Long-Term Lysosomes Tracking with a Water ... - ACS Publications

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Long-Term Lysosomes Tracking with a Water-Soluble Two-Photon Phosphorescent Iridium(III) Complex Kangqiang Qiu, Huaiyi Huang, Bingyang Liu, Yukang Liu, Ziyi Huang, Yu Chen, Liangnian Ji, and Hui Chao* MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, P. R. China S Supporting Information *

ABSTRACT: Lysosomes are the stomachs of the cells that degrade endocytosis and intracellular biomacromolecules and participate in various other cellular processes, such as apoptosis and cell migration. The ability of long-term tracking of lysosomes is very important to understand the details of lysosomal functions and to evaluate drug and gene delivery systems. For studying lysosomes, we designed and synthesized a water-soluble triscyclometalated iridium(III) complex (Ir-lyso) attaching morpholine moieties. The phosphorescent intensity of Ir-lyso is responsive to pH and decreases with an increase in the pH but not quenching in high pH. With excellent two-photon properties, Ir-lyso was used to light up the lysosomes in living cells and 3D tumor spheroids. Moreover, Ir-lyso could label lysosomes more than 4 days, so we developed this complex to act as a long-term probe for tracking lysosomes during cell migration and apoptosis. To the best of our knowledge, this is the first paradigm of metal complexes as the two-photon phosphorescent probe for long-term lysosomes tracking. KEYWORDS: lysosomes, two-photon imaging, iridium(III) complex, long-term tracking, 3D tumor spheroid

1. INTRODUCTION Lysosomes are often described as the stomachs of the cells to degrade biomacromolecules delivered by ways of phagocytosis, autophagy, and endocytosis.1,2 Additionally, lysosomes have a critical role in metabolism, cell antigen processing, cell migration, intracellular transport, plasma membrane repair, apoptosis, and exosome release.3−5 Lysosomal dysfunction is associated with diverse diseases, including silicosis, neurodegenerative disease, lysosomal storage diseases, inflammation, and cancer.6−9 Therefore, to understand the details of lysosomal functions and to evaluate drug and gene delivery systems, long-term tracking of lysosomes is needed.10,11 For studying lysosomes, fluorescence imaging techniques, such as confocal fluorescence microscopy, have been developed to visualize lysosomes.12 In confocal fluorescence imaging, the fluorescent probe is one of the key factors. The fluorescent probe used for long-term tracking of lysosomes is different from that used for long-term cell tracking,13,14 in addition to the need of photostability and safety, as well as the need of longterm specifically localized lysosomes. However, most of the commercially available fluorescent probes for lysosomes are © XXXX American Chemical Society

organic dyes and have a notable shortcoming of poor photostability,15,16 which limits researchers to observe the dynamic change of lysosome during a designated time period. Furthermore, the reported long-term lysosomal fluorescent probes, such as dextran-conjugated fluorophores,17 inorganic or hybrid fluorescent nanoparticles,11,18 and organic fluorophores,19,20 were excited by the short wavelength and led to cellular autofluorescence, photodamage, photobleaching as well as shallow penetration depth.21 To solve the above problems, great effort has been made to find the two-photon fluorescent materials because they exhibit lower phototoxicity, excellent photostability, greater penetration depths, and near-infrared (NIR) or longer excitation wavelengths.22−24 Having advantageous photophysical properties, transitionmetal complexes were used as two-photon luminescent probes.25,26 A more interesting thing is that some Ir(III) complexes have been used successfully as two-photon Received: March 21, 2016 Accepted: May 6, 2016

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DOI: 10.1021/acsami.6b03422 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

spectrometer (Mercury-Plus 300, Varian, U.S.A.) at 25 °C, respectively. For the 1H NMR spectrum, all chemical shifts are given relative to tetramethylsilane (TMS). The UV−vis spectrum, emission spectra, and time-resolved emission measurement were recorded on a PerkinElmer Lambda 850 spectrophotometer, a PerkinElmer LS 55 spectrofluorophotometer, and a FLS 920 combined fluorescence-lifetime and steady-state spectrometer at 25 °C, respectively. Quantum yield of phosphorescence was calculated by using [Ru(bpy)3]2+ (Φ = 0.028 in aerated aqueous solution) as the reference emitter according to literature procedures at room temperature (25 °C).40 The Origin 8 software package was used to process all data. 2.2. Synthesis of Ir-lyso. A mixture of water and 2ethoxyethanol (1:3, v/v) was added to a three-necked flask containing 4-(4-(pyridin-2-yl)benzyl)morpholine (pbm, 533 mg, 2.1 mmol) and IrCl3 (298 mg, 1.0 mmol) and then was refluxed for 24 h. After cooling, the solvent was evaporated under reduced pressure to give cyclometalated Ir(III) chlorobridged dimer, which can be utilized for the next step without further purification. The chloro-bridged dimer (147 mg, 0.1 mmol), pbm (254 mg, 1.0 mmol) and AgCF3SO3 (51.2 mg, 0.2 mmol) were added to a 50 mL three-necked flask with 10 mL of ethylene glycol. Under argon, the mixture was heated at 180 °C for 5 h. The reaction product was cooled to room temperature and poured into 100 mL of water after 5 h. To this solution, CH2Cl2 was added and extracted using a separatory funnel. Upon solvent evaporation from the organic layer, the crude product was then purified using silica gel (100−200 mesh size) column chromatography (CH2Cl2/ethanol) to get iridium complexes. Yield: 64.7 mg, 34%. Anal. Calcd for C48H51IrN6O3: C, 60.55; H, 5.40; N, 8.83. Found: C, 60.29; H, 5.62; N, 8.71. 1 H NMR (300 MHz, CDCl3) δ 7.81 (d, J = 9 Hz, 3H), 7.56− 7.60 (m, 6H), 7.51 (d, J = 3 Hz, 3H), 6.82−6.91 (m, 6H), 6.66 (s, 3H), 3.64 (s, 12H), 3.18 (d, J = 12 Hz, 6H), 2.30 (s, 12H). ESI-MS: m/z = 477.2 [M + 2H]2+, 953.8 [M + H]+. 2.3. X-ray Crystallography. A Rigaku R-AXIS SPIDER Image Plate diffractometer was used to perform X-ray diffraction measurement with Mo Kα radiation (λ = 0.71073 Å). The full-matrix least-squares refinement and structure solution based on F2 for Ir-lyso were performed with SHELXL 2013 program packages and the SHELXL 2013, respectively. All non-hydrogen atoms were refined with anisotropic thermal parameters and isotropic thermal parameters riding on those of the parent atoms were applied to all hydrogen atoms, which were included in calculated positions. Table S1 shows the crystal parameters and details of the data refinement and collection. Selected bond angles (o) and bond length (Å) are shown in Table S2. The supplementary crystallographic data (CCDC 1411837) for the present paper have been deposited with the Cambridge Crystallographic Data Centre and can be obtained free of charge via https://summary.ccdc.cam.ac.uk/ structure-summary-form. 2.4. Two-Photon Absorption Cross Sections. A broad spectral region by the typical two-photon induced phosphorescent (TPP) method, which is relative to Rhodamine B in methanol as the standard, was used to determine the twophoton absorption spectra of Ir-lyso. Opolette 355II (pulse width ≤100 fs, 80 MHz repetition rate, tuning range 710−840 nm, Spectra Physics Inc., U.S.A.) was used to acquire the twophoton phosphorescent data. Two-photon phosphorescent measurements were conducted with negligible reabsorption processes and were performed in fluorometric quartz cuvettes.

phosphorescent probes for subcellular organelles imaging27−31 and molecule detection32−34 in living cells. Because of these studies, it emerged that Ir(III) complexes could be promising candidates for two-photon phosphorescent imaging. Nevertheless, the reported two-photon phosphorescent Ir(III) complexes for lysosomes have high phototoxicity and cause cell autophagy, which could lead to cell death.30,31 In order to observe normal physiological activities of lysosome, a safe twophoton phosphorescent Ir(III) complex is needed. Until now, most of the two-photon phosphorescent Ir(III)based probes are biscyclometalated complexes.28−34 The triscyclometalated iridium analogues are much less studied because of their electronic-neutral molecules and limited water solubility.27,35−37 Herein, we developed a water-soluble triscyclometalated Ir(III) complex attaching morpholine moieties, Ir-lyso (Figure 1), as a safe two-photon probe for

Figure 1. Crystal structure of Ir-lyso. The hydrogen atoms were omitted for clarity.

long-term tracking of lysosomes. For the specific localization in lysosomes and hydrophilicity, the morpholine moiety within Irlyso is crucial.38 Once morpholine was protonated in lysosome, Ir-lyso would be retained in the lysosome because it is more hydrophilic. The protonated morpholine is a lock that allows Ir-lyso to accumulate in acidic lysosomes. The response capability of pH for Ir-lyso was inversely related to pH and the phosphorescence of Ir-lyso was still exhibited in high pH. With two-photon properties, the lysosomes of the HeLa cells and 3D tumor spheroids were lighted up by Ir-lyso. After a long-term tracking study, we found Ir-lyso could label lysosomes more than 4 days, so the probe was used for observing the dynamic changes of lysosomes during cell migration and apoptosis.

2. MATERIALS AND METHODS 2.1. Materials and General Instruments. IrCl3 and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma. LysoTracker Red DND99 (LTR) and MitoTracker Red FM (MTR) were purchased from Invitrogen. Fetal bovine serum (FBS) and Dulbecco’s modified eagle medium (DMEM) were purchased from Gibco. Phosphate-buffered saline (PBS) was prepared as follows: 1000 mL of H2O, 9 g of NaCl, 1.65 g of KH2PO4, 11.65 g of Na 2 HPO 4 . The compound 4-(4-(pyridin-2-yl)benzyl)morpholine (pbm) was prepared as the literature methods.39 Microanalysis (C, H and N), electrospray ionization mass spectrum (ESI-MS), and 1H NMR spectrum were measured with PerkinElmer 240Q elemental analyzer, LCQ system (Finnigan MAT, U.S.A.), and nuclear magnetic resonance B


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Scheme 1. Synthetic route to Ir-lyso: (i) HOCH2CH2OCH2CH3−H2O (3:1, v/v), IrCl3, reflux, 24 h; (ii) pbm, AgCF3SO3, ethylene glycol, 180 °C, 5 h, 34%

suspended in 150 μL of culture media and were left in a 37 °C humidified incubator with 5% CO2 for 72 h. For imaging procedures and subsequent drug treatments, spheroids with an average diameter of 450−550 μm were carefully transferred from 96-well plates to cell culture dishes for the experiments. Confocal laser scanning microscopy was used for imaging the spheroids. 2.7. Cell Uptake Pathway Studies. For the pathway study of cellular entry, HeLa cells were placed in two 35 mm2 Petri dishes (MatTek, U.S.A.) and incubated at 37 °C for 24 h. The dish was incubated with 2 μM Ir(III) complex at 37 or 4 °C for 30 min. Zeiss LSM 710 NLO confocal microscope (63×/NA 1.4 oil immersion objective) was used to image the cells after fresh PBS being washed with three times. At 750 nm (for Irlyso), the phosphorescence was excited. At 520 ± 20 nm (for Ir-lyso), the emission signal was collected. 2.8. Long-Term Tracking of Lysosomes. HeLa cells was seeded in 35 mm2 Petri dish (MatTek, U.S.A.) and incubated at 37 °C for 24 h. The cells were treated with 2 μM Ir-lyso (or LTR) for 30 min at 37 °C. The media was removed , and PBS buffer was used to remove the remaining probes by washing with three times. Then the cells were divided into two dishes and further incubated for 24 h. At the end of the 24 h incubation (the first passage), one dish was designated for photos, and 25% of the cells with fresh growth medium in another dish was transferred to the third dish. Photos were taken of the third dish after the end of 24 h (the second passage). HeLa cells in the second dish were further divided to the fourth dish as the third passage. The fourth passage was the last passage. For tracking of lysosomes during cell migration, the Petri dish with HeLa cells was prepared before 24 h. Then the cells were incubated with 2 μM Ir-lyso for 30 min at 37 °C. The dish was placed in a mini incubator after washing with PBS buffer three times, and the lysosomes were monitored by confocal laser scanning microscopy. For tracking the lysosomes of apoptosis, the Petri dish with HeLa cells was prepared before 24 h. After treatment with 2 μM Ir-lyso for 30 min and MTR for another 30 min at 37 °C, the dish was washed three times with PBS buffer and was placed in the mini incubator. A photo of the cells was taken by confocal laser scanning microscopy, and 20 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to the dish, after which the tracking of lysosomes began.

The quadratic dependence between the emission intensity and the incident power was verified at an excitation wavelength of 750 nm. The two-photon absorption cross sections of each wavelength for Ir-lyso were calculated according to eq 141 δ2 = δ1

ϕ1C1I2n2 ϕ2C2I1n1

(1)

where I denotes the integrated phosphorescent intensity, C denotes the concentration, n denotes the refractive index, and Φ denotes the quantum yield. Subscript “1” represents reference samples, and “2” represents samples. 2.5. Cell Culture Conditions and In Vitro Cytotoxicity. The culture conditions of HeLa cells were 37 °C and 5% CO2 in DMEM which contains 10% FBS. Seeded in triplicate into 96-well plates at 1 × 104 cells/well for 24 h, the exponentially grown HeLa cells were treated with increasing concentrations of Ir-lyso of 24 and 48 h. Twenty microliters of MTT (5 mg/mL) was added to each well to stain the viable cells, and then the plates were placed at 37 °C for 4 h. DMSO (200 μL) was added to each well after the media had been carefully removed. The optical density of each well was measured by a Tecan Infinite M200 monochromator-based multifunction microplate reader with background subtraction at 595 nm. It was considered 100% cell survival of the control wells without complex. 2.6. One- and Two-Photon Cellular Imaging. One milliliter of 1 × 105 HeLa cells was placed in 35 mm2 Petri dish (MatTek, U.S.A.) and incubated at 37 °C for 24 h. The cells were treated with 2 μM Ir-lyso for 30 min at 37 °C. The media was removed, and PBS buffer was used to remove the remaining probes by washing three times, the cells were further treated with LTR (or MTR) for another 30 min. The cells were imaged on a Zeiss LSM 710 NLO confocal microscope (63×/ NA 1.4 oil immersion objective) after fresh PBS being washed with three times. At 405 nm (for Ir-lyso) or 543 nm (for MTR or LTR), the phosphorescence (fluorescence) was excited. And at 520 ± 20 nm (for Ir-lyso) or 640 ± 20 nm (for LTR or MTR), the emission signal was collected. The excitation wavelength of the laser was 750 nm for two-photon imaging. We used the liquid overlay method to culture 3D multicellular spheroid. 42 In order to gain single cell suspensions, trypsin was used to dissociate the HeLa cells in the exponential growth phase. Subsequently, 96-well plates, which were previously coated with 50 μL of a sterile 1.0% solution of agarose in DMEM, were seeded with 6000 cells C


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3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization. Scheme 1 was the synthetic route of Ir-lyso. We started from 4-(4-(pyridin-2-

Figure 5. Viability of HeLa cells incubated with Ir-lyso for 24 and 48 h. Figure 2. Absorption spectrum and emission spectrum of Ir-lyso (5 μM) in disodium hydrogen phosphate/citric acid buffer solution (pH = 5.5).

yl)benzyl)morpholine (pbm), which was obtained according literature methods.39 The triscyclometalated iridium(III) complex was synthesized via a two-step procedure. First, [Ir(pbm)2Cl]2 was prepared through the literature procedure.43 The bis-iridium chloro-bridged dimer reacted with an excess of pbm in the presence of AgCF3SO3. The complex was purified using column chromatography to produce a yield of 34% and characterized via 1H NMR spectrum, elemental analysis, and ESI-MS (Figure S1−S2). Single-crystal X-ray diffraction analysis was used to confirm the molecular structure of Ir-lyso (Figure 1). Table S1 shows the structural refinements and the crystal data. Selected bond angles and lengths are shown in Table S2. The coordination of the iridium atom of Ir-lyso is octahedral. The Ir−N distance is 2.162 Å and the distance of Ir−C is 2.024 Å. The angles of Irlyso are 79.4°, 90.4°, 95.3°, and 172.6°. 3.2. Photophysical Properties of Ir-lyso. The pH of the lysosome is 4.5−5.5.30 Thus, we studied the optical properties of Ir-lyso in a disodium hydrogen phosphate/citric acid buffer solution at pH 5.5 (Table S3). Ir-lyso exhibited good solubility. The bands at 270−320 nm and 350−450 nm (Figure 2) were assigned to the spin-allowed ligand-centered (LC) π−π* transition of ligands and to the metal-to-ligand charge-transfer (MLCT) transitions.44 The emission spectra of Ir-lyso exhibited the maximum value at 524 nm with a quantum yield of 0.15, which was determined by the standard of [Ru(bpy)3]2+.40 The phosphorescent intensity of Ir-lyso responses to pH and reduces with the increase of pH but not quenching in high pH (Figure 3). This was due to the electron donor morpholine quenching the Ir(III) phosphorescence through photoinduced electron transfer (PET) under basic conditions. Morpholine has a pKa of 5−6,45 and the electron pair of each amine is protonated in acidic media. PET was prevented, and emission from Ir(III) phosphorescence occurred. The lifetime of Ir-lyso was determined to be 39.3 ns by the phosphorescence decay experiment via fitting the data to a single exponential decay function. The Stokes shift of Irlyso, which was measured to be 153 nm, offers advantages over the commercial LysoTrackers such as LysoTracker Green DND-26 (LTG, Stokes shift = 7 nm) and LTR (Stokes shift = 13 nm).46 The maximum two-photon absorption cross-section for Ir-lyso was 69.5 Göppert-Mayer (GM) units at 750 nm (Figure 4). A log−log linear relationship for Ir-lyso was

Figure 3. pH-sensitive emission spectra of Ir-lyso (5 μM) in disodium hydrogen phosphate/citric acid buffer solution. Inset: a plot of emission intensity of Ir-lyso at 524 nm versus different pH values.

Figure 4. Two-photon absorption cross sections of Ir-lyso at different excitation wavelengths. Inset: the power-dependence curve of Ir-lyso at an excitation wavelength of 750 nm.

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Figure 6. One-photon microscopy (OPM) and two-photon microscopy (TPM) images of HeLa cells colabeled with Ir-lyso (2 μM, 0.5 h) and LTR (50 nM, 0.5 h). Ir-lyso was excited at 405 nm (OPM) or 750 nm (TPM). LTR (OPM) was excited at 543 nm. The phosphorescence/fluorescence was collected at 520 ± 20 nm and 620 ± 20 nm for Ir-lyso and MTR, respectively. Overlay 1: overlay of the 1st and 2nd columns. Overlay 2: overlay of the 3rd and 4th columns. Scale bar: 20 μm.

Figure 7. TPM images of living HeLa cells incubated with 2 μM Irlyso under different conditions. (a) The cells were incubated with 2 μM Ir-lyso at 37 °C for 0.5 h. (b) The cells were incubated with 2 μM Ir-lyso at 4 °C for 0.5 h. Scale bar: 20 μm.

observed to confirm the two-photon process, with a slope of 2.13 between the emission intensity and the incident power. 3.3. Cytotoxicity, Cell Imaging, and Cellular Uptake. High cell viability is essential for long-term tracking of lysosomes.20 The MTT assay was used to determine the cytotoxicity of Ir-lyso with HeLa cells. The result showed that HeLa cells grew normally (Figure 5). Evidently, Ir-lyso did not interfere with the cell proliferation and physiology within the tested concentration range (1−10 μM). The pKa of morpholine is 5−6, and it can be protonated only in acidic lysosomes but not in other subcellular organelles or in the cytosol. Once the morpholine unit was protonated, Ir-lyso was retained in the lysosomes because it is more hydrophilic. The protonated morpholine is a lock that allows Ir-lyso to accumulate in acidic lysosomes. Additionally, the electron donor morpholine unit quenches the Ir(III) phosphorescence by PET in basic conditions, and the phosphorescence enhancement results from the suppression of PET in acidic lysosomes. Thus, background fluorescence of the trace amount Ir-lyso distributed outside the lysosomes is negligible.38,45,47,48 As shown in Figure 6, the colocalization experiment of Ir-lyso with LTR in the HeLa cells under one- and two-photon excitation demonstrated high overlap between the dye, complex, and lysosomes. The Pearson’s colocalization coefficient of Ir-lyso with LTR was 0.93 with two-photon excitation. Meanwhile, little colocalization for Ir-lyso with MTR was observed (Figure S3), indicating that Ir-lyso specifically localized in the lysosomes. We then used confocal microscopy to investigate the mechanism of cellular uptake of Ir-lyso (Figure 7). HeLa cells incubated with either Ir-lyso at 37 or at 4 °C were used to determine whether Ir-lyso entered the cell via an energydependent or an energy-independent transport pathway. Figure

Figure 8. (a) Quantitative photobleaching results of Ir-lyso and LTR in HeLa cells. (b) Confocal images of Ir-lyso (2 μM, OPM: λex = 405 nm, TPM: λex = 750 nm, λem = 520 ± 20 nm) and LTR (50 nM, λex = 543 nm, λem = 620 ± 20 nm) for photobleaching in HeLa cells. Scale bar: 20 μm.

7b showed that phosphorescence was observed, suggesting the Ir-lyso uptake followed an energy-independent pathway. 3.4. Photostability of Ir-lyso. Commercial lysosomal probes are not suitable for long-term tracking of the dynamic changes of lysosomes due to their poor photostability.15 A photobleaching experiment was used to examine the photostability between Ir-lyso and commercially available LTR in living HeLa cells. Figure 8a showed that under irradiation the E


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Figure 9. (a) Two-photon phosphorescent images of 3D tumor spheroids after incubation with Ir-lyso (2 μM) for 6 h. (b) The two-photon 3D Zstack images of an intact spheroid. (c) The two-photon Z-stack images were taken of every 3 μm section from the top to bottom. The images were taken using a 10× objective. λex = 750 nm; λem = 520 ± 20 nm.

Figure 10. Normalized phosphorescent/fluorescent intensities and images of living HeLa cells that stained with 2 μM of Ir-lyso and 2 μM of LTR at different passages. Scale bar: 100 μm.

fluorescent intensity of LTR decreased by 90% after 44.2 s (and 91.4% after 231.5 s). In contrast to this nearly complete photobleaching, the one-photon excited phosphorescent intensity of Ir-lyso decreased by only 44% after 231.5 s, and the two-photon excited phosphorescent intensity of Ir-lyso changed minimally after photobleaching. The intensity of Irlyso and LTR in the images (Figure 8b) and videos (Videos S1 and S2) showed a clear dynamic difference and provided strong evidence that Ir-lyso exhibits outstanding photostability relative to LTR for bioimaging. 3.5. Two-Photon Phosphorescent Imaging in 3D Spheroid Tumor Model. Three-dimensional multicellular tumor spheroids (3DMTSs), a valid intermediate that lies between monolayer in vitro cells and in vivo tissue, were a heterogeneous cell aggregate. With increasing imaging depths, the microscopic study of live tumor spheroids still have many significant technical challenges.42 Compared with one-photon fluorescence microscopy, two-photon fluorescence microscopy exhibits deeper tissue penetration depths, so 3DMTS was used to confirm the penetration depth of Ir-lyso. A HeLa multicellular spheroid was incubated with 2 μM Ir-lyso and

Figure 11. TPM images of HeLa cells received 1st passage (Psg, 24 h), 2nd passage (48 h), 3rd passage (72 h), and 4th passage (96 h), respectively. Lysosomes labeling was done with LTR (50 nM, 0.5 h) just 0.5 h before cell imaging. Ir-lyso (2 μM, 0.5 h) was excited at 750 nm. LTR was excited at 543 nm. The phosphorescence/fluorescence was collected at 520 ± 20 nm and 620 ± 20 nm for Ir-lyso and LTR, respectively. Overlay 1: overlay of the 1st and 2nd columns. Overlay 2: overlay of the 3rd and 4th columns.

was imaged using confocal laser scan microscopy. The phosphorescent intensity was observed from the spheroid to 105 μm depth for the two-photon excitation (Figure 9) and only 39 μm for the one-photon excitation (Figure S4). Phosphorescent images of every 3 μm along the Z-axis were captured. Compared with one-photon excitation, two-photon excitation exhibited much stronger phosphorescence in the deeper cell layers. The results indicated the two-photon excitation light possesses deeper penetration. 3.6. Long-Term Specifically Localized Lysosomes Study. In order to be applied in long-term lysosome tracking studies, it is important for probes possess the ability to stay in F


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Figure 12. Two-photon phosphorescent images of HeLa cells stained with Ir-lyso (2 μM) during cell migration.

Figure 13. Two-photon phosphorescent images of CCCP (20 μM) treated living HeLa cells stained with Ir-lyso (2 μM) with increasing scan time.

lysosomes for a long time.11 Imaging of Ir-lyso was compared with LTR (Figures 10 and 11). The HeLa cells that were stained with Ir-lyso after the second passage (48 h) were more emissive compared with the corresponding cells with LTR. The cells stained with LTR had almost no fluorescent signals after only two passages (48 h), whereas fluorescence was still visible in the Ir-lyso-stained cells even after four passages (96 h). It means Ir-lyso could track the lysosomes in living cells for a period of 4 days, satisfying the stable observation of most physiological activities of the lysosomes. 3.7. Long-Term Lysosomal Tracking during Cell Migration. Cell migration is central to wound healing, immune responses, and embryonic development; at the same time, it also plays a role in pathological processes, such as cancer.49 For delivery of cell adhesion regulators during cell migration, transport of late endosomes or lysosomes to the cell periphery is required, so cell migration could partly influence the positioning of lysosomes within the cytoplasm.50 In Figure 12 and Video S3, the green lysosomes were monitored during cell migration. In the brightfield, the action-rich lamellipodium of the cell extended from the cell body onto the surface, while the tail was contracted.51 At the leading edge, new adhesion pointed to the substrate formed. At the same time, the trailing edge was endocytosed, allowing the cell to detach,52 and lysosomes were found in the trailing edge. 3.8. Long-Term Lysosomal Tracking during Apoptosis. Lysosomes participate in apoptosis, and cell death leads to lysosomal membrane permeabilization,53 and therefore, we track the lysosomes during apoptosis. CCCP, an uncoupler of

oxidative phosphorylation that abolishes the mitochondrial membrane proton gradient,54 was used for apoptosis. MTR, targeted the mitochondria dependent on mitochondrial membrane potential,55 was used to stain the mitochondria. As shown in Figure 13 and Video S4, in front of CCCP adding, the red of MTR was observed. CCCP was added, and MTR disappeared after 7 min; this means the mitochondria experience a loss in membrane potential and apoptosis began. After 2 h and 45 min, an apoptosis body was found. Meanwhile the pH increased due to lysosomal membrane permeabilization, and the intensity of Ir-lyso was found to be less in one cell. More of an apoptosis body was found in 3 h and 25 min, and the intensity of green phosphorescence was found to be less in more cells. In the last measurement, most of observed cells have an apoptosis body, and green phosphorescent intensity was weakened in most of the cells.

4. CONCLUSION In conclusion, we have designed and synthesized a watersoluble triscyclometalated iridium complex that was confirmed to be an effective two-photon phosphorescent lysosomal probe for long-term tracking. Due to its large Stokes shift, low cytotoxicity, superior photostability, long-term specifically localized in the lysosomes, and excellent two-photon properties, Ir-lyso has been used to track lysosomes during cell migration and apoptosis. Further studies for other organelle imaging and biomedical applications with the two-photon phosphorescent iridium(III) complexes are ongoing in our laboratories. G


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b03422. 1 NMR spectra, ESI-MS spectra, confocal images, onephoton imaging in 3D multicellular spheroids, and photophysical data (PDF) Crystal data (CIF) Video S1 (AVI) Video S2 (AVI) Video S3 (AVI) Video S4 (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-20-84112245. Tel: +86-20-86110613. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 program (No. 2015CB856301) and the National Science Foundation of China (Nos. 21171177, 21471164, and 21525105).

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REFERENCES

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