Nucleolus-Targeted Red Emissive Carbon Dots with Polarity-Sensitive

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Biological and Medical Applications of Materials and Interfaces

Nucleolus-Targeted Red Emissive Carbon Dots with Polarity-Sensitive and Excitation-Independent Fluorescence Emission: High-Resolution Cell Imaging and in Vivo Tracking Xian-Wu Hua, Yan-Wen Bao, Jia Zeng, and Fu-Gen Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09590 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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ACS Applied Materials & Interfaces

Nucleolus-Targeted Red Emissive Carbon Dots with Polarity-Sensitive and Excitation-Independent Fluorescence Emission: High-Resolution Cell Imaging and in Vivo Tracking

Xian-Wu Hua,† Yan-Wen Bao,† Jia Zeng,† and Fu-Gen Wu*,†,‡

†State

Key Laboratory of Bioelectronics, School of Biological Science and Medical

Engineering, Southeast University, 2 Sipailou Road, Nanjing 210096, P. R. China ‡Key

Laboratory of Developmental Genes and Human Disease of the Ministry of Education

of China, Institute of Life Sciences, Southeast University, 2 Sipailou Road, Nanjing 210096, P. R. China

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ABSTRACT Red-emitting carbon dots (CDs) have attracted tremendous attention with wide applications in areas including imaging, sensing, drug delivery, and cancer therapy. However, it is still highly challenging for red-emitting CDs to simultaneously achieve high quantum yield (QY), nucleus targeting, and super-resolution fluorescence imaging (especially the stimulated emission depletion (STED) imaging). Here, it is found that the addition of varied metal ions during the hydrothermal treatment of p-phenylenediamine (pPDA) leads to the formation of red emissive CDs with QYs varying from 1.6% to 45.6%. Strikingly, although metal ions play a crucial role in the synthesis of CDs with varied QYs, they are absent in the formed CDs; that is, the obtained CDs are metal-free and the metal ions play a role similar to a “catalyst” during the CD formation. Besides, using pPDA and nickel ions (Ni2+) as raw materials, we prepare Ni-pPCDs which have the highest QY and exhibit various excellent fluorescence properties including excitation-independent emission (at ~605 nm), good photostability, polarity sensitivity, and ribonucleic acid responsiveness. In vitro and in vivo experiments demonstrate that Ni-pPCDs are highly biocompatible and can realize real-time, wash-free, and high-resolution imaging of cell nuclei and high-contrast imaging of tumor-bearing mice and zebrafish. In summary, the present work may hold great promise in the synthesis and applications of red emissive CDs.

KEYWORDS: carbon quantum dots, polarity-responsive, cell imaging, super-resolution imaging, nuclear targeting

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INTRODUCTION Fluorescent carbon dots (CDs), which are commonly synthesized via hydrothermal and solvothermal methods,1–3 have found numerous applications in bioimaging,4–9 drug delivery,10,11

catalysis,12–14

sensing,15–21

light-emitting

devices,22,23

nanomaterial

fabrication,24,25 and cancer therapy26–28 due to their characteristics including facile preparation, good biocompatibility, prominent optical properties, and proper surface chemistries. In particular, the red/near-infrared (NIR) emissive CDs have attracted tremendous attention due to their superior properties resulting from their long-wavelength emission, including large tissue penetration depths, low levels of light scattering/absorption, and negligible interference from the background autofluorescence of biological tissues.29–51 To design and fabricate red emissive CDs, heteroatom (usually nitrogen and sulfur) doping has been adopted.52–61 For instance, Jiang et al. used three different phenylenediamine isomers to prepare red, green, and blue emissive nitrogen-doped CDs via a solvothermal route for full-color displays and cell imaging.29 Among them, the CDs made from p-phenylenediamine (pPDA) have red fluorescence emission and display a fluorescence quantum yield (QY) of 26.1% in ethanol solution. Bao et al. developed the sulfur and nitrogen co-doped NIR emissive CDs for photoluminescence and photoacoustic imaging-guided photothermal cancer therapy.53 Liu et al. prepared red emissive nitrogen-doped carbonized polymer dots with a QY of 31.54% in ethanol, displaying excitation-independent and narrow bandwidth emission.54 Yang et al. synthesized sulfur and nitrogen

co-doped

hydrophobic

CDs

with

blue

dispersed

emission

and

red

aggregation-induced emission for the development of reversible two-switch-mode

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luminescence ink.58 Very recently, Jia et al. obtained bright red emissive nitrogen-doped CDs with a very high QY of 86.0% (in ethanol), which hold great promise for developing the high-performance electroluminescent warm white light-emitting diodes.59 Although huge progress has been made for the optimization of the fluorescence emission of the red emissive CDs,

the

tedious

purification

steps,

excitation-dependent

fluorescence,

poor

water-dispersibility, and/or the inability to target specific cellular organelles still hamper the further applications of CDs in bioimaging. To data, only a few reports focus on the specific organelle-targeting

ability

of

CDs.10,11,62–64

For

instance,

Liu

et

al.

developed

lysosome-targeted CDs and realized ratiometric imaging of formaldehyde.64 In our previous work, we prepared two types of fluorescent CDs for mitochondrial and nuclear imaging, respectively.10,11 Nevertheless, the low QYs, excitation-dependent emission, and/or short-wavelength fluorescence of these organelle-targeting CDs cause many obstacles in the costaining of various subcellular structures and in vivo tracking. The exploration on the interaction between CDs and metal ions has been a hot topic, such as the detection of metal ions and the preparation of metal-based nanomaterials.15–21,24,25 For the former, CDs exhibit different affinities for metal ions because of their diverse surface functional groups (which may come from the different carbon sources) and the interaction between CDs and metal ions usually leads to fluorescence quenching of the CDs.65–67 For the latter, we synthesized two non-fluorescent copper-based therapeutic agents for efficient cancer therapy via the interaction between CDs and Cu2+ in aqueous solution.24,25 Moreover, we further investigated the influence of various metal ions on the preparation of CDs via the hydrothermal method; unexpectedly, a series of non-fluorescent metal-doped nanomaterials

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were obtained.68–70 Collectively, the above two exploration aspects point out that metal ion addition can weaken the fluorescence of CDs. Up to now, few studies have reported the enhancement of the CD QYs with the assistance of metal ions, which remains a research area to be explored. As is well known, the lateral resolution limit of conventional fluorescence microscopy is > 200 nm as determined by the Abbe criterion.71 In the last two decades, the emergence of many super-resolution imaging techniques such as stimulated emission depletion (STED) microscopy, photo activated localization microscopy, stochastic optical reconstruction microscopy, structured illumination microscopy, and others, has overcome the diffraction limit with greatly improved spatial resolution.72–77 Among them, STED is one of the major super-resolution imaging techniques with unique and straightforward approach achieving super-resolution imaging without postreconstruction and chemical reactions.78–82 In recent years, various organic molecules and inorganic nanomaterials (especially quantum dots) have been developed and widely used for STED microscopy due to their high QYs and good photostability, respectively.83–91 However, the poor photostability (restricting the long-term imaging) of organic molecules and the potential toxicity of heavy metal elements in inorganic nanomaterials still seriously hinder their STED imaging application. In fact, CDs with high QYs, excitation-independent and long-wavelength emission, desirable cellular uptake, and unique localization can be excellent candidates for STED imaging of subcellular organelles. In this research, we find that red emissive CDs with different QYs can be prepared by the hydrothermal treatment of pPDA, or the binary mixtures of pPDA–Ag+, pPDA–Cu2+, pPDA–PtCl42–, pPDA–Fe3+, pPDA–PdCl42–, and pPDA–Ni2+, and the resultant CDs are

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termed pPCDs, Ag-pPCDs, Cu-pPCDs, Pt-pPCDs, Fe-pPCDs, Pd-pPCDs, and Ni-pPCDs, respectively (Scheme 1). We surprisingly find that all the above CDs do not contain metal elements, which ensures their excellent biocompatibility. In addition, the different QYs of the CDs indicate that these different metal ions can exert indispensible but different influences on the structure of the CDs. Among them, Ni-pPCDs show the highest red fluorescence QY of 45.6% and have various other advantages including good water-dispersibility and photostability, excitation-independent and polarity-responsive fluorescence emission, and nucleolar targeting performance, which enable the CDs to realize the high-quality STED imaging of the nucleoli. We have also confirmed the excellent in vivo imaging performance of Ni-pPCDs in mice and zebrafish. Compared with the commercial nucleolar dye, SYTO RNASelect, Ni-pPCDs exhibit various advantages, such as real-time live cell imaging capability, higher photostability, and lower cost.

Scheme 1. Schematic Illustrating the Synthetic Methods of A Series of Red Emissive CDs (Au/Cu/Pt-pPCDs, pPCDs, and Fe/Pd/Ni-pPCDs) and the Bioimaging Applications (Including the STED-Based Nucleolar Imaging in Mammalian Cells and in Vivo Imaging in Tumor-Bearing Mice and Zebrafish) Using Ni-pPCDs

RESULTS AND DISCUSSION

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Preparation and Characterization of CDs. We prepared 7 kinds of CDs including pPCDs (prepared from pPDA alone), Ag-pPCDs, Cu-pPCDs, Pt-pPCDs, Fe-pPCDs, Pd-pPCDs, and Ni-pPCDs, which showed a similar maximum emission peak at ~605 nm (Figure S1). To investigate the possible solvent polarity-sensitive fluorescence properties of the obtained CDs, we measured the QYs of the CDs in dichloromethane (DCM), dimethyl sulfoxide (DMSO), and water, respectively. As shown in Table 1, pPCDs show the QYs of 23.7%, 15.8%, and 0.6% in DCM, DMSO, and water, respectively. In comparison, Ni-pPCDs exhibit the highest QYs of 64.9% in DCM, 45.6% in DMSO, and 1.2% in water, while Ag-pPCDs possess the lowest QYs of 2.3% in DCM, 1.6% in DMSO, and 0.1% in water. The above results indicate the metal ions can significantly affect (either increase or decrease) the fluorescence properties of CDs, which is quite different from the common phenomenon that metal elements usually result in the fluorescence quenching of CDs. Additionally, the fluorescence emission of these CDs in the two organic solvents (DCM and DMSO) is much stronger than that in water, and the QYs of these CDs in DCM are the highest, indicating that solvent polarity also influences the fluorescence properties of these CDs.

Table 1. QYs of the CDs dispersed in DCM, DMSO, or water. QY in DCM (%) QY in DMSO (%) QY in water (%)

pPCDs

Ag-pPCDs

Cu-pPCDs

Pt-pPCDs

Fe-pPCDs

Pd-pPCDs

Ni-pPCDs

23.7

2.3

6.9

6.9

28.3

33.5

64.9

15.8

1.6

4.6

4.4

19.2

24.6

45.6

0.6

0.1

0.2

0.2

0.7

0.9

1.2

Next, we selected Ag-pPCDs (with the lowest QY), pPCDs (synthesized without metal 7



ions), and Ni-pPCDs (with the highest QY) as the three typical CDs for further investigation. As shown in Figure 1a–g, the sizes of Ag-pPCDs, pPCDs, and Ni-pPCDs are 2.0 ± 0.4, 2.5 ± 0.5, and 2.9 ± 0.5 nm, respectively, and the zeta potentials of these CDs are +26.3 ± 2.5, +25.3 ± 1.5, and +23.7 ± 2.1 mV, respectively, indicating that a larger size and a lower surface potential may be beneficial for achieving a higher QY. The ultraviolet–visible (UV–vis) absorption spectra (Figure 1h) of these CDs show three sharp peaks at 204, 243, and 286 nm and one broad band centered at ~510 nm. The former three peaks originate from the π–π* transition of aromatic C=C bond, and the latter band is ascribed to the n–π* transition of C=N, C–N–C, C=O, and C–O bonds. Fourier-transform infrared (FTIR) spectra of the three CDs (Figure 1i) reveal that all the samples exhibit similar absorption bands at 3450−2850 cm−1 (N−H and/or O−H stretching vibrations) and peaks at ~1260 cm−1 (C−N stretching vibration), ~1140 cm−1 (C−O stretching vibration), and ~825 cm−1 (C−N stretching vibration from C−NO2). However, Ni-pPCDs and pPCDs show two peaks at 1650 and 1515 cm−1 and Ag-pPCDs exhibit two peaks at 1606 and 1480 cm−1, indicating the presence of C=O/N−H and C=C/N=O in these three kinds of CDs (the C=C bonds originate from the benzene ring of pPDA). Besides, Ni-pPCDs exhibit a higher C=O stretching vibration absorption at 1650 cm−1 than pPCDs, indicating an increase in the degree of oxidation accompanying a higher QY. Compared to Ag-pPCDs and pPCDs, Ni-pPCDs exhibit stronger O−H/N−H bands at 3450–2850 cm−1, suggesting the presence of more N−H and/or O−H groups on the surface of Ni-pPCDs.

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Figure 1. Characterization of CDs. (a–c) TEM images and (d–f) corresponding size distribution histograms of Ag-pPCDs, pPCDs, and Ni-pPCDs. (g) Zeta potential histogram, (h) UV–vis absorption spectra, and (i) FTIR spectra of the three kinds of CDs. X-ray photoelectron spectroscopy (XPS) was used to further investigate the chemical structures and compositions of the CDs. The full curves presented in Figure 2a, e, and i show three typical peaks at 285 eV (C 1s), 400 eV (N 1s), and 531 eV (O 1s). Surprisingly, in the curves of Ag-pPCDs (Figure 2a) and Ni-pPCDs (Figure 2i), no Ag or Ni element can be found, verifying the metal-free characteristic of the resultant CDs. In the high-resolution curves, the C 1s bands of Ag-pPCDs/pPCDs/Ni-pPCDs can be deconvoluted into three peaks at 284.5/284.5/284.5, 285.6/285.5/285.5, and 288.4/287.2/287.4 eV, corresponding to sp2 carbons (C−C/C=C), sp3 carbons (C−N/C−O), and carbonyl carbons (C=O), respectively (Figure 2b, f, and j). The N 1s bands of Ag-pPCDs/pPCDs/Ni-pPCDs can be deconvoluted into three peaks at 398.1/398.4/398.2, 399.6/399.6/399.3, and 401.4/401.0/400.9 eV,

9



representing pyridinic N, amino N, and pyrrolic N, respectively (Figure 2c, g, and k). The O 1s bands contain two peaks at 530.9/530.8/531.0 and 532.2/532.6/532.4 eV originating from C=O/N=O and C−O/N−O, respectively (Figure 2d, h, and l). Meanwhile, the peak intensities at 288.4/287.2/287.4 eV gradually increase from Ag-pPCDs to pPCDs and to Ni-pPCDs, implying a corresponding increase in the content of carbonyl groups in the CDs. The sequence of the relative amino N content in the three CDs is Ni-pPCDs > pPCDs > Ag-pPCDs. Moreover, in the O 1s high-resolution curves, pPCDs and Ni-pPCDs exhibit a higher C=O/N=O content than Ag-pPCDs, indicating the higher surface oxidation degree in Ni-pPCDs and pPCDs, which will possibly induce a larger amount of surface defects. As is well known, the size, surface state, and crosslinking enhanced emission effect are the main factors determining the fluorescence properties of the corresponding CDs.1,2,45,92,93 For Ni-pPCDs, similar to a previous report that Fe3+ catalyzes the formation of red-emissive CDs,93 the added Ni2+ ions during the hydrothermal reaction may also serve as the catalyst to promote the formation of Ni-pPCDs. As shown in Table 1 and Figures 1 and 2, the CDs prepared without metal ions and the CDs prepared by other metal ions (except Ni2+ ions) have either smaller sizes or lower amino/pyrrolic N and carbonyl contents, indicating the indispensible role of Ni2+ ions in the formation of the CDs. We propose that the Ni2+ ions can have strong coordination interaction with the functional groups (i.e., the primary amine groups) of the raw materials (the pPDA molecules) to form Ni2+–pPDA networks, as demonstrated by the different colors of the NiCl2 solution, the pPDA solution, and the mixed solution of NiCl2 and pPDA (see Figure S2). The formed Ni2+–pPDA networks may be responsible for the formation of larger CDs with extended carbogenic cores and crosslinking

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enhanced fluorescence emission effect, and promotion of the surface oxidation degree (proved by the higher contents of N=O and C=O groups), which are beneficial for the fluorescence emission of the formed CDs. Consequently, the larger size, higher surface oxidation degree, and crosslinking enhanced emission effect are responsible for the significantly increased QY of Ni-pPCDs.

Figure 2. XPS curves of (a–d) Ag-pPCDs, (e–h) pPCDs, and (i–l) Ni-pPCDs, including survey scans (a, e, and i) and the high-resolution XPS peaks of C 1s (b, f, and j), N 1s (c, g, and k), and O 1s (d, h, and l), respectively. Nucleolus Imaging in Live/Fixed Cells. Benefiting from their high QYs, Ni-pPCDs were chosen for further investigation. As shown in Figure 3a, Ni-pPCDs exhibit unique excitation wavelength-independent fluorescence emission ranging from 550 to 700 nm, which can largely avoid the fluorescence interference with other fluorescent dyes and hold great promise in STED imaging. The fluorescence results correspond to the Commission Internationale del’Eclairage (CIE) color coordinate of (0.58, 0.42) (Figure 3b), indicating the red emission of Ni-pPCDs. Besides, as revealed in Figure S3, Ni-pPCDs also exhibit good fluorescence 11



stability (Em: 605 nm) in solutions with pH values of 3–10 or in solutions containing different concentrations (0–1000 mM) of NaCl. Considering the possible polarity-dependent fluorescence emission of Ni-pPCDs, the fluorescence emission spectra of Ni-pPCDs dispersed in different solvents including water, trichloromethane (TCM), DCM, DMSO, ethanol, and methanol were also collected. From Figure S4, the fluorescence excited at 500 nm exhibits blue-shifted emission (from red emission to green emission) and its intensity decreases in solvents with decreasing polarity (the sequence of solvent polarity: water > methanol > ethanol > TCM > DCM). Although the polarity of DMSO is larger than that of ethanol and methanol, the CDs display higher fluorescence intensity in DMSO than in the other two solvents, which is possibly due to the superior dispersity of the CDs in DMSO. Moreover, the fluorescence intensity of the CDs increased when they were transferred from an aqueous solution to a more hydrophobic intracellular environment, and such a polarity-responsive fluorescence emission property of the CDs is important for them to realize wash-free fluorescence imaging (Figure S5). Next, we explored the intracellular localization of the fluorescent Ni-pPCDs. After treating A549 cells (human lung cancer cells) with Ni-pPCDs (5 μg/mL) for different time periods (0.5, 1, 2, 4, 6, 9, 12, and 24 h), the wash-free confocal imaging and flow cytometry analysis were carried out for the treated cells. As shown in Figure S6, the nucleoli (the 1–4 spherical structures in the cell nucleus) were lit up with bright red fluorescence at each time point, which can be explained by the selective accumulation of the CDs in nucleoli and/or the turn-on fluorescence of the CDs due to their improved dispersion in nucleoli. The fluorescence intensity of the stained cells first sharply increased, reached the maximum at 4 h,

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and then maintained stable, demonstrating the rapid and massive cellular uptake of the CDs. Besides, the cellular uptake of Ni-pPCDs increased with increasing CD concentration when the incubation time was fixed at 30 min (Figure S7). To investigate the nucleolus imaging mechanism, we conducted the costaining experiments of Ni-pPCDs and SYTO RNASelect dye (a commercial dye for nucleolus imaging). First, live cells were simultaneously treated by Ni-pPCDs and SYTO RNASelect for 30 min. The results in Figure 3c and d reveal that Ni-pPCDs stained the nucleoli with bright red fluorescence; however, SYTO RNASelect could not stain the nucleoli of live cells, which severely limits its application for real-time nucleolus tracking. Afterwards, we carried out the costaining experiments with Ni-pPCDs and SYTO RNASelect on fixed cells. As shown in Figure 3e and f, the red fluorescence signals (from Ni-pPCDs) in the nuclei of the fixed cells, which were sequentially stained with Ni-pPCDs and SYTO RNASelect, well overlaid with the green fluorescence signals (from SYTO RNASelect) with comparable fluorescence intensities. However, when the fixed cells were simultaneously stained by SYTO RNASelect and Ni-pPCDs or first stained by SYTO RNASelect and then by Ni-pPCDs, the red fluorescence intensity of nucleoli was much lower than the green fluorescence intensity, indicating a competitive interaction of Ni-pPCDs and SYTO RNASelect with nucleoli. The results above suggest that Ni-pPCDs share the same nucleolus imaging mechanism with SYTO RNASelect—the selective binding between ribonucleic acid (RNA) and Ni-pPCDs/SYTO RNASelect leads to the “turn-on” fluorescence of the two dyes within nucleolus.

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Figure 3. (a) Fluorescence emission spectra of Ni-pPCDs (dispersed in water; 20 μg/mL) collected at different excitation wavelengths (420–620 nm). (b) CIE chromaticity diagram of Ni-pPCDs dispersed in water. (c) Confocal images of live A549 cells simultaneously treated with Ni-pPCDs and SYTO RNASelect for 30 min. (d) Line-scan fluorescence intensity profile of the marked region (the white arrow) in (c). (e, g, and i) Confocal images of fixed A549 cells costained by Ni-pPCDs and SYTO RNASelect with different staining procedures: The fixed cells in (e) were sequentially stained with Ni-pPCDs (30 min) and SYTO RNASelect (30 min); the fixed cells in (g) were simultaneously treated with Ni-pPCDs and SYTO RNASelect for 30 min; the fixed cells in (i) were sequentially stained with SYTO RNASelect (30 min) and Ni-pPCDs (30 min). (f, h, and j) Line-scan fluorescence intensity profiles of the marked regions (the white arrows) in (e, g, and i). Interactions between CDs and DNA/RNA. Considering that most RNAs (especially the ribosomal RNA, rRNA) in nucleus accumulate in the nucleoli (which also contain some DNAs), the mechanism of Ni-pPCDs for nucleolus targeting was further evaluated. First, we studied the effect of DNA/RNA digestion on the cellular localization of Ni-pPCDs. The A549 cells were first treated with deoxyribonuclease (DNase, which hydrolyzes DNA) and then fixed/permeabilized, the Ni-pPCDs and SYTO RNASelect could still stain the nucleolus with marked contrast to other regions in the nucleus, respectively (Figure 4a), indicating the negligible influence exerted by DNA digestion. In contrast, when the cells were first treated by ribonuclease (RNase, which hydrolyzes RNA) and then fixed/permeabilized, the nucleolar region could not be stained clearly by Ni-pPCDs or SYTO RNASelect, which was ascribed to 14


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the decrease of RNA content in the nucleoli. To further evaluate the fluorescence response of Ni-pPCDs to RNA/DNA binding, the fluorescence of Ni-pPCD dispersion before and after the addition of RNA or DNA was measured. For comparison, the fluorescence of SYTO RNASelect solution before and after the addition of RNA or DNA was also measured. As shown in Figure 4b and c, both Ni-pPCDs and SYTO RNASelect had the fluorescence “turn-on” response to the two types of nucleic acids, and the fluorescence increase of the two reagents was higher after binding with RNA than with DNA. The different fluorescence responses of Ni-pPCDs to RNA and DNA may be due to the structural diversity of two types of nucleic acids, which affects the electrostatic interaction between the positively-charged CDs and the negatively-charged nucleic acids. In summary, benefiting form their polarity-sensitive and selective RNA-responsive fluorescence properties, Ni-pPCDs show highly enhanced fluorescence emission when entering the hydrophobic intracellular environment, especially the nucleolus, where they can bind to RNA and emit strong fluorescence. Hence, Ni-pPCDs can successfully realize wash-free and selective nucleolus-targeted imaging in live mammalian cells.

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Figure 4. (a) Confocal images of A549 cells before (control) and after treatment with DNase (25 μg/mL) or RNase (25 μg/mL) at 37°C for 2 h. Before imaging, the cells were fixed by methanol, permeabilized by 0.5% Triton X-100, and costained by Ni-pPCDs (5 μg/mL, Ex: 552 nm), SYTO RNASelect (1 μM, Ex: 488 nm), and Hoechst 33342 (5 μg/mL, Ex: 405 nm) for 15 min. (b and c) Fluorescence spectra of Ni-pPCDs (5 μg/mL, Ex: 500 nm) and SYTO RNASelect (1 μM, Ex: 488 nm) in water, RNA solution, or DNA solution (RNA or DNA concentration: 150 μg/mL), respectively. Photostability and Endocytosis Mechanism of Ni-pPCDs. As displayed in Figure 5a and b, the red fluorescence from Ni-pPCDs remained bright (> 65% of its original intensity) even after 1-h continuous 532 nm laser irradiation. In contrast, the green fluorescence from SYTO RNASelect significantly decreased to 16% of its original intensity after the 1-h 488 nm laser irradiation. These results above confirm the excellent anti-photobleaching property of Ni-pPCDs, ensuring their suitability for photostable and long-term fluorescence imaging applications.

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Considering the high cellular uptake efficiency of Ni-pPCDs, we also investigated their cellular internalization pathways. Commonly, there are four types of endocytosis including macropinocytosis, clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and lipid raft-mediated endocytosis (LrME). We evaluated the influences of 4°C (inhibiting

the

energy

(5-(N,N-dimethyl)-amiloride

supply)

and

hydrochloride

the

four

(amiloride),

endocytosis

inhibiting

inhibitors

macropinocytosis;

chlorpromazine (CPZ), inhibiting CME; genistein, inhibiting CvME; methyl-β-cyclodextrin (MβCD), inhibiting LrME) on the cellular internalization of Ni-pPCDs. Compared with the fluorescence in the control group (in which the cells were stained with Ni-pPCDs without any other treatments), the fluorescence in the 4°C group significantly declined by ~90% (Figure 5c and d). Meanwhile, the fluorescence in the amiloride, CPZ, genistein, and MβCD groups declined by 11%, 8%, 28%, and 70%, respectively. These results demonstrate that the cellular internalization of Ni-pPCDs mainly involved temperature-dependent transport and LrME.

Figure 5. (a) Confocal fluorescence images of Ni-pPCDs (red)- and SYTO RNASelect 17



(green)-stained A549 cells after continuous irradiation (552 nm for Ni-pPCDs-stained cells and 488 nm for SYTO RNASelect-stained cells) for different time periods. (b) Photostability evaluation results of Ni-pPCDs and SYTO RNASelect. The fluorescence intensities were obtained from the confocal images shown in (a). *P < 0.05. (c) Confocal fluorescence images of Ni-pPCD-treated A549 cells and (d) corresponding fluorescence intensities. Control: Cells were stained with Ni-pPCDs (5 μg/mL) for 30 min without other pretreatments; 4°C, amiloride, CPZ, genistein, or MβCD: Cells were first incubated at 4°C for 30 min, or treated with amiloride, CPZ, genistein, or MβCD for 2 h, and then stained with Ni-pPCDs (5 μg/mL) for another 30 min. STED Imaging of Ni-pPCD-Treated Cells. For the selection of suitable probes for STED imaging, the wavelength of depletion laser must be set at the red tails of fluorescence emission spectra of CDs. In addition, the CDs should also possess excellent fluorescence properties including good photostability, high QY, and unique intracellular performance such as organelle-specific targeting. All the above-mentioned requirements make our CDs unique and especially suitable for STED imaging compared with other red-emitting CDs. Specifically, in our case, the unique fluorescence emission of Ni-pPCDs endowed them with the ability of STED imaging using a commercial 660 nm depletion laser equipped with the Leica TCS SP8 STED 3X microscope (which has an imaging resolution of ~80 nm), which can realize clearer fluorescence imaging with a remarkably improved resolution. Thus, we treated the live cells with Ni-pPCDs (5 μg/mL) for 30 min followed by wash-free STED imaging using a 552 nm laser for excitation and a 660 nm laser for stimulated depletion. The confocal and STED images of a cell and its enlarged nucleoli are shown in Figure 6. The fluorescence intensity analysis of the marked positions (white arrows) of the enlarged nucleoli show the full-width-at-half-maximum values of 172, 146, and 164 nm in the STED images (Figure 6I1–III1 and I2–III2) compared to 443, 852 and 426 nm in the conventional confocal images (Figure 6IV1–VI1 and IV2–VI2). These values are in good agreement with

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imaging resolution parameter of the microscope. To the best of our knowledge, it is the first time that super-resolution imaging of nucleolus is realized by red-emitting CDs without washing treatment, showing great potential of the CDs in the applications of precise bioimaging and nucleolus-related researches.

Figure 6. (a) STED image and (b) confocal image of A549 cells stained by Ni-pPCDs. (I1, II1, and III1) Enlarged STED images of the nucleoli of the A549 cell in (a), and (I2, II2, and III2) corresponding fluorescence intensity analysis results of the marked lines in (I1, II1, and III1). (IV1, V1, and VI1) Enlarged confocal images of the nucleoli of the A549 cell in (b), and (IV2, V2, and VI2) corresponding fluorescence intensity analysis results of the marked lines in (IV1, V1, and VI1). In Vivo Imaging Using Ni-pPCDs. Considering that long wavelength-emitting dyes can minimize the phototoxicity to biological samples and the interference from the background autofluorescence of cells and tissues,94,95 the red-emitting Ni-pPCDs may hold great promise

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for in vivo fluorescence imaging. Before in vivo imaging, the biocompatibility of Ni-pPCDs was evaluated. Figure S8 reveal that Ni-pPCDs showed low toxicity to the AT II normal lung cells and A549 lung cancer cells because more than 80% cells were still alive when the Ni-pPCD concentration was as high as 50 μg/mL. Afterwards, we evaluated the in vivo imaging performance of Ni-pPCDs in tumor-bearing mice and zebrafish. As shown in Figure 7a and b, the tumor region of the mouse emitted bright red fluorescence after the intratumoral injection of NipPCDs (dose: 0.5 mg/kg). As time went by, the fluorescence intensity of the tumor region gradually decreased. The high signal-to-noise ratio of the tumor fluorescence was attributed to the excellent fluorescence properties of the CDs. We further investigated the biodistribution of Ni-pPCDs. The major organs and tumor tissues of the treated mice were excised at 1, 3, and 7 d post-injection and then imaged by the in vivo fluorescence imaging system. As shown in Figure 7c and d, Ni-pPCDs mainly accumulated in the tumor sites at 1, 3, and 7 d post-injection with negligible distribution in heart, liver, spleen, lung, and kidneys, demonstrating the good biosafety of Ni-pPCDs. Because of the presence of a protective mucus layer covering the epidermal cells in zebrafish, it is highly difficult for most hydrophobic dyes to image the whole zebrafish.96 With the great significance of developing novel fluorescent dyes for zebrafish imaging,96 we further investigated the imaging performance of Ni-pPCDs in zebrafish. At 48 hours post fertilization (hpf), the zebrafish was incubated in Ni-pPCD (10 μg/mL)-containing medium for 30 min and then imaged by confocal microscopy. As shown in Figure 7e and f, the whole zebrafish was well stained by Ni-pPCDs with bright red fluorescence, indicating that Ni-pPCDs can successfully pass through the mucus layer and interact with the cells in

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zebrafish, which may be attributed to their ultrasmall size, positively charged surfaces, and amphiphilicity (coming from the benzene rings and amino groups). The excellent zebrafish imaging achieved by the CDs also proves the the great potential for zebrafish-related researches.

Figure 7. (a) In vivo fluorescence images and (b) corresponding fluorescence intensity results of the tumors after intratumoral injection of Ni-pPCDs (dose: 2 mg/kg). The blue dotted circles in (a) indicate the tumor areas. “Pre” indicates the mice before injection. (c) Ex vivo fluorescence images and (d) quantitative fluorescence intensity results of Ni-pPCDs in heart (H), liver (L), spleen (S), lung (Lu), kidneys (K), and tumor (T) of the mice after intratumoral injection of Ni-pPCDs (dose: 2 mg/kg) for different time periods (1, 3, or 7 days). **P < 0.01, ***P < 0.001. (e) Confocal fluorescence and (f) bright field images of zebrafish stained by Ni-pPCDs (10 μg/mL) for 30 min. Scale bar = 500 μm.

CONCLUSIONS In summary, we for the first time synthesized a series of red-emitting CDs with varied QYs via the hydrothermal treatment of pPDA and metal ions. It was found that the metal ions could significantly affect the properties of the obtained CDs, althouth the metal ions were all absent in the formed CDs. Among these CDs, Ni-pPCDs with various merits showed the

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highest red fluorescence QY (45.6%, in DMSO) and could realize wash-free, high-resolution, and high-quality nucleolar imaging. The high affinity for nucleolus, good photostability, and polarity/RNA binding-responsive turn-on fluorescence endow Ni-pPCDs with excellent nucleolar imaging performance, showing great advantages over the commercial nucleolar dye, SYTO RNASelect, which emits green fluorescence and can only stain the nucleolus of fixed dead cells. Besides, benefiting from their unique fluorescence excitation/emission wavelengths and excitation-independent fluorescence, Ni-pPCDs could realize STED imaging with an imaging resolution as high as 146 nm. Finally, with their red fluorescence emission and good biocompatibility, Ni-pPCDs also achieved satisfactory in vivo imaging both in mice and zebrafish models.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Materials and methods, fluorescence spectra, photograph, photostability assay results, confocal images, flow cytometric results, and cell viability results (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (21673037) and Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX18_0159).

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Nucleolus-Targeted Red Emissive Carbon Dots with Polarity-Sensitive

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