Plausible Mechanistic Insights in Biofilm Eradication Potential against Candida spp. Using In Situ-Synthesized Tyrosol-Functionalized Chitosan Gold Nanoparticles as a Versatile Antifouling Coating on Implant Surfaces.

In the present study, tyrosol-functionalized chitosan gold nanoparticles (Chi-TY-AuNPs) were prepared as an alternative treatment strategy to combat fungal infections. Various biophysical techniques were used to characterize the synthesized Chi-TY-AuNPs. The antifungal and antibiofilm activities of Chi-TY-AuNPs were evaluated against Candida albicans and C. glabrata, and efforts have been made to elucidate the possible mechanism of action. Chi-TY-AuNPs showed a high fungicidal effect against both sessile and planktonic cells of Candida spp. Additionally, Chi-TY-AuNPs completely eradicated (100%) the mature biofilms of both the Candida spp. FESEM analysis highlighted the morphological alterations in Chi-TY-AuNP-treated Candida biofilm cells. The effect of Chi-TY-AuNPs on the ECM components showed significant reduction in protein content in the C. glabrata biofilm and substantial decrease in extracellular DNA content of both the Candida spp. ROS generation analysis using DCFDA-PI staining showed high ROS levels in both the Candida spp., whereas pronounced ROS production was observed in the Chi-TY-AuNP-treated C. glabrata biofilm. Biochemical analysis revealed decreased ergosterol content in Chi-TY-AuNP-treated C. glabrata cells, while inconsequential changes were observed in C. albican s. Furthermore, the transcriptional expression of selected genes (ergosterol biosynthesis, efflux, sterol importer, and glucan biogenesis) was reduced in C. glabrata in response to Chi-TY-AuNPs except ERG11 and CDR1. Conclusively, the result showed the biofilm inhibition and biofilm eradication efficacy of Chi-TY-AuNPs in both the Candida spp. Findings of the present study manifest Chi-TY-AuNPs as a potential therapeutic solution to Candida biofilm-related chronic infections and overcome biofilm antifungal resistance.

Introduction

Candidiasis is among the utmost prevalent nosocomial pathogenic infections triggered by Candida, primarily in immune-compromised patients with an approximately 40% mortality rate.[1]Candida is a commensal polymorphic yeast responsible for superficial skin infections to deep tissue invasions. Candida albicans is the most ubiquitous and menacing pathogen followed by non-albicans Candida spp., where C. glabrata and C. tropicalis stand out as the most prevalent species.[2] Epidemiological reports from North America suggest that there are frequent incidences of candidiasis due to C. glabrata, also prevalent in the Asia-Pacific region.[3] The virulence properties of Candia spp. are attributed to its capability to form biofilm, a sessile, multicellular community where microbes encapsulate themselves in a self-secreted extracellular matrix (ECM).[4] This ECM is a conglomeration of biomolecules and hydrolytic enzymes, specifically lipase, proteinase, and phospholipase. Besides biofilm, adhesin proteins and persister cells further boost their resistant nature and contribute to recurrent candidiasis.[5] Persister cells belong to the biofilm community exhibiting lower growth rates, active drug efflux, and high resistance to antimicrobial treatment and are entirely different from free-floating planktonic cells.[6] These biochemical events are attributed to biofilm growth and act cohesively to combat the antifungals’ therapeutic action.

Inside the biofilm, cells communicate with each other harmonically, and this cellular signaling is known as quorum sensing (QS), medicated by quorum sensing molecules (QSMs). Tyrosol (2-(4-hydroxyphenyl)ethanol) is a QSM molecule produced by C. albicans that stimulates the formation of germ tubes and initiates the development of hyphae to facilitate biofilm formation, whereas its exogenous administration acts antagonistically.[7,8] The antibacterial and antifungal properties of tyrosol (TY) have been substantially explored in recent years; however, these investigations unravel insufficient mechanistic insight into tyrosol’s mode of action in Candida.[9,10] Increasing incidences of Candida-related nosocomial infections and emerging multidrug-resistant (MDR) Candida spp. are a global concern due to the high mortality rate. Rapidly increasing resistance to major antifungals facilitated through multiple mechanisms limited the treatment options for Candida-related infections.[11] Therefore, the development of novel antifungals, combinatorial therapies, and the quest for alternative therapeutic strategies are crucial to mitigate the recalcitrant biofilms and repercussions of rapidly growing antimicrobial resistance.

In recent years, nanoparticles emerged as an invincible tool in intracellular delivery of antimicrobials due to their minimal size, high surface-to-volume ratio, enhanced cellular uptake, and sub-cellular drug retention properties.[12] Among various metallic nanoparticles, gold in a nanoparticulated form has shown immense potential in antimicrobial drug delivery owing to their high therapeutic efficacy against bacteria and fungi followed by low cytotoxicity and high biocompatibility against mammalian cells.[13] Chitosan is a natural cationic polysaccharide comprising glucosamine and N-acetyl glucosamine units. The presence of different functional groups such as hydroxyl (−OH), amine (−NH2), and carbonyl (>C=O) renders chitosan an extensively used biopolymer for the synthesis of colloidal nanoparticles acting as a reducing and stabilizing agent.[14] The broad-spectrum antimicrobial action of chitosan against a wide variety of microbes is attributed to its polycationic nature contributed by NH3+ groups of glucosamine units. The cationic tail of chitosan interacts with the negatively charged cytoplasmic membrane of microbes via electrostatic interactions, leading to extensive cell surface modifications. Subsequently, this results in cellular internalization of nanoparticles and leakage of intracellular contents, ultimately resulting in inhibition of DNA transcription as well as RNA and protein synthesis.[15] In addition, chitosan’s toxicity toward bacterial cell and mammalian cell safety has ruled out its biocompatibility issues.[16]

A combinatorial drug delivery system that enables easy transport of drugs to the target site is a potential strategy in treating Candida infections.[17] Commonly used antifungals such as azoles, polyenes, etc. can be metamorphosed into nanoparticles to improve antifungal agents’ efficacy compared to the conventional therapeutic regimen.[12] In view of the exceptional physicochemical characteristics of chitosan and gold, these render them as excellent biomaterials in the fabrication of effective nanoformulation. In addition, chitosan’s biocompatibility and gold being inert along with antimicrobial efficacy offer a promising horizon in the development of noncytotoxic chitosan-gold nanoparticles.[18] In addition, chitosan-based nanoformulations possess a high positive surface charge, which helps in the transportation of molecules/drugs across the cell membrane, high drug payload, and enhanced cellular uptake. Such carrier systems would enhance the cellular internalization of the active molecule/drug due to the electrostatic interaction between nanoparticles and the cell membrane, leading to enhanced penetration in the Candida biofilm and its membrane disruption, thus providing a smart strategy to combat the menacing effect of biofilm. The all-pervading chitosan-gold nanoparticle’s ability inside the biofilm niche is very high; therefore, they possess high antimicrobial activity, which is crucial for combating Candida spp.-mediated superficial or systemic infections.

In the present study, TY-functionalized chitosan gold nanoparticles (Chi-TY-AuNPs) were synthesized by an in situ facile method to harness a synergistic effect via targeting both the fungal cells and biofilm matrix. The antifungal and antibiofilm potency of synthesized Chi-TY-AuNPs was investigated against C. albicans and C. glabrata. Further, efforts have been made to elucidate the mode of action of Chi-TY-AuNPs by examining their impact on ROS generation, cell surface hydrophobicity, ECM composition, and membrane ergosterol content in biofilms of both the Candida spp.; in addition, transcriptional expression of selected C. glabrata genes were also evaluated.

Results

Physicochemical Characterization of Chi-TY-AuNPs

The physicochemical characterization of Chi-TY-AuNPs was carried out by analyzing the size, zeta potential, aggregation behavior, and chemical interactions that were studied with electron microscopy, Zetasizer, and FTIR analysis. Synthesis of Chi-TY-AuNPs was carried out using chitosan, which displays both the stabilizing and reducing properties, and the size of the average nanoparticles was determined, as shown in Figure . Confirmation of synthesized Chi-AuNPs was ascertained by UV–visible spectroscopy. The bioreduction of Au+ ions into Au0 by chitosan was determined by the change in color from transparent to a wine red/ruby red color (Figure A). The reaction mixture was allowed to cool at 25 °C and analyzed by UV–vis spectroscopy at subsequent time intervals from 0 to 168 h at 531 nm. The appearance of a ruby red color and consistent sharp peaks at 531 nm confirms the formation of Chi-AuNPs. The presence of sharp peaks in the visible range is attributed to the excitation of its SPR, which in turn is dependent on the shape and size of the nanoparticles.[19,20] Furthermore, no significant variation was observed upon drug loading in the absorption spectra of Chi-TY-AuNPs (Figure A). Zeta potential is a significant physiochemical attribute for nanosystems, which plays a crucial role in the drug delivery mechanism (Figure B). The nanoparticulate system’s solubility, cellular absorption, and release rate are also influenced by zeta potential. Our findings showed that Chi-AuNPs and Chi-TY-AuNPs possess a charge of +62 mV (Figure B) and + 45.5mV (Figure B), respectively.[20] DLS has been used to estimate the diameter of Chi-TY-AuNPs, and the average hydrodynamic diameter was found to be 46.96 nm (Figure C), and a low PDI of 0.171 substantiates greater colloidal stability in the aqueous environment.

Figure 1

Physicochemical analysis of Chi-TY-AuNPs: (A) UV–visible spectroscopic analysis, (B) zeta potential, (C) dynamic light scattering, (D) apparent zeta potential of Chi-AuNPs, and (E) apparent zeta potential of Chi-TY-AuNPs.

Chi-TY-AuNP HRTEM images showed that the nanoparticles were in the range 10.345 ± 2.684 nm in diameter with a sphere-shaped morphology (Figure A–D). AFM investigation revealed the spherical shape of Chi-TY-AuNPs with an average diameter in the range 10–15 nm (as shown through nanoparticle color scripting and 3D image) (Figure E,F). Therefore, from the findings of TEM, AFM, and zeta potential, we can infer that the size obtained is efficacious in harnessing the antifungal property of Chi-TY-AuNPs owing to its enhanced permeability and retention (EPR) effect.[20] The selected-area electron diffraction (SAED) pattern of Chi-TY-AuNPs reveals a transition state of a polycrystalline nature. The broad spheres present in Chi-TY-AuNPs are attributes of the chitosan matrix, and rings are made of crystalline gold nanoparticulate (Figure H).

Figure 2

Morphological analysis of Chi-TY-AuNPs: TEM images at (A) 100 nm, (B) 50 nm, (C) 20 nm, and (D) 5 nm; (E) 2D AFM; (F) 3D AFM; (G) particle size distribution curve; (H) SAED pattern of synthesized Chi-TY-AuNPs.

Fourier-Transform Infrared (FTIR) Analysis

A comparative FTIR analysis was performed for the chemical characterization and functional group validation in Chi-TY-AuNPs with respect to chitosan and TY (Figure ). During the FTIR analysis, the carbonyl, C–O–NHR, NH2 and ammonium, NH3+ band, OH, and CH deformation in the region 100–2400 cm–1 have been considered as critical analytical peaks. Functional groups were assigned to chitosan such as N–H, O–H, and NH2 peaks at 3357–3290 cm–1 followed by a tiny peak of 2879 cm–1 assigned to −CH2 and −CH3 (Figure A).[24] However, the amide II bands (C–N stretching coupled to NH bending) and amide I peak (C=O stretching), as represented in Figure A, were also observed around 1513 and 1643 cm–1, respectively.[25]

Figure 3

FTIR analysis of (A) chitosan, (B) tyrosol, (C) gold chloride, (D) Chi-AuNPs, and (E) Chi-TY-AuNPs.

Nevertheless, the FTIR analysis of TY shows a small peak of C=C aromatic stretching at 1513 cm–1. The standard TY peaks are as follows: two bands from 1600–1400 cm–1 correlated to C=C (benzene ring) and aromatic C–H (in para position) forms the bending vibration in the 900–800 cm–1 range (Figure B).[26] To ascertain the interactions between chitosan functional groups and gold nanoparticles, the FTIR spectra of Chi-AuNPs were also assessed. The chemical interactions indicate the shift in the characteristic peaks of the spectrum. The FTIR analysis of Chi-AuNPs, obtained with nearly identical peaks of chitosan, shows a uniform chitosan deposition over gold nanoparticles (Figure D).[20] However, in the Chi-TY-AuNPs, as in Figure E, both the peaks of chitosan and TY were evident. This shows the successful adsorption of TY over the surface of Chi-AuNPs. The apparent disappearance of TY-associated peaks substantiates our finding in drug loading efficiency and pH-triggered release profile.

Drug Loading Efficiency

The drug loading efficiency of Chi-TY-AuNPs was found to be 46.08% as obtained by UV–visible analysis in 100 mg of Chi-TY AuNPs.

Chi-TY-AuNPs Showed Fungicidal Activity against Candida spp.

The planktonic growth of both the Candida spp. employed in this study was inhibited in a concentration-dependent manner (Figure A). The MIC80 value of Chi-TY-AuNPs was 200 and 400 μg/mL for C. albicans and C. glabrata growth, respectively. Chi-TY-AuNPs effectiveness was substantially greater against the growth of C. albicans as compared to C. glabrata. Nevertheless, the MFC value of Chi-TY-AuNPs for both C. albicans and C. glabrata was 800 μg/mL (Figure B). The MIC and MFC values of Chi-AuNPs against C. albicans and C. glabrata was considerably lower than those of Chi-TY-AuNPs, suggesting that tyrosol potentiated the antifungal activity of Chi-AuNPs (Table S1).

Figure 4

Determination of (A) MIC and (B) MFC of Chi-TY-AuNPs against C. albicans and C. glabrata planktonic cells; error bars represent SD (n = 3), *P < 0.05 considered as statistically significant.

Effect of Chi-TY-AuNPs on Candida Biofilms

The concentration-dependent activity of Chi-TY-AuNPs enabled it to inhibit biofilm development and eradicate the mature biofilms of C. albicans and C. glabrata (Figure ). The BIC80 of Chi-TY-AuNPs against both Candida spp. viz., C. albicans and C. glabrata were 200 and 400 μg/mL, respectively. At the highest concentration (800 μg/mL), Chi-TY-AuNPs inhibited 95.98 and 96.34% of C. albicans and C. glabrata biofilms, respectively (Figure A). In contrast, Chi-AuNPs showed 11.36% inhibition of the C. albicans biofilm and 14.21% inhibition of the C. glabrata biofilm with BIC80 and BEC80 values far higher than those of Chi-TY-AuNPs (Table S1). The biofilm eradicating efficiency of Chi-TY-AuNPs, measured in terms of BEC80, was 400 μg/mL against both the Candida spp. used in this study (Figure B). The biofilm eradicating efficacy of Chi-TY-AuNPs was found to be equal against both C. albicans and C. glabrata biofilms, and the BEC80 value was found to be 800 μg/mL for both species. Further, the morphological alterations in Chi-TY-AuNP-treated C. albicans and C. glabrata biofilm cells were visualized by FESEM analysis (Figure C). The FESEM micrographs of the untreated Candida biofilm showed a compact network of C. albicans hyphal cells, whereas C. glabrata cells appeared as healthy elongated and oval with no alteration in the surface topology of cells (Figure C, micrographs i and iii). However, the biofilm of C. albicans, in the presence of 400 μg/mL Chi-TY-AuNPs, exhibited the absence of a hyphal network and wrinkled cells, while the C. glabrata-treated biofilm showed cells with pores on the surface (Figure C, micrographs ii and iv).

Figure 5

Effect of Chi-TY-AuNPs on C. albicans and C. glabrata. (A) Biofilm eradication and (B) biofilm inhibition. (C) FESEM images representing the Chi-TY-AuNP-induced morphological changes in C. albicans and C. glabrata: (i, iii) untreated biofilms of C. albicans and C. glabrata, respectively, and (ii, iv) Chi-TY-AuNP-treated biofilms of C. albicans and C. glabrata, respectively; error bars in the graph represent SD (n = 3), magnification: 1000–5000×.

Chi-TY-AuNPs Inhibited C. albicans Germ Tube Formation

Since Chi-TY-AuNPs worked extremely well against Candida biofilms, to explore the possible antibiofilm mode of action of synthesized nanoparticles, their impact on hyphae was also investigated. However, this study was limited to C. albicans because C. glabrata do not form germ tubes. Figure S1 clearly shows the inhibition of germ tube development in C. albicans by Chi-TY-AuNPs at a subinhibitory concentration (200 μg/mL). The germ tubes were induced by exposing cells to 10% FBS. Negative control cells remained in the yeast and budding yeast form, while the positive control showed long hypha formation (Figure S1A,B). At a subinhibitory concentration, Chi-TY-AuNPs completely inhibited yeast to hyphae transformation and reduced the number of cells (Figure S1C). Chi-AuNPs did not show any alteration in the C. albicans morphology (Figure S1D).

Besides hyphal development, surface hydrophobicity also has a role in biofilm establishment and hence, the effect of Chi-TY-AuNPs in modulating hydrophobicity of the cells was evaluated in terms of HI using a two-phase system. The hydrophobicity evaluation results suggested no significant change in the HI value of both the Candida spp. compared to control cells upon Chi-TY-AuNP treatment. While the HI value of C. albicans cells increased in response to TY treatment, the value remained nearly the same in Chi-AuNP- and Chi-TY-AuNP-treated cells (Figure S2). Therefore, hydrophobicity was not observed to be responsible for mediating the antibiofilm activity of Chi-TY-AuNPs.

Effect of Chi-TY-AuNPs on Viability of Candida Biofilm Cells

Further, FDA-PI staining was used to assess the live and dead cells in Chi-TY-AuNP-treated C. albicans and C. glabrata biofilms and to visualize using a fluorescence microscope (Figure ). The FDA binds with the cell membrane polysaccharides of living cells and emits green fluorescence, while lysed/ dead cells fluoresce red due to PI’s binding with DNA. The C. albicans control biofilm and the one treated with Chi-AuNPs emitted green fluorescence indicating no cell death, whereas Chi-TY-AuNP-treated cells exhibited both red and green fluorescence indicating cell lysis (Figure A). Likewise, no red fluorescence was observed in the C. glabrata control biofilm and the biofilm treated with Chi-AuNPs; however, intense red fluorescence with dull green light was observed in the Chi-TY-AuNP-treated biofilm (Figure B).

Figure 6

FDA/PI-stained live and dead cells: (A) C. albicans; (B) C. glabrata; (i, iv) control (untreated) C. albicans and C. glabrata cells, respectively; (ii, v) Chi-TY-AuNP-treated C. albicans and C. glabrata, cells, respectively; (iii, vi) Chi-AuNP-treated C. albicans and C. glabrata cells, respectively; magnification: 40×, scale bar: 100 μm.

Effect of Chi-TY-AuNPs on the Biofilm Extracellular Matrix (ECM)

To gain insight into Chi-TY-AuNP-mediated effects on biochemical components of the C. albicans and C. glabrata biofilm ECM, a spectrophotometric study was performed. Insignificant changes were observed in the protein content of C. albicans as compared to the control in Chi-AuNPs and Chi-TY-AuNPs, while it was significantly increased in TY. However, in C. glabrata, the protein content was considerably reduced in Chi-TY-AuNPs as compared to the control and remained unchanged in the rest of the samples (Figure A). The eDNA content of the C. glabrata ECM was relatively much higher than that of the C. albicans control biofilm. The eDNA content in the C. albicans biofilm was increased in TY, whereas it was substantially decreased in Chi-TY-AuNPs and remained unchanged in Chi-AuNPs. In C. glabrata, the content of eDNA was decreased in all samples with the highest reduction in Chi-TY-AuNPs (Figure B).

Figure 7

Biochemical quantification of TY-, Chi-AuNP-, and Chi-TY-AuNP-treated C. albicans and C. glabrata: (A) estimation of protein, (B) estimation of eDNA, and (C) estimation of ergosterol; error bars represent SD (n = 3), *P < 0.05 considered as statistically significant.

Analysis of ROS Production in Candida Biofilms Treated with Chi-TY-AuNPs

ROS generation is one of the most widely adopted strategies of drug molecules to mount antimicrobial activity and sometimes promote cell death. To estimate the amount of ROS produced by TY, Chi-AuNPs, and Chi-TY-AuNPs in Candida biofilms, PI and DCFDA were used. PI binds with DNA, manifesting cell lysis, while DCFDA determines the ROS level generated within the cells. The C. albicans biofilm treated with Chi-TY-AuNPs and Chi-AuNPs exhibited a significantly elevated level of ROS in comparison to the control; however, ROS elevation was more pronounced in the C. glabrata biofilm (Figure A,B). The interaction of PI with the DNA of lysed cells determined the detrimental effect of ROS accretion on cells; a high fluorescence intensity of intercalated PI was observed in both the Candida spp. biofilms treated with Chi-TY-AuNPs (Figure C,D).

Figure 8

Measurement of ROS generation in C. albicans and C. glabrata cells exposed to TY, Chi-AuNPs, and Chi-TY-AuNPs. The ROS level is represented in terms of fluorescence intensity of (A) DCFDA and (B) PI. Microscopic fluorescence images of (C) C. albicans and (D) C. glabrata; error bars in the graph represent SD (n = 3), *P < 0.05 considered as statistically significant; magnification: 40×, scale bar: 100 μm.

Chi-TY-AuNP Differentially Modulated C. glabrata Transcriptional Expression

The cell membrane of the Candida is the first line of defense against any drug molecule, like all other organisms. Ergosterol is a critical component of the Candida cell membrane, which plays a remarkable role in its susceptibility to drug molecules and, thus, a primary target for antifungal drugs. Therefore, the change in ergosterol content of the plasma membrane was estimated upon exposure of both the Candida spp. to Chi-TY-AuNPs. C. albicans exhibited no change in ergosterol content in the presence of Chi-TY-AuNPs, while C. glabrata showed a decreased ergosterol concentration in comparison to the control (Figure C).

Therefore, the transcriptional expression study of the ergosterol pathway and ABC transporters was performed in C. glabrata cells. The transcriptional expression of the sterol importer (AUS1), multidrug transporter (CDR1), 1,3-β-glucan synthase (FKS1), ergosterol synthetic pathway (ERG11, ERG3, ERG2, ERG10, and ERG4), and GPI-anchored cell wall protein (KRE1) genes was studied using RT-PCR. Except for ERG11, expression of AUS1, KRE1, FKS1, and all genes of the ergosterol pathway were significantly downregulated upon Chi-TY-AuNP treatment (Table ). The trend in the gene expression pattern of TY-treated C. glabrata cells was similar to Chi-TY-AuNP-treated cells. However, the fold change value of TY-treated C. glabrata cells was lesser than that of the Chi-TY-AuNP-treated cells.

Table 1

Up- and Downregulation of C. glabrata Genes in Response to the Subinhibitory Concentration of Chi-TY-AuNPsa

		fold expression
C. glabrata genes	function	TY	Chi-AuNPs	Chi-TY-AuNPs
CDR1	ABC multidrug transporter regulated by Pdr1p	1.19	12.15	10.00
ERG2	C-8 sterol isomerase activity	no change	1.4	–2.0
ERG3	C-5 sterol desaturase activity	–1.67	1.1	–2.0
ERG4	C-24 sterol reductase activity	1.7	2.1	–2.0
ERG10	acetyl-CoA acetyltransferases have a role in sterol biosynthesis	–1.42	2.00	–3.34
ERG11	cytochrome P-450 lanosterol 14-alpha-demethylase role in ergosterol synthesis	1.2	43.41	12.2
AUS1	ABC transporter involved in sterol uptake	–2.0	–1.42	–10.0
KRE1	role in cell wall biogenesis and organization	–2.5	–2.0	–2.5
FKS1	component of 1,3-beta-glucan synthase	–2.5	–3.34	–10

Genes showing a fold expression of ≥1.5 were only considered to assess the changes. A fold expression of 1.5–5.0 was considered as significant.

Cytotoxicity Assay

The effect of fabricated Chi-AuNP and Chi-TY-AuNP formulations to assess cytocompatibility for biomedical application was determined by MTT assay. The nanoparticles can increase their effectiveness in real time by pursuing a preferred tolerance limit of nanoparticles by cells. The biocompatibility of Chi-AuNPs and Chi-TY-AuNPs in the presence of in NIH-3T3 cells was tested to evaluate the cytotoxicity activity of Chi-AuNPs and Chi-TY-AuNPs. The Chi-AuNPs showed a concentration-dependent decrease in cell viability probably due to the high cationic surface charge potential, whereas Chi-TY-AuNPs (low surface charge potential) manifested no reduction in cell viability and even in higher concentrations, only negligible cytotoxicity was observed in the NIH-3T3 mouse fibroblast cell line, represented in Figure S3. As compared to Chi-AuNPs, the drug-encapsulated formulation Chi-TY-AuNPs showed enhanced biocompatibility (>90%) in the treatment system. The results obtained from this assay give insight into the potential improvement in the in vivo application at the site of infection.

Discussion

Members of genus Candida are commensal, opportunistic fungal pathogens with biofilm-forming ability as the most prominent virulence feature responsible for the evolution of MDR Candida strains and therapeutic failure of conventional antifungals.[11] Due to its recalcitrant feature, complete removal of biofilms is a grave challenge. Nanocarrier drug delivery systems emerged as a promising strategy due to their biofilm barrier-penetrating capacity owing to their smaller size and their localization into cellular and subcellular compartments for site-specific antibiotic delivery. Engineering of biopolymer-catalyzed metal nanoparticles with antibiotics/small molecules will improve their intracellular delivery and antibiofilm effect via preventing the microorganism’s surface adherence and internalization into microbial cells resulting in the destruction of intracellular architecture.[27,28] Polymeric metal nanoparticles offer excellent drug loading efficiency manifesting a greater therapeutic index and improved pharmacokinetic profile in low doses compared to the free form of the drug with minimal associated side effects.[29]

TY, as an indigenous QSM, is well known to induce the yeast-to-hypha transformation via concentration-dependent fungal growth in a culture medium. Recent investigations showed the fungicidal and antibiofilm effects of exogenously administered TY in different Candida spp., but these studies were insufficient to prove the therapeutic potential of TY in complete eradication of a biofilm and its mechanistic insight.[7,30] In view of the antimicrobial property and biocompatibility of both chitosan and gold combined with the increased susceptibility of TY toward Candida, we synthesized Chi-TY-AuNPs and evaluated their antifungal and antibiofilm activities against C. albicans and C. glabrata. Further, we unraveled the mechanistic insights of Chi-TY-AuNPs by assessing their effect on ROS generation, cell surface hydrophobicity, ECM composition, and membrane ergosterol content in biofilms of both the Candida spp.; along with this, transcriptional expression of selected C. glabrata genes was also evaluated.

The physicochemical analysis revealed the spherical architecture of synthesized Chi-TY-AuNPs in a size range of 10.345 ± 2.684 nm in diameter (Figure G). The higher the zeta potential, the greater will be the stability of colloidal nanoparticles due to electrostatic repulsion.[13] In our study, the zeta potential of Chi-TY-AuNPs was found to be significantly higher, which is attributed to the cationic characteristic of chitosan, signifying greater colloidal stability (Figure B). Conclusively, from the findings of zeta potential, HRTEM, and AFM, we can infer that the high positive surface charge and smaller size obtained is efficacious in harnessing the antifungal property of Chi-TY-AuNPs owing to their EPR effect toward the negatively charged cytoplasmic membrane.[20] Further, FTIR analysis showed the functionalization of TY over the surface of nanoparticles (Figure E) and a high surface-to-volume ratio of Chi-TY-AuNPs, signifying high drug payload.

Chi-TY-AuNPs have efficiently reduced the fungal growth and killed the sessile as well as planktonic cells of both Candida spp. in a concentration-dependent manner (Figure A,B). The observed fungicidal action of Chi-TY-AuNPs may be a result of their smaller size and the ability of gold to its preferential binding with the cell surface, leading to intracellular localization of Chi-TY-AuNPs within the cytoplasm. Furthermore, the fungicidal effect of Chi-TY-AuNPs may be attributed to the interaction of −NH2 groups present on the polycationic chitosan with the negatively charged cytoplasmic membrane via electrostatic bond formation. This interaction leads to the disruption of the fungal cell membrane resulting in leakage of intracellular components.[31] The higher positive surface charge of Chi-TY-AuNP leads to a strong electrostatic interaction between nanoparticles and fungal cells, resulting in increased nanoparticle penetration inside the microbial cell, corroborating higher fungicidal activity.[13,32] Moreover, chitosan-gold nanoparticles and tyrosol worked synergistically; chitosan and Au altered the cellular morphology and mediated TY delivery in cytoplasm. TY, upon reaching the inside of the cell, altered intercellular signaling pathways and disturbed balance between cellular metabolic pathways, leading to loss of fungal cell viability. These results are in line with the previous studies where TY alone or with other antifungals upon exogenous administration has inhibited the biofilm formation in different Candida spp., including C. albicans and C. glabrata.[7,33]

Chi-TY-AuNPs efficiently inhibited and eradicated the biofilms of C. albicans and C. glabrata in a concentration-dependent manner. (Figure A,B). In the present study, the Chi-TY-AuNP concentration, which inhibited the biofilm formation of both the Candida spp., was much lower than that of TY used in earlier studies.[7,30] Strong electrostatic attractive forces and high positive charge exhibited by the −NH2 groups of polycationic chitosan present in Chi-TY-AuNPs are expected to interact with the negatively charged cell membrane leading to the migration and internalization of Chi-TY-AuNPs to the subcellular environment of cytoplasm.[34] This cell surface–nanoparticle interaction is not only mediated by the physicochemical characteristics of nanoparticles such as size, surface-to-volume ratio, surface functionalization, zeta potential etc. The structural components of the biofilm matrix also equally contributed to this event.[35] Encapsulation of TY in chitosan-Au nanoparticles enhanced its penetrability inside the biofilm community and thus enhanced its efficacy as compared to free TY. Furthermore, all constituents of internalized Chi-TY-AuNPs worked synergistically by interacting with different subcellular components (ergosterol, DNA, and RNA) and interfered in working of organelle cellular components, resulting in the modulation of transcription, translation, and other cellular component synthesis events. All these events might be collectively responsible for the inhibition of early formation of biofilms and eradication of mature biofilms.[34] Considering the potency of Chi-TY-AuNPs for complete eradication of biofilms of both the Candida spp., which may be the net therapeutic effect of tyrosol, chitosan and gold were coupled in a nanocarrier system. The morphological alterations and germ tube inhibition were also observed in response to Chi-TY-AuNP-treated C. albicans and C. glabrata cells (Figure S1 and Figure C). The FESEM observations are in line with the previous study conducted by Arias et al. where similar morphological distortions in TY (50–200 mmol–1 L–1)-exposed C. albicans biofilms were evident.[30] The assessment of cell damage and topological distortions mediated by Chi-TY-AuNPs on treated biofilms was carried out by FDA-PI staining. All these observations supported the fungicidal and antibiofilm efficacy of Chi-TY-AuNPs against Candida.

Adhesion of microbes to the cell surface is an early stage event and a major determinant in biofilm formation, which depends on several physiological factors, including physical and chemical surface characteristics of the cell, nutritional growth factors, and viability of microbial cells. Early inhibition of microbial adhesion has been shown to prevent further formation of biofilms.[36] Cell surface hydrophobicity has a significant role in the interactions between the bacterium and host cell and further in microbial biofilm matrix formation. Targeting HI is a novel strategy to combat biofilm formation and maturation.[37] In our study, we did not observe any significant change in the HI value of both Candida spp. upon Chi-TY-AuNP treatment compared to the control. (Figure S2), suggesting that Chi-TY-AuNPs are not participating in any kind of cell surface hydrophobicity-related activity in Candida cells.

The antimicrobial resistance of Candida biofilms is multifactorial, with additional protection facilitated by the biofilm ECM assembly. Structural components of Candida ECM such as hydrolytic enzymes, polysaccharides, protein, β-glucans, and eDNA contribute to tissue penetration and invasion and maintenance of structural integrity and stability of Candida biofilms.[38] These structural components of ECM collectively restrict the diffusion of antifungals to biofilms, leading to reduced therapeutic effectiveness and incomplete eradication of biofilms.[39] Since ECM components are major contributing factors in the formation of Candida biofilms, we studied the effects of Chi-TY-AuNPs on C. albicans and C. glabrata biofilm ECM to determine their interference with ECM components. We observed a significantly reduced protein content in the Chi-TY-AuNP-treated C. glabrata biofilm, while this effect was insignificant in C. albicans (Figure A). The functional role of eDNA has been correlated with the hyphal growth in C. glabrata, while it protects and stabilizes mature biofilms of C. albicans.[38,40] Additionally, eDNA acts as a master regulator of biofilm formation and antifungal resistance.[38] In our findings, the substantially decreased eDNA content of both the Chi-TY-AuNP-treated Candida spp. biofilms (Figure B) can be correlated with the reduced hyphal development as seen in the germ tube formation assay and biofilm susceptibility to Chi-TY-AuNPs (Figure S1). eDNA being polyanionic, its electrostatic interaction with polycationic chitosan present in Chi-TY-AuNPs alongside hydrophobic interactions of leached gold covalently bound to Chi-TY-AuNPs with the oxygen and nitrogen atoms of eDNA results in eDNA damage and inhibition of biofilm formation.[41,42] Another possible explanation is that gold has the affinity for the proteins and phosphate groups present in the DNA, and their interaction with gold might have resulted in the fungal cell lysis and consequent release of intracellular content and DNA damage (Figure D).[43]

Figure 9

Schematic representation of possible antifungal and biofilm inhibition mechanisms of Chi-TY-AuNPs. (A) Cellular uptake and internalization of Chi-TY-AuNPs with the cell membrane resulting in pore formation, (B) Chi-TY-AuNP-mediated ROS generation leading to cell apoptosis, (C) inhibition of ergosterol and glucan biosynthesis resulting in increased susceptibility of the cell membrane to external stress, and (D) modulation of ECM composition leading to inhibition of cellular communication between biofilm cells and finally resulting in architectural collapse.

Chi-TY-AuNPs were found to promote ROS generation in biofilms of both the Candida spp. (Figure ). In our study, we hypothesized that various intracellular events mediated the increased levels of ROS production in the Chi-TY-AuNP-treated Candida biofilms. Positively charged Chi-TY-AuNPs could be easily attracted and adsorbed by the negatively charged cell membrane through an electrostatic interaction resulting in pore formation. This leads to the increased permeation and internalization of the Chi-TY-AuNPs within the mitochondria followed by the mitochondrial dysfunction and activation of molecular signaling pathways responsible for ROS generation and cell apoptosis (Figure C).[43] The findings of Chi-TY-AuNP-mediated ROS generation in biofilms of both the Candida spp. were also evident by the presence of high fluorescence intensity in microscopic fluorescence images (Figure ).

It is known that ergosterol provides stability to the fungal cell wall and maintains its integrity. In addition to intracellular stress, the potential of Chi-TY-AuNPs in disrupting cell wall integrity of Candida spp. was also analyzed in terms of ergosterol content. Furthermore, a previous study reported the synergistic effect of TY and azoles against Candida spp. and showed a possible interaction of TY with ergosterol.[7] Thus, the present investigation was extended to estimate ergosterol content in both the Candida spp. in response to Chi-TY-AuNPs. In C. glabrata cells, the ergosterol content was significantly decreased in response to Chi-TY-AuNPs, while it remained unchanged in C. albicans (Figure C). This variation in the ergosterol content between Candida spp. in response to Chi-TY-AuNPs might be due to differences in their phylogenetic origin, ploidy, morphology, cell membrane composition, and mitochondrial functions.[44]

Although C. albicans remains the predominant cause of Candida-related infections, over the past decade, prevalence of C. glabrata-mediated infections has considerably increased, resulting in a high mortality rate.[3,45] Previous studies showed an azole-like function of exogenously administered tyrosol, resulting in inhibition of Candida spp. but lacks mechanistic elucidation of its action.[7,29] Therefore, it is imperative to address the void in treatment strategies for non-C. albicans spp. and asserted to explore the effective therapeutic regimen for C. glabrata-related infections. Since, in our study, Chi-TY-AuNPs have significantly affected the majority of biochemical parameters (ROS generation, cell surface hydrophobicity, ECM composition, and membrane ergosterol content) in C. glabrata related to biofilm inhibition for this reason, transcriptional expression analysis of selected genes was studied in C. glabrata only. All selected genes of ergosterol biosynthesis, efflux, sterol transporter, and glucan biogenesis were downregulated in response to TY and Chi-TY-AuNPs except ERG11 and CDR1, which were upregulated (Table ). The data suggested a direct effect of Chi-TY-AuNPs on ergosterol, glucan synthesis, and efflux pumps of C. glabrata and indicated its targeted attack on the C. glabrata cell membrane, resulting in increased susceptibility against external stress and weak cellular defense leading to biofilm inhibition and eradication (Figure C).

Conclusions

In the present investigation, Chi-TY-AuNPs were synthesized and characterized via various biophysical techniques. Synthesized Chi-TY-AuNPs showed fungicidal and biofilm eradication potential against both the Candida spp. Further, biochemical studies revealed the interference of Chi-TY-AuNPs with generated ROS, ECM components, and ergosterol content in biofilms of both the Candida spp. Transcriptional analysis of selected genes of C. glabrata manifested downregulation of genes involved in the maintenance of cell wall biosynthesis. Finally, a limitation of this study is that we performed the transcriptional analysis of C. glabrata only and further research is warranted to elucidate the therapeutic and mechanistic effect of Chi-TY-AuNPs against different biofilm-forming pathogens. Our findings confirm the effectiveness of an alternative therapeutic system that may control Candida-associated infections. From the above study, we may conclude that Chi-TY-AuNPs may act effectively in biofilm eradication when applied/coated on clinically relevant biomaterials and eventually will help in preventing implant-associated fungal infections.

Future research needs to be comprehensively focused on the critical evaluation of cytotoxicity, biodistribution, pharmacodynamics, and pharmacokinetics of such nanocarrier systems while investigating their efficacy against biofilm-associated infections.

Materials and Methods

Chemicals and Reagents

Chitosan (low molecular weight), glacial acetic acid, and gold(III) chloride trihydrate (HAuCl4·3H2O) (≥ 99.9%) were procured from Sigma-Aldrich, St. Louis, USA. Tyrosol (purity > 98.0%) was purchased from TCI Chemicals (India), Pvt. Ltd. All medium components were procured from Himedia, India. BCA protein assay kit (Sigma-Aldrich, USA), RNeasy kit (Qiagen, Germany), Verso cDNA synthesis kit (Thermo Fisher Scientific, USA), and other chemicals were obtained from Sigma-Aldrich, USA. All glassware was treated with aqua regia and rinsed with Milli-Q water, and before proceeding with the experiments, the glassware was dried in an hot-air oven for 5 h.

Synthesis of Chi-TY-AuNPs

Chitosan flakes were dissolved in 100 mL of Milli-Q water in 1% CH3COOH to prepare 0.2% (w/v) chitosan solution followed by the addition of 91.9 μL (136 mM) of gold(III) chloride trihydrate (HAuCl4·3H2O). Subsequently, it was heated for 15 min at 90 °C in a heating mantle accompanied by continuous stirring. The change in color of the mixture from colorless to ruby-red indicated the formation of Chi-AuNPs. For drug loading, Chi-AuNPs were stirred magnetically with 1 mg/mL TY for 24 h at 25 °C and further incubated at 4 °C for 48 h. The mixture was then subjected to ultracentrifugation for 30 min at 30,000 rpm, and Milli-Q water was used for pellet redispersion.[19,20]

Characterization of Synthesized Chi-TY-AuNPs

A Shimadzu-1700 UV–visible spectrophotometer (resolution 1 nm; scanning λ = 400–800 nm) was used to determine the surface plasmon resonance (SPR) of Chi-TY-AuNPs. “Image J 1.49” software (National Institute Health, USA) was used to calculate the distribution of the Chi-TY-AuNP’s diameter. The polydispersity index (PDI), dynamic light scattering (DLS) (hydrodynamic size), and surface charge of Chi-TY-AuNPs were estimated with a Zetasizer (Malvern Zetasizer Nano ZS90, UK) at 25 °C. High-resolution transmission electron microscopy (HRTEM, FEI Tecnai G2, USA), operating at a voltage of 200 kV, was utilized to determine the morphology of Chi-TY-AuNPs. For atomic force microscopy (AFM, AFM-STM, Ntegra T-150, Ireland) analysis, Chi-TY-AuNPs were diluted (10 times) with Milli-Q water and dried out onto a clean glass slide under vacuum at 25 °C for 24 h. Further, dried Chi-TY-AuNPs were examined for morphological analysis.

Fourier-Transform Infrared (FTIR) Analysis

The functional group characterization of Chi-AuNPs and Chi-TY-AuNPs, along with TY and chitosan, was performed by FTIR spectroscopy. Vibrational frequencies in the infrared (IR) region (4000–400 cm–1) were analyzed by the KBr pellet method using a Thermo Nicolet spectrometer, determining the formation and chemical modifications of Chi-TY-AuNPs.

Tyrosol Loading Efficiency of Chi-TY-AuNPs

After the synthesis of Chi-TY-AuNPs, the unreacted drug and other substrates were removed by continuous dialysis in a 14 kDa cutoff membrane for 72 h. A known volume of aqueous dispersed Chi-TY-AuNPs was subjected to probe sonication for 15 min, provided with 2 s “on” and 3 s “off” pulse duration. Using a UV–visible spectrophotometer, the amount of TY released from the Chi-TY-AuNPs was extrapolated using the standard curve of TY at 274 nm (0–100 μg/mL, r2 = 0.9934) and plotted the UV–visible absorbance value. Finally, the drug loading efficiency (DLE) was calculated using the following formula:

Strains and Culture Conditions

The strains of C. albicans (ATCC SC5314) and C. glabrata (MTCC 3019) used in this study were a kind gift from Dr. Navin Kumar, Graphic Era University, Dehradun, India. Yeast extract peptone dextrose agar (YPD) media plates comprising 2% dextrose, 2% peptone, 2% agar, and 1% yeast extract were used for routine maintenance of strains at 37 °C, which were cultured in YPD broth. A Roswell Park Memorial Institute (RPMI) medium was used for in vitro biofilm studies.

Estimation of Minimum Inhibitory and Fungicidal Concentration

The minimum inhibitory concentration (MIC) of Chi-TY-AuNPs against C. albicans and C. glabrata planktonic growth was studied in a flat-bottom 96-well multitier plate (MTP) as mentioned in M27-A2 micro-broth dilution guidelines of CLSI.[21] Briefly, 100 μL of cell suspension was obtained from a suspension of log-phase cells (2.5 × 103 cells/mL) prepared using the RPMI medium and added into the wells of MTP. Then, 100 μL of RPMI medium containing Chi-TY-AuNPs in various concentrations of 0, 25, 50, 100, 200, 400, and 800 μg/mL was later added into the MTP. Absorbance was taken at 600 nm after 48 h of incubation at 37 °C. The inhibition of growth by Chi-TY-AuNPs was described in MIC80 in comparison to the control. Chi-TY-AuNPs inhibited 80% growth of both Candida spp. (C. albicans and C. glabrata). The minimum fungicidal concentration (MFC) of Chi-TY-AuNPs was determined by spotting 5 μL of the MIC sample from MTP on the YPD medium plates followed by incubation at 37 °C for 18 h. The plates were then photographed for growth analysis. The concentration at which no growth was observed was considered as the MFC.

Germ Tube Formation Assay

To study the development of germ tubes in C. albicans, the protocol earlier described by Gupta et al. was followed.[22] Briefly, log-phase cells were incubated for 4 h at 37 °C with and without Chi-TY-AuNPs (200 μg/mL) in a YPD medium supplemented with 10% FBS. Inhibition of C. albicans hyphal development was visualized using a fluorescence microscope (EVOS-FL, Advanced Microscopy Group, USA) at 60× and compared with the positive and negative C. albicans control groups.

Effect of Chi-TY-AuNPs on Candida Biofilm Inhibition and Eradication

The method to determine the effectiveness of Chi-TY-AuNPs in the inhibition and eradication of the Candida (C. albicans and C. glabrata) biofilm was adapted from Gupta et al.[22] After attaining the log phase, cells of both Candida spp. were suspended in PBS (pH 7.0) to attain a cell count of 1 × 107 cells/mL, following which 100 μL of cell suspension was added to each well of MTP. For the attachment of cells (adhesion phase) to the MTP wells, the suspension was subjected to incubation at 37 °C for 1.5 h. MTP wells were then washed two times with PBS followed by the addition of RPMI (200 μL) media containing different concentrations of Chi-TY-AuNPs (0, 25, 50, 100, 200, 400, and 800 μg/mL) for biofilm formation assay. XTT reduction assay was employed to quantify the developed biofilms followed by a 24 h incubation period.

For biofilm eradication studies, the wells of MTP plates were supplemented with 200 μL of RPMI media without nanoparticles after the adhesion phase. The plates were incubated for 48 h at 37 °C for mature biofilm development. After incubation, the wells were washed with PBS and RPMI media containing Chi-TY-AuNPs were added and further incubated for 20 h. The wells were again washed with PBS, and the biofilm was quantified by XTT reduction assay. Biofilm inhibition and eradication efficacy of Chi-TY-AuNPs were described in terms of biofilm inhibitory concentrations 80 (BIC80) and biofilm eradication concentration 80 (BEC80), at which 80% biofilm growth was inhibited.

Field Emission Scanning Electron Microscopy (FESEM) Analysis

Morphological alterations in Chi-TY-AuNP-treated Candida (C. albicans and C. glabrata) biofilms were visualized using FESEM. The biofilms were developed on a polystyrene disk of 1 cm2, placed in a 24-well plate, and incubated with FBS for 24 h. The wells were added with 1 × 107 cells/mL of cell suspension and were incubated at 37 °C for 48 h. Later, each well having PBS washed disks were added with RPMI containing Chi-TY-AuNPs. Disks were washed with PBS, incubated for 4 h in the dark in 2.5% glutaraldehyde, and then dehydrated using a gradient of ethanol. Samples were air-dried and mounted on stubs for gold sputtering. Visualization of samples was performed using FESEM (voltage: 20 kV; magnification: 1000–5000×).[22]

Fluorescence Microscopy Analysis

Live and dead cells in Chi-TY-AuNP-treated biofilms of both Candida spp. were visualized under a fluorescence microscope using fluorescein diacetate (FDA) and propidium iodide (PI) staining. For this study, biofilms of both Candida spp. were developed in MTP, as described earlier in the presence of Chi-TY-AuNPs. The biofilms were washed with PBS after treatment with Chi-TY-AuNPs (100 μg/mL) and stained with the FDA and PI at concentrations of 2 and 0.6 μg/mL, respectively. After 20 min of incubation in the dark, PBS-washed wells were visualized under a fluorescence microscope at 40× magnification.

Determination of Ergosterol Content

For spectrophotometric determination of ergosterol concentration in the cell membrane, the log-phase cells of both Candida spp. were incubated in Sabouraud dextrose broth consisting of TY, Chi-AuNPs, and Chi-TY-AuNPs for 20 h at 37 °C.[22] Subsequently, the cells were pelleted down for 5 min at 6000 rpm. The wet weight was measured after washing the cell pellet with Milli-Q water followed by dissolving in 3 mL of 25% alcoholic potassium hydroxide (lysing agent) and vortexing. Incubation of the cell suspension was done in a water bath for 1 h at 85 °C, and a mixture of n-heptane and distilled water (1:3 ratio) was added followed by vigorous vortexing. The mixture was left undisturbed for 20 min at room temperature. The sterol containing the heptane layer was pipetted gently into the glass tube and was stored at −20 °C. After that, a mixture of absolute ethanol (100 μL) and sample (20 μL) was added to analyze the sterol extracts using a UV–visible spectrophotometer (230–300 nm). Ergosterol content was quantified with the help of the following equation:

where the dilution factor, E value for crystalline ergosterol, and E value for 24(28)-dehydro ergosterol were represented by F, 290, and 518, respectively.

Reactive Oxygen Species (ROS) Generation Assay

The level of ROS in both Candida spp. biofilms upon exposure to TY, Chi-AuNPs, and Chi-TY-AuNPs were determined using 2,7-dichlorodihydrofluoroscein diacetate (DCFDA) and PI.[22] Briefly, 48 h of mature biofilms were treated with TY, Chi-AuNPs, and Chi-TY-AuNPs for 4 h. A mixture of DCFDA (10 μM) and PI (1 mg/mL) was added to the MTP wells with biofilms. After 30 min of incubation in the dark, the fluorescence of DCFDA (λex = 520 nm and λem = 485 nm) and PI (λex = 617 nm and λem = 543 nm) was measured to assess the level of ROS. Further, a fluorescence microscope was used to capture the microscopic fluorescent images at 40× magnification.

Estimation of Biochemical Composition of ECM

For biochemical characterization of ECM, Candida biofilms were developed in a 24-well plate for 48 h and then treated with TY, Chi-AuNPs, and Chi-TY-AuNPs for 18 h. After incubation, wells were washed, and the attached biofilms were scrapped with a sterile scrapper in PBS. The biofilms were sonicated in ice using a Q700 sonicator (QSonica, 35 W) for 5 cycles (30 s each) followed by the centrifugation of suspension for 5 min at 12,000 rpm, after which the supernatant was collected. The estimation of protein and eDNA in ECM was performed with phenol and chloroform and isoamyl alcohol (PCI) and a BCA kit, respectively. For protein estimation, bovine serum albumin was employed as the standard, and the absorbance of samples was measured at 562 nm. For eDNA estimation, the samples were added with 1/10th volume of 3 M sodium acetate followed by the addition of PCI (25:24:1), resulting in the formation of an aqueous layer. The eDNA was precipitated with ethanol (2.5 volumes), and the aqueous layer was collected in a fresh tube. A Nanodrop (Thermo Fisher Scientific, USA) spectrophotometer (A260/280) was used to check the purity of eDNA.

Hydrophobicity Assay

The hydrophobicity of both Candida spp. was measured by exposing overnight-grown cells to a sublethal concentration of TY, Chi-AuNPs, and Chi-TY-AuNPs at 37 °C for 24 h. Subsequently, after incubation, the PBS-washed cells were suspended in 50 mM sodium phosphate buffer (3 mL; pH 7.0) at a 2 × 106 cells/mL concentration after the addition of 500 μL of octane. The cells were then vortexed for 1 min that resulted in the formation of an aqueous layer. The cells of the aqueous layer were observed at OD600, and the hydrophobicity index (HI) was calculated using the following formula:

where A1 and A2 indicate the absorbance of the inoculum and aqueous phase, respectively.

Transcriptional Analysis

The effect of the subinhibitory concentrations of TY, Chi-AuNPs, and Chi-TY-AuNPs on selected gene expression of C. glabrata was assessed with the help of qRT-PCR.[22] Briefly, log-phase C. glabrata cells were subjected to 3 h of incubation with TY, Chi-AuNPs, and Chi-TY-AuNPs. The RNeasy kit (Qiagen, Germany) was used for extraction of total RNA following the manufacturer’s protocol. The Nanodrop spectrophotometer was utilized for qualitative and quantitative analysis of RNA. A Verso cDNA synthesis kit (Thermo Fisher Scientific, USA) was used to synthesize cDNA from 1 μg of extracted RNA. The primers of selected genes were procured from Integrated DNA Technologies, India, and RT-PCR was performed using an SYBR green master mix for 100 ng of cDNA template and 300 nM of gene-specific primers to make each reaction. For RT-PCR, the following conditions were used: the first denaturation cycle was performed at 95 °C/3 min followed by annealing at 60 °C/30 s and extension at 72 °C/30 s, and the process was repeated for 40 cycles; melting-curve analysis starting from the initial temperature 45 to 95 °C, with a gradual increase in 0.5 °C/15 s. Melt-curve analysis was used as an indicator of primer specificity. The cycle threshold (CT) values of the housekeeping ACT1 gene were used to normalize the CT values of target genes. The ΔΔCT method using the 2–ΔΔ formula was utilized to evaluate the relative expression fold changes.

To determine the cytotoxic effect of the prepared nanoformulation, the mouse fibroblast NIH3T3 cell line was utilized. The cell line NIH3T3 was procured from National Centre for Cell Science (NCCS) Pune, India. The cell line was cultured in DMEM high glucose media (Himedia, AT007) supplemented with 10% FBS (Sigma, Germany) and 1% antibiotic solution (penicillin, streptomycin, and amphotericin B; Himedia). NIH3T3 was maintained at a 37 °C temperature in a carbon dioxide (CO2) incubator supplied with 5% CO2. For cell viability assessment, about 5000 cells were seeded in a 96-well plate and incubated with various concentrations of Chi-TY-AuNPs and Chi-AuNPs (prepared in a DMEM high glucose medium; final volume: 100 μL) for 24 h, after which they were stained with 10 μL of MTT dye (stock concentration: 5 mg/mL). The reaction was terminated after 4 h with the addition of DMSO. The absorbance of purple formazan particles was measured at a 570 nm wavelength in a multiwell plate reader (Cytation3, Biotek, USA), and the results were obtained as a percentage compared to the control.[23]

Statistical Analysis

All experiments were performed in triplicate, and the values were presented as the mean ± standard deviation (SD) obtained from three different observations for each assay. Student’s t-test was used for the statistical analysis, and a value of *P < 0.05 was considered statistically significant, **P < 0.01 as highly significant, and ***P < 0.001 as extremely significant.