Candida albicans (ATCC 90028, American Type Culture Collection, Manassas, VA, United States) was received from the Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. After collection, the culture was revived in YPD (Yeast extract peptone) broth (supplemented with 1% glucose) at neutral pH (7.2) and preserved in 25% glycerol at-80oC for further investigation. Polyethyleneimine, silver nitrate (AgNO₃), calcofluor white, bovine serum albumin (BSA), tryptophan, tyrosine, amphotericin B, and other routine chemicals were obtained from Sigma Aldrich Chemicals Private Limited (Bangalore, Karnataka, India). RPMI (Rosewell Park Memorial Institute media) and other media constituents were obtained from Hi-Media Laboratories Ltd. (Mumbai, Maharashtra, India). Plasticware was obtained from Tarsons Products Limited (Kolkata, West Bengal, India). The solvents, including ultra-purified water, were obtained from Merck Life Science Private Limited (Bangalore, Karnataka, India). All of the reagents were of analytical grade. The generated experimental data were plotted and analyzed using Origin 8.5 software (Origin Lab Corporation, Northampton, MA, United States).

The polyethyleneimine (PEI)-functionalized Ag-NPs were synthesized as reported previously with slight modifications (Tiwari et al., 2020). In a 2 mL glass vial, ethylene glycol diacetate (8–10%) and a methanolic solution of 1-vinyl 2-pyrrolidone (VPP, 50 μL from a 50 mM stock solution) were combined; a methanolic solution of AgNO₃ (10 mM solution), cyclohexanone (20 μL), and PEI (16.4 mg/mL; 40 μL) was added to the mixture. The mixture was thoroughly agitated on a vortex mixer for 30 s and subsequently placed in a microwave oven; for complete reduction of the Ag + cations, the 5 s cycle was repeated four to six times. The resulting mixture exhibited a deep brown color, which denoted the creation of PEI-f-Ag-NP. The nanoparticles were reconstituted in ultra-pure water to conduct additional characterization studies.

The synthesized Ag-NPs were characterized using a U-2900 UV–Vis spectrometer (Hitachi, Tokyo, Japan); 200–800 nm wavelengths were used for data collection. Transmission electron microscopy of the Ag-NPs was performed using a Tecnai G2 20 Twin (FEI, Hillsboro, OR, United States); the sample was diluted in methanol and then drop-cast over 300 mesh carbon-coated copper grids. Zeta potential analysis of the samples was accomplished with a Zetasizer instrument (Malvern Panalytical, Malvern, UK). X-ray diffraction analysis of synthesized nanoparticles was performed by forming a film of the sample on a glass coverslip and drying the sample at 70°C for 2 h. Spectra were recorded using a MiniFlex 600 X-ray diffractometer (Rigaku, Tokyo, Japan).

The agar well diffusion method was utilized to evaluate the antifungal activity of functionalized silver nanoparticles. In short, log-phase cells of C. albicans in RPMI media were adjusted to 10⁶ cfu/mL, swabbed on MHA (Muller Hinton ager) plates with a sterile cotton swab, followed by surface drying for 10 min. Agar wells were formed through the wide mouth of the sterile pipette tip. A 20 μL of constituted silver nanoparticles was poured into an agar well and incubated at 28°C for 48 h.

The broth microdilution method was used to obtain the MIC (Minimum Inhibitory Concentration) value of PEI functionalized Ag-NPs against chosen fungus; as described previously, a sterile flat-bottom 96-well microtiter plate was used for the study (Andrews, 2001; Cavaleri et al., 2005; Li et al., 2005). In short, log-phase cells of C. albicans grown in RPMI broth were centrifuged, resuspended in a fresh RPMI medium, and adjusted 0.5 MacFarland standard fungal suspension for MIC determination measurements (McFarland, 1907). An active suspension of 50 μg/mL of functionalized Ag-NPs was prepared in ultra-pure water and diluted serially; the final concentration was obtained as 0.43–50 μg/mL. Hundred microliter of the fungal suspension was then dispensed into each well. Amphotericin B was used as a positive control in this study. The microtiter plate was incubated at 28°C for 24 h; a visual demonstration of complete fungal inhibition (i.e., a visually clear well) was assigned the MIC value. Subsequently, a 5 μL suspension from a visually clear well was sub-cultured on the YPD plates for 24 h and fungal growth was noted. To ensure the accuracy and minimizing the handling errors, all measurements were performed in triplicate.

The log phase culture of C. albicans in RPMI media was obtained; it was centrifuged at 3500 rpm for 4 min, washed twice with PBS (Phosphate Buffer Saline) to remove media traces, and resuspended in phosphate buffer. The least cell count (5×10² cells/mL) was adjusted in a 1 cm quartz cuvette to avoid an inner filter effect. A F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) was used to obtain fluorescence emission (FL emission) spectra at room temperature. The routine excitation wavelength for proteins (λ = 280 nm) was used to obtain the emission spectra of C. albicans cells and bovine serum albumin V (BSA) (10 μL of 1 μg/mL). In comparison, λ = 270 nm was utilized for tryptophan and tyrosine (10 μL of 1 μg/mL) in phosphate buffer (pH 7.2). The slit width values for excitation and emission were set to 5.0/10 nm, respectively. The 3D FL spectra were recorded at λex/em = 280/300 nm matrix. The number of scans was 20 at 10 nm per scan increment. Further, FL spectra were recorded as a control to observe the effect of UV exposure on the C. albicans cells. The measurements were made in triplicate in phosphate buffer (pH 7.2).

To investigate the antifungal mechanism, the intracellular ROS level in treated cells was evaluated using an oxidation-sensitive fluorescent dye called 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA, Molecular Probes, Eugene, OR, United States). The cells were washed with PBS buffer and incubated with PEI-f-Ag-NPs for 2 h at 28°C. After centrifugation, the cells were incubated with H₂DCFDA for 30 min in the dark and washed twice. Ten thousand counts were collected; the FL intensity was measured using a cytoFLEX LX flow cytometry instrument (Beckman Coulter Inc., Pasadena, CA, United States) (Seong and Lee, 2018).

The silver nanoparticle was stabilized and functionalized by branched polyethyleneimine with a molecular weight of 60,000 Da. The synthesized silver nanoparticles were characterized using UV–Vis spectroscopy, TEM, zeta potential, and X-ray diffraction methods. Figure 1A shows the UV–Vis absorption spectra at 419 nm; these results are consistent with the characteristic absorption band of Ag-NPs from surface plasmon resonance (SPR). The size of the PEI-functionalized silver nanoparticles in a water solution was recorded using TEM (Figures 1B,D). The average size of the PEI-f-Ag-NPs was 5.6 ± 1.2 nm (mean ± SD) with a spherical shape; the corresponding histograms revealed that the particle size distributions were between 3–9 nm and revealed near monodispersed distribution (Figure 1D). In addition, the ζ potential of silver nanoparticles was recorded as 18.2 ± 2 mV, indicating high stability (Figure 1E). The colloidal stability of synthesized silver nanoparticles was ensured by interparticle electrostatic repulsion. The XRD pattern was recorded for the 2θ angle from 5 to 80 degrees. The generated diffractogram was compared with the standard powder diffraction card of JCPDS, silver file No. 04–0783. The observed Peaks at 2θ degrees of 38.116, 44.583, 64.418, and 77.438 degrees in the diffractogram were noted to correspond to (hkl) values of the (111), (200), (220), and (311) planes of silver as indicated in Figure 1C. Thus, the XRD study confirmed that the functionalized silver nanoparticles possess a face-centered cubic crystal structure.

The synthesized PEI-f-Ag-NPs were evaluated for their antifungal activity by the agar well diffusion method. The 16 ± 2 mm zone of inhibition indicated potent antifungal activity against C. albicans (Figure 2A). Further, the observed MIC value of PEI-f-Ag-NPs against C. albicans was 5 ± 0.5 μg/mL. Furthermore, the cells were treated with PEI-f-Ag-NPs at their MIC value (5 μg/mL) in a time-dependent manner (5, 15, 30, 60, 120, and 240 min); the cell morphology was observed using a compound light microscope (Figures 2B–H). The cell wall/membrane was damaged (a visual pit hole formation) over time and showed a strong antifungal activity within 30 min of treatment (a reduction of ~70% cell viability). Complete damage to cellular architecture was observed at 240 min of treatment.

All of the FL spectroscopic experiments were performed at room temperature and in phosphate buffer having neutral pH. The number of C. albicans cells and the concentration of functionalized silver nanoparticles per ml were kept low to avoid any inner filter effect. A control experiment was performed by exposing cells for a short time to evaluate the effect of UV light on proteins, which shows a slight effect (Figure 3). The FL result showed that PEI-f-Ag-NPs quenched the autofluorescence of C. albicans surface protein with an increasing time of incubation (incubated for 5, 15, 30, 60, 120, and 240 min) (Figure 4A); a similar result was noted with an increasing concentration (1, 2, 3 and 5 μg/mL) (Figure 4C). The characteristic FL quenching by PEI-f-Ag-NPs indicates strong binding with C. albicans cells at the nano-bio interface. The autofluorescence of the C. albicans cell surface protein quenching ratio by PEI-f-Ag-NPs was assessed with an increasing incubation time and concentration of silver nanoparticles, which showed rapid binding (Figures 4B,D). The plot of the quenching ratio against incubation time indicated multiple binding sites on the C.albicans surface for PEI-f-Ag-NPs and binding saturation over time. FL quenching achieves a steady state at 60 min of incubation because of binding site saturation (Figure 4B). Similarly, with an increasing concentration of PEI-f-Ag-NPs, the FL quenching ratio was reached at a steady state at 5 μg/mL. This result can be interpreted as saturation of binding sites on the cell surface (Figure 4D). A similar saturation effect was previously described by Wilhelm et al. (2002); however, they studied HeLa cells to understand the interaction dynamics of anionic super magnetic iron oxide nanoparticles. Further, Zheng et al. reported that smaller hematite nanoparticles had faster adsorption than larger ones on the E. coli surface (Zhang et al., 2010). These studies strongly support the rationality and underlying binding mechanism and the adsorption dynamics of functionalized silver nanoparticles on C. albicans cells surface.

Fluorescence involves short-term emission of light (of no greater than 10⁻⁸ s after excitation), which is associated with the absorption of electromagnetic radiation (e.g., infrared visible, or ultraviolet light) (Monici, 2005). The term “autofluorescence” is used to differentiate the intrinsic fluorescence associated with cells and tissues from the fluorescence associated with exposing samples to exogenous fluorescent markers that attach to cell and tissue components. The autofluorescence properties of biomolecules have been previously utilized to identify pathogenic microorganisms and cellular biomolecules (Richards-Kortum and Sevick-Muraca, 1996; Fusi et al., 2002).

FL quenching describes phenomena that are associated with a reduction in the FL intensity (Monici, 2005). These phenomena can be associated with intermolecular interactions (e.g., energy transfer, molecular rearrangements, and excited-state reactions); moreover, the quenching phenomena can exhibit dynamic (e.g., related to diffusive encounters between the quencher and the fluorophore within the excited state lifetime) or static (e.g., related to complex formation between the quencher and the fluorophore in the ground state (Monici, 2005). C. albicans exhibits an outer rigid cell wall structure covered by the cell membrane, which is composed of complex sugar moieties such as β-glucans and several anchored proteins that act as receptors for exogenous ligands. When excited with UV irradiation, these surface-anchored proteins fluoresce due to the aromatic amino acid tyrosine, tryptophan, and phenylalanine and act as a binding site for PEI-f-Ag-NPs. The fluorescence quenching data were correlated with the time-dependent kill kinetics of the cell; the results indicate that cellular architecture was destabilized as treatment time increased from 5–240 min (Figures 2, 4).

To better understand the interaction of PEI-f-Ag-NPs with molecules other than proteins, the cells were stained with Evans blue and calcofluor white for 15 min and then washed with water; 2D and 3D fluorescence spectra were obtained in the presence of PEI-f-Ag-NPs at various times. Calcofluor white (CFW) is a fluorescent blue-colored dye that is used for diagnosing fungal onychomycosis, which binds to the 1–3 and 1–4, β-linkage of chitin and cellulose in fungal, plant, and algal cell walls. The excitation wavelength for CFW is 380 nm; the emission maxima are between 440 and 475 nm (Shetty et al., 2019). The results showed that PEI-f-Ag-NPs quenched the fluorescence of CFW within 5 min; however, complete quenching was recorded at 120 min of incubation (Figure 5).

The function of electrostatic interactions between the cell surface and charged nanoparticles has previously been described for cellular uptake (Farquhar, 1978; Ghinea and Simionescu, 1985; Mutsaers and Papadimitriou, 1988). Cationic liposomes were noted to bind more efficiently than anionic and neutral ones (Lee et al., 1993; Miller et al., 1998; Chenevier et al., 2000). Cationic ferritin particles were noted to uniformly adsorb to the plasma membrane of fixed mammalian cells; this phenomenon was attributed to the many large anionic domains on the cell surface (Farquhar, 1978; Mutsaers and Papadimitriou, 1988). These previous findings, strongly supporting our approach to study the inter-molecular interactions at nano bio interface. Since, our present study was dedicated to qualitative investigation of functionalized silver nanoparticle binding on living system surface biomolecules.

Additional studies were carried out to investigate the specific binding of PEI-f-Ag-NPs with the protein BSA and selected aromatic amino acids, tryptophan and tyrosine residues. The FL emission of the BSA-PEI-f-Ag-NP system was measured in phosphate buffer at neutral pH and room temperature. The BSA was incubated with functionalized silver nanoparticles at various times and concentrations similar to C. albicans cells. BSA (Bovine serum albumin) is comprised of 607 amino acids (66 kDa), with 24 tyrosine residues and two tryptophan residues (Ray et al., 2009). Tyrosine and tryptophan residues exhibit intrinsic fluorescence; the tryptophan emission dominates the UV fluorescence spectra for BSA (Ray et al., 2009). The results indicated that the silver nanoparticles bound and quenched FL of BSA with increasing contact time and PEI-f-Ag-NP concentration similar to C. albicans cells with a comparable dynamic (Figure 6). The characteristic emission band at 340 nm decreased as increment in concentration of PEI-f-Ag-NPs; this correlation suggested a strong interaction between nanoparticles and BSA. Mariam et al. performed a similar study in which 10 mg/mL BSA and 90–812 mL Ag-NPs were used (Mariam et al., 2011). In another study, Huang et al. described the interaction between gold nanoparticles and BSA (Huang et al., 2014). The quenching ratio was studied; with an increase in incubation time and concentration, the binding of BSA on nanoparticles achieved a steady state (Figure 6); this result indicated the saturation of vacant binding sites on silver nanoparticles. Additional studies are underway to estimate the number of bindings of BSA molecules on the surface of a single PEI functionalized silver nanoparticle.

The interaction of PEI-f-Ag-NPs with tryptophan and tyrosine residues was investigated in a more detailed study as a standard fluorophore. Tryptophan and tyrosine exhibit intrinsic emission upon excitation on 280 and 270 nm, respectively. Figure 7 shows the FL emission spectra and quenching ratio of tryptophan and tyrosine recorded with various PEI-f-Ag-NPs concentrations. The fluorescence intensity of tryptophan and tyrosine was noted to decrease as the silver nanoparticle concentration was increased. Silver nanoparticles were able to interact with tryptophan and tyrosine, thus quenching the fluorescence intensity. PEI-f-Ag-NPs exhibit strong interactions with tyrosine compared to tryptophan residues, indicating a strong probability for interactions with tyrosine-rich proteins. The presented work conclusively, demonstrating a complex formation between cell surface anchored proteins and functionalized silver nanoparticle and quenched the fluorescence of protein with a saturation kinetic model. At this point, it is obvious to study, whether, functionalized nanoparticles are binding on specialized cell surface domains or binding uniformly. Depending on the type of cells, the distribution and nature of surface biomolecules varies. Thus it is interesting to note the cell and functionalizing agent specific binding kinetics of nanoparticle interaction with cells.

The modified Stern-volmer equation (double logarithm regression curve) can be used to obtain the binding constant as well as the number of binding sites/macromolecules, in which it is presumed that PEI-f-Ag-NPs bind independently to cell surface macromolecules (equivalent sites) (Hu et al., 2004; Xu et al., 2011). log F 0 − F / F = log K b + n log Q .

In this equation, Kb represents the binding constant, F₀ represents fluorescence intensity without a quencher, F represents fluorescence intensity with a quencher, and n represents the number of binding sites (Xu et al., 2011). The number of binding sites and the binding constant were obtained for C. albicans cells. Plots of log [(F₀–F)/F] versus log [PEI-f-Ag-NP] are shown in Figure 8A. The values of Kb and n were determined from the intercept on the y-axis and the slope of this plot, respectively. The Kb and n values of PEI-f-Ag-NPs for C. albicans cells were 1× 10⁵ M⁻¹ and 1.01, respectively (Figure 8A). The high binding constant values shows strong binding between PEI-f-Ag-NPs and C. albicans cell surface. Also, it is indicated that each interacting cell anchored protein can bind to 1.0 PEI-f-Ag-NP at a time. However, this phenomenon may be different for various types of cells because different types of cells have variable biomolecules composition with distinct physico-chemical properties. The apparent binding constant is dependent on several physical parameters, including the concentration of Ag-NPs and temperature; further, it is also dependent on the size-shape and surface tuning parameters of the Ag-NPs, which in turn are dependent on the synthesis protocol (Dasgupta et al., 2016). It should be noted that the apparent association constant is also dependent on the temperature; the role of temperature was not considered in this study as the focus of the study was to determine the nature and dynamics of the interaction. The binding constants (Kb) indicate that the affinity of PEI-f-Ag-NPs for BSA is very low in comparison to the reported binding constants, which range from 10⁴ to 10⁸; this finding supporting previous reports that serum albumin contains few binding sites for endogenous and exogenous ligands, which are commonly bound in a reversible manner (Rahman and Sharker, 2009). Hence, the above result concluded that, PEI-f-Ag-NPs have a strong binding tendency with complex cell wall polysaccharides and surface anchored proteins. The microscopic demonstration of surface binding of PEI-f-Ag-NPs on the C. albicans cell can be seen in Figure 8B.

Evidence of the antimicrobial use of silver ions appears in the historical record (Alexander, 2009). The antimicrobial mechanism of silver, either in a free ionic form or in a nanoparticle form, is not clearly defined. The known mechanism of silver indicates a multidimensional action to disrupt many fundamental functions of the microbial cell (Rai et al., 2012; Mikhailova, 2020). The antimicrobial activity of Ag-NPs is dependent on their shape and size; some of the observed effects can be associated with the release of Ag⁺ ions from the nanoparticle surface (Kędziora et al., 2018; Tang and Zheng, 2018). The cationic Ag-NPs exhibit a high affinity for the anionic surface of microorganisms (Mikhailova, 2020). Once nanoparticles adhere to the microbial surface, they initiate a “pitting” effect in the cell wall, which is associated with architectural collapse; this process results in leakage of intracellular contents, dissipation of the transmembrane potential, as well as denaturation to membrane-associated proteins (Tang and Zheng, 2018; McNeilly et al., 2021). Silver nanoparticles also initiate the production of toxic intracellular ROS that result in cell damage (McNeilly et al., 2021). The antifungal mechanism in PEI-f-Ag-NPs treated C.albicans cells in this work is associated with a huge generation of endogenous ROS accumulation and pit hole formation in cell wall (Figure 9). As seen in Figure 8B, it appears that intracellular ROS was the main cause of cell damage. Our previous study found that PEI-f-AgNP-1 interacts with Rhizopus arrhizus sporangiospores by generating stress, followed by the generation of ROS species, which ultimately damages the spore wall (Tiwari et al., 2022). A schematic representation of antifungal mechanism associated with the PEI-f-Ag-NPs against C. albicans cells can be seen in Figure 10.