Engineering Lysosome-Targeting BODIPY Nanoparticles for

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Engineering Lysosome-Targeting BODIPY Nanoparticles for Photoacoustic Imaging and Photodynamic Therapy under Near-Infrared Light Wenbo Hu, Hengheng Ma, Bing Hou, Hui Zhao, Yu Ji, Rongcui Jiang, Xiaoming Hu, Xiaomei Lu, Lei Zhang, Yufu Tang, Quli Fan, and Wei Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02721 • Publication Date (Web): 28 Apr 2016 Downloaded from http://pubs.acs.org on May 4, 2016

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ACS Applied Materials & Interfaces

Engineering Lysosome-Targeting BODIPY Nanoparticles for Photoacoustic Imaging and Photodynamic Therapy under Near-Infrared Light Wenbo Hu,a Hengheng Ma,a Bing Hou,a Hui Zhao,a Yu Ji,a Rongcui Jiang,a Xiaoming Hu,a Xiaomei Lu,b Lei Zhang,a Yufu Tang,a Quli Fan,* a and Wei Huang*b a

Key Laboratory for Organic Electronics and Information Displays & Institute of

Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing 210023, China. E-mail: [email protected] b

Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials

(IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, China. E-mail: [email protected].

KEYWORDS near-infrared absorption, theranostic nanoparticles, lysosome-targeting, photoacoustic imaging, photodynamic therapy

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ABSTRACT

Developing lysosome-targeting organic nanoparticles combined with photoacoustic imaging (PAI) and photodynamic therapy (PDT) functions toward personalized medicine

are

highly

desired

yet

challenging.

Here,

for

the

first

time

lysosome-targeting BODIPY nanoparticle was engineered by encapsulating near-infrared (NIR) absorbed BODIPY dye within amphiphilic DSPE-mPEG5000 for high-performing lysosomal PAI and acid-activatable PDT against cancer cells under NIR light.

INTRODUCTIONS

The development of organic nanoparticles combining optical imaging and photodynamic therapy (PDT) functions for theranostic applications are highly desired owning to the relatively better biocomapabiltiy of organic nanoparticles in comparison with that of inorganic nanoparticles, as well as the advantages of light in terms of noninvasive properties, high spatiotemporal sensitivity.1-4 However, frequently used optical imaging techniques in these nanoparticles mainly focused on fluorescent imaging, which suffers from the general limitations such as shallow imaging penetration depth.5 For example, fluorescent imaging in near-infrared (NIR, 650-900nm) region could effectively reduce the autofluorescence of endogenous fluorophores in comparison with imaging in the visible range,6 but its imaging penetration depth is still very shallow (~ 1 cm).7 In comparison, newly emerging 2 ACS Paragon Plus Environment

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photoacoustic imaging (PAI) technology that coupling the advantages of optical imaging and ultrasound imaging allows an fascinating imaging paradigm with higher spatial resolution and deeper imaging depth (5-6 cm).8-11 Thus, it is preferred as a highly promising imaging modality to be integrated into PDT system for determining the degree of photosensitizer (PS, which generates cytotoxic species upon light irradiation for PDT) uptake by deep diseased tissue, and final evaluation of the therapeutic outcome.12-13 Although small molecular organic dyes with NIR-absorption and emission, such as Food and Drug Administration (FDA) approved indocyanine green (ICG), have exhibited attractive properties for PAI or PDT,14-16 the serious photobleaching,

and

difficulty

of

functionalization

particularly

limit

their

applications.17 In comparison, semiconducting polymer nanoparticles with high optical stability have recently been proposed as PA contrary for PAI,5, 18-19 but these nanoparticles lack PDT ability. Despite of the fascinating PAI property, delivering organic nanoparticles with concurrent PAI and PDT ability into the location of interest is also a key factor for the theranostic applications to reduce nonspecific phototoxicity.20-21 Lysosome-targeting has attracted numerous attentions toward personalized medicine for the development of novel cancer therapeutics.22-23 Moreover, the significantly increased acidity in lysosome of cells (pH 4.5-5.0) also provides a promising pH-sensitive site for activatable PDT in which the ability of PDT is switched on at an acidic pH but is deactivated at physiological pH.24-25 Therefore, developing lysosome-targeting and



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NIR-absorptive organic nanoparticles that integrate recently advanced PAI with acid-activatable PDT is highly important yet remaining a challenge. BODIPY dyes, as a class of representative organic small molecules, has been widely used as fluorescent probe or PS due to its excellent optoelectronic property, and easy of functionalization.26-28 However, currently available BODIPY dyes for PDT or PAI mainly require UV-Vis light,29-30 which is inapplicable for bioapplications in deep tissue. In this study, we first demonstrated the use of NIR-absorptive bis-styryl BODIPY dyes for developing lysosome-targeting organic nanoparticles (BODIPY NPs) through conveniently nanoprecipitation with concurrent PAI and acid-activatable PDT ability under NIR light (Scheme 1). The as-prepared BODIPY NPs exhibited excellent biocompatibility, outstanding photostability, and great physiological stability. Furthermore, we demonstrated the potential application of BODIPY NPs for lysosome-targeting PAI and acid-activatable PDT. Given its excellent performance, BODIPY NPs showed high potential for further theranostic applications.

Scheme 1. Schematically illustrating the fabrication of lysosome-targeting BODIPY NP and their use in lysosomal PAI and pH-activatable PDT under NIR-light. 4 ACS Paragon Plus Environment

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EXPERIMENTAL SECTION

Materials and Instruments. All the reagents were purchased from Sigma-Aldrich and

used

directly.

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (DSPE-mPEG5000) was purchased from Avanti Lipids. Dulbecco’s modified Eagle’s medium (DMEM, Gibco, America) was purchased from Gene Tech Co. (Shanghai, China). Lysosome isolation kit was purchased from Sigma-Aldrich. The Annexin V-VFITC/propidium iodide (PI) cell apoptosis kit was purchased from KeyGen Biotech. Co. Ltd. (Nanjing, China). NMR spectra were recorded on a Bruker Ultra Shield Plus 400 MHz spectrometer (1H: 400 MHz,

13

C: 100 MHz) using

tetramethylsilane (TMS) as the internal standard, the following abbreviations (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet) represent the multiplicities. The steady-state ultraviolet-visible-near-infrared (UV-Vis-NIR) absorption and photoluminescence

spectra

were

measured

by

a

Shimadzu

ultraviolet-visible-near-infrared (UV-Vis-NIR) spectrophotometer (UV-3600) and Edinburgh FLSP920 fluorescence spectrophotometer, respectively. Dynamic light scattering (DLS) was performed on a particle size analyzer (NanoBrook 90Plus, Brookhaven Instruments Corporation). Transmission electron microscopy (TEM) images were performed on a HT7700 transmission electron microscope operating at 100 kV. 730 nm semiconductor laser was purchased from Changchun New Industries Optoelectronics Technology Co., Ltd. The methyl thiazolyl tetrazolium (MTT) assay was performed by a PowerWave XS/XS2 microplate spectrophotometer (BioTek, 5 ACS Paragon Plus Environment


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U.S.). PA data were collected by commercial Nexus-128 PAI tomography system (Endra Inc., Michigan, USA) equipped with a tunable nanosecond pulsed laser (680-950 nm, 5 ns pulses, 20 Hz pulse repetition frequency). Flow cytometry experiments were performed by FlowSight® Imaging Flow Cytometer (Merck Millipore, Darmstadt, Germany).

Material synthesis. Compounds of monomer 1, monomer 2 and monomer 3 were synthesized according to previous procedures with modification.31-33 The final bis-styryl

BODIPY

was

synthesized

by

reaction

of

monomer

3

with

4-(diethylamino)benzaldehyde.27 Scheme 2 presented the synthetic route to target compound and detail experimental procedures were given below.

Scheme 2. Synthetic route for bis-styryl BODIPY dye

Monomer 1. To a solution of 2,4-dimethyl-1H-pyrrole (1.19 g, 12.5 mmol) and 4-methylbenzaldehyde (0.60 g, 5 mmol) in dry CH2Cl2 (100 mL) is added a solution 6 ACS Paragon Plus Environment

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of trifluoroacetic acid (50 µL, 0.65 mmol) in dry CH2Cl2 (2.5 mL) slowly at room temperature.

After

3

h

stirring

under

ice

bath,

2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (1.128 g, 5 mmol) is added and stirred for additional 1 h at room temperature. Then, triethylamine (NEt3) (10 mL, 72 mmol) is added, followed by slow addition of boron trifluoride diethyl etherate (BF3.Et2O) (10 mL, 81 mmol). The reaction mixture is washed after 2 h of stirring at room temperature with saturated aqueous Na2CO3 solution (350 mL), dried over Na2SO4, and concentrated on a rotary evaporator for further purified by column chromatography on silica with petroleum ether/CH2Cl2 = 2:1. The product fraction is dried to yield a red-brown solid (Yield: 1.47g, 91%). 1H NMR (400 MHz, CDCl3): δ = 7.28 (d, 2H), 7.14 (d, 2H), 5.97 (s, 2H), 2.55 (s, 6H), 2.43 (s, 3H), 1.39 (s, 6H). 13C NMR (100 MHz, CDCl3): δ = 155.01, 143.19, 142.17, 138.68, 131.96, 131.62, 129.78, 127.78, 121.08, 21.36, 14.44. MALDI-TOF-MS (m/z): Calcd for C20H23BF2N2 [M+2H]+, 340.18; found, 339.91.

Monomer 2. A 100 mL round-bottom flask was first charged with monomer 1 (1.65g, 5 mmol), N-Iodosuccinimide (NIS) (3.38 g, 5 mmol) and THF (50 ml). After the addition was complete, the solution was heated to 45

。

C and refluxed for 24 h.

The reaction mixture is washed with saturated aqueous NaHSO3 solution (350 mL), dried over Na2SO4, and concentrated on a rotary evaporator for further purified by column chromatography with petroleum ether/CH2Cl2 = 2:1. The final product was dried to obtain a metallic dark-red solid (Yield: 2.5g, 68%). 1H NMR (400 MHz, CDCl3): δ = 7.32 (d, 2H), 7.11 (d, 2H), 2.64 (s, 6H), 2.46 (s, 3H), 1.40 (s, 6H).

13

C 7

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NMR (100 MHz, CDCl3): δ = 156.56, 145.40, 141.87, 139.57, 131.69, 130.12, 127.62, 85.54, 21.49, 17.05, 16.01. MALDI-TOF-MS (m/z): Calcd for C20H19BF2I2N2 [M]+, 590.00; found, 590.70.

Monomer 3. A 250 mL round-bottom flask was first charged with monomer 2 (2.95 g, 5 mmol), 1-(tert-butyl)-4-ethynylbenzene (2.37 g, 15 mmol), Pd (PPh3)4 (9.5 mg, 0.05 mmol), Triphenylphosphane (9.5 mg, 0.05 mmol) and CuI (9.5 mg, 0.05 mmol). Under nitrogen protection, added degaussed diisopropylamine (10 mL) and THF (20 mL). The reaction mixture was stirred at reflux for 24 h under nitrogen protection. After removal of excess diethylamine, the mixture was filtered, the combined organic layer was dried over Na2SO4, filtered and the solvent was removed by rotary evaporation. After removing the solvent, the residue was purified by chromatography using petroleum ether/CH2Cl2 = 2:1. Remove eluent by rotary evaporation to give red metallic powers (Yield: 2.93g, 90%). 1H NMR (400 MHz, CDCl3): δ = 7.39 (d, 4H), 7.33 (t, 6H), 7.15 (d, 2H), 2.71 (d, 6H), 2.47(s, 3H), 1.54(d, 6H), 1.31(s, 18H). 13C NMR (100 MHz, CDCl3): δ = 158.18, 151.45, 143.91, 142.80, 139.32, 131.09, 129.98, 127.70, 125.35, 120.42, 96.45, 80.94, 34.79, 31.17, 29.71, 21.48, 13.68, 13.48. MALDI-TOF-MS (m/z): Calcd for C44H45BF2N2 [M]+, 650.66; found, 650.61.

Bis-styryl

BODIPY

dye.

The

monomer

2

(0.65

g,

1

mmol),

4-(diethylamino)benzaldehyde (0.885 g, 5 mmol), acetic acid (3 mL), and piperidine (3 mL) were dissolved in 100mL of benzene refluxed for 72 h by using a DeanStark apparatus. The mixture was cooled to room temperature, the reaction mixture is 8 ACS Paragon Plus Environment

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washed with saturated aqueous NaHCO3 solution (350 mL), dried over Na2SO4, and concentrated on a rotary evaporator for further purified by column chromatography on silica with petroleum ether/CH2Cl2 = 6:1. The final product is dried to yield a brown powers (Yield: 0.43 g, 42%). 1H NMR (400 MHz, CDCl3): δ = 8.41 (d, 2H), 7.65 (d, 2H), 7.56 (d, 4H), 7.41 (d, 4H), 7.37 (d, 4H), 7.31 (d, 2H), 7.18 (d, 2H), 6.70 (d, 4H), 3.43 (d, 2H), 2.45 (d, 2H), 1.31 (d, 2H), 1.20 (d, 2H). 13C NMR (100 MHz, CDCl3) δ 151.26, 148.64, 139.28, 135.22, 131.36, 130.86, 130.74, 130.58, 129.68, 129.61, 128.64, 128.06, 125.47, 125.05, 120.91, 111.57, 97.88, 83.98, 44.56, 44.46, 32.22, 31.21, 29.71, 26.42, 23.44, 12.74, 12.56. MALDI-TOF-MS (m/z): Calcd for C66H73BF2N4 [M+2H]+, 971.13; found, 971.74.

Preparation and characterization of water-soluble BODIPY NPs. The preparation of water-soluble BODIPY NPs was according to our previous report.17 The bis-styryl BODIPY (2.0 mg) in 2 mL THF was swiftly dropped into the DSPE-mPEG5000 aqueous solution (5.0 mg in 10 mL H2O) under sonication. THF was then removed by argon blowing on the solution surface under stirring at 40 oC. A brown green aqueous solution was then obtained. The aqueous solution was further centrifuged using a centrifugal-filter (Amicon centrifugal filter device, MWCO = 100 kDa) and washed several times with deionized water. The resultant products, BODIPY NPs, were concentrated by the centrifugal-filter and further reconstituted into 2 ml PBS after filtering through a 0.22 µm filter.

Cell viability assay. The MTT assay was used to determine the in vitro cytotoxicity of BODIPY NPs in 3T3 and A549 cells. 3T3 and A549 cells were incubated 9 ACS Paragon Plus Environment


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respectively in DMEM supplemented with medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. Then, the cells were further inculcated in medium containing different doses of BODIPY NPs for 24 h. After that, 10 µL MTT (0.5 mg/mL) solution was added into each well. After 3 h incubation at 37 °C, the supernatant was removed and 200 µL of dimethylsulfoxide (DMSO) was added. A PowerWave XS/XS2 microplate spectrophotometer was used to record the absorbance intensity at 490 nm. The cellular viability relative to the untreated cells (control group) was calculated as

‫ܣ‬௦௔௠௣௟௘ ൘‫ܣ‬ , in which Asample and Acontrol respectively represent the ௖௢௡௧௥௢௟

average absorption of groups containing BOIDPY NPs and untreated cells.

PA spectrum and comparison of PA intensity. The PA spectrum of BODIPY NP was acquired via a point-to-point method. Briefly, the PA signal of BODIPY NPs in aqueous solution at different excitation wavelength (700， 710， 720， 730， 740， 750， 760， 770， 780， 790， 800，810， 820，830， 840 nm) were recorded respectively. Then PA signal intensities were measured by region of interest (ROI) analysis using the Vevo LAZR imaging system software package or OsiriX. The finally diagram of PA signal intensity vs excitation wavelength was considered as PA spectrum. The diagram of concentration-dependent PA signal was obtained through the similar method.

Lysosome isolation and PAI analysis of different fractions. The PAI properties of BODIPY NPs were further investigated in living cells. A549 cells were cultured in 25 cm2 flasks to 80% confluence in complete DMEM medium. Then, the cells were 10 ACS Paragon Plus Environment

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washed with PBS and treated with medium containing 10 µg/mL BODIPY NPs 6 h for the uptake of the material. Then, the BODIPY NPs stained cells were divided into lysosomal and cytoplasmic fractions by using lysosome isolation kit carefully following manufacturer’s guidelines. The as-prepared lysosomal and cytoplasmic fractions were condensed to the exactly same volume by centrifugation and then transferred into 200 µl sample tubes for further study. Finally, the PAI and UV-Vis-NIR spectrum of the cells were captured by a Nexus-128 PAI tomography system (Endra Inc., Michigan, USA) under 730 nm pulsed laser irradiation (6.1 mJ cm-2) and UV-Vis-NIR spectrophotometer. PA signal intensities were measured by region of interest (ROI) analysis using the Vevo LAZR imaging system software package or OsiriX.

Cell apoptosis assay. A549 cells were inculcated in 6-well plates in DMEM supplemented with medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. Each well were added BODIPY NPs (10 µg/ml) were added into each well. After incubation for 12 h, the excess BODIPY NPs was washed with fresh complete medium, and then 730 nm laser light (200 mW cm−2) was directed into each well for 10 min irradiation. After 4 h further incubation, the cells were stained by Annexin V-FITC/propidium iodide (PI) according to the product’s protocol, washed with PBS twice for flow cytometer analysis using FlowSight® Imaging Flow Cytometer.

Animals and tumor model. All A549 tumor bearing nude mice were purchased from Nanjing Mergene Life Science Co., Ltd and used according to the guideline of the 11 ACS Paragon Plus Environment


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Laboratory Animal Center of Nanjing Mergene Life Science Co., Ltd. A549 cells (1 × 106) were subcutaneously injected into the wanted region of the nude mice (6 weeks of age) to obtain A549 tumor-bearing mice. The tumor volume was calculated as

ܸ=

௅×ௐ మ ଶ

, in which L and W respectively represent the longitudinal and transverse

diameter of tumor.

In vivo PAI study. When the tumor volume reached 200-400 mm3, the A549 tumor-bearing mice were intravenously injected by BODIPY NPs (10 µg/ml, 150 µL). The real-time in vivo PAI was performed using Endra Nexus 128 PA tomography system (Endra Inc., Michigan, USA). The excitation wavelength was fixed at maximum absorption of BODIPY NPs (760 nm) with laser energy ~6.9 mJ/cm2 on the animal surface, and 128 ultrasonic transducers with 5.8 MHz center frequency were arranged in hemispherical fashion in a chamber containing water 37°C water. A high-performance graphics unit (GPU) was used for volume reconstruction. The reconstructed raw data was analysed using software OsiriX Lite. The quantitative PA signal intensity of region of tumor was measured using OsiriX Lite.

In vivo PDT efficacy. When the tumor volume reached to 50 mm3, the A549 tumor-bearing mice were weighed and randomly divided into two groups, and accepted the following treatments: 1. saline (150 µL); 2. BODIPY NPs (10 µg/ml, 150

µL). 30 min later, the tumor region of above-mentioned two groups were exposed to 730 nm continuous laser for 10 min at the power density of 200 mW cm−2. The tumor volume was determined every other day for 14 days. At day 14, the mice were euthanized, and the tumors were harvested for hematoxylin-eosin (H&E) and the 12 ACS Paragon Plus Environment

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terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining assay under IX71 optical microscope (Olympus, Japan).

Statistical analysis. Statistical analyses were performed by student’s t-test using a statistics program (GraphPad Prism; GraphPad Software). For animal experiments (6 mice per group), One-way analysis of variance (ANOVA) was utilized to compare the therapeutic outcome. Data were expressed as means ± s.d. Statistical significance was set at P < 0.05.

RESULTS AND DISCUSSION

To

obtain

NIR-absorptive

BODIPY

dyes,

strong

electron-donating

dimethylaminophenyl moiety was decorated into electron-withdrawing BODIPY core to form donor-π-acceptor bis-styryl BODIPY dye (Scheme. 2). The structure was characterized via 1H-NMR and matrix-assisted laser desorption/ionization time of flight

mass

spectrometry

(MALDI-TOF-MS,

Figure

S1).

Importantly,

dimethylaminophenyl is a representative acid-sensitive group which can selectively accumulate into acidic lysosome while provide an acid-activatable PDT.34 Our bis-styryl BODIPY dye displayed strong absorption at 760 nm in DMF with molar absorption coefficient ε of 1.81 × 104 M-1 cm-1 (Figure S2). In comparison with most available BODIPY dyes having absorption in visible light region,26 this bis-styryl BODIPY dye exhibited longer wavelength absorption in NIR region, which is more appropriate for bioapplications in deep tissue. As bis-styryl BODIPY dye was hydrophobic, a biocompatible DSPE-mPEG5000 was used to solubilize hydrophobic 13 ACS Paragon Plus Environment


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bis-styryl BODIPY dye, affording water-soluble BODIPY NPs. The as-prepared NPs exhibited narrow size distribution with average hydrodynamic diameter of 35.3 nm (Figure 1a). TEM image revealed their spherical morphology with high monodispersity (Figure 1b). Moreover, no naked-eye observed precipitates were observed after storage in refrigerator for at least three month (insert of Figure. 1b), suggesting great chemical stability of as-prepared BODIPY NPs. To further study the physiological stability of BODIPY NPs, we tested the hydrodynamic diameter of BODIPY NPs in serum. With time, the size of BODIPY NPs in serum remained unchanged (Figure S3), indicating good physiological stability of BODIPY NPs without aggregation. In addition, the dark-cytotoxicity of BODIPY NPs was determined by employing the standard MTT assay according to our previous protocol,35 and no significant cytotoxicity was observed in Figure S4. These data indicated great chemical stability, excellent biocompatibility and physiological stability of BODIPY NPs for imaging or therapeutic applications.


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Figure 1. a) Size distribution of BODIPY NPs. b) TEM image of BODIPY NPs. The insert picture: BODIPY NPs in PBS (pH = 7.4). c) Normalized UV-Vis-NIR absorbance spectra of BODIPY NPs in water. d) Photostability of BODIPY NPs and commercial ICG at each maximum absorption peak.

We then studied the optical properties of our BODIPY NPs. Compared with bis-styryl BODIPY dye in DMF, BODIPY NPs in aqueous solution showed slightly red-shifted NIR absorption at 775 nm (Figure 1c), owning to the intermolecular interactions between bis-styryl BODIPY dye inside NPs.36 To probe the photostability of BODIPY NPs, the commercial PA contrast, ICG was taken as a control due to the similar absorption spectral profile of ICG with that of BODIPY NPs (Figure 1c). As



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shown in Figure 1d, BODIPY NPs still exhibited excellent photostability after 60 min of continuous laser irradiation at 730 nm (laser density of ca 400 mW cm-1), while commercial PA contrast (ICG) exhibited considerably reduced absorption (>80%). The strong NIR absorption and outstanding photostability of BODIPY NPs made it an encouraging organic nanoparticles for NIR-response bioapplications.

Figure 2. a) PA spectrum of BODIPY NPs in water. b) PA amplitudes of BODIPY NPs in water after 730 nm pulsed laser irradiation (laser fluence of 6.1 mJ cm-2) vs mass concentration (R2 = 0.996).

The PA spectrum of BODIPY NPs in aqueous solution was then studied for PAI application. Figure 2a exhibited the PA spectral profile of BODIPY NPs with maximum PA signal at 775 nm, which is consistent with its UV-Vis-NIR absorption spectrum. Besides, the PA amplitudes of BODIPY NPs at 730 nm (consistent with the excitation wavelength used for subsequent PDT) were measured at a series of concentration from 10 to 250 µg mL-1, and a linearly increase of PA amplitudes with concentration was observed, providing the possibility to quantitatively analysis of the distribution of PSs. Although BODIPY dye has been observed to generate PA signals


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at 532 nm recently,37 the excitation wavelength still located in the visible light region, which is thus unsuitable for bioapplications in deep tissues. Moreover, the high laser fluence needed to generate PA signals greatly exceeded the American National Standards Institute (ANSI) recommended maximum permissible exposure (MPE) limits (20 mJ cm-2 for 400-700 nm pulsed laser with pulse duration range of 1-100 nanosecond).38 In this study, the laser fluence at 730 nm nanosecond pulsed laser (5 ns pulses, 20 Hz pulse repetition frequency) was as low as 6.1 mJ cm-2, which is far lower than the ANSI recommended MPE limits (23 mJ cm-2 for 730 nm pulsed laser with pulse duration range of 1-100 nanosecond, the pulse duration of the pulsed laser in this study is 5 nanosecond).38 Such a highly efficient PA signal of BODIPY NPs probably resulted from the strong fluorescence quenching of bis-styryl BODIPY dyes within BODIPY NPs (Figure S5), because it is generally accepted that reducing fluorescent emission of a dye will be beneficial for its PA signal enhancement.37 Given these merits of BODIPY NPs, it is therefore reasonable to predict the high potential of BODIPY NPs for PAI.

Figure 3. a) Two-dimensional PA signal of lysosomal and cytoplasmic fractions after 730 nm pulsed laser irradiation, the ca 6-folded higher PA signal (245 vs 39) of 17 ACS Paragon Plus Environment


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lysosomal fractions relative to cytoplasmic fractions indicated that BODIPY NPs mainly accumulated into lysosome. b) UV-Vis-NIR absorbance spectra of lysosomal and cytoplasmic fractions.

To examine the lysosome-targeting ability of BODIPY NPs, we broke BODIPY NPs stained cancer cells (A549) into lysosomal and cytoplasmic fractions by using lysosome isolation kit (Sigma-Aldrich) following manufacturer’s guidelines (Figure

3a). The PA signal of lysosomal fraction is obviously brighter than that of cytoplasmic fraction after a 730 nm pulsed laser irradiation, and the quantitative PA amplitude of lysosomal fraction was found to be about 6-folded higher (245 vs 39) than that of cytoplasmic fraction (Figure 3a). This remarkably strong PA signal indicated that BODIPY NPs mainly accumulate in lysosome rather than cytoplasm, confirming the excellent lysosome-targeting ability of BODIPY NPs. Furthermore, the UV-Vis-NIR absorbance intensity of BODIPY NPs of the lysosomal fraction was higher than that of the cytoplasmic fraction, clearly demonstrating the presence of BODIPY NPs mostly in lysosomal fraction (Figure 3b). It is thus highly anticipated to use BODIPY NPs as a lysosome-targeting PA contrast or PS for further applications.


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Figure 4. a) Absorbance of ADMA at 259 nm vs irradiation time (irradiation at 730 nm with power density of 200 mW cm-2). b) The time-dependent absorption of ADMA at 259 nm under different irradiation intensity (730 nm continuous laser). The cytotoxic 1O2 is one of the most vital stimulus for cancer death during PDT process, and thus a chemical 1O2 probe, 2,2′-(anthracene-9,10-diylbis(methylene)) dimalonic acid (ADMA), was utilized to verify whether BODIPY NPs can be applied as an acid-activatable PS.39 As shown in Figure 4a, BODIPY NPs in neutral water exhibited negligible consumption of ADMA because of the strong quenching of excited singlet state in neutral condition,34 while in acidic water displayed enhanced consumption, suggesting an acid-activatable 1O2 generation (detailed discussions were shown in Figure S6). In comparison, commercial ICG showed negligible consumption of ADMA, presumably due to the low power density of irradiation light. Furthermore, obvious irradiation intensity-dependent 1O2 release of BODIPY NPs in acidic water was found (Figure 4b), revealing the strong 1O2 generation ability of BODIPY NP in acidic environment. Coupling with the high-selectivity of BODIPY



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NPs in acidic lysosome, it was reasonable to conclude that BODIPY NPs could be used for acid-activatable 1O2 release.

Figure 5. a) Cell viability of A549 cells incubated with BODIYPY NPs and ICG for 12 h, 24 h and 36 h without laser irradiation. b) Irradiation time dependent cell viability of A549 cells incubated with BODIYPY NPs and ICG with laser irradiation. c) Flow cytometry analysis of A549 cells death induced by BODIYPY NPs mediated PDT.

The PDT effect of BODIPY NPs was studied by incubating A549 cells with BODIPY NPs. The cell viability of ICG and BODIPY NPs without laser irradiation was determined from MTT assay and no obvious cytotoxicity was observed, indicating that neither ICG nor BODIPY NPs is benign in the absence of laser 20 ACS Paragon Plus Environment

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irradiation. Furthermore, the cell viability that exposed to continuous laser irradiation at 730 nm with power density of 200 mW cm−2 was determined (Figure 5b). In order to exclude the influence of viability induced by laser irradiation, the control experiment without BODIPY NPs was performed and no obvious cytotoxicity was observed (Figure 5b), suggesting that A549 cells were resistance to the laser irradiation. Meanwhile, photothermal effects induced by BODIPY NP after laser irradiation were also excluded (Figure S7), because the temperature of BODIPY NPs-dissolved aqueous solution exhibited negligible enhancement after the laser irradiation (same parameters as PDT experiment). As expected, the viability of A549 cancer cells incubated with BODIPY NPs was dramatically reduced to 47.3%, probably because the more acidic environment (pH 5.0-5.5) within lysosome compared with cytoplasmic or other subcellular components (pH ca. 7.4) efficiently activated the PDT ability of BODIPY NPs.40 On the contrary, the viability of A549 cancer cells incubated with ICG remained nearly 100%, indicative of the low efficient of ICG for PDT. All these results indicated that BODIPY NPs can be applicable as a highly efficient PS in comparison with commercial ICG. To visualize the cell apoptosis during PDT, Annexin V-FITC/propidium iodide (PI) cell apoptosis kit was utilized to distinguish viable cells from dead cells of different stages according to previous report (Figure 5c).25 After the cells were treated with either laser or BODIPY NPs alone, most of the A549 cells are viable with a cell viability >90% (Annexin V-FITC−/PI−), further demonstrating the resistance of cells to the 730 nm continuous laser irradiation and low dark cytotoxicity of BODIPY NPs. In sharp 21 ACS Paragon Plus Environment


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contrast, consist with above cell viability, the sum population of early apoptotic (Annexin V-FITC+/PI−) and late-stage apoptotic (Annexin V-FITC+/PI+) A549 cells significantly increased (87%) upon the combining utilization of BODIPY NPs and laser, further confirming the promising application of the BODIPY NP as a highly efficient PS for PDT.

Figure 6. a) In vivo PAI of A549 tumor-bearing mice at a different time after intravenous injection of BODIPY NPs. b) The quantitative PA signal analysis of the region of tumor at different time after intravenous injection of BODIPY NPs. c) The A549 tumor growth curves after treatment with saline and BODIPY NPs. d) Representative images of the A549 tumors after treatment with Saline and BODIPY NPs at day 14. e) The body weight variation of A549 tumor-bearing mice during treatment. f) The H&E stained tumor tissues after treatment with saline and BODIPY NPs. g) Apoptotic analysis of TUNEL stained tumor tissues after treatment with 22 ACS Paragon Plus Environment

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Saline and BODIPY NPs. Error bars is the standard deviation from mean (6 mice per group), *** is P < 0.001. To test the in vivo PAI ability of BODIPY NPs, BODIPY NPs (10 µg/mL, 150 µL) were administrated intravenously into the A549 tumor-bearing mice via tail vein. The tumor region of mice were imaged at different time (pre-injection, 20, 30, 50, 120 min, and 24 h) after injection of BODIPY NPs. In Figure 6a, major blood vessels in the thoracic wall and intercostal vessels were observed at pre-injection due to the endogenous contrast of hemoglobin with an absorption peak at NIR region.[41] At 30 min after injection, the PA enhancement reached a maximum (Figure 6a, b), suggesting an excellent enhanced permeability and retention (EPR) effect of BODIPY NPs. After reaching maximum, the PA signal decreased gradually over time. At 120 min post-injection, the intensity of PA signal decreased by nearly 50% versus 30 min post-injection and almost vanished at 24 h post-injection.

The in vivo PDT efficacy of BODIPY NPs was also investigated by monitoring the relative tumor volumes of A549 tumor-bearing mice. Based on above in vivo PAI that BODIPY NPs reach its maximum accumulation in tumor (Figure 6a, b), the tumor was exposed to 730 nm laser with power density of 200 mW cm−2 for 10 min at 30 min post-injection of BODIPY NPs (10 µg/mL, 150 µL) via tail vein. As shown in

Figure 6c, d, the growth of the tumor after the treatment of BODIPY NPs and laser was significantly inhibited in comparison with that of saline and laser. Meanwhile, the body weight of the mice treated with BODIPY NPs or saline remained stable, revealing a negligible side effect of BODIPY NPs.[42] We further applied the H&E 23 ACS Paragon Plus Environment


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and TUNEL staining assay at 14 d after treatment to evaluate the PDT efficacy. Compared with the control group under the treatment of saline and laser, the images of H&E-stained tumor tissues treated by BODIPY NPs and laser exhibited prominent necrosis of the tumor cells (Figure 6f), suggesting a successful destroy of the tumor cells. The TUNEL stained images displayed a higher level of cell apoptosis in the tumor tissue under the treatment of BODIPY NPs and laser than that of control groups (Figure 6g). Take together, these preliminary results demonstrated that BODIPY NPs can efficiently accumulated at the tumor site and thereby realized the in vivo PAI and highly efficient PDT in living mice. Further investigations are still needed to comprehensively evaluate the BODIPY NPs toxicity.

CONCLUSION

In summary, for the first time we engineered a lysosome-targeting organic nanoparticle by conveniently encapsulating single NIR-absorbed bis-styryl BODIPY. They can exhibit high-performing lysosomal PAI at ultralow nanosecond pulsed laser fluence while serve as an acid-activatable nanophotosensitizer for PDT against cancer cells under NIR light. More importantly, not only does our study highlights flexibility and convenience of such conveniently approach toward engineering pure organic multifunctional nanoparticles with excellent biocompatibility but also provides a new paradigm with concurrent new emerging diagnostic (PAI) and acid-activatable PDT uses under NIR light. ASSOCIATED CONTENT 24 ACS Paragon Plus Environment

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Supporting Information. MALDI-TOF-MS spectrum of bis-styryl BODIPY dye, detailed

optical

performance,

physiological

stability

of

BODIPY

NPs,

dark-cytotoxicity of BODIPY NPs, and time-dependent photothermal curves of BODIPY NPs. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author *Email: [email protected].

*Email: [email protected].

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Basic Research Program of China (No. 2012CB933301 and 2012CB723402), the National Natural Science Foundation of China (No. 21574064, 51173080, 61378081, 51503103, 61136003 and 21222404), Synergetic Innovation Center for Organic Electronics and Information



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Displays and the Natural Science Foundation of Jiangsu Province of China (No. BZ2010043, BM2012010, NY211003).

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Table of Contents Graphic


Engineering Lysosome-Targeting BODIPY Nanoparticles for

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