Anticandidal and In vitro Anti-Proliferative Activity of Sonochemically synthesized Indium Tin Oxide Nanoparticles

The present work demonstrates the synthesis, characterization and biological activities of different concentrations of tin doped indium oxide nanoparticles (Sn doped In2O3 NPs), i.e., (Sn/In = 5%, 10% and 15%). We have synthesized different size (38.11 nm, 18.46 nm and 10.21 nm) of Sn doped In2O3 NPs. by using an ultra-sonication process. The Sn doped In2O3 NPs were characterized by by x-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) which confirmed the successful doping of tin (Sn) with Indium oxide (In2O3). Anticandidal activity was performed by standard agar dilution method using Candida albicans for the study. The minimum inhibitory/fungicidal concentration (MIC/MFC) values recorded were, 8 & >8 mg/ml for pure In2O3 NPs, 4 & 8 mg/ml for 5%, 2 & 8 mg/ml for 10%, whereas 1 & >4 mg/ml for 15% Sn doped In2O3 NPs, respectively. The topographical alteration caused by Sn doped In2O3 NPs on Candida cells, was clearly observed by SEM examination. A significant enhancement in anticandidal activity was seen, when Candida cells were exposed to (Sn/In = 5%, 10% and 15%). Moreover, we have also evaluated the impact of Sn-In2O3 NPs on human colorectal carcinoma cells (HCT-116). The results demonstrated that Sn-In2O3 NPs (Sn/In = 5%, 10% and 15%), caused dose dependent decrease in the cancer cell viability as the low dosage (2.0 mg/mL) showed 62.11% cell viability, while 4.0, 8.0, 16.0, 32.0 mg/mL dosages showed 20.45%, 18.25%, 16.58%, and 15.58% cell viability. In addition, the treatment of Sn-In2O3 NPs also showed significant cellular and anatomical changes in cancer cells as examined by microscopes. We have also examined the impact of Sn-In2O3 NPs (5%, 10%, 15%) on normal cells (HEK-293) and the results demonstrate that Sn-In2O3 NPs did not reduce the cell viability of normal cells.

them distinct from their corresponding bulk size material 3 . The NPs have been utilized in the preparation of drugs, detection of protein and pathogens, treatment of different cancers, separation and purification of biological molecules and cells 4 . The main reason for considering NPs, as an effective and alternative therapeutics is that, it can help in preventing the drug resistance. The unchecked use of antibiotics, has resulted in the emergence of several health hazards, like extended drug resistant superbugs 5 . To combat the drug resistance, there is a need to search and modulate new therapeutics as antimicrobials and anticancer agents. Therefore, NPs have offered a potential solution to this problem 2,6 .
Indium oxide nanoparticles (In 2 O 3 ) is an essential and interesting nanomaterial for a number of applications, including solar cells, photocatalysts, organic light emitting diodes, architectural glasses, panel displays, etc. [7][8][9] . Number of studies on the synthesis of different structured In 2 O 3 like nanotubes, nanowires, nanobelts, nanofibers, have been reported for wide applications 10 . Although, there is no information on In 2 O 3 as antimicrobial agent to best of our knowledge. Sn is reported to possess antimicrobial activities and has been widely used as a promising dopant with oxides like, In 2 O 3 and ZnO, for enhancing the antimicrobial, electrical, optical and structural properties [11][12][13][14] . There are several reports which suggested that ITO possess toxicity action on the cells and organs [15][16][17][18][19][20] . However, the database on deep and persistent toxicity, carcinogenicity, genotoxicity, reproductive toxicity, besides skin or eye irritation and sensitization is very inadequate or even missing. In our study, we have made an attempt to study the impact of tin (Sn) doped indium oxide (Sn-In203) nanoparticles (NPs) on human colon cancer cells . Sn is one of the important metals often investigated, as its doping is known to increase the carrier lifetime 21 . Different synthetic approaches have been used for the preparation of Sn-In 2 O 3 nanostructure, like chemical vapor deposition, calcinations, pulsed laser deposition, reactive thermal deposition and sol-gel process 22 . The synthesis of Sn-In 2 O 3 NPs by wet chemistry techniques shows an effective control over the morphology, crystallinity and size of the particles. In recent years, sonochemical reaction has become one of the most important wet chemistry method, applied for preparation of ultrafine nano-structured materials 12 .
In the current study, we have synthesized different percentage of Sn doped In 2 O 3 NPs (Sn/In = 5%, 10% and 15%) by a sonication method. To the best of our knowledge, the study of impact of Sn content over indium oxide on the biological properties has not been reported so far. The present study investigates the effect of Sn doping on structural properties by X-ray diffraction XRD, TEM, and TEM. Further, biological activities of the materials were assessed by using both plate assays and morphological analysis by microscopy.

Results
Synthesis and characterization of Sn doped in 2 o 3 nps. X-ray analysis. Fig. 1 The average crystallite size (D) was calculated by Scherer's equation 26 where K is the Scherer constant (0.9), λ is the wavelength (1.5406 nm), θ is the diffraction angle, β is the full width at half maximum (FWHM). The values of lattice parameter a and crystallite size D, were calculated from high www.nature.com/scientificreports www.nature.com/scientificreports/ intensity (222) plane with respect to Sn proportion and tabulated in Table 1. The lattice parameter a was found at10.15 Å, 10.08 Å and 10.04 Å for 5%, 10% and 15% for Sn doped In oxide, respectively, which shows an decreasing trend with increase of Sn construction.
Morphological analysis. Fig. 2(a-d) presents, TEM images of pure In 2 O 3 NPs and Sn doped In 2 O 3 NPs (Sn/ In = 5%, 10%, 15%). TEM micrographs shows the prepared NPs are irregular in shape and with almost uniform distribution. It was seen that the particle size decreased with the increasing concentration of dopant. Crystallite sizes noted from XRD correlates very well with the results of TEM. The high crystalline order of the samples was further confirmed by selected area electron diffraction (SAED) pattern presented in (Fig. 2(e-g)), where we witnessed the presence of diffused rings, indexed to body-centered cubic In 2 O 3 27 . Figure 3, on the other hand, depicts SEM morphology with low and high magnification. For In 2 O 3 NPs, small particles appeared separately, while the surface features of the Sn doped In 2 O 3 NPs samples showed clearly more agglomeration of particles with different shapes and sizes compared with a pure In 2 O 3 NPs.

Study of effect of Sn doped In 2 o 3 nps on the topology of C. albicans by SeM analysis.
The morphogenesis caused by Sn doped In 2 O 3 NPs, were examined by SEM. The untreated Candida cells (control), were normal in shape, intact with regular and smooth cell surface (Fig. 5a). However, the treated cells were found to be no more intact and cells were irregular in appearance (Fig. 5). The cells treated with pure In 2 O 3 NPs and 5% Sn doped In 2 O 3 NPs, were moderately affected and altered in cell morphology (Fig. 5b,c). However, Candida cells treated with 10% Sn doped In 2 O 3 NPs showed significant alteration, because of the enhanced attachment of NPs to the cell surface (Fig. 5d,e). Further, it has been observed, that the treatment of cells with 15% Sn doped In 2 O 3 NPs displayed severely damaged cells, due to the profuse attachment and penetration, which led to the formation of pits. Deformation and distortion of cell wall and membrane can be clearly seen in Fig. 5e, indicating loss of membrane integrity, that could possibly cause the cell death.
Study on hyphal growth in liquid medium and biofilm study of treated C. albicans by SeM has also been carried out. The details are included in supplementary file. In vitro antiproliferative activity of Sn-In 2 O 3 NPs on cancerous cells . The effect of Sn-In 2 O 3 NPs on cancerous cells was examined by both morphological analysis and MTT assay. Post 48 h treatments, we have found that (5%) Sn-In 2 O 3 NPs induced dose-dependent response on the cancer cells as the low dosage (2.0 mg/mL) showed 62.11% cell viability (Fig. 6A), while 4.0, 8.0, 16.0, 32.0 mg/mL dosages reduced the cancer cell viability to 20.45%, 18.25%, 16.58%, and 15.58% respectively. With a view to examine the morphologically changes in cancer cells, we have found that Sn-In 2 O 3 NPs (5%) treated cancer cells showed moderate impact on cancer cells membrane and nucleus ( Fig. 6B) with compare to control group cells (Fig. 6Ba). The treatment of 4.0 mg/mL has shown significant impact on cancer cell morphology, such as the cell membrane disruption, and nuclear condensation (Fig. 6B,Ba). The treatment of higher dosages (16.0 mg/mL and 32.0 mg/mL) showed most significant effect on the cancer viability and morphology ( Fig. 6B-d).

Discussion
At present, the treatment of fungal infections and cancer, are considered among the most important challenges across the world. The problems encountered with the treatment includes, high doses of available drugs and an escalating resistance to these drugs. In this regard, present study demonstrated the successful doping of Sn doped indium oxide NPs (5%, 10%, and 15%) by sonication technique. The results obtained by XRD, SEM, and TEM, proved the favorable doping of tin (Sn) with Indium oxide (In 2 O 3 ). Synthesis of different crystal sized NPs, was achieved. It was seen that by increasing the constriction of Sn, the crystallite size also decreased, markedly to be 38.11 nm, 18.46 nm and 10.21 nm, for 5%, 10% and 15%, respectively, which goes in compliance with the reported work of Katsube et al. and Ogihara et al. 28,29 .
Further, it was observed that the anticandidal activity was significantly enhanced with increased tin content from 5 to 15% over indium oxide. The anticandidal activity was found in the order of Sn doped, 15% >10% > 5% > undoped In 2 O 3 NPs. This finding is well supported by a previous studies conducted by Martínez et al. 2011, who reported the use of indium as dopant for titanium NPs, resulting in the decrease of size of NPs and increase in their antimicrobial properties 30 . There is almost no information available in the literature, regarding the Sn doped In 2 O 3 NPs against Candida. Although, some previous reports about the NPs have examined, that this activity could be associated to size and surface area and the amount of dopant added to the NPs. 31,32 . Moreover, all fungal species including Candida, possess a cell membrane, consisting of phospholipids and a cell wall made up of mannoproteins, β-glucan-chitin, β-glucan. Therefore, the NPs need to bind and interact with the macromolecules of cell wall for interaction with the phospholipids, integral and peripheral proteins, and also ionic channels 6,33 . Studies also support the generation of ROS, due to oxidative stress on the microbial cell leading to disruption of cell membrane and cease of cellular activity, which finally leads to cell death 31,34,35 . Presently, the development of new therapeutics for application in biomedicine, like infectious diseases, mainly nosocomial type, and cancer treatment has caused a meaningful interest in the search of nanomaterials with biomedical applications.
Additionally, the cytotoxic impact of Sn-In 2 O 3 NPs was morphologically and quantitatively evaluated, using human colorectal carcinoma cells (HCT-116). The findings demonstrated that Sn-In 2 O 3 NPs (5%, 10%, 15%) induced dose dependent manner reduction in cancer cell viability. For example, the lower dosages showed an average 71.10% cell survivability, whereas higher dosages 32.0 mg/mL showed 20.96% reduction in cancer cell viability. In addition, the treatment of Sn-In 2 O 3 NPs also showed significant cellular and anatomical changes in cancer cells as examined by microscopes. We have used specific colon cancer cell line (HCT-116) to study the impact of different concentrations of Sn-In 2 O 3 NPs and examined their impact on proliferation of cancerous cells, which we have found that concentration dependent decrease in the cancer cell proliferation as revealed by MTT assay. The prominent changes were which we have observed the disruption of cell membrane, condensation and augmentation of cell nucleus. Moreover, we have also observed significant cell death. There are couple of studies, which have demonstrated cytotoxic effects of NPs, where NPs induced cancer cell membrane disruption, nuclear fragmentation, and disintegration 36,37 . We have also examined the impact of Sn-In 2 O 3 NPs (5%, 10%, 15%) on normal cells (HEK-293) to explore whether Sn-In 2 O 3 NPs (5%, 10%, 15%) selectively target the cancerous cells or not. Our results demonstrate that Sn-In 2 O 3 NPs (5%, 10%, 15%) did not reduce the cell viability of HEK-293 cells (details in supplementary file). Therefore, the results suggested that Sn-In 2 O 3 NPs (5%, 10%, 15%) possess strong anti-cancer capabilities and could be considered for treatment of cancer.

Methodology
Synthesis of Sn doped in 2 o 3 nps. Sn doped In 2 O 3 samples were synthesized through the sonication process. High purity of tin acetate and indium acetylacetonate were obtained from Sigma-Aldrich, and prepared through different dissolved concentrations of the metal precursors (Sn/In = 5%, 10% and 15%) in 36 mL of distilled water. During the sonication process, 4 mL ammonium hydroxide (Sigma-Aldrich) was added slowly to adjust the pH to 9-10. Through direct immersion method, the solution was irradiated for 1 hr via a high intensity ultrasonic processor (750 W; 20 kHz and 6 mm Ti-probe tip). The temperature was increased to 80-90 °C, using a water bath during the sonication system. A homogeneous and transparent solution was obtained. The precipitates were washed and centrifuged number of times with distilled water and later dried at 70 °C. Finally, the products were calcined at 500 °C for 2 h 12 .   evaluation of Mic/Mfc. The MIC of pure In 2 O 3 NPs and Sn doped In 2 O 3 NPs (Sn/In = 5%, 10% and 15%) was evaluated by standard agar dilution method. SDA plates were streaked using serial dilutions of the test compound in concentration ranging from 16 to 1 mg/ml, incubated with C. albicans. The MIC is defined as the least amount of compound, at which no apparent growth is seen. The lowest concentration of pure In 2 O 3 NPs and Sn doped In 2 O 3 NPs (Sn/ In = 5%, 10% and 15%), at which no cell was obtained or less than three CFUs was present, was recorded as MFC 38 .

Effect on candida topography by SEM analysis. The effect of pure In 2 O 3 NPs and Sn doped In 2 O 3 NPs
(Sn/In = 5%, 10% and 15%) on the topography of C. albicans cells were further investigated by SEM. Briefly, 10 6 CFU/ml cells were treated with 4 mg/ml (conc was selected on the basis of MIC) of pure In 2 O 3 NPs and Sn doped In 2 O 3 NPs and incubated at 28 °C for 24 h. Untreated C. albicans was also included in the experiment as a control. After incubation, the treated and untreated cells were obtained by centrifugation at 12000 rpm for 10 min. Multiple washing with PBS was done and subsequently, fixed primarily with 2.5% glutaraldehyde, followed by 1% osmium tetroxide for secondary fixation. The fixed samples were again washed several times and dehydrated by 30, 50, 70, 90, 99% ethanol. The cells were placed on the stubs and dried, and gold coated, for examination by SEM, at an accelerating voltage of 20 kV 35 .  www.nature.com/scientificreports www.nature.com/scientificreports/ In vitro antiproliferative activity of Sn-in 2 o 3 nps by Mtt assay. Cell culture. In vitro cell culture was done as per method described by Khan et al., 2018 and in brief, human colorectal carcinoma (HCT-116) cells were grown in the media containing DMEM, (5%) L-glutamine, (10%) FBS, (5%) selenium chloride, penicillin and streptomycin. Cells were kept in 5% CO 2 incubator with 37 °C conditions. Cell morphology. In order to study the impact of Sn doped In 2 O 3 NPs on cancerous cells morphology, we have grown HCT-116 cells in 6 well culture dishes and when the cells reached to 80% confluency, they were treated with different concentrations of Sn doped In 2 O 3 NPs. The cells were treated with Sn doped In 2 O 3 NPs (Sn/ In = 5%, 10% and 15%) with these concentrations (2.0 to 32.0 mg/mL). Both control and Sn-In 2 O 3 NPs-treated cells were analyzed after 48 h intervals. Post 48 h treatment of Sn-In 2 O 3 NPs, HCT-116 cells were microscopically (TS100F Eclipse, Nikon) examined to evaluate any morphological changes in cells. Statistical analysis. The mean ± standard deviation (SD) from control and Sn-In203-treated groups were calculated. All statistical analyses were completed with GraphPad Prism 6 (GraphPad Software) and the difference between control and Sn-In203-treated groups were calculated by a one-way ANOVA test (P < 0.05). Where P < 0.05 was considered as statistically significant.

conclusion
The obtained results revealed that the doping of Sn (5-15%) over indium oxide was effective enough to enhance the invitro biological properties of these nanomaterials. The ability of the NPs, to inhibit the pathogenic Candida and cancer cell proliferation, has been demonstrated. The obtained results proved that these materials possess anticandidal and anticancer activities and hold promise for treatment of fungal infections and colon cancer.

Data availability
The data analyzed are available from the corresponding author upon a request.