DOI: 10.1002/chem.201406330
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& Fluorescent Carbon Dots
Carbon Dots (C-dots) from Cow Manure with Impressive Subcellular Selectivity Tuned by Simple Chemical Modification Cintya D’Angelis do E. S. Barbosa,[a] Jos R. CorrÞa,[b] Gisele A. Medeiros,[b] Gabrielle Barreto,[a] Kelly G. Magalh¼es,[b] Aline L. de Oliveira,[b] John Spencer,[c] Marcelo O. Rodrigues,*[a] and Brenno A. D. Neto*[b]
Abstract: Improved cellular selectivity for nucleoli staining was achieved by simple chemical modification of carbon dots (C-dots) synthesized from waste carbon sources such as cow manure (or from glucose). The C-dots were characterized and functionalized (amine-passivated) with ethylenediamine, affording amide bonds that resulted in bright green fluorescence. The new modified C-dots were successfully ap-
Introduction Carbon quantum dots, also known as C-dots, are small carbon nanoparticles with a quantum-confinement effect, as recently reviewed.[1] C-dots generally consist of C, H, and O atoms found in a quasi-spherical structure with crystalline graphite character,[2] as such, they represent a class of new materials, which was discovered only a few years ago.[1] They offer the advantages of both good biocompatibility coupled to virtually non-existent toxicity. Heteroatoms have already been used to dope C-dots and the overall net result was improved photophysical properties.[3] It is pertinent to highlight the fact that Cdots can be synthesized from a variety of sources, some of which are rather unconventional.[4] This feature opens up the opportunity of using cheap and waste materials to synthesize C-dots by using a direct and simple chemical oxidation methodology.[5]
[a] C. D’Angelis do E. S. Barbosa, G. Barreto, Prof. Dr. M. O. Rodrigues LIMA-Laboratrio de Inorgnica e Materiais Campus Universitrio Darcy Ribeiro, CEP 70904-970 P.O. Box 4478, Brasilia-DF (Brazil) Also, Graduate Program from DQF-UFPE E-mail: [email protected] [b] Dr. J. R. CorrÞa, G. A. Medeiros, Dr. K. G. Magalh¼es, Dr. A. L. de Oliveira, Prof. Dr. B. A. D. Neto Laboratory of Medicinal and Technological Chemistry Institute of Chemistry, University of Braslia, UnB Campus Universitrio Darcy Ribeiro, CEP 70904-970 P.O. Box 4478, Braslia, DF (Brazil) E-mail: [email protected] [c] Prof. Dr. J. Spencer Department of Chemistry, School of Life Sciences University of Sussex, Falmer, Brighton, East Sussex, BN1 9QJ (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201406330. Chem. Eur. J. 2015, 21, 5055 – 5060
plied as selective live-cell fluorescence imaging probes with impressive subcellular selectivity and the ability to selectively stain nucleoli in breast cancer cell lineages (MCF-7). The Cdots were also tested in four other cellular models and showed the same cellular selection in live-cell imaging experiments.
C-dots have been already applied as probes in bioimaging experiments with partial success, but such that they remain very promising materials, as recently reviewed.[6] Some C-dot derivatives have been used, for instance, as bioprobes capable of staining the cell membrane and the cytoplasm without reaching the nucleus to any significant extent in MCF-7 cells.[7] In another example, the cytoplasm was labelled, but without any organelle selectivity.[3] In a different description,[8] C-dot derivatives were capable of staining the cell membrane, the cytoplasmic area, and the nucleus (with weak fluorescence); therefore, showing no cellular selection. An interesting report from Ray and co-workers showed that small C-dots, produced by using 5 m nitric acid (a general procedure irrespective of the carbon source), with no further functionalization, were capable of penetrating the nuclei of Ehrlich ascites carcinoma (EAC) cell lineages;[9] however, it was not possible to locate C-dots in the cytoplasm with any organelle selectivity. Cellular selection is one of the most attractive attributes of new fluorescent probes;[10] therefore, this represents a limiting gap between a tangible successful application of C-dots and translational science; mostly because cellular selection is indispensable for such applications. Achieving efficient organelle selection and an accurate subcellular localization of fluorescent bioprobes is the prime consideration for more rationally designed markers.[11] Today, for a more effective, broad, and practical application of C-dots, there is still a need for effective subcellular selection and the chemical modifications that are necessary to reach this goal still lack in-depth understanding for more rational design. Landmark work of Ma and co-workers demonstrated that C-dots conjugated with folic acid were capable of distinguishing folate-receptor-positive cancer cells from healthy cells,[12] despite displaying no organelle selectivity. Bright fluorescence was also observed for both the cell membrane and the cytoplasm.[12] C-dots have also been tagged
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Full Paper with known and commonly used organic dyes (e.g., fluorescein, rhodamine B, and a-naphthylamine); the tagged C-dots were able to enter into the erythrocyte-enriched fraction of human blood cells,[13] but, once more, no organelle selection was seen. Recent breakthrough work by Kang and co-workers[14] showed small C-dots synthesized from ethylene glycol and polyethylene glycol displayed a tendency to stain nucleoli, but some leakage was still noted. Based on our interest in the development of more selective bioprobes,[15] herein we report that C-dots synthesized from cow manure (or from glucose) by chemical oxidation are capable of selectively staining the cell nuclei of the breast cancer cell lineage MCF-7 after a simple chemical modification with ethylenediamine. Four other cellular models were also tested and showed excellent results with the modified C-dots.
Results and Discussion Cow manure is known to be a rich carbon source owing to its high cellulose content. We therefore considered it to be an ideal source of carbon from waste material. Glucose is a highly abundant and cheap material, especially in Brazil. For these reasons, we decided to use these two sources of carbon to synthesize C-dot derivatives. C-dots produced from both sources of carbon gave similar and reproducible results, indicating that the results presented herein can be achieved irrespective of the carbon sources used to make the C-dots. C-dots from glucose are already known; therefore, we focused on the use of cow manure as the main carbon source. C-dots were synthesized by using the classical chemical oxidation procedure (see Scheme 1 and further details in the Supporting Information). The modified C-dots were produced through a simple chemical modification of the acidic C-dots. Interestingly, the modified C-dots are capable of selectively staining only nucleoli, with impressive selection and subcellular localization, with an intense bright green fluorescence, as will be discussed in due course. Figure 1 shows the emission spectra of the C-dots and of the modified C-dots (treated with ethylenediamine), the luminescence images of the samples under UV-light irradiation
Figure 1. Emissive behavior and morphology of the synthesized C-dots. Emission spectra of the C-dots (A) and modified C-dots (B) acquired at room temperature upon progressive excitation wavelength (from 310 nm to 450). (C) and (D) Nanostructures under white light and UV-light irradiation (lex = 365 nm). Note the fluorescence emission for the modified C-dots is much more pronounced. (E) HR-TEM images for the modified C-dots showing several nanodots. The insets show a high-resolution image of the fringe pattern of an individual particle and the interlayer distance (3.334 ). (F) Honeycomb architecture of a C-dot particle. The inset displays the lattice spacing of the (001) facet of graphite.
(365 nm), and the high-resolution TEM (HR-TEM). The fluorescence associated with the C-dots has been attributed to radioactive recombination arising from electron/hole pairs.[1, 4a] The emission spectra of the samples show a gradual decrease in fluorescence as a function of excitation wavelength. The high bright fluorescence emission, especially that exhibited by the modified C-dot, may be justified by the presence of surface energy trapping sites, which were stabilized by the passivating agent and the net result is the known phenomenon of lem dependent lex.[8] The intense fluorescence emission of the modified C-dots is correlated with a quantum yield of fluorescence of 0.65 upon excitation at 360 nm. The fluorescence properties observed for both type of C-dots are in accordance with previous reports, which have already demonstrated that the passivation strategy with amino reagents such as PEG 1500N, 4,7,10-trioxa-1,13-tridecanediamine (TTDDA), and poly-(ethyleneglycol)diamine, is critical for the optical properties of the Cdots.[16] The modified C-dots showed exceptional photostability and, Scheme 1. C-dots synthesized and tested in this work as selective probes for cell-imaging experiments. Note the over 8 h of irradiation, virtually presence of amide and free amine groups in the modified C-dots. Chem. Eur. J. 2015, 21, 5055 – 5060
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Full Paper no loss of fluorescence was observed (see Figure S1 in the Supporting Information). The temperature effect was also evaluated (Figure S2 in the Supporting Information) and a normal exponential decrease was noted at temperatures ranging from 5 8C to 80 8C. Although the fluorescence intensity decreases with increases in temperature, at temperatures below 40 8C the fluorescence signal is still intense, allowing for good cellular image acquisitions. The effect of pH was also evaluated (Figure S3 in the Supporting Information) and between pH 3–9 the modified C-dots showed very intense signals. The HR-TEM images (Figure 1 E, F) show that the modified Cdots display spheroidal morphologies with narrow size distributions of 4 nm (2–7 nm in the average, also see Figure S4 in the Supporting Information). The insets in Figure 1 E, show the crystalline structure of the C-dots and the lattice fringe with interlayer spacing distance of 3.334 0.047 , similar to that found in graphite 002.[17] In addition, these carbogenic nanocrystals present a lattice space of 2.10 0.034 (Figure 1 F), which is in good agreement with the (001) facet of graphite.[18] Some larger size hollow C-dots could also be isolated (Figure S5 in the Supporting Information) and washed off before the cellular experiments. The infrared spectra (Figure S6 in the Supporting Information) are also in full accordance with the as-prepared and amine-passivated C-dots.[4a] Bioimaging experiments were conducted, with impressive results, by using breast cancer cell (MCF-7) lineages (Figures 2 and 3). Figure 2 shows that MCF-7 cells treated with the unmodified C-dots showed diffuse cytoplasm staining, whereas Figure 3 (modified C-dots) shows a specific and clear staining pattern associated with the nucleoli regions. The intensity of the fluorescent signal emitted (bright green) was very similar for both test conditions, that is, live and adhered cells. Cells that had undergone a fixation procedure with the unmodified C-dots (Figure 2 A, B), clearly showed a more disperse fluores-
Figure 2. Cellular bioimaging experiments for the unmodified C-dots with the MCF-7 cell line. (A) and (B) show the unmodified C-dots’ stain pattern (green) on fixed cells. (C) and (D) show the unmodified C-dots’ stain pattern (green) for the non-fixed (live) cells. Note that for the fixed cells there is an increase in the fluorescence emission through the cytoplasm because of improved C-dots association with the cytoplasm targets. (B) and (D) show the overlay between the C-dots and commercially available DAPI (blue) emissions. Reference scale bar 25 mm. Chem. Eur. J. 2015, 21, 5055 – 5060
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cent emission through the cytoplasm region. On the other hand, experiments in live cells did not show this pronounced effect (Figure 2 C, D). The modified C-dots, interestingly, showed unique fluorescence features and very precise subcellular localization with real and impressive nucleoli selection (Figure 3). The predomi-
Figure 3. Cellular bioimaging experiments for the modified C-dots with the MCF-7 cell line. (A) and (B) show the modified C-dots’ stain pattern (green) for the fixed cells, whereas (C) and (D) show the staining for live cells (). The fluorescent signals are shown to be mostly associated with the nucleoli of all cells. (B) and (D) also show the nucleus staining with commercially available DAPI (blue). Note the arrows to indicate voids (“holes”) in the cytoplasm, thereby showing no association with these organelles. The cytoplasmic associations (pattern and distribution) are with ribosomal components. Reference scale bar 10 mm.
nance of acidic regions found in the nucleoli machinery is most probably responsible for the subcellular direction of the modified C-dots, a result of the presence of basic amine from the ethylene amine moiety of the modified C-dots, as seen in Figure 3. Nucleoli are known to be nuclear sites of ribosomal RNA transcription, processing, and also ribosome assembly, and are difficult to be selectively stained.[19] Due to the specific fluorescent pattern associated with the nucleoli region and the high quality cell images acquired by using the modified Cdots, this novel class of nucleoli marker is suitable for single or multicolor bioimaging (as seen in Figures 2 and 3). No photobleaching was noted for the tested C-dots during the cellular experimental time period, thus being an additional attractive feature of the new bioprobe. The fixed samples showed a diffuse fluorescence pattern throughout the cytoplasm. The cell organelles that had no affinity with these probes appeared as black voids on the cytoplasm (indicated by white arrows in Figure 3). The modified Cdots are likely to have affinity with ribosomal components; furthermore, there is no staining on cytoplasmic organelles. This feature can be explained by the absence of ribosome association with specific cell organelles such as endosomes, lysosomes, and Golgi complex. Ribosomal affinity also explains the diffuse fluorescent pattern observed in the cytosol region. The high fluorescent signal observed in the fixed-cell samples may
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Full Paper be associated with structural changes on the biomolecules created by the standard formalin fixation procedure. After the procedure, the C-dots are likely to be capable of cross-linking primary amino groups with proteins or DNA; therefore, exposing ribosome protein regions with affinity for the tested compound. The explanation provided herein may also explain the leakage observed in the bioimaging experiments of Kang and co-workers.[14] The nucleolus has a different biochemical composition to the rest of the nucleus and these properties can produce different patterns when nucleoli are stained. The typical refractive index associated with its composition makes the nucleoli easily visible by phase contrast or differential interference contrast microscopy. Interestingly, when the cellular nucleus is stained by a DNA-specific dye such as the commercially available DAPI (or Hoechst), the image obtained by epifluorescence microscopy shows the nucleoli as voids in the nuclei, indicating it lacks DNA (see Figure 4) and it is the exact region where the modi-
Figure 5. HUVEC cells staining profile. (A), (B), and (C) show fixed cells, whereas (D), (E), and (F) show live cells. The modified C-dots were found to be most accumulated on specific spots inside the nucleus (indicated by the arrows) correlated with nucleoli. Note the arrows in (A) and (B) show a homogeneous distribution of the C-dots on the cells in prophase stage of mitosis. (C) and (F) show the normal morphological aspects of these cells by phase contrast microscopy. Reference scale bar 25 mm.
Figure 4. DNA stained with DAPI shows voids regions (indicated by yellow arrows) because of the lack of DNA components in MCF-7 cell line. Reference scale bar 10 mm.
fied C-dots are emitting a bright green fluorescence by a highly specific association (Figure 3). The nucleolus is also known to be the region of the nucleus with the most transcriptional activity and it contains several active genes denoting highly decondensed DNA, which is hard to stain. Typically, nucleoli-staining procedures are time consuming and laborious such as fluorescence in situ hybridization (FISH) or the use of fluorescent antibody targeted RNA binding proteins.[20] The bioprobe tested herein is capable of overcoming the aforementioned drawbacks without the prolonged time typically required for this procedure. Lastly, most commercially available dyes that target nucleoli display small Stokes shifts, in contrast with this excellent feature of the tested Cdots, which improves the fluorescence detection and image analyses.[21] Moreover, the C-dots can be stored at ambient temperature without interfering with their capacity for targeting the nucleolus or fluorescence fading. Most commercial nucleoli staining kits must be stored between 20 to 80 8C and are therefore increasingly prone to degradation by repeated freeze–thaw cycles.[22] Finally, other cellular models were tested in bioimaging experiments using the modified C-dots (Figure 5–Figure 8) in Chem. Eur. J. 2015, 21, 5055 – 5060
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order to gain insights as to the generality of the bioprobe. Results demonstrated that the bioprobe is found inside the nucleus, more precisely, located in the specific area associated with the nucleoli. Interestingly, human umbilical vein endothelial cells (HUVEC) were photographed during the mitosis prophase; the bright green fluorescent signal is also dispersed throughout the cytosol region (Figure 5). The fluorescent signal was intense and did not show any photobleaching even after the total image-acquisition time period by using standard operational conditions of the laser beam of the confocal microscope. Also, cytotoxic effects when using the modified C-dots were not statistically significant (by MTT experiments, see Figure S7 in the Supporting Information). The lack of cytotoxicity at low concentrations, that is, at concentrations required for fluorescence bioimaging, is indeed expected for C-dots, as described elsewhere.[5, 6] The staining experiments using MDA-MB-231 (breast cancer cells Figure 6), Caco-2 (colorectal cancer cells, Figure 7) and DU145 (prostate cancer cells, Figure 8) returned similar results and the nucleoli of these cell lineages could be efficiently stained by using the modified C-dots. The analyses already described for the MCF-7 cells were confirmed by the data obtained by these cellular models.
Conclusion We have performed the synthesis and simple chemical modification (passivation, that is, amide formation) of C-dots from
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Figure 8. DU145 staining profile. (A), (B), and (C) show fixed cells, whereas (D), (E), and (F) show live cells. (C) and (F) show the normal morphological aspects of these cells by phase contrast microscopy. The arrows indicate the nucleoli position. Reference scale bar 25 mm.
Figure 6. MDA-MB-231 staining profile. (A), (B), and (C) show fixed cells, whereas (D), (E), and (F) show live cells. (C) and (F) show the normal morphological aspects of these cells by phase contrast microscopy. The arrows indicate the nucleoli position. Reference scale bar 25 mm.
the modified C-dots can be used to stain either live cells or cells submitted to fixative procedures. The C-dots were finally applied to other cell lineages (HUVEC, MDA-MB-231, Caco-2, and DU145) with the same impressive results as those described for MCF-7 cells. As recently reviewed,[1] it is the development of C-dots that enable specific targeting in cellular imaging is highly desirable. With the current work, this gap begins to be filled, opening new avenues for the development of this new and exciting field.
Acknowledgements The authors gratefully acknowledge CAPES, FAPDF, FINATEC, CNPq, INCT-Inami, FACEPE(APT-0859-1.06/08), LabMIC(UFG), DPP-UnBand SMSdrug.net for partial financial support. B.A.D.N. also thanks the INCT-Transcend group.
Keywords: carbon dots · cell imaging · fluorescence · functionalized c-dots · nucleoli
Figure 7. Caco-2 staining profile. (A), (B), and (C) show fixed cells, whereas (D), (E), and (F) show live cells. (C) and (F) show the normal morphological aspects of these cells by phase contrast microscopy. The arrows indicate the nucleoli position. Reference scale bar 25 mm.
cheap and waste carbon sources (e.g., cow manure), leading to an impressive cellular selection in MCF-7 cancer cells towards nucleoli. The cells stained with the modified C-dots proved to be a reliable methodology for visualizing nucleoli with a specific and reproducible staining pattern. In addition, Chem. Eur. J. 2015, 21, 5055 – 5060
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Received: December 3, 2014 Published online on February 18, 2015
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& Fluorescent Carbon Dots
Carbon Dots (C-dots) from Cow Manure with Impressive Subcellular Selectivity Tuned by Simple Chemical Modification Cintya D’Angelis do E. S. Barbosa,[a] Jos R. CorrÞa,[b] Gisele A. Medeiros,[b] Gabrielle Barreto,[a] Kelly G. Magalh¼es,[b] Aline L. de Oliveira,[b] John Spencer,[c] Marcelo O. Rodrigues,*[a] and Brenno A. D. Neto*[b]
Abstract: Improved cellular selectivity for nucleoli staining was achieved by simple chemical modification of carbon dots (C-dots) synthesized from waste carbon sources such as cow manure (or from glucose). The C-dots were characterized and functionalized (amine-passivated) with ethylenediamine, affording amide bonds that resulted in bright green fluorescence. The new modified C-dots were successfully ap-
Introduction Carbon quantum dots, also known as C-dots, are small carbon nanoparticles with a quantum-confinement effect, as recently reviewed.[1] C-dots generally consist of C, H, and O atoms found in a quasi-spherical structure with crystalline graphite character,[2] as such, they represent a class of new materials, which was discovered only a few years ago.[1] They offer the advantages of both good biocompatibility coupled to virtually non-existent toxicity. Heteroatoms have already been used to dope C-dots and the overall net result was improved photophysical properties.[3] It is pertinent to highlight the fact that Cdots can be synthesized from a variety of sources, some of which are rather unconventional.[4] This feature opens up the opportunity of using cheap and waste materials to synthesize C-dots by using a direct and simple chemical oxidation methodology.[5]
[a] C. D’Angelis do E. S. Barbosa, G. Barreto, Prof. Dr. M. O. Rodrigues LIMA-Laboratrio de Inorgnica e Materiais Campus Universitrio Darcy Ribeiro, CEP 70904-970 P.O. Box 4478, Brasilia-DF (Brazil) Also, Graduate Program from DQF-UFPE E-mail: [email protected] [b] Dr. J. R. CorrÞa, G. A. Medeiros, Dr. K. G. Magalh¼es, Dr. A. L. de Oliveira, Prof. Dr. B. A. D. Neto Laboratory of Medicinal and Technological Chemistry Institute of Chemistry, University of Braslia, UnB Campus Universitrio Darcy Ribeiro, CEP 70904-970 P.O. Box 4478, Braslia, DF (Brazil) E-mail: [email protected] [c] Prof. Dr. J. Spencer Department of Chemistry, School of Life Sciences University of Sussex, Falmer, Brighton, East Sussex, BN1 9QJ (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201406330. Chem. Eur. J. 2015, 21, 5055 – 5060
plied as selective live-cell fluorescence imaging probes with impressive subcellular selectivity and the ability to selectively stain nucleoli in breast cancer cell lineages (MCF-7). The Cdots were also tested in four other cellular models and showed the same cellular selection in live-cell imaging experiments.
C-dots have been already applied as probes in bioimaging experiments with partial success, but such that they remain very promising materials, as recently reviewed.[6] Some C-dot derivatives have been used, for instance, as bioprobes capable of staining the cell membrane and the cytoplasm without reaching the nucleus to any significant extent in MCF-7 cells.[7] In another example, the cytoplasm was labelled, but without any organelle selectivity.[3] In a different description,[8] C-dot derivatives were capable of staining the cell membrane, the cytoplasmic area, and the nucleus (with weak fluorescence); therefore, showing no cellular selection. An interesting report from Ray and co-workers showed that small C-dots, produced by using 5 m nitric acid (a general procedure irrespective of the carbon source), with no further functionalization, were capable of penetrating the nuclei of Ehrlich ascites carcinoma (EAC) cell lineages;[9] however, it was not possible to locate C-dots in the cytoplasm with any organelle selectivity. Cellular selection is one of the most attractive attributes of new fluorescent probes;[10] therefore, this represents a limiting gap between a tangible successful application of C-dots and translational science; mostly because cellular selection is indispensable for such applications. Achieving efficient organelle selection and an accurate subcellular localization of fluorescent bioprobes is the prime consideration for more rationally designed markers.[11] Today, for a more effective, broad, and practical application of C-dots, there is still a need for effective subcellular selection and the chemical modifications that are necessary to reach this goal still lack in-depth understanding for more rational design. Landmark work of Ma and co-workers demonstrated that C-dots conjugated with folic acid were capable of distinguishing folate-receptor-positive cancer cells from healthy cells,[12] despite displaying no organelle selectivity. Bright fluorescence was also observed for both the cell membrane and the cytoplasm.[12] C-dots have also been tagged
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Full Paper with known and commonly used organic dyes (e.g., fluorescein, rhodamine B, and a-naphthylamine); the tagged C-dots were able to enter into the erythrocyte-enriched fraction of human blood cells,[13] but, once more, no organelle selection was seen. Recent breakthrough work by Kang and co-workers[14] showed small C-dots synthesized from ethylene glycol and polyethylene glycol displayed a tendency to stain nucleoli, but some leakage was still noted. Based on our interest in the development of more selective bioprobes,[15] herein we report that C-dots synthesized from cow manure (or from glucose) by chemical oxidation are capable of selectively staining the cell nuclei of the breast cancer cell lineage MCF-7 after a simple chemical modification with ethylenediamine. Four other cellular models were also tested and showed excellent results with the modified C-dots.
Results and Discussion Cow manure is known to be a rich carbon source owing to its high cellulose content. We therefore considered it to be an ideal source of carbon from waste material. Glucose is a highly abundant and cheap material, especially in Brazil. For these reasons, we decided to use these two sources of carbon to synthesize C-dot derivatives. C-dots produced from both sources of carbon gave similar and reproducible results, indicating that the results presented herein can be achieved irrespective of the carbon sources used to make the C-dots. C-dots from glucose are already known; therefore, we focused on the use of cow manure as the main carbon source. C-dots were synthesized by using the classical chemical oxidation procedure (see Scheme 1 and further details in the Supporting Information). The modified C-dots were produced through a simple chemical modification of the acidic C-dots. Interestingly, the modified C-dots are capable of selectively staining only nucleoli, with impressive selection and subcellular localization, with an intense bright green fluorescence, as will be discussed in due course. Figure 1 shows the emission spectra of the C-dots and of the modified C-dots (treated with ethylenediamine), the luminescence images of the samples under UV-light irradiation
Figure 1. Emissive behavior and morphology of the synthesized C-dots. Emission spectra of the C-dots (A) and modified C-dots (B) acquired at room temperature upon progressive excitation wavelength (from 310 nm to 450). (C) and (D) Nanostructures under white light and UV-light irradiation (lex = 365 nm). Note the fluorescence emission for the modified C-dots is much more pronounced. (E) HR-TEM images for the modified C-dots showing several nanodots. The insets show a high-resolution image of the fringe pattern of an individual particle and the interlayer distance (3.334 ). (F) Honeycomb architecture of a C-dot particle. The inset displays the lattice spacing of the (001) facet of graphite.
(365 nm), and the high-resolution TEM (HR-TEM). The fluorescence associated with the C-dots has been attributed to radioactive recombination arising from electron/hole pairs.[1, 4a] The emission spectra of the samples show a gradual decrease in fluorescence as a function of excitation wavelength. The high bright fluorescence emission, especially that exhibited by the modified C-dot, may be justified by the presence of surface energy trapping sites, which were stabilized by the passivating agent and the net result is the known phenomenon of lem dependent lex.[8] The intense fluorescence emission of the modified C-dots is correlated with a quantum yield of fluorescence of 0.65 upon excitation at 360 nm. The fluorescence properties observed for both type of C-dots are in accordance with previous reports, which have already demonstrated that the passivation strategy with amino reagents such as PEG 1500N, 4,7,10-trioxa-1,13-tridecanediamine (TTDDA), and poly-(ethyleneglycol)diamine, is critical for the optical properties of the Cdots.[16] The modified C-dots showed exceptional photostability and, Scheme 1. C-dots synthesized and tested in this work as selective probes for cell-imaging experiments. Note the over 8 h of irradiation, virtually presence of amide and free amine groups in the modified C-dots. Chem. Eur. J. 2015, 21, 5055 – 5060
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Full Paper no loss of fluorescence was observed (see Figure S1 in the Supporting Information). The temperature effect was also evaluated (Figure S2 in the Supporting Information) and a normal exponential decrease was noted at temperatures ranging from 5 8C to 80 8C. Although the fluorescence intensity decreases with increases in temperature, at temperatures below 40 8C the fluorescence signal is still intense, allowing for good cellular image acquisitions. The effect of pH was also evaluated (Figure S3 in the Supporting Information) and between pH 3–9 the modified C-dots showed very intense signals. The HR-TEM images (Figure 1 E, F) show that the modified Cdots display spheroidal morphologies with narrow size distributions of 4 nm (2–7 nm in the average, also see Figure S4 in the Supporting Information). The insets in Figure 1 E, show the crystalline structure of the C-dots and the lattice fringe with interlayer spacing distance of 3.334 0.047 , similar to that found in graphite 002.[17] In addition, these carbogenic nanocrystals present a lattice space of 2.10 0.034 (Figure 1 F), which is in good agreement with the (001) facet of graphite.[18] Some larger size hollow C-dots could also be isolated (Figure S5 in the Supporting Information) and washed off before the cellular experiments. The infrared spectra (Figure S6 in the Supporting Information) are also in full accordance with the as-prepared and amine-passivated C-dots.[4a] Bioimaging experiments were conducted, with impressive results, by using breast cancer cell (MCF-7) lineages (Figures 2 and 3). Figure 2 shows that MCF-7 cells treated with the unmodified C-dots showed diffuse cytoplasm staining, whereas Figure 3 (modified C-dots) shows a specific and clear staining pattern associated with the nucleoli regions. The intensity of the fluorescent signal emitted (bright green) was very similar for both test conditions, that is, live and adhered cells. Cells that had undergone a fixation procedure with the unmodified C-dots (Figure 2 A, B), clearly showed a more disperse fluores-
Figure 2. Cellular bioimaging experiments for the unmodified C-dots with the MCF-7 cell line. (A) and (B) show the unmodified C-dots’ stain pattern (green) on fixed cells. (C) and (D) show the unmodified C-dots’ stain pattern (green) for the non-fixed (live) cells. Note that for the fixed cells there is an increase in the fluorescence emission through the cytoplasm because of improved C-dots association with the cytoplasm targets. (B) and (D) show the overlay between the C-dots and commercially available DAPI (blue) emissions. Reference scale bar 25 mm. Chem. Eur. J. 2015, 21, 5055 – 5060
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cent emission through the cytoplasm region. On the other hand, experiments in live cells did not show this pronounced effect (Figure 2 C, D). The modified C-dots, interestingly, showed unique fluorescence features and very precise subcellular localization with real and impressive nucleoli selection (Figure 3). The predomi-
Figure 3. Cellular bioimaging experiments for the modified C-dots with the MCF-7 cell line. (A) and (B) show the modified C-dots’ stain pattern (green) for the fixed cells, whereas (C) and (D) show the staining for live cells (). The fluorescent signals are shown to be mostly associated with the nucleoli of all cells. (B) and (D) also show the nucleus staining with commercially available DAPI (blue). Note the arrows to indicate voids (“holes”) in the cytoplasm, thereby showing no association with these organelles. The cytoplasmic associations (pattern and distribution) are with ribosomal components. Reference scale bar 10 mm.
nance of acidic regions found in the nucleoli machinery is most probably responsible for the subcellular direction of the modified C-dots, a result of the presence of basic amine from the ethylene amine moiety of the modified C-dots, as seen in Figure 3. Nucleoli are known to be nuclear sites of ribosomal RNA transcription, processing, and also ribosome assembly, and are difficult to be selectively stained.[19] Due to the specific fluorescent pattern associated with the nucleoli region and the high quality cell images acquired by using the modified Cdots, this novel class of nucleoli marker is suitable for single or multicolor bioimaging (as seen in Figures 2 and 3). No photobleaching was noted for the tested C-dots during the cellular experimental time period, thus being an additional attractive feature of the new bioprobe. The fixed samples showed a diffuse fluorescence pattern throughout the cytoplasm. The cell organelles that had no affinity with these probes appeared as black voids on the cytoplasm (indicated by white arrows in Figure 3). The modified Cdots are likely to have affinity with ribosomal components; furthermore, there is no staining on cytoplasmic organelles. This feature can be explained by the absence of ribosome association with specific cell organelles such as endosomes, lysosomes, and Golgi complex. Ribosomal affinity also explains the diffuse fluorescent pattern observed in the cytosol region. The high fluorescent signal observed in the fixed-cell samples may
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Full Paper be associated with structural changes on the biomolecules created by the standard formalin fixation procedure. After the procedure, the C-dots are likely to be capable of cross-linking primary amino groups with proteins or DNA; therefore, exposing ribosome protein regions with affinity for the tested compound. The explanation provided herein may also explain the leakage observed in the bioimaging experiments of Kang and co-workers.[14] The nucleolus has a different biochemical composition to the rest of the nucleus and these properties can produce different patterns when nucleoli are stained. The typical refractive index associated with its composition makes the nucleoli easily visible by phase contrast or differential interference contrast microscopy. Interestingly, when the cellular nucleus is stained by a DNA-specific dye such as the commercially available DAPI (or Hoechst), the image obtained by epifluorescence microscopy shows the nucleoli as voids in the nuclei, indicating it lacks DNA (see Figure 4) and it is the exact region where the modi-
Figure 5. HUVEC cells staining profile. (A), (B), and (C) show fixed cells, whereas (D), (E), and (F) show live cells. The modified C-dots were found to be most accumulated on specific spots inside the nucleus (indicated by the arrows) correlated with nucleoli. Note the arrows in (A) and (B) show a homogeneous distribution of the C-dots on the cells in prophase stage of mitosis. (C) and (F) show the normal morphological aspects of these cells by phase contrast microscopy. Reference scale bar 25 mm.
Figure 4. DNA stained with DAPI shows voids regions (indicated by yellow arrows) because of the lack of DNA components in MCF-7 cell line. Reference scale bar 10 mm.
fied C-dots are emitting a bright green fluorescence by a highly specific association (Figure 3). The nucleolus is also known to be the region of the nucleus with the most transcriptional activity and it contains several active genes denoting highly decondensed DNA, which is hard to stain. Typically, nucleoli-staining procedures are time consuming and laborious such as fluorescence in situ hybridization (FISH) or the use of fluorescent antibody targeted RNA binding proteins.[20] The bioprobe tested herein is capable of overcoming the aforementioned drawbacks without the prolonged time typically required for this procedure. Lastly, most commercially available dyes that target nucleoli display small Stokes shifts, in contrast with this excellent feature of the tested Cdots, which improves the fluorescence detection and image analyses.[21] Moreover, the C-dots can be stored at ambient temperature without interfering with their capacity for targeting the nucleolus or fluorescence fading. Most commercial nucleoli staining kits must be stored between 20 to 80 8C and are therefore increasingly prone to degradation by repeated freeze–thaw cycles.[22] Finally, other cellular models were tested in bioimaging experiments using the modified C-dots (Figure 5–Figure 8) in Chem. Eur. J. 2015, 21, 5055 – 5060
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order to gain insights as to the generality of the bioprobe. Results demonstrated that the bioprobe is found inside the nucleus, more precisely, located in the specific area associated with the nucleoli. Interestingly, human umbilical vein endothelial cells (HUVEC) were photographed during the mitosis prophase; the bright green fluorescent signal is also dispersed throughout the cytosol region (Figure 5). The fluorescent signal was intense and did not show any photobleaching even after the total image-acquisition time period by using standard operational conditions of the laser beam of the confocal microscope. Also, cytotoxic effects when using the modified C-dots were not statistically significant (by MTT experiments, see Figure S7 in the Supporting Information). The lack of cytotoxicity at low concentrations, that is, at concentrations required for fluorescence bioimaging, is indeed expected for C-dots, as described elsewhere.[5, 6] The staining experiments using MDA-MB-231 (breast cancer cells Figure 6), Caco-2 (colorectal cancer cells, Figure 7) and DU145 (prostate cancer cells, Figure 8) returned similar results and the nucleoli of these cell lineages could be efficiently stained by using the modified C-dots. The analyses already described for the MCF-7 cells were confirmed by the data obtained by these cellular models.
Conclusion We have performed the synthesis and simple chemical modification (passivation, that is, amide formation) of C-dots from
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Figure 8. DU145 staining profile. (A), (B), and (C) show fixed cells, whereas (D), (E), and (F) show live cells. (C) and (F) show the normal morphological aspects of these cells by phase contrast microscopy. The arrows indicate the nucleoli position. Reference scale bar 25 mm.
Figure 6. MDA-MB-231 staining profile. (A), (B), and (C) show fixed cells, whereas (D), (E), and (F) show live cells. (C) and (F) show the normal morphological aspects of these cells by phase contrast microscopy. The arrows indicate the nucleoli position. Reference scale bar 25 mm.
the modified C-dots can be used to stain either live cells or cells submitted to fixative procedures. The C-dots were finally applied to other cell lineages (HUVEC, MDA-MB-231, Caco-2, and DU145) with the same impressive results as those described for MCF-7 cells. As recently reviewed,[1] it is the development of C-dots that enable specific targeting in cellular imaging is highly desirable. With the current work, this gap begins to be filled, opening new avenues for the development of this new and exciting field.
Acknowledgements The authors gratefully acknowledge CAPES, FAPDF, FINATEC, CNPq, INCT-Inami, FACEPE(APT-0859-1.06/08), LabMIC(UFG), DPP-UnBand SMSdrug.net for partial financial support. B.A.D.N. also thanks the INCT-Transcend group.
Keywords: carbon dots · cell imaging · fluorescence · functionalized c-dots · nucleoli
Figure 7. Caco-2 staining profile. (A), (B), and (C) show fixed cells, whereas (D), (E), and (F) show live cells. (C) and (F) show the normal morphological aspects of these cells by phase contrast microscopy. The arrows indicate the nucleoli position. Reference scale bar 25 mm.
cheap and waste carbon sources (e.g., cow manure), leading to an impressive cellular selection in MCF-7 cancer cells towards nucleoli. The cells stained with the modified C-dots proved to be a reliable methodology for visualizing nucleoli with a specific and reproducible staining pattern. In addition, Chem. Eur. J. 2015, 21, 5055 – 5060
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Received: December 3, 2014 Published online on February 18, 2015
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