Near-infrared-emitting squaraine dyes with high 2PA cross-sections for multiphoton fluorescence imaging.

Experimental Materials and Methods Synthesis General

All reagents and solvents were used as received from commercial suppliers. Reactions were conducted under N 2 or Ar atmosphere. Melting points are uncorrected. 1 H and 13 C NMR spectra were recorded on a NMR spectrometer at 300 and 75 MHz, respectively. MS analyses were performed at the University of Florida. (Z)-2-(4-(Dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate ( 2 ) was prepared as reported previously. 21 Cy5 (DiD oil, D 307) was obtained from Invitrogen (Carlsbad, CA) 1-(2-(2-Methoxyethoxy)ethyl)-1H-pyrrole-2-carbaldehyde 3 A mixture of 1 H -pyrrole-2-carbaldehyde (0.476 g, 5.0 mmol), KOH (0.28 g, 5.0 mmol), and 18-crown-6 (0.04 g, 0.15 mmol) in C 6 H 6 was refluxed for 2 h, then BrCH 2 CH 2 OCH 2 CH 2 OCH 3 (1.14 g, 6.25 mmol) in C 6 H 6 was added and the mixture was refluxed for 4 h. Upon cooling, water was added and the organic phase was separated, washed with water and dried by MgSO 4 . Concentration and purification by column chromatography using CH 2 Cl 2 /MeOH (100/1) as eluent gave 0.78 g product 3 as a liquid (yield 79%). 1 H NMR (300 MHz, CDCl 3 ) δ 9.49 (s, 1H), 7.04 (m, 1H), 6.91 (m, 1H), 6.18 (m, 1H), 4.48 (t, J = 6.0 Hz, 2H), 3.73 (t, J = 4.5 Hz, 2H), 3.46 (m, 2H), 3.42 (m, 2H), 3.31 (s, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ 179.4, 132.8, 125.1, 110.0, 109.5, 71.8, 70.7, 70.5, 59.0, 48.8. HRMS (APCI) theoretical [M+Na] + = 220.0944, found [M+Na] + = 220.0941. 1-(2-(2-Methoxyethoxy)ethyl)-2-(3,4,5-trimethoxystyryl)-1H-pyrrole 4 5-(Chloromethyl)-1, 2, 3-trimethoxybenzene (0.86 g, 4.0 mmol) was refluxed with triethyl phosphite (1.5 mL) for 2 h, the excess triethyl phosphite was distilled off and the residue was dried under vacuum. The product was then mixed with compound 3 in dry DMF (3 mL) under Ar. NaH (0.48 g, 20.0 mmol) was added and the mixture was stirred at room temperature for 20 h. The mixture was diluted with water and the product was extracted with ethyl acetate. Purified by column chromatography using ethyl acetate as eluent gave 0.7 g of product 4 (yield 48%) as a liquid. 1 H NMR (300 MHz, CDCl3) δ 6.91 (d, J = 9.0 Hz, 1H), 6.82 (d, J = 9.0 Hz, 1H), 6.75 (m, 1H), 6.68 (s, 2H), 6.48 (m, 1H), 6.18 (m, 1H), 4.20 (t, J = 4.5 Hz, 2H), 3.93 (s, 6H), 3.87 (s, 3H), 3.76 ( J = 4.5 Hz, 2H), 3.56 (m, 2H), 3.51 (m, 2H), 3.36 (s, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ 153.4, 137.5, 133.7, 131.6, 123.7, 123.1, 122.9, 108.8, 108.6, 106.7, 106.5, 103.1, 71.9, 71.1, 70.7, 61.0, 59.1, 56.3, 56.1, 46.7. HRMS (APCI) theoretical [M+H] + = 362.1962, found [M+H] + = 362.1971. (E)-2-(1-(2-(2-Methoxyethoxy)ethyl)-5-(3,4,5-trimethoxystyryl)-1H-pyrrol-2-yl)-4-(1-(2-(2-methoxyethoxy)ethyl)-5-(3,4,5-trimethoxystyryl)-2H-pyrrolium-2-ylidene)-3-oxocyclobut-1-enolate (1) Compound 4 (0.70g, 2.00 mmol) and squaric acid (0.11 g, 0.97 mmol) in a BuOH/toluene mixture (2/1 v/v, 60 ml) were refluxed with a Dean-Stark apparatus for 6 h. The precipitated product was collected by filtration giving 0.31 g product 1 . (40% yield) m. p. 203–204 °C. 1 H NMR (300 MHz, CDCl 3 ) δ 7.84 (d, J = 3.0 Hz, 1H), 7.24 (d, J = 9.0 Hz, 1H), 7.16 (d, J = 9.0 Hz), 6.89 (d, J = 3.0 Hz, 1H), 6.77 (s, 4H), 4.96 (m, 4H), 3.94 (s, 12H), 3.89 (m, 6H+4H), 3.53 (m, 4H), 3.41 (m, 4H), 3.20 (s, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ 177.0, 167.2, 153.5, 147.8, 139.2, 135.4, 134.9, 132.1, 129.9, 124.2, 116.2, 115.7, 113.9, 105.0, 104.3, 103.7, 72.08, 71.9, 70.7, 61.1, 58.8, 56.3, 56.2, 47.1. HRMS (ESI-TOF) theoretical [M+Na] + = 823.3412, found [M+Na] + = 823.3356.

Methods

Linear optical properties Steady-state linear absorption was measured with an Agilent 8453 UV-vis spectrophotometer. Fluorescence emission and excitation spectra were measured using a PTI Quantamaster spectrofluorimeter equipped with a Hamamatsu R928 photomultiplier tube (PMT) in solvents of varying polarity. Fluorescence quantum yields were relative to cresyl violet as a reference. 30 Excitation anisotropy spectra were measured using a PTI Quantamaster spectrofluorimeter fitted with two Glan-Thomson polarizers in an L-format method in high viscosity solvent (glycerol, Acros) to avoid reorientation, and in low concentration solutions (C ~ 10 −6 M) to avoid reabsorption. 31 Lifetime measurements were performed using a tunable Ti:sapphire laser system (Coherent Verdi-V10 and MIRA 900, pulse duration ~200 fs/pulse (FWHM), and repetition rate 76 MHz). The polarization of the excitation beam was linear (690 nm) and oriented by the magic angle to avoid molecular reorientation effects. 30 A broad band-pass filter (FF01-694/SP-25, Semrock) was placed in front of the avalanche photodiode detector (APD, PicoQuant GmbH, LSM_SPAD) allowing the collection of emission >700 nm. Data was acquired with a time-correlated single photon counting system (PicoHarp300). The optical density of all the solutions did not exceed 0.12 at the excitation wavelength to avoid reabsorption. Measurements were conducted in 10 mm path length quartz cuvettes at room temperature. Linear photophysical properties of squaraines 1 and 2 were measured in acetonitrile (ACN), 1, 2-dichloromethane (DCM), dimethyl sulfoxide (DMSO), methanol (MeOH), tetrahydrofuran (THF), and Pluronic micelles that encapsulated the squaraine dye. All solvents were spectroscopic grade. Nonlinear optical properties The open aperture Z-scan method was performed using linear polarized excitation from a Clark-MXR, CPA2010, Ti:sapphire amplified system followed by an optical parametric generator/amplifier (model TOPAS 4/800, Light Conversion) providing laser pulses of 140 fs (FWHM) duration with 1 kHz repetition rate. 32 The tuning range was 520–2100 nm while 10 −3 M ≤ C ≤ 10 −2 M concentration solutions were used in a 1 mm quartz cuvette at room temperature between 780 and 860 nm. For the case of squaraine 1 , the same laser system coupled with a PTI Quantamaster Spectrofluorimeter was used for two-photon absorption (2PA) spectrum measurements of upconverted fluorescence under two-photon excitation over a broad spectral region from 840 to 1140 nm with 10 −5 M ≤ C ≤ 10 −3 M concentration DMSO solution in a 10 mm quartz cuvette at room temperature. 26 The 2PA spectrum of squaraine 1 was calibrated with ZnSe as a standard in the spectral region from 780 to 860 nm. 32 Photochemical and thermal stability Photochemical stability was evaluated for SQ 1 , SQ 2 , and Cy5 in DMSO and aqueous micelle media by irradiating the solution in a 10 mm path length quartz cuvette using a 650 nm diode laser. 33 – 37 Time-dependent absorption spectra upon irradiation at 650 NM were obtained with an Agilent 8453 UV/Vis spectrophotometer and photodecomposition quantum yields were determined. Thermostability was conducted by thermogravimetric analysis (TGA, Q 5000 TA Instruments) and differential scanning calorimetery (DSC, Q1000 TA Instruments).

Cell lines

HCT116 and COS 7 were purchased from America Type Culture Collection (ATCC, Manssas, VA, USA). All cells were incubated at 37 °C in a 95% humidified atmosphere containing 5% CO 2 in cell media (RPMI-1640, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA, USA), and 1% penicillin-streptomycin (Atlanta Biologicals, Lawrenceville, GA, USA). 0.25% Trypsin-EDTA (Invitrogen, Carlsbad, CA, USA) was used for cell splitting. Micelle encapsulation A concentrated micelle stock solution was prepared using 1 mg/mL of the dye by using a small amount of CH 2 Cl 2 to dissolve the dye. 29 Water was added with 2 wt% of surfactant (Pluronic F 127 Prill, BASF Corporation) and stirred overnight. The crude solution was filtered before using. The concentration of the micelle-encapsulated probe stock solution was determined via UV-vis spectrophotometry using the molar absorptivity (SQ 1 ε 853 = 0.6 × 10 5 M −1 cm −1 and SQ 2 ε 601 = 1.0 × 10 5 M −1 cm −1 ).

Cytotoxicity assay

HCT116 and COS 7 were placed in 96 well plates 3×10 3 cells per well in 90 μ L and incubated until there were 6×10 3 cells per well for the cytotoxicity assays. 38 The cells were incubated for additional 20 h with Pluronic micelle-encapsulated SQ dye in different concentrations. Subsequently, 10 μ L of CellTiter 96® Aqueous One Solution reagent (MTS assay) was added into each well, followed by further incubation for 4 h at 37 °C. The relative viability of the cells incubated with SQ dyes to untreated cells was determined by measuring the MTS-formazan absorbance on a microplate reader (Spectra Max M5, Molecular Devices) at 490 nm with subtraction of the absorbance of the cell-free blank volume at 490 nm. The results from three individual experiments were averaged.

Cell culture and incubation

HCT116 and COS 7 cells were placed onto poly-D-lysine coated glass cover slips (12 mm, #1) in 24-well plates, 3×10 4 cells per well, and the cells were incubated for 48 h before incubating with the micelle-encapsulated dyes. The filtered stock solution was diluted with complete growth medium, RPMI-1640, and then incubated for 1.5 h. 20 μL of 5 μg/mL Hoechst nuclear stain was added followed by incubation for another 30 min. After incubation, the cells were washed with PBS (× 5) and fixed using 3.7% formaldehyde solution for 15 min at 37 °C. NaBH4 (1 mg/mL, prepared by adding a couple of drops of 0.1 M NaOH) solution in PBS (pH = 8.0) was added to each well (0.5 mL/well) for 15 min. The plates were then washed with PBS (× 2) and water (× 1). Finally, glass cover slips were mounted using Prolong Gold mounting media (Invitrogen) for microscopy. One- and two-photon fluorescence microscopy (1PFM) and (2PFM) imaging Conventional one-photon fluorescence images were obtained using an inverted microscope (Olympus IX70) equipped with a QImaging cooled CCD (Model Retiga EXi) and mercury lamp 100 W. In order to improve the fluorescence background-to-image ratios, customized filter cubes were used for the 1PFM images. The specifications of the filter cube were tailored to match the excitation wavelength of the probe, and to capture most of the probe’s emission profile. Two-photon fluorescence microscopy (2PFM) images were obtained with a modified Olympus Fluoview FV300 microscope system combined with a tunable Coherent Mira 900F Ti:sapphire laser, pumped by a 9 W Coherent Verdi 10 frequency doubled Nd:YAG laser. The femtosecond NIR laser beam (with ~200 fs pulse width and 76 MHz repetition rate) was tuned to ~ 850 nm and used as the two-photon excitation source. Two-photon induced fluorescence was collected by a 60× microscopic objective (UPlanSApo 60×, NA = 1.35, Olympus). A high-transmittance broad short-pass filter (FF01-694/SP-25, Semrock) was placed in front of the PMT detector in the FV300 scanhead in order to filter out background radiation from the laser source.

Supplementary Material 1_si_001