Barium Titanate (Batio3) Nanoparticles Exert Cytotoxicity Through Oxidative Stress in Human Lung Carcinoma (A549) Cells

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Article Barium Titanate (BaTiO3) Nanoparticles Exert Cytotoxicity through Oxidative Stress in Human Lung Carcinoma (A549) Cells

Maqusood Ahamed 1,* , Mohd Javed Akhtar 1 , M.A. Majeed Khan 1, Hisham A. Alhadlaq 1,2 and Aws Alshamsan 3

1 King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] (M.J.A.); [email protected] (M.A.M.K.); [email protected] (H.A.A.) 2 Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia 3 Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] * Correspondence: [email protected]; Tel.: +96-61-1469-8781

Received: 15 October 2020; Accepted: 18 November 2020; Published: 22 November 2020

Abstract: Barium titanate (BaTiO3) nanoparticles (BT NPs) have shown exceptional characteristics such as high dielectric constant and suitable ferro-, piezo-, and pyro-electric properties. Thus, BT NPs have shown potential to be applied in various fields including electro-optical devices and biomedicine. However, very limited knowledge is available on the interaction of BT NPs with human cells. This work was planned to study the interaction of BT NPs with human lung carcinoma (A549) cells. Results showed that BT NPs decreased cell viability in a dose- and time-dependent manner. Depletion of mitochondrial membrane potential and induction of caspase-3 and -9 enzyme activity were also observed following BT NP exposure. BT NPs further induced oxidative stress indicated by induction of pro-oxidants (reactive oxygen species and hydrogen peroxide) and reduction of antioxidants (glutathione and several antioxidant enzymes). Moreover, BT NP-induced cytotoxicity and oxidative stress were effectively abrogated by N-acetyl-cysteine (an ROS scavenger), suggesting that BT NP-induced cytotoxicity was mediated through oxidative stress. Intriguingly, the underlying mechanism of cytotoxicity of BT NPs was similar to the mode of action of ZnO NPs. At the end, we found that BT NPs did not affect the non-cancerous human lung fibroblasts (IMR-90). Altogether, BT NPs selectively induced cytotoxicity in A549 cells via oxidative stress. This work warrants further research on selective cytotoxicity mechanisms of BT NPs in different types of cancer cells and their normal counterparts.

Keywords: BaTiO3 nanoparticles; selective cytotoxicity; oxidative stress; antioxidant enzymes; cancer therapy

1. Introduction

Barium titanate (BaTiO3) nanoparticles (BT NPs), a perovskite-type ceramic material, has exceptional characteristics such as high dielectric constant and suitable ferro-, piezo-, and pyro-electric properties. BT NPs are broadly used in the manufacture of multilayer ceramic capacitor, thermistors, transducers, infrared detectors, sensors, and electro-optical devices [1,2]. Studies also suggested that BT NPs have potential to be applied in biomedical ﬁelds, including tissue engineering, drug delivery, and cancer therapy [3–6]. For instance, BT NPs enhance the cellular uptake of the anticancer drug doxorubicin [7]. BT NPs are also being investigated as bone repair material due to their excellent osteoinductivity [6,8]. Nevertheless, research on the underlying mechanisms of

Nanomaterials 2020, 10, 2309; doi:10.3390/nano10112309 www.mdpi.com/journal/nanomaterials Nanomaterials 2020, 10, 2309 2 of 13 biological effects of BT NPs at cellular and molecular levels is scarce. Therefore, thorough study on the interaction of BT NPs with human cells is indispensable before their applications in biomedical field. Investigation on underlying mechanisms of toxicity of NPs is still in progress. However, excessive production of reactive oxygen species (ROS) and oxidative damage of cell macromolecules have been established now as some of the key toxicity mechanisms of NPs. Oxidative stress happens when the intracellular level of ROS increases beyond a certain level and limits the antioxidant defense ability of cells. Several recent studies suggested that NPs induce cytotoxicity through ROS generation and oxidative damage of cellular constituents [9–11]. Recent research also indicated that generation of intracellular ROS levels in a controlled way can be exploited as a therapeutic tool for cancer treatment [12,13]. In this regard, relatively higher levels of ROS observed in cancer cells as compared to their normal counterparts represent a promising tool to target cancer cell selectively [12]. Studies on nanotechnology-based novel drug delivery and cancer therapy are now gaining recognition [14–16]. Several studies have demonstrated that defined dosages of semiconductor NPs potentially killed cancer cells, while not much affecting the non-cancerous normal cells [17–19]. For example, ZnO NPs have shown inherent properties of killing cancer cells while sparing the normal cells [20,21]. Our recent work indicated that SnO2 NPs have inherent characteristics of exerting cytotoxicity to human breast cancer cells while sparing the normal human lung fibroblasts (IMR-90) [22]. Metal doped TiO2 NPs also showed selective toxicity against cancer cells [19,23]. These semiconductor NPs may utilize the extreme conditions of oxidative stress of cancer cells. Increases in intracellular ROS to a certain level due to NP exposure may damage the already ROS-stressed cancer cells without much affecting the relatively less stressed normal cells. This work was designed to investigate the cytotoxicity mechanisms of BT NPs in human lung carcinoma (A549) cells. The objective of the present study was conducted by estimating the cell viability, cellular morphology, activity of apoptotic enzymes (caspase-3 and -9), and mitochondrial membrane potential in A549 cells following exposure to BT NPs for 24 h. Cytotoxicity mechanisms of BT NPs were further delineated by measuring the several markers of oxidative stress, such as ROS, hydrogen peroxide (H2O2), and various antioxidant enzymes. Human lung cancer cell line (A549) was selected in this study because lung cancer is a leading cause of cancer-related mortality among men and women [24]. To check the selective cytotoxicity of BT NPs against A549 cancer cells, we also assessed the cytotoxic potential of BT NPs in normal human lung fibroblasts (IMR-90).

2. Materials and Methods

2.1. Synthesis of BaTiO3 Nanoparticles

Barium titanate (BaTiO3) nanoparticles were prepared by the facile sol–gel hydrothermal method using titanium tetrachloride (TiCl4) and barium chloride (BaCl2) as precursor materials. In brief, 1 mL of TiCl4 was diluted in 2 mL of HCl (2M) to prepare a slightly yellow solution. Then, 2.5 g of BaCl2 was dissolved in 20 mL of deionized water. The two solutions were mixed to form a barium titanium solution. Under stirring and N2 bubbling, 15 mL of NaOH (6M) was added to this solution to form a homogeneous colloidal barium titanium slurry. The colloidal solution of barium titanium was transferred into 50 mL Teﬂon-lined stainless autoclave and heated at 100 ◦ C for 2 h. After the completion of reaction, the autoclave was allowed to cool to room temperature. The prepared solid white powder of barium titanate was collected from the autoclave, washed several times with distilled water and ethanol, and dried at 60 ◦ C for h in a vacuum.

2.2. Characterization of BaTiO3 Nanoparticles Crystallinity and phase purity of prepared BT nanopowder were examined by powder X-ray diffraction (PXRD) (Malvern, WR14 1XZ, UK) equipped with a PanAnalytic X’Pert Pro X-ray (Malvern, UK) diffractometer with nickel filter using Cu-Kα radiation (λ = 0.154 nm at 45 kV Nanomaterials 2020, 10, 2309 3 of 13 and 40 mA) as the X-ray source. BT NPs were also characterized by micro-Raman spectroscopy (Horiba Raman system, IY-Horiba-T64000) (Northampton, NN3 6FL, UK). Surface morphology of BT nanopowder was assessed by field emission scanning electron microscopy (FESEM, JSM-7600F, JEOL, Inc., Tokyo, Japan) at an accelerating voltage of 15 kV. Shape and size of BT NPs were determined by field emission transmission electron microscopy (FETEM, JEM-2100F, JEOL, Inc., Tokyo, Japan) at an accelerating voltage of 200 kV. Briefly, a diluted suspension of BT NPs (50 µg/mL in ethanol) was sonicated in a sonicator bath for 10 min at 40 W to obtain a homogenous mixture. Then, a drop of suspension was placed onto a TEM grid, air dried, and observed with FETEM. Hydrodynamic size and zeta potential of BT NPs in distilled water and culture media were estimated by dynamic light scattering (DLS) (ZetaSizer Nano-HT, Malvern Instruments, UK). In brief, BT nanopowder was suspended in distilled water and culture medium at a concentration of 50 µg/mL. Then, the suspension was sonicated by a sonicator bath at room temperature for 10 min at 40 W, and DLS experiments were performed. We chose a 50 µg/mL concentration of BT NPs for DLS measurement, because this concentration was used in most of the biochemical studies.

2.3. Cell Culture and Exposure of BaTiO3 Nanoparticles Human lung cancer cells (A549) and non-cancerous human lung fibroblasts (IMR-90) (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) with the supplementation of 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen) at 37 ◦C in a humidified 5% CO2 incubator. To remove endotoxin contamination (if present), prepared NPs were autoclaved at 121 ◦C for 30 min using saturated steam under 15 pounds per square inch (psi) of pressure. An endpoint chromogenic limulus amebocyte lysate (LAL) assay further confirmed that prepared BT NPs were not contaminated from endotoxins (Figure S1 of Supplementary Materials). Cells were exposed for 24, 48, or 72 h to different concentrations (5–200 µg/mL) of BT NPs. Briefly, BT NPs were suspended in culture medium (DMEM + 10% FBS) and diluted to desired concentrations (5–200 µg/mL). Different dilutions of NPs were further sonicated utilizing a sonicator bath at room temperature for 10 min to avoid agglomeration of NPs before exposure to cells. The ZnO NPs (25 µg/mL) was used as a positive control [22,25]. Preparation of ZnO NPs suspension was similar to BT NPs. In some experiments, cells were co-exposed with 2 mM of N-acetylcysteine (NAC) (Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of BT NPs/ZnO NPs. Cells without NP exposure served as controls in each experiment.

2.4. Cytotoxicity Study Both MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [26] and NRU (neutral red uptake) [27] methods were used for the cell viability assay with some important changes [28]. Mitochondrial membrane potential was recorded with a microplate reader (Synergy-HT, BioTek, Winooski, VT, USA) and a ﬂuorescent microscope (DMi8, Leica Microsystems, GmbH, Wetzlar, Germany) using the probe rhodamine-123 (Rh-123) (Sigma-Aldrich) [29]. Caspase-3 and caspase-9 enzyme activities were determined using colorimetric assay kits of BioVision kits. Morphologies of cells were captured through a phase-contrast microscope (DMIL, Leica Microsystems). Brief descriptions of each parameter of cytotoxicity are given in the Materials and Methods section of the Supplementary Materials.

2.5. Oxidative Stress Study Intracellular levels of reactive oxygen species (ROS) were recorded with a microplate reader (Synergy-HT, BioTek, Vinooski, VT, USA) and a fluorescent microscope (DMi8, Leica Microsystems, Wetzlar, Germany) using the fluorescent probe 2’-,7’-dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma-Aldrich) [29,30]. Intracellular hydrogen peroxide (H2O2) level was estimated by a MAK164 fluorescence kit (Sigma-Aldrich, St. Louis, MO, USA). Glutathione content was assayed Nanomaterials 2020, 10, x 4 of 14 Nanomaterials 2020, 10, 2309 4 of 13 using Ellman’s method [31]. The activity of glutathione reductase (GR) enzyme was assayed according to the procedure described by Carlberg and Mannervik [32]. Rotruck’s protocol was using Ellman’s method [31]. The activity of glutathione reductase (GR) enzyme was assayed according applied for the glutathione peroxidase (GPx) enzyme assay [33]. A colorimetric assay of the to the procedure described by Carlberg and Mannervik [32]. Rotruck’s protocol was applied for the superoxide dismutase (SOD) enzyme was done using a kit from the Cayman Chemical Company glutathione peroxidase (GPx) enzyme assay [33]. A colorimetric assay of the superoxide dismutase (Michigan, OH, USA) [34]. Protein estimation was done by Bradford’s method [35]. Brief procedures (SOD) enzyme was done using a kit from the Cayman Chemical Company (Michigan, OH, USA) [34]. of each parameter of oxidative stress are given in the Materials and Methods section of the Protein estimation was done by Bradford’s method [35]. Brief procedures of each parameter of oxidative Supplementary Materials. stress are given in the Materials and Methods section of the Supplementary Materials.

2.6.2.6. StatisticalStatistical AnalysisAnalysis One-wayOne-way analysisanalysis ofof variancevariance (ANOVA)(ANOVA) followedfollowed byby Dunnett’sDunnett’s multiplemultiple comparisoncomparison teststests werewere usedused forfor statisticalstatistical analysis.analysis. AA pp-value-value <<0.050.05 was assigned as statistically signiﬁcant.significant.

3.3. ResultsResults andand DiscussionDiscussion

3.1.3.1. CharacterizationCharacterization ofof BaTiOBaTiO33 NanoparticlesNanoparticles FigureFigure1 1AA represents represents the the XRD XRD spectra spectra of of BT BT NPs. NPs. All All the the peaks peaks were were well-matched well-matched with with JCPDS JCPDS no.no. 892475,892475, exhibitingexhibiting thethe formationformation ofof thethe cubiccubic phasephase ofof BTBT NPs.NPs. TheThe peakspeaks atat 31.8131.81°,◦, 39.1239.12°,◦, 45.4445.44°,◦, 51.0851.08°,◦, 56.3456.34°,◦, and and 66.06 66.06 were were attributed attributed to (110), to (111),(110), (200),(111), (210), (200), (211), (210), and (211), (220) planes,and (220) respectively. planes, Averagerespectively. particle Average size was particle determined size was from determine the strongestd from peakthe strongest (110) using peak Scherrer’s (110) using equation Scherrer’s [19]. Theequation average [19]. particle The average size was particle estimated size towas be estimated around 15 to nm. be around Raman 15 spectroscopy nm. Raman is spectroscopy a very sensitive is a techniquevery sensitive to probe technique atomic to structuresprobe atomic of materials. structures Raman of materials. spectra Raman of prepared spectra BT of NPsprepared are given BT NPs in 1 1 −1 1−1 −1 1 −1 Figureare given1B. Peaksin Figure at 281 1B. cmPeaks− , at 305 281 cm cm− , 514, 305 cm cm− ,, and 514 720cm cm, and− presented720 cm presented Raman shifts Raman of BTshifts NPs. of TheseBT NPs. peaks These suggested peaks thesuggested crystalline the cubic crystalline phase ofcubi BTc NPsphase [36 ,of37 ].BT This NPs result [36,37]. was This in agreement result was with in XRDagreement spectra. with XRD spectra.

FigureFigure 1.1.( A(A) XRD) XRD and and (B) ( RamanB) Raman spectra spectra of barium of barium titanate titanate nanoparticles nanoparticles (BT NPs). (BT XRD: NPs). X-ray XRD: diffraction. X-ray diffraction. Morphological characterization was done by FESEM and FETEM. Figure2A–C represent the typical SEM andMorphological TEM images characterization of BT NPs. The was average done size by calculatedFESEM and from FETEM. TEM imageFigure (>2A–C100 particles)represent was the aroundtypical 16SEM nm, and which TEM was images well-matched of BT NPs. with The theaverage size calculatedsize calculated from from XRD TEM spectra. image A high(>100 resolution particles) TEMwas around image (Figure 16 nm,2 D)which suggested was well-matched the clear lattice with fringes the size and calculated crystallinity from of BTXRD NPs, spectra. supporting A high theresolution XRD data. TEM The image distance (Figure between 2D) adjacentsuggested planes the clear was foundlattice tofringes be 0.286 and nm, crystallinity corresponding of BT to NPs, the (110)supporting plane of the the XRD cubic BTdata. NPs The (Figure distance2D). Characterizationbetween adjacent data planes of BT NPswas werefound in accordanceto be 0.286 with nm, recentlycorresponding reported to studiesthe (110) [6 ,38plane]. of the cubic BT NPs (Figure 2D). Characterization data of BT NPs were in accordance with recently reported studies [6,38].

Nanomaterials 2020, 10, 2309 5 of 13 Nanomaterials 2020, 10, x 5 of 14

FigureFigure 2. (A 2.) ( FESEM,A) FESEM, (B (Band andC C)) lowlow resolution FETEM, FETEM, and and (D) (highD) high resolution resolution FETEM FETEM images imagesof BT of BT NPs.NPs. FESEM:FESEM: ﬁeldfield emissionemission sca scanningnning electron electron microscopy, microscopy, FETEM: FETEM: field ﬁeld emission emission transmission transmission electron microscopy. electron microscopy. Physicochemical characterization of BT NPs in distilled water and culture medium is given in Physicochemical characterization of BT NPs in distilled water and culture medium is given in Table 1. Hydrodynamic size of BT NPs was 6–7 times higher than the size of nano-powder (primary Tablesize)1. Hydrodynamic calculated from size XRD of and BT NPsTEM. was Higher 6–7 timeshydrodyn higheramic than size the of BT size NPs of nano-powderwas due to the (primarytendency size) calculatedof NPs from to agglomerate XRD and TEM.in aqueous Higher medium hydrodynamic [19]. Zeta size potential of BT results NPs was indicated due to that the BT tendency NPs of NPs tosuspended agglomerate in water in aqueoushad positive medium surface [ 19charges,]. Zeta whereas potential BT resultsNPs suspended indicated in culture that BT medium NPs suspended had in waternegative had positivesurface charges. surface Negative charges, surface whereas charge BT NPss of BT suspended NPs in culture in culture media medium could be had due negative to surfaceadsorption charges. of Negativenegatively surfacecharged proteins charges on of the BT surface NPs in of culture NPs. Physicochemical media could characterization be due to adsorption of of negativelyZnO NPs charged(positive proteinscontrol) in on distilled the surface water ofand NPs. culture Physicochemical medium is also given characterization in Table 1 [25]. of ZnO NPs (positive control) in distilled water and culture medium is also given in Table1[25]. Table 1. Physicochemical characterization of BaTiO3 NPs and ZnO NPs.

TableBaTiO 1. Physicochemical3 NPs characterization ZnO of BaTiO NPs [25]3 NPs and ZnO NPs. Parameters Mean Value Parameters Mean Value BaTiO NPs ZnO NPs [25] Primary3 size Primary size ParametersTEM Mean16 Valuenm ParametersTEM Mean41 nm Value PrimaryXRD size 15 nm PrimaryXRD size 43 nm HydrodynamicTEM size # 16 nm Hydrodynamic TEM size # 41 nm DistilledXRD water 105 15 ± nm 5 nm Distilled XRD water 113 43± 7 nmnm Culture medium 114 ± 3 nm Culture medium 155 ± 6 nm Hydrodynamic size # Hydrodynamic size # # # DistilledZeta potential water 105 5 nmZeta Distilled potential water 113 7 nm ± ± CultureDistilled medium water 114 23 ±3 2 nmeV Culture Distilled medium water 17 155 ± 3 eV6 nm Culture medium −27± ± 3 eV Culture medium −19 ±± 2 eV Zeta potential # Zeta potential # TEM: transmission electron microscopy, XRD: X-ray diffraction, culture medium: DMEM + 10% fetal Distilled water 23 2 eV Distilled water 17 3 eV bovine serum, # data presented are mean± ± SD of three independent experiments (n =± 3). Culture medium 27 3 eV Culture medium 19 2 eV − ± − ± TEM: transmission electron microscopy, XRD: X-ray diﬀraction, culture medium: DMEM + 10% fetal bovine serum, # data presented are mean SD of three independent experiments (n = 3). ± Nanomaterials 2020, 10, 2309 6 of 13 Nanomaterials 2020, 10, x 6 of 14

3.2.3.2. Cytotoxic Cytotoxic Response Response of of BaTiO BaTiO3 3Nanoparticles Nanoparticles CellsCells were were exposed exposed to ditoﬀ erentdifferent concentrations concentrations (5–200 (5–200µg/mL) µg/mL) of BT of NPs BT forNPs di ﬀforerent different time intervals time (24,intervals 48, and (24, 72 48, h), and and 72 cell h), and viability cell viability was measured was measured by MTT by MTT and and NRU NRU assays. assays. The The MTT MTT assayassay is utilizedis utilized to determine to determine metabolic metabolic activity activity as as an an indicator indicator of ofcell cell viability.viability. The The NRU NRU assay assay is isbased based on on the capability of live cells to integrate and bind the supravital dye neutral red (NR) in the lysosomes. the capability of live cells to integrate and bind the supravital dye neutral red (NR) in the lysosomes. MTT results showed that BT NPs decreased cell viability in a concentration (25–200 µg/mL)- and time MTT results showed that BT NPs decreased cell viability in a concentration (25–200 µg/mL)- and time (24–72 h)-dependent manner (Figure 3A). In agreement with MTT data, NRU results also (24–72 h)-dependent manner (Figure3A). In agreement with MTT data, NRU results also demonstrated demonstrated that BT NPs decreased cell viability in a concentration (25–200 µg/mL)- and time (24– that BT NPs decreased cell viability in a concentration (25–200 µg/mL)- and time (24–72 h)-dependent 72 h)-dependent manner (Figure 3B). Moreover, the positive control ZnO NPs (25 µg/mL) also manner (Figure3B). Moreover, the positive control ZnO NPs (25 µg/mL) also decreased the cell viability decreased the cell viability time-dependently. A recent study also reported that nitrogen-doped TiO2 time-dependently.NPs inhibit the proliferation A recent study of malignant also reported MDA-MB-231 that nitrogen-doped breast cancer TiO epithelial2 NPs inhibit cells the[39]. proliferation Based on of malignantcell viability MDA-MB-231 results, we utilized breast cancer the 25–100 epithelial µg/mL cells concentration [39]. Based onrange cell viabilityfor further results, apoptosis we utilized and theoxidative 25–100 µ stressg/mL studies. concentration range for further apoptosis and oxidative stress studies.

FigureFigure 3. Cell3. Cell viability viability of A549of A549 cells cells exposed exposed for 24,for 48,24, and48, 72and h to72 dih fftoerent different concentrations concentrations (5–200 (5–200µg/mL) of BTµg/mL) NPs. of (A BT) MTT NPs. cell(A) viability.MTT cell (viability.B) NRU ( cellB) NRU viability. cell viability. ZnO NPs ZnO (25 NPsµg/mL (25 forµg/mL 24 h) for were 24 h) used were as a positiveused as control. a positive Data control. are provided Data are as provided mean SDas mean of three ± SD independent of three independent experiments experiments (n = 3). * Indicates(n = 3). ± statistically*Indicates significant statistically di significantfference from difference the control from the group control (p < group0.05). (p < 0.05).

ApoptoticApoptotic response response of BTof BT NPs NPs in A549 in A549 cells cells was was examined examined by measuring by measuring the MMP the MMP level andlevel activity and of caspase-3activity of andcaspase-3 -9 enzymes. and -9 enzymes. Apoptosis Apoptosis is triggered is tr byiggered various by factors various such factors as stress, such as nutrient stress, nutrient deﬁciency, starvation,deficiency, foreign starvation, particles, foreign and particles, certain drugsand certain [40]. drugs MMP [40]. is a reliableMMP is indicatora reliable indicator of cellular of stresscellular and ongoingstress and processes ongoing of apoptosis.processes of Our apoptosis. quantitative Our quan analysistitative suggested analysis that suggested BT NPs that induced BT NPs MMP induced loss in

Nanomaterials 2020, 10, 2309 7 of 13 Nanomaterials 2020, 10, x 7 of 14 a dose-dependentMMP loss in a dose-dependent manner (25–100 mannerµg/mL) (25–100 (Figure µg/m4A). FluorescentL) (Figure 4A). microscopy Fluorescent data microscopy also depicted data that thealso brightness depicted ofthat the the Rh-123 brightness probe of (red the fluorescence)Rh-123 probe (red was fluorescence) lower in BT NP-treated was lower in cells BT inNP-treated comparison tocells controls in comparison (Figure4B). to controls The brightness (Figure 4B). of theThe redbrightness fluorescence of the red of thefluorescence Rh-123 probe of the decreasedRh-123 probe with decreasingdecreased MMPwith decreasing level. ZnO MMP NPs alsolevel. decreased ZnO NPs the also MMP decreased level of the A549 MMP cells. level Caspases of A549 belongs cells. to theCaspases proteases belongs family to present the proteases in mitochondria family present and playin mitochondria crucial roles and in theplay apoptotic crucial roles pathway in the [41 ]. Amongapoptotic these pathway proteases, [41]. caspase-3 Among these and -9proteases, are known casp toase-3 be critically and -9 are involved known into initiationbe critically and involved execution ofin the initiation apoptotic and process. execution Cytochrome of the apoptotic c releases process. from Cytochrome the mitochondrial c releases intermembranefrom the mitochondrial space and bindsintermembrane to apoptotic space protease and binds activating to apoptotic factor-1 protease (Apaf-1) activating that further factor-1 binds (Apaf-1) with pro-caspase-9 that further binds to form a complexwith pro-caspase-9 called apoptosome. to form a complex This complex called apopto cleavessome. the This pro-caspase-9 complex cleaves to its activethe pro-caspase-9 form, caspase-9, to whichits active further form, activates caspase-9, caspase-3. which further Activated activates caspase-3 caspase-3. can cleaveActivated several caspase-3 structural can cleave and regulatoryseveral structural and regulatory proteins involved in the apoptotic process [42]. In the present study, we proteins involved in the apoptotic process [42]. In the present study, we observed that BT NPs induced observed that BT NPs induced the activity of caspase-3 and -9 enzymes in a dose-dependent manner the activity of caspase-3 and -9 enzymes in a dose-dependent manner (Figure4C,D). Positive control (Figure 4C,D). Positive control ZnO NPs also decreased the MMP level and increased the activity of ZnO NPs also decreased the MMP level and increased the activity of caspase-3 and -9 enzymes in caspase-3 and -9 enzymes in A549 cells. Marino and co-workers reported that remote ultrasound- A549 cells. Marino and co-workers reported that remote ultrasound-mediated piezo-stimulation of mediated piezo-stimulation of BT NPs allowed in vitro growth of glioblastoma cells to be BTsignificantly NPs allowed decreasedin vitro [4].growth Another of glioblastoma study reported cells that to BT be significantlyNPs in combination decreased with [ 4tumor-treating]. Another study reportedfields exhibited that BT NPsantitumor in combination activity against with breast tumor-treating cancer cells fields by ma exhibitednipulating antitumor the cell activitycycle-related against breastapoptosis cancer pathway cells by [3]. manipulating The apoptotic the cellresponse cycle-related of other apoptosisceramic NPs pathway were also [3]. Thepreviously apoptotic reported response of[9]. other ceramic NPs were also previously reported [9].

FigureFigure 4. 4.( A(A)) MMP MMP levellevel ofof A549A549 cells exposed exposed for for 24 24 h h to to different different concentrations concentrations (25–100 (25–100 µg/mL)µg/mL) of of BTBT NPs. NPs. (B ()B Fluorescent) Fluorescent images images of of the the Rh-123 Rh-123 probe probe (indicator (indicator of of MMP MMP level) level) after after exposure exposure to to BT BT NPs (50NPsµg /(50mL) µg/mL) for 24 h.for ( C24and h. (CD and) Caspase-3 D) Caspase-3 and caspase-9 and caspase-9 enzyme enzyme activity activity of A549 of A549 cells exposedcells exposed for 24 h tofor di ff24erent h to different concentrations concentrations (25–100 (25–100µg/mL) µg/mL) of BT NPs.of BT NPs. ZnO ZnO NPs NPs (25 µ(25g/ mLµg/mL for for 24 24 h) h) was was used used as a positiveas a positive control. control. Dataare Data provided are provided as mean as meanSD of ± threeSD of independent three independent experiments experiments (n = 3). (n * = Indicates 3). * ± statisticallyIndicates statistically significant significant difference difference from the controlfrom the group control (p group< 0.05). (p < 0.05).

3.3. Oxidative Stress Response of BaTiO 3 Nanoparticles Studies have suggested that NPs induce cytotoxicity through oxidative stress. Oxidative stress is the state where excess pro-oxidant generation limits the antioxidant defense capacity of the cells [43].

Nanomaterials 2020, 10, x 8 of 14

Nanomaterials3.3. Oxidative2020, Stress10, 2309 Response of BaTiO3 Nanoparticles 8 of 13 Studies have suggested that NPs induce cytotoxicity through oxidative stress. Oxidative stress is the state where excess pro-oxidant generation limits the antioxidant defense capacity of the cells Excessive generation of ROS leads the disturbance of cellular redox homeostasis and/or injury to [43]. Excessive generation of ROS leads the disturbance of cellular redox homeostasis and/or injury cell macromolecules [44]. In this study, we assessed the ROS, H2O2, GSH, and activity of several to cell macromolecules [44]. In this study, we assessed the ROS, H2O2, GSH, and activity of several antioxidant enzymes in A549 cells exposed for 24 h to 50 µg/mL of BT NPs in the presence or absence antioxidant enzymes in A549 cells exposed for 24 h to 50 µg/mL of BT NPs in the presence or absence of NACof NAC (ROS (ROS scavenger). scavenger). Fluorescence Fluorescence microscopy microscopy data demonstrated that that the the brightness brightness of ofthe the DCF DCF probeprobe (marker (marker of of ROS ROS generation) generation) increased increased inin cellscells exposed to to BT BT NPs NPs or or ZnO ZnO NPs NPs as as compared compared to to controlcontrol cells cells (Figure (Figure5A). 5A). These These images images also also suggested suggested thatthat thethe increased level level of of ROS ROS in in BT BT NPs NPs or or ZnOZnO NP-treated NP-treated cells cells was was e ffeffectivelyectively abrogated abrogated byby NACNAC co-exposure (Figure (Figure 5A).5A). Quantitative Quantitative data data of ROSof ROS generation generation also also indicated indicated that that significantsignificant increases in in ROS ROS levels levels due due to to BT BT NP NP or or ZnO ZnO NP NP exposureexposure were were effi efficientlyciently alleviated alleviated by by NAC NAC co-exposure co-exposure (Figure 55B).B). Pro-oxidant H H2O2O2 levels2 levels in inBT BT NPNP or or ZnO ZnO NP NP exposed exposed cells cells were were significantly significantly higherhigher as compared compared to to controls controls (Figure (Figure 5C).5C). Again, Again, BTBT NP- NP- or or ZnO ZnO NP-induced NP-induced H H2O2O22level level waswas successfullysuccessfully mitigated mitigated by by NAC NAC co-exposure. co-exposure.

FigureFigure 5. Pro-oxidant5. Pro-oxidant levels levels of of A549 A549 cells cells exposed exposed for for 24 24 h to h 50to µ50g /µg/mLmL of BTof NPsBT NPs with with orwithout or without NAC (2 mM).NAC ZnO(2 mM). NPs (25ZnOµg /NPsmL for(25 24 µg/mL h) were for used 24 ash) a were positive used control. as a positive (A) Fluorescent control. microscopic (A) Fluorescent images of DCFmicroscopic intensity images in cells of (anDCF indicator intensity of in ROS cells generation). (an indicator (B )of Quantitative ROS generation). analysis (B) ofQuantitative intracellular ROSanalysis level. (ofC )intracellular Quantitative ROS analysis level. of(C intracellular) Quantitative H analysisO level. of Dataintracellular are provided H2O2 level. as mean Data SDare of 2 2 ± threeprovided independent as mean experiments ± SD of three (n independent= 3). * Indicates experiments statistically (n = significant3). * Indicates diff statisticallyerence from significant the control groupdifference (p < 0.05). from# theIndicates control that group NAC (p < e ff0.05).ectively # Indicates abrogated that theNAC eff ecteffectively of BT NPs abrogated or ZnO the NPs. effect of BT NPs or ZnO NPs. We further examined the effect of BT NPs on the antioxidant defense system of A549 cells. Cells were exposedWe further for 24 examined h to 50 µ theg/mL effect of BTof BT NPs NPs with on or the without antioxidant NAC. defense Results system demonstrated of A549 cells. that BTCells NPs or ZnOwere NPsexposed significantly for 24 h to decreased 50 µg/mL theof BT GSH NPs level with and or without as well NAC. as the Results activity demonstrated of several antioxidant that BT enzymesNPs or (GPx, ZnO GR,NPs and significantly SOD) in A549 decreased cells as the compared GSH le tovel controls and as (Figurewell as6A–D). the activity We further of several noticed thatantioxidant the effects enzymes of BT NPs (GPx, or ZnOGR, and NPs SOD) on antioxidant in A549 cells defense as compared systems to were controls successfully (Figure 6A–D). reverted We by further noticed that the effects of BT NPs or ZnO NPs on antioxidant defense systems were NAC co-exposure. These results suggested that BT NPs have potential to generate oxidative stress in successfully reverted by NAC co-exposure. These results suggested that BT NPs have potential to A549 cells in a similar fashion as in ZnO NPs. Barium oxide (BaO) NPs also exhibited genotoxic and generate oxidative stress in A549 cells in a similar fashion as in ZnO NPs. Barium oxide (BaO) NPs apoptotic effects in L929 cells, most likely due to generation of ROS [45]. An earlier study demonstrated also exhibited genotoxic and apoptotic effects in L929 cells, most likely due to generation of ROS [45]. that Ag-doped TiO2 NPs induced reactive oxygen species generation and oxidative stress in various types of human cancer cells [19]. Nanomaterials 2020, 10, x 9 of 14

NanomaterialsAn earlier2020 study, 10, 2309 demonstrated that Ag-doped TiO2 NPs induced reactive oxygen species generation 9 of 13 and oxidative stress in various types of human cancer cells [19].

FigureFigure 6. Antioxidant 6. Antioxidant levels levels of A549of A549 cells cells exposed exposed for for 24 24 h h to to 50 50µ gµg/mL/mL of of BT BT NPs NPs with withor orwithout without NAC (2 mM).NAC ZnO (2 mM). NPs ZnO (25 µ NPsg/mL (25 for µg/mL 24 h) for was 24 usedh) was as used a positive as a positive control. control. (A) GSH (A) GSH level. level. (B) GPx(B) GPx enzyme activity.enzyme (C) activity. GR enzyme (C) GR activity. enzyme ( Dactivity.) SOD ( enzymeD) SOD enzyme activity. activity. Data are Data provided are provided as mean as meanSD ± SD of three ± independentof three independent experiments experiments (n = 3). * ( Indicatesn = 3). * Indicates statistically statistically significant significant diff erencedifference from from the the control control group # (p < 0.05).group #(pIndicates < 0.05). Indicates that NAC that e ffNACectively effectively abrogated abrogated the e fftheect effect of BT of NPsBT NPs or ZnOor ZnO NPs. NPs.

3.4. BaTiO3.4. BaTiO3 Nanoparticles3 Nanoparticles Induced Induced Cytotoxicity Cytotoxicity via Oxidative Oxidative Stress Stress We examinedWe examined the the role role of of oxidative oxidative stress stress inin BT NP-induced NP-induced cytotoxicity cytotoxicity in A549 in A549 cells. cells. It has It been has been reported that oxidative damage of cells due to particles or drugs can be minimized by reported that oxidative damage of cells due to particles or drugs can be minimized by supplementation supplementation of external antioxidants such as vitamins and NAC. For example, NAC has shown of external antioxidants such as vitamins and NAC. For example, NAC has shown potential to mitigate potential to mitigate the oxidative damage of cells through ROS scavenging and GSH replenishment the oxidative[46]. Our data damage also showed of cells that through cytotoxicity ROS exer scavengingted by BT andNPs or GSH ZnO replenishment NPs was effectively [46]. attenuated Our data also showedby NAC that cytotoxicityco-exposure. exertedAs we can by see BT NPsin Figure or ZnO 7A, NPscell damage was effectively induced attenuatedby BT NPs was by NAC significantly co-exposure. As wereverted can see by NAC in Figure co-exposure.7A, cell MTT damage data (Figure induced 7B) byalso BT demonstrated NPs was significantlythat reduction revertedin cell viability by NAC co-exposure.after exposure MTT to data BT (FigureNPs was7B) successfully also demonstrated reverted when thatreduction co-exposed in with cell viabilityNAC. These after results exposure to BTsuggested NPs was that successfully BT NP-induced reverted cytotoxicity when was co-exposed mediated through with NAC. oxidative These stre resultsss. Most suggested importantly, that BT NP-inducedthe cytotoxicity cytotoxicity mechanism was mediatedof BT NPs throughwas similar oxidative in mode stress. of action Most to that importantly, of ZnO NPs. the Copper cytotoxicity mechanismdoping ofalso BT enhances NPs was oxidative similar inst moderess-mediated-cytotoxicity of action to that of ZnOof TiO NPs.2 NPs Copper in A549 doping cells [47]. also enhancesOur previous study also showed that Zn-doped TiO2 induced cytotoxicity in human breast cancer (MCF- oxidative stress-mediated-cytotoxicity of TiO NPs in A549 cells [47]. Our previous study also showed 7) via oxidative stress [23]. Elucidating the possible2 mechanisms of selective toxicity of BT NPs against that Zn-doped TiO induced cytotoxicity in human breast cancer (MCF-7) via oxidative stress [23]. cancer cells still 2remains a daunting task. ElucidatingIt cannot the possible be dismissed mechanisms that NPs, of particularly selective toxicity when they of BTwere NPs notagainst stored under cancer sterile cells condition, still remains a dauntingmay task.be contaminated with endotoxins during preparation or handling. Therefore, endotoxin contaminationIt cannot be might dismissed influence that the NPs, toxicity particularly of NPs [48]. whenEndpoint they chromogenic were not limulus stored amebocyte under sterile condition,lysate (LAL) may assay be contaminated further confirmed with that endotoxins prepared BT duringNPs were preparation not contaminated or handling. from endotoxins Therefore, endotoxin(Figure contamination S1 of Supplementary might Materials). influence the toxicity of NPs [48]. Endpoint chromogenic limulus amebocyte lysate (LAL) assay further confirmed that prepared BT NPs were not contaminated from endotoxins (Figure S1 of Supplementary Materials).

Nanomaterials 2020, 10, 2309 10 of 13 Nanomaterials 2020, 10, x 10 of 14

Nanomaterials 2020, 10, x 10 of 14

FigureFigure 7. BT 7. NP-induced BT NP-induced cytotoxicity cytotoxicity was was mediated mediated throughthrough oxidative oxidative stress. stress. Cells Cells were were exposed exposed for for 24 h to 50 µg/mL of BT NPs with or without NAC (2 mM). ZnO NPs (25 µg/mL for 24 h) were used 24 h toFigure 50 µ 7.g/ mLBT NP-induced of BT NPs cytotoxicity with or without was mediated NAC (2 through mM). ZnOoxidative NPs stress. (25 µ gCells/mL were for 24exposed h) were for used as a positive control. (A) Cell morphology. (B) MTT cell viability. * Indicates statistically significant as a positive24 h to 50 control. µg/mL of (A BT) Cell NPs morphology. with or without (B NAC) MTT (2 cellmM). viability. ZnO NPs * (25 Indicates µg/mL statisticallyfor 24 h) were significant used difference from the control group (p < 0.05). # Indicates that NAC effectively abrogated the cytotoxic differenceas a positive from the control. control (A) group Cell morphology. (p < 0.05). #(BIndicates) MTT cell that viability. NAC * eIndicatesffectively statistically abrogated significant the cytotoxic effect of BT NPs or ZnO NPs. effectdifference of BT NPs from or ZnOthe control NPs. group (p < 0.05). # Indicates that NAC effectively abrogated the cytotoxic effect of BT NPs or ZnO NPs. 3.5. Effect of BaTiO3 Nanoparticles on Non-Cancerous Normal Cells 3.5. Effect of BaTiO3 Nanoparticles on Non-Cancerous Normal Cells 3.5. EffectTo see of BaTiOthe selective3 Nanoparticles toxicity on of Non-Cancerous BT NPs, we Normalassessed Cells the effects of BT NPs in non-cancerous Tohuman see the lung selective fibroblasts toxicity (IMR-90). of BT Results NPs, weshowed assessed that theBT NPs effects did of not BT exert NPs toxicity in non-cancerous to IMR-90 cells human To see the selective toxicity of BT NPs, we assessed the effects of BT NPs in non-cancerous lung fibroblasts(Figure 8). As (IMR-90). expected, Results ZnO NPs showed also did that not BT induce NPs cytotoxicity did not exert to IMR-90 toxicity cells. to IMR-90 The benign cells nature (Figure 8). human lung fibroblasts (IMR-90). Results showed that BT NPs did not exert toxicity to IMR-90 cells As expected,of ZnO NPs ZnO was NPs reported also did previously not induce [20,21]. cytotoxicity Dubey and to IMR-90co-workers cells. found The that benign BT NPs nature did not of ZnO (Figure 8). As expected, ZnO NPs also did not induce cytotoxicity to IMR-90 cells. The benign nature NPs wasinduce reported any systemic previously toxicity [20 in,21 BALB/c]. Dubey mice and [49] co-workers. Selective toxicity found of that semiconductor BT NPs did NPs not induceagainst any of ZnO NPs was reported previously [20,21]. Dubey and co-workers found that BT NPs did not cancer cells is now being investigated by researchers [50,51]. systemicinduce toxicity any systemic in BALB toxicity/c mice in [ 49BALB/c]. Selective mice [49] toxicity. Selective of semiconductor toxicity of semiconductor NPs against NPs cancer against cells is now beingcancer investigatedcells is now being by researchers investigated [ by50 ,researchers51]. [50,51].

Figure 8. Effect of BaTiO3 NPs in non-cancerous human lung fibroblasts (IMR-90). Cells were exposed for 24, 48, and 72 h to different concentrations (5–200 µg/mL) of BT NPs, and cytotoxicity was Figure 8. Effect of BaTiO3 NPs in non-cancerous human lung fibroblasts (IMR-90). Cells were exposed Figuredetermined 8. Effect of by BaTiO MTT assay.3 NPs ZnO in non-cancerous NPs (25 µg/mL) human was us lunged as fibroblastsa positive control. (IMR-90). Data Cellsare provided were exposed as for 24, 48, and 72 h to different concentrations (5–200 µg/mL) of BT NPs, and cytotoxicity was for 24,mean 48, and ± SD 72 of h three to diff independenterent concentrations experiments (5–200 (n = µ3).g /mL) of BT NPs, and cytotoxicity was determined by MTTdetermined assay. ZnO by MTT NPs assay. (25 µ gZnO/mL) NPs was (25 used µg/mL) as a was positive used as control. a positive Data control. are provided Data are asprovided mean asSD of mean ± SD of three independent experiments (n = 3). ± three independent experiments (n = 3).

Nanomaterials 2020, 10, 2309 11 of 13

4. Conclusions We observed that BT NPs induce a dose- and time-dependent cytotoxicity to human lung carcinoma (A549) cells. MMP loss and higher activity of caspase-3 and -9 enzymes due to BT NP exposure were also observed. Moreover, BT NP-induced cytotoxicity, pro-oxidant generation, and antioxidant depletion were eﬀectively attenuated by co-exposure of an antioxidant (N-acetyl-cysteine), suggesting that BT NP-induced toxicity was mediated through oxidative stress. Importantly, cytotoxicity mechanisms of BT NPs against human lung cancer cells were similar in mode of action to those of ZnO NPs. Overall, this preliminary study indicated that BT NPs have potential to kill cancer cells while sparing their normal counterparts. This preliminary report warrants further research on cytotoxicity mechanisms of BT NPs against various types of cancer cells and their normal counterparts.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/10/11/2309/s1. Author Contributions: Conceptualization, M.A.; investigation and methodology, M.A., M.J.A., M.M.K., H.A.A. and A.A., writing—original draft preparation, M.A.; writing—review and editing, M.A. and M.J.A.; funding acquisition, M.A. All authors have read and agreed to the published version of the manuscript. Funding: The authors extend their appreciation to the Deputyship for Research and Innovation, “Ministry of Education “in Saudi Arabia for funding this research work through the project number IFKSURG-1439-072. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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