Study on the Regulated Cell Death of Hypertrophic H9c2 Cells Induced by Au:Ag Nanoparticles

Introduction

Cardiometabolic disease is a cluster of interrelated conditions that increase the risk of cardiovascular diseases (CVDs), coronary artery disease and metabolic disorders.¹ These conditions often overlap and include insulin resistance, hypertension, dyslipidemia, systemic inflammation, and obesity.^2–4 In this regard, obesity is linked to the progression of several CVDs, the primary cause of death worldwide, being the cause of death of approximately 20.5 million people in 2021 alone,^5,6 including hypertrophic cardiomyopathy.⁷ Hypertrophy is an adaptive response to CVDs, which involves an increase in muscle mass. When this occurs in the cardiac system, it causes the cardiomyocytes to enlarge, allowing the heart to reduce wall tension.^8,9 Furthermore, CVDs are frequently linked to metabolic disorders that undermine the immune system through persistent chronic inflammation.¹⁰ This chronic inflammatory state leads to an increase in the number of metabolically activated (MA) macrophages, which in turn aggravate metabolic disorders and their presence is highly correlated with CVDs such as coronary heart disease.^11–13 MA macrophages play a crucial role in the pathophysiology of these conditions by perpetuating inflammation and contributing to tissue remodeling and fibrosis. Macrophages, especially those in a metabolically activated state, result critically in the progression of obesity and CVDs due to their involvement in lipid metabolism, and interaction with other cell types within the immune system. It is believed that, unlike healthy cells, cells with alterations such as hypertrophic cells could be more susceptible when exposed to nanomaterials due to their altered cellular metabolism.^14,15

In recent years, nanomaterials have been employed in medical applications for imaging purposes, due to their unique characteristics such as high mobility throughout the body, large surface area-to-volume ratio, and imaging versatility.^8,9,16 Particularly, metallic nanomaterials have been employed in the diagnosis of CVDs,¹⁷ and more recently, have been extensively investigated for therapeutic purposes.^18,19 In this regard, metallic nanoparticles (NPs) have gained significant interest in the medical field for pharmaceutical applications, due to their unique optical and electrical properties, as well as their excellent biocompatibility.²⁰ However, it has been reported that the exposure to some of these NPs may cause a breakdown of the cellular buffering system by interfering with cellular mechanisms, such as by impairing mitochondrial function.²¹ Particularly, silver (Ag) NPs have shown to decrease the mitochondrial membrane potential (∆Ψm), which leads to a decrease in ATP production as well as an increase in mitochondrial superoxide generation on diverse cells, including those of cancerous and cardiac nature.^22,23

Among the various types of metallic NPs, Ag and gold (Au) NPs stand out, due to their antibacterial and biocompatibility properties, respectively.^24–27 Historically, Au NPs have been used for cancer-related applications in the cardiovascular area, taking advantage of their bioactivity.²⁸ Au NPs also have exhibited inherent antioxidant properties, such as catalase-mimic and peroxidase-like activities that may be useful for CVDs control.^20,29 However, to the best of our knowledge, the cytotoxicity of Au and Ag NPs under pathological conditions such as cardiometabolic disease has not been described yet. Furthermore, Ag NPs are an attractive approach to destroy bacterial drug resistance as they present inhibitory and anti-microbial activity.^30,31 Bimetallic Au and Ag NPs (Au:Ag NPs) have opened a new field of research due to their broad-spectrum of capabilities in the biomedical field, such as antibacterial and wound healing, owing to the synergy between the metals that make up their composition.³² Au:Ag NPs exhibit electronic, optical, and catalytic properties that surpass monometallic nanostructures, making the use of Au attractive to modulate Ag toxicity, such as in cardiometabolic diseases.²⁴

Hence, to associate possible applications of noble metal-based NPs in metabolic disorders, it is first important to explore their nanotoxicology under pathological conditions. In this regard, here we investigated the cytotoxicity and cell death mechanism induction of mono- and bimetallic Au:Ag NPs on in vitro cellular models that are related to metabolic disorders, ie, hypertrophic cardiac cells, and metabolically activated macrophages. We also compared the NP cytotoxic susceptibility of these models with their healthy counterpart.

Materials and Methods

Reagents and Materials

Sodium tetrachloroaurate (NaAuCl₄) and sodium bicarbonate (NaHCO₃) were purchased from Sigma Aldrich (St. Louis, MO, USA). Silver nitrate (AgNO₃ 99%) and nitric acid (HNO₃ 70%) were purchased from Fermont (Monterrey, N.L, Mexico). Starch, sodium hydroxide (NaOH), sodium carbonate (Na₂CO₃) and potassium sodium tartrate (KNaC₄H₄O₆) were purchased from CTR Scientific (Monterrey, N.L., Mexico). All chemicals were used without any purification step, Au:Ag NPs synthesis and characterization were carried out using deionized (DI) water, while biological experiments were conducted using double distilled water.

Prior to their use, all glassware and magnetic stirring bars used were kept in a 1:8 hydrochloric acid (HCl, Sigma Aldrich, ST. Louis, MO, USA) solution for 24 h, then they were transferred to a 1 M NaOH solution for 24 h and were thoroughly rinsed with DI water. Both solutions were made with DI water.

Au:Ag Nanoparticle Synthesis

The synthesis of colloidal monometallic (Au NPs and Ag NPs) and bimetallic Au:Ag NPs was carried out using a modified version of previously reported synthesis methods.^33,34 First, a 1% w/v starch solution was added to 9.5 mL of DI water at 70 ± 1°C with an adjusted pH of 11. Afterwards, the corresponding metal precursor solution ratio was added using the amounts shown in Table 1. The reaction occurred under constant stirring for 2 h in a water bath at a reaction temperature of 70 ± 1 °C. The resulting colloidal NPs were washed by centrifugation and re-dispersed for further characterization and biomedical tests.

Table 1 Experimental Conditions for the Synthesis of Colloidal Au, Ag, and Au:Ag NPs. In All Cases, 500 μL of a 1% w/v Starch Solution Was Used

UV-Visible Spectroscopy

UV-visible spectra of the colloidal NPs were obtained using a PerkinElmer 365 spectrometer (PerkinElmer Inc, Waltham, Ma, USA). The colloidal samples were diluted in a 1:5 ratio with DI water. The absorption spectra of the samples were measured in the range of 300–800 nm with a scan rate of 300 nm/min and a scan step of 0.5 nm. The absorption spectra of each sample were measured at the same day of synthesis and tracked at 14 and 26 days after synthesis to monitor the colloidal stability.

Powder X-Ray Diffraction Analysis

Powder XRD patterns of the Au:Ag NPs were analyzed with a Rigaku Miniflex 600 diffractometer (Rigaku, Tokyo, Japan) using a 40 kV voltage, a current of 15 mA, and a Cu Kα radiation source (λ=1.542 Å). All patterns were recorded at room temperature using a scan speed of 3°/min and a step width of 0.05° in the range of 25–80° (2θ). The XRD samples were prepared by drying dropwise 3–5 mL of the colloidal suspensions on a glass sample holder at ~100 °C.

The lattice parameter (a) was calculated using Bragg’s law (λ = 2 d_hkl sinθ_hkl)³⁵ and the relation between d_hkl and a, where λ is the radiation wavelength, d_hkl corresponds to the interplanar distance for parallel planes with Miller indices (hkl), and θ_hkl is the angle of diffraction. The average crystallite size (L) was calculated using the Debye-Scherrer equation (L=k λ/[Bcosθ_hkl]),³⁴ where L is the nanoparticle’s crystallite size, k represents the Scherrer constant, assuming k=0.94 (for spherical particles), and B is the full width at half maximum (FWHM).

Fourier Transform Infrared (FT-IR) Spectroscopy

FT-IR spectra of the Au:Ag NPs were obtained using a PerkinElmer Spectrum 400 FT-IR/FT-NIR (Perkin-Elmer, Waltham, MA, USA) in attenuated total reflectance (ATR) mode. The spectra were measured between 600 and 4000 cm⁻¹.

Dynamic Light Scattering (DLS)

Hydrodynamic diameter (D_H) and particle size distribution (PSD) analysis by dynamic light scattering (DLS) were carried out using a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Measurements were carried out without diluting the colloidal NP samples, at 25 ± 0.1°C, using 1 mL disposable ζ-potential cells (Malvern, DTS 1070), and a He-Ne laser (λ = 633 nm) with a backscattering detection configuration (173°). The Au NP sample measurements were done using a refractive index of 0.700 and an absorption of 3.320, while the Ag NP sample was measured using a refractive index of 0.135 and an absorption of 3.390. All the bimetallic nanoparticles (Au75:Ag25, Au50:Ag50 and Au25:Ag75) samples were measured using a refractive index of 0.200 and an absorption value of 3.320.

Electrophoretic Light Scattering (ELS)

Electrophoretic light scattering (ELS) was used to measure the ζ-potential of the NPs. Measurements for the determination of colloidal stability were carried out using a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Measurements were carried out without diluting the colloidal NP samples, at 25 ± 0.1°C, using 1 mL disposable ζ-potential cells (Malvern, DTS 1070) and a He-Ne laser (λ = 633 nm) with a backscattering detection configuration (173°).

Transmission Electron Microscopy (TEM)

The size and shape of the bimetallic Au:Ag NPs were analyzed by transmission electron microscopy (TEM) with a JEOL JEM-2010 equipment, operated with an acceleration voltage of 200 kV. The specimen was prepared by placing a drop of the colloid directly on a formvar-coated copper grid and allowing the dispersion medium to evaporate at room temperature. The TEM images were processed by ImageJ software to obtain the size distributions of nanoparticles.

H9c2 Cell Culture

Neonatal rat ventricular myoblast H9c2 cell line (ATCC, CRL-1446) was purchased from ATCC. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Sigma Aldrich, ST. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Biowest, Riverside, MO, USA), and penicillin-streptomycin (Gibco^® by Life Technologies, Carlsbad, CA, USA) which contained 100 U/mL penicillin and 100 μg/mL streptomycin. The cell cultures were incubated in a Sanyo MCO-18AIC (UV) CO₂ Incubator at 37°C in an air-humidified 5% CO₂ atmosphere. Cell detachment was performed using 0.25% trypsin-EDTA (Gibco^® by Life Technologies, Carlsbad, CA, USA) and cell quantification using a trypan blue solution (Sigma Aldrich, St. Louis, MO, USA) in a Neubauer-improved counting chamber.

H9c2 Hypertrophy Induction

The H9c2 cells were cultured as previously described, cells were seeded at 4 × 10³ per well in 24-well plates. Cells were deprived to 1% FBS supplemented DMEM for 24 h, afterwards, they received two stimuli of angiotensin II (1μg/μL; TOCRIS, Bristol, UK) each 24 h during two consecutive days.

U937 Differentiation Into Macrophages

Macrophages were derived from the U937 human lymphoblast monocytes cell line (ATCC, CRL-1593). They were cultured in RPMI-1640 medium, supplemented with 10% FBS (Biowest, Riverside, MO, USA), and penicillin-streptomycin (Gibco^® by Life Technologies, Carlsbad, CA, USA) which contained 100 U/mL penicillin and 100 μg/mL streptomycin. The cell cultures were incubated in a Sanyo MCO-18AIC (UV) CO₂ incubator at 37 °C in an air-humidified 5% CO₂ atmosphere. In order to induce differentiation of U937 monocyte cells to the macrophage phenotype, 2 × 10⁵ cells were incubated with culture media supplemented with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich, ST. Louis, MO, USA) for 72 h. Subsequently, the cells were washed twice with 100 μL of sterile phosphate buffer solution (PBS, Sigma Aldrich, ST. Louis, MO, USA) and incubated for an additional 24 h in RPMI-1640 without PMA.

Macrophages Induction for Metabolic Activation

Macrophages were differentiated from U937 cells, as described in the previous section. Afterwards, to induce a metabolically activated macrophage state, they were treated with Bovine Serum Albumin (BSA) (Sigma Aldrich, ST. Louis, MO, USA) conjugated with palmitic acid (PA) (Sigma Aldrich, ST. Louis, MO, USA) at a 1:6 molar ratio equivalent to 200 μM, for 24 h, and were kept in incubation at 37 °C in an air humidified 5% CO₂ atmosphere.

Protein Quantification

Cells were washed before being detached using trypsin and lysed with Triton X-100 solution. Protein content was determined by Lowry assay. This assay is based on the biuret reaction, where Cu⁺ ions react with peptides to form a copper-protein complex, and the subsequent reaction of said copper-protein complex with the Folin-Ciocalteu reagent (Merck, Darmstadt, Germany), resulting in a blue color that is proportional to the protein concentration. Samples were incubated with biuret solution (4 mg/mL NaOH, 20 mg/mL Na₂CO₃, 10 mg/mL KNaC₄H₄O₆, and 5 mg/mL CuSO₄), and finally samples were incubated with Folin solution in a 1:10 (v:v) ratio (Merck, Darmstadt, Germany). Bovine serum albumin (Sigma Aldrich, St. Louis, MO, USA) was used as a standard curve. The quantification was read on the spectrophotometer BioTek Synergy HT (BioTek, Winooski, VT, USA) by fluorescence excitation and emission measured at 530 and 590 nm, respectively.

Cell Vitality Assay

For the in vitro cell vitality assay, H9c2 and hypertrophic H9c2 cells were seeded at 4×10³ cells/well in 100 μL DMEM media, and were treated with different concentrations (0.1, 0.5, 1, 5 and 10 ppm) of mono- and bimetallic NPs for 24 h. Macrophages were seeded at 2 × 10⁵ cells/well in 200 µL of RPMI media underwent the same concentrations (0.1, 0.5, 1, 5 and 10 ppm) of metallic NPs for 24 h. Afterwards, 5 mg/mL of alamar blue (Thermo Fisher Scientific, Waltham, MA, USA) was added to each cell well, and was incubated for 4 h at 37 °C without exposure to light. Fluorescence excitation and emission were measured at 530 and 590 nm, respectively, using a microplate BioTek Synergy HT (BioTek, Winooski, VT, USA).

The half maximal inhibitory concentration (IC₅₀) was calculated for each type of nanoparticle treatment and for both cell types by analyzing the inhibitor concentration versus the normalized response. The vitality percentage was calculated using the following equation:

where sample, control and blank represent the average fluorescence for each NP treatment, non-treated cells and wells with no cells, respectively.

The normalized response described by the Hill-Langmuir equation was fitted to the following equation through a nonlinear regression:

where Max and Min are the maximum and minimum cell vitality, and are set to 100 and 0, respectively, x corresponds to NPs concentration (ppm) and H is the Hill coefficient.

Cell Viability Analysis

Cell viability analysis was analyzed using Ghost dye satin (eBioscience, Waltham, MA, USA). In brief, cells were seeded in 12-well plates (Corning Inc. Costar^®, Corning, NY, USA) and treated with the previously calculated IC₅₀ of Ag and Au25:Ag75 NPs for 24 h. Afterwards, cells were collected and incubated with Ghost dye satin (eBioscience, Waltham, MA, USA) for 15 min at 37 °C in darkness. Then, cells were washed with PBS and analyzed using flow cytometry (CytoFLEX, Beckman Coulter, California, USA).

Mitochondrial Membrane Potential Analysis (∆Ψm)

∆Ψm was measured using JC-1 staining (Thermo Scientific, Waltham, MA, USA). H9c2 cells were seeded in 12-well plates (Corning Inc. Costar^®, Corning, NY, USA) and treated with the previously calculated IC₅₀ for Ag and Au25:Ag75 NPs for 24 h. Afterwards, cells were collected and incubated with the medium containing JC-1 (1 μM) for 20 min at 37 °C in darkness. Then, cells were washed with PBS and stained with Ghost dye satin (eBioscience, Waltham, MA, USA) for 15 min at 37 °C in darkness. Finally, cells were washed with PBS and analyzed using flow cytometry (CytoFLEX, Beckman Coulter, California, USA), as previously described. The levels of ∆Ψm are represented in percentage of red fluorescence intensity and green fluorescence intensity.

Mitochondrial Reactive Oxygen Species (mROS) Analysis

The mROS production levels were assessed with MitoSOX^TM Red mitochondrial superoxide indicator (Thermo Fisher Scientific, Waltham, MA, USA). Cells at a density of 1 × 10⁵ cells/well were seeded in 24-well plates (Corning Inc. Costar^®, Corning, NY, USA) and treated with the IC₅₀ of Ag and Au25:Ag75 NPs, and 50 μM H₂O₂ as a positive control, all of them for 24 h. Afterwards, the medium was removed and washed with DMEM before detaching the cells with 0.25% trypsin-EDTA, cells were then centrifuged for 5 min at 500 g. MitoSox^TM Red (200 μL) was added for 20 min at 37 °C. MitoSox^TM Red was analyzed using 510 nm and 580 nm for excitation and emission wavelengths, respectively, using a BD FACS CANTO II flow cytometer (BD Biosciences, Heidelberg, Germany). Data was analyzed with the FlowJo Software (Tree Star Inc., Ashland, OR, USA).

Cell Death Analysis

H9c2 cell death was determined by analyzing phosphatidylserine (PS) exposure and cell-membrane permeabilization, using Annexin V-allophycocyanin (APC) (AnnV, 0.25 µg/mL; BD Biosciences Pharmingen, San Jose, CA, USA) and propidium iodide (PI; 0.5 µg/mL; Milliporesigma, Eugene, OR, USA), respectively. H9c2 and hypertrophic H9c2 cells, at a density of 1×10⁵ cells/well, were cultured and treated with the previously calculated IC₅₀ of Ag and Au25:Ag75 NPs for 24 h. After treatment, cells were washed and resuspended in binding buffer (10 mm HEPES/NaOH pH 7.4, 140 mm NaCl, 2.5 mm CaCl₂), and stained with AnnV (0.1 µg/mL) and PI (0.5 µg/mL) for 30 min at 4 °C. Cells were analyzed by flow cytometry (FACS CANTO II, BD Biosciences, Heidelberg, Germany). Data was analyzed with the FlowJo Software (Tree Star Inc., Ashland, OR, USA).

Statistical Analysis

The data were analyzed using the software GraphPad Prism (GraphPad Software, San Diego, CA, USA). Results are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. Statistical analysis was done using a paired Student’s t-test. The statistical significance was defined as p < 0.05.

Results

Physicochemical Characterization of Mono- and Bimetallic Au:Ag NPs

The eco-friendly synthesis of the colloidal Au:Ag NPs was performed employing starch as the reducing and capping agent for the Ag⁺ and AuCl₄⁻ ions through a wet chemical reduction.^33,34 In Supplementary Figure S1, a shift in coloration from red (Au NPs) to brown (Ag NPs) depending on the amount of Au and Ag added can be observed, which indicates the synthesis of colloidal Au:Ag NPs with different chemical compositions. To investigate the optical properties of the synthesized NPs, UV-vis spectroscopy was performed as shown in Figure 1A. The obtained optical spectra exhibited the characteristic localized surface plasmon resonance (LSPR) bands observed in Au:Ag NPs, which describe the collective oscillation of the free surface electrons in quasi-spherical NPs induced by an electromagnetic wave in the UV-Vis range.^36–39 A linear increase in the position of the LSPR band (λ_max) with the increment of the Au molar ratio indicates the formation of an Au:Ag nanoalloy rather than a core-shell growth, as shown in Figure 1B.⁴⁰ Moreover, these results encompass 10 independent samples of each atomic composition, having standard deviations of ± 3 nm per NP type, indicating a high level of reproducibility of the synthesis method.³⁴ Additionally, Supplementary Table S1 shows a comparison of λ_max obtained in this work compared to the values reported in previous studies. In the cases of Au, Ag and Au50:Ag50 NPs the values are in close agreement. Stability studies were conducted to verify that the colloids did not lead to NPs agglomeration over time. The absorption spectra of the colloidal NPs were measured on the same day of their synthesis (day 0), on day 14, and again on day 26 after their synthesis to demonstrate the stability of the colloids (Supplementary Figure S2A). The results showed that the synthesis method yielded samples that showed colloidal stability for at least 26 days.

Figure 1 Physicochemical characterization of Au:Ag NPs. (A) Representative UV-vis absorption spectra. (B) Relation of the position of the LSPR band (λmax) and atomic composition of the NPs. The graph was constructed using 10 independent samples for each NPs system. (C) representative hydrodynamic diameter distribution obtained by DLS. (D) FT-IR spectra of the Au:Ag NPs compared with pure starch.

The XRD patterns for Au NPs, Ag NPs, and bimetallic Au:Ag NPs are shown in Supplementary Figure S2B along with the calculated XRD patterns for bulk face-centered cubic (FCC) Au and Ag.³⁵ The observed diffraction peaks at around 2θ = 38.2°, 44.4°, 64.6°, 77.6°, and 81.8° for all the Au:Ag samples correspond to the crystallographic planes (111), (200), (220), (311), and (222), respectively, of Ag or Au with an FCC crystal structure.⁴¹ By analyzing the diffraction peaks associated with the (111) plane, it is possible to determine the cubic lattice parameter (a) of the FCC structure and the average crystallite size (L). The results obtained are presented in Supplementary Table S2. It is noteworthy that the results fall within the expected range, with values close to 4.07 ų³ and average values of the crystallite size (L) are relatively close to the corresponding average particle size found in DLS and TEM measurements (vide infra).

DLS is a widely employed technique to determine the hydrodynamic diameter of NPs in colloidal suspensions.⁴² As shown in Figure 1C, the number-based measurements exhibited single peaks for the NPs, with D_H around 10 nm, excepting Au NPs, for which D_H was ~20 nm. The ζ-potential measurements were performed using the principles of ELS to determine electrostatic repulsion between particles, thus influencing their stability in suspension. Generally, a colloid is considered stable if the ζ-potential is above a critical value of ± 30 mV.⁴³ Table 2 presents the obtained ζ-potential of Au, Ag, and Au:Ag NPs, whose values were around −30 mV, indicating that the colloids are near their critical threshold for stability. As the proportion of Ag increases, the absolute value of the ζ-potential decreases, suggesting that the addition of Au may improve the stability of the NPs.

Table 2 Hydrodynamic Diameter (DH) and ζ-Potential of the Au:Ag NPs as Determined by DLS and ELS Measurements, Respectively

The starch-coated NPs were investigated using FT-IR spectroscopy. The FT-IR spectra obtained from the Au, Ag and Au:Ag NP samples (Figure 1D) show the skeletal mode vibrations of the glucose pyranose ring, and markers of vibrations involving glycosidic linkage. Specifically, the stretching vibrations of the C-O-C glycosidic bridge can be observed between the ranges of 1160–1130 cm⁻¹ and 999–965 cm⁻¹, while C-O-H bending and CH₂ twisting can be observed at 1344 cm⁻¹. The peak observed at 1640 cm⁻¹ is characteristic of water adsorption in the amorphous regions of starch.^44,45 These results are similar to the FT-IR spectrum of the powder starch used in the synthesis, thereby confirming the presence of a starch coating in the analyzed NPs.

Au:Ag NPs Showed Quasi-Spherical Morphology

The particle size and morphology of the synthesized Au, Ag, and Au:Ag NPs were investigated through TEM analysis. Figure 2 displays representative TEM images, which illustrate the quasi-spherical shape and uniformity of the synthesized NPs and their respective size distribution analysis. In the left insets of each type of NP in Figure 2, the particle size distribution is showed, presenting distributions of 22.68 ± 1.7 nm, 13.30 ± 3.6 nm, 12.43 ± 4.1 nm, 12.35 ± 4.3 nm, and 25.57 ± 7.7 nm for Au NPs, Au75:Ag25, Au50:Ag50, Au25:Ag75 and Ag NPs, respectively.

Figure 2 TEM analysis of mono- and bimetallic Au:Ag NPs. Representative electronic micrographs of: (A) Au, (B) Au75:Ag25, (C) Au50:Ag50, (D) Au25:Ag75, and (E) Ag NPs. For each NP, left insets show the particle size distribution histograms with a normal distribution fitting, and right insets show a representative particle magnification.

Ag and Au:Ag NPs Reduce Mitochondrial Metabolism of Healthy and Hypertrophic H9c2 Cells

Previous reports demonstrated the cytotoxicity of Ag NPs on H9c2 cells,²² however, to the best of our knowledge, there are no reports regarding the cytotoxicity of noble metal-based NPs against cells under pathologic hypertrophic conditions, as well as on immune cells associated to metabolic disorders, such as MA macrophages. Therefore, here we evaluated the cytotoxic effect of Au NPs, Ag NPs and multiple bimetallic Au:Ag NPs alloys on hypertrophic H9c2 cells and MA macrophages. The hypertrophy on H9c2 cells were induced by Angiotensin-II as described in the methodology. The Angiotensin-II treated H9c2 cells showed more protein content per cell (Supplementary Figure S3) compared to untreated group, demonstrating that Angiotensin-II stimuli induced hypertrophy on H9c2 cells, in agreement with previous reports.^46,47 In Figure 3, it can be observed the cytotoxic effect of Au:Ag NPs in different concentrations, from 0.1 to 10 ppm on macrophages (Figure 3A), MA macrophages (Figure 3B), H9c2 cells (Figure 3C) and hypertrophic H9c2 cells (Figure 3D) after 24 h of treatment. Our results exhibit that all NPs did not reduce more than 20% of the relative cell vitality on macrophages. Interestingly, on MA macrophages, all NPs between 1 and 5 ppm showed an apparent increase in cell vitality. On other hand, Ag NPs showed significant inhibition of the cell vitality in a concentration-dependent manner on H9c2 cells, contrary to Au NPs and the Au:Ag NPs alloys, where the cell vitality was not reduced more than 20%, ie, they were not cytotoxic. Table 3 shows the IC₅₀ of Ag and Au25:Ag75 NPs on H9c2 cells, for which values of 7.06 ppm and >10 ppm, respectively, after 24 h were calculated. Another important finding was that on hypertrophic H9c2 cells the Ag NPs and Au25:Ag75 NPs decreased the relative cell vitality in a dose-dependent manner, while Au, Au75:Ag25 and Au50:Ag50 NPs did not significantly affect the relative cell vitality. The IC₅₀ was 5.3 ppm and >10 ppm for Ag and Au25:Ag75 NPs after 24 h of stimuli (Table 3), respectively. These findings support the cytotoxicity of Ag NPs on H9c2 cells and highlight their higher cytotoxic effect against hypertrophic H9c2 cells, highlighting toxicological differences. Thus, we decided to elucidate the mechanism of cytotoxicity of the NPs on hypertrophic H9c2 cells based on the IC₅₀, here representing the threshold for 50% inhibition of the metabolic function of the cell culture.

Table 3 The Half of Inhibitory Concentrations (IC50) for Ag and Au25:Ag75 NPs Indicated in ppm on H9c2 and Hypertrophic H9c2 Cells After 24 h of Stimuli

Figure 3 Cell vitality analysis on macrophages, metabolically activated (MA) macrophages, H9c2 and hypertrophic H9c2 cells. Relative cell vitality analysis quantification by Alamar blue assay on: (A) macrophages, (B) metabolically activated macrophages, (C) H9c2 and (D) hypertrophic H9c2 cells, treated at different concentrations with different concentrations (0.1, 0.5, 1, 5 and 10 ppm) of Au NPs, 75:25, 50:50, 25:75 and Ag NPs after 24 h. The percentages refer to relative cell vitality represented as percentage of control (non-treated cell vitality=100%). Data are presented as mean ± SEM of triplicates of at least three independent experiments.

To further visualize the mechanism of the NPs cytotoxicity, we first confirmed the cytotoxicity by analyzing the cytoplasmic esterase activity using ghost dye stain by flow cytometry at the IC₅₀ of Ag and Au25:Ag75 NPs. Based on our previous cell vitality assay, where the IC₅₀ Au25:Ag75 NPs was >10 ppm, we decided to estimate theoretically the IC₅₀ value. In both H9c2 and hypertrophic H9c2 cells, the estimated IC₅₀ was 12 ppm, which was the concentration employed for the following experiments. In Figure 4A it is observed that the percentage of viable H9c2 cells treated with Ag and Au25:Ag75 NPs was 87.57 ± 8.6%, and 84.84 ± 9.7%, respectively, whereas untreated cells showed 97.97 ± 1.3% of viable cells. Conversely, the percentage of viable hypertrophic H9c2 cells treated with the IC₅₀ of Ag NPs and Au25:Ag75 NPs was 55.22 ± 13.2%, and 56.01 ± 13.6%, respectively, while the untreated cells showed 95.89 ± 1.6%. Representative dot plots are found in supplementary Figure S4. These results confirm the high cytotoxicity of Ag and Au25:Ag75 NPs against hypertrophic H9c2 cells compared to non-hypertrophic (healthy) H9c2 cells.

Figure 4 Biochemical implications of the cytotoxicity induced by Ag NPs and Au25:Ag75 NPs on healthy- and hypertrophic H9c2 cells. (A) Cell viability quantification using Ghost dye stain and, (B) mitochondrial membrane potential (∆Ψm) quantification using JC-1 stain by flow cytometry on H9c2 cells and hypertrophic H9c2 cells treated for 24 h with IC50 of Ag NPs and Au25:Ag75 NPs, and, CCCP (80 nM) as a positive control. (C) Mitochondrial reactive oxygen species (mROS) analysis and quantification using MitSox stain by flow cytometry on H9c2 cells and hypertrophic H9c2 cells treated with IC50 of Ag NPs, Au25:Ag75 NPs and H2O2 (50 μM) after 24 h of stimuli. (D) Regulated cell death analysis (left) and quantification (right) by flow cytometry using Annexin-V and PI stain on H9c2 cells and hypertrophic H9c2 cells treated with IC50 Ag NPs and Au25:Ag75 NPs at 24 h. Data are presented as mean ± SEM of triplicates of at least three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 compared to Control group (untreated cells).

Mitochondria is the major bioenergetic center of cells and the main reservoir of proteins involved in intrinsic apoptotic pathway. Mitochondrial alterations, such as loss of ∆Ψm or exacerbated mROS production, are associated with regulated cell death (RCD) or apoptosis.⁴⁸ To elucidate the mechanism of cytotoxicity of Ag and Au25:Ag75 NPs, we analyzed mitochondrial alterations on healthy and hypertrophic H9c2 cells. First, we evaluated the ∆Ψm using JC-1 stain by flow cytometry. The JC-1 stain allowed the differentiation of polarized mitochondria such as those high ∆Ψm, versus depolarized mitochondria, such as those with low ∆Ψm. Representative dot plots are found in supplementary Figure S5. As shown in Figure 4B, the control group showed 8.31 ± 1.8% of low ∆Ψm, whereas treated cells with Ag and Au25:Ag75 NPs exhibit 50.13 ± 24.5% and 45.12 ± 22.7% of low ∆Ψm, respectively. The positive control CCCP (80 nM) revealed 29.04 ± 16.2% of low ∆Ψm. Similar trends were observed on hypertrophic H9c2 cells, ie, untreated cells showed 16.34 ± 3.9% of low ∆Ψm, whereas 66.06 ± 15.1% and 68.92 ± 16.3% of low ∆Ψm were observed on Ag and Au25:Ag75 NPs-treated cells. CCCP treatment revealed 41.75 ± 17.2% of low ∆Ψm. Thus, these results highlight that Ag and Au25:Ag75 produce loss of ∆Ψm. Furthermore, we analyzed mROS production using MitoSox stain by flow cytometry. In Figure 4C, we can observe representative histograms (left) and quantification (right) of the fold change of MitoSox MFI with respect to the control group on H9c2 cells, where the Ag NPs showed a 1.65 ± 0.8 fold increase, the Au25:Ag75 NPs revealed a 1.58 ± 0.6 fold increase, and the positive control H₂O₂ (50 μM) exhibited 1.81 ± 0.8 fold increase, all compared to the control group. On hypertrophic H9c2 cells, Ag NPs showed a 1.19 ± 0.1 fold increase, Au25:Ag75 NPs exhibited 1.28 ± 0.1 fold increase, and H₂O₂ (50 μM) exposed cells presented a 1.67 ± 0.3 fold increase, all compared to the control group. Overall, these data demonstrated that Ag and Au25:Ag75 NPs induce mitochondrial damage on healthy and hypertrophic H9c2 cells, suggesting an RCD.

Finally, to correlate the mitochondrial alterations induced by the Ag and Au25:Ag75 NPs with apoptosis, we analyzed phosphatidylserine exposure and membrane permeability by flow cytometry using Ann-V and PI stains, respectively. Positive cells for Ann-V, PI or both were considered as dead (RCD), and negative cells as live. In Figure 4D, we can observe that the control group showed 7.12 ± 2.7 %, Ag NPs 37.60 ± 5.5 %, and Au25:Ag75 NPs 21.54 ± 3.0 % of RCD. On hypertrophic H9c2 cells, control cells showed 8.08 ± 2.3 %, Ag NPs 29.17 ± 2.5 %, and Au25:Ag75 NPs 17.97 ± 2.0 % of RCD. These results demonstrated that both metallic NPs induce RCD on healthy and hypertrophic H9c2 cells, albeit higher for Ag NPs.

Discussion

Nowadays, metallic NPs have gained significant attention for their wide biomedical applications due to their physicochemical properties. Understanding the mechanisms underlying the cytotoxicity of metallic NPs upon metabolic disorders, such as cardiometabolic disease, is essential for mitigating potential risks and optimizing their use for biomedical applications. In this work, we analyzed the cytotoxicity of mono- and bimetallic Au:Ag NPs with different chemical compositions on healthy and hypertrophic H9c2 cells, and healthy and MA macrophages.

Thus, we first determined the physicochemical properties of mono- and bimetallic NPs. In the case of the bimetallic Au:Ag NPs, the presence of single bands in the respective UV-Vis spectra is a strong indicator of the formation of nanoalloys (Figure 1A), and the absence of any additional signals or shoulders discard the presence of impurities or agglomerations.⁴⁹ Furthermore, the observed narrow bandwidth of the UV-Vis spectra indicates a low level of particle size polydispersity, suggesting a predominant proportion of NPs with similar particle sizes within a system.^40,50 Furthermore, a linear monotonic increase of the position of the LSPR band (λ_max) with the atomic increment of Au indicates the formation of an Au:Ag nanoalloy rather than a core-shell growth.⁴⁰

As previously mentioned, the presence of starch in the NP samples was confirmed by FT-IR spectroscopy (Figure 1D), showing results consistent with those previously reported in the literature.^33,34,36 Additionally, the stability studies previously described (Supplementary Figure S2A) demonstrate that starch effectively stabilizes the NPs in water for at least 26 days after their synthesis. The hydrodynamic diameter (D_H) distributions of the Au:Ag NPs systems lied within a range of 6–27 nm (Table 2). TEM images of Au75:Ag25 NPs showed a chain-like structure, similar to previous results reported by Nieto-Argüello et al.³⁴ However, its corresponding absorption spectrum shown in Figure 1A, displays only a single LSPR absorption band, ruling out the possibility of an anisotropic nanostructure. The mean diameter of the NPs (D_mean) as determined by TEM for Au and bimetallic Au:Ag NP systems are similar to the D_H and crystallite size (L) as determined by DLS and XRD, respectively. The increase in ζ-potential can be explained by the relatively poor stability of Ag due to its ease of ionization,⁵¹ as can be seen in Figure 2, where the monometallic Ag NPs and bimetallic Au:Ag NPs present agglomerations, unlike the Au NPs.

After characterizing the mono- and bimetallic Au:Ag NPs, we determined their cytotoxicity on healthy and MA macrophages, and healthy and hypertrophic H9c2 cells. In our results, we did not observe significant cytotoxicity in any macrophage (Figures 3A and 3B). These results are similar to previous studies which demonstrated the non-cytotoxic effect of Ag⁵² and Au NPs upon human peripheral mononuclear cells (primary culture of macrophages and lymphocytes),^53,54 and on either pro- (M1) and anti- (M2) inflammatory macrophages, and Ag NPs modulated pro- and anti-inflammatory cytokines release.⁵⁵ Moreover, Ag NPs with smaller particle sizes (< 4 nm) induced more pro-inflammatory cytokines release on U937 cells than bigger Ag NPs (> 4 nm).⁵⁶ Our Au and Au:Ag NPs have smaller particle sizes than the Ag NPs; thus, the former NPs may promote pro-inflammatory cytokines release. This assumption could explain the slight cell vitality increase on MA macrophages induced by Au and Au:Ag NPs but not by Ag NPs.

On the other hand, Ag NPs showed significant cytotoxicity in a concentration-dependent manner on both healthy and hypertrophic H9c2 cells, but the Au NPs and bimetallic Au:Ag NPs do not show more than 20% cytotoxicity (Figure 3). Previously, the cytotoxicity of Ag NPs was demonstrated against H9c2 cells,²² whereas Au NPs with a mean particle size of 20 nm did not show cytotoxicity on the same cell line, contrary to Au NPs with particle sizes of 5, 40 and 100 nm that decreased cell vitality on H9c2 cells.⁵⁷ However, alloying Ag with platinum (Pt) increased the toxicity of NPs in vivo tests on zebrafish embryos.^58,59 In other CVD contexts, Au NPs based on curcumin demonstrated cardio-protective effects in an in vivo model upon cardiac injury by a conventional chemotherapy (doxorubicin).⁶⁰ Thus, the reduction of the cytotoxic effect of the Ag NPs on healthy and hypertrophic H9c2 cells when alloying them with Au to form Au:Ag NPs could suggest a cardioprotective effect of the bimetallic nanomaterials.

Additionally, we demonstrated that Ag and Au25:Ag75 NPs induce mitochondrial alterations, specifically with regards the loss of ∆Ψm, which is exacerbated on hypertrophic H9c2 cells and the mROS production that is attenuated on hypertrophic H9c2 cells (Figure 4C). This loss of ∆Ψm is likely due to excess of Ag ionization, increasing mROS, an effect similar to that produced by SiO₂ NPs,⁶¹ which under certain conditions could lead to cardiac fibrosis.⁶² This effect could be counteracted with antioxidants capable of reaching mitochondria such as quercetin in polymeric NPs⁶³ or mitochondrial antioxidant compounds.⁶¹ It is conceivable that mitochondrial damage could be attenuated by loading the metallic NPs with antioxidant molecules, even as a cardioprotective strategy.

Also, we revealed that the mechanism of cytotoxicity of Ag and Au25:Ag75 NPs induced the increase of mROS production and the loss of ∆Ψm, associated to diminution of esterase activity and high RCD (Figure 4). Similarly, it has been previously demonstrated that Ag and Au NPs have shown to induce mitochondrial dysfunction such as ROS production, overexpression of pro-apoptotic proteins, loss of ∆Ψm and autophagy in variety of human, mouse and rat cells, including H9c2 cells.^53,54,64,65 Such mitochondrial dysfunction is correlated with a dysregulation of Ca²⁺-ROS homeostasis⁶⁶ and thus, the demodulation of the mitochondrial Ca²⁺ dynamics, which are essential to bioenergetic balance and are likely a key player behind CVDs.^46,67 Mitochondrial disruptions result in decreased cellular metabolism and bioenergetics activity, which accelerates cell death and autophagy.⁶⁸ Higher expression of genes associated with intrinsic apoptosis (p53, caspase-3 and Bax/Bcl-2) is correlated with high molar ratio of Ag in the Au:Ag alloy on human cancer cell lines (HCT116, 4T1 and HUH7).²⁵ Higher levels of ROS production were observed on Ag NPs compared to Au NPs and Au:Ag bimetallic NPs on the human breast cell line MCF-10A.⁶⁹

ROS are second messengers in a variety of signaling cascades⁷⁰ at low ROS levels, they have been shown to play a role in hypertrophy signaling, and at high levels induce cell death.⁷¹ Also, ROS accumulation directly contributes to Angiotensin II–induced hypertrophic cardiomyocyte by saturation and decrease of antioxidant enzymes activity.^72,73 The fact that Ag NPs increase mROS production compared to Au25:Ag75 NPs on healthy and hypertrophic H9c2 cells suggests the possibility that Ag NPs induce higher levels of RCD, whereas the Au25:Ag75 NPs could ameliorate hypertrophy as remodeling to prevent cytotoxicity. The cardiac cells under pathologic hypertrophy condition revealed prominent susceptibility to reduce mitochondrial metabolism and function (Figure 4), leading to a slight increase in mROS production compared to healthy H9c2 cells. Regarding regulated cell death mechanism, both healthy and hypertrophic cardiac cells showed comparable results, showing a higher effect for Ag NPs than Au25:Ag75 NPs (Figure 4). The implication of alloying Ag with Au to create bimetallic Au:Ag NPs to attenuate their cytotoxicity encourages the potential application of such advantageous NPs in biomedical applications, including obesity-associated disorders. For example, the generation of safer and low-cost Au-based NPs for cardiac laser ablation in cardiac pathologies, or photo-thermal therapy on cancers, such as those of breast, liver, and pancreas.^26,74,75 In general, there are many clinical applications of metallic NPs in the context of CVDs. For example, for imaging and detection Au:Ag NPs could be used as a contrast agent in imaging technologies.⁷⁶ Also, therapeutic agents as a drug delivery system that could be conjugated with cardiovascular drugs,⁷⁷ and anti-inflammatory applications, where Au NPs could act as an immunomodulator agent.^78,79 But the first step towards an application is to identify the cytotoxicity of the noble metal-based NPs in pathological hypertrophy conditions. In this context, we have characterized bimetallic Au:Ag NPs with low nanotoxicity that may be employed for clinical application, such in the treatment of metabolic disorders.

Conclusion

Overall, our results demonstrate for the first time the cytotoxicity of the Ag and alloyed bimetallic Au25:Ag75 NPs against MA macrophages and cardiac cells under pathological hypertrophic conditions. Ag and Au25:Ag75 NPs induce regulated cell death, interestingly, with a major sensitivity on hypertrophic H9c2 cells, as evidenced by a marked reduction in metabolism and ∆Ψm. This work helps to understand the potential cytotoxicity of mono- and bimetallic Au:Ag NPs on macrophages and cardiac cells under pathological conditions, highlighting their potential biomedical uses of those NPs with low Au-content. Finally, this study is the first step to provide evidence that pathological conditions related to metabolic disorders, such as obesity-associated cardiometabolic diseases, may have a higher susceptibility to certain noble metal-based NPs.