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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08148 • Publication Date (Web): 08 Aug 2017 Downloaded from http://pubs.acs.org on August 9, 2017

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ACS Applied Materials & Interfaces

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 200234, China. KEYWORDS. lysosome-target probe, carbon dots, surface functionalization, photostability, live cell imaging.

ABSTRACT: Development of suitable fluorescent probe for the specific labeling and imaging of lysosomes through the direct visual fluorescent signal is extremely important for understanding the dysfunction of lysosomes which might induce various pathologies, including neurodegenerative diseases, cancer, and Alzheimer’s disease. Herein, a new carbon dot based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. In this probe, PEI (polyethylenimine) is introduced to improve water solubility and provide abundant amine groups for the as-prepared CDs-PEI, the morpholine group (ML) serves as a 1 ACS Paragon Plus Environment


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targeting unit for lysosomes. More importantly, passivation with PEI could dramatically increase the fluorescence quantum yield of CDs-PEI-ML as well as their stability in fluorescence emission under different excitation wavelength. Consequently, experimental data demonstrated that the target probe CDs-PEI-ML has low cytotoxicity and excellent photostability. Additionally, further live cell imaging experiment indicated that CDs-PEI-ML is a highly selective fluorescent probe for lysosomes. We speculate the mechanism for selective staining of lysosomes that CDs-PEI-ML was initially taken up by lysosomes through the endocytic pathway and then accumulated in acidic lysosomes. It is notable that there was less diffusion of CDs-PEI-ML into cytoplasm which could be ascribed to the presence of lysosome target group morpholine on surface of CDs-PEI-ML. The blue emission wavelength combined with the high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML become an alternative fluorescent probe for multicolor labeling and long-term tracking of lysosomes in live cells and the potential application in super-resolution imaging. To best of our knowledge, there is still limited carbon dots based fluorescent probes were studied for specific lysosomal imaging in live cells. The concept of surface functionality of carbon dots will also pave a new avenue for developing carbon dots based fluorescent probes for subcellular labeling.

INTRODUCTION Lysosome, one of the subcellular organelles, which play vital roles in protein degradation, cell signal transduction, plasma membrane repairment, homoeostasis, and autophagy1-2. As the digestive system of the cell, lysosomes contain about 50 acid hydrolases that are responsible for hydrolyzing biomacromolecules such as proteins, 2 ACS Paragon Plus Environment

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nucleic acids, lipids and polysaccharides3. The deficiency of these enzymes are closely related to inflammation1, tumor2, and lysosomal storage diseases4. Additionally, the increased lysosomal enzyme expression presumably results in the tumor invasion and metastasis5. Thus, development of new tools for tracing and visualizing the lysosomes are significant for understanding the functionality of lysosomes and related diseases. A variety of different fluorescent probes have been used for lysosomal imaging6-8. And most of the developed probes for lysosomes are small molecular probes, which could differentiate lysosomes from other organelles in live cells through the clear fluorescence signal readout. However, these small molecular probes might have relatively low stability against photobleaching which was ascribed to that a number of conventional organic dyes for constructing these probes would photodegrade under the continuous irradiation. This limitation will prevent the practice use of the small molecular fluorescent probes for real-time tracing and visualizing the lysosomes with microscopy in live cell imaging. Additionally, to overcome the diffraction limit (about 200-250 nm) which would decrease the spatial and temporal resolution in visualizing much clearer subcellular features, such as the mammalian cellular lysosome (the size is between 0.1 and 1 µm), the super-solution imaging has recently been developed9-10. The key factor in breaking the lateral resolution diffraction limit is the super-resolution fluorophore, which should feature a high photostability, brightness10-11. Thus, development of more photo-stable fluorescent probes will make the live cell imaging at single-molecule level which is significant for unveiling much more detailed features of subcellular organelles as well as the specific functionality of some biomolecules including proteins, DNA and RNA etc12. Meanwhile, the fluorescent probes with distinct colors are also urgently needed in multicolor imaging in order to discriminate different subcellular organelles or 3 ACS Paragon Plus Environment


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biomolecules with multicolor labeling13. Fluorescent probes with different emission wavelengths are also required in the multicolor super-resolution imaging12. Hence, to overcome the photoblinking of some fluorescent dyes as well as to provide much more fluorescent probes with distinct colors, there is an increasing interest in the rationale for use of fluorescent quantum dots for biological application14-19. Due to the bright fluorescence and high stability against photobleaching as well as their tuned emission wavelength, the past decades have witnessed the great progress in the development of fluorescent quantum dots for fluorescence sensing and biological imaging19-21. But most previous research focus on semiconductor quantum dots that exhibited high toxicity and thus limited their further use in live cell and in vivo imaging. Thus, there is still limited fluorescent probes exhibited more photo-stability and biocompatibility in highly selective labeling lysosomes in live cells22-24. Recently, fluorescent carbon dots (CDs), with the high biocompatibility and exceptionally low in vivo toxicity has exhibited great potential uses in biological systems25-27. Lots of efforts have been devoted to preparing fluorescent carbon dots including the “top-down” and “bottom-up” approaches28, optical properties25, surface functionality29, fluorescence sensing30-33 and biological imaging34-35. However, to best of our knowledge, there is still limited numbers of CDs based probes for specific lysosomal imaging36-37. Despite the good performance in visualizing lysosomes, the Pearson’s correlation coefficient (about 0.7892) suggested these CDs based probe might diffuse out of the lysosomes, which could decrease the specificity in lysosomal imaging. Herein, we designed and synthesized a new carbon dots based probes (CDs-PEIML, Figure 1) for highly selective lysosomal imaging in live cells. Compared to 4 ACS Paragon Plus Environment

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previously reported probes for fluorescence imaging of intracellular lysosomes, CDsPEI-ML contains the following properties: i) the high photostability and could be used for long-term irradiation under microscopy; ii) bright fluorescence (relatively high fluorescence quantum yield) in live cell imaging; iii) biocompatibility for live cells (including high water solubility and low cytotoxicity); iv) high selectivity for targeting lysosomes; v) blue emission wavelength can be an alternative probe for multicolor labeling of lysosomes in live cells.

Figure 1. Synthetic procedures for the Carbon-dots-based fluorescent probe and schematic illustration of imaging lysosomes in living cells. EXPERIMENTAL SECTION Materials and Apparatus. All commercially available chemical reagents were used directly without further purification. Citric acid monohydrate, 4-(25 ACS Paragon Plus Environment


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Aminoethyl)morpholine and polyethylenimine were used as the starting materials. EDC and NHS were used for conjugation reagents (the detailed chemical name of these commercial reagents and their

provider were displayed in the supporting

information). The reaction was monitored by thin-layer chromatography (TLC) on silica gel plates. The column chromatography was introduced for purification of the target compounds (detailed information please see the supporting information). The cytotoxicity of the target compounds was evaluated with Cell Counting Kit-8 (CCK8). 1H and

13

C NMR spectra were obtained from Bruker AV-400 spectrometer. The

size and morphology of the dried Carbon dots were tested by HRTEM Zeta potential of target compounds solutions was measured with dynamic light scattering (DLS). The protocol for fluorescence and absorbance spectra were described in details in the supporting information. Please see the detailed information of the commercial reagents providers and the apparatus in the supporting information. Synthesis of CDs-PEI-ML. EDC and NHS were introduced for covalent conjugation38 between compound ML and the surface of CDs-PEI. 745 mg compound ML and as-prepared CDs-PEI were suspended in 30 ml ultrapure water containing 539 mg NHS and 2.69 g EDC. Then the reaction mixture was stirred at romm temperature for 24 h. After completion of the reaction, 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 afford the brown solid. RESULTS AND DISCUSSION Design and Synthesis. Firstly, we try to design a lysosome-target fluorescent probe (CDs-PEI-ML, Figure 1) which has low cytotoxicity, excellent photostability, good membrane permeability and high fluorescence quantum yield (Φ) for live HeLa cells. 6 ACS Paragon Plus Environment

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Although the last decades have witnessed a great advance in synthesis of carbon dots, most of carbon dots have relatively low Φ values which were often less than 10%39-43. Such obstacle limited their practice use in fluorescence imaging in biological systems, especially for the real-time observation through the direct fluorescence signal readout. Thus, development of new method for carbon dots with high fluorescence quantum yield is urgently needed44. To date, doping and passivation are common approaches to enhance the quantum yield. Qian et al. found the Φ of carbon dots can be improved when doping nitrogen atoms in carbon dots to adjust the surface features.45-46 Zhu and co-workers also attributed to improve the Φ of carbon dots. They found that the Φ of the prepared carbon dots are less than 10% if the reactant contained –COOH or –OH only. However, the Φ can be more than 10% if molecules with amino group were used in the synthesis process47. Hence, during the pyrolysis procedure, the introduction of surface-passivation reagents particularly amine group containing molecules would pave the way of increasing the Φ of carbon dots29. According to this hypothesis, we first prepared the carbon dots (CDs, Scheme S1) through pyrolysis method with citric acid as the starting material. Then, the same approach was used to synthesize CDs-PEI with citric acid as the starting material and branched polyethyenimine (PEI) as the passivation reagent. As the PEI is an abundant amine groupscontaining polymer with extremely high density of ethylenediamine units, which can enhance the Φ of carbon dots. In addition, introduction of hydrophilic PEI onto CDs could increase the water solubility of CDs-PEI. And the abundant amine could also be used for further chemical modification.38,

48-49

Finally, to ensure the highly

selectivity for lysosomal localization, the morpholine derivative ML, serving as a lysosome-target group50, was modified onto the surface of CDs-PEI through covalent conjugation to produce target lysosome specific fluorescent probe CDs-PEI-ML. 7 ACS Paragon Plus Environment


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Meanwhile, the control probe CDs-MP without PEI-passivation treatment was also prepared. Compound MP was also modified onto the surface of the CDs through covalent conjugation between the primary carboxylic groups of CDs and amine groups of MP in the presence of the coupling reagents EDC/NHS (Figure 1 and Scheme S1). Characterization of CDs, CDs-MP and CDs-PEI-ML. As indicated in Figure 2af, all the synthesized carbon dots were spherical-shaped and had similar sizes (that is, 4-nm in diameter), which was observed by high resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS). In details, the CDs exhibited a relatively narrowly distribution in diameters, with an average value of 3.15 nm (Figure 2a, b). There was only a little enhancement in diameter after compound ML, a morpholine derivative for targeting lysosome, was modified onto the surface of the CDs-PEI by the covalent reaction between carboxylic groups of compound ML and primary amine groups of CDs-PEI (Figure 2e, f). Similarly, no obvious morphology change in diameter for as the control probe CDs-MP after the CDs was modified with compound MP. The successful surface functionalization and passivation processes were also further monitored and identified through nuclear magnetic resonance (NMR) spectroscopy (Figure S1 and Figure S2). The NMR spectrum of CDs displayed two separated groups of peaks at 2.75 and 2.55, respectively, which could be ascribed to the methylene of citric acid (Figure S1a). After passivation with PEI, these two separated peaks changed to one low-resolution splitting peak at around 2.10-3.50 (Figure S2b), indicating the successful passivation of PEI. In comparison to CDs-PEI, CDs-PEI-ML displayed two new groups of peaks at approximate 3.31 and 2.35 respectively after conjugation with ML group (Figure S2c). These two peaks are similar as the peaks from ML group (Figure S2). All these 8 ACS Paragon Plus Environment

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NMR results demonstrated the successful synthesis of carbon dots and their surface modification. Moreover, zeta potential assay was used for characterization of the prepared CDs based probes. As shown in Figure 1 and Figure 2g, there are a number of exposed carboxyl groups on the CDs surface, which makes the CDs become negatively. The zeta potential of CDs-MP increased to -3.8 mV relative to the CDs (21.0 mV), which could be ascribed to the formation of amide bonds between carboxyl acids of the CDs and the amine groups of MP. After CDs were passivated with PEI, the zeta potential of the CDs changed from -21.0 mV to +28.0 mV. The high-positive charges of CDs-PEI ascribed from the abundant amine groups on the surface of CDsPEI, indicating the PEI layer was successful coated onto the CDs. When the surface of CDs-PEI was conjugate with compound ML, the zeta potential of the CDs-PEIML decreased to +9.3 mV, which is much lower compared with the value of bare CDs-PEI (+28 mV). The cause for this phenomenon is the successful conjugation of ML onto the CDs-PEI surface. Finally, the FTIR, Elemental analysis and XPS assay also were conducted to characterization of these carbon dots (Figure S3 and S4, Table S1, the detailed information please see the supporting information). All above results demonstrated the successful synthesis of CDs, CDs-MP and CDs-PEI-ML.

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Figure 2. Left: HRTEM spectra of as-prepared CDs (a), CDs-MP (c), and CDs-PEIML (e). Right: histograms of particles size distribution of CDs (b), CDs-MP (d), and CDs-PEI-ML (f). (g) Zeta potential of CDs, PEI, CDs-PEI, CDs-MP and CDs-PEIML, respectively. The insets at the right bottom of 2a, 2c and 2e show the magnification of a single nanoparticle, respectively. Photophysical performance of CDs, CDs-MP and CDs-PEI-ML. Then, to further investigate the photophysical performance of the prepared carbon dots, the UV-visible (UV-vis) absorption spectroscopy of CDs, CDs-MP and CDs-PEI-ML were initially determined. As shown in Figure 3a, the maximal absorption value of CDs was nearly 322 nm and CDs-MP showed a relatively weak absorption intensity at 336 nm corresponding to the strong absorption intensity at 359 nm that was ascribed to CDs-PEI-ML. The red shifting in the absorbance spectra of CDs-PEIML is presumably from the increase in size32, verifying successful PEI passivation and surface functionalization with lysosome-targeting group ML. Similarly to most reported carbon dots,51-54 CDs, CDs-MP and CDs-PEI-ML also exhibited the excitation-dependent emission, which were assigned not only to the various surface states but also to the diversified size distribution.32, 51, 55-56 Among the three types of carbon dots, a remarkable multi-emission peaks were observed for CDs, which displayed significant red shift in the maximum emission when excitation wavelength changes from 300 nm to 420 nm (Figure 3c). Meanwhile, it is interesting that both CDs-MP and CDs-PEI-ML showed single emission peak, extending from the 370 nm (ultraviolet) to the 550 nm (green-yellow regions) when the excitation wavelength was beyond 400 nm (Figure 3d, e). Especially for CDs-PEI-ML, there is a relatively stable fluorescence emission wavelength under different excitation wavelength, which is a vital factor for live cell imaging by using the multi-color labeling to discriminate 11 ACS Paragon Plus Environment


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various subcellular organelles. Finally, all the carbon dots (CDs, CDs-MP and CDsPEI-ML) emitted the blue solid-state fluorescence under UV excitation with microscope and hand-held UV lamp (Figure 3b and Figure S5).

Figure 3. (a) UV-vis spectra of CDs (7 mg ml-1), CDs-MP (1 mg ml-1), and CDsPEI-ML (1 mg ml-1). (b) The microscopy images of CDs, CDs-MP and CDs-PEIML (left: bright field, right: fluorescence photos under UV light excitation), scale bar, 0.5mM. (c) Fluorescence emission spectra of CDs (0.2 mg ml-1). (d) Fluorescence spectra of CDs-MP (0.2 mg ml-1). (e) Fluorescence spectra of CDs-PEI-ML (0.2 mg 12 ACS Paragon Plus Environment

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ml-1). The insets are the photographs of the carbon dots obtained under bright and hand-held UV lamp (365nm), respectively. As indicated in the insets in Figure 3c, d, e, the clear solution of CDs, CDs-MP and CDs-PEI-ML was observed under bright field, respectively. Similarly, solutions with CDs, CDs-MP and CDs-PEI-ML also give rise to the blue fluorescence. All these results suggested the water solubility of these carbon dots. It is notably that the blue fluorescence of CDs-PEI-ML in solution was brighter compared to the solutions of CDs and CDs-MP, respectively. Herein, the fluorescence quantum yield (Φ) was introduced as an important factor for quantitatively evaluating fluorescent intensity of CDs, CDs-MP and CDs-PEI-ML. As shown in Figure 4, the quinine sulfate was taken as the standard sample for determining the Φ, then the Grade values were calculated to be 1802790, 219337, 364435, and 1242230 for quinine sulfate, CDs, CDs-MP and CDs-PEI-ML, respectively. The Φ of the CDs was nearly 7% (6.5 ± 0.4%, n = 3) and the Φ of the CDs-MP was about 11% (11.0 ± 0.4%, n = 3). It is notable that the Φ of CDs-PEI-ML was higher than 35% (37.7 ± 0.6%, n = 3), which was 3 times as much as the Φ of CDs-MP and 5-fold increase in Φ compared to CDs. These results indicated surface functionalization with abundant amine group containing-PEI can increase the quantum yield of carbon dots. The brighter fluorescence of CDs-PEI-ML makes it more suitable for live cell imaging through eliminating the background signal under the confocal microscopy. More importantly, this facile one-pot approach towards preparing the highly fluorescent CDs-PEI through hydrothermal treatment of PEI and citric acid is feasible and less timeconsuming on a large scale25, 57-59.



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Figure 4. Quinine sulfate was taken as a fluorescence standard reference reagent, plot of integrated fluorescence vs absorbance. The dark lines were obtained with the linear fitting of the data of quinine sulfate and red lines is obtained by the linear fitting of the data of CDs (a), CDs-MP (b), CDs-PEI-ML (c), respectively. (d) Fluorescence quantum yield (Φ) of three different carbon dots. Additionally, fluorescent probes for lysosomal labeling requires a stable fluorescence singal readout22-23. Hence, the pH titration assay was conducted to investigate whether the acid environment of lysosomes could interfere with fluorescent intensity of CDs-PEI-ML. As suggested in Figure S6, the fluorescence intensity of the bare carbon dots (CDs) changes as the function of pH in water, which is consistent with previous report that some type of synthesized carbon dots were sensitive to pH43. The maximum of fluorescence intensity reaches to the maximal value at pH 5.0 for CDs, then a significant fluorescence decrease was observed when 14 ACS Paragon Plus Environment

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the pH value increased to 7 (Figure S6a). However, through the functionalization of CDs with MP group, the fluorescence intensity of CDs-MP decreased slowly with pH changes during the range of 4-7 (Figure S6b). Furthermore, it is interesting that the target probe CDs-PEI-ML with PEI passivation exhibited a relatively stable fluorescence intensity during the pH range of 4-7 (Figure S6c), which covers the pH of lysosomes (Figure S6d, intracellular lysosomal pH value is during the range of 4660). Thus, the surface passivation of carbon dots with PEI could increase the fluorescence stability of CDs-PEI-ML during the pH range of 4-7, which makes CDs-PEI-ML suitable for fluorescence imaging of lysosomes in live cells. In order to investigate whether some commonly metal ions in biological systems and other bioactive small molecules could interfere with the fluorescence of the prepared probes, the interference assay was conducted. 50 µM of different reagents were added into solutions of the CDs, CDs-MP, and CDs-PEI-ML, respectively. Then, the maximum fluorescence intensity (IF 425 nm, IF 454 nm, IF 441nm) were monitored respectively. There were no significant fluorescent intensity changes when the solutions with CDs and CDs-MP were treated with 50 µM of different interference reagents, respectively (Figure 5). Additionally, except for Cu2+ treated solution, no obvious fluorescence intensity changes were found for CDs-PEI-ML containing solution. The decrease of fluorescence intensity in Cu2+ treated CDs-PEIML solution was about 16%, which is mainly associated with the formation of coordination complex between Cu2+ and the amino groups on the surface of the CDsPEI-ML whereby the Cu2+ has an inner filter effect because of its paramagnetic property48. However, the concentration of endogenous free Cu2+ in the biological system is much lower than that of 50 µM used in this interference assay61-62. Thus, the



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intracellular endogenous Cu2+ would not interfere with the live cell imaging with CDs-PEI-ML.

Figure 5. Fluorescence intensity changes of 0.2 mg ml-1 CDs (λex = 320 nm, , λem = 425 nm), 0.2 mg ml-1 CDs-MP (λex = 360 nm, λem = 454 nm) and 0.2 mg ml-1 CDsPEI-ML (λex = 320 nm, λem = 441 nm) in water after addition of different metal ions and other interfered reagents, respectively. From 1 to 20 : blank probe; Cu2+, Zn2+, Ca2+, Al3+, Fe2+, Fe3+, Mg2+, NH4+, F-, Cl-, Br-, I- , SO42- , Cysteine, Homocysteine, Glycine, Arginine, Histidine, Glutathione. The concentration for all the interfered reagents were 50 µM. Error bars represent s.d. Meanwhile, the photostability of CDs, CDs-MP and CDs-PEI-ML was evaluated respectively by determining the changes in maximum fluorescence intensity (IFmax) 16 ACS Paragon Plus Environment

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when these carbon dots were excited with over 2 h (Figure S7). There were no remarkable IFmax changes among the three varieties of carbon dots, demonstrating the high photostability against the bleaching. Moreover, the photostability of CDs-PEIML was further investigated by comparison with the photostability of commonly used organic dyes including fluorescein, rhodamine B and commercially available lysosomal probes (LysoTracker DND-99 and LysoTracker Deep Red) under the continuous irradiation with 100W soft white bulb. As shown in Figure S8, IFmax of fluorescein, rhodamine B, LysoTracker DND-99, and LysoTracker Deep Red decreased remarkably within 60 h, indicating there is a photobleaching under the continuous irradiation. Especially, the fluorescence intensity of both LysoTracker DND-99 and LysoTracker Deep Red decreased significantly within 10 h. The relative fluorescence intensity of these commercial lysosomal probes decreased to lower than 10 percent of its initial intensity when exposed to light for 34 h. By contrast, the fluorescence intensity of CDs-PEI-ML remined almost no changes even within 60-h continuous irradiation, demonstrating higher photostability of CDs-PEI-ML. All above results indicate the CDs-PEI-ML is a highly water soluble, brighter fluorescence and more photo-stable probe, which promote us to further investigate the subcellular imaging with CDs-PEI-ML in live cells. Live Cell Imaging with CDs, CDs-MP, and probe CDs-PEI-ML, respectively. Since the carbon dots entered into cells through the endocytosis process43, and the lysosomes play a principal role in this pathway. We deduced the carbon dots could be endocytosed by lysosomes and the surface basic amino groups of CDs-PEI-ML allows it largely accumulate in the acidic lysosomes. Thus, the lysosomal imaging with CDs-PEI-ML was conducted in live Hela cells. Initially, the cytotoxicity of CDs, CDs-MP, and CDs-PEI-ML were evaluated by conducting the CCK-8 assays. 17 ACS Paragon Plus Environment


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There is no significant cytotoxicity (over 80% cell viability) after the HeLa cells were incubated with three different types of carbon dots respectively for 12 h at the concentrations from 0 to 900 µg ml-1 (Figure S9). Then, the kinetics of internalization process of target probe CDs-PEI-ML into Hela cells was tested. As shown in Figure S10, there is a visible fluorescence in live Hela cells after 30 min incubation of CDsPEI-ML with cells. And then the fluorescence signal of labeled cells enhanced with increased times within 2 h. The fluorescence intensity reached a plateau after 2.5 incubation (Figure S10a). Additionally, the increase of the intracellular fluorescence intensity with time was measured by semi-quantitative analysis was conducted to evaluate the averaged fluorescence intensity in each cell from the displayed images (Figure S10b). All the results demonstrated the fluorescence of labeled cells reached a plateau after 2.5h-incubation with CDs-PEI-ML. Thus, the incubation time of carbon dots with live Hela cells was fixed to 2.5h. Next, we investigated the fluorescence response of all carbon dots in live Hela cells. CDs-PEI-ML was loaded into the cells and incubated for 2.5 h. Then cells were washed with DMEM (without serum) for three times. The fluorescence in live cells was collected with confocal microscope after the washing medium was replaced with fresh DMEM. As the control probes, the fluorescence response of CDs and CDs-MP was also determined after they were loaded into cells, respectively. As shown in Figure S11a, a relatively weak fluorescence signal in dark field (green, 420-580 nm) of CDs and CDs-MP but strong of CDs-PEI-ML was obtained from the intracellular zone. By the semi-quantitative analysis of the averaged fluorescence intensity (FI) from live cells with Image J software, we could obtain that FI from CDs-PEI-ML labeled cells were 4-fold and 12-fold higher than CDs-MP and CDs labeled cells (Figure S11b). This result demonstrated that the target probe CDs-PEI-ML with PEI passivation exhibited a 18 ACS Paragon Plus Environment

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remarkable brighter fluorescence in live cell imaging, which allows CDs-PEI-ML become more suitable fluorescent reagent for intracellular tracing in live cell imaging. Additionally, it is remarkable that the labelled fluorescence signal in the live cells displayed preferentially in the spherical zone, suggesting the lysosomal localization. We deduced that carbon dots entered into cells through the endocytosis process which translocated carbon dots from the extracellular environments into the intracellular lysosomes.63 Finally, to further study the specificity in lysosomal imaging with CDs, CDs-MP, and CDs-PEI-ML, the co-localization experiments with the commercial lysosome probe (LysoTracker® Deep Red) in live HeLa cells were conducted. As shown in Figure 6a, b, the remarkable orange fluorescence signal was obtained in the live cells when the green images from CDs-PEI-ML labelled signal were merged with red images from LysoTracker® Deep Red labelled signal. Furthermore, through analyzing the region of interest (ROI) in the live cells in Figure 6b, it is obvious that CDs-PEIML stained location (green line in Figure 6d) overlapped well with LysoTracker® Deep Red (red line in Figure 6d) stained lysosomes. Then, through the 3D surface plot analyzing images of Figure 6b, we could clearly observe the co-localization images of CDs-PEI-ML and commercial lysosomal probe co-stained living HeLa cells. Finally, the Pearson’s correlation coefficient of 0.87 ± 0.02 for CDs-PEI-ML was also obtained by using the ImageJ software analysis. All these results implied that CDsPEI-ML mainly localized in the lysosomes and could be used as a highly selective fluorescent probe for lysosomal imaging. Meanwhile, the overlaid images of the control probe CDs and CDs-MP treated cells were also obtained. As displayed in Figure S12, despite a significant proportion of orange signal appearing in the 19 ACS Paragon Plus Environment


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lysosomes of CDs treated cells, there was still a diffuse distribution of the fluorescence signal within the cells, indicating parts of the CDs might diffuse out of the lysosomes. However, it is interesting that the fluorescence signal from the overlaid images of CDs-MP treated cells distributed predominantly in a granular pattern, indicating presumably lysosomal localization (Figure S13). Furthermore, the Pearson’s correlation coefficient for CDs, CDs-MP and CDs-PEI-ML treated cells was 0.68 ± 0.03, 0.84 ± 0.03 and 0.87 ± 0.02, respectively, by using the ImageJ software analysis (Figure 7). Hence, the introduction of the lysosome target group alkylmorpholine (MP and ML) onto the surface of carbon dots could decrease the diffusion of the carbon dots from the lysosomes after they were translocated into lysosomes via endocytic pathway, which could increase the specificity of CDs-PEIML in lysosomal imaging.


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Figure 6. Evaluation of the lysosomal localization of CDs-PEI-ML in live Hela cells. (a, b) The colocalization images of CDs-PEI-ML and commercial lysosomal probe (Lyso-Tracker Red) co-stained Hela cells. CDs-PEI-ML channel: CDs-PEIML stained signal collected at 420-580 nm. Lyso-Tracker channel: commercial lysosomal probe stained signal obtained at 650-800 nm (Pearson's correlation coefficient R = 0.87 ± 0.02). Excitation wavelength: 405 nm. Scale bar:10 µm. (d) 21 ACS Paragon Plus Environment


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Plot analysis of ROI (region of interest) across HeLa cells. green line: CDs-PEI-ML labeled location; red line: commercial lysosomal probe (Lyso-Tracker) labeled location. (c, e-g) 3D surface plot analyzing the colocalization images of CDs-PEIML and Lyso-Tracker Red co-stained HeLa cells. This analysis was conducted with the function of interactive 3D surface plot in the ImageJ software.

Figure 7. Pearson's correlation coefficient of each carbon dots. Pearson’s correlation coefficient of CDs, CDs-MP and CDs-PEI-ML is 0.68±0.03, 0.84±0.03 and 0.87 ± 0.02, respectively. The intracellular photostability of CDs-PEI-ML was further evaluated by comparison with Lyso-Tracker Deep Red. In this experiment, HeLa cells were incubated with Deep Red and CDs-PEI-ML, and 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. Figure S14 showed that the fluorescence signal of Deep Red bleached quickly in the first 4-minute continuous irradiation, and the fluorescence intensity decreases nearly to 20% of the initial 22 ACS Paragon Plus Environment

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intensity after 30-minute continuous irradiation. 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. Finally, the ability for long-lasting cell imaging is important for long-term lysosomal tracking study in live cells. Thus, the experiments were conducted to estimate the potential of CDs-PEI-ML as a long-life lysosome marker. Figure S15 demonstrated the strong fluorescence signal in live Hela cells remained almost unchanged for at least 48 h. It is particular that the labelled fluorescence signal in the live cells is still preferentially localized in the spherical zone, suggesting the labeled fluorescence signal is mainly from lysosomes. In addition, there is no fluorescence signal was observed in extracellular zone, demonstrating the labeled CDs-PEI-ML did not leak into the extracellular zone even after 48 h staining of the live Hela cells. All the results revealed that CDs-PEI-ML could be used a long-life lysosome marker for long-lasting labeling of lysosomes in live cells, which could be significant for continuous visualizing of the specific biological functions of lysosome in live cells. Meanwhile, these results also verify that CDs-PEI-ML exhibited the potential application in super-resolution imaging. CONCLUSIONS In summary, a new carbon dot based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. Compared to previously reported small-molecule fluorescent probes for lysosomes, CDs-PEI-ML embodies the relatively high photostability, making it suitable for long-term observation in live imaging with microscopy. Additionally, the introduction of 23 ACS Paragon Plus Environment


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lysosome target group alkylmorpholine onto the surface of carbon dots decrease the diffusion of the carbon dots from the lysosomes after they were translocated into lysosomes via endocytic pathway, which allows CDs-PEI-ML to exhibit a high specificity in lysosomal imaging. And the well overlap with the LysoTracker® Deep Red labelled signal and higher Pearson’s correlation coefficient in lysosomal labeling (0.87 ± 0.02) further verified that CDs-PEI-ML was a highly selective fluorescent probe for lysosomes. To best of our knowledge, there is still limited carbon dots based fluorescent probes were studied for highly selective lysosomal imaging in live cells. Meanwhile, through the surface passivation with PEI, the fluorescence quantum yield of CDs-PEI-ML was increased. Especially, CDs-PEI-ML exhibited a relatively stable fluorescence emission under different excitation wavelength, which is a vital factor for live cell imaging by using the multi-color labeling to discriminate various subcellular organelles. And the blue emission wavelength can be an alternative probe for multicolor labeling of lysosomes in live cells. The high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML the potential application in superresolution imaging. Finally, the starting material for preparing CDs-PEI-ML is citric acid the synthetic procedures were convenient, which ensures the higher water solubility and low cytotoxicity of CDs-PEI-ML. Thus, this study presents a highly selective carbon dots based fluorescent probes for lysosome labeling and imaging. The concept of surface functionality of carbon dots will also pave a new avenue for developing carbon dots based fluorescent probes for subcellular labeling.


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ASSOCIATED CONTENT Supporting Information. Additional materials and methods including detailed protocol for synthesis, characterization of target compounds, cell cultures, and detailed experiments for live cell imaging were in the supporting information. AUTHOR INFORMATION Corresponding Author *Chusen Huang, E-mail: [email protected]. Fax: 86-21-64321833. * Nengqin Jia, E-mail: [email protected]. Fax: 86-21-64321833. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank National Natural Science Foundation of China (Grants 21672150, 21302125 and 21373138), Alexander von Humboldt Foundation (AvH), Doctoral Fund of Ministry of Education of China (Grant No. 20133127120005), Shanghai “Chenguang” Program (Grant 14CG42), and Program for Changjiang Scholars and Innovative (IRT_16R49). ABBREVIATIONS CDs, carbon dots; PEI, polyethyenimine; Φ, fluorescence quantum yield; TLC, thin layer chromatography; CCK-8, Cell Counting Kit-8. 25 ACS Paragon Plus Environment


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