Carbonization of Human Fingernails: Towards the Sustainable

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Functional Nanostructured Materials (including low-D carbon)

Carbonization of Human Fingernails: Towards the Sustainable Production of Multifunctional Nitrogen and Sulfur co-Doped Carbon Nanodots with Highly Luminescent Probing and Cell Proliferative/Migration Properties Theodoros G. Chatzimitakos, Athanasia Kasouni, Anastassios N. Troganis, and Constantine D. Stalikas ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03263 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018

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ACS Applied Materials & Interfaces

Carbonization of Human Fingernails: Towards the Sustainable Production of Multifunctional Nitrogen and Sulfur co-Doped Carbon Nanodots with Highly Luminescent Probing and Cell Proliferative/Migration Properties Theodoros G. Chatzimitakos,† Athanasia I. Kasouni,†† Anastassios N. Troganis,†† Constantine D. Stalikas*,† †

Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece

††

Laboratory of Biophysical Chemistry, Department of Biological Applications and Technologies, University of Ioannina, 45110, Ioannina, Greece

*Corresponding author, e-mail: [email protected]

KEYWORDS: Carbon nanodots, Cr(VI) probing, cell proliferation, bioimaging, cell cycle, cell migration, blood compatibility.

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ABSTRACT A simple, yet effective method is employed to prepare multifunctional fluorescent carbon nanodots (CNDs) from human fingernails. The results demonstrate that the CNDs have excellent optical properties and a quantum yield of 81%, which is attributed to the intrinsic composition of precursor material itself. The CNDs are used to develop an ultra-sensitive fluorescent probe for the detection of hexavalent chromium (limit of detection: 0.3 nM) via a combined inner- filter and static mechanism. Moreover, the toxicity of the CNDs over four epithelial cell lines is assessed. A negligible toxicity is induced on the three of the cell lines whereas an increase in HEK-293 cell viability is demonstrated, granting cell proliferation properties to the as-synthesized CNDs. According to cell cycle analysis, cell proliferation is achieved by enhancing the transition of cells from S phase to the G2/M one. Interestingly, CNDs are found to significantly promote cell migration, maybe due to their free radical scavenging ability, rendering the CNDs suitable for wound healing applications. In addition, relevant experiments have revealed blood compatibility of the CNDs. Finally, the CNDs were found suitable for cell imaging applications while all of the aforementioned merits make it possible for them to be used for extraordinary, more advanced biological applications.

1.

INTRODUCTION

Carbon nanodots (CNDs) are a rising star among carbon-based nanomaterials. Their eminence over common semiconductor quantum dots is widely acknowledged, since the superiority of most of them lies within their unique fluorescent characteristics, accompanied by several alluring properties like splendid dispersibility, photobleaching resistance, chemical inertness, low cytotoxicity etc.1-2 Thus, an ever growing interest in this new class of fluorescence nanomaterials is expressed. Hitherto, a lot of studies have been published, aiming to produce CNDs and examine their properties, enabling their use, primarily, in various sensing and biomedical applications. The fluorescence of CNDs, in their native form or after targeted modifications, is sensitive to several experimental chemical factors that, if controlled, transform CNDs into nanoprobes with great analytical potential. Although several techniques3-4 and a variety of natural or waste resources5-9 have been exploited for the production of CNDs, the demand for synthesis routes that adhere to the principles of green chemistry is still high. To fulfill this requirement, researchers have used human 2 ACS Paragon Plus Environment

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hair as a precursor for the synthesis of CNDs10, since hair is rich in protein (∼65-95% of their content) and suitable for such reasons.11 Keratin, the main protein of hair, is also ubiquitous in human fingernails.12 However, studies have demonstrated that human fingernails contain both hair and epidermal keratins, rendering them qualitatively different from human hair.13 The distinguishing feature of keratin is the presence of large amounts of the sulfur-containing amino acid cysteine required for the disulfide bridges. Therefore, nails contain, mainly, carbon, nitrogen, oxygen, sulfur and hydrogen, as a result of the proteins they consist of. The high content of protein renders them a promising raw material for the synthesis of CNDs, essentially at no cost, since nails can easily be obtained from nail salons waste. Thus, it is conceivable that this natural material could be a promising candidate precursor for synthesizing nitrogen and sulfur co-doped CNDs. In biomedical applications, fluorescent carbon dots are usually administered in vivo through various routes. However, biocompatibility is a requisite for their safe application. The misconception that CNDs are “biocompatible” without appropriate cell cycle data at hand in order to monitor alterations in cell cycle phases (e.g. Sub-G1 phase), weakens this notion.14 Cell cycle analysis offers an insight into cell division phases and provides essential information about the occurrence of apoptosis, cell cycle arrest and progression, etc. that correlate with cell apoptosis, toxicity or proliferation. On the other hand, increasing/promoting cell proliferation and/or cell migration induced by growth factors is a benefit, which can be reaped in tissue engineering and ultimately in regenerative medicine.15 Despite the low in-vitro cytotoxicity of CNDs reported so far in previous studies, cell proliferative effects are not commonly observed. On the contrary, in some cases, inhibition of cell proliferation accompanied by cell cycle arrest is apparent.16-18 Hence, the use of CNDs in tissue engineering and regenerative medicine applications is limited. Even though trivalent chromium (Cr(III)) is a rather harmless, essential trace element species for physiological body functions (e.g. glucose tolerance), hexavalent chromium (Cr(VI)) is highly toxic, carcinogenic and linked with numerous health issues.19-21 In order to assess real-life effects of chromium, speciation analysis is deemed necessary and new methods, able to discriminate between the chromium species have been developed.22-23 Herein, we describe the utilization of multifunctional CNDs produced from human fingernails via a one-step, simple



pyrolysis procedure. The strong photoluminescent properties of the synthesized CNDs make them suitable for the sensitive probing of Cr(VI), its discrimination from Cr(III) and the efficient imaging of cells. Interestingly, our efforts towards developing novel CNDs for analytical and bioimaging applications revealed their unprecedented biological properties, relating to the enhancement of cell proliferation and cell migration, which make feasible their use as a growth factor for tissue regeneration. To the best of our knowledge, this is the first time that cell proliferation validated by cell cycle analysis is reported for CNDs. Due to these promising properties, further experiments regarding free radical scavenging ability and blood compatibility of the CNDs were performed and the results are discussed herein.

2.

EXPERIMENTAL SECTION

Chemicals. All information about chemicals and materials is provided in the Supporting Information. Synthesis of the CNDs. Fingernails were collected from two adult volunteers (male and female) using cosmetic nail clippers. Nail clippings were rinsed extensively with DDW and dried with a paper towel. The pooled nails were placed in a porcelain crucible and heated for 3 h in a pre-heated furnace, at 200°C. The black residue was ground into fine powder followed by the addition of 25 mL of DDW and ultrasonication, for 5 min. After centrifugation at 8000 rpm for 10 min, the supernatant was retracted, dialyzed against water for 24 h using a benzoylated dialysis tubing membrane (MWCO: 2.000 Da) and finally freeze-dried, resulting in a yellowbrown residue. Instrumentation. All instruments used are listed in Supporting Information. Quantum yield measurements. The method used to determine quantum yield is given in Supporting Information. Detection of Cr(VI) ions. To detect Cr(VI), the following procedure was followed: 1 mL of Cr(VI) working standard solution or sample solution was added to 2 mL of ammonia-ammonium chloride buffer solution (5 mM, pH 9.0) containing 0.5 µg of the CNDs. After vortexing for 30 sec, the fluorescence emission was recorded at λex/λem: 330/380 nm. A calibration curve was drawn to calculate the concentration of Cr(VI).


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Biomedical applications study. All information regarding in vitro experiments (i.e. cell culture conditions, cell viability, trypan blue assay, cell cycle analysis, cellular imaging and scratch assay) are given in details in the Supporting Information DPPH free radical scavenging assay. The DPPH free radical scavenging assay was performed according to a previous method applied by our group.24 Blood compatibility. Experimental details of blood compatibility-related experiments (i.e. blood clot time and hemolysis assay) are given in Supporting Information. Statistical analysis. All assays were repeated three times to ensure reproducibility. Results were expressed as mean ± standard deviation (SD).One-way analysis of variance (ANOVA) using Student's t-test, as post-hoc test for comparison of means was used to compare the levels of significance between samples for cell proliferation. A two-way ANOVA with replication using Duncan’s multiple range test as post-hoc test was used to compare the means for cell migration assay.

3.

RESULTS AND DISCUSSION

Carbon nanodots synthesis. To maximize the fluorescence intensity of the synthesized CNDs, two major parameters, i.e. temperature and time, which directly affect the carbonization of fingernails, were studied. For the purpose of obtaining comparable results, a constant amount of nails (10 mg) was used in all experiments and the resulting solution (following the procedure explained in part 2.2) was diluted up to a constant volume. The temperatures tested were 200, 250 and 300°C, at three different time periods (1, 2 and 3 h), resulting in a total of nine experiments. The results are depicted in Fig S1 of Supporting Information. It is noteworthy that for all CNDs produced from the above conditions, the same excitation and emission fluorescence spectra were recorded. This is in agreement with other reports that CNDs exhibit size-independent photoluminescence, in a reasonable size range.25 Also, the results revealed that the product prepared after carbonization of nails at 200°C, for 3 h had the highest fluorescence intensity. Heating the nails at the same temperature for 2 h resulted in a product with ∼25% less intensive fluorescence, while heating for 1 h was unable to yield fluorescent product. Even though heating at 250°C for 1 h resulted in a fluorescent product, an additional 1-h heating resulted in a product with increased fluorescence intensity by ∼50%. Still, the product was ∼20% less fluorescent than that carbonized at 200°C, for 3 h. It is notable that the product after 3 5 ACS Paragon Plus Environment


h of heating at 250°C did not exhibit any fluorescence. This can be attributed to the complete ashing of the nails, as evidenced by their white-silver color. The same observation was made with products originating from heating at 300°C, after 2 and 3 h, while after 1 h the nails were white-silver colored with some black spots, thus, justifying the weak fluorescence of the product. Characterization of CNDs. Figure 1a shows the deconvoluted C1s peak of the synthesized CNDs. The peak is analyzed into the following components: carboncarbon bonds, at 284.6 eV and 285.5 eV (sp2 and sp3 bonding, respectively), carbonoxygen bonds at 286.5 eV (epoxides end hydroxides) and 288.2 eV (carbonyls).26 Figure 1b shows the deconvoluted O1s XPS spectrum. The peak is deconvoluted into two components assigned to single oxygen-carbon bonds -C-O(H) or O-C-O at 532.6 eV and double oxygen-carbon bonds C=O(H) or O-C=O(H) at 531.6 eV.26 Figures 1c and 1d show the N1s and S2p peaks of the CNDs. The peak centered at 399.6 eV is assigned to amine/amide or pyrolic moieties27 and the peak centered at binding energy of 163.7 eV is assigned to sulphur-carbon bonds (C–Sn–C, n = 1 or 2).28 Using the total peak area of O1s, C1s, S2p and N1s peaks and the appropriate sensitivity factors (based on Wagner’s collection and adjusted to the transmission characteristics of analyser EA10) and equations, the % average relative atomic concentration in the analysed region was found to be (within experimental error 10%): Carbon: 55.8%, Oxygen: 23.5%, Nitrogen: 19.2% and Sulphur: 1.5%.


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Figure 1. (a) C1s, (b) O1s, (c) N1s and (d) S2p XPS spectra of the CNDs from the carbonization of human nails. A wide diffraction peak located at ca. 22o can be seen in the XRD pattern of the CNDs (Figure S2 of Supporting Information), which corresponds to an interlayer spacing of about 0.402 nm and is indicative of an amorphous carbon structure.8 Moreover, TEM images (Figure S3 of Supporting Information) revealed that the synthesized CNDs have an almost circular shape, with a diameter of around 3.5 nm and a narrow size distribution. The FT-IR spectra of the synthesized CNDs (Figure S4 of Supporting Information) confirm the presence of functional groups such as: O−H, N−H (stretching vibrations at 3100-3500 cm-1), C−H, −NH2, −NH3+ (stretching vibration at around 2900 cm-1)29, C=C and N−H (stretching and bending vibrations, respectively, at 1645 cm-1)6, C−N (stretching vibration at 1390 cm-1) and C−O (bending vibrations between 1000 and 1100 cm-1).30 Optical properties and stability of CNDs solutions. The UV-Vis spectrum (Figure S5 of Supporting Information) of the CNDs shows two characteristic absorption bands, appearing at around 275 and 330 nm. The former is due to the presence of aromatic π orbitals or n-π* transitions30 and the latter is commonly observed in cases 7 ACS Paragon Plus Environment


of highly fluorescent CNDs, as a result of trapping excited state energy from surface states. Therefore, high-fluorescence/high-quantum yield CNDs is anticipated.31-32 The as-prepared CNDs, are highly hydrophilic, resulting in highly dispersed solutions with transparent yellow color (under daylight), while irradiation with UV light results in strong, bright blue fluorescence. Figure 2 shows the tunable double-emissive nature of the CNDs, since two fluorescence emission peaks are recorded: the first one at 380 nm, after excitation at 330 nm and the second one at 450 nm, after excitation at 370 nm. The first emission peak can be attributed mainly to the excitation of high energy n-π* transitions, which results in emission at short wavelengths.33 The simultaneous decrease and red shift of the fluorescence intensity is attributed to the heterogeneity of the organic groups on the surface of the CNDs, whereby intermediate states between the HOMO and LUMO orbitals are generated.34 Between the two fluorescence maxima, the first one, bearing the highest density was selected for the analytical study although for purposes of intra- and extra-cellular probing this can potentially pose a problem. Using quinine sulfate, as reference, the quantum yield of the CNDs was calculated to be 81.4%, which is much higher than that of most of synthesized CNDs obtained by other syntheses.30,

35

Molecules with the structure RHN–C–C–SH (e.g. cysteamine,

cysteine) were found to be suitable for the production of highly fluorescent CNDs.36 For instance, Yang et al synthesized CNDs by hydrothermal treatment of citric acid and cysteamine for 12 h, resulting in products with QY up to 81%.37 Despite the high quantum yield, cysteamine is relatively costly, while 12 h were taken for the production of the CNDs. In our case, the presence of cysteine in the keratins of human fingernails facilitates the synthesis of high QY CNDs. Furthermore, a natural waste resource is used and the synthesis is completed in 3 h, thus, providing a low-cost, energy-efficient and time-saving synthesis. Overall, it can be inferred that human fingernails are suitable to produce CNDs of high quality via the proposed procedure.


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Figure 2. (A) Fluorescence emission spectra of the CNDs at excitation wavelengths between 300 and 390 nm, (B) Excitation spectrum for emissions at 380 nm.

Photoluminescence stability of the CNDs in various conditions is favorable for applications. Relevant experiments revealed that aqueous solutions of the synthesized CNDs emit stable fluorescence within a wide pH range (Figure S6 of Supporting Information) and at high ionic strength and they were also resistant to photobleaching (Details can be found in Supporting Information). It can be concluded that the synthesized CNDs exhibit excellent photostability, making them suitable for a wide range of applications. Cr(VI) detection. Screening experiments revealed that the fluorescence emitted by the CNDs could be quenched in the presence of Cr(VI) ions and hence, they can be utilized as a fluorescent probe. A more pronounced decrease in the fluorescence intensity is recorded on the first emission peak, compared to the second one (as seen in Figure S7 of Supporting Information), indicating the suitability of the selected wavelengths for the sensitive detection of Cr(VI). The most pivotal factor that may affect the response to Cr(VI) is the pH, since CrO42−, Cr2O72−, HCrO4− and H2CrO4 species coexist in chemical equilibrium in a solution, with the position of the equilibrium to be dependent on the pH and the concentration of Cr(VI).38 As the fluorescence of the as-synthesized CNDs is constant at pH 9 ACS Paragon Plus Environment


between 4 and 9, the quenching of the fluorescence was examined in this range. Highest fluorescence quenching was achieved at pH > 7 (based on Figure S8 of Supporting Information), with minor increase in the fluorescence quenching as the ambience was rendered more alkaline. This can be justified by the high abundance of CrO42− at pH 9 (> 97%), compared to that at pH 7 (~ 75%), highlighting the high sensitivity of the CNDs towards CrO42− species. The high performance of probe in alkaline environment necessitated the use of an alkaline buffer solution. Finally, relevant experiments revealed that the emitted fluorescence of the CNDs is instantaneously quenched in the presence of Cr(VI) and ionic strength up to 1.0 M did not affect it. The quenching of CNDs fluorescence caused by the presence of 100 nM of various metal species is depicted in Figure S9 of Supporting Information. Obviously, the proposed fluorescent probe was not only highly selective towards Cr(VI) over other metal ions, but also it was selective over Cr(III). Therefore, it is suitable for the speciation of chromium. Selectivity was also assessed over certain major classes of compounds, such as amino acids, vitamins, pharmaceuticals and pesticides; no fluorescence quenching was recorded. Figure 3 shows the fall of fluorescence intensity (as F0/F) vs Cr(VI) concentration in the range of 1.7 to181 nM. The analytical figures of merit (provided in Supporting Information and Tables S1 and S2) highlight the potential of the probe for environmental detection applications of Cr(VI).

Figure 3. Fall of the fluorescence intensity at various concentrations of Cr(VI). Inset: linear range for Cr(VI) probing.


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Mechanism of interaction. Quenching of the emitted fluorescence of a fluorophore can be the result of various potential reasons, including static or dynamic quenching, inner-filter effect, fluorescence resonance energy transfer (FRET), electron transfer etc.39 Figure S10 of Supporting Information demonstrates the normalized fluorescence and absorption spectra of the CNDs and Cr(VI) (at pH=7). It is obvious that both the excitation and emission spectra of the CNDs and the molecular absorption spectrum of Cr(VI) markedly overlap. This is directly associated with the pH value of the solution, since different Cr(VI) species have different absorption behavior (a red-shift of the maximum absorption wavelength is noticed, in more acidic solutions). Moreover, the absorption of Cr(VI)-CNDs (which is lower than the sum of the individual spectra of Cr(VI) and CNDs) increases, as the concentration of the quencher increases, both at the excitation and emission wavelength of CNDs (Figure S11 of Supporting Information). All of the above suggest that the inner-filter effect is responsible, to a certain degree, for the observed quenching. Any contribution of FRET mechanism should be ruled out since the distance (r) between the donor and the acceptor was found to be 16.6 nm, namely, higher than 8 nm (details can be found in Supporting Information). Aside from inner-filter effect, one should also examine the static and dynamic quenching mechanism for the system CNDs-Cr(VI), by means of the well-known Stern-Volmer equation

= 1 + where F0 and F are the fluorescence intensities in the absence and presence of the quencher (Cr(VI)), [Q] is the concentration of the quencher and KSV is the SternVolmer constant. Due to the different dependence of the static and dynamic quenching on temperature, Stern-Volmer plots at three different temperatures (i.e. 25, 35 and 45 oC) were constructed over the whole range of Cr(VI) concentrations of the calibration curve. The dependence of quenching on temperature is more obvious at high concentrations of Cr(VI) (Figure S12 of Supporting Information). An increase in temperature leads to a reduction in fluorescence quenching, indicating the dominance of a static quenching mechanism.39 This can be explained by the fact that increased temperatures favor the decrease of complex stability and this is verified by the decrease of the Stern-Volmer constants in the linear region (inlet of Figure S12 of Supporting Information). Moreover, from Figure S7 of Supporting Information, a 11 ACS Paragon Plus Environment


blue-shift of the maximum emission, close to 8 nm is observed, which is also hinting at a static quenching mechanism.39 Although the principles of inner-filter effect differ from static and dynamic quenching, it does not pose a problem for analytical purposes.40 However, it causes interferences in the calculation of the static quenching constants, since an increase in the concentration of Cr(VI) is accompanied by a increasing fluorescence quenching. For the calculation of the quenching constants, correction of the inner-filter effect was carried out for each tested concentration, using the following equation:

= × 10( )/ where Fcorr is the corrected fluorescence value, Fobs is the measured fluorescence value in the presence of various Cr(VI) concentrations and Aex and Aem are the absorption values at the excitation and emission wavelength, respectively. The corrected fluorescence values were plotted against the concentration of Cr(VI) resulting in the fitted Stern-Volmer plot (Figure S13 of Supporting Information), whereof the Ksv value of the system is calculated to be 9.9×102 M-1. It can be seen that the upward curvature of the non-fitted Stern-Volmer plot no longer exists, strengthening the hypothesis that static quenching is applicable in our case. These findings are in accordance with a previous study dealing with a CNDs-Cr(VI) system, which revealed through thermodynamic studies, that interactions arise mainly from hydrogen bonds or Van der Waals forces.41 Promotion of cell proliferation. Examining the cytotoxicity of a material is of high importance, especially, when it is intended for biological applications, such as labeling of cellular or subcellular targets with high resolution. In order to further examine the applicability of the synthesized CNDs as cell imaging agents, we evaluated their induced cytotoxicity over cell lines. The results of the short-term cell viability assay revealed that nearly all four epithelial cells were viable after 24 h of incubation with CNDs, at concentrations between 25 and 200 µg mL-1 (Figures S14 of Supporting Information). Aside from cell proliferation, cell mortality was also investigated using the trypan blue assay, so as to further examine the effect of CNDs on the studied cell lines. In all cases, no obvious mortality was induced, since differences between the CNDs-treated and control cells were non-significant (p

Carbonization of Human Fingernails: Towards the Sustainable

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