Organic solvents were obtained from Chengdu KeLong Chemical Co., Ltd. (Chengdu, China) and were used directly without further purification unless specified. Anhydrous dichlormethane was further purified by distillation and was dried over molecular sieves prior to use. Aqueous solutions were all prepared using phosphate-buffered saline (PBS; pH 7.4). Cyclohexanone, 2,3,3-trimethylindolenine, iodoethane, oxalyl chloride, trimethylamine, oxalyl chloride and (3-carboxypropyl) triphenylphosphonium bromide were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), and 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-bromide (MTT) was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The green fluorescent mitochondria tracker (MitoTracker^® Green) and nucleic acid stain (Hoechst 33342) were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA), and used according to the manufacturer's protocols. B16 melanoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Cy7.Cl was synthesized by our laboratory according to the previously described method (14). The chemical structure was further identified by proton nuclear magnetic resonance spectroscopy (¹HNMR; Bruker-400 MHz NMR; Bruker Corporation, Billerica, MA, USA) and Time of Flight Mass Spectrometer (TOF-MS; Agilent Technologies, Inc., Santa Clara, CA, USA). The chemical shifts of Cy7.Cl reported in ppm (DMSO-d6; TMS as internal standard) were as follows: ¹HNMR (400 MHz, DMSO-d₆) δ=8.27 (d, J=14.0 Hz, 2H), 7.65 (d, J=7.4 Hz, 2H), 7.53–7.38 (m, 4H), 7.30 (t, J=6.6 Hz, 2H), 6.33 (d, J=14.2 Hz, 2H), 4.26 (d, J=6.9 Hz, 4H), 2.73 (s, 4H), 1.89 (d, J=20.2 Hz, 2H), 1.67 (s, 12H), 1.31(t, J=7.1 Hz, 6H). TOF-MS: calculated for C₃₄H₄₀ClN₂⁺, [M+H]⁺=512.1,575, found 512.2,881.

Cy7.Cl (639.0 mg, 1.0 mmol) and AcONa (820.0 mg, 10.0 mmol) were dissolved in 10 ml anhydrous N,N-dimethylformamide (DMF) under 100% nitrogen atmosphere for 3 h at 80°C. Then the mixture was washed three times with saturated KI solution, and extracted using CH₂Cl₂ (50 ml) for 3 times. All organic mixtures were dried using anhydrous sodium sulfate, concentrated on a rotary evaporator and the residues were purified by silica chromatography (500–800 mesh) eluted with petroleum ether: Ethyl acetate=1:1 (v/v) to give a dark red solid 209.0 mg. The yield of Cy-OH was 42.5% The H-shifts of Cy-OH were as follows: ¹H NMR (400 MHz, DMSO-d₆) δ 7.93 (d, J=13.3 Hz, 2H), 7.33 (d, J=7.2 Hz, 2H), 7.19 (t, J=8.0 Hz, 2H), 6.90 (dd, J=7.3, 4.4 Hz, 2H), 5.48 (d, J=13.4 Hz, 2H), 3.80 (dd, J=14.0, 7.0 Hz, 4H), 2.58–2.53 (t, 4H), 1.79–1.69 (m, 4H), 1.56 (s, 12H), 1.16(t, J=7.0 Hz, 6H). TOF-MS: Calculated for C₃₄H₄₀N₂O, [M+H]⁺=492.7,070, found 492.7,128.

(3-Carboxypropyl) triphenylphosphonium Bromide (443.0 mg, 1.0 mmol) and 2 drops of DMF were dissolved in 10 ml anhydrous CH₂Cl₂, prior to the addition of chloroglyoxylate (1 ml). The mixture was stirred at 0°C for 4 h. Then the solvent was evaporated for further use without additional purification.

Under N₂ conditions, Cy-OH (246.0 mg, 0.5 mmol) and triethylamine (50 µl) were dissolved in 5 ml anhydrous CH₂Cl₂. TPP⁺ (462.0 mg, 1.0 mmol) was dissolved in 5 ml anhydrous CH₂Cl₂ and added into the reaction mixture dropwise at 0°C for 30 min; the mixture was maintained at 25°C for 12 h. The mixture was washed 3 times with saturated KI solution, and extracted using CH₂Cl₂ (50 ml) for 3 times. The organic mixture solvent was dried using anhydrous sodium sulfate, evaporated on a rotary evaporator and the residues were purified by silica chromatography (500–800 mesh) eluted with dichloromethane:methanol (5:1, v/v) to give 164.5 mg of green solid. The yield of Cy-TPP was 35.0%. The H-shift of Cy-TPP were as follows: ¹H NMR (400 MHz, DMSO-d₆) δ 8.32 (d, J=13.3 Hz, 2H), 7.33–7.38 (m, 17H), 7.53–7.38 (t, J=8.0 Hz, 4H), 7.32 (d, J=7.3, 4.4 Hz, 2H), 6.34 (d, J=13.4 Hz, 2H), 4.27 (d, J=14.0, 7.0 Hz, 4H), 2.58–2.53 (t, 8H), 1.79–1.69 (m, 4H), 1.56 (s, 12H), 1.16 (t, J=7.0 Hz, 6H). TOF-MS: calculated for C₅₆H₆₁N₂O₂P²⁺, [M+H]⁺=825.0887, found 825.0596.

The UV-Vis absorption and fluorescence emission (B) spectra of Cy-TPP (2 µM) were conducted in MeOH, PBS and 100% FBS. Fluorescence spectra was obtained by fluorescence spectrometer (F7000 Fluorescence Spectrophotometer; Hitachi High-Technologies Corporation, Tokyo, Japan) with a Xenon lamp and 1.0-cm quartz cells at the slits of 5.0/5.0 nm. Absorption spectra was recorded by UV-Vis spectrophotometer (DU800; Beckman Coulter. Inc., Brea, CA, USA). The responses of Cy-TPP (5 µM) with different analytes (50 mM for K⁺, Na⁺, 10 mM for Arg, Lys, Glu, 1 mg/ml for BSA, 25 mM for Ca²⁺, Mg²⁺, Al³⁺, Cu²⁺, Fe³⁺, Cr³⁺, Co³⁺, Mn²⁺, S₂O₃²⁻, H₂O₂) were conducted by incubating them for 30 min at 37°C and measured by fluorescence spectrometer. The fluorescence responses of Cy-TPP to various concentrations of BSA (0.1–1 mg/ml) were also measured by fluorescence spectrometer.

In order to evaluate the cellular toxicity of different doses of Cy-TPP, the compound treated cells were illuminated using series of concentrations. Typically, B16 cells (5×10⁴ cells/well) were cultured in RPMI-1640 medium (Hyclone; GE Healthcare Life Sciences, Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences) in an incubator with 5% CO₂ at 37°C for 24 h. Prior to any experiments, the cell medium was removed and an increasing concentration (0.05, 1, 2, 5 and 10 µM) of probe Cy-TPP was added. The cells were then incubated for a further 24 h, and MTT assays were performed according to the manufacturer's instructions. Briefly, the medium was replaced with fresh medium containing 10% MTT (v/v). Following 3 h, the medium was replaced with 100 µl DMSO to solubilize the formed formazan. Absorption was measured at 490 nm for each well using a microplate reader. An untreated assay with RPMI-1640 was also conducted under the same conditions, which was recorded as the relative cell viability.

Fluorescent images were acquired on a confocal laser-scanning microscope (Nikon Eclipse Ti; Nikon Corporation, Tokyo, Japan) with an oil lens (magnification, ×60). B16 cells (5×10⁴ cells/well) were plated in cell culture Petri-dishes (F=20 mm) in RPMI-1640 supplemented with 10% FBS in an incubator with 5% CO₂ at 37°C. Following 24 h, the medium was removed and the cells were washed three times with PBS buffer. The cells were then incubated with the probe Cy-TPP (2.0 µM) in serum-free medium. Following 2 h, MitoTracker^® Green (200 nM) was added, followed by Hoechst 33342 (1 µM). Following incubation for 15 min, the medium was removed. Cell imaging was carried out following cell washing with PBS three times to remove the excess compounds. Hoechst fluorescence was analyzed with an excitation at 405 nm and a scan range of 440 to 500 nm; MitoTracker Green fluorescence was analyzed with an excitation at 488 nm and a scan range of 520 to 570 nm; probe Cy-TPP was analyzed with an excitation at 770 nm and a scan range of 790 to 810 nm. Images were processed using Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

All data are presented as the mean ± standard error of the mean. The data were analyzed by two-way analysis of variance followed by Dunnett's tests using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Cy-TPP was designed and synthesized by adding TPP onto the Cy skeleton. The details of the synthetic routes and characterizations of Cy-TPP are outlined in Fig. 1. This design was chosen for two purposes. Firstly, it is well known that many lipophilic cations, such as TPP and Cy, possess an overall positive charge, which facilitates their uptake and accumulation in the mitochondria (15). Thus, heptamethine Cy was chosen as a signal transducer due to its favorable stability and NIR absorption and emission profile. Secondly, the TPP moiety has also been confirmed to be essential for antitumor activity (16). With an aim to increase the cytotoxic activity of heptamethine cyanine, TPP was used to enhance its lipophilic nature and cationic charge, which can facilitate the mitochondrial membrane potential dependent accumulation of TPP within the negatively charged mitochondrial matrix (17). The structure of Cy-TPP was identified by ¹HNMR and MS.

Optical properties of Cy-TPP. The optical properties of Cy-TPP in methanol, 100% FBS and PBS were then determined (pH=7.4). The absorption and emission peaks of Cy-TPP were all in the NIR region (700 to 900 nm), providing 17 to 26 nm Stokes shifts (Fig. 2A and B). The fluorescence responses of Cy-TPP (5 mM) to various species, including K⁺, Na⁺, Ca²⁺, Mg²⁺, Al³⁺, Cu²⁺, Fe³⁺, Co²⁺, Mn²⁺, arginine, lysine, glucose and bovine serum albumin (BSA) was then investigated. The present study demonstrated that the fluorescence intensity of Cy-TPP markedly increased following the addition of BSA (Fig. 2C). Therefore, the effect of different doses of BSA on the fluorescence intensity of Cy-TPP was further investigated (Fig. 2D). Serum significantly increased the intensity fluorescence intensity of Cy-TPP, indicating it may have potential for biomedical application.

An MTT assay in B16 cells was performed to evaluate the ability of Cy-TPP to inhibit cell proliferation. As displayed in Fig. 3A, the inhibition rate of cell viability in B16 cells incubated with 2.0 µM Cy-TPP was <50%, which is essential for its further applications in the anti-proliferation of cancer cells. Fig. 3B reveals cellular morphological alterations in B16 cells following the addition of Cy-TPP at doses 0–10 µM. An IC₅₀, the half-maximal inhibitory concentration, value of 3.04 µM also indicated that Cy-TPP significantly inhibits the proliferation of B16 cells.

The mitochondria-targeting properties of the Cy-TPP were determined by incubating the probes with B16 cells (Fig. 4A-D). The confocal laser scanning microscopy analysis of the cells treated with Cy-TPP revealed accumulation in the mitochondria (Fig. 4E and F). Quantitative analysis using the Imagine-Pro Plus 6.0 ‘colocalization analysis’ tool revealed the significant colocalization of Cy-TPP with MitoTracker Green in the mitochondria of the cells (Pearson's correlation coefficient=8.1). The highly efficient mitochondrial targeting ability of Cy-TPP may be attributed to their high buffering capacity, which is generated by lipophilic TPP.