Instituto Tecnológico y de Estudios Superiores de Monterrey Campus Monterrey
School of Medicine and Health Sciences
“Characterization of the Cytotoxicity of Graphene Oxide and Reduced Graphene Oxide in Hypertrophic Cardiomyocytes”
Dissertation presented by
Estefanía Luna Figueroa
Submitted to
School of Medicine and Health Sciences
In partial fulfillment for the requirements for the degree of
Master’s of
Biomedical Sciences
Dedication
Este estudio lo dedico a mi familia.
Gracias mamá y papá por su constante apoyo y por siempre creer en mi.
Gracias a mi hermana por impulsarme a ser mejor persona.
Ustedes han sido mi inspiración para seguir adelante.
Acknowledgments
I would like to extend my thanks to Tecnológico de Monterrey and CONACYT, this work would not have been possible without their support.
To my advisors, Dr. Gerardo Garcia-Rivas and Dr. Flavio Contreras-Torres, for their continuous feedback, valuable suggestions, and never-ending patience, but most importantly, I would like to
thank them for giving me the opportunity carry out this project.
I would also like to thank the rest of my thesis committee: Dr. Fabiola Castorena Torres, Dr.
Omar Lozano Garcia, and Dr. Francisco Servando Aguirre Tostado, for their insightful comments and advice.
My sincere thanks also go to my co-workers form the Hospital Zambrano-Hellion and the School of Engineering and Sciences; specifically, Dr. Eduardo Vasquez, Ing. Fausto Abril, Dr. Nestor Rubio, and Dr. Carlos Campero. Without their help, it would not have been possible to conduct
this research.
Finally, I must express my very profound gratitude to Dr. Alejandro Torres and M. S. Anay Lazaro, and M.S. Romina Duarte, thank you for sharing your knowledge and providing me with unfailing support and continuous encouragement throughout these two years. You were a crucial
element to the development and completion of this project.
Summary
Graphene oxide (GO) and reduced graphene oxide (RGO) are carbon nanomaterials, which stand out for their industrial and biomedical use due to their extraordinary
physicochemical properties. Nevertheless, possible health risks call into question the benefits derived from its use. In particular, our interest is focused on cardiovascular tissue. Accumulation of particles in the myocardium may be feasible in this type of tissue, a risk that is more severe in tissues with a predisposition to damage. Even at low concentrations of particles, the risk ratio indicates the possibility of cardiometabolic disorders. The present study analyzes the cytotoxicity of GO and RGO in healthy cardiomyoblasts and cardiomyoblasts with cellular damage, using a pathological model of angiotensin II-induced hypertrophy. From the results obtained, we proposed possible mechanisms of cellular damage.
Table of Contents
Summary ... 1
1.Introduction... 2
2.Background ... 6
Hypothesis ... 11
General Objective... 11
Specific Objectives... 11
3.Materials and Methods ... 12
3.1 Nanomaterial Synthesis ... 12
3.2 Characterization of the nanomaterials ... 14
3.3 Biological Assays ... 17
4.Results and Discussion ... 24
4.1 Physicochemical characterization of GO and rGO ... 24
4.2 Toxicity analysis of GO and rGO in cardiomyocytes ... 30
4.3 Toxicity analysis of GO and rGO in hypertrophic cardiomyocytes ... 35
5.Conclusion ... 40
6.Perspectives ... 41
References ... 42
Resume ... 51
1. Introduction
Nanotechnology is an emerging field that entails the manipulation of nanometric
materials at the nanoscale (1-100 nm). In particular, the use of carbon-based nanomaterials such as graphene oxide (GO) has increased exponentially for biomedical applications, especially in the form of multifunctional nanocomposites (Krishnan et al., 2019). In particular, GO is used in biomedicine for drug delivery, cancer therapy, bio-imaging, and biosensors. This wide range of applications is due to the GO’s molecular structure. The oxidized honeycomb-like carbon structure attributes to GO high electron conductivity, mechanical flexibility, strength, and a sizeable surface-volume ratio for further chemical functionalization with compounds of medical interest (Novoselov et al., 2012). For instance, the structure of GO favors the pi-pi stacking interactions for loading anticancer drugs (Campbell et al., 2019; Chen et al., 2019). Such a loading capacity can also be improved due to surface functionalization and electrostatic interactions among moieties and active drugs. This scenario appears technically feasible to develop several GO-based materials in the future, providing novel applications for the new era of biomedicine. However, there exist concerns about the toxicity of GO and its derivatives,
especially in specific cases involving a dose range, size specifications, and a comparative chemical nature of materials. In this regard, the biological interactions of GO are closely related to its molecular properties, which include the C/O ratio, oxidation degree, particle size,
dispersibility in physiological conditions, methods of synthesis, purification, among others (Zhu et al., 2010). Several crucial aspects need to be considered before toxicological protocols can define the safety of GO. Therefore, these points deserve special attention, providing exhaustive discussions about all the possible toxic effects. Here, we are focused on the toxicity of GO and a
chemicaly reduced compound from GO in the heart as an emerging response to previous studies that revealed the accumulation of GO could occur in this organ (Kanakia et al., 2014).
GO also showed to trigger toxicity in zebrafish. It was suggested that GO entered
embryos via endocytosis, primarily distributed to the heart, and caused pericardial edema (Chen et al., 2016) with a significant reduction of heart rate(D’Amora et al., 2017). One of the most frequent GO’s properties associated with toxicity is the oxidation degree of GO. Depending on the amount of the C/O atomic ratio, GO can convert to a graphene analog called reduced-GO (rGO). Previous studies reported rGO exerts a strong toxic effect in cardiomyocytes after a 24- hour exposure, five times greater than GO. (Contreras-Torres et al., 2017). rGO toxicity appears to be influenced by its ability to internalize more readily. These findings were complemented using molecular insights such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Arbo et al., 2019). It seems that ROS are involved in the cytotoxic mechanisms caused by GO, increasing in a dose-dependent manner.
GO, and rGO toxicity can be amplified in pathological conditions, as well as in hearts with a predisposition to damage. A previous study showed that hearts with previous conditions present an exponential increase in nanoparticle accumulation (Ruiz-Esparza et al., 2016).
Another study revealed that exposure to naturally occurring nanoparticles provides a strong risk factor for cardiovascular diseases (Downward et al., 2018; Liang et al., 2020). Additionally, the observed heightened tendency of nanoparticles to accumulate in damaged hearts suggests that nanoparticle toxicity may be amplified in populations with current heart conditions.
The importance of preventing GO and rGO nanoparticle toxicity in the cardiovascular system highlights how critical comparative research is in healthy and hypertrophic
cardiomyocytes. Thus, the main goal of this study is to evaluate the cytotoxicity of both GO and
rGO in healthy and hypertrophic cardiac myocytes. Moreover, a complete physical
characterization of these two materials was carried out to facilitate comparison with similar materials. We hypothesized that hypertrophic cardiomyocytes should be more prone to display toxicity to GO as compared to control cardiomyocytes. The mechanisms of GO toxicity are thought to impact on with the same mechanisms that regulate hypertrophy, making
cardiomyocytes more susceptible to damage. In this sense, rGO should be more toxic than GO in both scenarios. Viability assessments of the toxicity of GO and rGO on cardiac myocytes were carried out for in vitro dose- and time-dependent experiments.
Cardiac hypertrophy arises from a set of stimuli that genetically reprogram proteins (MacDonnell et al., 2009). In particular, Ca2+ has been described as the key signaling ion that modulates hypertrophy by the activation of transcription factors that stimulate the renin- angiotensin-aldosterone pathway (Fiedler et al., 2002). Consequently, the activation of α1 AR and the angiotensin type-1 receptors stimulates the production of inositol 1,4,5 triphosphate (IP3), from phospholipase C, which in turn elevates the nuclear Ca2+ concentration. As a result, the production of hypertrophic signaling molecules, calcineurin, and NFAT is induced through the CaMKII/MEF2 pathway (Gomez et al., 2013).
In this regard, a model of heart damage mediated by Angiotensin (Ang II), an inducer of remodeling transcription factors that ultimately lead to hypertrophy in cardiac cells, was
established and characterized for the toxicity studies (Sadoshima & Izumo, 1993). This model was considered appropriate as it has been shown to resemble hypertrophy induced by heart failure. Viability studies were employed to identify the concentrations at which the
nanomaterials cause toxicity. With a detailed analysis of the methods mentioned above and a
comprehensive review of the literature available, the toxicity of GO and rGO in healthy and hypertrophic cardiomyocytes is studied and possible mechanism of action are proposed.
2. Background
Nanomaterials are those materials which show a range of 1-100 nm in at least one of its dimensions. In particular, carbon-based nanomaterials (CBNs) are nanostructures of carbon with different atomic arrangements that arise from the capability of carbon to form sp, sp2, and sp3 hybridization orbitals (Siqueira & Oliveira, 2017). Graphene, carbon nanotubes, fullerenes, and nanodiamonds are attractive CBNs for different applications owing to their unique properties (Siqueira et al., 2016; Kumar et al., 2017). In particular, the development of innovative devices for biomedical applications widespread the attention on the use of CBNs. However, cellular studies on the response mechanisms to CBNs are still incomplete, and thus the toxicity of these nanostructures is still under debate.
The possibility that CBNs can exhibit a predominantly toxic behavior is a primary
concern since the real impact of these nanostructures varies depending on the allotrope as well as their broad spectrum of properties, including purity and reactivity (Sanchez et al., (2012);
Sukhanova et al., 2018). Numerous studies, both in vivo and in vitro, have focused on
determining the biocompatibility and toxicological effects of CBNs (Kasai et al., 2015; Khosravi et al., 2018; Leite et al., 2015; Patlolla et al., 2017; Yu et al., 2018; Zare-Zardini et al., 2018).
Here, we focused on the toxicity of graphene oxide nanostructures. Graphene oxide (GO) is the product of a strong oxidation of graphite resulting in the incorporation of several oxygen- containing hydroxyl and epoxy groups into the basal surface of graphite (see Figure 2.1). To some extent, the physical properties of GO resemble those of graphene, whose thermal
conductivity, flexibility, strength, and large surface area properties (Novoselov et al., 2012) have inspired a particular interest and expectation to develop “a truly building-block” of
nanotechnology. A less oxidized graphene nanostructure is the so-called reduced GO (rGO), in
which the chemical composition and structure can be regarded as graphene particles but showing several defects in the crystal lattice. Both GO, and rGO can be used as sensing matrix element bases for biosensors for treatment of diseases (S. L. Chen et al., 2019) and detection of
biomolecules (Sun et al., 2019), imaging techniques (Campbell et al., 2019), drug and gene delivery conjugatesfor site-specific targets (Kim et al., 2014) and radical scavengers (Awan et al., 2016; Lin et al., 2017). In recent years GO and rGO nanostructures have also gained interest in biomedicine. Additionally, these materials have been shown to stimulate the growth and differentiation of bone marrow stem cells (Jin et al., 2016; Liu et al., 2017) neuroblastoma cells (Kuzmenko et al., 2018), PC12 neural cells (Chen et al., 2016), and human dermal fibroblasts (Pal et al., 2017). Interestingly, they have also been used in wound healing in vivo (Wang et al., 2019). In the cardiology research area, GO, and rGO also promoted the growth of vascular endothelial cells, enhanced angiogenesis and arteriogenesis in vivo in a chick chorioallantoic membrane model (Chakraborty et al., 2018), improved the electrical conductivity of the heart (Chakraborty et al., 2018; Park et al., 2015; Saravanan et al., 2018), stimulate cardiac tissue regeneration (Zhou et al., 2018), promote mechanical support (Choe et al., 2019) and modulate inflammatory responses after myocardial infarction (Han et al., 2018).
Figure 2.1. Graphene as the building block for the composition of GO and rGO. (figure
Data available about the toxicity of GO and rGO on the heart is not currently conclusive, mainly due to research that has not explored mechanisms in detail. The hypothesis of GO and rGO toxicity arises from well-known biodistribution analysis of graphenic materials (Kanakia et al., 2014), in which (in vivo) results demonstrate that the heart is among the organs that can accumulate the highest concentration of these nanomaterials upon administration. As a result of exposure to GO (and rGO) when nanomaterials are incorporated in biomedical scaffolds, there are claimed concerns about the possible adverse consequences that its use may cause on the heart. Other studies indicate that these nanostructures of carbon can cause a prolonged inflammation and alveolar damage in the lungs (Bengtson et al., 2017; Horie et al., 2012; K.
Wang et al., 2011), immune responses through the induction proinflammatory cytokines (Rodrigues et al., 2018), nephrotoxicity (Patlolla et al., 2016), morphological alterations in the liver (Patlolla et al., 2017) and inflammation of the spleen (Li et al., 2016).
Insights into the cellular mechanisms of GO and rGO in cardiac H9c2 myocytes have been recently pioneered by the “Group of Cardiology in the Hospital Zambrano-Hellion.” To test for viability, a dose-dependent toxicity study of GO and rGO assessed the metabolic activity of myocytes upon treatment with these nanomaterials (Contreras-Torres et al., 2017). It was observed that rGO showed a five-fold toxicity concerning GO. A high degree of molecular internalization of rGO into the myocytes was related to the mechanism of toxicity. Finally, the proportion of oxygen functionalities and graphitic domains present in these nanostructures were suggested as the structural properties that drive the differential toxicity among GO and rGO. A dose-dependent toxicity study was conducted using Catla Catla cardiac cells (Xing et al., 2016).
Interestingly, those results showed that GO is comparatively more toxic than rGO. It was suggested the toxicity of rGO was attenuated as a result of contaminants in the sheets, remaining from the reduction process. These studies support the claim that chemical composition is a potent regulator of the toxicity of GO and rGO.
Dutra Arbo et al. (Arbo et al., 2019a) also investigated the toxicity effects of GO using H9c2 cells. It was shown that GO could increase the generation of ROS and RNS. An assessment of the mitochondrial membrane potential additionally demonstrated the hyperpolarization of the mitochondria and thus opening a question about if GO could damage the ATP synthase.
Furthermore, genotoxicity assays suggested that the mechanisms involved in the toxicity of GO can cause breaks in the DNA (possibly inducing death by apoptosis or necrosis). Accordingly, the genotoxicity of GO could be mediated by the oxidative stress being generated as a result of the increased ROS.
The potential toxicity of these particles is thought to escalate when they are exposed to preexisting heart conditions. Studies conducted after the administration of particles on diseased hearts concluded that damaged hearts undergo a tendency to accumulate higher concentrations.
Quantitative studies revealed that the accumulation of nanomaterials in failing myocardium is approximately 14 times greater in comparison to healthy hearts (Ruiz-Esparza et al., 2016). A more recent study also provided evidence of the increased incidence of cardiovascular diseases in populations exposed to higher concentrations of naturally occurring particles in the
environment (Liang et al., 2020). These studies suggest damage to the myocardium is plausible and ongoing conditions can aggravate the toxicity of GO and rGO nanoparticles.
To better comprehend the toxicity effects of both, GO and rGO in myocardial cells, in this thesis, we are focused on elucidating possible intracellular mechanisms by which these
nanostructures can cause damage. To this end, we carried out the experimental synthesis of the two nanomaterials to ensure complete control over their physical and chemical characteristics. In particular, we reproduced a model of cardiac damage induced by Ang II, a neurohumoral agent which indirectly modulates the release of Ca2+ from the SR through the IP3 receptor. This event results in Ca2+ overload, and the simulation of TGF-B, NFAT, GATA-4, and other growth factors which lead cytoskeleton rearrangement and myocyte enlargement, characteristic features of hypertrophy (Ruzicka et al., 1999; Sadoshima et al., 1993). Thus, this model simulates malignant hypertrophy in cardiac failure.
Lastly, we studied the toxicity of GO and rGO in hypertrophic and healthy H9c2 cardiac myoblasts through a quantitative assay of cellular viability. These studies were conducted in a dose and time-dependent manner to assess variables in the toxicity.
Hypothesis
Hypertrophic cardiomyocytes exhibit greater toxicological susceptibility to graphene oxide and reduced graphene oxide nanoparticles, compared to control cardiomyocytes
General Objective
To evaluate quantitatively, the cytotoxicity of graphene oxide and reduced graphene oxide in a dose-dependent manner in control and hypertrophic cardiac cells.
Specific Objectives
1. To synthesize and characterize the GO and rGO nanoparticles.
2. To conduct in vitro assessments of the toxicity of GO and rGO in both control and hypertrophic cardiomyocytes.
3. To elucidate possible mechanisms of GO and rGO toxicity.
3. Materials and Methods
3.1 Nanomaterial Synthesis Synthesis of Graphene Oxide
Graphene Oxide (GO) was synthesized using Tour’s method through the oxidation of pure synthetic graphite power (<20μm, Sigma-Aldrich LOT# MKBW7613V). Briefly, 50 ml of phosphoric acid (H3PO4, Sigma-Aldrich, 85% purity) were mixed in 350 ml of sulfuric acid (H2SO4, Sigma-Aldrich, 98% purity) in a 1000 ml beaker and stirred for 15 minutes. Graphite powder (4.0 g) was slowly added to the mixture and allowed to incorporate fully while
continuously stirring. The mixture was stirred for 2 hours, and then 25 mg of KMnO4 was added in a carefully controlled rate to keep the reaction below 70 °C while stirring continuously for 8 hours. As oxygen atoms were incorporated in the graphite, the reaction is observed to turn from black to brown. After the 8 hours, the solution was treated with 1 ml of hydrogen peroxide (H2O2, 1 M) to eliminate any excess KMnO4. It was added in a dropwise manner into the mixture and stirred for 5 minutes. Then, the beaker was transferred to an ice-cold bath, and the mixture is slowly diluted with 500 mL of deionized H2O. After cooling down, the GO suspension was poured into 50 mL tubes, and washed by centrifugation three times (5 min, 5000 rpm), every time decanting the supernatant and refilling the tubes with clean deionized H2O. The resulting mixture was then transferred to a 12-14 kD cellulose dialysis membrane (Spectra/Por) and placed in a 1000 mL beaker with 800 mL of deionized H2O to wash away any remaining byproducts of the synthesis reaction. Five days later, the contents of the dialysis tubing were removed and transferred to plastic tubes, frozen and subsequently lyophilized for two days to obtain GO powder.
Mechanical exfoliation of Graphene Oxide sheets
The mechanical exfoliation of GO sheets proceeded using ethanol (EtOH, Sigma-Aldrich, 98%) and mechanical force to intercalate molecules in between the layers and separate them. The exfoliation preceded by pouring 50 mg GO powder together with 1 ml of EtOH. The mixture was manually grounded with the pestle until most of EtOH seemed to evaporate. This last step was repeated twice. Afterward, the GO was transferred to a 20 ml glass vial, and 15 ml deionized H2O was added. The vial was vortexed (5 min, 4000 rpm) and then ultrasonicated for 5 min.
Then, the mixture was maintained unmoved for 24 hours. After the allotted time, GO was visibly seen to have separated into two phases; a top portion in the form of solution and a precipitate.
The two phases were carefully separated and transferred to a second 20 ml glass vial. The second vial was vortexed, ultrasonicated and maintained unmoved as described before. Twenty-four hours later, the mixture was separated again, and the supernatant was placed in a third vial. In this last vial, GO was observed to remain constant (minimal to no precipitate) up to 72 hours after being separated. Finally, the dissolution was frozen and then lyophilized for two days to obtain exfoliated GO powder.
Synthesis of reduced Graphene Oxide
The reduction of GO to obtain rGO consisted of removing oxygen from the structure of GO. To this, a hydrothermal process proceeded using a green reduction agent, which prevents the synthesis of toxic byproducts. In this regard, ascorbic acid was proven to be a suitable yet- nontoxic reducing agent. Exfoliated GO powder (19 mg) was dispersed in 90 ml of deionized H2O and stirred for 15 minutes in a 250 ml beaker. The dissolution was poured into a 150 ml Teflon chamber, to which 352 mg of L-Ascorbic Acid (C6H8O6, Sigma-Aldrich, LOT #
SLBC8161V) were added while stirring. Next, ammonium hydroxide (NH4OH, Sigma-Aldrich,
≥25%) was added to the mixture, as needed, to raise the pH to 10. The Teflon chamber was then placed inside into a hydrothermal autoclave reactor and was tightly sealed. The reactor was heated to 200°C for 4 hours. After heating, the resultant black rGO suspension was taken out of the reactor, washed and centrifuged three times (8 min, 5000 rpm). The supernatant was
discarded and replaced with deionized H2O every time after centrifugation of rGO. Finally, rGO was transferred to 20 ml vials, frozen and then lyophilized for two days.
3.2 Characterization of the nanomaterials
The following characterization techniques comprise an in-depth study of the chemical and physical characteristics of the synthesized GO and rGO.
UV-Vis
The synthesis of GO and rGO were confirmed by UV-vis spectra. Dissolutions of exfoliated GO and rGO were prepared by mixing 1200 µg of nanomaterial and 2.5 ml of deionized H2O. The mixtures were vortexed for 1 min and further sonicated for 5 min. Once a dissolution was acquired, 2.5 ml was pipetted into a 10 mm quartz cuvette, with attention to preventing the formation of air bubbles. Absorption spectra for the materials were obtained in a PerkinElmer Spectrophotometer at a wavelength range of 200-800 nm.
Scanning Electron Microscopy (SEM)
In an aluminum stub, 20 µl of a GO dissolution, in H2O, was pipetted and allowed to dry in air. The stub was then fixed to a filter disk holder and placed in the SEM (JEOL JSM-7100F).
Micrographs were acquired at a magnification of 20X, 35X, 43X, and 100X using 30 kV.
Micrographs of exfoliated GO and rGO samples were obtained and visualized in dry and in solution. SEM images of the dry samples were prepared by placing the material into double- sided adhesive tabs and adhering them to the SEM stub. The solutions were prepared by mixing the nanoparticles in 1 mL of isopropanol (70% in H2O) and 5 min for ultrasonication.
Approximately 40 µl of each solution were placed on the stub, the solutions were allowed to air dry, and micrographs were obtained at x30000, x50000 x10000 and 150000 magnifications at 10 kV.
Dynamic Light Scattering (DLS)
The samples prepared for dynamic light scattering (DLS) were obtained from dissolutions of GO and rGO at a concentration of 300 ppm in deionized H2O at room temperature (24 °C). Quartz cuvettes (10 mm) were filled with 2.5 ml of the solutions, respectively, and inserted into the DLS (Zetasizer Nano ZS90) to analyze. The average
hydrodynamic diameter was obtained from three independent measurements, at a count rate of 314.6 kcps, a measurement position of 4.65 mm, and for 50 sec, with 15 runs for each
measurement.
Zeta potential
The zeta-potential analysis of GO and rGO particles was carried out by using a dissolution of the nanoparticles in H2O at a concentration of 200 µg/ml. Posteriorly, 1mL of the nanoparticle solution was pipetted into a folded capillary cuvette and then inserted into the Zeta potential analyzer (Zetaizer nano ZS90). The measurement was repeated three independent times, with fifteen rounds each one, using different samples.
Atomic Force Microscopy
Exfoliated sheets of GO were solubilized in deionized H2O using vortex (1 min) and ultrasonication (5 min). A small silica wafer was placed on the sample holder for the acquisition of micrographs. A small drop of the prepared solution was then placed atop the silica wafer and then allowed to dry in air. The sample was placed in the AFM (AFM Digital Instruments 4.31), and micrographs were obtained using a tapping mode at different magnifications.
Fourier Transformed Infrared Spectroscopy (FTIR) study
FTIR spectra were recorded using 100 µl of GO and rGO solutions, prepared as described before. Each solution was placed in the FTIR sample holder, allowed to dry and placed inside the FTIR analyzer (PerkinElmer Spectrum One). The sample measurements were recorded in
transmittance mode at a resolution of 1 cm-1 in the range of 525-4000 cm-1 wavenumber.
X-ray Diffraction (XRD) analysis
For the analysis, two drops of GO solution at a concentration of 500 ppm were loaded into a silica sample holder. The solution was allowed to dry to generate a thin film of GO, which was analyzed. The diffraction patterns were obtained using an X-ray diffractometer (Bruker, D2- phaser) for 5 min (CuKa1, l=1.54 Å, 10 mA, 30 kV).
Raman Spectroscopy
The analysis was performed using dry samples of GO and rGO placed on a glass
substrate and covered with aluminum foil. The samples were placed in the sample compartment of the Raman spectrometer (Anton-Paar Cora 5600), and the spectra were obtained at an
excitation wavelength of 532 nm at 50 mW for 10000 ms. For each material, two traces were obtained, one demonstrating the raw data and another with baseline correction, additionally a
smoothing of the curve executed for rGO (the GO spectrum could not be smoothed due to carbonization of the sample) .
X-ray photoelectron spectroscopy (XPS)
XPS measurements were carried out using ion etching to detach O2 adsorbed from the environment. Thus, superficial sheets often provide results of higher oxygen content as compared to more profound material. The samples were placed in the sample holder to obtain the XPS spectra, and the analysis was performed in an ultra-high vacuum system scanning XPS (XPS microprobe PHI 5000 VersaProbe II). The data was obtained at 45 degrees to the surface with etching for 15 sec with 0.5 kV 500 nA in 9 mm2 using Argon to detach the excess of physisorbed oxygen onto the surface of the sample.
3.3 Biological Assays In vitro cell culture
Neonatal rat ventricular cardiomyocyte-derived cell line H9c2 was used throughout the study for cytotoxicity assays. Previous to any experiments, H9c2 cells were cultured in 25 mL flasks in high-glucose DMEM cell culture media and incubated at 37 °C in 95% of O2 and 5% of CO2. The DMEM used was prepared and supplemented with 10% of fetal bovine serum (FBS) and streptomycin (100 µg/ml) and penicillin (100 U/ml). The cell culture media was changed every three days, and cells were subcultured upon reaching 70-80% confluence. For all analyses, this cell line was used between passages 9 and 25.
Nanoparticle suspensions in DMEM
Suspensions of GO and rGO in DMEM media were prepared for the cellular application process. A stock suspension with the highest concentration was prepared by weighing 10,000 μg
of the particles and resuspending them in a 2 ml plastic tube with 1 ml of DMEM supplemented with 10% of FBS. The mixture was vortexed (5 min, 4000 rpm) and then ultrasonicated for 5 min. For the serial dilutions, the amount of solute was transferred from the preceding higher concentrated suspension, starting from the stock to the lesser concentrated microtube. Then, each concentration was diluted with supplemented DMEM to end with 1.0 ml for each suspension, as stated in Table 1, making sure to vortex each suspension for 30 sec before transferring it on to the next tube.
Table 3.1. Serial dilutions for the viability assays.
Concentration (μg/ml) Volume from previous higher
concentrated suspension (μl) Amount of supplemented DMEM (μl)
10,000 * 1000
5,000 500 500
1,000 200 800
600 600 400
500 833.3 166.7
400 666.6 333.4
300 750 250
200 666.6 333.4
100 500 500
10 100 900
1 100 900
Cytotoxicity studies
H9c2 cells were plated at a density of 5 × 103 per well in 96-well plates in 100 µl DMEM supplemented with 10 % FBS. Twenty-four hours post-seeding, the cells were treated with GO or rGO suspensions at different concentrations ranging from 10,000 ppm to 1.0 ppm. In this experiment, two control groups were used, a positive control group (“Ctrl +”) which received no
treatment, and a Negative control group (“Ctrl –”), which received a treatment with Doxorubicin (50 μM), a substance with well-reported toxicity. Furthermore, the plates were incubated for 24 hours, as previously described. After the incubation period, the cells were removed from the incubator, and the treatment was carefully removed. Then, each well was carefully washed 2-3 times with PBS solution to remove any remaining traces of the particles. The cells were then resuspended in 100 μl of 10% FBS supplemented DMEM and viability assay was applied
AlamarBlue viability assay
Previously treated cardiomyocytes were resuspended in culture media, and 10 μl of alamarBlue™ was pipetted into each well, avoiding light exposure to prevent the reagent’s degradation. Additionally, 100 µl of supplemented DMEM were added to three extra wells, and 10 μl of alamarBlue™ were added onto each one, to correct the background fluorescence. The culture plates were then incubated for 3 hours, as previously described. During the incubation period, viable cells can reduce resazurin, the dye that composes alamarBlue, into resorufin. After the allotted time, the oxidation-reduction reaction can be visualized in the change of color from blue to pink, the fluorescence was read in a fluorometer (Synergy HT, BioTeK) at 525 nm excitation and 590 nm emission wavelength.
Time-dependent viability assays
Exposure to GO was analyzed along different periods to have a better understanding of nanoparticle behavior. Cells were cultured in 96-well plates in 100 µl of 10% FBS supplemented DMEM with varying cell concentrations, which depended on the exposure times to prevent overcrowding of cells, as follows: 12 hours with 1×104 cells, 24 hours with 5×103 cells, 48 hours with 3×10 cells, and 72 hours with 3×10 cells. The cells were then incubated for 24 hours at 37
°C in 95% of O2and 5% of CO2. After 24 hours, the cells were removed from the incubator, and the DMEM was removed from each well. The cells were resuspended in 100 µl of a GO
nanoparticle DMEM solution, at a concentration equal to the half-inhibitory concentration obtained after 24 hours of incubation. The cells were incubated with the GO particles for 12, 24, 48, and 72 hours. The GO solutions were removed after their respective allotted periods; each well was very carefully washed 2-3 times with PBS solution, and the cells were resuspended in 10% FBS supplemented DMEM. The viability of the cells was assayed using alamarBlue™.
Induction of hypertrophy on cardiomyocytes
H9c2 cells were seeded at a density of 7 × 103 atop 25 mm2 coverslips in six-well plates with 500 µl of DMEM culture media supplemented 10% FBS. After 24 hours, the cell culture media was removed and exchanged for 1 ml of DMEM supplemented with 1% FBS,
streptomycin (100 µg/ml) and penicillin (100 U/ml). After 24 hours of exchanging the cell culture medium, an Angiotensin II (Ang II) stimulus at a concentration of 1µM was applied and maintained for 48 hours to induce hypertrophy in the myocytes, making sure to avoid light exposure to prevent deterioration of the reagent. After the allotted time, each cover glass with the fixed cells was washed with 1 ml of Tyrode’s Solution supplemented with 5 mM of glucose. The cells were then characterized by confocal microscopy.
Characterization of Ang II-induced Hypertrophy
Before evaluation under confocal microscopy, each cover glass was incubated for 30 min, preventing light exposure, at room temperature with calcein (5mM), a cell-permeable
fluorophore that dyes cytosolic calcium. After 30 min, the cells were washed with 400µl of Tyrode’s solution and subsequently analyzed under the microscope (Leica TCS-SP5). More than
100 cells per group were reviewed to assess for hypertrophy. The micrographs obtained from confocal microscopy were analyzed in ImageJ software. The perimeter of each cell was outlined individually to obtain the area of the control and the hypertrophic cells. The data was further analyzed in GraphPad Prism to obtain the average area of the cells, and the results were graphed.
MitoSOX ROS Assay
After 48 hours with a 1µM stimulation of Angiotensin II, H9c2 cells were harvested with 0.5 mL of Trypsin-EDTA (0.25%), centrifuge washed in 2.0 mL of Tyrode’s solution (1500 rpm, 5 min) and incubated with 5 µM MitoSOX (Thermo Fisher) at 37 °C for 15 minutes. Afterward, the cells were washed twice with Tyrode’s solution supplemented with 5mM glucose for 10 minutes, supernatants were discarded, and the pellets were resuspended in 150μL of
supplemented Tyrode’s solution and read in the flow cytometer. Cells were analyzed in a FACSCanto II cytometer and gated, according to FSC and SSC morphology of live cells. We recorded at least 10,000 events discarding doublets by FSC-A/FSC-W. All data was recorded uncompensated and analyzed in an external software. MFI from the PE (FL2) channel of the population was compared against the treated samples and controls.
Toxicity Studies of GO and rGO on Hypertrophic Cells
H9c2 cells were seeded in 96-well plates at a concentration of 4 × 103 cells per well in 100 µl of DMEM media supplemented 10% FBS. Subsequently, hypertrophy was induced on the myocytes, as previously described. Briefly, the culture media was removed after 24 hours and substituted for cell culture media supplemented with 1% FBS and maintained for 24 hours. The next day, 1 µl of Ang II (1mM) was added to each well. 24-hours later, the Ang II stimulus the cell culture media was replaced with 100 µl of nanoparticle solutions in 1% FBS supplemented
DMEM at varying concentrations supplemented with µl of Ang II (1mM) as added. The
nanoparticle treatments were incubated for 24 hours, as aforementioned. After the allotted time, the nanoparticle solutions were removed, each well washed 2-3 times with 100 µl of PBS
solution, and the cells were resuspended in 100 µl of 1% FBS supplemented DMEM. Lastly, cell viability was measured using alamarBlue™.
Data Analysis for Viability Assays
The data obtained for the fluorometer was analyzed in Microsoft Excel, firstly averaging the values obtained for the “Blank” wells. The “Blank” value obtained was then deducted from all the other measured values. The rest of the values were averaged, respectively, and the “Ctrl +” group was taken as the 100% viability. Each group was normalized against the “Ctrl +” group average using the following equation:
The final value obtained represents the percentage of living cells per group. The data was analyzed in GraphPad Prism to obtain the half inhibitory concentrations, and nonlinear regressions were adjusted to the data.
Statistical Analysis
Independent results are presented as mean ± standard error (SE). Viability slopes and half inhibitory concentrations were obtained using nonlinear regression analysis. A curve fitted to the log of the concentrations (dependent variable) and the viability reported, the data was normalized to the maximum viability reported for the positive control group (“Ctrl +”). To determine
significant differences were determined using one-tailed t-tests were performed for two data set
comparisons and one-way ANOVA with Tukey posthoc test for multiple data set comparisons.
Adjusted p-value <0.01 (*), p-value <0.005 (**) and p-value <0.0001 (***) were considered significant.
4. Results and Discussion
4.1 Physicochemical characterization of GO and rGO
GO powder was synthesized through the Tour’s method (Marcano et al., 2010). This method uses graphite as the carbon source and KMnO4 as an oxidizing agent. The as-obtained GO material is a dark brown powder that visibly forms aggregates non-soluble in water due to the hydrophobic nature of graphite layers not oxidized so far. Such GO aggregates were observed by AFM (see Figure 4.1-a). The topographic images were obtained using the tapping mode. The size of GO aggregates is above 500 nm in length and width. However, the measured thickness was observed to be slightly smaller than 20 nm, as a result of a high grade of
disorganized stacking of GO sheets.
Mechanical exfoliation in GO aggregates were applied to obtain GO sheets by a constant intercalation of ethanol molecules in between individual layers. The separation of layers during the exfoliation process arises from overpassing the van der Waals forces in graphite layers (Chiou et al., 2018). SEM images monitored the changes in the morphology of GO before and after the exfoliation process. While opaque lamellar structures were observed before exfoliation (see Figure 4.1-b), the micrographs post-exfoliation resulted in a significant decrease in the GO’s size, indicating the peeling of layers (see Figures 4.1-d).
A change in the particle’s hydrodynamic diameter was also observed as a result of the exfoliation process. GO powder was measured in about 1180 nm, while GO sheets showed to be made up of two sized population, namely, population sizes in about 494 nm and 771 nm (Table 4.2). The smaller particles (Figure 4.1-d) were observed to remain in a stable colloidal solution
for extended periods. Henceforth, it was decided to use this particle population to favor the solubility in physiological media and application in cells.
Figure 4.1. Characterization of GO before and after exfoliation. (a) Atomic force microscopy topographic map of the as-synthesized GO powder following the synthesis reaction. (b) FE-SEM characterization of GO powder showing a typical agglomerate (scale bar, 1 µm); the dispersion of these poorly solubilized aggregates are shown in the vial containing GO powder. (c) FE-SEM characterization of GO sheets after mechanical exfoliation (scale bar, 1 µm) and the
corresponding dispersion of these solubilized sheets that still contain several aggregates. (d) FE- SEM characterization of GO sheets after mechanical exfoliation (scale bar, 100 nm), and the dispersion is showing a transparent solution.
Table 4.2. Separation of GO nanoparticles populations after exfoliation Before Exfoliation After Exfoliation Entire population size:
~1182 nm
Population 1: ~449 nm Population 2: ~771 nm
rGO was synthesized hydrothermally by the chemical reduction of GO using ascorbic acid as a reducing agent due to its non-toxic nature to the environment and biological systems.
The importance of this green method for rGO synthesis relies on the fact that toxic byproducts are not formed to interfere in the viability assays (Khosroshahi et al., 2018).
Both GO sheets to rGO nanoparticles were analyzed by UV-vis spectroscopy. The spectrum for GO (Figure 4.2-a) showed a typical peak at 230 nm (Lai et al., 2012) This peak represents the summation of energy that require chromophores in the surface of the GO sheets (e.g., C=C, C=O, and C–O) to do the transition from p-bonding orbital (HOMO) up to p*
antibonding orbital (LUMO) (Huang et al., 2011). In the case of rGO, the peak is shifted from 230 nm to 260 nm due to the partial removal of oxygen functional groups which reconstitutes C=C bonds (Abid et al., 2018). The FTIR spectra show vibrations from the main functional groups in GO and rGO samples. From the spectrum of GO, there are observed peaks located at 1723 cm-1, 1586 cm-1, and 1250 cm-1, for C=O, C=C, and C-O bonds, respectively (Figure 4.2-b).
The spectrum for rGO showed a decrease in the number of bands (Guex et al., 2017), but the vibrations for C=C bonds were observed to increase substantially due to the increased formation of carbon bonds after the partial reduction of GO.
The obtained Raman spectra for GO and rGO is shown in Figure 4.2-c. There is observed the typical bands D and G representative for graphenic materials as reported elsewhere (Hidayah et al., 2017). The G band (or graphitic band) is located at 1580 cm-1, which results from in-plane vibrations (stretching) of the carbon atoms in the sp2 plane. While, the D band (or disordered band) represents the out-of-plane vibrations that are related to the structural defects on sp2- hybridized carbon. The higher D band in the GO correlates to the disruption of sp2 bonds as a result of the oxygen functional groups attached to the surface, which results in increased sp3 bonds (Mkhoyan et al., 2009). The ratio of the intensities of the D and G band characterize the disorder of the GO and rGO sheets. The decreased D/G band ratio in rGO is known to result
from the reduction in the domain size present in the carbon structures (Roscher et al., 2019).
Similar peak intensities in the D and G bands in rGO also indicate structural defects by the presence of point dislocations (Muzyka et al., 2018). Moreover, the difference in the intensity of the peaks observed suggests that GO is thicker, meaning that these nanoparticles are more agglomerated than rGO.
Finally, the X-ray diffraction patterns of GO and rGO are shown in Figure 4.2-d. For GO, there is observed a broad peak at 11.8 – 16.0 (2θ°) typical for amorphous materials. The XRD pattern from rGO closely resembles previous diffractograms reported for graphene (Mishra et al., 2014), suggesting the restoration of the aromatic rings after the reduction with ascorbic acid. In the two cases, the peak broadening observed is due to the presence of nano-size crystallite domains in the particles as well as micro tensions due to deformation in the crystalline lattice. It is well-known that sulfur used in the reduction process is thought to promote these defects (Hayes et ail., 2014).
Figure 4.2. Chemical characterization of GO (red lines) and rGO (green lines). (a) UV-vis absorbance spectra. (b) ATR-FTIR transmittance spectra; primary changes in the functional groups are pointed out. (c) Raman characterization with and without baseline correction and smoothing for rGO. (d) X-ray diffraction of these materials.
For XPS measurements, an ion etching pre-treatment was used to provide the inner composition of the sample. Ion etching is typically recommended when analyzing graphenic samples (Robinson et al., 2013); this delivers results that are more accurate given that
environmental oxygen can be physisorbed, thus oxidizing the outer layers of the particles. The data displays the major elemental compositions present on the GO and rGO. The two significant peaks present in Figure 4.3 correspond to the carbon and oxygen in the samples; a quantification of the elements revealed the reduction with ascorbic acid caused an increase in carbon of 7.1%
and a decrease in the oxygen of 8.2%. Additionally, an extra peak is observed in the rGO
nanoparticles, which corresponds to nitrogen as an impurity from the synthesis. However, its amount in the composition is minimal and only represents 1.1%. A deconvolution of the peaks reported in Figure 4.3, using the binding energy reported for C=C and C=O bonds, reported these bonds constitute 68.9% and 25.1%, respectively, of all the bonds present in the GO sample. The subsequent reduction, with ascorbic acid, increased the C=C to 73.5%, and the C=O decreased to 24.0%. These results are consistent with the chemical structures of these materials in which the green hydrothermal synthesis of rGO was noted to reduce the oxygen content significantly.
Figure 4.3. XPS spectra of GO (left) and rGO (right).
Table 4.3. Chemical composition of GO obtained by XPS analysis with argon ion etching.
Element/Bond Pos. FWHM Area At %
C 285.338 3.05969 691.8 78.44
O 532.338 4.07219 444.0 21.56
C = O 531.93 1.86 25.10 4.19
C = C 284.78 1.44 68.90 2.85
Table 4.4. Chemical composition of rGO obtained by XPS analysis with argon ion etching.
Element/Bond Pos. FWHM Area At %
C 285.405 2.60364 784.1 85.52
O 532.405 3.20088 285.0 13.32
N 400.405 2.0379 17.0 1.16
C = O 531.96 1.48 24.02 6.06
4.2 Toxicity analysis of GO and rGO in cardiomyocytes
Here we provide a comparative study of the toxic effects of GO and rGO in H9c2 cardiomyoblasts, which is a well-known in vitro model used to test myocardial responses (Zordoky et al., 2007).
The cytotoxicity of GO and rGO was assessed using alamarBlue™. A dose-dependent toxicity is shown in Figure 4.4. The results indicate that after 24-hour exposure to either GO or rGO, these two nanomaterials induce cell death as observed by a decrease of metabolic activity.
The half-inhibitory concentration of GO (676.0 ± 80.3 ppm) and rGO (152.9 ± 40.1 ppm) is shown in Table 4.5. According to these results, rGO induces toxicity on H9c2 cells in smaller concentrations of about five-fold half-inhibitory concentration in comparison with GO.
Figure 4.4. Viability percentage of H9c2 cells after 24-hour exposure to different GO and rGO nanomaterials concentrations. Values represent the mean +/- SE; viability is normalized to the control mean, n=5 independent experiments.
Table 4.5. Half-inhibitory concentrations (IC50) of GO and rGO after 24-hour incubation with cardiomyocytes. Values represent the mean +/- SE, n=5 experiment.
Nanomaterials IC50
GO 152.9 ± 40.1 ppm rGO 676.0 ± 80.3 ppm ***
*** P <0.0001 vs control, T-test.
Time-dependent viability assays of cardiomyoblasts were conducted using the half inhibitory concentration determined for GO. After the treatment, most of the damage is seen to occur within the first 12 hours of exposure (see Figure 4.5). It was observed that a half-inhibitory time occurs at 28.28 ± 4.05 hours. A continuous decrease in the metabolic activity was observed with increased exposure time that reaches about 90% of death by 72 hours. These results indicate that GO induces toxicity in a time-dependent manner, in which half of the cardiomyocyte
population is expected to die about 28-hours post-incubation with GO (700 ppm). These time- dependent viability assays results follow similar observation as previous in vivo studies (Souza et al., 2018), where chronic exposure of GO resulted in increased mortality rates in Ceriodaphnia dubia.
Figure 4.5. Nonlinear regression of the toxicity of GO (700 ppm) at different exposure times.
Viability is normalized to the control mean. Values represent the mean +/- SE. n=3 independent experiments.
Carbon-based nanomaterials such as GO and rGO have gained attention in the biomedical field for applications in nano constructs to improve the functioning of the heart. Previous studies reported by our group arrived at similar conclusions, in which GO is comparatively a less toxic nanomaterial than rGO. This study (Contreras-Torres et al., 2017) suggested that the difference in cytotoxicity can be correlated to the material’s internalization, as it was observed that rGO could internalize in a more significant proportion, according to flow cytometry data. The increased cytotoxicity observed for rGO was attributed to its physicochemical characteristics, including the level of oxidation and the presence of graphitic domains. Another study (Xing et al., 2016) observed that after coating the surface of rGO with biomolecules lead to a significant decrease in the toxicity in Catla Catla cardiac cells.
The toxicity of GO in cardiac cells was assessed through the kinetics of ROS production (Arbo et al., 2019) A 24-hour exposure of GO on cardiac myocytes was seen to cause an increase in the generation of ROS in a dose-dependent manner. The integrity of mitochondrial
mechanisms was evaluated through measurements of the mitochondrial membrane potential
resulting in mitochondrial hyperpolarization (Arbo et al., 2019). This phenomenon is known to be associated with disruptions in the ATP synthase, preventing the proton conductance across the electrochemical gradient, and thus, the generation of ATP. This study also demonstrated that the hyperpolarization of the mitochondria increases ROS production, further damaging in mouse hearts (Mourier et al., 2014). These observations concomitantly imply the mitochondria is a significant source of damage in the toxicity mechanisms of GO. It was observed that GO acts as an electron donor in complex I of the electron transport chain (ETC), causing its overstimulation and subsequently an increase in the production of ROS, as previously shown in macrophages (Duch et al., 2011). GO also demonstrated to induce mitochondrial alterations by affecting canonical pathways of the oxidative phosphorylation, which resulted in its downregulation (Ghanbari et al., 2017) The downregulation in the oxidative phosphorylation process correlates with a hyperpolarization of the mitochondrial membrane, followed by a decrease in the ATP production. It is proposed the overstimulation of ETC and the downregulation of the oxidative phosphorylation act simultaneously. The functioning of the ETC generates a proton gradient that cannot be consumed in the process of oxidative phosphorylation as a result of its
downregulation; thus, the accumulation of protons in the intermembrane space elevates the mitochondrial membrane potential. A prolonged hyperpolarization of mitochondria is known to sensitize myocytes to necrosis due to a lack of ATP (Eguchi et al., 1997).
The excess of ROS leads to oxidative stress resulting in an impaired balance between ROS and antioxidants. The set of reactions that comprise oxidative stress result in the
detrimental modification of proteins, lipids, and nucleic acids. The latter was confirmed by genotoxicity assays showing that GO causes breaks in the DNA of cardiac myocytes (Arbo et al., 2019), and inducing death by apoptosis or necrosis. In this regard, cytotoxicity studies analyzing
cellular death pathways of GO (and rGO), mitochondrial respiration, and production of ATP in cardiac cells are needed to confirm the hypothesis.
4.3 Toxicity analysis of GO and rGO in hypertrophic cardiomyocytes
Previous reports (Ruiz-esparza et al., 2016) suggest that the toxicity of nanoparticles could be intensified in conditions of cardiac damage. To test this hypothesis, in this study, we replicated a model of heart disease using cardiomyocytes treated with Ang II. Treatments with Ang II (1mM) demonstrated to develop hypertrophy after 48-hours. In particular, confocal micrographs show a significant increase in the relative surface area of the myocytes (Figure 4.6- A). These morphological observations indicate an irregular round shape in the Ang II-stimulated cells, indicative of hypertrophy.
Figure 4.6. Characterization of Ang II-induced hypertrophy in cardiomyocytes. A)
Representative confocal images of calcein stained for control H9c2 cells (left) and H9c2 cells treated with Ang (right) (1 µM, 48 h); scale bar represents 25 µm. B) Dispersion of the measured cell area. Values represent the mean +/- SE, N=2 experiment,>1.0 x 10³cells analyzed per group.
*** P <0.0001 vs control, t-test.
Figure 4.7 shows a 48-hour treatment with Ang II stimulated the overproduction of ROS in cardiomyocytes mitochondria, which is consistent with previous results (Tian et al., 2018) (Figure 4.7-A). Tian et al. observed that the upregulation of ROS was primarily mediated via the AT2 receptor. Consequently, this induces the activation of NADPH, which generates O− and
inflammation (Chetsawang et al., 2010). Additionally, Ang II can trigger apoptosis via the mitochondrial-dependent pathway. This is scribed to elevated concentrations of cytochrome c as a result of the opening of the mitochondrial transition pore (Ou et al., 2016). However, even though an increase in cell size and overproduction of ROS was observed, no significant changes were found in the viability of the cardiomyoblasts after treatment with Ang II (Figure 4.7-B)
Figure 4.7. Effects of Ang II on H9c2 cells. A)Reactive oxygen species production in the mitochondria of hypertrophic cardiomyocytes as measured with MitoSOX. n = 4 experiments.
*P <0.01 vs control, t-test. B) Viability of cardiomyoblast after treatment with Ang II.
Ang II, a neurohumoral agent, is known to act by activating the AT1 receptor that couple to Gq protein intracellularly and activates phospholipase C. This activation triggers the release of Ca2+ from the sarcoplasmic reticulum via the IP3 receptor. The increased Ca2+ content in the cytoplasm leads to the activation of calcineurin and, subsequently, the induction of inflammatory cytokines and transcription factors that prompt hypertrophy and inflammation (Sadoshima et al., 1993)
Studies of GO and rGO nanoparticles in hypertrophic cardiomyocytes elucidated an increase in the toxicity of GO, however, the toxicity of rGO did not seem to be significantly affected (Figure 4.6, thus making GO more toxic to hypertrophic cells. This increased toxicity of
GO is thought to be a result of the sensitization of the myocytes caused by the detrimental effects of Ang II. The precursory damage, in consequence, could predispose these cells to have a
heightened GO nanoparticle toxicity because of the accumulation of detrimental events. In particular, the accumulation of ROS generated by nanoparticles and Ang II, can generate an excessive pro-oxidative environment that can lead the cell to undergo mechanistic impairments and death more readily. Additionally, previous studies have associated the toxicity of GO in healthy cells with being limited to a low internalization. This dramatic increase in the toxicity of GO could also be related to an increased permeability in the plasma membrane, caused by Ang II, as described elsewhere (Gómez et al., 2018). The peroxidation, peroxidation of lipids, influenced by the increase in ROS, is known to further impact permeability of the membrane (Catalá et al., 2016)
Interestingly, the toxicity of rGO was not affected in hypertrophic cells, while the toxicity of GO was seen to increase fifty-fold (Figure 4.8). Here, we propose the redox environment present in the mitochondria could reduce GO (Hu et al., 2008)., thus generating a reduced form of GO, comparable to the rGO that we synthesized This reasoning would explain why the
toxicity of GO and rGO in the presence of hypertrophic conditions are not significantly different.
(Figure 4.9).
Altogether, these analyses assure the toxicity of GO and rGO in myocardial cells. Results also confirm the hypothesis that GO and rGO are more toxic in cells with previous damage. This suggests that vulnerable populations with a predisposition to cardiac damage may be at higher risk of nanoparticle-induced toxicity. In this account, the regulation of these particles should be considered carefully to prevent future adverse health consequences.
Figure 4.8. Toxicity of GO and rGO on hypertrophic cardiomyocytes at 24 hrs. Nonlinear regression of dose-dependent toxicity of hypertrophic cardiomyocytes. Viability normalized to the control mean. Values represent the mean +/- SE. n=4 independent experiments.
Table 4.6. Half inhibitory concentrations (IC50) of graphene oxide and reduced graphene oxide after 24-hour incubation with hypertrophic cardiomyocytes. Values represent the mean +/- SE, n=5 experiment;
Nanomaterials IC50
GO 12.60 ±10.7 ppm
rGO 86.33.0±12.9 ppm ***
*** P <0.0001 vs control, t-test.
Figure 4.9. Half-inhibitory concentration values of GO and rGO after 24-hour incubation in healthy and hypertrophic cardiomyocytes. Values represent the mean +/- SE; ** P <0.005 vs control; *** P <0.0001 vs control, one-way ANOVA with posthoc Tukey.
5. Conclusion
In summary, the synthesis and an in-depth physicochemical characterization of graphene oxide and reduced graphene oxide nanoparticles facilitates their molecular comparison and serves as a guide to give an account of their differences in toxicity. We demonstrated that these nanomaterials significantly decrease the metabolic activity in control and hypertrophic
cardiomyocytes in a dose-dependent manner. We also observed that the toxicity of graphene oxide Ang II-induced hypertrophic cardiomyocytes was exacerbated 50-fold, possibly as a result of increased internalization; while the toxicity of rGO was seen to remain unchanged.
Possible mechanisms of toxicity regard the mitochondria as a central source of damage can induce the overproduction ROS, and thus stimulates detrimental mechanisms to lead to cellular damage. Further investigation must be done to elucidate mechanisms of action with precision.
6. Perspectives
We acknowledge additional studies that could complement the results obtained in this project could provide strong evidence for the mechanisms of toxicity. However, in accordance with the regulations imposed with the contingency caused by the COVID pandemic,
experimental procedures were halted without previous notice. Thus, all of the proposed
objectives could not be met. Nevertheless, to the best of our efforts, mechanisms were proposed complementing them with the literature available in the subject.
Other studies that could enrich and give more significance to the project include assays evaluating the type of death of cellular death, which is essential to backtrack sources of the cell damage. Association studies from flow cytometry and confirmation of internalization via TEM could also provide significant knowledge about knowing if the toxicity is related to the amount of material entering the cell. TEM analysis can also illustrate if the material is physically damaging the cell.
Differential analysis of the production of ROS in the mitochondria and the cytosol could be an excellent roadmap to determine if oxidative stress is involved in the toxicity of the
materials. Studies involving the functioning of the different complexes of the electron transport chain, the mitochondrial membrane potential, and essays on the production of ATP could add information on the state of the mitochondria. Analysis of an increase in lipid peroxidation could also indicate if the membranes of the cell are being damaged. Quantitative analysis of the
expression of proinflammatory biomolecules and remodeling biomarkers could be increasing the hypertrophy or using the same pathway as Ang II. It is proposed, these studies in conjunction could serve as a guide to the toxicity of GO and rGO.
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