ROS-dependent anticandidal activity of zinc oxide nanoparticles synthesized by using egg albumen as a biotemplate

Clinical isolate of C. albicans 077 was obtained from Department of Microbiology, JN Medical College, Aligarh, UP, India. Stock culture was maintained on slants of SD agar (containing dextrose 40 g l⁻¹, mycological, peptone 10 g l⁻¹ and agar 15 g l⁻¹) at 4 °C. The primary culture of the C. albicans 077 was prepared from the stock slant into the SD broth medium and incubated at 37 °C for 48 h (stationary phase, 10⁸ cfu ml⁻¹). The primary culture (∼1 ml) was re-inoculated into the 50 ml fresh SD broth and grown for ∼12 h up to mid-log phase (∼10⁵ cfu ml⁻¹) at 37 °C. All experiments were performed from the mid-log phase (∼10⁵ cfu ml⁻¹) freshly grown C. albicans 077 culture in triplicates.

For agar disc diffusion assay, 5 ml mid-log phase grown of C. albicans 077 was centrifuged at 4000 rpm for 5 min at 4 °C. Then, the pellet was washed with 1 × phosphate buffer saline (PBS) and resuspended in 500 μl normal saline solution (NSS). A 100 ml of the suspended cells were spread uniformly on SD agar plates and the plates were incubated at 37 °C for 30 min. The seeded petri plates were used for loading various concentrations of EtZnO NPs (0, 5, 10 and 15 μg ml⁻¹) onto the pre-sterilized filter paper discs. The petri plates were incubated at 37 °C for 40 h, after that zone of inhibition was recorded.

Cell suspension of C. albicans 077 was obtained similarly as described in the agar disc diffusion assay and suspension (100 μl) was dispensed into the 96-well microtiter plate in triplicates. Various concentrations of EtZnO NPs (0, 5, 10 and 15 μg ml⁻¹) diluted in sterile SD broth medium were added and incubated at 37 °C for 2 h. The whole suspension of the plate wells was spread on the SD agar plate and incubated at 37 °C for 40 h. Anticandidal activity was detected by the dose dependent reduction in cfu ml⁻¹.

To see the effect of EtZnO NPs on the growth kinetics of C. albicans, 50 ml of SD broths in individual flask were inoculated with 100 μl of the NSS suspended cells. Different concentrations of EtZnO NPs (0, 5, 10 and 15 μg ml⁻¹) to be tested were applied in the individual flask. The flasks were incubated at 37 °C for 40 h and time dependent growth kinetics were recorded turbidometrically at A_595nm. The turbidity backgrounds induced by EtZnO NPs were subtracted from the final reading.

The crystal structure of EtZnO NPs was characterized by XRD with CuK_α radiation (λ = 0.15418 nm). The data revealed that the well resolved 11 XRD peaks were obtained at 2θ = 31.01°, 34.21°, 35.64°, 47.10°, 56.02°, 62.15°, 65.68°, 67.51°, 69.01°, 72.08° and 76.24° which correspond to the crystal planes [100], [002], [100], [102], [110], [103], [200], [112], [201], [004] and [202] of polycrystalline wurtzite structure (Zincite, JCPDS 5-0664), respectively (figure 2(a)). The XRD data of EtZnO NPs indicated the absence of any impurities, which attested its high quality. The average crystallite size (D) of EtZnO NPs was calculated following the Debye–Scherrer formula

$$\begin{eqnarray*} &&D = \frac{{0.9\lambda }}{{B\cos \theta }},\end{eqnarray*}$$

where constant 0.9 is the shape factor, λ is the x-ray wavelength of CuK_α radiation (1.54 Å), θ is the Bragg diffraction angle and β is the full-width at half-maximum (FWHM) of the (101) plane diffraction peak. The calculated average particle size was found to be ∼16 nm.

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The nanostructure of synthesized EtZnO NPs was analyzed by SEM, TEM and AFM. The figure 2(b) showing the scanning electron micrograph recorded from EtZnO NPs film deposited on a carbon tape clearly demonstrates the formation of secondary EtZnO NPs (average size ∼1 μm). The elemental analysis using EDAX indicates the presence of only zinc and oxygen in as-synthesized EtZnO NPs (figure 2(c)). Moreover, the EDAX mapping result again shows that there are no other elemental impurities present in the synthesized EtZnO NPs (figure 2(d)). The TEM image clearly showed the EtZnO NPs to be actually composed of several particles of different sizes grouped in clusters (figure 2(e)). It can also be seen that EtZnO NPs have a spherical morphology with a size range of 10–20 nm, which is consistent with XRD and optical results [38].

TEM characterization of EtZnO NPs revealed that all NPs were stable, well dispersed, smooth and the agglomeration might be due to the preparation technique. The particles were deposited on a copper grid and drying promotes agglomeration [37, 39]. The AFM images also display the spherical morphology of EtZnO NPs in the size range of ∼8–22 nm (figures 2(f) and (g)), which is consistent with results obtained by the XRD and TEM. Thus, the data confirmed that the EtZnO NPs were successfully synthesized using the egg albumen as a biotemplate.

The comparative analysis of the FTIR spectra revealed possible interactions between egg albumen and ZnO NPs. The FTIR spectrum of EtZnO NPs shows absorption band at 511 cm⁻¹, which corresponds to E₂ mode of hexagonal ZnO wurtzite structure [40]. The band at the position 511 cm⁻¹ reflects EtZnO NPs stretching frequency of Zn–O bonds. The intermediate product hydrozincite (without sintering) shows the absorption band at 677 cm⁻¹ due to the presence of the hydroxide phase of the EtZnO NPs (figure 3). The sintering process also indicated the disappearance of the egg albumen signature peaks which confirmed its decomposition from the EtZnO NPs. The comparative FTIR spectra confirmed that the transformation of the hydroxide to the oxide phase occurred during the sintering process of EtZnO NPs. The bands at ∼1557 and 3452 cm⁻¹ were due to the stretching frequency of hydroxyl groups of absorbed water from ambient atmosphere [41, 42].

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The electronic structure of ZnO NPs is characterized by the band gap (E_g), which is essentially the energy interval between the valence band (E_v) and the conduction band (E_c), each of which has a high density of states [28]. The generation of a specific type of ROS such as ^•OH, ¹O₂, or O₂^•– is governed by the metal oxide NPs related to the electronic structures as well as the redox potentials (EH) of different ROS generation reactions [28, 43, 44]. The oxidative stress induced by ZnO NPs is thought to be the main mechanism of their antimicrobial activity [1, 9, 28, 43–45]. Therefore, we calculated the electronic band gap energy (E_g) of EtZnO NPs because of their broad application in antimicrobial properties. The ZnO-NPs (10 μg ml⁻¹) were dispersed in ethanol by using ultra sonication and then the solution was used to perform the UV–Vis measurement (figure 4(a)). The spectrum reveals a characteristic absorption peak of EtZnO NPs at wavelength of ∼A_360nm which can be assigned to the intrinsic band-gap absorption of EtZnO NPs due to the electron transitions from the valence band to the conduction band (O_2p → Zn_3d) (figure 4(a)) [46, 47]. The sharp absorption peak of EtZnO NPs also indicated the narrow nanosize particle distribution. Moreover, the egg albumen showed the absorbance at wavelength of ∼A_280nm due to the presence of the proteins in their composition (figure 4(a)). Thus, results indicate that the egg albumen does not influence the absorption of EtZnO NPs, suggesting that EtZnO NPs were fully functional. The electronic band gap (E_g) of the EtZnO NPs was determined by employing Tauc relationship as follows:

$$\begin{eqnarray*} &&\alpha h\nu = A(h\nu - E_{\rm{g}} )^n , \end{eqnarray*}$$

where α is the absorption coefficient (2.303A/t), h is Planck's constant, ν is the photon frequency, and E_g is the elctronic band gap. The value of n = 1/2, 3/2, 2 or 3 depending on the nature of the electronic transition responsible for absorption and n = 1/2 for direct band gap semiconductor. An extrapolation of the linear region of a plot of (αhν)² on the y axis versus photon energy (hν) on the x-axis gives the value of the E_g as shown in figure 4(b). The E_g of EtZnO NPs was determined to be 3.55, which was the higher than bulk ZnO NPs powder (E_g =3.37 eV) [48]. The high value of the E_g is possibly attributed due to the quantum confinement effect of the NPs. The widening effect might be related to the influence of numerous factors such as structural parameters (size and pH), carrier concentrations and the presence of defects (oxygen vacancies), which may lead to the Burstein–Moss shift [49, 50].

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The optical properties of ZnO are more interesting since confinement of charge carriers in the restricted volume of the small particles can lead to effects such as widening of E_g [51]. Since the EtZnO NPs have a wide E_g = 3.55 eV good electron transporting properties [52], they can be utilized as an anticandidal agent, which can efficiently kill the C. albicans via ROS production. The data suggest that the egg albumen facilitates the electronic band gap widening effect via controlling the nucleation and surface capping of the intermediate products (hydrogencite) [51]. The amino acid moiety in the egg albumen is sufficient to form proper capping, resulting in the formation of smaller sized EtZnO NPs, so as to keep the wide E_g [53].

The photoluminescence behavior of EtZnO NPs could give information on energies and dynamics of photogenerated charge carriers as well as on the nature of the emitting states [54]. Figure 4(c) shows photoluminescence emission spectra in the visible range of EtZnO NPs and monitoring by measuring the dose-dependent changes in the intensity of EtZnO NPs. The emission spectra have a broad band with a maximum ∼532 nm which can be ascribed to the singly ionized oxygen vacancy with exited EtZnO NPs at ∼A_370nm. The green emission in the visible region arises when a photogenerated hole (O^–) trapped at a deep level above the valence band recombines with an electron trapped at a shallow level below the conduction band [55]. Usually, the emission intensity and band width are related to the size and nature of the carrier trapped states located at the surface of the nanocrystals [56]. From figure 4(d) we can see that about ∼5% of the total weight loss of EtZnO NPs might be due to the evaporation of water adsorbed on the surface of NPs [57, 58]. The differential thermal analysis (DTA) of EtZnO NPs shows the endothermic reaction peak at 135 °C possibly due to change of phases [59].

In light of the evidence, the rapid global spread of resistant in clinical isolates of C. albicans and new families of antimicrobial agents have a short life assurance, thus, the need to find new anticandidal agents is of supreme importance [61]. Researchers are increasingly turning their attention to nanomaterials, looking for new leads to develop better nano-antimicrobial drugs against MDR strains of C. albicans. In the present study we assessed the anticandidal activity of EtZnO NPs against MDR strain 077 of C. albicans. Anticandidal assays revealed that the EtZnO NPs efficiently suppressed the growth of C. albicans 077 in a dose dependent manner (figures 6(a)–(c)). The cells treated with the EtZnO NPs (15 μg ml⁻¹) also exhibited cavity formation, examined by SEM analysis (figure 6(d)). These cavities possibly reflected the formation of apoptosome in the C. albicans 077 cells, indicating promising anticandidal activity. The untreated sample cells showed a normal pattern of growth with a lag phase of ∼4 h, active exponential phase of 8 to ∼21 h before attaining stationary phase. However, EtZnO NPs led to the suppression of growth and delay exponential phases of C. albicans 077 with minimum inhibitory concentration (MIC) ∼29.7 μg ml⁻¹ (figure 6e), again proving strong antimicrobial activity of EtZnO NPs against C. albicans 077. The MIC is the lowest concentration of the compound at which the microorganism tested does not demonstrate visible growth. The almost complete cessation of growth was observed at 34.1 μg m1^-1 concentration. The obtained anticandidal activity of EtZnO NPs is corroborated with previous published reports on anticandidal nanomaterials [62, 63].

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Recently, our group synthesized biosurfactant stabilized anticancer CdS QDs. This effect was associated with the production of ROS [37]. In light of these results, we concluded that the anticandidal effect of EtZnO NPs may be ROS dependent. As shown in figure 7(a), exposure of C. albicans cells to EtZnO NPs increased the intracellular ROS production in a time- and dose-dependent manner, when compared to untreated control. ROS-mediated oxidative stress is a well known inducer of cytotoxicity and apoptotic cell death in C. albicans [4]. Therefore, we sought to examine the possible role of ROS in anticandidal activity of EtZnO NPs. C. albicans were pretreated with 5 mM of histidine, a known scavenger of hydroxyl radicals (^•OH) and singlet oxygen (¹O₂). The data revealed that the histidine completely abrogates the antimicrobial property of EtZnO NPs (figure 7(b)). This clearly indicates that the anticandidal activity of EtZnO NPs is due to ROS production. The electronic band gap (E_g) structures of the metal oxides NPs with the redox potentials (EH) of the different ROS generation reactions have been proposed [23]. The metal oxide NPs when excited with energy higher than the E_g, the electrons (e^-) of metal NPs were promoted across the band gap to the conduction band (E_c), which creates a hole (h⁺) in the valence band (E_c). The electrons in the E_c and holes in the E_v exhibit high reducing and oxidizing power, respectively [28, 64]. The e^- reacted with molecular oxygen to produce superoxide anion (O₂^•–) through reductive reactions. The h⁺ can extract electrons from water and/or hydroxyl ions to generate ^•OH (figure 8(a)). Taken together, results have confirmed that the ROS principally contributes the anticandidal activity of the EtZnO NPs (figure 8(b)). However, various research articles in the last five years have been published on generation of ROS by various metal-oxide NPs. The literature survey revealed that very limited research has been done on the role of the electronic band gap property of metal-oxide NPs in ROS generation [28, 65].

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