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. 2011 Spring;16(1):6–10.

Efficacy of Pt-modified TiO2 nanoparticles in cardiac cells

Adiel Mallik 1, Sean Bryan 1, Stephanie Puukila 1, Aicheng Chen 2, Neelam Khaper 3,✉
PMCID: PMC3076159  PMID: 21523200

Abstract

BACKGROUND:

The application of TiO2 nanoparticles promises to revolutionize cardiac imaging and targeted medical treatment.

METHODS:

A novel type of platinum-modified TiO2 (Pt-TiO2) nanoparticle was synthesized and characterized. Commercially available P25 TiO2 nanoparticles were used for comparison. Cellular toxicity and its mechanisms were evaluated by analyzing nanoparticle uptake, oxidative stress and mitochondrial membrane potential in rat cardiac (H9c2) cells.

RESULTS:

There was greater cellular uptake of Pt-TiO2. Furthermore, Pt-TiO2 caused a greater increase in oxidative stress and greater decrease in mitochondrial membrane potential. These data suggest that Pt modification of TiO2 nanoparticles rendered them more cytotoxic than their commercial counterparts at lower, more physiologically relevant concentrations.

CONCLUSION:

Despite the functional advantages of Pt modification, which results in increased uptake at a lower concentration, the corresponding increase in cardiotoxic effect indicates that a thoughtful, cautious approach to cardiac nanotechnologies is required.

Keywords: Nanoparticles, Oxidative stress, Platinum, Titanium dioxide, Toxicology

The potential for the medical application of nanotechnologies is enormous, particularly in the areas of cardiovascular imaging and treatment of cardiovascular diseases (1). Nanoparticles offer novel modalities for targeted drug delivery to the cardiovascular system. Indeed, a recent study (2) demonstrated the use of tissue factor-targeted nanoparticles containing doxorubicin in targeting vascular smooth muscle cells to abrogate restenosis in an angioplasty model. TiO2 and modified TiO2 nanomaterials have great potential as drug delivery and molecular imaging vehicles in the cardiovascular system due to their high photocatalytic activity (3,4). TiO2 nanoparticles are also currently being used as bactericidal agents in artificial heart valves (5). However, studies have shown that TiO2 nanoparticles can be cytotoxic (6) – a feature that is likely due to the different physical and chemical properties of TiO2 nanoparticles versus their bulk counterparts.

Recent studies have shown that the smaller size and greater surface area of TiO2 nanoparticles result in increased toxicity compared with their micrometre-sized counterparts (7,8). In addition, surface properties such as crystal phase have also been shown to have an impact on toxicity (9). In the case of TiO2 nanoparticles, the most toxic crystal phase is amorphous, followed by the anatase and rutile phases (9). The reason for this toxicity may be the generation of reactive oxygen species (ROS) because the amorphous and anatase crystal phases were found to generate more ROS ex vivo (10).

Given the widespread use of anatase-based TiO2 nanoparticles in food additives, sunscreens and drug additives, most toxicology studies have focused on commercially available P25 TiO2 nanoparticles. P25 TiO2 nanoparticles are 80% anatase and 20% rutile, 30 nm in diameter and photocatalytic. Recent in vivo studies (11,12) have confirmed that anatase-based TiO2 nanoparticles accumulate in the heart, liver and lungs. However, the results of in vitro toxicological studies vary based on the cell type investigated. For instance, in the case of Neuro-2A cells, TiO2 nanoparticles did not cause any change in viability at concentrations as high as 300 μg/mL (13). However, a concentration of 100 μg/mL was sufficient to cause a significant decrease in the viability of rat liver cells (14). The use of TiO2 nanoparticles was approved based on data in the material safety data sheets of the bulk form of TiO2 nanoparticles (15) despite no existing toxicology studies concerning the heart. Given the rapid pace of nanomaterial development, modifications to P25 TiO2 nanoparticles are being widely explored. One such modification involves adding a thin layer of platinum (Pt) to the surface of the P25 TiO2 nanoparticle. Recent studies have shown that TiO2 nanoparticles modified with Pt (Pt-TiO2) exhibit higher photocatalytic activity for several chemical reactions, leading to their candidacy for use in molecular imaging and drug delivery (16–20). Despite their promise and, to the best of our knowledge, the efficacy of Pt-TiO2 nanoparticles has not been characterized in any in vitro or in vivo system, including the heart.

In the present study, we report on the heretofore unknown efficacy of TiO2 and Pt-TiO2 nanoparticles in rat cardiac (H9c2) cells. P25, one of the best commercially available TiO2 photocatalysts, was chosen due to its widespread use (21). The Pt-TiO2 nanoparticles were prepared using a photoassisted reduction method. The physicochemical properties of the TiO2 and Pt-TiO2 nanoparticles were characterized using atomic force microscopy (AFM), x-ray diffraction (XRD) and Brunauer-Emmett-Teller (BET) surface area measurements. We systematically investigated nanoparticle uptake by H9c2 cells and the mechanism(s) and extent of nanoparticle-induced cell damage, including the involvement of oxidative stress and mitochondrial damage.

METHOD

Synthesis and characterization of Pt-TiO2 nanoparticles

A commercially available TiO2 powder (P-25, Degussa AG, Germany) containing approximately 80% anatase phase and 20% rutile phase was used as a standard in the present study. To prepare rutile phase-dominated TiO2 nanoparticles, P25 was heated at 750°C in air for 3 h. The Pt-TiO2 nanoparticles were prepared using a photoassisted reduction method wherein P25 (50 mg) was added to CH3OH (50% by volume [5 mL]) containing 5.0×10−4 M H2PtCl6. After purging with pure Ar for 30 min, the mixture was irradiated with ultraviolet light (Oriel Systems Ltd, UK) equipped with a 300 W xenon arc lamp for 4 h. The Pt-modified P25 was separated using a high-speed Sorvall Biofuge Stratos centrifuge (Thermo Fisher Scientific Inc, Canada). The unmodified P25, the heated P25 and the Pt-TiO2 were characterized by XRD (PW 1050-3710 diffractometer [Philips Healthcare, USA] with Cu-K alpha radiation) and AFM (Agilent Technologies Canada Inc). Their specific surface areas were determined using a Nova 2200 BET meter (Quantachrome Instruments, USA) by nitrogen adsorption/desorption at the temperature of liquid nitrogen.

Cell culture

Rat cardiac (H9c2) cells were obtained from the American Type Culture Collection (Virginia, USA) and cultured in 75 cm3 and 150 cm3 sterile cell culture flasks as per the recommended protocol: Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, USA) supplemented with 10% fetal calf serum (Hyclone, USA) and 1% penicillin-streptomycin (Invitrogen, USA), and incubated at 37°C at 5% CO2 and 100% humidity. Cells were seeded at 3×105 cells/cm2 of growth area in the appropriate volume of medium and grown for 24 h before nanoparticle exposure.

Nanoparticle treatment and exposure

Fresh nanoparticle stock solutions were prepared weekly to ensure consistency of the nanoparticles’ physical and chemical properties (22). The nanoparticle stock solutions were sonicated for 1 min in a 400 W sonicating bath (Sonic Dismembranator, Fisher Scientific, USA) before preparing the final working nanoparticle dispersions in DMEM. Nanoparticle dispersions were prepared immediately before use and were vortexed thoroughly before being added to the experimental cell cultures. Cells were treated with different concentrations of P25 TiO2 (10 μg/mL, 30 μg/mL and 100 μg/mL) and Pt-TiO2 (5 μg/mL, 30 μg/mL and 50 μg/mL) nanoparticles for a period of 24 h. Each experiment was repeated three times with two replicates per experiment.

Assessment of cell viability

H9c2 cells were seeded at a density of 3×104 cells/cm2 into sterile, flat-bottom, six-well plates 24 h before treatment. After a 24 h nanoparticle exposure period, the viability of the cells was assessed using the Vi-CELL automated cell viability analyzer (Beckman Coulter Inc, USA). Control samples consisting of cell-free nanoparticles and cell-free media were used to ensure that particle-dye interactions did not affect the data.

Assessment of cellular nanoparticle uptake

Cells were treated with different concentrations of P25 TiO2 (10 μg/mL, 30 μg/mL and 100 μg/mL) and Pt-TiO2 (5 μg/mL, 30 μg/mL and 50 μg/mL) nanoparticles for a period of 24 h. Cells were then washed with phosphate-buffered saline (PBS), trypsinized, suspended in medium, washed again with PBS and resuspended in PBS. Using a FACSCalibur flow cytometer (Becton Dickinson Biosciences, USA), the level of cellular nanoparticle uptake was assessed via the side scatter (SSC) light pattern generated by the laser beam’s (488 nm) passage through the cell. SSC is related to the internal granularity, complexity and density of a given particle, and cellular uptake has previously been evaluated in this manner (23). In that study, sectional scanning was performed using confocal laser scanning microscopy, which showed that the nanoparticles were internalized to the cytoplasm and not merely attached to the surface of the cell membrane (23). This demonstrated that the SSC light pattern generated by the flow cytometer is a valid measure of intracellular nanoparticle internalization.

Oxidative stress assay

Nanoparticle-induced oxidative stress was quantified using the CM-H2DCFDA assay (Molecular Probes, USA), a cell-permeant indicator of ROS. After 24 h of nanoparticle treatment, the cells were washed with PBS and incubated for 30 min with CM-H2DCFDA according to the manufacturer’s instructions. The CM-H2DCFDA was then aspirated and the cells were washed with PBS. The cells were removed from the culture dish by careful scraping, and the relative fluorescence of untreated and nanoparticle-treated cells was measured using flow cytometry as per the manufacturer’s instructions.

Mitochondrial membrane potential assay

Nanoparticle-induced changes in mitochondrial membrane potential were quantified using the MitoProbe JC-1 assay (Molecular Probes, USA). To measure mitochondrial membrane potential, sterile 25 cm3 cell culture flasks were seeded with H9c2 cells at a density of 3×105 cells/cm2. After 24 h, the cells were treated with P25 TiO2 or Pt-TiO2 nanoparticles and incubated for a further 24 h. Following treatment, the cells were washed with PBS, trypsinized, collected and quantified using the Vi-CELL cell viability analyzer (Beckman Coulter Inc, USA). Following quantification, 1×106 cells from each treatment were resuspended in 1 mL of DMEM warmed to 37°C. The JC-1 dye was prepared and added to each treatment tube according to the manufacturer’s instructions, and the treatment tubes were incubated in the dark for 30 min (at 37°C and 5% CO2). The cells were centrifuged at 500 g for 5 min and resuspended in 1 mL of PBS. The cells were analyzed via flow cytometry (FACSCalibur flow cytometer) as per the manufacturer’s instructions.

Statistical analysis

Data are presented as mean ± SD. Each experiment was repeated three times, with two replicates per experiment. A one-way ANOVA test was used to analyze the statistical significance of the data (SigmaPlot 11 [Systat Software Inc, USA] for Windows [Microsoft Corporation, USA]). In cases in which data passed the normality test, the Holm-Sidak method for pairwise comparisons was used. In cases in which the data failed to pass the normality test, the Student-Neuman Keuls test for pairwise comparisons was used. Differences were considered to be statistically significant at P<0.05.

RESULTS

AFM images of the TiO2 and Pt-TiO2 sample revealed that the particle size was approximately 30 nm (data not shown). Figure 1A displays the XRD patterns of the P25 TiO2 nanoparticles and Pt-TiO2 nanoparticles. As expected, both P25 TiO2 and Pt-TiO2 nanoparticles were primarily anatase phase. It is interesting to note that the XRD patterns of the TiO2 and Pt-TiO2 nanoparticles were almost identical; no obvious Pt peaks were observed. This is consistent with our AFM study, in which no differences in TiO2 particle size were observed following Pt modification. In contrast, the colour of the particles differed (Figures 1B and 1C), wherein the colour of the TiO2 nanoparticles changed from white to black on Pt modification. Similarly, the BET surface area measurements also differentiated the Pt-TiO2 from the P25 TiO2 nanoparticles because the Pt-TiO2 nanoparticles featured a slightly smaller surface area (45 m2/g versus 56.4 m2/g).

Figure 1).

Figure 1)

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A X-ray diffraction patterns of (a) P25 TiO2 nanoparticles and (b) platinum-modified TiO2 nanoparticles. B Photograph of P25TiO2 nanoparticles. C Photograph of platinum-modified TiO2 nanoparticles. a.u. Arbitrary units

The determination of nanoparticle-induced cytotoxicity was evaluated with a trypan blue exclusion assay using the Vi-CELL automated cell viability analyzer. This assay distinguishes viable from nonviable cells according to membrane integrity because necrotic and late-apoptotic cells feature compromised, trypan blue dye-permeable membranes. Based on initial cell viability studies, the maximal concentration of the Pt-TiO2 was halved to 50 μg/mL due to excessive cell death (data not shown) and to achieve toxicological equivalency to the 100 μg/mL concentration of the TiO2 nanoparticle. For both the P25 TiO2 and Pt-TiO2 nanoparticles, there were significant concentration-dependent decreases in viability following 24 h nanoparticle treatment. Interestingly, the relatively low concentration of P25 TiO2 (10 μg/mL) did not greatly affect cell viability, while the higher concentrations (30 μg/mL and 100 μg/mL) significantly decreased cell viability (Figure 2). Similarly, relatively low concentrations of Pt-TiO2 (5 μg/mL and 30 μg/mL) did not cause nearly the reduction in cell viability as the 50 μg/mL concentration (Figure 2).

Figure 2).

Figure 2)

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Trypan blue exclusion assay showing cell viability after 24 h treatment with P25 TiO2 and platinum-modified TiO2 (Pt-TiO2) nanoparticles. *Significant difference from the control treatment (P<0.05); †Significant difference from the P25 TiO2 10 μg/mL treatment (P<0.05); ‡Significant difference from the P25 TiO2 30 μg/mL treatment (P<0.05); §Significant difference from the Pt-TiO2 5 μg/mL treatment (P<0.05)

The degree of P25 TiO2 and Pt-TiO2 nanoparticle uptake by H9c2 cells was determined via flow cytometric SSC analysis (Figure 3). Cellular nanoparticle uptake was scaled with increasing concentrations for both nanoparticle types. However, after controlling for the concentration difference between the high-concentration Pt-TiO2 and P25 TiO2 (50 μg/mL versus 100 μg/mL, respectively), the Pt-TiO2 was more readily incorporated by the cells in terms of absolute particles per cell (Figure 3).

Figure 3).

Figure 3)

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A Flow cytometry side scatter analysis indicating the amount of nanoparticle uptake in H9c2 cells after 24 h treatment with P25 TiO2 and platinum-modified TiO2 (Pt-TiO2) nanoparticles. *Significant difference from the control treatment (P<0.05); †Significant difference from the P25 TiO2 10 μg/mL treatment (P<0.05); ‡Significant difference from the P25 TiO2 30 μg/mL treatment (P<0.05); §Significant difference from the Pt-TiO2 5 μg/mL treatment; Significant difference from the Pt-TiO2 30 μg/mL treatment (P<0.05). a.u. Arbitrary units. B to D Representative dot plots showing cell population of control, P25 TiO2 (30 μg/mL) and Pt-TiO2 (30 μg/mL) treatments, respectively. FSC-H Height of forward scatter; SSC-H Height of side scatter

Oxidative stress is known to cause DNA damage, lipid peroxidation and protein damage which, if not effectively countered by cellular antioxidant defenses, can lead to cell death (24). The CM-H2DCFDA fluorescent dye assay is widely used to determine cellular ROS levels, in which the relative level of fluorescence corresponds to the relative level of ROS present within the cell (25). It was found that treatment with P25 TiO2 and Pt-TiO2 nanoparticles each resulted in increased ROS generation (Figure 4). In P25 TiO2 nanoparticle-treated cells, a concentration-dependent increase in ROS production up to and including the 30 μg/mL concentration was observed. In contrast, in Pt-TiO2 nanoparticle-treated cells, concentration-dependent increases in ROS production were observed up to and including the highest concentration (50 μg/mL). Moreover, treatment with the Pt-TiO2 nanoparticles resulted in higher absolute ROS levels than their P25 TiO2 counterparts (Figure 4).

Figure 4).

Figure 4)

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A CM-H2DCFDA assay indicating reactive oxygen species (ROS) levels in H9c2 cells after 24 h treatment with P25 TiO2 and platinum-modified TiO2 (Pt-TiO2) nanoparticles. *Significant difference from the control treatment (P<0.05). a.u. Arbitrary units. B to D Representative dot plots showing FL1 fluorescence versus forward scatter (FSC) of the cell population of control, P25 (30 μg/mL) and Pt-TiO2 (30 μg/mL) treatments. H Height

Given that mitochondria are a key site of ROS generation and are highly susceptible to redox imbalances, the mitochondrial membrane potential of nanoparticle-treated cells was investigated. Mitochondria require sufficient membrane potential to function and, thus, this feature can be used to assess mitochondrial activity. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria, and its concentration-dependent formation of red fluorescent J-aggregates causes a shift from green (monomeric form) to red (aggregated form) fluorescence emission. Mitochondrial permeability results from the collapse of the electrochemical gradient across the mitochondrial membrane during early cellular apoptosis and, thus, a low red/green fluorescence intensity ratio is indicative of a lower mitochondrial membrane potential (26).

In both the P25 TiO2 and Pt-TiO2 nanoparticle-treated cells, concentration-dependent decreases in mitochondrial membrane potential were observed (Figure 5). Approximately equivalent mitochondrial membrane potential levels were observed in the 10 μg/mL of P25 TiO2 and 5 μg/mL of Pt-TiO2 nanoparticle-treated samples (Figure 5). Taken together, the similarity in mitochondrial membrane potential despite the differences in absolute nanoparticle concentration indicates that Pt-TiO2 nanoparticles caused greater mitochondrial membrane damage than their P25 TiO2 counterparts.

Figure 5).

Figure 5)

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A JC-1 assay indicating mitochondrial membrane potential in H9c2 cells after 24 h treatment with P25 TiO2 and platinum-modified TiO2 (Pt-TiO2) nanoparticles. *Significant difference from the control treatment (P<0.05). B to D Representative dot plots showing FL2 versus FL1 fluorescence of the cell population of control, P25 (30 μg/mL) and Pt-TiO2 (30 μg/mL) treatments. H Height

DISCUSSION

These findings represent the first investigation of Pt-TiO2 nanoparticle toxicity in cardiac cells. Given the novel nature of the present study, characterization of the physicochemical properties of these nanoparticles was essential. Despite plainly visible colour differences between the P25 TiO2 and the Pt-TiO2 nanoparticles (Figures 1B and 1C), no obvious deviations in XRD (Figure 1A) were observed. This can be attributed to the very thin layer of Pt formed on the TiO2 nanoparticles during the photoassisted reduction process.

Our data support the hypothesis that Pt-TiO2 nanoparticles are more cytotoxic than their non-Pt-modified counterparts, as evidenced by the 50 μg/mL Pt-TiO2 causing similar decreases in cell viability to the much higher concentration of P25 TiO2 (100 μg/mL) (Figure 2). Moreover, this is further supported by our findings that the Pt-TiO2-treated cells exhibited higher particle uptake and ROS levels than the P25 TiO2-treated cells (Figures 3 and 4). Indeed, controlling for the concentration difference between the high concentration Pt-TiO2 and P25 TiO2 (50 μg/mL versus 100 μg/mL, respectively), the Pt-TiO2 was more readily incorporated by the cells (Figure 3). Given that the Pt-TiO2 were the same size as the P25 TiO2 nanoparticles and were of the same crystal phase (80% anatase and 20% rutile), it is clear that other factors were responsible for the observed differences in cellular nanoparticle uptake and toxicity. One potential factor was surface area – the P25 TiO2 nanoparticles featured a greater surface area than the Pt-TiO2 nanoparticles. However, in light of a previous study (23), this difference would suggest that the P25 TiO2 nanoparticles should have demonstrated higher cellular uptake, while the opposite was noted in the present study. Thus, we suggest that the surface chemistry of the Pt-TiO2 nanoparticles remains the most likely explanation for the observed differences.

A recent study showed that Pt-modified TiO2 material catalyzed the water-splitting reaction, producing hydrogen and hydroxyl radicals more effectively than P25 TiO2 (27). As has been suggested (28), it is possible that the Pt-TiO2 nanoparticles entered the cell and produced sufficiently increased ROS though catalysis of this reaction to compromise the cell membrane and allow further nanoparticle incorporation. Validation of this mechanism will require further directed study, and could prove to be a significant advance in our basic understanding of nanoparticle biocompatibility.

It was found that ROS generation was increased in P25 TiO2 nanoparticle-treated cells in a concentration-dependent manner up to and including 30 μg/mL, but a clear decrease in ROS was evident at a concentration of 100 μg/mL (Figure 4). This decrease was likely due to the decrease in cell viability that occurred after treatment with 100 μg/mL P25 TiO2 (Figure 2), resulting in fewer viable cells being present in which to measure ROS levels. Importantly, it was also found that Pt-TiO2 nanoparticles generated much higher levels of ROS than did P25 nanoparticles (Figure 4). This may be due to Pt-TiO2 nanoparticles having been shown to more effectively catalyze the electrolysis reaction than P25 nanoparticles, as well as being more effective oxidizers of nitric oxide (NO) species, phenol and acridine orange (17,18,20,29). Indeed, the presence of phenol red in the experimental cell culture media and the fact that NO species are an important class of signalling agents in the heart lend credence to this assertion (30).

Interestingly, Pt-TiO2 nanoparticles caused more cell death than the P25 TiO2 nanoparticles. This is evidenced by the 50 μg/mL Pt-TiO2 treatment having shown a level of cell viability similar to the 100 μg/mL P25 TiO2 treatment. In a similar fashion, the 5 μg/mL Pt-TiO2 treatment had a level of viability similar to the 10 μg/mL P25 TiO2 treatment (Figure 2). This may be due to the Pt-TiO2 likely having more surface defects due to its thin layer of Pt. Such additional surface defects could hinder the recombination rate of excited electron hole pairs, which may be responsible for the increased ROS generation (10,31).

The mature heart is one of the most metabolically active organs of the body, and is very rich in mitochondria (32). Small changes in mitochondrial membrane potential can lead to substantially decreased cardiomyocyte energy production and can ultimately lead to cellular apoptosis (32). We investigated the effect of these nanoparticles on mitochondrial membrane potential. We found that Pt-TiO2 nanoparticles caused greater decreases in mitochondrial membrane potential than P25 TiO2 (Figure 5), given the concentration difference between the high-concentration Pt-TiO2 and P25 TiO2 (50 μg/mL versus 100 μg/mL, respectively). These data are consistent with our observation that Pt-TiO2 generated more ROS than P25 TiO2, which likely resulted in the decreased mitochondrial membrane potential. This has significant implications for cardiac cells because lower mitochondrial membrane potential can lead to cytochrome c release and induce cellular apoptosis through calcium-sensitive proteases, or through coupling proteins that coordinate the activation of caspases and DNA fragmentation enzymes (32).

Taken together, these results suggest that at lower, more physiologically relevant concentrations, the Pt-TiO2 nanoparticles caused greater cell death in H9c2 cells than did the P25 TiO2 nanoparticles, which may be due to the higher cellular uptake of Pt-TiO2 and/or their greater ROS-inducing effect, which may itself result from their increased capacity to oxidize phenol, NO and other compounds versus P25 TiO2. Moreover, Pt-TiO2 nanoparticles may have been more toxic due to the greater number of surface defects versus P25 TiO2 resulting in more reactive sites at which proteins and other biomacromolecules could interact to produce toxic byproducts (10).

SUMMARY

Our study has shown that the relatively thin surface layer of Pt on the TiO2 nanoparticle appears to greatly affect a variety of its cellular interactions. Given the biomedical significance of these nanomaterials as biosensors and in molecular imaging, effective means of mitigating their toxic effects while maintaining their functionality are required. This information will be critical to appropriately modify nanoparticle surfaces so they are safer and more effective in biomedical applications. The results obtained in the present study have significant implications for the heart because cardiac cells are considered terminally differentiated, and their nanoparticle-induced depletion could lead to cardiovascular complications when using this increasingly popular technology.

Acknowledgments

The authors thank Samantha Nigro for synthesizing the Pt-TiO2 nanoparticles, Dr Guosheng Wu for the XRD analysis, Jiali Wen for the AFM image and BET analysis and Ghislaine Pilot-Attema for editing the manuscript.

Footnotes

FUNDING: This work was funded by the Natural Sciences and Engineering Research Council of Canada (AC) and the Northern Ontario School of Medicine (NK). Studentship support was provided by the Undergraduate Student Research Awards (NSERC) (AM) as well as the Heart and Stroke Foundation of Ontario (SB).

CONFLICTS OF INTEREST: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

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