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Keywords:

  • BODIPY;
  • tetraphenylethene;
  • aggregation-induced emission;
  • nanoparticle;
  • biological imaging

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and Discussion
  5. 3 Conclusions
  6. 4 Experimental Section
  7. Acknowledgements
  8. Supporting Information

BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) is an emissive chromophore in solutions but suffers from fluorescence quenching when aggregated due to its flat molecular conformation and small Stokes shift. To create aggregate-state emissive BODIPY luminogens, tetraphenylethene (TPE), which is a popular luminogen with intriguing aggregation-induced emission (AIE) characteristic, is introduced as periphery to a methylated BODIPY core. Three TPE-BODIPY adducts are synthesized and characterized, and their photophysical properties and electronic structures are investigated. The incorporation of AIE-active TPE units alleviates aggregation-caused quenching of BODIPY core, furnishing emissive nanoparticles based on TPE-BODIPY adducts. Significantly, the two-photon absorption (TPA) and two-photon excited fluorescence (TPEF) properties are improved as more TPE units are attached. The luminogens with 3TPE units (3TPE-BODIPY) shows the strongest TPA and TPEF in the wavelength range of 750–830 nm, with cross-section values of 264 and 116 GM at 810 nm, respectively. Red emissive nanoparticles with a Stokes shift of 60 nm and a fluorescence quantum yield of 16% are attained by encapsulating 3TPE-BODIPY with 1,2-sistearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. The nanoparticles are biocompatible and function well in TPEF cellular imaging and mouse brain blood vascular visualization.

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and Discussion
  5. 3 Conclusions
  6. 4 Experimental Section
  7. Acknowledgements
  8. Supporting Information

Currently, extensive researches are focused on the fabrication of biocompatible nanoparticles (NPs) with fluorescent dyes by various methods, such as encapsulation and dispersion, for biological applications.[1] The red/near-infrared emissive NPs are of particular interest in order to mitigate the interference of biological autofluorescence, deepen tissue penetration, and reduce the damage to cells.[2] In comparison with conventional nanoprobes, as represented by fluorescent proteins[3] and inorganic quantum dots,[4] organic fluorescent NPs based on small organic chromophores or polymers are becoming more and more popular, due to their noteworthy merits of synthetic versatility, high fluorescence, easily modulated emission colors, low cytotoxicity, strong photobleaching resistance, and facile surface functionalization.[5-7]

BODIPY (4,4-difluoro-4-bora-3a,4a-diaza- s-indacene) is a classical chromophore synthesized by Treibs and Kreuzer in 1968.[8] Recently, it has regained considerable research attention owing to its excellent photophysical properties, including large molar extinction coefficients, intense photoluminescence (PL), narrow absorption and emission bandwidths, high photostability, etc. Its attractive chemical nature allows facile modifications at the meso-, 2,6- and 3,5-positions of BODIPY ring or fusing other aromatic rings to pyrroles,[9] which offers an abundant possibility to fulfill specific requirements of practical applications. To date, a great variety of BODIPY dyes have been explored and have found promising applications in light harvesting,[10] solar cells,[11] fluorescent bioprobes,[12] chemosensors,[13] solid-state lasers,[14] electroluminescent devices,[15] etc.

For bioimaging, many BODIPY dyes, however, have some disadvantages. For instance, their emission wavelengths, typically ≈500 nm, are far away from the red/near-infrared region. The Stokes shifts are very small (≈10 nm), leading to self-quenching through energy transfer in the solid state. The crosstalk among their emission and absorbance also causes strong interference signals from excitation light. In addition, the planar BODIPY ring favors tight π–π stacking interactions, which results in nonradiative decay of the excited state. Therefore, the emission of many BODIPY dyes is quenched greatly when fabricated into NPs. Various bulky substituents such as 4-tritylphenylethynyl,[16] triphenylsilylphenyl,[17] paracyclophane,[18] and tert-butylphenyl[19] were incorporated into the BODIPY core to alleviate the π–π stacking interactions, and surmount emission quenching of BODIPY dyes in the solid state. These attempts, however, ended with limited success. BODIPY dyes with efficient red emission in the aggregate state are still rare.[20]

In 2001, Tang's group reported a novel phenomenon of aggregation-induced emission (AIE): a series of propeller-like luminogens that are non-fluorescent in solutions is induced to emit strongly by aggregate formation.[21-23] Based on AIE motif, we established an effective strategy to create efficient solid-state emitters by introduction of AIE-active groups, typically tetraphenylethene (TPE), into conventional chromophores that are subject to aggregation-caused quenching (ACQ). The generated luminescent materials show excellent emission efficiencies up to unity in the solid state[24] and perform remarkably in bioprobes[6, 25] and electronic devices.[26, 27] In this work, we extend the research on BODIPY dyes by the design and synthesis of a new series of robust luminogens consisting of methylated BODIPY and TPE units (Figure 1). Different connection patterns between the two segments are applied, for the sake of structure–property relationship understanding. The electronic structures and energy levels are calculated and the linear and nonlinear optical properties are presented. Biocompatible NPs are fabricated by encapsulating BODIPY luminogens in matrix of 1,2-sistearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) (Figure 1). The applications of the synthesized NPs are demonstrated in two-photon excited cellular imaging and blood vascular imaging in the brain of living mice.

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Figure 1. Chemical structures of Ph-BODIPY, TPE-decorated BODIPY, and DSPE-PEG2000.

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2 Results and Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and Discussion
  5. 3 Conclusions
  6. 4 Experimental Section
  7. Acknowledgements
  8. Supporting Information

2.1 Synthesis

The synthetic routes to TPE-decorated BODIPY luminogens are illustrated in Scheme 1. The detailed procedures and characterization data are given in the Experimental Section. Briefly, the preparation of the key intermediate, 4-(1,2,2-triphenylvinyl)phenylboronic acid (3) was described in our previous publication.[26a] The parent Ph-BODIPY and its iodide derivatives (46) were synthesized according to the methods in literatures.[12e,[28]] Suzuki couplings of 3 with 46, respectively, afforded the target compounds in moderate yields (45–63%). All the TPE-decorated BODIPY luminogens are readily soluble in common organic solvents, such as THF, dichloromethane, and chloroform, but insoluble in water.

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Scheme 1. Synthetic routes to TPE-decorated BODIPY luminogens.

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2.2 Linear Optical Properties

The one-photon absorption (OPA) spectra of TPE-decorated BODIPY luminogens in THF solutions are shown in Figure 2a. 3TPE-BODIPY and 2TPE-BODIPY that bear two TPE units at the 2,6-positions of BODIPY ring exhibit almost the same spectral profiles with absorption maxima at 534 nm, associated with the π–π* transition. TPE-BODIPY containing one TPE moiety linked through a phenyl bridge at the meso-position of BODIPY core shows absorption maximum at 500 nm, which is close to that of Ph-BODIPY (495 nm).[29] The absorption spectra of 3TPE-BODIPY and 2TPE-BODIPY are bathochromically shifted by 34 nm than that of TPE-BODIPY, demonstrating that the conjugation length of luminogen is noticeably more extended by covalently attaching π-conjugated substituents at the 2,6-positions of BODIPY ring than at the meso-position. Figure 3b displays the one-photon excited fluorescence (OPEF) spectra of the luminogens in THF solutions. 3TPE-BODIPY and 2TPE-BODIPY show fluorescence peaks at 584 and 583 nm, respectively, which are much redder than those of TPE-BODIPY (516 nm) and Ph-BODIPY (507 nm).[29] The Stokes shifts of 3TPE-BODIPY and 2TPE-BODIPY are 46 nm, which is much larger than those of TPE-BODIPY (16 nm) and Ph-BODIPY (12 nm), evidently disclosing that incorporation of TPE units at the 2,6-positions of the BODIPY ring is an effective approach to create BODIPY derivatives with large Stokes shifts.

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Figure 2. a) Absorption and b) fluorescence spectra of TPE-decorated BODIPY luminogens in dilute THF solutions.

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The good conjugation and large Stokes shift enable 3TPE-BODIPY to fluoresce efficiently. The fluorescence quantum yield (ΦF) of 3TPE-BODIPY is 44%, which is higher than those of 2TPE-BODIPY (38%) and TPE-BODIPY (28%), measured in THF solutions by the integrating sphere method. Although many luminogens consisting of TPE units fluoresce faintly in the solution state due to exciton annihilation by intramolecular rotation,[23, 24] there are also some exceptions. Several TPE-modified heterocyclics including benzo-2,1,3-thiadiazole,[6c,26e] 2-(4H-pyran-4-ylidene)malononitrile,[6g] etc., were found to exhibit good PL properties in the solution state. Since BODIPY core is an excellent energy acceptor due to its low-lying lowest unoccupied molecular orbital (LUMO),[10] rapid energy transfer from peripheral TPE units to BODIPY core is feasible. Therefore, the nonradiative energy decay by the intramolecular rotation of TPE units is decreased, rendering good emission of the molecule.[6c,10a,[30]]

2.3 Nonlinear Optical Properties

Significantly, these TPE-decorated BODIPY luminogens show good two-photon absorption (TPA) and two-photon excited fluorescence (TPEF) within the window of biomedical interest, in particular at the wavelength range of 750–830 nm. The cross-sections of TPA (σTPA) and TPEF (σTPEF) of these luminogens vary with the molecular structure as well as wavelength of the laser excitation (Figure 3). The values become augmented obviously with the increase of TPE units in the luminogens. 3TPE-BODIPY shows the best nonlinear optical property, with the highest σTPA and σTPEF values of 264 and 116 GM, respectively, when its THF solution is pumped with laser pulses at wavelength of 810 nm. These values are much higher than those of Ph-BODIPY[29] and other BODIPY dyes in the literatures (4–128 GM).[31] Figure 4a compares the OPEF spectrum with TPEF spectrum of 3TPE-BODIPY excited at 375 and 800 nm, respectively. The TPEF spectrum resembles the OPEF one except for a tiny bathochromic shift of ≈3 nm, suggesting that the red emissions of both TPEF and OPEF originate from the same decay of singlet excitons.

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Figure 3. a) TPA and b) TPEF dispersion curves of TPE-decorated BODIPY luminogens dissolved in THF.

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Figure 4. OPEF and TPEF spectra of 3TPE-BODIPY a) in THF solution and b) in aqueous NP suspension.

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2.4 Electronic Structures

To gain a better understanding of the optical properties of TPE-decorated BODIPY luminogens at molecular level, density functional theory calculations were carried out using a suite of Gaussian 09 program. The nonlocal density functional of B3LYP with 6–31G(d) basis sets was used for the calculation. The optimized structures and orbital distributions of HOMOs and LUMOs of the luminogens are shown in Figure 5. It can be seen that the four BODIPY luminogens show similar orbital amplitude plots of LUMOs, which are centered on the BODIPY core and less located at the phenyl fragments adjacent to BODIPY ring. The phenyl ring at the meso-position is located in a nearly perpendicular pattern to the BODIPY ring, and contributes little to the HOMOs of the luminogens. No electron density is found at the TPE units that are linked to the BODIPY core through the phenyl bridge at the meso-position neither. On the contrary, the TPE units that are attached at the 2,6-positions of the BODIPY ring show remarkable contribution to the HOMOs of 2TPE-BODIPY and 3TPE-BODIPY. The energy levels of these BODIPY luminogens are listed in Table 1. Whereas the four luminogens show close LUMO energy levels ranged from −2.31 to −2.38 eV, 2TPE-BODIPY (−5.13 eV), and 3TPE-BODIPY (−5.12 eV) possess much higher HOMO energy levels than Ph-BODIPY (−5.33 eV) and TPE-BODIPY (−5.31 eV). Thus, the energy bandgaps of 2TPE-BODIPY (2.75 eV) and 3TPE-BODIPY (2.75 eV) become much narrower than those of Ph-BODIPY (3.00 eV) and TPE-BODIPY (3.00 eV), which is in good agreement with the optical bandgaps calculated from the onesets of absorption spectra. These results suggest that the substitutions at the 2,6-positions of BODIPY ring have decisive impact on the optical properties of these BODIPY luminogens.

Table 1. Energy levels of BODIPY luminogens calculated using B3LYP/6–31G(d) basis set with G09 program
 HOMO [eV]LUMO [eV]Eg [eV]Eg (opt)a) [eV]
  1. a)Optical bandgap calculated from the onset of absorption spectrum.

Ph-BODIPY−5.33−2.333.002.28
TPE-BODIPY−5.31−2.313.002.34
2TPE-BODIPY−5.13−2.382.752.10
3TPE-BODIPY−5.12−2.372.752.10
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Figure 5. Molecular orbital amplitude plots of HOMOs and LUMOs of BODIPY luminogens calculated using B3LYP/6–31G(d) basis set with G09 program.

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2.5 Nanoparticles of 3TPE-BODIPY

In order to investigate the emission behaviors of TPE-decorated BODIPY luminogens in the aggregate state, PL spectra of the luminogens in THF/water mixtures are recorded. Figure 6 shows the PL spectra of 3TPE-BODIPY in THF/water mixtures as an example. It can be seen that emission intensity is decreased slightly when a large amount of water is added, where the 3TPE-BODIPY molecules have formed NPs in the mixture with high water fractions, due to the immiscibility of the hydrophobic molecule with the hydrophilic medium. The AIE effect of multiple TPE units has partially alleviated the ACQ effect of BODIPY ring, making 3TPE-BODIPY emit strong fluorescence in the aggregate state. 3TPE-BODIPY NPs in aqueous solution also show good nonlinear optical property. Intense TPEF peaked at 590 nm is detected under excitation of laser pulses at wavelength of 800 nm, which is similar to OPEF spectrum excited at 375 nm (Figure 4b).

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Figure 6. a) OPEF spectra of 3TPE-BODIPY in THF/water mixtures with different water fractions (fw). b) Plots of (I/I0)–1 versus water fractions in THF/water mixtures, where I0 is the PL intensity in pure THF solution.

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The large Stokes shift, high fluorescence efficiency and good linear and nonlinear optical properties of 3TPE-BODIPY encourage us to explore its potential applications in cellular imaging. To improve the biocompatibility, NPs of 3TPE-BODIPY loaded in DSPE-PEG2000 matrix (3TPE-BODIPY-DSPE NPs) are fabricated by a modified nanoprecipitation method.[32a,b] The size of the generated 3TPE-BODIPY-DSPE NPs remains unchanged by laser light scattering monitoring (Figure S1, Supporting Information), indicating their good colloidal stability. The high-resolution transmission electron microscopy (HR-TEM) reveals that the NPs have a spherical shape with diameters in the range of ≈30–50 nm. 3TPE-BODIPY-DSPE NPs show absorption maximum at 536 nm and fluorescence peak at 596 nm, presenting a large Stokes shift of 60 nm (Figure 7a). The absorption maximum is close to the wavelength of the confocal microscopy laser (543 nm), which is ideal for cellular imaging. The ΦF value measured for NPs in water using rhodamine 6G in ethanol as standard is 16%, being much higher than those of commercially available red dyes, such as Nile red (0%) and DCM (4-(dicyanomethylene)-2-methyl-6-[4-(dimethylaminostyryl)-4H-pyran]) (5%), in aggregates.

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Figure 7. a) Absorption and PL spectra and b) HR-TEM image of 3TPE-BODIPY-DSPE NPs.

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2.6 One- and Two-Photon Excited Fluorescence Cellular Imaging

To demonstrate the potential of 3TPE-BODIPY-DSPE NPs for fluorescence imaging in living cancer cells, MCF-7 breast cancer cells were incubated with the NPs for 4 h in culture medium containing 1 × 10−6 m 3TPE-BODIPY, and then used for one-photon confocal imaging. Figure 8a shows the OPEF imagings of the cells, taken under 543 nm with a 580–800 nm bandpass filter. Intense intracellular red fluorescence was observed from 3TPE-BODIPY-DSPE NPs in the cell cytoplasm. To identify their intracellular location, MCF-7 cancer cells were further costained with LysoTracker Green and 3TPE-BODIPY-DSPE NPs. The good overlap between LysoTracker Green and NPs (Figure 9) indicates that 3TPE-BODIPY-DSPE NPs are mainly accumulated at the endosomes of MCF-7 cancer cells.

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Figure 8. a) OPEF and b) TPEF images and c) bright-field images of MCF-7 breast cancer cells after 4 h incubation with 3TPE-BODIPY-DSPE NPs suspension with 1 × 10−6m 3TPE-BODIPY at 37 °C. All the images share the same scale bar of 40 μm.

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Figure 9. Colocalization of 3TPE-BODIPY-DSPE NPs (a, λex = 543 nm, 580–800 nm band pass filter) and LysoTracker Green DND-26 (b, λex = 488 nm, 500–560 nm band pass filter). Image c is the overlap of images a and b. All the images share the same scale bar of 20 μm.

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Compared to conventional one-photon confocal laser scanning microscopy, two-photon microscopy shows many advantages including deeper tissue penetration, reduced substrate autofluorescence and self-absorption, less photobleaching, higher spatial resolution, etc.[32, 33] In view of this, TPEF images of MCF-7 breast cancer cells after incubation with 3TPE-BODIPY-DSPE NPs in culture medium with 1 × 10−6m 3TPE-BODIPY are investigated and the results are shown in Figure 8b. The images were obtained by excitation at 800 nm with a 560–760 nm bandpass filter. Red fluorescence signal from cytoplasm can be clearly observed to distinguish the cell profile in a similar pattern as compared to OPEF images in Figure 8a. These results indicate that 3TPE-BODIPY-DSPE NPs can serve as a promising two-photon fluorescent probe for imaging.

2.7 Cytotoxicity of 3TPE-BODIPY-DSPE NPs

The cytotoxicity of 3TPE-BODIPY-DSPE NPs was investigated by studying the metabolic viability of MCF-7 breast cancer cells after incubation with these NPs at various dye concentrations. Figure 10 shows the cell viability after incu­bation with the NP suspension at 1 × 10−6, 5 × 10−6, and 10 × 10−6m dye concentrations for 36, and 72 h, respectively. Cell viability remains ≈98% within 72 h under the experimental conditions, which indicates that 3TPE-BODIPY-DSPE NPs are of good biocompatibility for bioimaging applications.

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Figure 10. Metabolic viability of MCF-7 breast cancer cells after incubation with 3TPE-BODIPY-DSPE NP suspensions at different dye concentrations for 36 and 72 h, respectively.

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2.8 Real-Time Two-Photon Brain Blood Vascular Imaging

To determine the applicability of 3TPE-BODIPY-DSPE NPs for in vivo applications, we performed TPEF in vivo imaging of the mouse brain, using the NPs as a blood vessel visualizing agent. 3TPE-BODIPY-DSPE NPs were excited at 800 nm and emitted signals that were collected at 542 ± 27 nm. Figure 11 shows representative images of a 30-min time-lapsed imaging of the brain vasculature, imaged through a cranial window (Movie S1, Supporting Information). Figure 11a shows a z-projected image depicting the overall blood vasculature that could be observed with the NPs, while Figures 11b–e showing the distinct types of blood vasculature at different depths. Besides the major blood vessels in the uppermost layer, capillaries in the pia matter (100–300 μm below surface of brain) could also be visualized with the NPs. The high-resolution, 3D reconstructed image (Figure 11f) further illustrates the applicability of 3TPE-BODIPY-DSPE NPs for TPEF in vivo imaging. As a control, no fluorescence was detected in the blood vessels prior to the injection of the NPs (Figure S2, Supporting Information).

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Figure 11. Intravital TPEF images of 3TPE-BODIPY-DSPE NPs stained blood vessels of brain at depth of a) 0 μm, b) 100 μm, c) 200 μm and d) 300 μm, and e) respective Z-projected image as well as f) 3D reconstructed image. All the images share the same scale bar of 50 μm.

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3 Conclusions

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and Discussion
  5. 3 Conclusions
  6. 4 Experimental Section
  7. Acknowledgements
  8. Supporting Information

In summary, a series of luminescent materials consisting of TPE peripheries and a BODIPY core were synthesized and characterized. The linear and nonlinear optical properties were investigated and the theoretical calculations were carried out to gain a deeper insight into the structure–property relationship of these BODIPY luminogens. The results reveal that the TPE substituents at the 2,6-positions of the BODIPY ring function much better in extending molecular conjugation, enlarging Stokes shifts, and shifting emission spectra into red region than that connected through a phenyl bridge at meso-position of BODIPY ring. The accumulation of TPE units can not only ameliorate the fluorescence emission in the aggregate state but also benefit to TPA and TPEF properties of the luminogens. Red emissive biocompatible NPs were fabricated by encapsulating 3TPE-BODIPY into DSPE-PEG2000 matrix and further applied in TPEF cellular and blood vascular imagings, demonstrating their great potential as two-photon excited contrast agents in biological system.

4 Experimental Section

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and Discussion
  5. 3 Conclusions
  6. 4 Experimental Section
  7. Acknowledgements
  8. Supporting Information

Materials and Instruments: 1H and 13C NMR spectra were measured on a Bruker AV 400 spectrometer in deuterated chloroform using tetramethylsilane (TMS; δ = 0) as an internal reference. The UV–vis spectra were recorded on a Shimadzu UV-1700 spectrometer. Photoluminescence was recorded on a PerkinElmer LS 55 spectrofluorometer. High-resolution mass spectra were recorded on a GCT premier CAB048 mass spectrometer operating in a MALDT-TOF mode. Average particle size and size distribution of the NPs were determined by laser light scattering (LLS) with particle size analyzer (90 Plus, Brookhaven Instruments Co., USA) at a fixed angle of 90° at room temperature. The morphology of NPs was also studied by field emission transmission electron microscope (FE-TEM, JEM-2010F, JEOL, Japan).

1,2-Sistearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly­ethylene glycol)-2000] (DSPE-PEG2000) was a gift from Lipoid GmbH (Ludwigshafen, Germany). Tetrahydrofuran (THF), 3-(4,5-dimethy­lthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), penicillin–streptomycin solution, and trypsin–EDTA solution were purchased from Sigma–Aldrich. Fetal bovine serum (FBS) was purchased from Gibco (Lige Technologies, Ag, Switzerland). LysoTracker Green DND-26 was provided by invitrogen. Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, Breford, USA). MCF-7 breast cancer cells were provided by American Type Culture Collection. All other chemicals and regents used in the synthetic procedures were purchased from Aldrich and used as received without further purification.

Synthesis: TPE-BODIPY: A mixture of 4 (0.5 g, 1.11 mmol), 3 (0.5 g, 1.33 mmol), Pd(PPh3)4 (0.069 g, 0.06 mmol), and potassium carbonate (0.608 g, 4.4 mmol) in 100 mL of toluene/ethanol/water (8/1/1 v/v/v) was heated to reflux for 12 h under nitrogen. The reaction mixture was cooled to room temperature and poured into water. The organic layer was extracted with dichloromethane and the combined organic layers were washed with saturated brine solution and water, and dried over anhydrous magnesium sulfate. After filtration and solvent evaporation, the residue was purified by silica-gel column chromatography using hexane/dichloromethane as eluent. Orange solid of TPE-BODIPY was obtained in 63% yield (0.46 g). 1H NMR (400 MHz, CDCl3), δ (TMS, ppm): 7.69 (d, 2H, J = 7.6 Hz), 7.44 (d, 2H, J = 7.6 Hz), 7.30 (d, 2H, J = 7.6 Hz), 7.14–7.10 (m, 13H), 7.07–7.03 (m, 4H), 5.98 (s, 2H), 2.56 (s, 6H), 1.43 (s, 6H). 13C NMR (100 MHz, CDCl3), δ (TMS, ppm): 155.5, 143.7, 143.5, 143.1, 141.6, 141.4, 141.2, 140.3, 137.6, 133.8, 132.0, 131.4, 131.3, 128.4, 127.8, 127.7, 127.3, 126.6, 126.5, 126.1, 121.2, 14.6. HRMS (MALDT-TOF) m/z: M+ calcd for C45H37BF2N2, 654.3018; found 654.3002.

2TPE-BODIPY: Red solid, yield 56%. 1H NMR (400 MHz, CDCl3), δ (TMS, ppm): 7.48–7.47 (m, 3H), 7.32–7.31 (m, 2H), 7.10–7.02 (m, 34H), 6.86 (d, 4H, J = 6.8 Hz), 2.48 (s, 6H), 1.24 (s, 6H). 13C NMR (100 MHz, CDCl3), δ (TMS, ppm): 154.3, 143.7, 143.6, 143.5, 142.5, 141.4, 140.8, 139.1, 136.8, 135.6, 133.6, 131.7, 131.3, 131.2, 129.4, 129.2, 128.2, 127.7, 127.5, 126.5, 13.4, 12.7. HRMS (MALDT-TOF) m/z: M+ calcd for C71H55BF2N2, 984.4426; found 984.4403.

3TPE-BODIPY: Red solid, yield 45%. 1H NMR (400 MHz, CDCl3), δ (TMS, ppm): 7.70 (d, 2H, J = 8.0 Hz), 7.44 (d, 2H, J = 8.4 Hz), 7.34 (d, 2H, J = 8.0 Hz), 7.15–6.99 (m, 51H), 6.86 (d, 4H, J = 8.4 Hz), 2.49 (s, 6H), 1.30 (s, 6H). 13C NMR (100 MHz, CDCl3), δ (TMS, ppm): 153.6, 143.0, 142.9, 142.8, 141.9, 141.0, 140.8, 140.7, 140.5, 140.0, 139.6, 138.4, 136.9, 133.6, 132.9, 131.3, 131.0, 130.7, 130.5, 128.7, 127.9, 127.2, 127.1, 127.0, 126.9, 126.8, 125.9, 125.5, 12.7, 12.4. HRMS (MALDT-TOF) m/z: M+ calcd for C97H73BF2N2, 1314.5835; found 1314.5845.

Determinations of Two-Photon Absorption and Fluorescence Cross Sections, and Absolute Fluorescence Quantum Yields: Two-photon excitation spectra of molecules dissolved in THF (1 × 10−5m) were measured using the TPEF technique. A mode locked Ti:Sapphire laser was employed as excitation source (pulses of 100 fs, repetition rate 80 MHz, and wavelength tunability in the range 750–830 nm). The laser beam was focused onto a quartz cell of 1 cm of path length (containing solutions of BODIPY luminogens) using a lens (5 cm focal length). To avoid linear absorption effects, the beam was focused close to the cell wall. Then the fluorescence emission from solutions was focused and recorded on a spectrometer. The measurements were performed in a low excitation intensity regime where the fluorescence signal showed a quadratic dependence on the intensity of the excitation beam. A methanol solution of Rhodamine 6G whose cross sections values have been completely characterized in the literature[35] was employed as a reference for calculations of σTPA and σTPEF. This laser dye was also employed as reference to determine the fluorescence quantum yield of samples at low concentrations (<1 × 10−5m) by using the integrating sphere method with excitation provided by a diode laser operating at 375 nm. The quantum efficiency of the Rhodamine 6G was considered as 95%.[36]

Fabrication of 3TPE-BODIPY-DSPE NPs: The 3TPE-BODIPY-loaded DSPE-PEG2000 NPs were prepared through a modified nanoprecipitation method. Briefly, 1 mL of THF solution containing 1 mg of 3TPE-BODIPY and 2 mg of DSPE-PEG2000 was poured into 9 mL of water. This was followed by sonicating the mixture for 60 s at 10 W output using a microtip probe sonicator (XL2000, Misonix Incorporated, NY). The mixture was then stirred at room temperature overnight to evaporate THF. The obtained solution was filtered using a 0.20-μm syringe-driven filter to collect the products.

Cell Culture: MCF-7 breast cancer cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS and 1% penicillin–streptomycin at 37 °C in a humidified environment containing 5% CO2. Before experiment, the cells were pre-cultured until confluence was reached.

Cell Imaging: MCF-7 breast cancer cells were cultured in the confocal imaging chambers (LAB-TEK, Chambered Coverglass System) at 37 °C. After 80% confluence, the medium was removed and the adherent cells were washed twice with 1× PBS buffer. 3TPE-BODIPY-DSPE NPs in FBS-free DMEM medium at 1 × 10−6 m of 3TPE-BODIPY were then added to the chambers. After incubation for 4 h, the cells were washed three times with 1 × PBS buffer and then fixed by 75% ethanol for 10 min, which were further washed twice with 1× PBS buffer. For costaining experiment, after 4 h incubation with NPs, the cell was further incubated with the LysoTracker Green suspension for 30 min before ethanol fixation. One- and two-photon fluorescence images of MCF-7 breast cancer cells after incubation with BODIPY luminogens loaded DSPE NPs were studied by Leica TCS SP 5X and multiphoton microscope equipped with two-photon Chameleon Ultra II, respectively. The one-photon fluorescence images were taken under 543 nm with a 580–800 nm bandpass filter. And two-photon fluorescence images were achieved by excitation at 800 nm with a 560–760 nm bandpass filter.

Cytotoxicity: The metabolic activity of MCF-7 breast cancer cells was evaluated using methylthiazolyldiphenyl-tetrazolium (MTT) assays. MCF-7 cells were seeded in 96-well plates (Costar, IL, USA) at an intensity of 4 × 104 cells mL−1. After 24 h incubation, the medium was replaced by the 3TPE-BODIPY-DSPE NP suspensions at different 3TPE-BODIPY concentrations in DMEM containing 10% FBS and 1% penicillin streptomycin, and the cells were then incubated for 36 and 72 h, respectively. After the designated time intervals, the wells were washed three times with 1 × PBS buffer and 100 μL of freshly prepared MTT (0.5 mg mL−1) solution in culture medium was added into each well. The MTT medium solution was carefully removed after 3 h incubation in the incubator. Dimethyl sulfoxide (DMSO, 100 μL) was then added into each well and the plate was gently shaken for 10 min at room temperature to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan) after subtracting the absorbance of the corresponding control cells incubated with 3TPE-BODIPY-DSPE NPs at the same concentration but without the addition of MTT to eliminate the absorbance interference from BODIPY luminogens. Cell viability was expressed by the ratio of absolute absorbance of the cells incubated with NP suspension to that of the cells incubated with culture medium only.

Brain Blood Vascular Imaging: The experimental setup for brain imaging is described elsewhere.[34] Briefly, a small 2-mm circular piece of parietal bone was excised using a dental drill, exposing the meninges and the brain of the immobilized mouse. For TPEF experiments, mice were anesthetized (150 mg kg−1 ketamine and 10 mg kg−1 xylazine) and placed on a heating pad to maintain a core body temperature of 37 °C throughout each imaging procedure. 200 μL of 3TPE-BODIPY-DSPE NPs at 50 × 10−6 m 3TPE-BODIPY was administered via retro-orbital injection prior to imaging. As a control, a group of mice without NP injection were also imaged under the same experimental conditions. All procedures were performed under the institution's IACUC (Institutional Animal Care and Use Committee) guidelines. A TriM Scope II single-beam two-photon microscope (LaVision BioTec) with a tunable 680–1080 nm laser (Coherent) was used to acquire the images. 3TPEBODIPY-DSPE NPs and second harmonic generation were excited at 800 nm, and emitted light was split by 520 and 640 nm long pass mirrors and detected through 542/27 nm filters.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and Discussion
  5. 3 Conclusions
  6. 4 Experimental Section
  7. Acknowledgements
  8. Supporting Information

We acknowledge the financial support from the National Natural Science Foundation of China (51273053 and 21104012), the Natural Science Foundation of Zhejiang Province (Y4110331), the Program for Changjiang Scholars and Innovative Research Teams in Chinese Universities (IRT 1231), the National Basic Research Program of China (973 Program, 2013CB834702) and the Project of Zhejiang Key Scientific and Technological Innovation Team (2010R50017). G.R.-O. thanks the support from CONACyT (Grant 132946). B.L. thanks Singapore National Research Foundation (R279–000–390–281) for support. B.Z.T. thanks the support of the Guangdong Innovative Research Team Program of China (20110C0105067115) and the Research Grants Council of Hong Kong (HKUST2/CRF/10).

Supporting Information

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and Discussion
  5. 3 Conclusions
  6. 4 Experimental Section
  7. Acknowledgements
  8. Supporting Information

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