Introduction

Synthesis of homogenously well-dispersed nanostructured metal oxide powder has attracted the attention due to their vast applications in our daily life based on novel physicochemical properties due to their defect chemistry, porosity, surface effects and its quantum confinement effects. To adopt a facile and green strategy for reliable and reproducible synthesis without phase separation remains an open challenge to nanochemists. The ability to control the particle size, shape, surface structure and morphology which play a crucial role both from functional and technological research perspective still remains challenging task. Synthesized ZnBaO2 nanopowder is a new material in the metal oxide series. The wet chemical method is used for its simplicity, low cost and use of non toxic solvents and gives crystalline reproducible yield at moderate conditions with controlled stoichiometric ratio without phase separation and ill-defined particle aggregations. It was observed that the homogenous distribution of bimetallic oxide depends on experimental conditions, type of molecular precursor used and controlled calcinations. The presence of two non-toxic elements helps to maintain colloidal stability under physiochemical condition in human cell for its wide use in screening at the particular pH, which depends on favorable shape and size retaining the surface morphology intact. The use of ZnBaO2 nanopowder has become a motivating area for research due to its volume–cost-effective relation with the macroscopic quantum tunnel effect (Trusova et al. 2012; Hayashi and Hakata 2010; Cao and Wang 2004; Vollath 2008; Lalena and Cleary 2010; Fan et al. 2015; Klabunde and Richards 2012; Muller et al. 2007; Betke and Kickelbick 2014; Athar 2008; Kickelbick and Schubert 2002; Prechtl and Campbell 2013; Naumann 2009; Mackenzie and Bescher 2007; Edler 2004; Klein 1988; Talapin 2012; Lee et al. 2013; Pang et al. 2013; Wang et al. 2005; Pilenic 2007).

It is assumed that due to non-toxic effect of Zn and Ba metal ions, it can be used for sensor and biological applications. However, these fundamental benefits could predictably correlate with changes in the biological properties of the cells. Based upon the analyses of cell adhesion, relative viability, proliferation and functional detoxification assays of human primary hepatocytes during exposure to ZnBaO2 nanoparticles for 72 h, we intended to find its biological safety for biomedical applications. All these considerations are broadly characterized and included in this paper.

Experimental procedures

The structural parameters play a crucial role in determining and optimizing the sensor-related properties (Calfen and Mann 2003; Tretyakov 2003; Rawle 2007; Perry and Carter 2006; Evans et al. 2010; Colfen and Antonietti 2005; Demortière et al. 2008; Shevchenko et al. 2006; Koziej et al. 2014; Ansari et al. 2014). In order to achieve desired shape and size with enhanced functional properties, bottom-to-top wet chemical approach was undertaken to synthesize functional nanopowder for their chemical and biological studies.

Characterization

FTIR spectra were recorded in the range of 4000–400 cm−1 using KBr pellets in Perkin–Elmer GX spectrometer with a wave number resolution of 4 cm−1. The optical properties were recorded by using GBC UV–Vis Cintra spectrophotometer with wavelength ranging from 200 to 800 nm. Thermal analysis was carried out by heating the sample at the rate of 10 °C/min from ambient to 1000 °C in dry air using Mettler Toledo star analyzer. X-ray powder diffraction patterns were taken in reflection mode of CuKα (λ = 1.5406 Å) radiation from 0θ to 80θ on a Siemens D5000 X-ray diffractometer by using continuous diffraction. Scanning microscopy image was taken by using Hitachi S520 scanning electron microscope. Philips Tecnai G2 FEI F12 transmission electron microscope was operated at 80–100 kV. The samples were collected on a carbon holey copper grid to see morphology and particle size.

DLS and laser Doppler velocimetry were used for the characterization of particle size and zeta potential in solution by using Malvern Instruments Zetasizer Nano-ZS instruments equipped with 4 mW He–Ne laser θ = 632.8 nm. Steady-state emission spectra were measured using a Shimadzu RF 510 spectrofluorophotometer connected to a Haake Ultra thermostat (Julabo F10) with temperature precision ±0.1 °C using a rectangular quartz cell of dimensions 0.2 cm × 1 cm to minimize the re-absorption.

Materials

Zinc chloride (97 %), barium hydroxide octahydrate (97 %) and solvents were purchased from Aldrich and used as such without further purification. Deionized water with an electrical resistivity of 18 MΩ−1 cm (Millipore, India) was used in the reaction.

Synthesis

Colloidal and monodispersable ZnBaO2 was synthesized via chimie douce. The ZnCl2 (5.74 g, 42.08 mmol) was dissolved in 25 ml deionized water and then heated with stirring from 25 to 130 °C for 6 h. No change in color was observed at neutral pH. The flask was cooled to room temperature, followed by the addition of Ba(OH)2·8H2O (13.29 g, 42.12 mmol), and again refluxed for 6 h with stirring. The progress of reaction was monitored with the help of pH. The pH remains unchanged 7.5, and it was filtered and a crystalline white powder was obtained after several washing with deionized water. After calcinations, 2.44 g of white powder was obtained. The nanopowder is UV active, and its fluorescence gives butter color.

figure a

In vitro cytotoxicity assessment of ZnBaO2 nanoparticles

In this study, we used primary human hepatocytes for in vitro investigation of ZnBaO2 nanoparticles for its cytotoxicity effects. Different biological methods were applied to measure the cellular parameters during nanoparticles exposure and compared with control. Each parameter was validated in triplicate and repeated at least three times.

Hepatocytes culture and ZnBaO2 nanoparticles exposure

Primary human hepatocytes were cultured as previously described (Vishwakarma et al. 2014). Briefly, 2 × 104 cells were cultured on 0.2 % gelatin-coated six-well plates in serum-free medium containing 20 ng/ml epidermal growth factor (EGF; PeproTech, London, UK), 10 ng/ml basic fibroblast growth factor (b-FGF; PeproTech), 20 ng/ml hepatocyte growth factor (HGF; Sigma, St. Louis, MO, USA) and 0.61 g/l nicotinamide (Sigma) with 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate. Cells were maintained in the appropriate culture medium at 37 °C, 95 % humidity and 5 % CO2. After reaching to 70–80 % confluency, cells were harvested by trypsinization and cultured in respective plates with and without nanoparticles for 72 h in the above-mentioned culture conditions. Cells without nanoparticles were considered as control.

Relative cell viability

Relative cell viability was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay which was further validated by trypan blue exclusion assay after 24, 48 and 72 h of exposure to ZnBaO2 nanoparticles at different concentrations ranging from 10 to 100 μg/ml (Athar 2015). The viability was checked after plating the cells with and/or without nanoparticles and incubating them for 72 h.

Cell adhesion assay

In the adhesion assay at 4 h after plating the cells, we estimated the number of cells adhered on the culture plate. Cell adhesion assay was carried out by plating 2 × 104 cells along with ZnBaO2 nanoparticles on 0.2 % gelatin-coated coverslips and incubating them for 4 h. After 4 h of incubation, non-adherent cells were removed and 50 μl of 1 mg/ml MTT solution was added to the remaining cells and then incubated at 37 °C, 5 % CO2 and 95 % air conditions for 4–5 h. The reaction was stopped by adding 100 μl of acidified isopropanol. The percentage of cell adhesion was calculated by taking absorbance at 570 nm by using a microplate reader (BioRad).

Cell proliferation assay

Hepatocytes proliferation was measured by using MTT cell proliferation assay. Briefly, 2 × 104 primary human hepatocytes were mixed with ZnBaO2 nanoparticles in variable concentrations as described above and cultured in a 96-well culture plate coated with 0.2 % gelatin. Cells were maintained in serum-free medium under standard condition as described in previous sections. Each variable was kept in triplicate for 24, 48 and 72 h. At each time point, 50 μl of 1 mg/ml MTT was added and incubated for 4–5 h. After incubation, purple formazan crystal was dissolved using 100 μl acidified isopropanol. Absorbance was measured at 570 nm and compared with the control.

LDH activity measurement

ZnBaO2 nanoparticles cytotoxicity on membrane organization was assayed by the amount of lactate dehydrogenase (LDH) released from dead cells using LDH assay kit (Promega, WI). For 24, 48 and 72 h, 2 × 104 viable hepatocytes were incubated with 100 µl of ZnBaO2 nanoparticles (10–100 μg/ml). It was then mixed with 50 µl of reagent. The mixture was incubated for 30 min at room temperature. The reaction was stopped by adding 50 µl of stop solution. Absorbance was measured at 490 nm using a microplate reader (BIORAD). Total cell lysate was used as positive control to assess kit sensitivity for 100 % with the release of LDH from the dead cells after incubation.

Detoxification functional assay

Functional toxicity of ZnBaO2 nanoparticles was assessed by using detoxifying functional assay with NH4Cl as a source for NH3 in human hepatocytes. After exposure of dispersed ZnBaO2 nanoparticles, 2.5 mM NH4Cl was used to assess the rate of ureagenesis in cultured human hepatocytes. The influence of nanoparticles concentration was tested for exposure to 10–100 µg/ml concentrations for 24, 48 and 72 h during in vitro proliferation. Briefly, one million human hepatocytes were incubated with different concentrations of ZnBaO2 nanoparticles for 72 h and used to estimate the efficiency of NH4Cl detoxification. Briefly, after finishing the incubation with nanoparticles, culture supernatants were collected from each group and then centrifuged at 5000×g for 5 min to obtain a cell-free supernatant. Urea formation was detected by urease method using a commercial kit (SIGMA).

Statistical analysis

All data were expressed as mean ± standard deviation. Results were compared by one-way and two-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. A value of p < 0.05 was considered statistically significant.

Results and discussion

Understanding the effect of electronegativity and ionization potential of metal ion helps to control the kinetic–thermodynamic parameters for the formation of primary nuclei and leads to growth process via self-assembly of nanobricks. The synthesis was carried out at moderate temperature by using single-source molecular precursor (Athar et al. 2015a, b, c, d, e; Wang 2000a, b; Mishra et al. 2007).

FTIR

The study was carried out after calcinations, thereby supporting the formation of self-assembled framework by observing the strong Zn–O–Ba symmetric and asymmetric peaks at 498.97 and 851.51 cm−1, respectively. After, there is no significant peak at the range from 1000 to 400 cm−1, which supports the formation of particle after calcinations with purity. At 1000 °C all the surface impurities were removed. Bending vibration of Zn–O–Ba occurs at 1421–61 cm−1, and formation of nanostructure framework takes place by retaining the nanopowder purity. The presence of symmetric and asymmetric peaks at 356 and 1750 cm−1 occurred due to absorption of atmospheric moisture during sample preparation. However, it presumed that nanostructure framework is retained without any change in shifting of frequencies after aging as shown in Fig. 1 (Pavia et al. 2009; Skoog et al. 2007; Stuart 2004).

Fig. 1
figure 1

FTIR study of ZnBaO2 nanoparticles

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UV studies

UV–Visible spectra were recorded in MeOH to study their electronic properties along with crystalline structure. The presence of excitonic absorption band at 214.4 nm and occurrence of broad transmittance peak at 257.6 nm can be assigned to π–π* and π–p bathochromic shift, which support the formation of monodispersable colloidal powder with well-defined nanostructured framework due to quantum size effect, which leads to control the surface morphology to enhance functional properties. Controlling the kinetic–thermodynamic parameters helps to shift bathochromic wavelength in the presence of lone pair of electron present in the bridging oxygen atom in between two different metal ions and leads to stabilization of electronic state from the ground state. The formation of self-assembled nanostructured gives crystalline shape due to quantum size effect and oxygen vacancy by using two different metal ions. As the particle size increases, the significant shifting of band occurs, thereby reducing the forbidden band due to the absence of defective surface morphology which leads to aggregation due to primary nucleation without effecting the chemical composition as shown in Fig. 2 (Skoog et al. 2007; Stuart 2004; Gabbott 2007).

Fig. 2
figure 2

UV-visible spectra of ZnBaO2 nanoparticles

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XRD studies

The presence of strong diffraction peaks confirms the formation of crystalline structure of nanostructure ZnBaO2 framework. The particle size was calculated with the help of Bragg–Scherer’s equation. It is difficult to assign diffraction peaks, because it is a new nanopowder, so far not reported in JCPDS data file. The presence of sharp peaks supports the formation of crystalline phase. The particle size was calculated by using Scherer equation and found to be 15.88 nm. All peaks can be assumed by assigning index to hexagonal phase, and no other phase was observed supporting the high purity of the sample as shown in Fig. 3 (Skoog et al. 2007; Stuart 2004; Mitra 2003; Guinier 1994).

Fig. 3
figure 3

XRD study showing strong diffraction peaks which confirm the formation of crystalline structure of nanostructure ZnBaO2 framework

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Thermal studies

The thermal analysis was carried out to understand the changes taking place in the particle shape, size, morphology and texture chemistry after the removal of surface impurities to give right prediction of physical properties. Thermal studies help in getting the information of how crystalline particle was built via self-assembly. The first step involves the removal of unbound water at 85 °C and then followed by removal of other volatile materials at 170 and 210 °C. Thereafter, formation of crystalline powder takes place as supported by no further change in weight. The same information was obtained from DTA studies. It is concluded that the formation of phase strongly depends on the type of molecular precursor used, mode of synthetic methodology and polarity of solvent. The detailed analysis is shown in Fig. 4 (Skoog et al. 2007; Stuart 2004; Mitra 2003; Gabbott 2007).

Fig. 4
figure 4

Thermal study showed that formation of phase strongly depends on the type of molecular precursor used, mode of synthetic methodology and polarity of solvent

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SEM studies

The shape of ZnBaO2 nanopowder obtained was granular and well dispersed with a square shape. SEM images show flake-like structure due to inter- and intramolecular forces with limited porosity. The small particle interaction takes place due to Ostwald ripening process and with high surface energy as shown in Fig. 5 (Mitra 2003; Davydov 2003; Zeng 2007).

Fig. 5
figure 5

SEM image of ZnBaO2 nanoparticles shows granular, well-dispersed, square and flake-like structure

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TEM studies

TEM investigation gives information about the structural and morphological features of nanoparticles. TEM image shows granular, non-spherical, round and smooth with different particle morphology due to aggregation after annealing and the presence of high surface energy with narrow particle size distribution. The nanoparticle agglomerates into several other primary particles which can be classified as secondary particles due to the presence of soft forces as well as hard forces as shown in Fig. 6 (Mitra 2003; Davydov 2003; Zeng 2007; Wang 2000a, b).

Fig. 6
figure 6

TEM image ZnBaO2 nanoparticles shows granular non-spherical round and smooth particle with different particle morphology

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DLS studies

DLS studies were preformed with scattering of light intensity with reference to time due to random Brownian motion which leads to stability of suspended particle size in water. The mean particle size diameter was found to be 300 nm as shown in Fig. 7 (Jillavenkatesa et al. 2001; Brillain 2001).

Fig. 7
figure 7

DLS study of ZnBaO2 nanoparticles

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Fluorescence studies

The fluorescence studies exhibit the presence of broad excitonic peak at 322 and 351 nm with excitation intensity (Y-axis) at 150 and 180 to give information about the formation of nanostructure framework with purity. The excitation intensity changes with temperature; however, intrinsic intensity remains constant with concentration. The following observation can be assumed: (1) the shape and band maxima of absorption and fluorescence spectra remain unchanged, (2) no other type of emission band was observed and (3) finally absorbance of the fluorophore does not change during experiment as shown in Fig. 8 (Lakowicz 2006).

Fig. 8
figure 8

Fluorescence studies of ZnBaO2 nanoparticles exhibited broad excitonic peak at 322 and 351 nm with excitation intensity (Y-axis) at 150 and 180 giving information of nanostructure framework

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Cell adhesion and changes in cell viability during exposure to ZnBaO2 nanoparticles

Relative cell viability and adhesion assays on human hepatocytes were performed using protocols described in Materials and methods, and the results are shown in Fig. 9a, b. During cell adhesion assay at 4 h after plating the cells, we could estimate that almost 80 % cells were adherent in control and nanoparticles were exposed to the cell with a concentration of 30 µg/ml. However, it was gradually decreased above 40 µg/ml and was significantly reduced at higher concentrations (Fig. 9a).

Fig. 9
figure 9

Effect of ZnBaO2 nanoparticles exposure on a cell adhesion after 4 h and b relative cell viability at 24–72 h

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Relative cell viability percentage was calculated for 24, 48 and 72 h of nanoparticles exposure to hepatocytes. Figure 9b shows that there was no change in cell viability at lower concentration up to 30 µg/ml. But it was significantly reduced with incubation time and increasing nanoparticles concentration. As compared to 24 h of incubation, at 48 h and 72 h significantly reduced viability was observed above 40 µg/ml nanoparticles exposure.

Assessment of cell proliferation

Cell counts at different time points after plating 2 × 104 cells with and without nanoparticles were estimated, and data are shown in Fig. 10. We observed continuous increase in cell number with time progression. However, proliferation rate of cells was decreased with increasing nanoparticles concentration with respect to time as compared to control which could probably be due to poorer viability of cells exposed to nanoparticles.

Fig. 10
figure 10

Changes in cell numbers during hepatocytes proliferation on exposure to different concentrations of ZnBaO2 nanoparticles for 24–72 h

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LDH activity and cell membrane permeability

In vitro LDH assay was performed in human hepatocytes with incubation time of 24–72 h by using ZnBaO2 nanoparticles. Figure 11 shows that there was steady release of LDH from cells treated with low concentration of ZnBaO2 nanoparticles which was almost similar to control up to 30 µg/ml concentration. However, LDH release was enhanced above 40 µg/ml nanoparticles concentrations and was significantly high at 50–100 µg/ml. In general, high concentration of nanoparticles causes LDH leakage from cells representing reduced cell viability as well as cell membrane integrity.

Fig. 11
figure 11

Effect on cell membrane permeability measured by the release of LDH from cells during exposure to various concentrations of ZnBaO2 nanoparticles

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Effect on hepatocytes detoxification

Human hepatocytes when incubated with ZnBaO2 nanoparticles at variable concentrations for 72 h showed decrease in efficiency for detoxification of NH4Cl in dose-dependent manner (Fig. 12). After 72 h, 100 μg/ml concentration was found to have highest impact on ammonia detoxification. Lower concentrations (<30 µg/ml) did not show adverse effects in hepatocytes.

Fig. 12
figure 12

In vitro ammonia detoxification assay showing changes in urea production after 24-, 48- and 72-h exposure to ZnBaO2 nanoparticles

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Conclusions

Wet chemical approach helps to control the size, shape, composition and structural particle properties at the inter-grain space which is not oxidized in the air leading to monodispersity with colloidal stability. Right chemical composition and microstructural properties help to fabricate sensor devices. The shape of a building block is the determining factor for structural and functional properties in nanostructure. Synthetic strategies based on topotactic wet chemical approach give information of state-of-the-art design to enhance functional materials. The wet chemical approach is novel process that does not involve high temperature, so is suitable for bulk production. It was demonstrated that the hepatocytotoxicity depends on varying concentrations of ZnBaO2 nanoparticles exposure at different time points. These nanoparticles may have potential applications in a variety of areas which need to be explored further.