Eco-synthesis and characterization of titanium nanoparticles: Testing its cytotoxicity and antibacterial effects

2.1 Preparation of the Lupinus extract

The Lupinus (Lupin bean) powder was procured from the local market. Ten grams of lupin bean powder was soaked in 20 mL deionized water overnight. Thereafter, the solution was filtered to prepare the lupin extract.

2.2 Eco-synthesis of TiO₂NPs

A volume of 20 mL of the prepared lupin bean extract was mixed with 2.5 mM of titanium(iv) isopropoxide (Merck Co., Germany) at a molar ratio of 2:1 under vigorous and continuous stirring at room temperature. On stirring the contents, a white paste started forming at the bottom of the flask, which indicated the formation of TiO₂NPs. The paste was heated on a hot plate at 80°C and then calcined in a muffle furnace at 450°C for 3 h, producing a beige powder including TiO₂NPs.

2.3 Characterization of eco-synthesized crystal TiO₂NPs

An extensive characterization of the synthesized bio-crystal TiO₂NPs was employed using various techniques. The crystal structure and crystalline grain size were determined by X-ray diffraction (XRD) (Bruker D8 Discover, UK). The average particle size was analyzed by a Zeta-sizer, (Nano series, HT Laser, ZEN3600 from Molvern Instrument, UK). Transmission electron microscopy (TEM) (JEM-1011, JEOL, Japan) was used to study the size distribution and shape. Scanning electron microscopy (SEM) (JEOL-FE, Japan) was employed to characterize the shape and morphology of the formed NPs. Energy dispersive spectrometer (EDS) (Oxford Instrument, UK) was used to characterize the elemental composition (Ti and O) of NPs.

2.4 Cytotoxicity assay

The cytotoxic effect of the synthesized NPs was assessed against the MCF-7 cell line (human breast cancer cell line), obtained from VACSERA Tissue Culture Unit. The cell culture was propagated in Dulbecco’s modified Eagle’s medium enriched with 10% heat-inactivated fetal bovine serum and 1% l-glutamine 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer; gentamicin was added at a concentration of 50 μg/mL to avoid contamination. The cell culture was maintained at 37°C in a humidified atmosphere supplemented with continuous aeration of 5% CO₂ and was subsequently subcultured twice a week. Cell toxicity was evaluated by determining the effect of the test samples on cell structure and viability. For cytotoxicity assay, the cells were seeded in a 96-well plate at a cell concentration of 1 × 10⁴ cells/well in 100 µL of growth medium. A fresh medium containing different concentrations of the test sample was added after 24 h of seeding to avoid depriving the cells of nutrients. Serial twofold dilutions of the test compound were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette. Microtiter plates were incubated at 37°C in a humidified incubator with 5% CO₂ for a period of 48 h. Three wells were used for each concentration of the test sample. Vinblastine (a known anticancer drug) was used as a positive control for the MCF-7 cell line. Control cells were incubated without the test sample and with or without dimethyl sulfoxide (DMSO). The concentration of DMSO was kept low (maximal 0.1%) and was found to have negligible effect on the course of the experiment. Post incubation of the cells for 24 h at 37°C, different concentrations of the test sample (TiO₂NPs) (100, 50, 25, 12.5, 6.25 and 3.125 µg) were added and incubated for another 48 h, and the yield of viable cells was determined by colorimetric method. After incubation, medium was aspirated, and the crystal violet solution (1%) was added to each well for at least 30 min for color development. The plates were rinsed with tap water until all excess stains were removed. Glacial acetic acid (30%) was then added to all wells and mixed thoroughly. After gentle shaking, the absorbance of the plates was read on a microplate reader (TECAN) at 490 nm. All results were calibrated, and the corrected absorbance was calculated. Treated samples were compared with the control in the absence of the tested sample. Internal triplicates for each concentration were included in each experiment. Finally, the cytotoxic effect of each tested concentration was calculated [18].

2.5 Antibacterial activity of TiO₂NPs

The bactericidal effect of eco-synthesized TiO₂NPs was investigated using disc diffusion method [19] against Enterococcus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria). Nutrient agar medium plates were prepared, sterilized and solidified. Bacterial cultures were swabbed on these plates after solidification of the agar medium. Using hole-punched agar plates, the TiO₂NPs solutions at different concentrations (5, 10, 15 µg/mL) were placed in the inoculated nutrient agar plates and kept for further incubation at 37°C for 24 h. The inhibitory potency of the NPs was visualized by the measured zones of inhibition. The experiments were conducted in triplicates, and the mean value of the measured diameter of the inhibition zone was recorded.

3.1 Characterization

Figure 1 shows X-ray diffractograms of TiO₂NPs synthesized using the lupin bean extract. The XRD pattern shows sharp peaks 2θ at about ≈25°, 37° and 48° indicating the existence of crystallite sized particles, with a semicrystalline nature. These results are in agreement with the previous study by Hudlikar et al. [20]. All the peaks confirmed the crystal anatase form [21].

Figure 1

XRD pattern of lupin TiO₂NPs.

The dynamic light scattering (DLS) Zetasizer was used to determine the average size of the eco-synthesized NPs. The Z-average mean diameter (d [nm]) of the green TiO₂NPs was 9.227 nm, and the polydispersity index (PDI) was 0.382 with intercept 0.244, with fairly stable condition. However, the peak strongly indicates that the particles are monodispersed as shown in Figure 2. Overall, the size and PDI of the eco-synthesized NPs revealed good stability. These results validated the results of XRD discussed earlier.

Figure 2

DLS measurement of lupin TiO₂NPs.

TEM is the most frequently reported technique to analyze the morphology and structure of the synthesized NPs. TEM illustrated the shape of the synthesized NPs as shown in Figure 3(a and b); it indicates uniform, almost spherical (Figure 3a) and crystallite rod-shaped particles (Figure 3b) with uniform and narrow size distribution. These electron micrographs of the synthesized TiO₂NPs further confirmed the results of the XRD. Thus, the titanium was formed as a small-sized NP using the lupin bean extract as a directing agent.

Figure 3

(a and b) TEM images of lupin TiO₂NPs.

The biopolymer, on being dispersed in the liquid media, formed an organic matrix with titanium ions being bound to its functional groups (hydroxylic or carboxylic groups). The association of titanium ions with polysaccharide created a uniform and homogenous dispersion of the ions in the available matrix. Thus, these binding sites provided nucleation centers and growth sites for the hydrolyzed Ti⁴⁺-containing particles, as a result of increased local supersaturation in titanium ions. The oxide precursor requires heating to be changed into oxide ion. The homogeneous dispersion of titanium cations within the matrix of the polysaccharide coupled with low temperature shifted the reaction from nucleation toward the increased crystal growth of the nanooxide particles [22,23].

The subnanostructure and morphology of the TiO₂NPs were assessed using SEM. The image showed relatively spherical shape and crystallite rods of the green-synthesized NPs (Figure 4a). The EDS results shown in Figure 4b confirm the presence and existence of TiO₂NPs in suspension. The spectrum analysis revealed signals in the titanium and oxygen regions, which confirmed the formation of TiO₂NPs, and the table represents the percentage of elements present in the Ti and O₂ suspension.

Figure 4

(a) SEM image. (b) EDS analysis results presented the percentage of TiO₂ element in green nanosuspension.

3.2 Evaluation of cytotoxicity and antibacterial activities of the green TiO₂NPs

The green NPs had a significant dose-dependent effect on the cell viability as observed in the MTT assay. The cytotoxic effect against MCF-7 cells with IC₅₀ of 41.1 µM was tested using the eco-synthesized TiO₂ crystal NPs. It was found that the cytotoxicity of green TiO₂NPs increased with increased concentration of NPs and demonstrated a 100% dose-dependent death in the MCF-7 cells. With the increase in the concentration of the sample, the ROS production also increases which in turn explains the oxidative damage caused to the cancer cells [24]. Thus, a greater number of NPs easily enter the cancer cells and generate free radicals which are responsible for the destruction of the cancer cells [25]. A similar pattern of results on the cell cytotoxicity of NPs was reported by Maheswaria et al. [26], Murugan et al. [27] and Sukirtha et al. [28]. In line with previous in vitro studies indicating that the TiO₂NPs are toxic to the mammalian cells [29], the results of the present study provide evidence of a profound cytotoxic effect of synthesized TiO₂NPs against the breast cancer MCF-7 cell line in comparison to Vinblastine. Moreover, the size of the nanoparticle and the cell type dominantly affected the cytotoxicity of NPs. Thus, a comparison of the results of the cytotoxicity of the green TiO₂NPs with those of Vinblastine showed that the NPs were more efficacious (Figure 5).

Figure 5

Cytotoxic activity of the green TiO₂NP suspension compared with that of Vinblastine against MCF-7 cell line.

Figure 6 shows the antibacterial activity of TiO₂NPs, which was evaluated via the disc diffusion method, against two bacterial strains: Enterococcus (Gram-positive) and E. coli (Gram-negative). The antibacterial effect of the NPs was visualized by the wide zone of inhibition around the TiO₂NPs-integrated discs. The basic mechanism of the antibacterial activity stems from the interaction between the positively charged surface of NPs with the net negative charge on the bacterial cell wall [30]. The differential adhering of the positively charged NPs to the bacterial strains, Enterococcus and E. coli, is due to the difference in the composition of the cell wall of the Gram-positive and Gram-negative bacteria. It has been reported that the magnitude of the negative charge varies in different strains. The Gram-negative bacteria (E. coli) has higher negative charge and adheres more strongly to positive surfaces than the Gram-positive bacteria (Enterococcus). This is attributed to the fact that the cell wall of the Gram-negative bacteria consists of an outer membrane composed of lipopolysaccharides followed by a thin layer of peptidoglycan, whereas the Gram-positive bacteria has only a thick layer of peptigdoglycan. Thus, the positively charged eco-synthesized TiO₂NPs are more active against the Gram-negative bacteria, regardless of their resistance level [31]. Based on previous literatures, it can be hypothesized that an electromagnetic attraction between microorganisms and metal oxides leads to oxidation and eventually the cell death of microorganisms [32]. Overall, the bactericidal effect of the tested NPs could also be attributed to the decomposition of bacterial outer membranes by ROS, primarily hydroxyl radicals (OH), which leads to phospholipid peroxidation and oxidative cell death [33,34].

Figure 6

Antibacterial activity of the green TiO₂NPs and the values of inhibition zones (mm).

The above-discussed findings of the present study showed a significant antibacterial effect of the eco-synthesized TiO₂NPs, particularly against opportunistic infections caused by multidrug-resistant Gram-negative organisms. Thus, it offers a vast potential for NPs in biomedical applications and cosmetology [35,36].