One-Step Synthesis of Ultrasmall and Ultrabright Organosilica

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One-Step Synthesis of Ultrasmall and Ultrabright Organosilica Nanodots with 100% Photoluminescence Quantum Yield: LongTerm Lysosome Imaging in Living, Fixed, and Permeabilized Cells Xiaokai Chen, Xiaodong Zhang, Liu-Yuan Xia, Hong-Yin Wang, Zhan Chen, and Fu-Gen Wu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b04700 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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One-Step Synthesis of Ultrasmall and Ultrabright Organosilica Nanodots with 100% Photoluminescence Quantum Yield: Long-Term Lysosome Imaging in Living, Fixed, and Permeabilized Cells

Xiaokai Chen,†,∥ Xiaodong Zhang,†,∥ Liu-Yuan Xia,† Hong-Yin Wang,† Zhan Chen,*,‡ and Fu-Gen Wu*,†

†

State Key Laboratory of Bioelectronics, School of Biological Science and Medical

Engineering, Southeast University, Nanjing 210096, China ‡

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann

Arbor, Michigan 48109, United States

Corresponding Authors *Fu-Gen Wu, E-mail: [email protected]. *Zhan Chen, E-mail: [email protected]. Author Contributions ∥

X.K.C. and X.D.Z. contributed equally.

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ABSTRACT Water-dispersible nanomaterials with superbright photoluminescence (PL) emissions and narrow PL bandwidths are urgently desired for various imaging applications. Herein, for the first time, we prepared ultrasmall organosilica nanodots (OSiNDs) with an average size of ~2.0 nm and ~100% green-emitting PL quantum efficiency via a one-step hydrothermal treatment of two commercial reagents (a silane molecule and rose bengal). In particular, the structural reorganization and halide loss of rose bengal during the hydrothermal treatment contribute to the ultrahigh quantum yield and low phototoxicity of OSiNDs. Owing to their low pH-induced precipitation/aggregation property, the as-prepared OSiNDs can be used as excellent lysosomal trackers with many advantages: (1) They have superior lysosomal targeting ability with a Pearson’s coefficient of 0.98; (2) The lysosomal monitoring time of OSiNDs is up to 48 h, which is much longer than those of commercial lysosomal trackers (< 2 h); (3) They do not disturb the pH environment of lysosomes and can be used to visualize lysosomes in living, fixed, and permeabilized cells; (4) They exhibit intrinsic lysosomal tracking ability without the introduction of lysosome-targeting ligands (such as morpholine) and superior photostability; (5) The easy, cost-effective, and scalable synthetic method further ensures that these OSiNDs can be readily used as exceptional lysosomal trackers. We expect that the ultrasmall OSiNDs with superior fluorescence properties and easily modifiable surfaces could be applied as fluorescent nanoprobes, light-emitting diode phosphor, and anti-counterfeiting material, which should be able to promote the preparation and application of silicon-containing nanomaterials.

Keywords: organosilica nanodots, metal-free fluorescent nanoprobe, ultrahigh quantum efficiency, lysosomal tracking, organelle-specific imaging

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Photoluminescent nanomaterials (PLNMs) are extensively used in various biomedical fields such as biosensing, bioimaging, diagnostics, and therapy.1–8 Compared to organic fluorescent molecules, PLNMs have higher photostability and larger tunable emission wavelength range, and have thus gained much attention in recent years.9–16 Although various PLNMs including semiconductor quantum dots,17–22 noble metal nanoclusters,23–25 upconversion nanomaterials,26,27 polymer dots,28 silicon-containing nanomaterials,29–42 and carbon/graphene quantum dots43–49 have been synthesized, their biomedical applications are usually limited because of many drawbacks such as potential cytotoxicity, low photoluminescence (PL) quantum efficiency, complicated synthetic procedures, broad PL emission peaks, poor water dispersibility, difficulty of modification, and high cost. Therefore, it is urgently needed to develop new PLNMs with superior physicochemical, optical, and biocompatible properties to overcome the above-mentioned shortcomings. On the other hand, lysosomes as the waste disposal system of cells are crucial regulators of many cellular processes such as energy homeostasis, autophagy, and cell death.50–52 Therefore, much effort has been devoted to tracking lysosomes for investigating their states and behaviors during the biological processes.53–56 Among various imaging technologies, fluorescence labeling technique is considered to be a powerful method.57–65 Generally, there are two types of lysosomotropic reagents including acidotropic dyes and some large molecules. Acidotropic molecules are usually

weak

basic

amines,

such

as

N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine

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morpholine,66 (DAMP),

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neutral red (NR), acridine orange (AO), and Lyso-Tracker/Lyso-Sensor probes, which can accumulate in lysosomes due to the acidic lumen (pH 4.5–5) of the lysosomes.67 Nonetheless, there are still some deficiencies with these compounds, including nonfluorescence (morpholine and DAMP), low specificity for lysosomes (NR and AO), and induction of lysosomal pH increase and morphological/physiological changes (Lyso-Tracker/Lyso-Sensor probe).54,67 The second category of the lysosomal markers was designed based on large molecules such as Alexa 594-conjugated dextran and BODIPY-conjugated bovine serum albumin,68 which are uptaken by cells through the endo-lysosomal pathway. However, the rapid degradation and the low photostability hinder the application of these biomarkers for long-time live cell imaging. In this research, for the first time, we synthesized green-emitting organosilica nanodots (OSiNDs) with photoluminescence quantum yields (PLQYs) of up to ~100% and narrow PL bandwidths (full width at half maximum (FWHM) ≈ 30 nm). As shown in Scheme 1, the fluorescent OSiNDs were synthesized via a simple one-step hydrothermal reaction between rose bengal (RB) and silane molecule. It can be seen that the characters “OSiNDs” written on filter paper using the OSiND solution (colorless in dilute form) displayed very bright green emission under 302 nm ultra violet (UV) light irradiation, suggesting the possible application of OSiNDs in anti-counterfeiting field. As we will discuss in detail below, after incubation with the cells, OSiNDs could visualize the lysosomes with many advantages compared with other materials discussed above, including high specificity, low toxicity, fixation- and

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permeabilization-tolerant capability, and universal lysosomal tracking ability for different cell lines (normal cells, cancerous cells, and macrophages). Furthermore, long-time lysosomal monitoring was achieved due to the good photostability and the precipitation/aggregation-induced lysosomal retention property of the OSiNDs. Finally, we should mention that the OSiNDs prepared in this work are different from carbon dots (see detailed discussion in the supporting information).

Scheme 1. Schematic illustrating the synthesis of green-emitting OSiNDs with ultrahigh quantum yields and low phototoxicity, and their application in long-time lysosomal imaging. The characters “OSiNDs” were written on filter paper by using OSiND solution, and were pictured under the 302 nm UV light irradiation.

The highly fluorescent OSiNDs were prepared by the hydrothermal treatment of the aqueous

mixture

containing

RB

and

3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (AEEA) at 160 oC for 4 h (Figure 1a). The OSiNDs were also synthesized from two other silane molecules, (3-aminopropyl)trimethoxysilane

(APTMS)

and

N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO), which were denoted as APTMS OSiNDs and DAMO OSiNDs, respectively. The OSiNDs obtained from 5


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AEEA (abbreviated as OSiNDs) were used as the representative in the following characterization

and

lysosomal

tracking

application.

Transmission

electron

microscopy (TEM) image shows that the average diameter of OSiNDs is ~2.0 nm (Figure 1b), which is similar to those of APTMS OSiNDs and DAMO OSiNDs (Figure S1). Selected area electron diffraction (SAED) pattern (Figure S2) and powder X-ray diffraction (XRD) spectrum (Figure S3) indicate the amorphous nature of OSiNDs. To explore the chemical composition and functional groups in OSiNDs, Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) experiments were carried out (Figure 1c–h). Four elements can be observed in the XPS spectrum of OSiNDs including C (58.3%), O (19.6%), N (15.8%), and Si (6.5%) (Figure 1d). Cl and I (from RB molecules) could not be detected, indicating the loss of halide atoms after the hydrothermal reaction (due to the instability of C–Cl and C–I bonds at high temperatures69) and dialysis. In the FTIR spectrum of OSiNDs (Figure 1c), the stretching vibrations of Si–O–Si and Si–O–H bonds are observed at 1112 and 1033 cm–1, respectively. The presence of these silicon-containing bonds can also be verified by the Si2p signal (Si–O–H: 102.5/103.1 eV and Si–O–Si/C: 103.3/103.6 eV, Figure 1e)70 and O1s signal (Si–O: 532.2 eV, Figure 1h) in the XPS spectra. These results indicate the formation of a silica-like structure in OSiNDs via the partial crosslinking between the hydrolyzed AEEA molecules during the hydrothermal reaction. Meanwhile, the two peaks at 1633 and 1573 cm–1 in the FTIR spectrum of OSiNDs (marked by arrows) are attributed to the vibrations of amide bonds,

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indicating that the reaction between the carboxyl group of RB and the amine group of AEEA during the hydrothermal treatment may also contribute to the formation of the final OSiNDs. Besides, unreacted amine groups of OSiNDs can be observed in both FTIR (~3300 cm–1, Figure 1c) and XPS (400.4 eV, Figure 1g) spectra, making the OSiNDs suitable for further chemical modifications. Based on these results, we could summarize the process of the formation of OSiNDs as following: During the hydrothermal reaction, the hydrolyzed silane molecules were partially crosslinked and the RB molecules lost their halide atoms. Meanwhile, the RB molecules were conjugated with the silane molecules via the reaction between the carboxyl and the amine groups at high temperatures.71 Such a series of structural reorganizations contribute to the final formation of OSiNDs with a silica-like structure. To further study

the

molecular

composition

of

OSiNDs,

the

matrix-assisted

laser

desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry experiment was performed. Several peaks in the region of m/z = 700 to 3500 can be observed in the MALDI-TOF mass spectrum. The strongest peak is located at ~1380, which can be assigned to [(hAEEA)5(hfRB)–(H2O)5+Na]+ (hAEEA: a hydrolyzed AEEA silane molecule, hfRB: a halogen-free RB molecule). Similarly, the peaks at m/z ~1585 and ~1790

can

be

attributed

[(hAEEA)6(hfRB)–(H2O)6+Na]+

to

and

[(hSi)7(hfRB)–(H2O)7+Na]+, respectively. Then the possible geometry structures of the main product [(hAEEA)5(hfRB)–(H2O)5] were optimized using MM2 force field for energy minimization (Figure S4). The total energy of each of the seven isomers of [(hAEEA)5(hfRB)–(H2O)5] ranges from 34.6 to 45.8 kcal/mol. The structure in Figure

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S4e with the lowest energy of ~34.6 kcal/mol may be the main form of the hdyrothermal product. Meanwhile, the MALDI-TOF mass spectra of APTMS and DAMO OSiNDs were also collected (Figure S5). The strongest peaks at ~1109 (Figure

S5a)

and

~1373

(Figure

S5b)

are

assigned

to

[(hAPTMS)4(hfRB)2–(H2O)7+Na]+ and [(hDAMO)6(hfRB)–(H2O)7+3CH2–H+2Na]+, respectively, indicating the number of RB molecules in an APTMS OSiND is larger than that in a DAMO or AEEA OSiND.

Figure 1. (a) Schematic illustration of the synthetic method of OSiNDs via hydrothermal reaction. (b) TEM image and corresponding size distribution histogram (inset) of OSiNDs. (c) FTIR spectra of AEEA, OSiNDs, and RB. (d) Survey XPS spectrum of OSiNDs and the high resolution XPS peaks of (e) Si2p, (f) C1s, (g) N1s, and (h) O1s. (i) MALDI-TOF mass spectrum of OSiNDs.

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Additionally, the reaction could be monitored through the color change of the reactants. The color of the reaction mixture turned from rose red to greenish orange after the hydrothermal reaction (Figure S6). Under 302 nm UV light irradiation, OSiNDs emitted strong green fluorescence. To further characterize the fluorescence properties of OSiNDs, ultraviolet–visible (UV–vis) absorption and fluorescence spectra were collected. OSiNDs have an absorption/excitation peak at ~511 nm and a very narrow excitation-independent fluorescence emission peak at 525 nm (FWHM ≈ 30 nm) (Figure 2b and Figure S7). The narrow PL bandwidth of OSiNDs may be mainly attributed to the similar property of the reactant RB molecule whose fluorescence emission FWHM is ~35 nm (Figure S8). Additionally, the as-prepared OSiNDs are uniform with a narrow size distribution (Figure 1b), which can decrease the effect of inhomogeneous broadening due to size fluctuations. The uniform size of the nanoparticles may also be responsible for the low FWHM value.72 The fluorescence results correspond to the Commission Internationale de l’Eclairage (CIE) color coordinates of (0.23, 0.72) (Figure 2c), which is close to the spectral locus, indicating the saturated green emission of OSiNDs and showing the potential application of OSiNDs as a novel green phosphor for light-emitting diodes (LEDs). Using quinine sulfate (dissolved in 0.01 M H2SO4, PLQY = 54%) or rhodamine B (dissolved in ethanol, PLQY = 68%) as references,73 the PLQY of OSiNDs was calculated to be nearly 100% (Table 1). Meanwhile, the absolute PLQY value of OSiNDs was measured to be ~99% using an absolute quantum yield measurement system (Figure S9), which agreed well with the calculated relative PLQY value.

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Meanwhile, UV–vis and fluorescence spectra of the reactant AEEA and RB aqueous solution before and after the hydrothermal treatment were measured (Figure S8). Only weak fluorescence was observed after the hydrothermal treatment of AEEA (PLQY < 1%) or RB aqueous solution (PLQY ~1%) alone (Figure S6, Table 1), indicating that both RB and silane molecules are essential for preparing OSiNDs. The RB molecule has a conjugated structure, but its low water-solubility and high content of halide atoms decrease its fluorescence efficiency due to the “heavy atom effect”, in which replacement of an atom with one of higher atomic number can lead to an increase in the probability of forming a triplet state, and thereby inducing a photosensitization reaction and reducing the PL efficiency and lifetime of the fluorescence process.74–76 After the hydrothermal treatment of RB and silane molecules, the product had excellent water-dispersity (Figure 1b and Figure S1), thereby decreasing the self-quenching effect. Meanwhile, the XPS result (Figure 1d) indicates the occurrence of the dehalogenation reaction during the hydrothermal treatment, which could significantly reduce the transition possibility of the excited state (S1) to the triple state (T1) in OSiNDs, leading to the stronger fluorescence emission and less singlet oxygen generation (Figure 2d). This was verified by the electron paramagnetic resonance (EPR) (Figure S10) and transient fluorescence studies (Figure 2e). For the RB solution, strong singlet oxygen (1O2) signal was observed after irradiation. By contrast, the OSiND solution could hardly produce singlet oxygen at the same condition. The fluorescence lifetime of OSiNDs was calculated to be 4.2 ns, which was much longer than 0.78 ns of RB (Figure 2e),

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further proving the “heavy atom effect”. On the other hand, resulting from the effect of silane molecules, the fluorescence lifetime of OSiNDs shows the same order of magnitude as those of other silicon-based nanomaterials,31,77 which can be explained by their similar silane molecules as the silicon sources. On the other hand, for APTMS OSiNDs and DAMO OSiNDs, their UV–vis absorption and fluorescence properties are analogous to those of AEEA OSiNDs (Figure S11), suggesting that these OSiNDs may have a similar structure. Nevertheless, the PLQY value of APTMS OSiNDs (~74%) is lower than those of DAMO OSiNDs (~100%) and AEEA OSiNDs (~100%) (Table 1), which could be explained by the following reasons: (1) The amine contents of DAMO and AEEA are higher than that of APTMS, and it has been reported that a higher amine content can enhance the PL of silicon-based nanodots;31 (2) The number of RB molecules in an APTMS OSiND is larger than that in a DAMO or AEEA OSiND (Figure 1i and S5), which can increase the possibility of interparticle collisions, leading to a lower PLQY for APTMS OSiNDs.

Table 1. PLQYs of AEEA, RB, and OSiNDs. Sample

PLQY (%)

AEEA after hydrothermal (4 h) RB before hydrothermal RB after hydrothermal (4 h) APTMS OSiNDs (4 h) DAMO OSiNDs (4 h) AEEA OSiNDs (4 h)

20 nm, Figure S12b; 24 h: > 50 nm, Figure S12c). The above results were also confirmed by dynamic light scattering (DLS) (Figure S12d). Further, the relative PLQYs of the hydrothermal products after different reaction times were measured. It can be seen that 4 h is the optimal reaction time for achieving the highest PLQY, and a shorter (1 h) or longer (12 or 24 h) hydrothermal reaction time can decrease the PLQYs of AEEA OSiNDs (Table S1). Then the in vitro dark toxicity and phototoxicity of OSiNDs were tested using MTT assay. It can be seen that without irradiation, RB had negligible cytotoxicity towards the lung cancerous A549 cells. However, its cytotoxicity increased significantly when the cells were exposed to white light for 4 h (Figure 2f). By comparison, when the concentration of OSiNDs was < 1000 µg/mL, the OSiNDs-treated cells without and with white light irradiation preserved > 85% viability, demonstrating the good biocompatibility and low phototoxicity of OSiNDs (Figure 2g). The lower phototoxicity of OSiNDs compared to RB is consistent with the EPR results (Figure S10). Furthermore, the real-time cell analysis (RTCA), apoptosis assay, cell cycle distribution, and ATP level detection experiments were carried out to evaluate the effect of OSiNDs on the cell proliferation and metabolic process. The results shown in Figure 2h and Figure S13–S15 reveal that OSiNDs at the concentrations lower than

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200 µg/mL did not affect the proliferation and metabolic process of A549 cells, indicating the good biocompatibility of OSiNDs and ensuring their safe use in long-term cell imaging.

Figure 2. (a) Photographs of RB and OSiND solutions (100 µg/mL) under white light and UV light (302 nm) irradiation, respectively. 1, 2, 3, and 4 represent RB solution, APTMS OSiND solution, DAMO OSiND solution, and AEEA OSiND solution, respectively. (b) UV–vis absorption, fluorescence excitation, and fluorescence emission spectra of OSiND solution. (c) CIE chromaticity coordinate of OSiNDs. (d) Energy level diagram of OSiNDs. (e) Time-resolved fluorescence decay curves of RB solution and OSiND solution. Cytotoxicity evaluation of (f) RB and (g) OSiNDs without and with white light irradiation. (h) Real-time cell viability of A549 cells after incubation with different concentrations of OSiNDs.

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Since the OSiNDs can emit superbright fluorescence light and exhibit low toxicity, we would like to see if they can be used for cell imaging. Figure 3a shows that OSiNDs could effectively enter the A549 cells within 2 h post-incubation. Before observation under a confocal microscope, the OSiNDs-treated cells were stained with Lyso-Tracker Red (LT-Red) and Hoechst 33342 for lysosome and nucleus labeling, respectively. The remarkable yellow signals in the merged channel indicated the excellent co-localization of intracellular OSiNDs (green fluorescence) and lysosomes (red fluorescence). The calculated Pearson’s colocalization coefficient is 0.98, indicating that OSiNDs can image lysosomes with extremely high selectivity. Furthermore, the long-term co-localization performance (Figure 3a) demonstrates that OSiNDs had a high affinity for lysosomes and they did not translocate to other organelles for at least 24 h. Meanwhile, the superior lysosomal imaging results of LT-Red (which was used to visualize lysosomes just before observation) during the 24 h period suggest that OSiNDs had negligible disturbance to the lysosomal environment. To further evaluate the lysosomal imaging ability of OSiNDs, two commonly used commercial lysosomal markers, LT-Red and Lyso-Tracker Green (LT-Green), were selected. Compared with OSiNDs, LT-Red had a significant reduction of lysosomal retention and the red dots were distributed on the nuclear membrane and even in the nucleus after 8 h incubation (Figure 3b). In addition, the lysosome-targeting stability was also tested. After pretreated with OSiNDs (2 h), LT-Green (30 min), and LT-Red (30 min), respectively, the A549 cells were washed

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with PBS solution to remove the extracellular probes and incubated in dark for different time periods (1, 2, 4, 12, 24, 36, or 48 h). The confocal images (Figure 3c) and the corresponding analyses by flow cytometry (Figure 3d) illustrate that the imaging performance of OSiNDs was much better than those of the representative commercial lysosomal dyes (LT-Green and LT-Red). The fluorescence intensities of LT-Green and LT-Red rapidly decreased within 2 h. In contrast, the fluorescence intensity of OSiNDs was still higher than 50% of its original value within the whole time period of 48 h. Similarly, to confirm that the OSiNDs were still in the lysosomes during the entire 48 h time period, the cells were co-stained with LT-Red just before confocal imaging. The co-localization results verify the high lysosome-targeting stability of OSiNDs (Figure S16). In addition, the resolution of OSiNDs during long-term fluorescence imaging was measured (Figure S16). For control purposes, the OSiNDs-stained lysosomes at different time points were also stained with LT-Red before the fluorescence images were collected. The resolution values of OSiNDs at different time points remained consistent in the range of 380–500 nm, which were similar to those of LT-Red. Also, the resolution matched well with that of confocal microscopy (~400 nm) and the size of lysosomes (100 to 1200 nm). Since OSiNDs had a potential for long-term lysosomal tracking, the intracellular photostability was also evaluated. The lysosomes were first co-stained with OSiNDs and LT-Red, and then exposed to a 488 nm laser irradiation (0.05 mW, objective: 63×/1.40, zoom in factor: 3.78) for different time periods. Compared with the relatively strong photobleaching effect of LT-Red, whose fluorescence intensity

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quickly decreased within 3 min, OSiNDs exhibited excellent photostability whose fluorescence signal could be observed even after 30 min of laser irradiation. The good photostability of OSiNDs may likely be attributed to their low singlet oxygen generation efficiency (Figure S10), their silica-like structure which can protect the fluorescent moieties from photobleaching by oxygen molecules,78 and the intrinsic optical properties of silicon-based nanomaterials.34,77

Figure 3. (a) Co-localization images of OSiNDs and lysosomes at different incubation time points in A549 cells. The cells were incubated with 20 µg/mL OSiNDs for 2, 4, 18, and 24 h, respectively. Before imaging, the cells were stained with LT-Red (for 30 min) and Hoechst 33342 (for 10 min) to visualize lysosomes and 16


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nuclei, respectively. (b) Confocal images of A549 cells incubated with LT-Green (1 µM) and LT-Red (1 µM) for 30 min, 4 h, or 8 h. (c) Confocal images and (d) corresponding analyses by flow cytometry of long-term lysosomal imaging performance of OSiNDs, LT-Green, and LT-Red in A549 cells. Before confocal and flow cytometric experiments, the cells were first treated with 20 µg/mL OSiNDs (for 2 h), 1 µM LT-Green (for 30 min), and 1 µM LT-Red (for 30 min), respectively, and then washed with PBS solution and further incubated for 0, 1, 2, 4, 12, 24, 36, or 48 h. (e) Photostability comparison of OSiNDs (20 µg/mL) and LT-Red (1 µM) under 488 nm irradiation.

Besides, the superb lysosomal imaging ability of OSiNDs was also tested using different cell lines including MCF-7 (cancerous human breast cell line), AT II (normal lung cell line), and RAW264.7 (macrophages) cells (Figure S17). The excellent co-localization between OSiNDs and lysosomes indicated that OSiNDs were capable of universal lysosomal staining, regardless of the cell types. The APTMS OSiNDs and DAMO OSiNDs also had the good lysosomal imaging performance (Figure S18). Collectively, with their low long-term cytotoxicity, highly specific lysosome-targeting ability, excellent anti-photobleaching property, and universal lysosomal imaging performance, the OSiNDs are especially suitable for long-term monitoring of lysosomes in living cells. Furthermore, the retention ability of OSiNDs in the lysosomes of fixed and permeabilized cells was also investigated. Fixation preserves the cellular structures and permeabilization makes the membrane permeable. Thus, in many immunostaining experiments, living cells after staining are usually required to be fixed and

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permeabilized for further studies. As shown in Figure 4, the lysosomes in A549 cells were first stained with OSiNDs, LT-Green, and LT-Red, respectively. All of these lysosomes in the living cells displayed bright fluorescence. However, after fixation by 4% glutaraldehyde, the fluorescence intensities of lysosomes labeled with LT-Green and LT-Red drastically decreased, and the fluorescence signals almost completely disappeared after the fixed cells were further permeabilized by 0.1% Triton X-100. However, the OSiNDs-labeled lysosomes after fixation and permeabilization were as bright as those before the treatments. These results, together with the above living cell results, demonstrate the universal lysosomal staining capability of OSiNDs for living, fixed, and permeabilized cells.

Figure 4. Confocal images of A549 cells incubated with OSiNDs, LT-Red, or LT-Green before the treatments, after the fixation treatment, and after the permeabilization treatment.

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To explore the lysosome-targeting mechanism, we next examined the dispersibility of OSiNDs under various pH conditions. The OSiND solutions were transparent at neutral and basic conditions (pH 6.5–8.0), while the solutions became turbid at weakly acidic environments (pH 3–6), indicating the formation of large aggregates (Figure 5a). The extinction at 650 nm (Figure 5b) and the TEM images of OSiNDs in solutions with different pH values (Figure S19) confirm the above observation. Furthermore, we observed many aggregates within the lysosomes extracted from the OSiNDs-treated cells and the Si content in these lysosomes is much higher than that in control lysosomes (as revealed by the corresponding energy dispersive spectra) (Figure S20). These results verify that the low pH-induced precipitation/aggregation property of OSiNDs is responsible for the long-term lysosomal imaging ability. Besides, the endocytosis pathway and mechanism of OSiNDs were studied. Endocytosis is known as an active transportation process, in which adenosine triphosphate (ATP) plays an important role.79 Thus, we first investigated the influence of energy on the internalization of OSiNDs. Two strategies were adopted in this study, including the treatment of cells with sodium azide for depleting the intracellular ATP and incubation of the cells under low temperature (4 oC) for decreasing the activity of enzymes to lower the production of ATP. Both the sodium azide and 4 oC treatments significantly inhibited the internalization of OSiNDs (Figure 5c), revealing that the endocytosis of OSiNDs was energy-dependent. Then, we used different inhibitors to further study the endocytosis pathway of OSiNDs. Chlorpromazine (CPZ), genistein, and amiloride were used to inhibit clathrin-mediated, caveolae-mediated, and

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macropinocytosis-dependent pathways, respectively.80 Only genistein remarkably reduced the internalization of OSiNDs, while other inhibitors had negligible inhibition effects, suggesting that OSiNDs were endocytosed through the caveolae-mediated pathway (Figure 5c–e). From the above results, we could infer the possible long-time lysosomal targeting of OSiNDs through the following sequential events: (i) After incubation with the cells, the extracellular OSiNDs were endocytosed into the cells via a caveolae-mediated pathway. (ii) Afterwards, the intracellular OSiNDs were accumulated into the acidic organelles (lysosomes) due to the presence of weak basic amine groups in OSiNDs. (iii) Finally, large aggregates were formed in the acidic environment of lysosomes, realizing long-time and stable lysosomal imaging (Figure 5f).

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Figure 5. (a) Photographs of OSiND solutions (10 mg/mL) under different pH conditions, and (b) the corresponding extinction values at 650 nm. (c) Confocal images, (d) flow cytometric results, and (e) the corresponding quantitative histogram of A549 cells treated without inhibitors and with CPZ, amiloride, genistein, sodium azide, and 4 oC incubation, respectively, before adding 20 µg/mL OSiNDs to the cells. (f) Schematic illustration of the long-time lysosomal tracking mechanism of OSiNDs. The OSiNDs first entered cells via caveolin-mediated endocytosis to form endosomes, and then the endosomes fused with lysosomes in which the OSiNDs were precipitated in the acidic lysosomes for long-time lysosomal tracking.

In summary, for the first time we successfully prepared ultrabright green-emitting OSiNDs with outstanding optical properties including ultrahigh PLQYs (~100%), 21


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narrow PL bandwidths (~30 nm), and high photostability. The halide loss and structural reorganization of reactant during the hydrothermal reaction are the two main causes of the ultrahigh photoluminescence efficiency and low phototoxicity of OSiNDs. Notably, the prepared OSiNDs show intrinsic lysosomal tracking ability without the introduction of lysosome-targeting ligands. With their advantages of high lysosomal selectivity, ultralong lysosomal retention time (up to 48 h), universal lysosomal imaging capability for cells in different states (such as living, fixed, and permeabilized cells), excellent photostability, and low cytotoxicity, the lysosomal imaging performance of the OSiNDs outperform that of commercial lysosomal trackers. The excellent long-term lysosomal imaging capability is attributed to the acid environment-induced precipitation/aggregation of OSiNDs within the acidic lysosomes. We believe that the ultrasmall and ultrabright OSiNDs can be used as outstanding fluorescent nanoprobes for many chemical and biomedical applications, and ideal candidates for green-emitting LED phosphor and anti-counterfeiting material.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed materials, methods, additional discussion, and supplementary figures.

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AUTHOR INFORMATION Corresponding Authors *Fu-Gen Wu, E-mail: [email protected]. *Zhan Chen, E-mail: [email protected]. Author Contributions ∥

X.K.C. and X.D.Z. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21673037), Natural Science Foundation of Jiangsu Province (BK20170078), Innovative and Entrepreneurial Talent Recruitment Program of Jiangsu Province, Six Talents Peak Project in Jiangsu Province (2015-SWYY-003), Graduate Students’ Scientific Research Innovation Project of Jiangsu Province Ordinary University (KYCX17_0155), Scientific Research Foundation of Graduate School of Southeast University (YBJJ1777), and Fundamental Research Funds for the Central Universities. Z.C. thanks the University of Michigan for the support.

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One-Step Synthesis of Ultrasmall and Ultrabright Organosilica

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