A New Approach to Sensitize Antitumor Monofunctional Platinum(II

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A New Approach to Sensitize Antitumor Monofunctional Platinum(II) Complexes via Short Time Photo-Irradiation Xuling Xue, Chengcheng Zhu, Huachao Chen, Yang Bai, Xiangchao Shi, Yang Jiao, Zhongyan Chen, Yupeng Miao, Weijiang He,* and Zijian Guo* State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China S Supporting Information *

ABSTRACT: Sensitizing the antitumor activity of monofunctional PtII complexes is a reliable approach to developing antitumor agents different from the classic Pt-based drugs. Considering the poor intracellular accumulation of monofunctional PtII complexes, in this study, the photosensitizing monofunctional PtII complex Pt-BA was derived from a weak BODIPY (boron-dipyrromethene)-derived photosensitizer BA, with the purpose to improve its antitumor cytotoxicity via enhancing its intracellular accumulation with a short time photo-irradiation. Photoinduced reactive oxygen species (ROS) determination indicated that the PtII center in Pt-BA is able to improve the photoinduced ROS production ability of BA, which makes Pt-BA a mild photosensitizer. Fluorescence imaging disclosed that dark incubation makes Pt-BA accumulate mainly on the surface of cell membrane, and the later short time photo-irradiation (5 min) promotes distinctly the intracellular accumulation of Pt-BA, which has been confirmed by inductively coupled plasma−mass spectrometry determination. Flow cytometric Annexin V-FITC assay indicated that the short time irradiation of Pt-BA induces in situ the cell membrane damage, which might finally enhance the intracellular accumulation of this monofunctional complex. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay confirmed that the short time photo-irradiation promotes distinctly the antitumor cytotoxicity of Pt-BA against MCF-7, SGC-7901, A549, and HeLa cell lines. The photopromoted antitumor activity of Pt-BA implies that modifying monofunctional PtII complex as a mild photosensitizer to promote its cell accumulation is a useful approach to sensitizing the antitumor activity of monofunctional PtII complex and renders the possibility of monofunctional PtII prodrugs for precise chemotherapy via only short time photoactivation.

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s one of the most important nonclassical PtII-based antitumor complexes,1 monofunctional PtII complexes characterized with only one leaving group show the antitumor mechanisms different from that of the classic PtII drugs, and this offers the favorable antitumor activity and advantages over the classic platinum drugs.2 However, most monofunctional PtII complexes display no or very low antitumor cytotoxicity, which might be originated from the poor cell uptake of the positively charged PtII center and the incapability to form bifunctional adducts with DNA.3 We have previously shown that the cell accumulation of two 4-nitrobenzo-2-oxa-1,3-diazole-derived (i.e., NBD-derived) fluorescent monofunctional PtII complexes is much slower (3−6 h) than that of neutral bifunctional PtII complex such as {1-([5-(and 6-)carboxyfluorescein diacetate]aminomethyl)-1,2-ethylenediamine}dichloroplatinum(II) (CFDA-Pt),4 although their NBD-derived ligands show the rapid cell uptake. On the one hand, screening the different cell lines found that the two compounds show the higher half maximal inhibitory concentration (IC50) values and slower cell accumulation simultaneously. On the other hand, pyriplatin, one of the most well-known monofunctional PtII complexes, demonstrates the high antitumor activity specifically against the colorectal cancer due to its promoted cell accumulation via © 2016 American Chemical Society

organic cation transporters (OCT) 1 and 2, which are abundantly expressed in human colorectal cancers.5 Therefore, improving the cell uptake of cationic monofunctional PtII complexes might be an effective approach to sensitizing their antitumor activity. Although the positively charged PtII center of monofunctional complexes disfavors the cell uptake of these Pt II complexes, their positive charge makes these complexes inclined to accumulate on the cell membrane.4a,b Recently, a cationic photosensitizer, the oligo(p-phenylenevinylene) derivative bearing 1,3-dialkyl imidazolium branches, was utilized to accumulate on the cell membrane to promote the cell accumulation of doxorubicin, paclitaxel, vincristine, 5-fluorouracil, and cisplatin,6 and the photosensitizer damaging the cell membrane in situ by the photoinduced reactive oxygen species (ROS) was proposed as the mechanism to promote the cell uptake. Therefore, modifying monofunctional PtII complex as a photosensitizer might be an effective approach to enhancing its cell accumulation and antitumor activity via photo-irradiation inducing the cell membrane damage. With such a modified Received: September 8, 2016 Published: December 6, 2016 3754

DOI: 10.1021/acs.inorgchem.6b02148 Inorg. Chem. 2017, 56, 3754−3762

Article

Inorganic Chemistry monofunctional PtII complex, the precise chemotherapy via only a short time photoactivation can be expected in the future. Therefore, the mild photosensitizing ability of the monofunctional PtII complex is required, which is sufficient to damage the cell membrane but display no distinct photodynamic therapeutic (PDT) activity. In this way, the antitumor effect relies on not the PDT activity but the chemotherapeutic activity, and the sustained photo-irradiation is not required. In this study, a new monofunctional PtII complex, Pt-BA (Scheme 1), was formed using α-(4-amino)styryl-4,4-difluoro-

were determined with an LCQ electrospray mass spectrometer (Thermo Finnigan). High-resolution mass spectra (HR-MS) were determined with an Agilent 6540Q-TOF HPLC-MS spectrometer. Elemental analysis was performed with a PerkinElmer 240C analytical instrument. Fluorescence measurements were recorded using FluoroMax-4 spectro-fluorometer with 1.5 nm slit for both excitation and emission. Absorption spectra were measured with a PerkinElmer Lambda 35 spectrophotometer. All spectroscopic measurements were performed at room temperature (25 °C). The confocal fluorescence imaging was performed on a Zeiss LSM 710 fluorescence confocal laser scanning microscope. Preparation of Monofunctional Pt(II) Complex trans-Pt-BA and Pt-Aniline. Synthesis of Ligand trans-BA. BODIPY derivative 1 was prepared according to the literature procedure.10 Then compound 1 (1 mmol, 0.382 g) and 4-aminobenzaldehyde (1.2 mmol, 0.145 g) were dissolved in toluene (15 mL) containing piperidine (1.0 mL) and acetic acid (1.2 mL) under nitrogen atmosphere at room temperature. Then the reaction mixture was stirred for 12 h at 120 °C. After the removal of solvent in vacuo, the residual was purified via column chromatography (silica gel, CH2Cl2), and the desired product trans-BA was obtained as dark blue solid (0.130 g). Yield, 27%. 1H NMR (500 MHz, CDCl3, ppm): δ 8.18 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 16.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 4H), 7.19 (d, J = 16.0 Hz, 1H), 6.67 (d, J = 8.5 Hz, 2H), 6.59 (s, 1H), 5.98 (s, 1H), 3.97 (s, 3H), 2.59 (s, 3H), 1.40 (s, 3H), 1.36 (s, 3H). 13C NMR (126 MHz, CDCl3, ppm): δ 14.42, 14.64, 14.80, 52.35, 115.01, 115.18, 117.90, 120.94, 126.97, 128.58, 128.80, 129.40, 130.25, 130.73, 132.52, 137.70, 137.84, 140.22, 141.07, 142.48, 147.86, 154.05, 154.78, 166.53. HR-MS (positive mode, m/z): Calcd 486.2169, found 486.2157 for [M + H]+; calcd. 508.1989 for [M + Na]+, found 508.1986 for [M + Na]+. Elemental analysis found (calcd) for C28H26BF2N3O2 (%): C, 69.38 (69.29); H, 5.26 (5.40); N, 8.80 (8.66). Synthesis of Complex trans-Pt-BA. Cisplatin (300 mg, 1 mmol) and AgNO3 (170 mg, 1 mmol) were stirred in anhydrous dimethylformamide (DMF; 3 mL) for 24 h at room temperature. After the removal of AgCl precipitate by filtration, the filtrate was added dropwise with BA (388 mg, 0.8 mmol) solution in DMF (20 mL), and the resulting mixture was stirred at room temperature for 48 h. Then DMF was removed in vacuo, and the residue was washed with methanol, acetone, and ether three times in sequence. The desired product trans-Pt-BA (0.130 g) was obtained as dark purple solid in a yield of 27%. 1H NMR (500 MHz, CD3OD, ppm): δ 8.13 (d, J = 8.0 Hz, 2H), 7.49−7.53 (m, 3H), 7.44 (d, J = 7.0 Hz, 2H), 7.28 (d, J = 15 Hz, 1H), 7.26 (d, J = 8 Hz, 2H), 6.70 (s, 1H), 6.04 (s, 1H), 3.87 (s, 3H), 2.45 (s, 3H), 1.37 (s, 3H), 1.33 (s, 3H). 195Pt NMR (86 MHz, deuterated dimethyl sulfoxide (DMSO-d6), ppm): δ −2299.65. HRMS (positive mode, m/z): calcd. 749.1978, found 749.1959 for [MNO3]+. Elemental analysis found (calcd) for C28H32BClF2N6O5Pt(%): C: 41.25(41.42); H: 4.01(3.97); N: 10.52(10.35). Synthesis of Complex Pt-Aniline. Cisplatin (150 mg, 0.5 mmol) and AgNO3 (80 mg, 0.5 mmol) were stirred in anhydrous DMF (3 mL) for 24 h at room temperature in dark. After the removal of AgCl precipitate by filtration, the filtrate was added dropwise with aniline (38 mg, 0.4 mmol) solution in DMF (6 mL), and the resulting mixture was stirred at room temperature for 48 h. Then DMF was removed in vacuo, and the residue was washed with CH2Cl2, acetone, and ether three times in sequence. The desired product Pt-Aniline (0.090 g) was obtained as white solid in a yield of 22%. 1H NMR (400 MHz, DMSO-d6, ppm): δ 7.41 (broad), 7.33−7.14 (m, 5H), 4.31−4.29 (broad), 4.00 (broad). 195Pt NMR (86 MHz, DMSO-d6, ppm): δ −2336.59. HR-MS (positive mode, m/z): calcd. 357.0446, found 357.0438 for [M-NO3]+. Elemental analysis found (calcd) for C 6 H 13 ClN 4 O3 Pt (%): C: 16.96 (17.17); H: 3.34(3.12); N: 13.46(13.35). Tracking the Photoinduced ROS Formation in Solution. The photoinduced ROS formation induced by BA or Pt-BA was tracked by recording the fluorescence (λem, 525 nm, λex, 488 nm) of fluorescent 2′,7′-dichlorodihydrofluorescein (2,7-DCF), which is formed in situ from nonfluorescent 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) via ROS oxidation.11 Therefore, the sample solutions

Scheme 1. Synthesis of trans-BA and trans-Pt-BA and Chemical Structure of Pt-Anilinea

a

(i) 4-Aminobenzaldehyde, acetic acid/piperidine, toluene, reflux 12 h; (ii) cis-[Pt(NH3)2(DMF)Cl]NO3, DMF, room temperature, 48 h.

4-bora-3a,4a-diaza-s-indacene (BA) as the third ligand, which is a very weak photosensitizer of low photo-ROS production ability. Since the heavy atom effect of PtII center is able to promote the photoinduced ROS production ability of photosensitizers such as chlorin via metal coordination,7 it was expected that the PtII center in Pt-BA is also able to enhance the ROS production ability of BA to provide Pt-BA the mild photosensitizing ability to damage in situ the cell membrane upon irradiation, and finally improves the cellular accumulation of monofunctional PtII complex. This synergic promotion effect between Pt center and BA was expected to sensitize the antitumor activity of monofunctional PtII complex via a short time photo-irradiation, which may provide a new photoactivation strategy for Pt-based prodrug.8 The high extinction coefficient of BA profiting from the internal charge transfer (ICT) effect from the amino group to pyrrole N atom favors also the photoactivation process.9 In this design, the photoactivation is just a method to promote cell uptake; therefore, the typical photosensitizer was not adopted as the third ligand to avoid the normal photodynamic therapeutic effect. In addition, Pt-Aniline, a triamminochloroplatinate analogue of Pt-BA, was also prepared as a control of monofunctional PtII complex without the photosensitizer.

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EXPERIMENTAL SECTION

Materials and Instruments. Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. The 1H NMR and 13C NMR spectra were recorded on Bruker DRX-500 with tetramethylsilane (Si(CH3)4) as internal standard. The 195Pt NMR spectra were recorded on Bruker DRX-400 with K2PtCl4 as internal standard. Mass spectrometric data 3755

DOI: 10.1021/acs.inorgchem.6b02148 Inorg. Chem. 2017, 56, 3754−3762

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Inorganic Chemistry

Figure 1. Absorption (a) and emission (b) spectra of Pt-BA (10 μM) and BA (10 μM) in HEPES buffer (50 mM, 100 mM KNO3, pH7.40) containing 10% DMSO (v/v), λex (BA): 592 nm, λex (Pt-BA): 566 nm. (inset, a) Photograph of BA and Pt-BA solutions. (inset, b) Photograph of BA and Pt-BA solutions upon UV irradiation at 365 nm. of DCFH-DA (10 μM) in HEPES buffer (50 mM, 10% DMSO, v/v, pH7.40) containing Pt-BA (10 μM) or BA (10 μM) were irradiated with the laser beams of 532 (for Pt-BA) or 635 nm (for BA) at a light power density of 3.5 mW·cm−2, and the fluorescence spectra of the sample solution were determined with a FluoroMax-4 spectrometer upon excitation at 488 nm after each irradiation, which lasted for 1 min. On the one hand, Pt-Aniline as the analogue of Pt-BA was also determined as a control for its photoinduced ROS production ability. On the other hand, the photoinduced singlet oxygen quantum yield of Pt-BA was also determined by measuring the absorbance (413 nm) of singlet oxygen scavenger 1,3-diphenyl isobenzofuran (DPBF), which has been mixed with Pt-BA, and Rose Bengal was utilized as a reference. The quantum yield was calculated with a standard equation.12 Therefore, the absorption spectra of DPBF (20 μM) in HEPES buffer (50 mM, containing 10% DMSO, pH = 7.40) in the presence of Pt-BA (10 μM) were determined after each irradiation (532 nm, 3.5 mW·cm−2). Each irradiation lasted for 5 s. Pt-Aniline (10 μM) was also determined as a control for its singlet oxygen quantum yield. Confocal Fluorescence Imaging of MCF-7 Cells. MCF-7 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5% CO2 and 95% air at 37 °C. Before the imaging, the phosphate-buffered saline (PBS)-rinsed cells were incubated with RPMI-1640 containing BA (5 μM) or Pt-BA (5 μM) at 37 °C in dark for different incubation time (0−5 h). For the BA-incubated cells, the fluorescence images were collected with a band path of 660−750 nm upon excitation at 633 nm. For Pt-BA-incubated cells, a band path of 560−600 nm upon excitation at 543 nm was adopted. To image the photopromoted cell uptake of Pt-BA, the PtBA-incubated (5 μM, 0−5 h) MCF-7 cells were exposed to a short time photo-irradiation (5 min, 532 nm, 3.5 mW·cm−2) at second hour of Pt-BA dark incubation. All the images were collected with a fluorescence confocal laser scanning microscope Zeiss LSM710. Intracellular Pt Content Determination in MCF-7 Cells. MCF-7 cells were seeded in 35 mm dishes for 24 h and then incubated in dark, respectively, with Pt-BA (5 μM), Pt-Aniline (5 μM), and cisplatin (5 μM) for 24 h. A short time photo-irradiation (5 min) at 532 nm with a light power density of 3.5 mW·cm−2 was given at second hour of incubation, and the incubation without irradiation was also performed as the control. After the removal of the culture media and rinse with 1 mL of PBS buffer (1X), the cells were treated with 500 μL of 0.25% trypsin and centrifuged at 1000 rpm. The supernatant was then removed, and the cells were rinsed with PBS twice. After being counted by a cell counter, the cells were treated with concentrated nitric acid (65%, 50 μL) at 95 °C for 2 h, hydrogen peroxide (30%, 20 μL) at 95 °C for 1.5 h, and concentrated hydrochloric acid (37%, 20 μL) at 37 °C for 0.5 h in sequence. The resulting solution was diluted to 1 mL with water from Milli-Q system (>18 MΩ), and Pt content was determined directly by the inductively coupled plasma mass spectrometer (ICP-MS; VG Elemental). The experiment was performed in triplicate, and the average of the data was obtained. The Pt content in MCF-7 cells induced by Pt-BA incubation was also determined with adding the ROS scavenger ascorbic acid (2 mM) 10

min before the short time photo-irradiation.13 The possible effect of the minor impurities induced by short time irradiation of Pt-BA was also determined via measuring the intracellular Pt content for the cells incubated with the Pt-BA pre-irradiated (532 nm, 3.5 mW·cm−2, 5 min) before incubation, and the final apparent concentration of the pre-irradiated Pt-BA in the culture medium is 5 μM. Antitumor Cytotoxicity Determination. MCF-7, HeLa, SGC-7901, and A549 cells were cultured, respectively, in RPMI-1640 or DMEM media, which was supplemented with 10% FBS (heat-inactivated) and 100 U·mL−1 penicillin, in 5% CO2 atmosphere at 37 °C. The cells were seeded in the 24-well plates with 100 000 cells per well. Solutions of Pt-BA, BA, or Pt-Aniline (0, 2, 4, 6, 8, 10 μM) were added into the wells and incubated for 24 h in dark at 37 °C with or without the photo-irradiation (5 min, 3.5 mW·cm−2; 532 nm for Pt-BA and PtAniline, 635 nm for BA) at second hour of incubation; the final concentration of DMSO is 2% (v/v). The PBS buffer (1X) containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 200 μL, 5 mg·mL−1) was then added into each well and incubated further for 4 h.14 Then the medium was discarded, DMSO (200 μL) was added to dissolve the purple formazan, and the absorbance at 570 nm of each well was recorded using a Varioskan Flash microplate reader (Thermo Scientific). Cisplatin was also determined as a control. All experiments were performed in triplicate to give the mean cell viability. In addition, the cytotoxicity of Pt-BA against MCF-7 cells was also determined upon irradiation with adding 2 mM ascorbic acid 10 min before the irradiation. The possible effect of the minor impurities induced by short time irradiation of Pt-BA was also determined via measuring the antitumor cytotoxicity of the Pt-BA pre-irradiated (532 nm, 3.5 mW·cm−2, 5 min) against MCF-7 cells. Then the preirradiated Pt-BA was added into each well to the desired apparent PtBA concentration, and the cytotoxicity was determined in the same procedure. Flow Cytometric Annexin V-FITC Assay of MCF-7 Cells. The cell membrane damage induced by Pt-BA was studied by the flow cytometric Annexin V-FITC assay, which has been widely used to monitor the destruction of cell membrane integrity and early apoptosis.15 Therefore, the MCF-7 cells seeded in dishes (35 mm) were incubated with Pt-BA (5 μM), and the incubation time lasted from 2 to 6 h. A short time of photo-irradiation (532 nm, 5 min) with a light power density of 3.5 mW·cm−2 was performed at the second hour of incubation. After the incubation medium was removed at the desired incubation time, the adherent cells were treated with 500 μL of trypsin (0.25%) and followed by rinse with PBS twice. Then the cells were centrifuged at 1000 rpm, and the supernatant was removed. The resulting cells were resuspended in 100 μL of binding buffer containing Annexin V-FITC (5 μL, BD) at room temperature for 1 h. The cells were determined by a flow cytometer (BD LSRFortessa) after the medium was removed and they were rinsed with PBS. The data were analyzed with the program FlowJo equipped with the instrument. On the one hand, the Pt-BA incubated cells without the photo-irradiation were determined for comparison. On the other hand, the cisplatin or Pt-Aniline (5 μM) incubated cells with/without 3756

DOI: 10.1021/acs.inorgchem.6b02148 Inorg. Chem. 2017, 56, 3754−3762

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Inorganic Chemistry

Figure 2. Photoinduced emission intensity of DCFH-DA (10 μM) at 525 nm (λex, 488 nm) in HEPES buffer (50 mM, containing 10% DMSO, pH 7.40) in the presence of Pt-BA (a; 10 μM) or in the presence of BA (b; 10 μM) determined after each irradiation (1 min) at 635 nm (for BA) or 532 nm (for Pt-BA) at a density of 3.5 mW·cm−2. Pt-BA and BA without irrdaition were also determined for comparison. Blank, HEPES buffer (50 mM, 10% DMSO, pH 7.40) containing only DCFH-DA.

Figure 3. Temporal tracking of the intracellular fluorescence in MCF-7 cells incubated (0−5 h, in dark) with 5 μM BA (a) and 5 μM Pt-BA (b, c) via confocal fluorescence imaging. (a) Images of the BA-incubated cells. Incubation times were given in the images, band path: 660−750 nm, λex, 633 nm; (b) images of the Pt-BA-incubated cells. Incubation times were given in the images, band path: 560−600 nm, λex, 543 nm; (c) images of the PtBA incubated cells photoirradiated (5 min, 532 nm, 3.5 mW·cm−2) at 2 h of incubation. Band path: 560−600 nm, λex, 543 nm. Scale bars, 20 μm. The times shown in (c) were timed from 2nd hour of Pt-BA incubation. The total Pt-BA incubation time for cells in each image in (c) is 1 h longer than the indicated time in the image. irradiation were also determined. Moreover, the blank control was also investigated.

determined with the normal procedure using DCFH-DA as ROS probe.11 The probe fluorescence at 525 nm (λex, 488 nm) is almost negligible if Pt-BA or BA (10 μM) in solution is not photo-irradiated, suggesting both Pt-BA and BA in dark cannot generate ROS. Upon the irradiation with laser at a light power density of 3.5 mW·cm−2 (532 nm for Pt-BA, ε532 = 4.02 × 104 M−1 cm−1, 635 nm for BA, and ε635 = 4.02 × 104 M−1 cm−1), the probe fluorescence increases almost linearly with the irradiation time, and the increase rate in the presence of Pt-BA is much higher than that for BA (Figure 2). The probe fluorescence intensity in the case of Pt-BA after 6 min of irradiation is almost fourfold higher than that for BA after 20 min of irradiation. Therefore, Pt-BA is a much more effective photoinduced ROS producer than its ligand BA considering the similar extinction coefficient of Pt-BA at 532 nm to that of BA at 635 nm, and Pt-BA itself can function as a mild photosensitizer due to the heavy atom effect of PtII center promoting the photoinduced ROS production ability of BA. The much higher photoinduced ROS production ability upon short time irradiation (5 min in Figure 2) confirmed Pt-BA possesses the higher photosensitizing ability than BA, which is a very weak photosensitizer. By measuring the DPBF absorbance

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RESULTS AND DISCUSSION Absorption and Fluorescence Spectra of Pt-BA and BA. The spectroscopic study of Pt-BA (10 μM) and BA (10 μM) in HEPES buffer (50 mM, 100 mM KNO3, pH 7.4) containing 10% DMSO (v/v) indicated that BA exhibits a distinct ICT absorption band centered at 592 nm (ε = 1.24 × 105 M−1·cm−1), while the absorption band of Pt-BA is shifted to 567 nm (ε = 1.37 × 105 M−1·cm−1, Figure 1a). BA shows an emission band centered at 720 nm upon excitation at its excitation maximum 592 nm (Figure 1b). This near-infrared emission originates from the extended ICT effect favored by α(4-aminostyryl) group.9a,16 Pt-BA shows a main emission band centered at 575 nm accompanied by a shoulder band at 625 nm upon excitation at 566 nm. The PtII-induced hypsochromic shift in both absorption and emission was also showed as the distinct color change in solutions, which suggests the ICT alteration in ligand BA upon PtII coordination. Photoinduced ROS Production of Pt-BA and BA. The photoinduced ROS production ability of Pt-BA and BA was 3757

DOI: 10.1021/acs.inorgchem.6b02148 Inorg. Chem. 2017, 56, 3754−3762

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Inorganic Chemistry with Rose Bengal as the reference, the photoinduced singlet oxygen quantum yield (ΦΔ) of Pt-BA was determined as 0.13 (Figure S12), which is distinctly less than that of the typical PDT agent hematoporphyrin (ΦΔ = 0.65).17 Therefore, the photoinduced ROS production ability of Pt-BA is mild and distinctly lower than normal PDT agent. This is consistent with that Pt-BA was expected to function mainly as a chemotherapeutic agent, and photo-irradiation is just as the activation method to promote cell uptake. For the control compound PtAniline, both the photoinduced singlet oxygen determination via measuring the DPBF absorption and the ROS determination via measuring the DCFH-DA emission indicate PtAniline possesses no photoinduced ROS production ability due to the scarcity of any photosensitizer in its structure (Figure S13). Confocal Fluorescence Imaging of MCF-7 Cells Incubated with Pt-BA. With the fluorescence of Pt-BA, the cell uptake process of Pt-BA with or without photo-irradiation was monitored in MCF-7 cells by confocal imaging, and BA was determined as the control. Then the cells were incubated respectively with Pt-BA and BA (5 μM) in dark and imaged every 0.5 h. Upon BA incubation without any photo-irradiation, the cells display clear but weak fluorescence in cytoplasm after 0.5 h of incubation, and the gradually enhanced fluorescence in cytoplasm can be observed in the following incubation (Figure 3a). The cells are in good shape even after 4 h of incubation. All these indicate that BA has sufficient cell membrane permeability and that its intracellular accumulation increases consistently. Upon Pt-BA incubation (5 μM), the cell image shows only the weak peripheral fluorescence even after 2 h of incubation, showing the cell membrane preferring affinity of PtBA. There is only very weak fluorescence in the cytoplasm even after 4 h of incubation, and the cells are still in good shape (Figure 3b). Considering the quick cell uptake of BA, the delayed cytosolic fluorescence enhancement upon Pt-BA incubation implies the monofunctional PtII center might be the origin for the delayed cell uptake. As the imaging result indicates Pt-BA takes 1−2 h to localize on the cell membrane, the photoactivation in the following study was all performed at second hour of Pt-BA incubation. Figure 3c shows that a short time photo-irradiation (532 nm, 3.5 mW·cm−2, 5 min) at second hour of Pt-BA incubation leads to the bright cytosolic fluorescence 25 min after the irradiation (Figure 3c), and the fluorescence can be observed even in nucleus ∼1 h after irradiation. On the one hand, the quick enhancement of intracellular fluorescence implies the cell membrane permeability of Pt-BA can be effectively improved via photoirradiation, although the irradiation time is only 5 min. In addition, the clear apoptotic bodies were also observed in the latter incubation. On the other hand, the cells incubated with only PBS buffer showed no distinct difference in cell morphology upon such a short time irradiation. It seems that such a short time irradiation is helpful to avoid the distinct PDT effect, and the photo-irradiation in the latter study for PtBA was all given in the same condition. Intracellular Pt Accumulation in MCF-7 Cells upon PtBA Incubation. The promoted cell accumulation of Pt-BA via photo-irradiation was confirmed further by the ICP-MS determination of Pt content in MCF-7 cells. The Pt-BAincubated (24 h) cells with or without 5 min of photoirradiation (532 nm) at second hour of dark incubation were digested to form sample solution. As can be seen from Figure 4, the Pt content in Pt-BA-incubated cells without any photo-

Figure 4. Pt content in 1 × 105 cells determined by ICP-MS measurement for the digestion solutions of MCF-7 cells incubated, respectively, with Pt-BA (5 μM), Pt-Aniline (5 μM), and cisplatin (5 μM) with or without photo-irradiation (5 min, 532 nm, 3.5 mW·cm−2) at 2nd hour of dark incubation. The incubated cells were digested for ICP-MS determination using a standard procedure after 24 h of incubation. Blank, Pt contents for the cells without any PtII complex incubation.

irradiation was determined as ∼12.3 ppb per 1 × 105 cells, while the Pt content in cells with 5 min of photo-irradiation was enhanced to ∼37.0 ppb per 1 × 105 cells, showing an approximately threefold enhancement factor. In addition, the PtII content in cells without Pt-BA incubation is negligible. The distinct photoinduced enhancement of PtII content in the PtBA-incubated cells implies that the short time of photoirradiation is really an effective method to promote the cell accumulation of this monofunctional PtII complex. As the analogue of Pt-BA without the photosensitizer, Pt-Aniline, shows that Pt-Aniline incubation leads to the low intracellular Pt content (∼13.3 ppb per 1 × 105 cells upon irradiation, 12.1 ppb per 1 × 105 cells without irradiation), no matter whether the complex is irradiated. This result showed that introduction of a photosensitizer is essential for the photoactivated cell uptake of Pt-BA. Without photo-irradiation, Pt-BA and PtAniline show the comparable low level of intracellular Pt content (∼12 ppb per 1 × 105 cells), while the intracellular Pt content induced by cisplatin incubation is ∼64 ppb/1 × 105 cells. This distinct difference might be ascribed to the positive charge in Pt-BA and Pt-Aniline delaying the cell uptake process. Similar to Pt-Aniline, there is no photosensitizer in cisplatin; the Pt content in the cisplatin-incubated MCF-7 cells is stabilized at higher level, no matter whether there is the photo-irradiation at second hour of incubation. Compared with the photo-independent cell uptake of cisplatin, the distinct photoinduced improvement in cell uptake of Pt-BA suggests also the introduction of a photosensitizer is essential. In addition, the adding of ROS scavenger ascorbic acid (2 mM) before the photo-irradiation leads to an intracellular Pt content of 18.5 ppb per 1 × 105 cells (Figure S14a), which is only slightly higher than that without photo-irradiation. This drop of the photoinduced enhancement of cell uptake suggests the photoinduced cell uptake enhancement is ROS-dependent, implying again the essential role of photosensitizer in Pt-BA. Cytotoxicity of Pt-BA against Tumor Cell Lines. With the confirmed enhancement of cell uptake upon photoirradiation, Pt-BA was further investigated with MTT assay (24 h) to determine its antitumor cytotoxicity. As shown in Figure 5a, the cell viability of MCF-7 cells is higher than 85% upon Pt-BA incubation, even when the incubation concentration reaches 10 μM. This indicates that Pt-BA possesses almost no cytotoxicity against MCF-7 cells in dark. When a short time photo-irradiation (532 nm, 3.5 mW·cm−2, 5 min) 3758

DOI: 10.1021/acs.inorgchem.6b02148 Inorg. Chem. 2017, 56, 3754−3762

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Inorganic Chemistry

(Figure S14b), which confirms that the photoinduced ROS should play a role in activating the antitumor activity of Pt-BA. For other tested tumor cell lines, SGC-7901, A549, and HeLa cells, Pt-BA displays almost no cytotoxicity upon dark incubation. On the one hand, the cytotoxicity was promoted distinctly when a short time photo-irradiation was given at second hour of incubation (Figure 5b−d), and the IC50 values against the three cell lines were ∼6.2, 9.7, and 7.4 μM, respectively (Table 1). On the other hand, the short time photo-irradiation itself does not induce any distinct any cytotoxicity against all four tested cell lines (Figure S15). According to the determined IC50 values (Table 1, 24 h), it is noted that the highest photoinduced cytotoxicity enhancement of Pt-BA was observed for MCF-7 cells among all the four tested cell lines. Compared with cisplatin, Pt-BA shows that its photoactivated cytotoxicities against MCF-7, SGC-7901, and A549 are higher than the cytotoxicity of cisplatin, except for the HeLa cells. For ligand BA, it shows almost no cytotoxicity against all the four tested cell lines, and the short time photo-irradiation at second hour of incubation does not induce any obvious difference. In addition, the analogue of Pt-BA without the photosensitizer Pt-Aniline shows also almost no cytotoxicity against all four tested cell lines even upon the short time photoirradiation, suggesting the photosensitizer BA is essential for the short time photoactivated antitumor cytotoxicity of Pt-BA; it is the integrated BODIPY-derived ligand that led to the photoactivated enhancement in the cytotoxicity of Pt-BA. Photoinduced Cell Membrane Damage in MCF-7 Cells. The photoinduced cell membrane damage of MCF-7 cells incubated with Pt-BA was studied effectively by the flow cytometric Annexin V-FITC assay, which is a typical method to monitor the destruction of cell membrane integrity in early apoptosis,18 since the emission band of Pt-BA is distinctly different from that of FITC. Without the photo-irradiation, the FITC fluorescence of the Pt-BA-incubated cells is stabilized at a low level during the incubation (Figure 6b,c). However, the photo-irradiation (5 min) at second hour of Pt-BA incubation results in the significant enhancement of FITC fluorescence. The FITC fluorescence temporal profile shows the quick enhancement within 1 h after the irradiation, followed by a short plateau (1−2 h after irradiation) and the subsequent quick enhancement (2−4 h after irradiation). This profile suggests that photoinduced ROS produced by Pt-BA would damage the cell membrane mainly within 1 h after irradiation. The followed plateau indicates there is no more membrane damage in this period, in which Pt-BA enters the cell smoothly. The fluorescence enhancement in the final 2 h might originate from the additional membrane damage induced by the internalized Pt-BA. Compared with the profile without the

Figure 5. Cytotoxicity of Pt-BA against MCF-7 (a), SGC-7901 (b), A549 (c), and HeLa (d) cell lines determined after 24 h of dark incubation with (red bar) or without (gray bar) the photo-irradiation (5 min, 3.5 mW·cm−2) at 2nd hour of incubation. BA, Pt-Aniline, and cisplatin were also determined as the controls.

was given at second hour of incubation, the distinctly enhanced cytotoxicity can be observed, and the cell viability is ∼10% at 10 μM of Pt-BA, while the IC50 value was determined as ∼3.8 μM (Table 1). Although confocal imaging has confirmed the sufficient cell accumulation of BA, BA displays almost no cytotoxicity against MCF-7 cells even with the photoirradiation (635 nm, 3.5 mW·cm−2, 5 min) at second hour of incubation. As a control, cisplatin incubation demonstrates the decreased cell viability upon increasing cisplatin concentration, and the same photo-irradiation (532 nm, 3.5 mW·cm−2, 5 min) does not induce any distinct difference in its cytotoxicity. Moreover, the cell viability of MCF-7 cells upon Pt-BA incubation with the irradiation is much lower than that of cisplatin at the same concentration, yet that induced by Pt-BA without irradiation is much higher than that of cisplatin. All these suggested the antitumor cytotoxicity of Pt-BA against MCF-7 cells was effectively promoted via the short time photoirradiation. In addition, the addition of ROS scavenger ascorbic acid (2 mM) before the irradiation makes the cytotoxicity enhancement induced by photo-irradiation almost disappear

Table 1. IC50 Values of Pt-BA, BA, Pt-Aniline, and Cisplatin against MCF-7, SGC-7901, A549 and HeLa Cell Lines after 24 h of Dark Incubation IC50 against MCF-7 (μM) irradiated Pt-BA BA Pt-Aniline cisplatin

a

3.8 ± 0.2 >50 >40 10.9 ± 1.2

IC50 against SGC-7901 (μM) a

non-irradiated

irradiated

>50 >50 >40 11.2 ± 0.5

6.2 ± 0.1 >50 >40 13.2 ± 2.2

IC50 against A549 (μM) a

non-irradiated

irradiated

>50 >50 >40 11.5 ± 1.8

9.7 ± 0.2 >50 >40 10.1 ± 0.6

IC50 against HeLa (μM)

non-irradiated

irradiateda

non-irradiated

>50 >50 >40 13.3 ± 2.7

7.4 ± 0.3 >50 >40 2.1 ± 3.3

>50 >50 >40 3.2 ± 1.6

Irradiation was given at 2nd hour of dark incubation and lasted for 5 min, and the power density is 3.5 mW cm−2. The irradiation wavelength for PtBA, Pt-Aniline, and cisplatin is 532 nm, and that for BA is 635 nm. a

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DOI: 10.1021/acs.inorgchem.6b02148 Inorg. Chem. 2017, 56, 3754−3762

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Inorganic Chemistry

even with the minor impurities induced by short time irradiation cannot lead to the enhanced cell uptake of Pt-BA. Therefore, the promoted cell uptake of Pt-BA upon short time irradiation at second hour of the dark incubation shown in Figure 4 cannot be induced by the minor impurites. On the one hand, just as shown by the flow cytometric assay, it was the photo-irradiation of the photosensitizer in Pt-BA that led to the cell membrane damage, which finally results in the promoted cell uptake of Pt-BA. On the other hand, the pre-irradiated PtBA before incubation was also investigated for its antotumor cytotoxicity agianst MCF-7 cells without the short time photoirradiation at second hour of dark incubation (Figure S21). Even with the minor impurity induced by the pre-irradiation, Pt-BA displays the poor antitumor acivity without the short time irradiation at second hour of dark incubation, which is comparable to that induced by Pt-BA without pre-irradiation before incubation. All these indictate that the minor impurites in Pt-BA induced by short time photo-irradiation are not involved in the photoactivated cell uptake and antitunor activity of Pt-BA.

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CONCLUSIONS

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ASSOCIATED CONTENT

In short, a novel monofunctional PtII complex Pt-BA was constructed by combining a BODIPY-derived fluorophore with a monofunctional PtII center, to sensitize the antitumor activity of monofunctional PtII complexes via improving their cell accumulation. The positively charged PtII center not only provides the cell membrane anchoring ability but also makes the complex a mild photosensitizer via the heavy atom effect, favoring the in situ cell membrane damage via photo-irradiation and the subsequent promoted cell uptake of PtII complex. This study implies a new approach to construct monofunctional PtII complexes via integrating a weak photosensitizer with the monofunctional PtII center. Although many issues such as whether the intracellular monofunctional PtII binds DNA to induce apoptosis are still not clear, this new strategy for monofunctional PtII complex implies also the possibility of new chemotherapy that can be activated by a short time photoirradiation, and the high spatial/temporal adjustability of photoirradiation provides the perspectives in the future for targeted activation and precise chemotherapy.8,19

Figure 6. Flow cytometric Annexin V-FITC assay for MCF-7 cells incubated with Pt-BA (5 μM). The FITC fluorescence determined at different incubation time with (a) or without (b) the photo-irradiation (532 nm, 3.5 mW·cm−2, 5 min) at 2nd h of Pt-BA dark incubation. (c) The related temporal FITC fluorescence profile determined in (a, b). The times shown here were timed from 2nd hour of Pt-BA incubation.

irradiation, this fluorescence profile of Pt-BA obtained upon irradiation indicates that the photo-irradiation is essential for the membrane damage. The longer incubation time (12 and 24 h) with 1 or 5 μM Pt-BA discloses also the distinct irradiationinduced cell membrane damage (Figure S16). As to the analogue of Pt-BA without the photosensitizer, Pt-Aniline stabilizes the FITC fluorescence at a low level similar to the blank control during the incubation process no matter whether there is the short time photo-irradiation at second hour of PtAniline incubation (Figure S17b). Blank control with PBS incubation discloses the similar results (Figure S17a). All these indicate that the short time photo-irradiation itself does not induce any membrane disruption, and it is the photosensitizer in Pt-BA that was associated with photoinduced cell membrane damage of Pt-BA. Stability of Pt-BA upon Short Time Irradiation. On the one hand, electrospray ionization mass spectroscopic (ESI-MS) study found that Pt-BA in HEPES buffer after 5 min of photoirradiation (532 nm, 3.5 mW·cm−2) displays a similar ESI mass spectrum to that of Pt-BA before the irradiation, besides the more noise signals. The main m/z signals 749.42 and 1497.92 can be assigned, respectviely, as [M-NO3]+ and [2M-2NO3− H]+ (Figure S19). On the other hand, HPLC analysis found the purity of Pt-BA after 5 min of photo-irradiation is ∼92%, and the main impurity is BA (∼6%). All these indicated that most Pt-BA still retains its structure after 5 min of photo-irradiation at 532 nm with a power density of 3.5 mW·cm−2. To determine the effect of the minor impurities in Pt-BA induced by the short time irradiation, the pre-irradiated Pt-BA before incubation was utilized to incubate the MCF-7 cells in dark with the apparent Pt-BA concentration of 5 μM, and the related intracelluar Pt content is exactly comparable to that induced by the dark incubation with Pt-BA, which has not been pre-irradiated before incubation (Figure S20). This result implies that Pt-BA

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02148. Details of the compounds characterization, NMR spectra, HR mass spectra, fluorescence excitation spectra, photoinduced ROS determination, control experiment data for cell uptake and cytotoxicity, flow cytometric data, stability data, cell uptake and cytotoxicity data of the pre-irradiated Pt-BA before incubation (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (W.H.) *E-mail: [email protected]. (Z.G.) ORCID

Weijiang He: 0000-0002-3157-5769 3760

DOI: 10.1021/acs.inorgchem.6b02148 Inorg. Chem. 2017, 56, 3754−3762

Article

Inorganic Chemistry Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Basic Research Program of China (No. 2015CB856300), National Natural Science Foundation of China (Nos. 21271100 and 21571099), and Natural Science Foundation of Jiangsu Province (No. BK20150054) for financial support. This work was also supported by the NSFC-ISF Research Program, jointly funded by the National Natural Science Foundation of China and the Israel Science Foundation (21361140352).

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