Enhanced antimicrobial and anticancer properties of ZnO and TiO2 nanocomposites.

The study describes the antibacterial and anticancer activities of a nanocomposite prepared by mixing zinc oxide and titanium dioxide nanoparticles. The particle mixtures were analyzed by X-ray diffraction, Field emission scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and dynamic light scattering techniques. Thus, analyzed samples were subject to disc diffusion method at various concentrations to analyze their antibacterial activities against two Gram-positive and two Gram-negative bacteria. The same samples were then analyzed for their anticancer activities on four different cell lines. The results indicate a synergistic effect of the nanocomposite on both antibacterial and anticancer properties when compared to their individual counterparts.


Introduction
Nanomaterials are currently influencing a lot of our daily lives (Abbas et al. 2009;Adeleye et al. 2016). Of these, inorganic nanoparticles comprise of those which do not directly influence but indirectly improve our interaction with the world around us (Biyela et al. 2014;Chen et al. 2011;Huang et al. 2015;Johnston 2005). Inorganic nanocomposites are a mixture of individually prepared nanoparticles combined at different concentrations (Ivanov et al. 2003) as opposed doping (Rajendar et al. 2013) or substitution (Ahmadipour et al. 2012;Rajendar et al. 2014aRajendar et al. , b, 2016a. These particles would gallop the properties of both the nanoparticles simultaneously while retaining their individual characteristics. There have been multiple reports of these composites which perform dually well when combined that in their individual states (Ivanov et al. 2003;Flahaut et al. 2000;Granqvist 1993). This property is of great use in the medical field where the use of nanocomposites could relatively increase the performance of two individual nanoparticles just by combining them. Synergistic application of multiple drugs are common among the medical field where two individual medications fail to produce a desired effect disjointedly, while inducing potent effects when combined together (Fried et al. 2002;Hurwitz et al. 2004).
The premise of using composite materials stems from the fact that zinc oxide (ZNPs) and titanium dioxide nanoparticles (TNPs) induce potent antibacterial and anticancer activities when applied in mill molar concentrations (Jones et al. 2008;Yamamoto 2001;Zhang et al. 2007;Brunet et al. 2009;Han et al. 2016;Montazer et al. 2011;Savvova and Bragina 2010). We have previously demonstrated potent antibacterial and anticancer activities of ZNPs and TNPs prepared by multiple synthesis routes & V. Rajendar rajendar.nano@gmail.com (Rajendar et al. 2014a(Rajendar et al. , 2016aRajendar et al. 2016b, c;Li et al. 2012). Of these, we have shown potent activity in case of those prepared using Tween 80 as the surfactant (Rajendar et al. 2016b, c). Thus, produced nanoparticles seem to perform to a greater extent that those previously studied.
In this study, we have used a mixture of ZNPs and TNPs prepared by TWEEN 80 surfactant method and observed their antibacterial and anticancer properties via zone of inhibition and MTT assays. The particle mixtures were analyzed for their physiochemical properties before they were evaluated for the above mentioned assays.

Synthesis of ZNP/TNP nanocomposites
The method of preparation of ZNPs and TNPs has previously been reported (Rajendar et al. 2016b, c). Thus, prepared nanoparticles were weighed and suspended in deionized (DI) water to initiate the mixing process. The particles were homogenized using Sonication for a period of 30 min at 20 kHz and Ultra Sound Power: 60-90 W. Mixing ratios of 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, and 3:2 wt% ZNPs: TNPs were used in this study. Post homogenization, the samples were centrifuged at 2000 rpm for 30 min to pellet the nanocomposite mixture. These samples were then dried overnight at 100°C in an oven to realize while crystalline nanocomposite powder.

Characterization techniques
Materials were characterized using field emission scanning electron microscopy and energy dispersive spectroscopy (FESEM&EDS) and images were taken at 1 lm magnification. JEOL JEM-2010 (HT) transmission electron microscopy (TEM) was used to analyze particle morphology and particle size analysis and the images were taken at 200 kV accelerating voltage. Dynamic light scattering (DLS) was performed on samples suspended in ethanol using a YD-laser (532 nm) and the mean value of the obtained histogram was considered the average particle size. Fourier transform infrared spectroscopy (FTIR) was performed for wavelengths 500-4000 cm -1 . X-ray diffraction studies were performed between 2h values 20°a nd 80°. The samples were then analyzed for their average crystallite using the modified Debye Scherrer's formula.

Antimicrobial assay
The antimicrobial assays were performed using standard disc diffusion assay method, the process for which has been explained elsewhere (Rajendar et al. 2016b, c). B. Subtilis, S. Aureus, E. Coli, and C. Albicans were used as model Gram-negative and Gram-positive bacteria for this study. The nanocomposite concentrations used were 0.02, 0.04, 0.06, 0.08, 0.1, and 0.12 mg/lL. Only 1:1 ZNP/TNP samples were utilized in this study.

Anticancer activity
The anticancer activity was evaluated by performing MTT assay on four different non-additive cell lines, namely: human cervical cancer cell line (HeLa), Chinese hamster ovary cells (CHO), human breast adenocarcinoma cell line (MD-231), and Mus musculus skin melanoma cell line (B16-F10). Briefly, cells were plated in a 96-well plate at a seating density of 2 9 10 4 per well and left
The average crystallite sizes for these samples were analyzed by Debye-Scherrer's formula [D = K 9 k/(b cos h)] and the results are indicated in Table 1. b is the full width half maximum (FWHM) of the XRD corresponding peaks, K is Debye-Scherer's constant, D is crystallite size, k is wave length of the X-ray, and h is Bragg angle.

Morphology and particle size analysis
The morphology of the nanocomposite at ZNP/TNP 1:1 ratio was evaluated by FESEM, TEM, and dynamic light scattering equipment. FESEM images (Fig. 2a, b) show globule-like particles along with unstructured particles present in the sample. The same is iterated in the TEM (Fig. 2d-g) showing an equal quantity of globule shaped particles with variable shaped particles. The globule shaped particles were confirmed to be ZNPs, while unstructured particles were shown to be TNPs by energy dispersive X-ray spectroscopy ( Fig. 2c; Table 2) provided along with the FESEM. It is interesting to note that the dynamic light scattering analysis revealed the particle size to be in the

Fourier transform infrared spectroscopy (FTIR) analysis
In Fig. 3, the FTIR spectra of ZNP/TNP nanocomposite can be observed. The wavelength range measured was from 500 to 4000 cm -1 . The broad absorption at low frequency at 500 cm -1 could be attributed to the Ti-O bonding. C-O stretching is observed at 1195 cm -1 , C-C stretching is observed at 1460 cm -1 , C=C stretch stretching is observed at 1690, 1730 cm -1 . Zn-O bonding is observed at 2850 and 2960 cm -1 , and O-H stretching at 3500 cm -1 (shown in Table 3). From this, we can confirm purity of the prepared samples without any unforeseen impurities. It can be hence stated that while varying wt% of ZNP/TNP nanocomposite, i.e., 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, and 3:2 wt%, same functional groups were observed in all.

Antimicrobial assay
Zone of inhibition for ZNPs alone, TNPs alone, and ZNP/ TNPs has been evaluated individually at different concentrations. The quantified values of the zone inhibition (ZI) are shown in Figs. 4, 5, and 6. It is clearly evident from the assays that ZNPs have a lower antibacterial action when compared to TNPs. This could be attributed to the spontaneous mutation caused by TNPs in bacterial samples as opposed to the reactive oxygen species-based mechanism explained for ZNPs (Li et al. 2012). However, it is interesting to note that the antibacterial activity of ZNP/ TNP nanocomposite significantly increases when compared to ZNPs alone while decreases to a small extent when compared to TNPs. This result is important, because TNPs are known to cause genetic mutations in human beings when applied in higher quantities as opposes to the biocompatible and non-toxic property of ZNPs. Hence, we could say that the use of ZNP/TNP composite would significantly improve the antibacterial property of ZNPs while lowering the toxic range of TNPs.

Anticancer activity
The MTT assay for the ZNP/TNP nanocomposite can be seen in the Fig. 7a and b. Similar to the antibacterial assay, the anticancer properties of the nanocomposite seem to complement the ZNPs while having lower activity when compared to those of TNPs. This would mean that a composite material would significantly alter the methodology through which anticancer activities could be governed without compromising on the biocompatibility issues. Another important thing to note through these experiments is that except for CHO cell line, TNPs showed lower anticancer activity when compared to ZNPs at all concentrations. Furthermore, ZNPs had really low anticancer activity against the CHO cell line when compared to other cell lines. However, ZNP/TNP nanocomposite showed significantly high and equivalent anticancer activity against all the cell lines. And     Table 4. This implies that through the use of nanocomposites, the disadvantages offered by one particle can be compensated by the other particle used in the sample.

Conclusions
Nanocomposites of zinc oxide nanoparticles and titanium dioxide nanoparticles were made and tested for their antibacterial and anticancer properties. The results indicated an elevated antibacterial property for the nanocomposite when compared to zinc oxide but diminished property compared to titanium dioxide. In contrast, a diminished anticancer property was observed for the nanocomposite compared to zinc oxide, while an increased anticancer property when compared to titanium dioxide. However, bacterial types and cancer cell lines which were unaffected by either of the nanoparticles individually had an elevated antibacterial or anticancer property for the nanocomposite. This implicates those similar to medical drugs, which have synergistic properties when used against a certain disease, nanoparticles also show a synergistic effect when applied as composites. Our goal would be to further this research by testing various other combinations of composites and compare them to their individual nanoparticles.