Subcellular localization of mitochondria targeted TPP-CDs and non-targeted CDs after 4 h incubation with HeLa cells and colocalization with MitoTracker Deep Red. Scale bar 1⁄4 20 m m. Excitation wavelength for MitoTracker is 635 nm. 

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Context 1

... absorption spectra were used to investigate the binding of TPP to CDs. As displayed in Fig. 2A, CDs show virtual absorption peak at around 340 nm. A  er conjugation with TPP, which has absorption peaks at 260, 267 and 275 nm, the UV-vis spectrum of TPP-CDs contains the typical peaks of both CDs and TPP, which suggests that TPP was successfully conjugated onto the CDs. The CDs display an excitation-wavelength dependent  uorescence property (Fig. 2B), which was consis- tent with the previous reports. 14,15 This phenomenon allows for multicolor emission under di ff erent excitation wavelengths, which is an important property for biomedical applications. 1 It should be noted that the maximum emission peak of TPP-CDs changed from 425 to 416 nm when it was excited at 340 nm because of the introduction of the TPP moiety on the surface of CDs (Fig. 2C). To con  rm whether TPP-CDs are useful in confocal imaging, the  uorescence spectra were investigated as displayed in Fig. 2D, which indicated that the TPP-CDs are still able to emit  uorescence at the excitation wavelength of 488 nm. Using quinine sulfate as a standard, the  uorescence QY of TPP-CDs was about 15%. In addition, two-photon  uorescence emission from the CDs was observed upon excitation at 760 nm with a femtosecond laser. To con  rm whether the  uorescence originates from the two-photon absorption process with laser excitation in the NIR, we examined the change of  uorescence intensity by varying the power of the 760 nm laser. As shown in Fig. S4, † the photoluminescence (PL) intensity and the excited laser power demonstrate an obvious quadratic relationship, which reveals that the two-photon excitation is truly responsible for the emission in nature. These results indicate that CDs and TPP-CDs could be expected for cell imaging with two-photon  uorescence microscopy. We also examined the e ff ect of pH on the PL property of CDs and TPP-CDs in Britton – Robinson bu ff er solution (Fig. S5 † ). There are 22% and 17% increases of PL intensity for CDs and TPP-CDs, respectively, when the pH values were changed from 2.0 to 12.0. The presence of many – NH 2 and – COOH groups on the surface of CDs may account for the properties, which is quite similar to amino acids. Amino acids are ampholytes and have varying isoelectric points and dissociation constants. 16 Moreover, the emission wavelength showed a blue-shi  for the PL spectra for both CDs and TPP-CDs (Fig. S6 † ). The pH e ff ect indicates that the  uorescent intensity would not be signi  cantly quenched in acidic or alkaline media. In addition, both CDs and TPP-CDs exhibit good photostability as compared with  uorescein (Fig. S7 † ). The  uorescence intensity of both CDs and TPP-CDs decreased about 10% a  er being irradiated for 110 seconds, whereas the emission intensity of  uorescein decreased 55% during the same period. The CDs and TPP-CDs derived from citric acid and urea with good photostability might have potential applications in a wide range of pH in various  elds of nano-biotechnology. The applicability for one- and two-photon bioimaging was assessed by incubating HeLa cells with the CDs and TPP-CDs, respectively. We observed that the HeLa cells became bright under excitation at 405 nm, 488 nm (one-photon) and 760 nm (two-photon) laser respectively (Fig. S8 † ). The cell images clearly showed a high contrast  uorescence of the CDs and TPP-CDs around each nucleus, indicating that these CDs can be used for living cells imaging without invading the nucleus in both the one- and two-photon models. 17 The location of CDs and TPP- CDs in live cells for both one- and two-photon imaging e ffi ciently matches. It should be noted that a low laser power of 0.27 W was su ffi cient to induce strong two-photon  uorescence of these CDs internalized in HeLa cells. The quantitative analysis with calibration curve shows that the uptake of TPP-CDs of each cell is around 7.17 Â 10 À 7 mg (Fig. S9 † ). The e ffi cient cellular uptake and strong one- and two-photon  uorescence emitting ability revealed that these carbon nanomaterials can be used as potential probes for high contrast bioimaging. The mitochondria-targeting properties of the TPP-CDs were investigated by incubating the probes with HeLa cells using non-targeted CDs as control. The confocal microscopy analysis of the cells treated with TPP-CDs showed a greater uptake of TPP-CDs than of CDs in the mitochondria of the cells (Fig. 3). The details of the experimental procedure are described in the ESI. † Quantitative analysis using the Olympus FluoView “ colocalization analysis ” plug-in revealed the signi  cant colocalization of the TPP-CDs with MitoTracker Deep Red in the mitochondria of the cells (Pearson's correlation coe ffi cient, r 1⁄4 0.62). In regards to the non-targeted CDs, a lower r value ( r 1⁄4 0.46) was obtained as demonstrated by the di ff erent posi- tion of the blue signals of CDs and the mitochondrial red staining (Fig. 3). An analogous result in TCA-8113 tongue squamous carcinoma cells treated with TPP-CDs was also obtained as shown in Fig. S10. † The signi  cant colocalization of the TPP-CDs with MitoTracker in the mitochondria of TCA-8113 was also found ( r 1⁄4 0.55, CDs control r 1⁄4 0.19). The highly e ffi cient mitochondrial uptake of the targeted TPP-CDs can be attributed to their high bu ff ering capacity provided by lipophilic TPP. 11 The delocalized positive charge of these cations enables them to easily permeate lipid bilayers and to accumulate within the mitochondria because of the large membrane potential ( À 150 to À 70 mV, negative inside). 11 The plasma membrane potential ( À 30 to À 60 mV, negative inside) also drives the accumulation of these molecules from the extracellular  uid into isolated cells, from where they are concentrated further within mitochondria. 11 The cellular uptake and intracellular tra ffi cking of nanoparticles usually occurs along with competing pathways. Colocalization with the lysosomes of both TPP-CDs and CDs was also investigated; TPP-CDs resulted in a lower accumulation ( r 1⁄4 0.17), whereas CDs showed greater uptake ( r 1⁄4 0.33) in the lysosomes of the HeLa cells (Fig. 4). The data show that the uptake of targeted TPP-CDs by mitochondria is considerably higher than that by lysosomes during the tra ffi cking process, suggesting the good mitochondria accumulation ability of TPP- CDs. All these results reveal that TPP-CDs target the cellular mitochondria in living samples. The biocompatibility of probes is one of the most important properties to consider their bio-applications. MTT (3-(4,5- dimethylthiazole-2-yl)-2,5-phenyltetrazolium bromide) assay in HeLa cells was carried out to investigate the cytotoxicity of the as-prepared CDs and TPP-CDs. As shown in Fig. S11, † cell viability remains more than 84% a  er incubation with a relatively high concentration of TPP-CDs (6 mg mL À 1 ) for 8 hours, demonstrating the lower toxicity of the NPs to living cells. In addition, the viability of HeLa cells incubated with CDs was more than 96%. It is clear that the cytotoxicity of both CDs and TPP-CDs was relatively low at shorter incubation time, which is essential for their further applications in living cells imaging. In summary, we have developed a general and facile method to prepare a mitochondria-targeted  uorescent probe based on TPP modi  ed  uorescent CDs derived from low-cost citric acid and urea. The resulting TPP-CDs were well-dispersed in water and possess excitation tunable photoluminescence, high photostability, low cytotoxicity and suitability for their use in various pH conditions. Signi  cantly, the TPP-CDs showed clear two-photon emission under the excitation of NIR  uorescence with the possibility for two-photon imaging of live cells. Moreover, the TPP-CDs were applied for mitochondria-targeted tumor cell imaging, which were capable of producing outstanding mitochondria accumulation. All these results demonstrated that the prepared TPP-CDs may have potential to be a  uorescent probes for biomedical applications. This work was supported by the National Nature Science Foundation of China (91227126), National Special Fund for Key Scienti  c Instrument and Equipment Development (2013YQ17046307) and the Nature Science Foundation of Liaoning Province, China (2013020177). 1 S. N. Baker and G. A. Baker, Angew. Chem., Int. Ed. , 2010, 49 , 6726 – 6744. 2 Y.-P. Sun, B. Zhou, Y. Lin, W. Wang, K. S. Fernando, P. Pathak, M. J. Meziani, B. A. Harru ff , X. Wang and H. Wang, J. Am. Chem. Soc. , 2006, 128 , 7756 – 7757. 3 X. Wang, K. Qu, B. Xu, J. Ren and X. Qu, J. Mater. Chem. , 2011, 21 , 2445 – 2450. 4 D. J. Campbell, M. J. Andrews and K. J. Stevenson, J. Chem. Educ. , 2012, 89 , 1280 – 1287. 5 M. J. Ruedas-Rama, J. D. Walters, A. Orte and E. A. Hall, Anal. Chim. Acta , 2012, 751 , 1 – 23. 6 H. Tao, K. Yang, Z. Ma, J. Wan, Y. Zhang, Z. Kang and Z. Liu, Small , 2012, 8 , 281 – 290. 7 Y. Wang, P. Anilkumar, L. Cao, J.-H. Liu, P. G. Luo, K. N. Tackett, S. Sahu, P. Wang, X. Wang and Y.-P. Sun, Exp. Biol. Med. , 2011, 236 , 1231 – 1238. 8 B. Kong, A. Zhu, C. Ding, X. Zhao, B. Li and Y. Tian, Adv. Mater. , 2012, 24 , 5844 – 5848. 9 L. Cao, X. Wang, M. J. Meziani, F. Lu, H. Wang, P. G. Luo, Y. Lin, B. A. Harru ff , L. M. Veca and D. Murray, J. Am. Chem. Soc. , 2007, 129 , 11318 – 11319. 10 S. Marrache and S. Dhar, Proc. Natl. Acad. Sci. U. S. A. , 2012, 109 , 16288 – 16293. 11 R. A. Smith, C. M. Porteous, A. M. Gane and M. P. Murphy, Proc. Natl. Acad. Sci. U. S. A. , 2003, 100 , 5407 – 5412. 12 B. C. Dickinson and C. J. Chang, J. Am. Chem. Soc. , 2008, 130 , 9638 – 9639. 13 B. Johnsson, S. Löf as and G. Lindquist, Anal. Biochem. , 1991, 198 , 268 – 277. 14 M. Tan, L. Zhang, R. Tang, X. Song, Y. Li, H. Wu, Y. Wang, G. Lv, W. Liu and X. Ma, Talanta , 2013, 115 , 950 – 956. 15 H. Yan, M. Tan, D. Zhang, F. Cheng, H. Wu, M. Fan, X. Ma and J. Wang, Talanta , 2013, 108 , 59 ...

Context 2

... NH 2 as compared with CDs. The broad peak at 3400 cm À 1 can be attributed to the O – H vibration stretch of the carboxylic moiety and N – H. The zeta potential of CDs is À 18.0 mV due to the existence of – OH/ – COOH groups on the surface of CDs. A  er conjugation with TPP, the zeta potential of TPP-CDs becomes À 6.3 mV (Fig. 1D). Because of the presence of a large amount of – COOH, – OH, and – NH 2 , the prepared TPP-CDs exhibit high water solubility, which is essential for further bio-imaging application. UV-vis absorption spectra were used to investigate the binding of TPP to CDs. As displayed in Fig. 2A, CDs show virtual absorption peak at around 340 nm. A  er conjugation with TPP, which has absorption peaks at 260, 267 and 275 nm, the UV-vis spectrum of TPP-CDs contains the typical peaks of both CDs and TPP, which suggests that TPP was successfully conjugated onto the CDs. The CDs display an excitation-wavelength dependent  uorescence property (Fig. 2B), which was consis- tent with the previous reports. 14,15 This phenomenon allows for multicolor emission under di ff erent excitation wavelengths, which is an important property for biomedical applications. 1 It should be noted that the maximum emission peak of TPP-CDs changed from 425 to 416 nm when it was excited at 340 nm because of the introduction of the TPP moiety on the surface of CDs (Fig. 2C). To con  rm whether TPP-CDs are useful in confocal imaging, the  uorescence spectra were investigated as displayed in Fig. 2D, which indicated that the TPP-CDs are still able to emit  uorescence at the excitation wavelength of 488 nm. Using quinine sulfate as a standard, the  uorescence QY of TPP-CDs was about 15%. In addition, two-photon  uorescence emission from the CDs was observed upon excitation at 760 nm with a femtosecond laser. To con  rm whether the  uorescence originates from the two-photon absorption process with laser excitation in the NIR, we examined the change of  uorescence intensity by varying the power of the 760 nm laser. As shown in Fig. S4, † the photoluminescence (PL) intensity and the excited laser power demonstrate an obvious quadratic relationship, which reveals that the two-photon excitation is truly responsible for the emission in nature. These results indicate that CDs and TPP-CDs could be expected for cell imaging with two-photon  uorescence microscopy. We also examined the e ff ect of pH on the PL property of CDs and TPP-CDs in Britton – Robinson bu ff er solution (Fig. S5 † ). There are 22% and 17% increases of PL intensity for CDs and TPP-CDs, respectively, when the pH values were changed from 2.0 to 12.0. The presence of many – NH 2 and – COOH groups on the surface of CDs may account for the properties, which is quite similar to amino acids. Amino acids are ampholytes and have varying isoelectric points and dissociation constants. 16 Moreover, the emission wavelength showed a blue-shi  for the PL spectra for both CDs and TPP-CDs (Fig. S6 † ). The pH e ff ect indicates that the  uorescent intensity would not be signi  cantly quenched in acidic or alkaline media. In addition, both CDs and TPP-CDs exhibit good photostability as compared with  uorescein (Fig. S7 † ). The  uorescence intensity of both CDs and TPP-CDs decreased about 10% a  er being irradiated for 110 seconds, whereas the emission intensity of  uorescein decreased 55% during the same period. The CDs and TPP-CDs derived from citric acid and urea with good photostability might have potential applications in a wide range of pH in various  elds of nano-biotechnology. The applicability for one- and two-photon bioimaging was assessed by incubating HeLa cells with the CDs and TPP-CDs, respectively. We observed that the HeLa cells became bright under excitation at 405 nm, 488 nm (one-photon) and 760 nm (two-photon) laser respectively (Fig. S8 † ). The cell images clearly showed a high contrast  uorescence of the CDs and TPP-CDs around each nucleus, indicating that these CDs can be used for living cells imaging without invading the nucleus in both the one- and two-photon models. 17 The location of CDs and TPP- CDs in live cells for both one- and two-photon imaging e ffi ciently matches. It should be noted that a low laser power of 0.27 W was su ffi cient to induce strong two-photon  uorescence of these CDs internalized in HeLa cells. The quantitative analysis with calibration curve shows that the uptake of TPP-CDs of each cell is around 7.17 Â 10 À 7 mg (Fig. S9 † ). The e ffi cient cellular uptake and strong one- and two-photon  uorescence emitting ability revealed that these carbon nanomaterials can be used as potential probes for high contrast bioimaging. The mitochondria-targeting properties of the TPP-CDs were investigated by incubating the probes with HeLa cells using non-targeted CDs as control. The confocal microscopy analysis of the cells treated with TPP-CDs showed a greater uptake of TPP-CDs than of CDs in the mitochondria of the cells (Fig. 3). The details of the experimental procedure are described in the ESI. † Quantitative analysis using the Olympus FluoView “ colocalization analysis ” plug-in revealed the signi  cant colocalization of the TPP-CDs with MitoTracker Deep Red in the mitochondria of the cells (Pearson's correlation coe ffi cient, r 1⁄4 0.62). In regards to the non-targeted CDs, a lower r value ( r 1⁄4 0.46) was obtained as demonstrated by the di ff erent posi- tion of the blue signals of CDs and the mitochondrial red staining (Fig. 3). An analogous result in TCA-8113 tongue squamous carcinoma cells treated with TPP-CDs was also obtained as shown in Fig. S10. † The signi  cant colocalization of the TPP-CDs with MitoTracker in the mitochondria of TCA-8113 was also found ( r 1⁄4 0.55, CDs control r 1⁄4 0.19). The highly e ffi cient mitochondrial uptake of the targeted TPP-CDs can be attributed to their high bu ff ering capacity provided by lipophilic TPP. 11 The delocalized positive charge of these cations enables them to easily permeate lipid bilayers and to accumulate within the mitochondria because of the large membrane potential ( À 150 to À 70 mV, negative inside). 11 The plasma membrane potential ( À 30 to À 60 mV, negative inside) also drives the accumulation of these molecules from the extracellular  uid into isolated cells, from where they are concentrated further within mitochondria. 11 The cellular uptake and intracellular tra ffi cking of nanoparticles usually occurs along with competing pathways. Colocalization with the lysosomes of both TPP-CDs and CDs was also investigated; TPP-CDs resulted in a lower accumulation ( r 1⁄4 0.17), whereas CDs showed greater uptake ( r 1⁄4 0.33) in the lysosomes of the HeLa cells (Fig. 4). The data show that the uptake of targeted TPP-CDs by mitochondria is considerably higher than that by lysosomes during the tra ffi cking process, suggesting the good mitochondria accumulation ability of TPP- CDs. All these results reveal that TPP-CDs target the cellular mitochondria in living samples. The biocompatibility of probes is one of the most important properties to consider their bio-applications. MTT (3-(4,5- dimethylthiazole-2-yl)-2,5-phenyltetrazolium bromide) assay in HeLa cells was carried out to investigate the cytotoxicity of the as-prepared CDs and TPP-CDs. As shown in Fig. S11, † cell viability remains more than 84% a  er incubation with a relatively high concentration of TPP-CDs (6 mg mL À 1 ) for 8 hours, demonstrating the lower toxicity of the NPs to living cells. In addition, the viability of HeLa cells incubated with CDs was more than 96%. It is clear that the cytotoxicity of both CDs and TPP-CDs was relatively low at shorter incubation time, which is essential for their further applications in living cells imaging. In summary, we have developed a general and facile method to prepare a mitochondria-targeted  uorescent probe based on TPP modi  ed  uorescent CDs derived from low-cost citric acid and urea. The resulting TPP-CDs were well-dispersed in water and possess excitation tunable photoluminescence, high photostability, low cytotoxicity and suitability for their use in various pH conditions. Signi  cantly, the TPP-CDs showed clear two-photon emission under the excitation of NIR  uorescence with the possibility for two-photon imaging of live cells. Moreover, the TPP-CDs were applied for mitochondria-targeted tumor cell imaging, which were capable of producing outstanding mitochondria accumulation. All these results demonstrated that the prepared TPP-CDs may have potential to be a  uorescent probes for biomedical applications. This work was supported by the National Nature Science Foundation of China (91227126), National Special Fund for Key Scienti  c Instrument and Equipment Development (2013YQ17046307) and the Nature Science Foundation of Liaoning Province, China (2013020177). 1 S. N. Baker and G. A. Baker, Angew. Chem., Int. Ed. , 2010, 49 , 6726 – 6744. 2 Y.-P. Sun, B. Zhou, Y. Lin, W. Wang, K. S. Fernando, P. Pathak, M. J. Meziani, B. A. Harru ff , X. Wang and H. Wang, J. Am. Chem. Soc. , 2006, 128 , 7756 – 7757. 3 X. Wang, K. Qu, B. Xu, J. Ren and X. Qu, J. Mater. Chem. , 2011, 21 , 2445 – 2450. 4 D. J. Campbell, M. J. Andrews and K. J. Stevenson, J. Chem. Educ. , 2012, 89 , 1280 – 1287. 5 M. J. Ruedas-Rama, J. D. Walters, A. Orte and E. A. Hall, Anal. Chim. Acta , 2012, 751 , 1 – 23. 6 H. Tao, K. Yang, Z. Ma, J. Wan, Y. Zhang, Z. Kang and Z. Liu, Small , 2012, 8 , 281 – 290. 7 Y. Wang, P. Anilkumar, L. Cao, J.-H. Liu, P. G. Luo, K. N. Tackett, S. Sahu, P. Wang, X. Wang and Y.-P. Sun, Exp. Biol. Med. , 2011, 236 , 1231 – 1238. 8 B. Kong, A. Zhu, C. Ding, X. Zhao, B. Li and Y. Tian, Adv. Mater. , 2012, 24 , 5844 – 5848. 9 L. Cao, X. Wang, M. J. Meziani, F. Lu, H. Wang, P. G. Luo, Y. Lin, B. A. Harru ff , L. M. Veca and D. Murray, J. Am. Chem. Soc. , 2007, 129 , 11318 – 11319. 10 S. Marrache and S. Dhar, Proc. Natl. Acad. Sci. U. S. A. , 2012, 109 , ...