with diabetes; this is expected to show a steady increase to 10.2% in 2030 and 10.9% in 2045.⁷ In Karnataka, the overall prevalence was found to be 16%.⁸ DS is observed in approximately 58% of denture wearers with diabetes mellitus as compared to any other systemic disease.⁹ The increased rate of progression of alveolar bone loss, as well as the enhanced harmful effects of mechanical irritation from the denture, will lead to a greater need for relining of dentures in such patients. Soft liners are more prone to microbial adhesion than acrylic resin denture base materials as their surface texture is conducive to interaction with oral microbes, efficient mechanical cleansing is difficult, and chemical solutions affect their physical properties.¹⁰^-¹²

The most common mode of treatment of DS is the application of topical antifungals, which not only have an unpleasant taste but are also not as effective as the required dosage of medication is not sustainable due to washing and ingestion of antifungal agents by saliva. Some side effects of antifungals include abdominal pain, diarrhea, flatulence, and, in rare cases, liver damage.¹³^,¹⁴ Hence, the incorporation of nanoparticles (NPs) that have antifungal activity within the denture soft liner could effectively combat the above-mentioned drawbacks by preventing colonization of C. albicans and, thus, DS. Prior studies conducted showed that copper oxide NPs¹⁵, titanium NPs,¹⁶ and silver and zinc oxide (ZnO) NPs¹⁷ showed anti-Candida efficacy. The biological synthesis of NPs are gaining importance over the chemical method of synthesis due to its simple methodological procedure and the use of nontoxic, environmentally friendly chemicals. ZnO-NPs, in particular, are known for their antifungal and photocatalytic properties due to the photon-induced generation of reactive oxygen species and the release of zinc ions resulting in disruption of cell membrane integrity, thus establishing its key role in antifungal activity. It is evidenced in the literature that ZnO-NPs can be valued for their minimum effects on human and animal cells.¹⁸ Several researchers have synthesized metal NPs from various plants using the biological method.¹⁹Azadirachta indica (A. indica), native to India, has a wide range of medicinal properties due to its vast array of biologically active compounds.²⁰ Extraction of ZnO-NPs from it is a cost-effective and eco-friendly method, and hence, it was chosen to biologically synthesize ZnO NPs from A. indica. Since colonization of C. albicans occurs more often in soft lining materials than acrylic resin denture base materials, the incorporation of an antifungal agent in soft lining material¹⁵^-¹⁷ will be able to decrease the colonization of C. albicans as well as combat the drawbacks faced with the current allopathic antifungal treatments.²¹ Therefore, the purpose of this in vitro study was to compare the efficacy of various concentrations of chemically and biologically synthesized ZnO NPs (BSZnO-NPs) incorporated in soft denture liners against C. albicans.

MATERIALS AND METHODS

An experimental in vitro study was performed to comparatively evaluate the efficacy of various concentrations of chemically synthesized ZnO NPs (CSZnO-NPs) and BSZnO-NPs incorporated in soft denture liner against C. albicans.

Sample Size Estimation

The sample size in this comparative in vitro study was 120 and was computed to be 60 per group at an assumed mean difference of 0.2 at 1% α-error and 95% power for an effect size of 0.8. The statistical methods were carried out with Statistical Package for the Social Sciences (SPSS) version 20.

Flowchart 1 for the methodology followed is provided below for easy understanding.

Flowchart 1: Methodology

Step 1: Procurement of Characterized CSZnO-NPs

These ZnONPs were white in appearance in powder form with an average particle size of 71 nm procured from Sigma Aldrich.

Step 2: Biological Synthesis and Characterization of ZnO-NPs from A. Indica Leaves

Step 2A: Biological synthesis.

Obtaining A. indica extract: The A. indica leaves were plucked from the A. indica tree in the month of October 2022 as guided by a botanical expert and cleansed with distilled water, dried under natural sunlight, and crushed with a mortar and pestle. A total of 10 gm of dried crushed leaves were added to 100 mL of distilled water (1:10) and boiled for 30–45 minutes with continuous stirring. This was then filtered using a Whatman No.1 filter paper (Cytiva, diameter 125 mm) to get 10% extract. This leaf extract was then refrigerated.²²^,²⁵
Obtaining ZnO-NPs from A. indica extract: A total of 10 mL of 10% neem extract was placed in a hot water bath. At 60°C, 1 gm of zinc nitrate (Sisco Research Laboratories Pvt. Ltd.) was added and stirred constantly using a magnetic stirrer till it reached the consistency of a paste. This was then heated in a silica crucible at 400°C for 2 hours in a muffle furnace and then cooled. The material obtained postcalcination was finely powdered in a mortar and pestle.²²

Step 2B: Characterization of BSZnO-NPs.

Scanning electron microscopy (SEM): It is a method used to gain surface topological information on nanomaterials. This experiment was conducted at an accelerating voltage of 10 kV. The slide was coated with gold, and SEM images were taken (Fig. 1).
Ultraviolet-visible spectroscopy (UV–vis). The optical properties of the BSZnO-NPs were evaluated using a double-beam UV-vis spectrophotometer in the range of 290–400 nm.
X-ray powder diffraction (XRD): This analytical technique was used for phase identification of the BSZnO-NPs (Fig. 2).
Fourier transform infrared spectroscopy (FTIR): The sample was prepared by the KBr pellet method in the range of 400–4000 cm⁻¹ using a Thomas Scientific Spectrometer for information on functional groups present.

Fig. 1: Scanning electron micrographs of BSZnO-NPs within 100 nm

Fig. 2: X-ray powder diffraction (XRD) spectra of BSZnO-NPs showing crystalline structure formation

Step 3: Incorporation of Various Concentrations of Zno-NPs in Soft Relining Material

A total of 120 samples were made. About 60 samples with soft liner incorporated with CSZnO-NPs (group I) and 60 samples with soft liner incorporated with BSZnO-NPs (group II). In group I, 10 samples each were incorporated with CSZnO-NPs in concentrations of 0 (baseline control), 3, 5, 7, 9, and 11 wt%, respectively. In group II, 10 samples each were incorporated with BSZnO-NPs in concentrations of 0 (baseline control), 3, 5, 7, 9, and 11 wt%, respectively, as depicted in Table 1. The soft liner used in this study was GC Soft-Liner (GC Soft Liner, GC India Dental). A probe sonication apparatus was used for 3 minutes to avoid clumping.¹⁶ This mixture was then cooled by placing it in a water bath so as to prevent bulk heating of the liquid during sonication. The polymer was added, and the mixture was placed into a prefabricated metal mold to get uniform discs of the desired size (5 × 1 mm). Soft liner discs without NPs served as the control samples. All samples were then sterilized on both sides with UV application for 2 minutes.

**Table 1:** Incorporation of CSZnO-NPs and BSZnO-NPs into soft liner
Weight% of ZnO-NPs added to soft liner	Recommended weight of polymer (gm)	Recommended weight of monomer (gm)	Group I No. of samples with chemically synthesized ZnO-NP incorporated in the soft liner (n)	Group II No. of samples with biologically synthesized ZnO-NP incorporated in the soft liner (n)
0 (control)	2.2 gm	1.8 gm	10	10
3			10	10
5			10	10
7			10	10
9			10	10
11			10	10

Step 4: Analysis for Evaluation of Antifungal Sensitivity by Disk Diffusion Method

Disk diffusion was carried out according to CLSI guidelines. The culture medium used for this test was Sabouraud dextrose agar (HiMedia Laboratories, Mumbai, Maharashtra, India), which was prepared as per the manufacturer’s instructions. C. albicans strain was procured from the American Type Culture Collection (ATCC 14053). The colonies were suspended in 5 mL of sterile 0.85% saline, and the turbidity was adjusted to yield 1 × 10⁵–1 × 10⁶ cells/mL (0.5 McFarland standard).

Sterile swabs were dipped into the C. albicans inoculum suspension, and excess fluid was pressed out. The surface of 20 agar plates were swabbed in three directions to ensure even growth of C. albicans and left for about 5 minutes to air dry. A total of 20 such agar plates were inoculated, and disks were placed as follows: 10 plates each contained one control disc and discs incorporated with 3, 5, 7, 9, and 11 wt% CSZnO-NPs. The other 10 plates each contained one control disk and disks incorporated with the same concentrations of BSZnO-NPs. The plates were then kept at room temperature for 120 minutes for diffusion and subsequently incubated aerobically for 24 hours at 37°C. A digital caliper was used to measure the zones of inhibition (ZOI), if any.

Statistical Analysis

After collating the data, observational descriptive statistics were applied to describe and summarize the data.

For intragroup comparison of BSZnO-NPs and CSZnO-NPs against baseline control samples, an analysis of variance (ANOVA) test was used, after which a post hoc analysis was performed.
For intergroup comparison of various concentrations of CSZnO-NPs vs BSZnO-NPs, an independent t-test was performed.

RESULTS

The study samples were fabricated according to the described methodology. The results of this experimental in vitro study, which was undertaken to compare the efficacy of CSZnO-NPs and BSZnO-NPs incorporated in soft denture liners against C. albicans, are as follows.

Results of Characterization of BSZnO-NPs

Results of SEM for surface topography: The results of the SEM study on BSZnO-NPs extract showed and substantiated the formation of NPs of approximately spherical morphology with irregular rough surfaces. The aspect ratio confirmed that the particles were <100 nm in size.
Results of UV-vis for optical properties: The spectrum revealed an absorption peak (λ_max) of ZnO at a wavelength of 220 nm. This denotes the intrinsic band-gap absorption of ZnO caused by electron transitions from the valence band to the conduction band.²⁵
Results of XRD for phase identification: The XRD patterns of the BSZnO-NPs showed definite line broadening of the X-ray diffracted peaks, suggesting that the prepared particles were in the nanoscale range. Further, the distinct peaks obtained for zinc and oxygen atoms represent the formation of ZnO-NPs. It is also seen that with an increase in the annealing temperatures, the diffracted peaks became narrower with higher intensity, indicative of good crystalline structure formation. A high crystallinity of ZnO was shown due to the presence of strong and narrow diffraction peaks in the XRD pattern. This showed conformity with the XRD of the Joint Committee on Powder Diffraction Standards card No. 36-1451.
Results of FTIR for the presence of functional groups: FTIR was performed in order to study and determine the functional groups of synthesized ZnO-NPs. FTIR spectrum analysis showed a broad absorption band was observed at 575 cm^–1 that is attributed to Zn-O stretching vibration. In some synthesis methods, a ZnO-NPs stretch is seen at around 400 cm^–1. However, given the method that was used in this study, a minor shift was expected in biologically synthesized NPs. A band observed at 1440.00 corresponds to (NH) C=O stretch and C-H stretch between 1710.02 and 2320.23 cm^–1

Comparison of Antifungal Efficacy of Various Concentrations of CSZnO-NPs Incorporated in Soft Denture Liner against Baseline Control Samples

The mean ZOI for soft denture liner with CSZnO-NPs incorporated were analyzed using descriptive statistics and found to be 8.1 mm for 3 wt%, 9.88 mm for 5 wt%, 13.96 mm for 7 wt%, 11.26 mm for 9 wt%, and 12.75 mm for 11 wt%. The baseline control (0 wt%) showed no ZOI. The above-mentioned ZOI are depicted in Figure 3.
The intragroup comparison showed that the most optimal concentration for inhibition of C. albicans was at 7 wt% incorporation of CSZnO-NPs in soft liner, as depicted in Table 2 and Figure 4. The ANOVA test applied showed that the difference in mean ZOI between the concentrations were statistically significant (p < 0.001) (Table 3). The least significant difference (LSD) post hoc analysis showed a significant difference between all concentrations, with the most significant difference seen at 7 wt% as the mean value was the highest (Table 4).

**Table 2:** Mean ZOI with the addition of 3, 5, 7, 9, and 11 wt% of CSZnO-NPs incorporated in soft denture liner
Weight%	Mean ZOI of 10 samples in mm	Standard deviation	Standard error	Minimum	Maximum
3	8.1000	0.29059	0.09189	7.70	8.60
5	9.8800	0.26162	0.08273	9.50	10.30
7	13.9600	0.29515	0.09333	13.40	14.40
9	11.2600	0.22706	0.07180	10.90	11.50
11	12.7500	0.26352	0.08333	12.30	13.10

**Table 3:** Analysis of variance (ANOVA) test for mean ZOI with addition of CSZnO-NPs incorporated in soft denture liner
Groups	Degree of freedom (df)	Mean square	F	Significance
Between the concentrations	4	53.439	740.152	0.0001
Within the concentrations	45	0.072
	49

**Table 4:** Least significant difference (LSD) *post hoc* analysis for mean ZOI with the addition of CSZnO-NPs incorporated in soft denture liner
Concentrations		Mean difference	Standard error	Significance	95% confidence interval
Concentrations		Mean difference	Standard error	Significance	Lower bound	Upper bound
3 wt%	5 wt%	−1.78000^*	0.12017	0.0001	−2.0220	−1.5380
	7 wt%	−5.86000^*	0.12017	0.0001	−6.1020	−5.6180
	9 wt%	−3.16000^*	0.12017	0.0001	−3.4020	−2.9180
	11 wt%	−4.65000^*	0.12017	0.0001	−4.8920	−4.4080

Figs 3A and B: Disk diffusion assay showing maximum zone of inhibition of (A) 7% for CSZnO-NPs; and (B) 9% for BSZnO-NPs

Fig. 4: Mean ZOI in mm for soft liner disks incorporated with CSZnO-NPs in various concentrations against C. albicans

Comparison of Antifungal Efficacy of Various Concentrations of BSZnO-NPs Incorporated in Soft Denture Liner against Baseline Control Samples

The mean ZOI for soft denture liner with BSZnO-NPs incorporated were 6.84 mm for 3 wt%, 7.46 mm for 5 wt%, 7.93 mm for 7 wt%, 11.08 mm for 9 wt%, and 8.98 mm for 11 wt%. The baseline control (0 wt%) showed no ZOI. The above-mentioned ZOI are depicted in Figure 3B.
Intragroup comparison showed that the most optimal concentration for inhibition of C. albicans was at 9 wt% incorporation of BSZnO-NPs in soft liner as depicted in Table 5 and Figure 5. The ANOVA test applied showed that the difference in mean ZOI between the concentrations was statistically significant (p < 0.001) (Table 6). LSD post hoc analysis showed a significant difference between all groups; the most significant difference was seen at 9 wt% as the mean value was the highest (Table 7).

**Table 5:** Mean ZOI with the addition of 3, 5, 7, 9, and 11 wt% of BSZnO-NPs incorporated in soft denture liner
Weight%	Mean ZOI of 10 samples in mm	Standard deviation	Standard error	Minimum	Maximum
3%	6.8400	0.15776	0.04989	6.60	7.10
5%	7.4600	0.11738	0.03712	7.30	7.60
7%	7.9300	0.18288	0.05783	7.60	8.20
9%	11.080	0.24404	0.07717	10.70	11.40
11%	8.9800	0.22998	0.07272	8.60	9.30

**Table 6:** Analysis of variance (ANOVA) test for mean ZOI with addition of BSZnO-NPs incorporated in soft denture liner
Groups	df	Mean square	F	Significance
Between the concentrations	4	27.600	747.748	0.0001
Within the concentrations	45	0.037
	49

**Table 7:** Least significant difference (LSD) *post hoc* analysis for mean ZOI with the addition of BSZnO-NPs incorporated in soft denture liner
Concentrations		Mean difference	Standard error	Significance	95% confidence interval
Concentrations		Mean difference	Standard error	Significance	Lower bound	Upper bound
3 wt%	5 wt%	−0.62000^*	0.08592	0.0001	−0.7931	−0.4469
	7 wt%	−1.09000^*	0.08592	0.0001	−1.2631	−0.9169
	9 wt%	−4.24000^*	0.08592	0.0001	−4.4131	−4.0669
	11 wt%	−2.14000^*	0.08592	0.0001	−2.3131	−1.9669

Fig. 5: Mean ZOI in mm for soft liner disks incorporated with BSZnO-NPs in various concentrations against C. albicans

Comparison of Antifungal Efficacy of Various Concentrations of CSZnO-NPs vs BSZnO-NPs Incorporated in Soft Denture Liner

Inferential statistics used for intergroup comparison were independent t-tests, which showed that incorporation of 3, 5, 7, and 11 wt% of CSZnO-NPs showed to be significantly more efficacious than the same concentrations of BSZnO-NPs against C. albicans. At 9 wt%, there was no significant difference between the two groups, even though the mean ZOI was greater with the addition of CSZnO-NPs (Table 8) (Fig. 6).

**Table 8:** Independent samples t-test for 3, 5, 7, 9, and 11 wt% of CSZnO-NPs and BSZnO-NPs
Concentration	Mean	Standard deviation	Standard error mean	t	Significance (two-tailed)
3 wt% (CSZnO-NPs)	8.1000	0.29059	0.09189	−12.050	0.0001
3 wt% (BSZnO-NPs)	6.8400	0.15776	0.04989	−12.050	0.0001
5 wt% (CSZnO-NPs)	9.8800	0.26162	0.08273	−26.688	0.0001
5 wt% (BSZnO-NPs)	7.4600	0.11738	0.03712	−26.688	0.0001
7 wt% (CSZnO-NPs)	13.9600	0.29515	0.09333	−54.919	0.0001
7 wt% (BSZnO-NPs)	7.9300	0.18288	0.05783	−54.919	0.0001
9 wt% (CSZnO-NPs)	11.2600	0.22706	0.07180	−1.708	0.105
9 wt% (BSZnO-NPs)	11.0800	0.24404	0.07717	−1.708	0.105
11 wt% (CSZnO-NPs)	12.7500	0.26352	0.08333	−34.085	0.0001
11 wt% (BSZnO-NPs)	8.9800	0.22998	0.07272	−34.085	0.0001

Fig. 6: Comparison of mean ZOI in mm of CSZnO-NPs vs BSZnO-NPs

DISCUSSION

Soft denture liners are widely used to distribute functional loads homogeneously on the denture-bearing tissues in cases of irregular bone resorption, bony undercuts, and abused tissue.²⁶ These materials are more prone to microbial adhesion, such as C. albicans, as the surface texture of soft liner favors interaction with oral microbes, and efficient mechanical and chemical cleansing is difficult.¹⁰^-¹² Hence, incorporation of an antifungal agent in soft lining material could decrease the colonization of oral C. albicans. ZnO-NPs were chosen over other metal NPs owing to their superior antifungal properties due to ion-shedding ability, photon-induced generation of reactive oxygen species, and release of zinc ions.²⁷^,²⁸ The conventional chemical method of synthesizing NPs uses toxic chemicals that are of serious concern to the environment, and hence, the biological method is gaining importance due to its simple methodological procedures and usage of nontoxic, environmentally friendly chemicals.²⁹^,³⁰ Although the value of antifungal efficacy of ZnO-NPs has been evidenced in the literature, this in vitro study aimed to compare the efficacy of various concentrations of CSZnO-NPs vs those of BSZnO-NPs incorporated in soft denture liner against C. albicans.

This study ascertained the sensitivity of various concentrations of CSZnO-NP and BSZnO-NPs against C. albicans using a disc diffusion assay as a preliminary step for future research toward the calculation of minimum inhibitory concentration.

In a previous study conducted by Homsiang³¹ where ZnO-NPs were incorporated into tissue conditioner, it was reported that 15 wt% ZnO-NPs provided the most optimum antifungal effect against C. albicans and another study conducted by Mousavi¹⁷ showed that the best growth inhibition occurred at 5 wt% concentration. A pilot study conducted by the authors for the current research showed a zone of inhibition at 3 wt%, and therefore, it was ascertained that antifungal efficacy of 3, 5, 7, 9, and 11 wt% required further research and evaluation.

Biological synthesis or green synthesis is an approach that bridges nanotechnology and plant biotechnology. The metabolites of plant extracts reduce metal ions to NPs. ZnO-NPs have been biologically synthesized from various plant sources, and in the current research, ZnO-NPs were synthesized from leaves of A. indica as it is recognized as an indigenous medicinal plant that is well known for its antifungal, immunomodulatory, anti-inflammatory, antihyperglycemic, antimalarial, antibacterial, antioxidant, antimutagenic and anticarcinogenic properties.³² Neem elaborates a vast array of biologically active compounds that are chemically diverse and structurally variable.²⁰ Quercetin and ß-sitosterol were the first polyphenolic flavonoids purified from neem fresh leaves shown to have antibacterial and antifungal properties.³³

In the current study, after following a standardized protocol, the calcinated powder was characterized by SEM, UV–visible spectroscopy, XRD, and FTIR, as evidenced in the literature.²⁵ The results of the SEM showed that particles synthesized in this study were of spherical morphology with a size of <100 nm, which was in accordance with a study conducted by Elumalai. Additionally, aggregation was also observed, which could be due to the high surface energy of ZnO-NPs that occurs when synthesis is carried out in aqueous medium or due to densification resulting in narrow space between particles.²⁵ Other studies conducted on ZnO-NPs synthesized from A. indica leaves showed²²^,³⁴ the formation of stable ZnO nanoflakes and spindle-shaped NPs of the size of <100 nm.

Ultraviolet visible (UV–vis) spectroscopy was carried out to evaluate the optical properties of the BSZnO-NPs. A study conducted by Rajendran³³ observed that the maximum absorption peak was found at 235 nm for ZnO-NPs. Other studies conducted by Elumalai and Velmurugan²⁵ and Singh et al.²⁴ showed the maximum absorption peak at 370 and 368 nm, respectively. The absorption behavior of ZnO suspension depends on the valence band to conduction band transition, and the highest light absorption occurs in the wavelength range of 220–350 nm.³⁵^,³⁶ The current study revealed an absorption peak (λ_max) of ZnO at a wavelength of 220 nm, which is well within the expected wavelength range. Further, the band gap energy of ZnO was calculated to be 5.6 eV.

X-ray powder diffraction (XRD) is used to identify the phase of nanomaterials by analyzing the broadening pattern. Prior studies carried out by Bhuyan et al.³⁷ showed that BSZnO-NPs were crystalline in nature. The results of the current study were in accordance with the above, wherein the broadening pattern showed the high crystalline nature of ZnO-NPs due to the presence of strong and narrow diffraction peaks in the XRD pattern.

Fourier transform infrared spectroscopy (FTIR) was used to assess the presence of functional groups, which determine the reducing property of the leaf extract. A prior study conducted ascertained the ZnO absorption band in the region between 400 and 600 cm^–1 for ZnO-NPs synthesized from neem leaves, confirming characterization. Similar results were obtained in the current study, which showed a broad absorption band observed at 575 cm^–1 that is attributed to ZnO stretching vibration. Hence, it can be inferred that the reduction/oxidation reaction is the main reaction involved in ZnO-NP biosynthesis from A. indica leaf extract. The reducing property of leaf extract is due to metabolites such as reducing sugars, phenolic groups, and terpenoids. These phytochemicals cause direct reduction of the Zn ions into ZnO-NPs.³⁷

In the current study, for the purpose of comparison, priorly characterized CSZnO-NPs as per product specifications from Sigma Aldrich were used, thus ensuring standardization. The CSZnO-NPs and BSZnO-NPs were incorporated into the GC Soft liner monomer using a method by Ahmed and Ali¹⁶ where a probe sonication apparatus was used for 3 minutes to avoid clumping and to break them into individual NPs.

Kirby-Bauer method is an agar diffusion method, which is a standardized method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing.³⁸ The lack of a chemical bond between ZnO-NPs and any component of the soft liner is a prerequisite for diffusion into the agar. Prior studies conducted by Homsiang et al.³¹ concluded that 15 wt% ZnO-NPs incorporated into the tissue conditioner provided an antifungal effect for up to 14 days, and another study conducted¹⁷ concluded that the most effective concentration of ZnO–Ag NPs incorporated in tissue conditioner was 5% with C. albicans. In the current study, the optimal concentration for inhibition of C. albicans was with incorporation of 7 wt% of CSZnO-NPs and 9 wt% of BSZnO-NPs in soft liner.

To our knowledge, this study is of value as it is a pioneer study that comparatively evaluates the efficacy of CSZnO-NPs and BSZnO-NPs incorporated in soft denture liners against C. albicans. Among the various concentrations tested, 7 wt% of CSZnO-NPs and 9 wt% incorporations of BSZnO-NPs may be used to effectively combat DS, which is the need of the hour. In the interest of protecting the environment and reducing our carbon footprint, BSZnO-NPs may be used as a viable alternative, most effectively at 9 wt%.

The limitations of the study include that, firstly, it necessitates an in vivo study to substantiate its antifungal value as various patient-related factors have to be taken into consideration. Secondly, further research is required to ascertain the effect of the addition of NPs on the physical and mechanical properties of denture liners.

Future Prospects

Chemically synthesized ZnO NPs (CSZnO-NPs) and BSZnO-NPs have shown antifungal efficacy against C. albicans. We recommend further in vivo studies and clinical trials to evaluate if these results substantiate in a real-world clinical scenario. Minimum inhibitory concentration (MIC) calculation may be considered for definitive concentrations of susceptibility.

CONCLUSION

Within the limitations of this study, it can be concluded that the optimal antifungal concentration of CSZnO-NPs and BSZnO-NPs against C. albicans was 7 and 9 wt%, respectively. CSZnO-NPs were proved to be more efficacious against C. albicans than BSZnO-NPs.

REFERENCES

1. Cunha-Cruz J, Hujoel PP, Nadanovsḱy P. Secular trends in socio-economic disparities in edentulism: USA, 1972-2001. J Dent Res 2007;86(2):131–136. DOI: 10.1177/154405910708600205

2. Daniluk T, Tokajuk G, Stokowska W, et al. Occurrence rate of oral Candida albicans in denture wearer patients. Adv Med Sci 2006;51(Suppl 1):77–80. PMID: 17458064.

3. Bhat V, Sharma SM, Shetty V, et al. Prevalence of Candida associated denture stomatitis (cads) and speciation of candida among complete denture wearers of South West coastal region of Karnataka. J Heal Allied Sci NU 2013;03(3):59–63. DOI: 10.1055/s-0040-1703679

4. Pereira-Cenci T, Del Bel Cury AA, Crielaard W, et al. Development of Candida-associated denture stomatitis: new insights. J Appl Oral Sci 2008;16(2):86–94. DOI: 10.1590/s1678-77572008000200002

5. Abualsaud R, Aleraky DM, Akhtar S, et al. Antifungal activity of denture base resin containing nanozirconia: in vitro assessment of Candida albicans biofilm. Scientific World Journal 2021;2021:5556413. DOI: 10.1155/2021/5556413

6. Contaldo M, Romano A, Mascitti M, et al. Association between denture stomatitis, Candida species and diabetic status. J Biol Regul Homeost Agents 2019;33(3):35–42. PMID: 31538448.

7. Saeedi P, Petersohn I, Salpea P, et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: results from the International Diabetes Federation Diabetes Atlas, 9th edition. Diabetes Res Clin Pract 2019;157:107843. DOI: 10.1016/j.diabres.2019.107843

8. Rao CR, Kamath VG, Shetty A, et al. A study on the prevalence of type 2 diabetes in coastal Karnataka. Int J Diabetes Dev Ctries 2010;30(2):80–85. DOI: 10.4103/0973-3930.62597

9. Dorocka-Bobkowska B, Budtz-Jörgensen E, Wtoch S. Non-insulin-dependent diabetes mellitus as a risk factor for denture stomatitis. J Oral Pathol Med 1996;25(8):411–415. DOI: 10.1111/j.1600-0714.1996.tb00288.x

10. Kreve S, Dos Reis AC. Denture liners: a systematic review relative to adhesion and mechanical properties. Sci World J 2019;2019:6913080. DOI: 10.1155/2019/6913080

11. Kusama T, Aida J, Yamamoto T, Kondo K, et al. Infrequent denture cleaning increased the risk of pneumonia among community-dwelling older adults: a population-based cross-sectional study. Sci Rep 2019;9(1):13734. DOI: 10.1038/s41598-019-50129-9

12. Vural C, Ozdemir G, Kurtulmus H, et al. Comparative effects of two different artificial body fluids on candida albicans adhesion to soft lining materials. Dent Mater J 2010;29(2):206–212. DOI: 10.4012/dmj.2009-081

13. Akpan A, Morgan R. Oral candidiasis. Postgrad Med J 2002;78(922):455–459. DOI: 10.1136/pmj.78.922.455

14. Mousavi SA, Ghotaslou R, Akbarzadeh A, et al. Evaluation of antibacterial and antifungal properties of a tissue conditioner used in complete dentures after incorporation of ZnO–Ag nanoparticles. J Dent Res Dent Clin Dent Prospects 2019;13(1):11. DOI: 10.15171/joddd.2019.002

15. Ansarifard E, Zareshahrabadi Z, Sarafraz N, et al. Evaluation of antimicrobial and antibiofilm activities of copper oxide nanoparticles within soft denture liners against oral pathogens. Bioinorg Chem Appl 2021;2021:9939275. DOI: 10.1155/2021/9939275

16. Ahmed AQ, Ali M. The influence of titanium dioxide nanoparticles incorporation into soft denture lining material on candida albicans adherence and some mechanical and physical properties. J Pure Appl Microbiol 2018;12(2):783–791. DOI: 10.22207/JPAM.12.2.39

17. Mousavi SA, Ghotaslou R, Khorramdel A, et al. Antibacterial and antifungal impacts of combined silver, zinc oxide, and chitosan nanoparticles within tissue conditioners of complete dentures in vitro. Ir J Med Sci 2020;189(4):1343–1350. DOI: 10.1007/s11845-020-02243-1

18. Hosseini SS, Ghaemi E, Koohsar F. Influence of ZnO nanoparticles on Candida albicans isolates biofilm formed on the urinary catheter. Iran J Microbiol 2018;10(6):424–432. PMID: 30873271.

19. Dobrucka R, Długaszewska J. Biosynthesis and antibacterial activity of ZnO nanoparticles using Trifolium pratense flower extract. Saudi J Biol Sci 2016;23(4):517–523. DOI: 10.1016/j.sjbs.2015.05.016

20. Mahmoud DA, Hassanein NM, Youssef KA, et al. Antifungal activity of different neem leaf extracts and the nimonol against some important human pathogens. Braz J Microbiol 2011;42(3):1007–1016. DOI: 10.1590/S1517-838220110003000021

21. Bajunaid SO. How effective are antimicrobial agents on preventing the adhesion of Candida albicans to denture base acrylic resin materials? A systematic review. Polymers (Basel) 2022;14(5):908. DOI: 10.3390/polym14050908

22. Karthika D, Vimala T. Green synthesis of zinc oxide nanoparticles using Azadirachta indica leaves extract and its characterization and application in the degradation of methylene blue. Int J Sci Res 2018;7(8):1175–1177. DOI: 10.21275/ART2019823

23. Pillai AM, Sivasankarapillai VS, Rahdar A, et al. Green synthesis and characterization of zinc oxide nanoparticles with antibacterial and antifungal activity. J Mol Struct 2020;1211:128107. DOI: 10.1016/j.molstruc.2020.128107

24. Singh A, Neelam, Kaushik M. Physicochemical investigations of zinc oxide nanoparticles synthesized from Azadirachta indica (Neem)leaf extract and their interaction with Calf-Thymus DNA. Results Phy 2019;13:102168. DOI: 10.1016/j.rinp.2019.102168

25. Elumalai K, Velmurugan S. Green synthesis, characterization and antimicrobial activities of zinc oxide nanoparticles from the leaf extract of Azadirachta indica (L.). Appl Surf Sci 2015;345:329–336. DOI: 10.1016/j.apsusc.2015.03.176

26. Hashem MI. Advances in soft denture liners: an update. J Contemp Dent Pract 2015;16(4):314–318. DOI: 10.5005/jp-journals-10024-1682

27. Moradpoor H, Safaei M, Mozaffari HR, et al. An overview of recent progress in dental applications of zinc oxide nanoparticles. RSC Adv 2021;11(34):21189–21206. DOI: 10.1039/d0ra10789a

28. Sirelkhatim A, Mahmud S, Seeni A, et al. Review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism. Nano-Micro Lett 2015;7(3):219–242. DOI: 10.1007/s40820-015-0040-x

29. Parveen K, Banse V, Ledwani L. Green synthesis of nanoparticles: their advantages and disadvantages. AIP Conf Pro 2016;1724. DOI: 10.1063/1.4945168

30. Gour A, Jain NK. Advances in green synthesis of nanoparticles. Artif Cells Nanomed Biotechnol 2019;47(1):844–851. DOI: 10.1080/21691401.2019

31. Homsiang W, Kamonkhantikul K, Arksornnukit M, et al. Effect of zinc oxide nanoparticles incorporated into tissue conditioner on antifungal, physical, and mechanical properties. Dent Mater J 2021;40(2):481–486. DOI: 10.4012/dmj.2020-095

32. Subapriya R, Nagini S. Medicinal properties of neem leaves: a review. Curr Med Chem - Anticancer Agents 2005;5(2):149–156. DOI: 10.2174/1568011053174828

33. Govindachari TR, Suresh G, Gopalakrishnan G, et al. Identification of antifungal compounds from the seed oil of Azadirachta indica. Phytoparasitica 1998;26(2):109–116. DOI: 10.1007/BF02980677

34. Noorjahan CM, Shahina SKJ, Deepika T, et al. Green synthesis and characterization of zinc oxide nanoparticles from neem (Azadirachta indicia). Int J Sci Eng Technol Res 2015;4(30):5757–5753.

35. Rajendran SP, Sengodan K. Synthesis and characterization of zinc oxide and iron oxide nanoparticles using sesbania grandiflora leaf extract as reducing agent. J Nanosci 2017;2017(17):1–7. DOI: 10.1155/2017/8348507

36. El Badri Nagla El Badri MS, Sulieman KM. Optical absorption measurement of synthesized ZnO using ultra violet-visible spectrophotometer. Int J Sci Res 2015;4(11):62–65.

37. Bhuyan T, Mishra K, Khanuja M, et al. Biosynthesis of zinc oxide nanoparticles from Azadirachta indica for antibacterial and photocatalytic applications. Mater Sci Semicond Process 2015;32:55–61. DOI: 10.1016/j.mssp.2014.12.053

38. Kreve S, Oliveira VC, Bachmann L, et al. Influence of AgVO3 incorporation on antimicrobial properties, hardness, roughness and adhesion of a soft denture liner. Sci Rep 2019;9(1):11889. DOI: 10.1038/s41598-019-48228-8

________________________
© The Author(s). 2023 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and non-commercial reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.