Antifungal activity of titanium dioxide nanoparticles against Candida albicans

Abstract

The unregulated release of titanium dioxide nanoparticles into the environment has raised concern, in particular due to the impact of the nanoparticles on indigenous micro-biome in our ecosystem. This paper reports a study on antifungal activity of titanium dioxide nanoparticles on a healthy growing fungi species, Candida albicans, a known opportunistic pathogen. A quantification of the total cell death was performed using a direct staining method, Trypan blue exclusion assay. Exposure to nanoparticles not only altered the growth rate, but also affected the onset and length of Candida albicans growth phases. The log and the onset of the death phase were shortened and accelerated, respectively. Up to 65% of the Candida albicans were killed after exposure to 100 μg/mL of the anatase titanium dioxide nanoparticles, while only 33% were killed with rutile. A higher dosage and incubation time of the nanoparticles increased their toxicity. Cells suffered from morphological changes upon the nanoparticle exposure, which correlates well with the results showing an altered growth phase culture.

Full Article

Antifungal Activity of Titanium Dioxide Nanoparticles against Candida albicans

Nurul Shahidah Ahmad,^a Norhafizah Abdullah,^a,* and Faizah Md Yasin ^a,b

The unregulated release of titanium dioxide nanoparticles into the environment has raised concern, in particular due to the impact of the nanoparticles on indigenous micro-biome in our ecosystem. This paper reports a study on antifungal activity of titanium dioxide nanoparticles on a healthy growing fungi species, Candida albicans, a known opportunistic pathogen. A quantification of the total cell death was performed using a direct staining method, Trypan blue exclusion assay. Exposure to nanoparticles not only altered the growth rate, but also affected the onset and length of Candida albicans growth phases. The log and the onset of the death phase were shortened and accelerated, respectively. Up to 65% of the Candida albicans were killed after exposure to 100 μg/mL of the anatase titanium dioxide nanoparticles, while only 33% were killed with rutile. A higher dosage and incubation time of the nanoparticles increased their toxicity. Cells suffered from morphological changes upon the nanoparticle exposure, which correlates well with the results showing an altered growth phase culture.

Keywords: Titanium dioxide; Trypan blue exclusion assay; Candida albicans; Antifungal activity; Growth rate

Contact information: a: Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, Serdang, 43400, Seri Kembangan, Selangor, Malaysia; b: Institute of Advanced Technology, Universiti Putra Malaysia, Serdang, 43400, Seri Kembangan, Selangor, Malaysia;

* Corresponding author: nhafizah@upm.edu.my

INTRODUCTION

In recent years there has been a rapid development and application of nanotechnology in a wide range of fields such as biosciences, information of technology, computers, and medicine. With the rapid emergence of nanomaterial incorporated in consumer products, as well as its uncontrolled release into the environment, it is essential to identify the factors associated with its toxicity. Despite their distinctive application and advantages in the industrial and domestic sectors, the use of materials with nanometer dimensions has raised issues of safety for both consumers and the environment. Investigation on the exposure effect of discrete nanoparticles (NPs), one of the many forms of nanomaterials, and their toxicity is very important as their small size causes more inflammation than bulk counterparts when delivered at the same mass dose (Zhang et al. 2007; Nair et al. 2009; Liu et al. 2011; Raghupathi et al. 2011). Because of their small size and their unique characteristics, NPs have the ability to harm humans (Chang et al. 2011; Akhavan et al. 2012, 2013), microorganisms (Stoimenov et al. 2002; Baek and An 2011; Azam et al. 2012), and other wildlife (Wang et al. 2009; Akhavan et al. 2015; Chen et al. 2016) by interacting through various mechanisms (Liu et al. 2011; Xiong et al. 2011).

Titanium dioxide (TiO₂) is among the most widely used material with its broad range of function, for both industry and consumer applications. There are three forms of TiO₂ crystal structures, which are anatase, rutile, and brookite. The first two forms play an important role in industrial applications, while the brookite form has no commercial value and is not being industrially produced (Diebold 2003). Each form has different chemical activities and properties, and hence different industrial uses. The rutile form is less reactive, has a greater ability to absorb ultraviolet (UV) radiation, and is thus suitable in the paints and cosmetic industries (Nowack and Bucheli 2007), while the anatase form is able to produce significant reactive oxygen species (ROS) levels under UV irradiation and hence is suitable for the water treatment industry (Ju-Nam and Lead 2008). TiO₂ NPs are organized either in fine (>100 nm) or ultrafine (<100 nm) sizes (Ma et al. 2010).

TiO₂ NPs also exhibited a value-added property of antimicrobial function. In previous studies, researchers reported that TiO₂ NPs exhibited significant toxicity effects against both Gram-positive (Adams et al. 2006; Yeung et al. 2009) and Gram-negative (Maness et al. 1999; Rincón and Pulgarin 2004; Fu et al. 2005) bacteria, as well as algae (Aruoja et al. 2009), zebrafish (Xiong et al. 2011), and mice (Ma et al. 2010). The usefulness of the antibacterial effect of TiO₂ NPs was used in the disinfection of water contaminated with Escherichia coli (E. coli) (Maness et al. 1999; Kubo et al. 2005). The already published studies on bacterial activity have demonstrated that TiO₂ NPs can kill bacteria even at low concentrations (Fu et al. 2005), in which the photocatalytic process of TiO₂ NPs causes fatal damage to microorganisms (Sunada et al. 1998).

Only a few studies on the antifungal effect of NPs were found in scientific publications. Nevertheless, researchers only focused on the silver (Ag) (Kim et al. 2009; Panáček et al. 2009; Monteiro et al. 2013) and zinc (Zn) (Lipovsky et al. 2011) NPs and their antifungal activity against fungal species, Candida albicans (C. albicans). Both NPs exhibited a strong antifungal effect toward the C. albicans in a concentration-dependent manner. By comparison, zinc is less potent than silver NPs in reducing C. albicans growth with a dose of 0.1 mg/mL (Lipovsky et al. 2011) and a dose of 0.4 μg/mL to 3.3 μg/mL (Monteiro et al. 2012), respectively. To date, access to the antifungal activity of most manufactured NPs remains extremely limited. Hence, in this paper, the antifungal activity of TiO₂ NPs against C. albicans, a prevalent human pathogen, was investigated using a trypan blue exclusion assay, which is a simple quantification method. The study covers the effect of different forms (anatase versus rutile) of TiO_2, their exposure times, and dosage concentrations on C. albicans culture.

EXPERIMENTAL

Materials

The sample of C. albicans was a donation from the Faculty of Medicine, Universiti Putra Malaysia (Serdang, Malaysia). The TiO₂ powders ranging from 70 nm to 200 nm (anatase and rutile) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The potato dextrose medium, trypan blue, and glycerol were also purchased from Sigma-Aldrich and used directly without any further purification. All aqueous solutions were prepared with deionized water.

Characterization of TiO₂ NPs

For imaging of the TiO₂ NPs, anatase and rutile TiO₂ NPs powders were mounted onto the stub and coated with gold. Then, the samples were analyzed with a SEM (scanning election microscope) (S-3400N, Hitachi, Tokyo, Japan) and EDX (energy dispersive x-ray) (S-3400N, Hitachi, Tokyo, Japan).

Preparation of TiO₂ Suspension

TiO₂ powder (50, 100, and 150 μg/mL) was suspended in deionized water. The suspensions were sonicated for 30 min to avoid any agglomeration. The suspension was freshly prepared, ensuring homogeneity when added to the growing C. albicans culture.

TiO₂ on C. albicans Culture

C. albicans was cultured in a potato dextrose medium at 37 °C and 180 rpm on an orbital shaker. When the culture reached a mid-log phase (approximately after 24 h of incubation time), the homogeneous TiO₂ suspension was added. The samples were incubated for 96 h. Sampling was done at 0, 6, 24, 48, 72, and 96-h time points. The numbers of viable and non-viable cells were counted and compared with a negative control (cell culture without TiO₂).

Quantification of Cell Viability using Trypan Blue Staining Method

Viable and non-viable cells were counted using a hemocytometer cell counter. An aliquot of the 100 μL sample was gently mixed with 100 μL of a 0.4% trypan blue dye and left to stand for 5 min at room temperature. Then, 20 μL of the cell suspension was loaded into each chamber of the hemocytometer and analyzed under a 40× magnification of light microscope (DM3000, Leica, Wetzlar, Germany). Both viable (unstained) and non-viable (stained) cells in each of the five quadrants (1, 2, 3, 4, and 5) as shown in Fig. 1 were counted.

Fig. 1. Hemocytometer grid

The averages of these five readings were calculated and multiplied by 10⁴ to obtain the number of cells per mL in the sample that were applied to the hemocytometer. The readings were multiplied by a ratio of trypan blue used in the solution and the dilution factors used in the original sample preparation on the cell suspension. The number of cells was calculated by Eq. 1.

Cell Morphology Observation

The cell culture was harvested via centrifugation. The samples were fixed in 2.5% glutaraldehyde for 6 h at 4 °C. The samples were centrifuged to remove the liquid form and washed three times with 0.1 M of a sodium cacodylate buffer. It was incubated for 10 min after each washing process. The samples were then post fixed in 1% of an osmium tetraoxide solution for two h at 4 °C and washed with 0.1 M of a sodium cacodylate buffer three times. Again, it was left to sit for 10 min after each washing process. It was then put into a critical dyer with CO₂ at 35 °C (EM CPD030, Leica) for 30 min, mounted onto the stub, and coated with gold. The samples were analyzed with a SEM (S-3400N, Hitachi, Tokyo, Japan). For the TEM (transmission electron microscope) analysis, the specimen were infiltrated with an acetone and resin mixture, placed into a beam capsule, and filled up with resin. It was then polymerized in an oven at 60 °C for 24 h. The specimen was cut and stained with uranyl acetate for 15 min before observation under a TEM (JEM-2100F, JEOL Ltd., Tokyo, Japan).

RESULTS AND DISCUSSION

The Effect of TiO₂ NPs on C. albicans Growth

The study on antifungal activity of TiO₂ (in a form of rutile and anatase) against C. albicans growth was performed by dosing the culture at their log phase of growth (Fig. 2) with 100 ppm of TiO₂. In the absence of TiO₂ (indicated as a control in Fig. 2), the culture showed a typical growth curve in which the sample continued to grow at an exponential phase pattern until 48 h, followed by a stationary phase at 48 h to 72 h, and subsequently entered the death phase with a gradual decrease in the viable cell count. The calculated specific growth and death rate for this control culture was 0.0146 h^-1 and 0.0188 h^-1, respectively (Table 1). The culture dosed rutile experienced some reduction in their log phase with until 48 h of incubation time, before entering the death phase. Anatase NPs were the most toxic toward the C. albicans culture as it had the highest death rate of 0.0223 h^-1 when compared to rutile (0.0119 h^-1) NPs. At the end of the incubation time, rutile TiO₂ NPs inhibited 33% of the C. albicans culture as compared to the control, while the anatase TiO₂ caused 65% of the growth inhibition.

Fig. 2. Growth of the C. albicans cells without and with anatase and rutile TiO₂ NPs exposure at 37 °C for 96 h and at a 180-rpm shaking speed; growth inhibition was determined by Trypan blue staining using a hemocytometer cell counting method and expressed as a percentage of the control; the variable, X, was defined as the time of the NPs dosage; error bars represent the standard error of the mean.

Table 1. Growth Phase and Kinetics of C. albicans Culture

Both the anatase and rutile TiO₂ NPs induced a toxicity effect at different magnitudes, leading to a reduction in the viable cell. Based on the result (Table 1), it can be clearly seen that anatase was the most toxic toward C. albicans as compared to rutile. Anatase had the least number of viable cells with a final cell concentration of 200±1.000 × 10⁷ cells/mL. Few researchers reported that anatase NPs tend to be more toxic than rutile when tested on different cell cultures, such as mammalian (Gurr et al. 2005), E. coli (Sunada et al. 1998), and Bacillus megaterium (B. megaterium) (Sunada et al. 1998). This statement agreed well with the findings in this study.

When comparing the toxicity of anatase and rutile, several factors can be considered. The SEM image (Fig. 3) showed the morphology of anatase and rutile NPs. It was found that the size of anatase NPs (70 nm to 130 nm) was generally smaller than the rutile (90 nm to 200 nm) particles. A similar observation was made by Reyes-Coronado et al. (2008) and Gopal et al. (1997), in which they observed that anatase was smaller than rutile NPs. Anatase NP had a mean particle diameter of 13 nm and a relatively narrow size distribution; whereas, rutile consisted of relatively large particles that can form spheroids, which were upwards of 200 nm. Note that, smaller particles have higher surface areas and particle numbers per unit mass, and this may contribute to the higher antifungal activity observed in this study. Previous work by Pal et al. (2007) found that smaller particles caused significant reduction in cell viability due to a larger contact surface area, and therefore have a higher tendency to induce oxidative damage (Gurr et al. 2005), causing more cell death. In a different study using a different organism, it was also found that the smaller size of TiO₂ NPs showed significant toxicity against nematode Caenorhabditis elegans, compared to the bulk particles which did not show any toxicity (Wang et al. 2009).

Fig. 3. Particle size of (a) anatase and (b) rutile TiO₂ NPs

Larger particles that consist of the aggregation of two or more NPs together might reduce the direct interaction between bacteria and NPs (Zhang et al. 2007; Das et al. 2011); hence they have fewer chances to mingle with cells, and as a consequence, reduce the cell wall penetration or membrane damage. An increase in the aggregation of the NPs will lead to a reduction in the surface area, which would reduce the surface available for the interaction of cells and NPs, resulting in a lower toxicity effect. This statement was in agreement with work presented by Zhang et al. (2007) and Das et al. (2011). Rutile was observed to form larger aggregates with relatively smaller surface areas, whereas anatase was indicated to have lower aggregates particles (Reyes-Coronado et al. 2008). This finding explained why anatase was more toxic to microbes in comparison to rutile under the same incubation conditions and agreed well with the current work.

The size of NPs alone is not the only factor contributing to the toxicity and explains why the rutile and anatase forms of TiO₂ behave differently. It seems likely that the particle chemistry played an important role in affecting the toxicity of the TiO₂ NPs, based on some findings by other researchers. Anatase with a band gap of ~3.2 eV (Luttrell et al. 2015) exhibited higher photocatalytic activity compared to rutile (Gurr et al. 2005) with a band gap of ~3.0 eV (Luttrell et al. 2015). The photocatalytic activity of the anatase was approximately 1.5 times higher than that of the rutile form (Kakinoki et al. 2004).

Concentration-dependent Antifungal Activity of TiO₂ NPs

TiO₂ NPs at different concentrations (50, 100, and 150 μg/mL) were dosed into C. albicans culture, and their effect was measured after 48 h. Figure 4 showed an increase in growth inhibition with the increase in the TiO₂ NPs dose for both anatase and rutile. At a low dosage (50 μg/mL) of rutile TiO₂, the cell exhibited tolerance with no significant cell loss at only 3%. Similarly, acceptable tolerance was observed for the culture dose with anatase TiO₂, with a slightly higher cell loss at 17%. However, at higher doses (100 and 150 μg/mL), both anatase and rutile TiO₂ caused a significant toxicity effect on the cells. Anatase at 100 μg/mL and 150 μg/mL resulted in a 35% and 57% cell loss, respectively. Whereas, for rutile, the dose of 100 μg/mL and 150 μg/mL lead to a lower cell loss as compared to anatase, at 8% and 48%, respectively.

Fig. 4. Growth inhibition of C. albicans treated with anatase and rutile TiO₂ NPs at 37 °C for 24 h at a 180-rpm shaking speed; growth inhibitions were determined by Trypan blue staining using a hemocytometer cell counting method and expressed as a percentage of the control; error bars represent the standard error of the mean.

The results agreed with findings previous research using different varieties of NPs on different living organism (Rincón and Pulgarin 2004; Brayner et al. 2006; Hu et al. 2010; Das et al. 2011; Tu et al. 2013; Akhavan et al. 2015). It was concluded that the antimicrobial activity of NP was concentration-dependent. Fu et al. (2005) reported that by increasing the concentration of anatase TiO₂ from 0.5 mM to 5 mM, the growth of both E. coli and B. megaterium were effectively inhibited. Research by Maness et al. (1999) revealed that in the presence of 0.1 mg of TiO₂, NPs killed 98% of E. coli cells, but lower concentrations did not kill the cells effectively. As suggested earlier, regarding the factors affecting the antibacterial activity of NPs, a generation of ROS also played an important role. Xiong and coworkers (2011) reported that the rate of hydroxyl (OH) radical generation by NPs increased with an increasing NP concentration. At a concentration of 50 mg/L, TiO₂ NPs did not produce mortality in zebrafish. However, 300 mg/L of TiO₂ NPs caused 100% mortality of the zebrafish.

In other research, C. albicans had an antifungal effect toward various NPs at different magnitudes (Kim et al. 2009; Panáček et al. 2009; Lipovsky et al. 2011; Monteiro et al. 2012; Li et al. 2013). The Ag NP exerted a strong antifungal effect against C. albicans cells at very low concentrations (0.4 μg/mL to 3.3 μg/mL) (Monteiro et al. 2012). Additionally, 97.5% of cell reduction was observed when C. albicans was exposed to 100 μg/mL of the zinc oxide (ZnO) NP, while almost complete cell reduction was recorded at 1000 μg/mL ZnO NP exposure (Lipovsky et al. 2011). When comparing the previous result with our findings, TiO₂ had the least antifungal activity as compared to Ag and ZnO because at 100 μg/mL, both anatase and rutile TiO₂ NPs only killed 35% and 8% of the C. albicans, respectively. Based on the previous findings, it was demonstrated that other materials may exert the same effect in different magnitudes towards the C. albicans cell, while some researchers also recorded similar trends over various types of cells using different NPs.

Time-dependent Antifungal Activity of TiO₂ NPs

The time-dependent antibacterial activity of NPs was evaluated by incubating the C. albicans culture with the addition of 50 μg/mL TiO₂ (anatase and rutile) NPs at the mid log phase of their growth (24 h) and leaving the sample to further incubate for 96 h. Figure 5 shows the percentage of cell viability versus time for C. albicans dosed with anatase and rutile NPs.

Fig. 5. Percentage of C. albicans remaining after treatment with rutile and anatase TiO₂ NPs at 37 °C and a 180 rpm shaking speed; cell viability was determined through a hemocytometer cell counting method and expressed as a percentage of the control; error bars represent the standard error of the mean.

After exposure of anatase in the C. albicans culture for 24 h of incubation time, only 56% of the cells remained viable. Prolonged incubation (72 h) led to further reduction in the cell viability to 36%. It was observed that this trend was also similar when C. albicans cells were exposed to rutile TiO₂ NPs. Cell viability reduced from 74% to 68% after 24 h of NP exposure. Cell viability continued to drop to 63% and 54% after 48 h and 72 h of NP exposure, respectively.

The viability of cells treated with NPs decreased with increasing incubation time. By increasing the incubation time, NPs had more of a chance to have contact with the cells, attach to the cell membrane, and therefore caused more membrane damage leading to cell death (Stoimenov et al. 2002). This trend was similar with the findings of other researchers (Liu et al. 2011; Raghupathi et al. 2011; Gurunathan et al. 2012; Li et al. 2013), which suggested that the antibacterial activity of NPs is time-dependent. Rincón and Pulgarin 2004 reported that for high cell mass populations, longer exposure time is required for bacterial inactivation.

Morphological Assessment of Cell Morphology When Exposed to NPs

Morphological changes in all samples were monitored after treatment with 100 μg/mL of NPs for 96 h. For normal C. albicans (Fig. 6a), its morphology consists of mycelia and rod shapes. Cell-treated NPs became abnormal in size, displaying cellular shrinkage, and had irregular shape. Cells treated with anatase TiO₂ (Fig. 6b) suffered from chronic cell membrane damage due to the high toxic impact that took place. Most of the cells become flattened and lost their cellular integrity after being exposed to anatase TiO₂. Meanwhile, rutile TiO₂ (Fig. 6c) had a minimal effect towards the C. albicans culture because some of the cells remained integral. Size of the treated C. albicans culture did not change, but only surface breakage occurred. To confirm this phenomenon, TEM analysis was conducted to examine the membrane defects upon treatment with NPs. From TEM analysis, untreated C. albicans consist of a rod shape with a smooth surface structure (Fig. 6d); whereas, cell-treated anatase NPs (Fig. 6e) cannot retain its original shape as compared to untreated cells. Direct contact of NPs could influence the cellular integrity of the cells and cause cell burst, with the cell membrane being severely destroyed, and the cytoplasm and cell constituents flowing out (Stoimenov et al. 2002; Kang et al. 2008; Tu et al. 2013). The degradation of the cell wall and the loss of intracellular materials were observed after the exposure of anatase NPs to the cell culture.

The presence of elemental titanium on the bacterial cells was shown on the EDX analysis as a green colored region on the image (Fig. 7). It was found that a strong physical attachment of TiO₂ was evident, even after an attempt to dissociate the NPs from the cell surface via a vigorous rinsing of the cell suspension took place. TiO₂ failed to dissociate the later from the cell surface due to opposite charges between the bacteria and NPs. One possible reason is that opposite charges caused adhesion of NPs to the cell surface. NPs were tightly bound with the cell surface due to the electrostatic force (Stoimenov et al. 2002; Hu et al. 2009; Nair et al. 2009). This result was correlated well with a study by Morones et al. (2005) in which the difference in charge between bacteria and NPs might have caused the strong interaction between them. However, based on the EDX mapping and analysis, the percentage of attachment for both of the NPs were not significant even though anatase exerted a stronger antifungal effect compared to the rutile TiO₂.

Fig. 6. Electron micrographs for a) surface scanning image of untreated C. albicans; b) surface scanning image of C. albicans after exposure with anatase TiO₂ NP; c) surface scanning image of C. albicans after exposure with rutile TiO₂ NP; d) transmission cross-sectional image of untreated C. albicans cell, and e) transmission cross-sectional image of C. albicans after exposure with anatase TiO₂ NP

Fig. 7. EDX mapping and spectrum of C. albicans exposed to (a) anatase and (b) rutile TiO₂ NPs

The mechanism by which the NPs are able to cause the cell membrane damage and lead to cell death is not fully understood, but the present study suggests that when cells were treated with NPs, changes took place in its membrane, leading to a major increase in its permeability. Nevertheless, different mechanisms of action taking place within the cells upon exposure to TiO₂ has been suggested by many researchers (Cho et al. 2004; Rincón and Pulgarin 2004; Yeung et al. 2009; Xiong et al. 2011). Cells were reported to generate higher amounts of ROS (Hussain et al. 2005; Yeung et al. 2009) and OH radicals (Cho et al. 2004; Rincón and Pulgarin 2004) when exposed to NPs. This lead to an increment in lipid peroxidation (Ma et al. 2010), inflammatory cells (Ma et al. 2010), and a reduction in the mitochondrial function of the cell (Hussain et al. 2005), eventually causing cell death (Cho et al. 2004).

CONCLUSIONS

1. This study demonstrated TiO₂ nanoparticles (NPs) in the form of anatase and rutile displayed excellent antifungal potential towards C. albicans.
2. The loss of cell viability was concentration- and time-dependent.
3. Morphological analysis suggests a direct interaction between TiO₂ and cells as the main mechanism causing cell death.
4. Although the findings of this study suggested that TiO₂ NPs had antifungal activity against C. albicans, their uncontrolled released to the environment may harm the ecosystem.

ACKNOWLEDGEMENTS

The work is financially supported by the Fundamental Research Grant Scheme (5524364) and Graduate Research Fellowship, Universiti Putra Malaysia.

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Article submitted: June 30, 2019; Peer review completed: August 26, 2019; Revised version received and accepted: September 6, 2019; Published: September 23, 2019.

DOI: 10.15376/biores.14.4.8866-8878