Synthesized tin oxide nanoparticles promote apoptosis in human osteosarcoma cells

2.1 Materials

Tin(II) chloride dehydrate (SnCl₂·2H₂O, ACS reagent, 98 %, product No. 243523), 2, 7-dichlorofluorescein diacetate (DCFDA), and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA).

2.2 Synthesis of SnO₂ NPs through combination method

SnCl₂·2H₂O (Merck) (1 M) suspended in 100 mL water–ethanol mixture (10 % ethanol, to reduce the polarity of water solvent) were stirred (500 rpm for 30 min) followed by irradiation by a high intensity ultrasonic processor (750 W; 20 kHz and 6 mm Ti-probe tip) for 1 h. The pH of the medium was regulated by ammonium hydroxide (Sigma Aldrich) and gradually added to reach a range between 9 and 10 to result in the formation of monodispersed NPs. The temperature was also set at 80–90 °C during the sonication system. The samples were then centrifuged (5 min, 3000 rpm/min), washed several times with deionized water, dried at 70 ˚C, and finally calcined at 500 ˚C for 2 h (Rehman et al., 2020).

2.3 Characterization of the synthesized SnO₂ NPs

The particle size and the morphological structure were investigated by transmission electron microscope TEM (FEI Tecnai G2 X-TWIN 100 kV) and scanning electron microscope (SEM, MIRA3). Fourier transform infrared (FTIR) was done on a spectrophotometer (FTIR- Tensor 27, Bruker, Germany) in the range of 4000–400 cm⁻¹). Phase identification and crystalline nature of the synthesized SnO₂ NPs were measured by X-ray diffraction (XRD) using a diffractometer system (PANalyticalX'pert PRO XRD), Cu as anodic materials [CuKα radiation (λ = 1.54178 Å), 40 kV and 30 mA] with scan step in 2θ range from 20° to 80°. The hydrodynamic radius and zeta potential distribution of SnO₂ NPs were measured using dynamic light scattering (DLS, Malvern Instruments, Worcestershire, UK, ZEN 3600 model Nano ZS).

2.4 Cell culture

Human osteosarcoma, U-2OS, and normal human osteoblast, NHOst, cell lines obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in DMEM supplemented with 10 % FBS, 1 % antibiotics, 1 mM l-glutamine (Gibco, Grand Island, NY, USA) and 100 µg/mL gentamicin (Gibco, Grand Island, NY, USA) at 37 °C with 5 % CO₂.

2.5 Cell viability assay

Cell viability assay was done using a conventional MTT assay. Briefly, cancerous and noncancerous cells seeded into 96-well culture plates with a density of 5 × 10³ cells/well were incubated for 24 h. Afterward, SnO₂ NPs with different concentrations ranging from 1 to 100 µg/mL were transferred and further incubated for 24 h. Then, 50 μl of MTT (5 mg/mL) was added into each well after removing the culture medium and incubated for 4 h at 37 °C. 100 μl of dimethyl sulfoxide was then added to the wells and the optical density (OD) of the solution was detected at 570 nm on a microplate ELISA reader.

2.6 Lactate dehydrogenase (LDH) release assay

LDH assay was used to evaluate the cytotoxic effects of SnO₂ NPs on U-2OS human osteosarcoma cells or NHOst normal cells. Cells seeded in a 96-well plate at a density of 5 × 10³ cells/well were exposed to IC₅₀ concentration of SnO₂ NPs (15.50 µg/mL) for 24 h. LDH Assay Kit (Shanghai Biyuntian Biotechnology Co., Ltd.) was used to detect the LDH release in cell culture medium according to the manufacturer's protocol and the absorbance of the sample was read at 490 nm using a microplate ELISA reader.

2.7 Reactive oxygen species analysis (ROS)

ROS assay was used to assess the oxidative stress induced by SnO₂ NPs in U-2OS human osteosarcoma cells. Cells seeded in a 6-well plate at a density of 3 × 10⁵ cells/well were incubated with IC₅₀ concentration of SnO₂ NPs (15.50 µg/mL) for 6 h. Afterward, cells were collected, washed, and suspended in a DCFH-DA solution (20 µM) for 30 min. The cells were then washed and the fluorescence intensity of fluorescent product 2′,7′-dichlorofluorescein (DCF) was read on a SYNERGY-HT multiwell plate reader, Bio-Tek (USA) at excitation and emission wavelengths of 485 and 528 nm, respectively.

2.8 Oxidative stress analysis

Cells with a density of ∼5 × 10⁶ in a 75 cm² culture flask exposed to IC₅₀ concentration of SnO₂ NPs (15.50 µg/mL) were scraped, washed with chilled 1 × PBS, and centrifuged at 500g for 15 min. The pellets were washed and then incubated on ice with 1 × Lysis Buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 0.5 % Triton-100) for 10 min supplemented with appropriate protease inhibitors. Then, the cells were centrifuged at 12 000 g for 10 min and the supernatant was collected. Protein concentration was determined by the Bradford assay (Bonjoch and Tamayo, 2001). Subsequently, superoxide dismutase (SOD, A001-1; Nanjing Jiancheng Bioengineering Institute) and catalase (CAT, A007-2; Nanjing Jiancheng Bioengineering Institute) activities as well as glutathione (GSH, A006-1; Nanjing Jiancheng Bioengineering Institute) and 3,4-Methylenedioxyamphetamine (MDA, A003-1; Nanjing Jiancheng Bioengineering Institute) contents were detected with relevant detection kits according to the protocols recommended by the manufacturer.

2.9 Activity of caspase-3, caspase-9, and caspase-8

The activity levels of caspase-3, caspase-9 and caspase-3 were measured according to the manufacturer's protocol provided with the caspase colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute). Briefly, an aliquot of cell suspension was mixed with the reaction buffers containing the substrates followed by incubating at 37 °C for 2 h and terminating by the addition of a stop buffer. Then, the optical density of samples was read at 405 nm using a microplate ELISA reader.

2.10 Mitochondrial membrane potential (MMP)

Cells were seeded in a 6-well microculture plate at a density of 3 × 10⁵ cells/well and after incubation with IC₅₀ concentration of SnO₂ NPs (15.50 µg/mL) for 24 h, then the medium was replaced with reaction buffer provided with MMP kit (Shanghai Yeasen Biotechnology Co., Ltd.). The relative ratio of red to green fluorescence intensity was determined as the level of MMP depolarization.

2.11 Total RNA extraction and quantitative real-time PCR (qRT-PCR)

Cells were seeded in a 6-well microculture plate at a density of 3 × 10⁵ cells/well and after incubation with IC₅₀ concentration of SnO₂ NPs (15.50 µg/mL) for 24 h total RNA was extracted by RNA isolation Kit (Huayueyang Biotechnology, China) according to the manufacturer's instructions. After checking the quantity and quality of RNA, the cDNA synthesis kit (Takara, Beijing, China) was used to reverse-transcribe RNA following the manufacturer's instructions. The reactions were done on a Bio-red IQ2 Sequence Detection System and Applied Biosystems software using SYBR Green PCR Master Mix (Applied Biosystems) following the standard protocols. Primer design was based on previous studies (Liang et al., 2012; Punzo et al., 2017). The comparative threshold cycle (Ct) values were calculated and analyzed using the 2^−ΔΔCT method, with β-actin as an internal control.

2.12 Statistics analysis

SPSS statistics package was used for statistical analysis between different groups by one-way analysis of variance (*P < 0.05, ** P < 0.01, *** P < 0.001).

3.1 Imaging analysis

SEM and TEM analyses were done to determine the shape and size of the synthesized SnO₂ NPs. The morphology of SnO₂ NPs was assessed, whereas the size of NPs was determined by the TEM test. SEM images showed that synthesized SnO₂ NPs provided monodispersed spherical- or quasi-spherical-shaped morphology (Fig. 1A). TEM analysis revealed that synthesized SnO₂ NPs show the average particle size around 10 nm (Fig. 1B). In other words, from TEM analysis, the probable uniform distribution of small sized SnO₂ NPs (about 10 nm) is revealed. This can be attributed to the cavitational effects stimulated by ultrasound, which results in the generation of extreme shearing forces, micromixing and turbulence derived from physical influences inserted by the transient deformation of cavities that appeared due to ultrasound (Deosarkar et al., 2013; Choudhari et al., 2020; Karunakaran,Raadha,and Gomathisankar, 2013). Also, solvent polarity reduction not only results in the dispersion of synthesized NPs but also a decrease in the size of NPs (Hussain et al., 2020).

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Fig. 1

SEM (A) and TEM (B) imaging of synthesized SnO₂ NPs through the combination of solvent polarity reduction and sonochemical method.

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Sery et al. (Sery et al., 2022) reported that synthesized SnO₂ NPs through the sonochemical method had a wide size range of 2 to 30 nm, which makes them complicated for biological applications Karunakaran et al. (Karunakaran,Raadha,and Gomathisankar, 2013) also showed that sonochemically synthesized SnO₂ NPs had an average diameter of 10 nm, which is in good agreement with our TEM result. However, they found that the prepared NPs lack finite shape or structure, which might be due to the generation of hotspots through the sonochemical method as the main destructors of the templating effect. Additionally, Zhu et al. reported that sonochemically synthesized SnO₂ NPs in an aqueous solution of SnCl₄ and azodicarbonamide were ∼3 − 5 nm in size, as detected by TEM analysis (Zhu et al., 2000). However, it should be indicated that ultrasmall NPs (around 3 to 5 nm) are easily filtered by liver and kidney tissues. Therefore, we can conclude that sonochemistry along with solvent polarity reduction could result in the synthesis of SnO₂ NPs with monodispersed distribution and a potential size of around 10 nm for biological implementations (Bartucci et al., 2020).

3.2 FTIR measurement

Fig. 2 depicts the FTIR spectrum for synthesized SnO₂ NPs in the wavenumber region of 4000–400 cm^{– 1}. The strong absorption peaks at 1635 cm^{– 1} and 3476 cm⁻¹ attribute to the stretching of H–O–H and O–H bonds, respectively deduced from water molecules (Garg et al., 2020). One broader absorption peak is also visible in the spectrum at 649 cm^{– 1}, corresponding to the stretching vibration of the O–Sn–O bond (Garg et al., 2020; Sery et al., 2022).

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Fig. 2

FTIR spectrum of synthesized SnO₂ NPs.

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This data showed the purity and the lack of crystal lattice defects or disorders in synthesized SnO₂ NPs (Garg et al., 2020; Sery et al., 2022).

3.3 XRD analysis

The XRD pattern of the synthesized SnO₂ NPs is shown in Fig. 3. The peaks at 2θ values of 27.75°, 33.80°, 37.80°, 52.05°, 54.15°, 62.35°, 66.40°, and 79.01° can be attributed to (1 1 0), (1 0 1), (2 0 0), (2 1 1), (2 2 0), (3 1 0), (3 0 1), and (3 2 1) plans, respectively (Patil et al., 2012; Sery et al., 2022). A matching of the detected and standard (hkl) planes proved that the particle is of SnO₂ with a tetragonal system of rutile-like structure, which is in good agreement with the literature values (JCPDS, PDF file No 41–1445, space group P42/MNM, lattice parameters a = 4.7420 Å and c = 3.1900 Å) (Sery et al., 2022).

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Fig. 3

XRD pattern of synthesized SnO₂ NPs.

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No other detectable XRD peaks were observed, revealing the high purity of the synthesized SnO₂ NPs (Sery et al., 2022). Also, XRD patterns of SnO₂ NPs demonstrated high peak intensities, suggesting ideal crystallinity and crystallite-size developments (Sery et al., 2022), which is consistent with the NP size observed by TEM analysis. Also, slight broad peaks were detected for synthesized SnO₂ NP samples corroborating the generation of fine nanosized samples (Sery et al., 2022). Therefore, it can be assumed that the sonication of the solution provides us with a high probability of synthesizing particles in the nanometer size.

3.4 Hydrodynamic radius and charge studies

DLS study was also done to measure the hydrodynamic radius and zeta potential values of the synthesized SnO₂ NPs. Fig. 4 A shows the hydrodynamic size of the dispersed synthesized SnO₂ NPs in solution determined by DLS which was found to be ranged between 34 and 60 nm with an average size of 43.93 nm. The differences in determined size between TEM and DLS techniques could be associated with the slight agglomeration of NPs in the solution. However, a monodisperse distribution of synthesized SnO₂ NPs was detected in Fig. 4A, which could be due to the cavitation process induced by the nonchemical approach, which results in large NPs breaking up into homogenous small particles and their slight aggregation (Barbhuiya et al., 2022).

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Fig. 4

DLS (A) and zeta potential (B) analyses of synthesized SnO₂ NPs.

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Fig. 4B depicts that the average zeta potential of synthesized SnO₂ NPs was −31.00 mV. This strong negative value indicates an apparent particle repulsion, corresponding to the stability of NPs (Manimaran,Muthuvel,and Said, 2023). Generally, zeta potential reveals how electrostatic forces determine the strength of repulsion forces between neighboring NPs. Following the adsorption of OH⁻ ions on the surface of NPs, their zeta potential values become negative, inhibiting the strong NP aggregation and maintaining the colloidal stability (Manimaran,Muthuvel,and Said, 2023; Srivastava and Mukhopadhyay, 2014).

3.5 SnO₂ NPs interaction with cells

The anticancer activity of synthesized SnO₂ NPs on U-2OS human osteosarcoma cells was evaluated by using the MTT assay. As depicted in Fig. 5A, treatment with synthesized SnO₂ NPs for 24 h significantly decreased the proliferation of U-2OS cells in a concentration-dependent fashion in comparison to control cells (*P < 0.05, **P < 0.01, ***P < 0.001), while the growth of human normal osteoblasts NHOst cells (Fig. 5B) was much less influenced by the synthesized SnO₂ NPs at the same studied concentrations.

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Fig. 5

Cytotoxic effects of SnO₂ NPs against U-2OS human osteosarcoma cells (A) and human normal osteoblasts NHOst cells (B) determined by MTT assay. Membrane leakage induced by SnO₂ NPs in U-2OS human osteosarcoma cells (C) and human normal osteoblasts NHOst cells (D) determined by LDH assay. All experiments were done after 24 h incubation of cells with SnO₂ NPs. *P < 0.05, ** P < 0.01, *** P < 0.001 relative to control. Data were presented as mean ± SD of three experiments.

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Therefore, the IC₅₀ concentration of synthesized SnO₂ NPs on U-2OS human osteosarcoma cells was determined to be about 15.50 µg/mL at 24 h.

A previous study has shown that biosynthesized SnO₂ NPs with particle size distribution ranging between 5 and 10 nm had significant antiproliferative effects against colorectal and lung cancer cells with IC₅₀ values of 165–208 µg/mL and 138–187 µg/mL, respectively (Tammina et al., 2017). Additionally, Hanna et al. (Hanna and R. Saad 2021) reported that the calculated IC₅₀ value for the sol–gel synthesized SnO₂ NPs with a size of around 79 nm against SKOV3 human ovarian cancer cells, was 47.87 µg/mL at 24 h incubation. However, Ahamed et al. (Ahamed et al., 2018) reported that the sol–gel synthesized SnO₂ NPs with a size of about 21 nm did not induce cytotoxicity in MCF-7 breast cancer cells up to the concentration 50 µg/mL at 24 and 48 h. Therefore, several parameters may play a key role in the NP-induced cytotoxicity such as the physicochemical properties of NPs, type of cancer or normal cells, and experimental procedures.

LDH enzyme release from cytoplasm into cell culture medium can be appointed as a useful marker of damage to cell membrane integrity (Ahamed et al., 2018). Higher levels of LDH enzyme activity in cell culture media reveal the higher damage of cell membranes induced by NP exposure (Ifijen et al., 2023). We also found that synthesized SnO₂ NPs prompted LDH leakage in U-2OS human osteosarcoma cells in a concentration-dependent manner (Fig. 5C), whereas these NPs did not induce a significant LDH release in normal human osteoblast, NHOst, below 100 µg/mL at 24 h (Fig. 5D). Altogether, these data proved that SnO₂ NPs show potential to trigger concentration-dependent cytotoxicity in the U-2OS cell line. Our findings are consistent with recent reports demonstrating SnO₂ NPs' anticancer effects in several cancer cells (Ahamed et al., 2018; Ahmadabad et al., 2021; Ifijen et al., 2023).

3.6 SnO₂ NPs induced oxidative stress in U-2OS human osteosarcoma cells

Induction of oxidative stress through inhibition of antioxidant enzyme activities and depletion of non-enzymatic antioxidants are the most common pathways through which NPs stimulate apoptosis in cancer cells (Akhtar et al., 2012; Rayzah et al., 2023). To maintain the redox balance in cells, antioxidant defense and strict control over the generation of free radicals are essential (Rayzah et al., 2023). The GSH, SOD, and CAT are serving as strong antioxidant molecules in protecting cells from oxidative stress prompted by ROS and other free radicals (Rayzah et al., 2023). Antioxidant enzymes, SOD and CAT, are crucial for shielding cells from the damaging effects of free radicals. GSH plays a crucial role in inhibiting lipid peroxidation (Rayzah et al., 2023). Because cancer cells can express antioxidant molecules, they are able to eliminate the excessive generation of ROS, extending their survival half-life through preventing apoptosis. On the other hand, elevated oxidative stress in cancerous cells may initiate anti-tumorigenic signaling cascades, resulting in tumor cell death (Zeng et al., 2023; Tuli, Kaur, et al. 2023).

Therefore, the oxidant and antioxidant levels were examined in the U-2OS cell line treated with SnO₂ NPs with a concentration of 15.50 µg/mL at 24 h. ROS (Fig. 6A) and MDA (Fig. 6B) levels were assessed as main biomarkers of oxidative stress, whereas antioxidant status was assessed by analyzing the SOD (Fig. 6C) and CAT (Fig. 6D) activities as well as GSH content (Fig. 6E). The obtained data displayed that oxidant (ROS and MDA) levels were higher in SnO₂ NPs-treated cancer cells compared to negative control cells (Fig. 6A and B).

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Fig. 6

Oxidative stress induction of SnO₂ NPs in U-2OS human osteosarcoma cells determined by ROS assay (A), MDA assay (B), SOD assay (C), CAT assay (D), and GSH assay (E). All experiments were done after 24 h incubation of cells with SnO₂ NPs. *P < 0.05, ** P < 0.01, *** P < 0.001 relative to control. Data were presented as mean ± SD of three experiments.

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Furthermore, CAT and SOD activities as well as the GSH content were remarkably reduced by the IC₅₀ concentration of SnO₂ NPs (Fig. 6C-E). These findings indicated that SnO₂ NP treatment collapses the antioxidant system of the cells while increasing the ROS and MDA levels, thereby accelerating oxidative stress-manipulated cell death in U-2OS human osteosarcoma cells. These data are in good agreement with a study reported by Ahamed et al. (Ahamed et al., 2018), indicating that SnO₂ NPs could induce oxidative stress-mediated anticancer effects through deregulation of enzymatic and non-enzymatic oxidants.

3.7 SnO₂ NPs activated both intrinsic and extrinsic signaling pathways

There are two possible ways for NPs to trigger apoptotic cell death: intrinsic or extrinsic pathways (Tuli,Joshi,et al., 2023). Intrinsic mechanisms of apoptosis are activated by non-receptor mediated stimuli that trigger the disruption of several mediators such as MMP, Bax/Bcl-2 ratio, and caspase-9 activity to stimulate apoptosis, whereas extrinsic apoptotic cell death is initiated upon the interaction of anticancer drugs with transmembrane receptors and activation of caspase-8 (Lossi, 2022; Fulda and Debatin, 2006). Metal NPs are potential candidates to induce apoptotic cell death in different types of cancers, including bone, brain, blood, liver, and so on (Tuli,Joshi,et al., 2023). Especially, functionalized SnO₂ NPs have been observed to promote anticancer activity in human ovarian cancer cells by overexpression of Bax, and cleaved caspase-3 (Hanna and R. Saad, 2021). In cervical cancer HeLa cells, cell SnO₂ NP treatment was shown to deregulate the Bax/Bcl-2 mRNA ratio (Bazsefidpar et al., 2021). Guo et al. explored the downregulation of PI3K/Akt/mTOR/Bax/Bcl-2 in SnO₂ NPs- treated breast cancer cells, leading to apoptotic cell death (Guo et al., 2021). Similarly, Jiang et al. (Jiang et al., 2022) observed the apoptotic effect of biosynthesized SnO₂-sodium alginate-polyethylene glycol-carvacrol NPs in esophagus squamous cells cancer via upregulation of Bax and caspase 3. Therefore, in this study, for 24 h to assess if synthesized SnO₂ NP solution at the IC₅₀ concentration (15.50 µg/mL) could induce apoptotic effects in U-2OS human osteosarcoma cells via intrinsic or extrinsic pathways, MMP, qRT-PCR, and caspase activity assays were performed.

As determined by the decreased ratio of red to green fluorescence by JC-1 staining, incubation with the SnO₂ NP solution at the IC₅₀ concentration (15.50 µg/mL) for 24 h significantly reduced MMP in U-2OS human osteosarcoma cells, compared with negative treated cells (Fig. 7A). Furthermore, All the caspase activities under study found to be increased (Fig. 7B-D) following incubation of U-2OS cells with 15.50 µg/mL SnO₂ NPs at 24 h, compared to control cells.

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Fig. 7

Apoptosis induction of SnO₂ NPs in U-2OS human osteosarcoma cells determined by different assays. MMP assay (A), caspase-9 activity assay (B), caspase-8 activity assay (C), caspase-3 activity assay (D), and qRT-PCR assay (E). All experiments were done after 24 h incubation of cells with SnO₂ NPs. *P < 0.05, ** P < 0.01, *** P < 0.001 relative to control. Data were presented as mean ± SD of three experiments.

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Additionally, the upregulated levels of Bax mRNA, caspase-9 mRNA, caspase-8 mRNA and caspase-3 mRNA, and the reduced level of Bcl-2 mRNA were observed in U-2OS human osteosarcoma cells following incubation with 15.50 µg/mL SnO₂ NPs at 24 h (Fig. 7E). These results confirmed the activation of both intrinsic and extrinsic apoptosis signaling pathways by SnO₂ NPs in the U-2OS cells. Using leukemia K562 cells, Ahmadabad et al. (Ahmadabad et al., 2021) showed that synthesized SnO₂ NPs through hydrothermal method with a size of 60 nm triggered overexpression of caspase-9, −8, −3 and Bax/Bcl2 mRNA ratio. However, the main drawback of this study was the heterogenous distribution of synthesized NPs in solvent as determined by the DLS study.