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. 2020 Dec 10;12(2):245–253. doi: 10.1039/d0md00317d

Normal breast epithelial MCF-10A cells to evaluate the safety of carbon dots†

Nuno Vale 1,2,✉, Sara Silva 1,3, Diana Duarte 1,3, Diana M A Crista 4, Luís Pinto da Silva 4,5,✉, Joaquim C G Esteves da Silva 4,5,✉
PMCID: PMC8128052  PMID: 34046613

Abstract

The human normal breast cell line MCF-10A is being widely used as a model in toxicity studies due to its structural similarity to the normal human mammary epithelium. Over the years, application of carbon dots (C-dots) in biomedicine has been increasing due to their photoluminescence properties, biocompatibility, biosafety and possible applications in bioimaging and as drug carriers. In this work we prepared three different C-dots from the same set of carbon and nitrogen precursors (citric acid and urea, respectively) via three distinct bottom-up synthetic routes and their safety was tested against the normal breast cell line MCF-10A. The characterization results demonstrated a similar size range and composition for all the C-dots. The MCF-10A cells were treated with different concentrations of C-dots for 24, 48 and 72 h to evaluate the cell viability over time. For the 24 h incubation, there were no significant decreases in the viability of the MCF-10A cells. For the 48 h treatment, there was a significant decrease in the viability of the cells treated with calcination-based C-dots, but without significant cellular viability changes for microwave and hydrothermal-based C-dots. For 72 h, cells treated with hydrothermal-based C-dots have the most promising viability profile. Also, compared with paclitaxel, these C-dots have a safety profile very close to that of an antineoplastic in non-tumor cells. Our results suggest that these new C-dots have potential as imaging candidates or biosensing tools as well as drug carriers, and further investigation in animal models is needed for future application in medicine.

The human normal breast cell line MCF-10A is being widely used as a model in toxicity studies due to its structural similarity to the normal human mammary epithelium.graphic file with name d0md00317d-ga.jpg

Introduction

Breast cancer is the most common type of cancer in women and the second leading cause of cancer death worldwide.1,2 The study of the mechanisms of development and progression of breast cancer is of particular importance and has led to the development of different in vivo and in vitro models. Although in vivo studies give results closer to reality, they are more complicated models and it is difficult to recapitulate between normal human breast and breast cancer development. Thus, in vitro studies have a complementary role in these studies and are widely used.3 Despite the recent technological advances, the fields of diagnostics and treatment in breast cancer still have a lot of potential to be explored. Novel nanomaterials can be essential tools for the diagnosis of diseases or transport/delivery of drugs in tumor cells.4,5

Fluorescent nanomaterials, such as carbon dots (C-dots),6 semiconductor carbon dots,7,8 polymer dots,9etc., have gained increasing interest due to their properties, namely environment-friendly and stability,7,10,11 and to their wide range of applications, such as fluorescence imaging, transport of drugs and genes by conjugation, controlled drug delivery, medical diagnosis, and biosensing, among others.12–17 Recently, these C-dots have already been applied as potential antiviral and antibacterial therapeutics.18–20 C-Dots were first reported in 2004 by Xu et al.21 and are small quasi-spherical photoluminescent nanomaterials (usually less than 10 nm)22 that have been used in imaging, biological labeling and analyte sensing (biosensing). The properties of C-dots include high photoluminescence,23 low cost (precursors might even be coffee grounds,24 tea,25 grass,26etc.), stability, high absorption, light-stable23 and environment-friendly features,27,28 and biocompatibility,29 which make them ideal for imaging and biological labeling.

C-Dots can be prepared from different materials and through different synthesis strategies, which are classified into top-down and bottom-up approaches.6,30 The top-down synthesis is based on large starting materials such as graphite powder and multi-walled carbon nanotubes (MWCNTs), with their subsequent breakdown into smaller carbon materials.31–33 Nevertheless, the bottom-up synthesis is the most widely used type of synthesis. Contrary to top-down procedures, bottom-up routes produce C-dots from smaller carbon sources, such as carbohydrates34,35 and citric acid,36–39via hydrothermal,36,40 calcination28 and microwave-assisted syntheses.37,41

The addition of other small molecules containing heteroatoms (such as N, S and B) leads to heteroatom doping of the C-dots.42 Among them, N-doping is the most often employed strategy, and nitrogen and carbon atoms have similar size. Moreover, N-doping has been linked to an increase of the fluorescence quantum yield of these nanomaterials.42,43 N-Doping can be achieved by adding a nitrogen-rich small molecule to the carbon precursor, with typical nitrogen sources being molecules such as urea and ethylenediamine.37

MCF-10A cells are the most common cell line used as a model for normal human breast cells.3 They are very structurally similar to normal human mammary epithelial cells and are adherent at normal calcium levels.44 In collagen, these cells grow in 3D structures and express sialomucins, keratins, hemidesmosomes and desmosomes.45,46 In addition, they are controlled by hormones and growth factors, although they do not show estrogen receptors or signs of terminal differentiation or senescence.47 In mice, these cells were also found to be non-tumorigenic.46 Studies suggest that these cells respond to insulin, glucocorticoids, cholera endotoxin and epidermal growth factor (EPG).47 Given that their characteristics are very similar to the normal human breast epithelium, these cells have been widely used in cytotoxicity studies as a control to evaluate the safety of different compounds, in comparison with breast tumor cell lines, such as MCF-7 or MDA-MB-231, among others.48–50 These studies included the evaluation of the cytotoxicity of different compounds, from natural molecules such as eupatorin51 to the study of antineoplastics, such as doxorubicin.52 In the last five years, promising carbon-based materials for breast cancer chemotherapy application have been reported.53–57 In the field of nanotechnology, MCF-10A cells also continue to be used to assess the toxicity of nanoparticles containing drugs.58 These cells have already been used to evaluate the toxicity of different carbon dots, since they are a good model to evaluate the biosafety and biocompatibility of these nanomaterials.

In a study with C-dots obtained from green tea, these nanoparticles were tested on 5 cell lines such as human breast cancer cell lines (MCF-7 and MDA-MB-231), normal mammary epithelial cells (MCF-10A), human cervical cancer cells (HeLa) and normal porcine kidney cells (LLC-PK1).25 The synthesized C-dots effectively decreased the viability of the tumor cell lines MCF-7, MDA-MB-231 and HeLa by 20%, 18% and 68%, respectively, for the highest concentration tested, while the normal cells (MCF-10A and LLC-PK1) only present a 5% inhibition, which demonstrates the high efficiency of these C-dots in tumor cells and their safety in normal cells.25 In addition, compared with antineoplastic drugs that are already on the market, such as doxorubicin and paclitaxel, it was found that these C-dots were less toxic.25 Paclitaxel is currently used against a wide range of solid tumors, including lung, ovarian, urothelial and breast tumors59 and its clinical use assumes that this antineoplastic is safe for non-tumoral cells.

Another study tested fluorescent C-dots obtained from ginger on different tumor cell lines, namely human lung cancer cell line A549, human breast cancer cell line MDA-MB-231, human cervical cancer cell line HeLa and human hepatocellular carcinoma cell line HepG2, with high suppression of the growth of these cells, mainly HepG2 cells.60 These C-dots have also been tested on normal lines such as normal mammary epithelial cells (MCF-10A) and normal liver cells (FL83B) to assess their biosafety and have demonstrated low toxicity to these cells.60 Ray et al. synthesized C-dots with diameters between 2 and 6 nm and it was also demonstrated that the toxicity of these nanomaterials was negligible at the concentrations normally used for bioimaging.61

A relatively recent in vivo study (conducted in rats) performed by Zhan et al. showed that aspirin-based C-dots had no significant cytotoxicity in the liver, spleen, lungs, kidneys or heart, while providing anti-inflammatory protection.62 Leblanc's group produced C-dots from carbon nanopowder, which were revealed to be non-toxic while possessing high affinity and specificity for bone binding.63 Du and co-workers produced gadolinium-doped C-dots with stable fluorescence that accumulated in the kidneys after vein injection and could be cleared away by urination, demonstrating relevant biocompatibility.64 Finally, a relevant amount of research supports the biocompatibility of C-dots in both mouse and zebrafish testing.65

Despite these results, it remains necessary to explore the cytotoxicity of these C-dots for more comprehensive applications, such as in vivo imaging and pharmaceuticals. This work aims to evaluate the safety and compatibility of these new nanoparticles in the MCF-10A cell line. To this end, three different C-dots were prepared from the same set of carbon and nitrogen precursors (citric acid and urea, respectively) via three distinct bottom-up synthesis routes: hydrothermal, calcination and microwave-assisted syntheses, following a previous report from our group.66 Their cytotoxicity toward the human normal breast cell line MCF-10A was subsequently tested. With this approach we will be able to analyze the biosafety and biocompatibility of these nanomaterials, when considering widely used starting materials and procedures, while providing insight into the different effects exerted by distinct bottom-up strategies.

Results and discussion

Characterization of C-dots

The different syntheses led to similar size distributions, which were obtained by AFM measurements (Fig. 1). Namely, we obtained the following average sizes (± standard deviation) for the three types of C-dots: 6.9 ± 2.0 nm (hydrothermal), 6.1 ± 1.7 nm (calcination) and 7.3 ± 1.7 nm (microwave-assisted synthesis). These are sizes typically associated with C-dots, and indicate that the obtained particles are indeed nano-sized. It should be noted that while microwave-based C-dots present higher average sizes for individual nanoparticles, calcination and hydrothermal-based C-dots appear to have higher tendency toward agglomeration under the employed AFM measurements, which explains some higher-size agglomerates for these types of CDs (Fig. 1).

Fig. 1. 3D AFM images relative to the size of hydrothermal-, calcination- and microwave-based C-dots.

Fig. 1

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An XPS analysis was made to analyze the surface compositions of the three C-dot samples (Fig. S1–S3†) and the XPS atomic composition (at wt%) for each sample (Table 1). As expected, all C-dots are composed mainly of C (60.0–62.0%), followed by O (24.7–28.8%) and N (9.1–13.5%). A detailed scan for internal levels of C 1s, O 1s and N 1s was subsequently made, towards deconvolution and chemical state and quantitative analysis (Fig. S1–S3†).

Atomic surface compositions (%) for hydrothermal-, calcination- and microwave-based C-dots, determined by XPS analysis.

Atomic surface composition (%) Hydrothermal-based C-dots Microwave-based C-dots Calcination-based C-dots
C (%) 62.0 60.0 61.9
N (%) 9.1 13.1 13.5
O (%) 28.8 26.9 24.7
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The C 1s spectra (Fig. S1†) could be split into three peaks for calcination- and microwave-based C-dots: at binding energies of ∼285 eV (attributed to C–C/C–H groups), ∼286 eV (attributed to C–N/C–O groups) and ∼289 eV (attributed to O–C Created by potrace 1.16, written by Peter Selinger 2001-2019 O groups). As for hydrothermal-based C-dots, the C 1s spectrum was split into four peaks instead, with the new one being found at a binding energy of ∼287 eV (attributed to C Created by potrace 1.16, written by Peter Selinger 2001-2019 O groups).

By their turn, the O 1s spectra (Fig. S2†) were split into two peaks for all syntheses: at binding energies of ∼531 eV (attributed to C Created by potrace 1.16, written by Peter Selinger 2001-2019 O) and ∼532 eV (attributed to C–O groups). Finally, the N 1s spectra were also split into two peaks: at binding energies of ∼400 eV (attributed to amine–amide groups) and ∼401 eV (attributed to protonated amides). Thus, the surface compositions of these C-dots appear to be similar.

The normalized excitation and fluorescence spectra of the three C-dots can be found in Fig. 2. The fluorescence quantum yields of these C-dots were calculated previously by our group66 and are: 3.7% for hydrothermal-based C-dots, 25.1% for microwave-based C-dots, and 29.3% for calcination-based C-dots. All nanoparticles present similar blue emission: maxima at 435 nm for hydrothermal- and calcination-based C-dots, and at 430 nm for microwave-based C-dots. The excitation maxima of these C-dots can be found in the ultraviolet A (UV-A) range. Nevertheless, the variation in the excitation wavelength maximum between the C-dots is greater than for the emission. Namely, hydrothermal-based C-dots can be excited at 325 nm, while microwave-based C-dots can be excited at 340 nm. Calcination-based C-dots present the most red-shifted excitation at 345 nm. All three C-dots present identical absorption spectra (Fig. S4†), which consist of a main band at ∼350 nm and a shoulder at ∼245 nm. These can be attributed to n → π* and π → π* transitions.52 Regarding the luminescence of C-dots, it should be noted that their emission cannot be explained by just a single-dominating mechanism, but rather by two or more processes (such as quantum size effects, bandgap transitions or defect-derived emission).67

Fig. 2. Normalized excitation and emission spectra (in water) for hydrothermal-, calcination- and microwave-based C-dots.

Fig. 2

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The effect of pH on emission of the three C-dots was also assessed (Fig. S5†). The pH affects all the C-dots in a similar manner. More specifically, both the emission intensity and wavelength are identical at neutral (pH 7) and basic (pH 10) pH. At acidic pH (pH 5) a very slight blue-shift with a relevant decrease in intensity occurs. These changes can be attributed to protonation of some functional groups of the C-dots, which leads to changes in fluorescence. In fact, previous studies have also observed that the fluorescence of C-dots produced from citric acid and urea can be pH-sensitive.68,69 These authors attributed this pH-sensitivity to the presence of surface moieties resembling molecular fluorophores (as citrazinic acid), which can be present in various molecular forms at different pH values.68,69 Nevertheless, the reduction in intensity is significantly lower (∼10%) for calcination-based C-dots than for both hydrothermal- and microwave-based C-dots (∼30%).

While there is a significant amount of work trying to evaluate the effect of using different carbon precursors and/or heteroatom dopants, there is limited information regarding which way different synthetic routes can affect the properties of C-dots (such as their biocompatibility). Thus, the next step of this study was to assess what way different bottom-up strategies can be used to optimize the biocompatibility of the new C-dots, which are produced from the same carbon precursor (citric acid) and N-dopant (urea).

Cytotoxic effect of C-dots

After the synthesis of C-dots, we intended to evaluate the cytotoxicity of these nanomaterials to normal breast cells. As the purpose of these nanoparticles is to assist in the diagnosis of breast cancer and transport of drugs to tumor cells, it is important to ensure, at an early stage, that the safety of these particles is guaranteed in normal cells. Although the formulations are different, our goal was to find the most appropriate (and safe) C-dots for this type of application. For this, the cytotoxicity of these three formulations of carbon dots was tested against the human normal breast cell line MCF-10A. The cells were incubated with the C-dots (0.01–1 g L−1) for 24, 48 and 72 hours. When selecting these incubation times, we ensure that the safety of these nanomaterials is guaranteed over time, with no cytotoxicity associated. To assess the cell viability, the MTT assay was performed. In this assay, the control well corresponds to 100% viable cells and the percentage of cell viability in each well is represented as relative to the control. In order to study the compatibility of these nanoparticles with this assay, incubation of the C-dots was performed with the MTT reagent. The results for the 24 h incubation confirm the low cytotoxicity of these nanoparticles (Fig. 3). When treated with hydrothermal-based C-dots, the cell viability significantly increases, which may be related to an increase in the number of cells in the well or higher metabolic activity of the cells. Due to natural variations of cellular metabolism or because slightly more cells were seeded in that well due to small pipetting errors, it is totally normal to have samples that exceed the 100% viability. In this case, we found out that these C-dots may also have an influence on the cell growth, confirmed by the cell count with trypan blue. The upper graphs allow faster reading of the results while the lower graphs allow evaluation of the precision and accuracy of the values obtained in the cellular assays.

Fig. 3. Relative C-dot cytotoxicity against MCF-10A cells after 24 h incubation through the MTT assay. The percentage of cell viability is represented as relative to the negative control. The values represent mean ± SEM (n = 3) (*p < 0.05).

Fig. 3

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As expected, after 48 h treatment, there was a general decrease in cell viability compared to the results obtained at 24 h, mainly for calcination and microwave-based carbon dots at the highest concentrations. For hydrothermal-based C-dots, the same trend was observed as that in 24 h treatment, but with an attenuated increase in cell viability (Fig. 4).

Fig. 4. Relative C-dot cytotoxicity against MCF-10A cells after 48 h incubation through the MTT assay. The percentage of cell viability is represented as relative to the negative control. The values represent mean ± SEM (n = 2) (* p < 0.05) (** p <0.01).

Fig. 4

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For the 72 h incubation, the decrease of cell viability was accentuated in the microwave-based formulation (Fig. 5) when compared with the same formulation for the 48 h (Fig. 4) and 24 h incubation (Fig. 3). In addition, the toxicity based on the MTT assay showed that there is no significant sign of toxicity observed for all concentrations of hydrothermal-based C-dots over time (Fig. 3–5).

Fig. 5. Relative C-dot cytotoxicity against MCF-10A cells after 72 h incubation through the MTT assay. The percentage of cell viability is represented as relative to the negative control. The values represent mean ± SEM (n = 2).

Fig. 5

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As hydrothermal-based carbon dots demonstrated less cytotoxicity, we compared the toxicity of these C-dots with an antineoplastic commonly used in clinical practice, paclitaxel. Paclitaxel has low toxicity, is considered safe and is approved by the FDA for tumor therapy. Since this drug has a safety profile compatible for its clinical use, we compared the toxicity of our C-dots with paclitaxel. The hydrothermal-based C-dots formulated in this study cause less decreases in cell viability than paclitaxel for the same range of concentrations, so their use in normal breast cells can be considered safe (Fig. 6).

Fig. 6. Relative viabilities of MCF-10A cells after 24, 48 and 72 h treatments with paclitaxel vs. hydrothermal-based C-dots. The percentage of cell viability is represented as relative to the negative control. The values represent mean ± SEM. The data for paclitaxel are in agreement with the literature.63.

Fig. 6

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In this work, the characterization results of the new C-dots showed similar particle size and surface composition. The structural properties of the synthesized C-dots proved to be favorable to the study of the bioapplication of these nanomaterials as non-toxic fluorescent agents for bioimaging. As C-dots usually have promising biocompatibility profiles, MTT assays were performed to assess the toxicity of these nanoparticles against MCF-10A cells (normal breast cells). The MTT assay is a colorimetric assay that is based on the ability of cell mitochondria to reduce the MTT dye to the purple-colored formazan. This reduction is greater as cells are more viable, so there is a correlation between the MTT signal and the cell viability. Thus, the greater the toxicity of a given agent, the less the reduction of MTT into formazan (less viable cells, less functional mitochondria). The MTT assay demonstrated that the prepared calcination, microwave and hydrothermal-based C-dots showed high cell viabilities for MCF-10A cells, even in high concentrations of 1 g L−1 for 24 h (Fig. 3). The most significant decrease in cell viability was observed for calcination-based C-dots in concentrations above 0.6 g L−1 for 48 h incubation (Fig. 4). The probable mechanism for this process can be associated with ROS produced in the mitochondria. The ROS generation process plays a decisive role in biological processes such as cell division, differentiation, apoptosis, cellular senescence and radical-mediated oxidative damage.

Despite the physicochemical similarities of all the C-dots, the hydrothermal-based C-dots were considered the best formulation, as they showed no effect on cell viability for all tested concentrations and incubation. For cross checking the toxicity, the cells were treated with paclitaxel, an antineoplastic widely used in breast cancer therapy that has a considered safe toxicological profile, so the toxicity of these C-dots was compared with the toxicity of paclitaxel to MCF-10A cells. In Fig. 6, it is possible to conclude that the hydrothermal-based C-dots are significantly less toxic than paclitaxel for the same range of concentrations. Two possibly complementary explanations can be given to justify the higher biocompatibility of the hydrothermal-based C-dots. Wang and co-workers have found for graphene quantum dots a positive correlation between a higher surface oxygen content and an improved antioxidant activity of the nanoparticles.70 In fact, the XPS analysis (Table 1) performed by our group has shown that hydrothermal-based C-dots have a higher surface oxygen content (28.8%) than both microwave- and calcination-based C-dots (24.7–26.9%). Thus, it is possible that by exhibiting a higher antioxidant activity, hydrothermal-based C-dots can provide some protection toward oxidative stress to the cells, and so, maintain their cellular viability. Liu et al.71 have also recently studied the influence of surface functional groups on the toxicity of carbon-based nanomaterials. Interestingly, they have found that the presence of epoxide (C–O–C) functional groups enhances the oxidation potential of these nanomaterials, which leads to higher toxicity.71 Consistent with this scenario, hydrothermal-based C-dots present a lower contribution of C–O functional groups (9.2%) to the C 1s spectra (Fig. S1†) than both microwave- (11.0%) and calcination-based (12.1%) C-dots. Taken together, both hypotheses might help to explain the higher biocompatibility of C-dots.

Conclusions

The increase in biomedical application of C-dots is due to their incredible photoluminescence properties, photostability, biocompatibility and biosafety. The carbon dots synthesized in this study are obtained through different synthesis strategies that reflect in the in vitro results. Among the C-dots tested in these study, hydrothermal-based carbon dots have the lowest toxicity against the normal breast cells, MCF-10A, demonstrating high biocompatibility and biosafety, with potential for use in medicine as bioimaging and biosensing agents and drug or gene delivery.72

Experimental section

Chemicals

DMEM/nutrient mixture F-12 Ham (DMEM-F12), fetal bovine serum (FBS) and penicillin–streptomycin solution were purchased from Millipore Sigma (Merck KGaA, Germany). Other cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc, MA, USA). Paclitaxel (cat. no. 1097, from Tocris) was obtained from Biogen Cientifica (Madrid, Spain). Thiazolyl blue tetrazolium bromide (MTT, cat. no. M5655) was obtained from Sigma-Aldrich (Merck KGaA, Germany).

Synthesis of C-dots

The C-dots were obtained by three different methods: hydrothermal, calcination and microwave-assisted syntheses, following a previous report from our group.66 A mixture of citric acid (0.75 g) and urea (0.25 g) was used as a starting material in all synthesis procedures. For the hydrothermal synthesis, the citric acid–urea mixture was dissolved in 5 mL of deionized water. The solution was placed in a Teflon-lined reactor and heated for 2 hours in an oven at 200 °C. For the calcination-based procedure, the citric acid–urea powder mixture was placed in a beaker and heated for 2 hours in an oven at 200 °C. Finally, for the microwave-assisted synthesis, the citric acid–urea mixture was dissolved in 5 mL of deionized water and subsequently placed in a glass petri dish. The reaction mixture was subjected to microwave irradiation (700 W in a domestic microwave) for 5 minutes.

The obtained C-dots were subsequently suspended in water and initially purified by centrifugation (10 minutes at 12000 rpm) to eliminate suspended impurities. The samples were further purified by dialysis for 24 hours, by using a Float-A-Lyzer®G2 Dialysis Device Spectrum® with a molecular weight cut-off of 500 Da.

The synthesis yields (in %) for the three C-dots were: 1.8% for hydrothermal-based C-dots, 28.5% for microwave-based C-dots and 26.9% for calcination-based C-dots.66

Characterization of C-dots

Atomic force microscopy (AFM) was carried out using a Vecco Metrology Multimode/Nanoscope IVA in tapping mode, using a Bruker silicon probe (model TESP-SS, resonant frequency 320 kHz, nominal force constant 42 N m−1, estimated tip radius 2 nm). X-ray photoelectron spectroscopy (XPS) was performed with a Fi Kratos Axis Ultra HAS-VISION, using monochromatic A1-Kα radiation (15 kV, 90 W). The spectra were analyzed and quantified using CasaXPS software employing sensitivity factors supplied by the manufacturer. Analysis included the subtraction of a linear background and charge referencing to the adventitious carbon signal at 285 eV. Fluorescence was measured in standard 10 mm fluorescence quartz cells by using a Horiba Jovin Yvon Fluoromax-4 spectrofluorometer. The spectra were obtained with a 1 nm interval and 5 nm slit widths. The average sizes were calculated by quantifying the size of 40 individual nanoparticles for each CD sample.

Cell incubation

The human normal breast MCF-10A cell line was obtained from the American Type Culture Collection (ATCC; Virginia, USA) and maintained according to ATCC's recommendations at 37 °C and 5% CO2 in DMEM-F12 medium supplemented with 10% fetal bovine serum, 100 μg mL−1 epidermal growth factor (EGF), 1 mg mL−1 hydrocortisone, 10 mg mL−1 insulin, 100 U mL−1 penicillin G and 100 μg mL−1 streptomycin. The cells were maintained in the logarithmic growth phase at all times. The medium was renewed every 2 days and the cells were trypsinized with 0.25% trypsin-EDTA and subcultured in the same medium. MCF-10A cells (5000 cells per well) were seeded in 96-well plates and allowed to adhere overnight prior to drug exposure. After that, the cell culture medium was replaced with C-dot-containing media. The cells were exposed to C-dots for 24, 48 and 72 h, followed by the MTT assay to evaluate the cell viability of these cells in the C-dot treatments.

Cytotoxicity assays

To determine the effect of C-dots on the viability of MCF-10A cells, the MTT assay was used. For the MTT protocol, after C-dot treatment, the cell medium was removed, and 100 μL per well of MTT solution (0.5 mg mL−1 in PBS) was added. The cells were incubated for 3 h, protected from light. After this period, the MTT solution was removed, and DMSO (100 μL per well) was added to solubilize the formazan crystals. Absorbance was measured at 570 nm using an automated microplate reader (Synergy HT, Biotek Instruments Inc., Winooski, VT, USA). The results are expressed as percentage of the respective control (vehicle). All conditions were performed in triplicate.

Statistical analysis

The results are presented as mean ± SEM for n experiments performed. Statistical comparisons between groups, at the same time point, were performed with one-way ANOVA, after Shapiro–Wilk test normality evaluation. Statistical significance was accepted at p values < 0.05. The Student–Newman–Keuls post-hoc test was used once a significant p value was achieved.

Funding

This work was financed by FEDER – Fundo Europeu de Desenvolimento Regional through the COMPETE 2020 – Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia, in the framework of CINTESIS, R&D Unit (reference UIDB/4255/2020), by project UIDB/00081/2020 (CIQUP) and project IF/00092/2014/CP1255/CT0004. Luís Pinto da Silva acknowledges funding from FCT, under the Scientific Employment Stimulus (CEECIND/01425/2017).

Conflicts of interest

The authors declare no conflict of interest.

Supplementary Material

MD-012-D0MD00317D-s001

Acknowledgments

The Laboratory of Computational Modelling of Environmental Pollutants-Human Interactions (LACOMEPHI) is acknowledged. We thank Centro de Materiais da Universidade do Porto (CEMUP) for performing the AFM and XPS measurements (www.cemup.up.pt). Diana Crista acknowledges FCT for funding her PhD grant (SFRH/BD/144423/2019). Sara Silva and Diana Duarte acknowledge FCT for funding their PhD grants (PD/BD/135458/2017 and SFRH/BD/140734/2018, respectively).

†

Electronic supplementary information (ESI) available: Fig. S1 – XPS C 1s spectra for calcination-, microwave- and hydrothermal-based C-dots. Fig. S2 – XPS O 1s spectra for calcination-, microwave- and hydrothermal-based C-dots. Fig. S3 – XPS N 1s spectra for calcinated- (top), microwave- (middle) and hydrothermal-based (bottom) C-dots. Fig. S4 – absorption spectra for calcination-, hydrothermal- and microwave-based C-dots. Fig. S5 – emission spectra for hydrothermal-, microwave- and calcination-based C-dots, at different pH values (5, 7 and 10). See DOI: 10.1039/d0md00317d

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