Supporting Information

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1 Supporting Information Morpholine Derivative-functionalized Carbon Dots Based Fluorescent Probe for Highly Selective Lysosomal Imaging in Living Cells Luling Wu, Xiaolin Li, Yifei Ling, Chusen Huang*, and Nengqin Jia* The Education Ministry Key Laboratory of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials, and Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors, Department of Chemistry, Shanghai Normal University, 100 Guilin Road, Shanghai , China. S-1

2 Table of Contents S2-S4 S4-S7 S7-S10 S10-S11 S12-S18 S19 S19-S20 1. Synthetic experiments 2. Characterization of synthesized carbon dots 3. Detailed protocols for evaluating the photophysical performance of CDs, CDs-MP, and CDs-PEI-ML 4. Detailed protocols for cell culture and CCK-8 assay 5. Detailed protocols for live cell imaging with CDs, CDs-MP, and CDs-PEI-ML 6. NMR spectrum 7. References 1. Synthetic experiments Materials and methods All chemical reagents and solvents were purchased from commercial sources and used without further purification. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxy-succinimide) were purchased from Sigma-Aldrich. Citric acid monohydrate, 4-(2-Aminoethyl)morpholine and polyethylenimine (PEI, M.W. 1800, 99%) were purchased from Aladdin (Shanghai, China). Thin-layer chromatography (TLC) was performed on silica gel plates and visualized by UV. Column chromatography was performed using silica gel (Hailang, Qingdao) mesh. Cell Counting Kit-8 (CCK-8) was purchased from SIGMA-ALDRICH. 1 H and 13 C NMR spectra were recorded employing a Bruker AV-400 spectrometer with chemical shifts expressed in parts per million (in deuteriochloroform, deuterium oxide, Me 4 Si as internal standard). The size and morphology of the Carbon dots in a dried state were measured by high resolution transmission electron microscopy (HRTEM) using a JEOL JEM-2100 microscope operating at 200 kv. Zeta potential of samples dispersed in water was measured with dynamic light scattering (DLS) using a MALVERN Nano ZS90 instrument. Fluorescence measurements were determined on a Hitachi Fluorescence Spectrophotometer F Excitation and emission slit widths were modified to adjust the fluorescence intensity to a suitable range. Absorption spectra were measured on a HitachiU-3900 UV/VIS spectrophotometer. S-2

3 Scheme S1. Synthesis of ML, CDs, CDs-MP, and CDs-PEI-ML. Synthesis of ML. A mixture of 2-morpholinoethan-1-amine (1.4 g, 16 mmol, 1.0 eq), 3-bromopropanoic acid (2.9, 19 mmol, 1.2 eq), potassium carbonate (3.3 g, 24 mmol, 1.5 eq) was dissolve acetonitrile (30ml) and then heated under 40 for 10 h under nitrogen. TLC showed the reaction was completed, filtrated. The solvent was evaporated in vacuo and the residue was purified by flash column chromatography eluting with CH 2 Cl 2 /CH 3 OH (8:1) to afford product as milky white solid (2.01 g, 79% yield). 1 H NMR (400 MHz, CDCl 3 ) δ (s, 1H), 3.82 (s, 4H), 2.86 (t, J = 6.0 Hz, 2H), 2.77 (s, 4H), 2.55 (t, J = 6.0 Hz, 2H). 13 C NMR (400 MHz, CDCl 3 ) δ , 65.88, 53.77, 52.30, Synthesis of CDs. CDs were prepared by the low temperature pyrolysis method with citric acid monohydrate as the carbon precursor. Acid monohydrate (5 g) was dissolved in 30 ml hot water, and then heated moderately (about 160 ) by a heating mantle to evaporate water until a uniform pale-yellow gel was obtained. Before the gel was sorched, 1ml cold water was added under heating continuously. The above procedure was repeated for 10 times (in 2 h). Finally, we got pale-yellow solid. Synthesis of CDs-MP. To a solution of CDs (0.7 g) and MP (1.2 g) in 25 ml ultrapure water, 1 g mg Nhydroxy-succinimide (NHS) and 5 g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were added. The reaction mixture was continued to stirring for 24 h at room temperature. Then, the final CDs-MP were dialyzed (M.W. 1000) with ultrapure water for about 3 h, and then drying at a low temperature to get brown oil. S-3

4 Synthesis of CDs-PEI. CDs-PEI were synthesized according to the reported procedures 1. In brief, branched polyethylenimine (1.8 g) and citric acid monohydrate (3.1 g) were dissolved in 30 ml hot water, and then heated moderately (about 160 ) by a heating mantle to evaporate water until a uniform pale yellow gel was obtained. Before the gel was sorched, 2ml water was added under heating continuously. The same procedure was repeated at least 10 times, the color of gel turned from pale yellow to orange, suggesting CDs-PEI were successfully synthsized. The cude product obtained was dialyzed (M.W. 3000) with ultrapure water to wash out unreacted reagents for about 12 h, and then drying at a low temperature to get orange solid. Synthesis of CDs-PEI-ML. Carbodiimide chemistry was employed to covalently conjugate 2 compound ML onto surface of CDs-PEI, where 745 mg compound ML and as-prepared CDs-PEI were suspended in 30 ml ultrapure water containing 539 mg Nhydroxy-succinimide (NHS) and 2.69 g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and were allowed to react at room temperature for 24 h under gentle stirring. Then, the final CDs-PEI-ML were extensively dialyzed (M.W. 1000) against water to wash out unreacted reagents for about 10 h, and then drying at a low temperature to get brown solid. 2. Characterization of synthesized carbon dots 2.1 The surface functionalization of carbon dots with small molecules (MP and ML, respectively) were identified by nuclear magnetic resonance (NMR) spectroscopy. Figure S1. 1 H NMR spectra of (a) CDs, (b) MP, (c) CDs-MP in 0.5 ml of D 2 O. S-4

5 Figure S2. 1 H NMR spectra of (a) ML, (b) CDs-PEI, (c) CDs-PEI-ML in 0.5 ml of D 2 O. 2.2 FT-IR spectra of CDs, CDs-MP, CDs-PEI and CDs-PEI-ML FT-IR spectra were recorded on a ThermoNicolet Avatar 370 FT-IR spectrometer. The broad band at cm -1 (ν N H ) and cm -1 (δ N H ) is ascribed to the vibration of NH 2 groups, indicating the amine groups on the surface of CDs-PEI (curve c). In comparison to CDs-PEI, the absence of peak at cm -1 (δ N H ) and the absorption peak of ν C=O with a slight shift from cm -1 to cm -1, indicating compound ML have successfully conjugated on the surface of CDs-PEI. Figure S3. FTIR spectra of (a) CDs, (b) CDs-MP, (c) CDs-PEI, and (d) CDs-PEI-ML. S-5

6 2.3 Elemental analysis of CDs, CDs-MP, CDs-PEI and CDs-PEI-ML Elemental analysis of four types of carbon dots was conducted by Vario EL cube elemental analyzer which is purchased from Germany. The difference of carbon dots in element distribution is obvious for CDs, CDs-MP, CDs-PEI and CDs-PEI-ML. Thus, after the element doping (e.g., O, N) and the groups on the surface of carbon dots could affect the physical and chemical properties and optical performance of CDs, CDs-MP, and CDs-PEI-ML, which makes our further understanding of the fluorescence performance of these carbon on Figure 3 and Figure 4 3. Table S1. Elemental analysis of CDs, CDs-MP, CDs-PEI and CDs-PEI-ML. 2.4 XPS spectra of CD, CDs-MP and CDs-PEI-ML X-ray photoelectron spectroscopy (XPS)measurements were performed on a Perkin-Elmer PHI 5000CESCA system with a base pressure of 10-9 Torr. In order to further investigate the optical properties of the surface groups and the distribution of elements on the carbon dots, XPS measurements were performed. As shown in Figure S4, CDs mainly composed of C and O, which could be ascribed to the characteristic peaks of C 1s (284.9 ev, Fig. S4b), O 1s (532.2 ev, Fig. S4d). Meanwhile, XPS results confirmed the presence of N on the surface of CDs-MP and the surface of CDs-PEI-ML (Figure S4a and 4c). S-6

7 Figure S4. (a) XPS of CDs, CDs-MP and CDs-PEI-ML. C 1s spectra (b), N 1s spectra (c), O 1s spectra (d). 3. Detailed protocols for evaluating the photophysical performance of CDs, CDs-MP, and CDs-PEI-ML. The quantum yield (Φ) is considered as an important factor for quantitatively evaluating fluorescent intensity of fluorescent materials. Φ is the ratio of photons absorbed to photons emitted through fluorescence and the number of absorbed photons. We measured the Φs according to A Guide to Recording Fluorescence Quantum Yields by Jobin Yvon Horiba Ltd at: pdf. Quinine sulfate dissolved in 0.1M H 2 SO 4 was taken as a standard, whose Φ is The Φs were determined by comparing the intergrated fluorescence intensity and the absorbance value (less than 0.1 at excitation). In our experiment, we used slope method to calculate the Φ of three kinds of carbon dots using the following equation: Φ f = Φ f (Grad sample /Grad f ) (n sample /n f ) 2 where Φ is fluorescence quantum yield, Grad is the slope by the carves in Figure 4 in the manuscript and n is refractive index (1.33 for water and a 0.1M H 2 SO 4 solution). The subscript f refers to the standards and sample refers to the unkown samples. For these solutions, n sample /n f = 1. Six different concentrations for the standard and Carbon dots were measured to obtain the slopes. S-7

8 Figure S5. The different color and solid-state fluorescence of (a) CDs, (b) CDs-MP, (c) CDs-PEI-ML. The photos were taken by camera directly under the irradiation with hand-held UV lamp (365nm) for solid-state fluorescence and for the bright field under natural light. S-8

9 Figure S6. Normalized fluorescence intensity of CDs (a), CDs-MP (b), CDs-PEI-ML (c) in water, respectively. (d) Comparison of fluorescence intensity changes between the prepared carbon dots in water with various ph values. Figure S7. The time courses of maximum fluorescence intensity of (a) CDs (0.2 mg ml -1, λ ex = 320 nm), (b) CDs-MP (0.2 mg ml -1, λ ex = 360 nm) and (c) CDs-PEI-ML (0.2 mg ml -1, λ ex = 350 nm). S-9

10 Figure S8. Photostability of CDs-PEI-ML, fluorescein, rhodamine B, LysoTracker DND-99, and LysoTracker Deep Red. Relative fluorescence intensity of 5 µm compounds in HEPES buffer plotted as exposure time. The fluorescent intensity was obtained at different time during the continuous irradiation with 100 W soft white bulb, respectively. The fluorescent intensity was obtained with Hitachi Fluorescence Spectrophotometer F-7000 under excitation at 356 nm for CDs-PEI-ML, 488 nm for fluorescein, 550 nm for rhodamine B, 570 nm for LysoTracker Deep and 635 nm for LysoTracker Deep Red. The experiment was processed according to reported literatures Detailed protocols for cell culture and CCK-8 assay Cell culture. HeLa cells were obtained from American Type Culture collection, and grown in DMEM (High glucose) medium supplemented with 10% FBS. Cells were incubated in a 5% CO 2 humidified incubator at 37 o C and typically passaged with sub-cultivation ratio of 1:4 every two days. CCK-8 assay. The CCK-8 (Cell Counting Kit - 8) assay was used to test the cytotoxicity of the three different Carbon Nanodots (CDs, CDs-MP, CDs-PEI-ML) to Hela cells. The cells with a density of cells per ml were cultured in a 96-well microplate to a total volume of 100 µl per well at 37 in a 5% CO 2 atmosphere. After 12 h, different concentrations of each kind of Carbon Nanodots (CDs, CDs-MP, CDs-PEI-ML) of 100 µg ml -1, 300 µg ml -1, 500 µg ml -1, 700 S-10

11 µg ml -1, and 900 µg ml -1 were incubated with Hela cells for 12 h in fresh medium, respectively. Cells in a culture medium without Carbon Nanodots (CDs, CDs-MP, CDs-PEI-ML) were used as the control. After removing the medium, 100 µl of CCK-8 solution (CCK-8/DMEM = 1/9) was added into each well of the 96-well microplate for another 0.5 h. Then, the cell viability was determined by measuring the light absorbance at 450 nm with a microplate reader. The cell viability was calculated by the following equation: % viability = [Σ(A i /A control 100)]/n 2 where A i is the absorbance of different concentrations of the probe of 10 µm, 30 µm, 40 µm, 50 µm, 60 µm and 70µM, respectively. A control is the average absorbance of the control well in which the probe was absent, and n (=5) is the number of the data point. Figure S9. Cell viability of CDs (0, 100, 300, 500, 700, and 900 µg ml -1 ), CDs-MP (0, 100, 300, 500, 700, and 900 µg ml -1 ), and CDs-PEI-ML (0, 100, 300, 500, 700, and 900 µg ml -1 ) at 12 h. S-11

12 5. Detailed protocols for live cell imaging with CDs, CDs-MP, and CDs-PEI-ML Figure S10. The kinetics of internalization process of target probe CDs-PEI-ML into Hela cells. (a) The fluorescent images of live cells after the incubation of CDs-PEI-ML with live Hela cells at different time, respectively; (b) The semi-quantitative calculation of averaged fluorescence intensity of each cell in the displayed images. Error bars represents S.E.M. Intracellular fluorescence imaging with CDs, CDs-MP, and CDs-PEI-ML. The Hela cells were seeded into confocal petri dwash in complete medium (90% DMEM and 10% FBS), and then incubated for 12 h under standard culture conditions (atmosphere of 5% CO 2 and 95% air at 37 C) to allow the cells attach. The cells were washed three times with DMEM, and then were incubated respectively with 2 ml of CDs, probe CDs-MP (500 µg ml -1 ) and probe CDs-PEI-ML (500 µg ml -1 ) for 2.5 h under standard culture conditions. Then the cells were washed once with fresh DMEM, and loaded with fresh DMEM for imaging. Fluorescence images were collected by Leica TCS SP5 II confocal laser scanning microscopy using HC PLAPO 63X oil objective (NA: 1.40), CDs, CDs-MP, and CDs-PEI-ML were excited at 405 nm and the emissions were collected in the range of nm (dark field). S-12

13 Figure S11. (a) Fluorescence imaging of Hela cells loaded with three types of Carbon dots (500 S-13

14 µg ml -1 ). Excitation wavelength for CDs, CDs-MP, and CDs-PEI-ML: 405 nm; Emission collection: nm. Scale bar: 25 µm. (b) The histogram shows the semi-quantitative calculation of averaged fluorescence intensity (FI) of each carbon dots in the displayed images. Co localization of three kinds of carbon dots respectively with commercial lysosome probe LysoTracker Deep Red. The Hela cells were seeded into confocal petri dish in complete medium, and then incubated for 12 h under standard culture conditions. After the cells had attached, the cells were washed with DMEM for three times, and then were incubated with 2 ml of probe CDs-PEI-ML under standard culture conditions for 2h. Probe CDs-PEI-ML first prepared as a aqueous stock solution with a concentration of 200 mg ml -1, followed by dilution with DMEM to afford the final concentration (500 µg ml -1 ) for cell incubation. Then, 14 µl of lysosome probe LysoTracker Deep Red stock solution (10-2 M) were added into the medium and were incubated for another 30 min. Finally, cells were washed three times with 2 ml of DMEM, and 2 ml of DMEM culture medium was added and then observed under confocal microscope. LysoTracker Deep Red was excited at 633 nm and the red emission was collected in the range of nm; probe CDs-PEI-ML was excited at 405 nm and the emissions were collected in the range of nm. The above method was also used to the co localization of CDs and CDs-MP respectively with LysoTracker Deep Red. S-14

15 Figure S12. Lysosome-targeting properties of CDs in Hela cells. (a, b) The colocalization images of CDs and Lyso-Tracker Red costained living Hela cells. Green image: CDs stained signal collected at nm. Red image: Lyso-Tracker labelled signal collected at nm (Pearson's correlation R = 0.683). Excited at 405 nm. Scale bar is 10 µm. (d) Intensity profile of ROI (regions of interest) analysis (green line, CDs location; red line, Lyso-Tracker Red location) across HeLa cells. (c, e-g) 3D surface plot analysis of the colocalization images of CD and Lyso-Tracker Red costained living HeLa cells with interactive 3D surface plot function in the ImageJ software. S-15

16 Figure S13. Lysosome-targeting properties of CDs-MP in Hela cells. (a, b) The colocalization images of CDs-MP and Lyso-Tracker Red costained living Hela cells. Green image: CDs-MP stained signal collected at nm. Red image: Lyso-Tracker labelled signal collected at nm (Pearson's correlation R = 0.841). Excited at 405 nm. Scale bar is 10 µm. (d) Intensity profile of ROI (regions of interest) analysis (green line, CDs-MP location; red line, Lyso-Tracker Red location) across HeLa cells. (c, e-g) 3D surface plot analysis of the colocalization images of CD-MP and Lyso-Tracker Red costained living HeLa cells with interactive 3D surface plot function in the ImageJ software. The photostability of CDs-PEI-ML as the lysosomal labeling probe in live Hela cells. The experiment was conducted according to previous reported protocol 5. HeLa cells were incubated with Deep Red and CDs-PEI-ML, the same group of cells was excited for 30 min by the successive intense irradiation, and the fluorescent images were acquired at 0 min, 1 min, 4 min, S-16

17 10 min and 30 min, respectively. The photostability of the two fluorescent probes in the cells was evaluated by comparing a simultaneous continuous excitation with λ = 405 nm and 635 nm lasers. And then with the corresponding intensity curves were recorded according to obtained fluorescent images. As shown in FigureS14, the fluorescence of Deep Red is bleached quickly in the first 4-minute continuous irradiation, and the fluorescence intensity decreases nearly to 20% of the initial intensity after 30-minute continuous irradiation under the wavelength of 635 nm. In contrast to this Lyso-Tracker Deep Red labeled cells, the cells labeled with CDs-PEI-ML give a stable fluorescence intensity, remaining almost constant (the fluorescence intensity decreases less than 10%) even after 30 minutes of continuous irradiation. Figure S14. Photostability comparison of CDs-PEI-ML and Lyso-Tracker Deep Red in HeLa cells. The images of Hela cells that were labeled with both CDs-PEI-ML and Lyso-Tracker Deep Red were taken at 0 min, 1 min, 4 min, 20 min and 30 min, respectively through continually intense excitation under laser scanning confocal microscopy. (a) CDs-PEI-ML labeled channel (λ em = nm). (b) Lyso-Tracker Deep Red (λ em = nm). (c) Semi-quantitative analysis of the corresponding fluorescence intensity changes of CDs-PEI-ML labeled cells (a) and Lyso-Tracker Deep Red labelled cells with increasing time. Error bars represents S.E.M. S-17

18 Long-lasting cell imaging with CDs-PEI-ML. The cells were washed three times with DMEM, and then were incubated respectively with 2 ml of CDs-PEI-ML (500 µg ml -1 ) for 2.5 h under standard culture conditions. Then the cells were washed once with fresh DMEM to remove extracellular free probes, and loaded with fresh culture medium for further incubation. After 2.5, 12, 24 and 48 h, respectively, the fluorescence images were collected by Leica TCS SP5 II confocal laser scanning microscopy using HC PLAPO 63X oil objective (NA: 1.40), excited wavelength is at 405 nm and the emissions were collected in the range of nm (dark field). Figure S15. Permanent staining of CDs-PEI-ML in live Hela cells by confocal microscopy (a) 2.5h, (b) 12, (c) 24 h, (d) 48 h. λ ex =405 nm, λ em = nm. Scale bar: 25 µm. S-18

19 6. NMR spectrum Figure S16. 1 H NMR spectrum of ML. Figure S C NMR spectrum of ML. 7. References: (1) Dong, Y.; Wang, R.; Li, G.; Chen, C.; Chi, Y.; Chen, G. Polyamine-functionalized Carbon Quantum Dots as Fluorescent Probes for Selective and Sensitive Detection of Copper Ions. Anal. Chem. 2012, 84, (2) Zhang, Z.; Shi, Y.; Pan, Y.; Cheng, X.; Zhang, L.; Chen, J.; Li, M.-J.; Yi, C. Quinoline Derivative-functionalized Carbon Dots as a Fluorescent Nanosensor for Sensing and Intracellular Imaging S-19

20 of Zn 2+. J. Mater. Chem. B 2014, 2, (3) Hu, S.; Trinchi, A.; Atkin, P.; Cole, I. Tunable Photoluminescence Across the Entire Visible Spectrum from Carbon Dots Excited by White Light. Angew. Chem. Int. Ed. 2015, 54, (4) Gao, J.; Wang, P.; Giese, R. W. Xanthamide Fluorescent Dyes. Anal. Chem. 2002, 74, (5) Shi, H.; He, X.; Yuan, Y.; Wang, K.; Liu, D. Nanoparticle-Based Biocompatible and Long-Life Marker for Lysosome Labeling and Tracking. Anal. Chem. 2010, 82, S-20