Luminescent Ruthenium Complexes for Theranostic Applications

Article pubs.acs.org/jmc

Luminescent Ruthenium Complexes for Theranostic Applications Carolina R. Cardoso,† Márcia V. S. Lima,† Juliana Cheleski,‡ Erica J. Peterson,§ Tiago Venâncio,† Nicholas P. Farrell,*,§,∥ and Rose M. Carlos*,† †

Departamento de Química, Universidade Federal de São Carlos, São Carlos, São Paulo CP 676, 13565-905, Brazil Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, São Paulo 13560-970, Brazil § Goodwin Laboratory, Massey Cancer Center, Virginia Commonwealth University, 401 College Street, Richmond, Virginia 23298, United States ∥ Department of Chemistry, Virginia Commonwealth University, 1001 W. Main Street, Richmond, Virginia 23284-2006, United States ‡

S Supporting Information *

ABSTRACT: The water-soluble and visible luminescent complexes cis-[Ru(L-L)2(L)2]2+ where L-L = 2,2-bipyridine and 1,10phenanthroline and L= imidazole, 1-methylimidazole, and histamine have been synthesized and characterized by spectroscopic techniques. Spectroscopic (circular dichroism, saturation transfer difference NMR, and diffusion ordered spectroscopy NMR) and isothermal titration calorimetry studies indicate binding of cis-[Ru(phen)2(ImH)2]2+ and human serum albumin occurs via noncovalent interactions with Kb = 9.8 × 104 mol−1 L, ΔH = −11.5 ± 0.1 kcal mol−1, and TΔS = −4.46 ± 0.3 kcal mol−1. High uptake of the complex into HCT116 cells was detected by luminescent confocal microscopy. Cytotoxicity of cis[Ru(phen)2(ImH)2]2+ against proliferation of HCT116p53+/+ and HCT116p53−/− shows IC50 values of 0.1 and 0.7 μmol L−1. Flow cytometry and western blot indicate RuphenImH mediates cell cycle arrest in the G1 phase in both cells and is more prominent in p53+/+. The complex activates proapoptotic PARP in p53−/−, but not in p53+/+. A cytostatic mechanism based on quantification of the number of cells during the time period of incubation is suggested.

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INTRODUCTION The antitumor properties of ruthenium complexes have attracted much attention primarily because some of them have shown favorable pharmacological profiles in vitro and in vivo in different models including platinum-resistant cell lines.1−11 The Ru(III) complexes NAMI-A, ([Him][transRuCl4(DMSO)(im)], im = imidazole), KP1019, ([Hind][trans-[RuCl4(ind)2], ind = indazole), and its Na+ analogue (KP1339) have been intensively investigated. NAMI-A and KP1339 are currently in clinical trials.12−14 The axial ligands play a fundamental role on the pharmacological properties of these complexes.15−20 For example, upon intravenous administration, both complexes bind to human serum albumin (HSA).21−26 KP1019 interacts with the hydrophobic domains of HSA in a noncovalent manner whereas NAMI-A binds strongly to HSA. The binding differences may play an important role in determining the pharmacologic properties and efficacy of these agents.27−31 Indeed, the looser association of KP1019 results in higher cellular absorption of KP1019 compared to NAMI-A.27−31 Moreover, KP1019 is active in © 2014 American Chemical Society

primary cancers and induces apoptosis in the colorectal tumor cell lines SW480 and HT29, being more active in these cells than the clinically used drugs 5-fluorouracil and cisplatin,32,33 whereas NAMI-A is antiangiogenic and shows antimetastatic activity in secondary tumors.34,35 Importantly, the cytotoxicity of cisplatin is mainly correlated to DNA binding while the biologic targets of NAMI-A and KP1019 have not yet been totally elucidated, as both compounds are able to target DNA and proteins, suggesting different, or multiple, pathways from those for cisplatin.36−38 Both complexes are in fact prodrugs and in biological medium are reduced to Ru(II) by reductant molecules such as glutathione, cysteine, and ascorbic acid.39−43 The many mechanistic studies support the “activation by reduction” hypothesis where by reduction of Ru(III) complex by redox biomolecules gives the Ru(II) active species that attacks the target cells.44 In accord, electrochemical experiments have Received: March 19, 2014 Published: May 15, 2014 4906

dx.doi.org/10.1021/jm5005946 | J. Med. Chem. 2014, 57, 4906−4915

Journal of Medicinal Chemistry

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shown that, in the hypoxic and acid medium environment of solid tumors, the Ru(III) complexes are reduced to Ru(II).45−47 In view of these properties, there is wide interest in Ru(II) complexes as antitumor agents with both Werner-type and organometallic compounds currently in development.48−51 Many Ru(II) compounds containing chelate ligands such as polypyridine ligands have been assayed and show interesting preclinical results.52,53 Moreover, the photophysical properties of polypyridine Ru(II) complexes open wide possibilities of using molecular systems in biological applications such as cellular accumulation, trafficking and biodistribution, and characterization of cancer cells.54,55 The key features are metal-to-ligand charge transfer (MLCT) absorption and emission of light at wavelengths in the visible to near-infrared region for depth penetration in the tissue, large Stokes shifts to minimize the incident light scattering, and long-lived emission that often increases the viability of the energy transfer process to a biologic target. In this respect, the luminescence of antitumor candidates based on Ru(II) polypyridine complexes has been used to determine the cellular localization and uptake of the complex.55 Small changes in the nature of the ligands and oxidation state of ruthenium can have a major effect on the properties of the complex tuning both the photophysical properties as well as the pharmacologic properties of the complex. These findings encouraged us to study the cytotoxicity and antitumor activity and diagnosis of a new Ru(II) series of general formula cis-[Ru(L-L)2(L)2]2+ where L-L = 2,2bipyridine (bpy) and 1,10-phenanthroline (phen) and L= imidazole (ImH), 1- methylimidazole (1MeIm), and histamine (Him) (Figure 1). Imidazole is an electron-rich ligand that behaves as a strong σ donor toward the Ru(II) atom. A

coordinated imidazole is a stronger hydrogen-bond donor than a free imidazole, as the pKa of coordinated imidazole exceeds that of free imidazole (∼7.0). A small environmental change can thus substantially change the hydrogen-bond donating or accepting properties of the imidazole ligand.56−58 Hydrogen bonds are essential in many biological and enzymatic processes and may play a role in defining anticancer activities.59,47 Furthermore, the rigid structure of the {Ru(phen)2}2+ moiety provides stability to the complex and π−π stacking interactions with aromatic residues found in the hydrophobic region of proteins. The {Ru(phen)2}2+ moiety also provides intense MLCT absorptions enabling design of a luminescent complex as a possible molecular biological probe in human cells, tissue, and organisms. This paper reports on the synthesis, characterization, and biological properties of cis-[Ru(L-L)2(L)2]2+ with special emphasis on the cis-[Ru(phen)2(ImH/Im-CH3)2]2+ pair (Figure 1). The binding interactions with HSA were investigated using isothermal titration calorimetry (ITC), circular dichroism (CD), saturation transfer difference (STD), and diffusion ordered spectroscopy (DOSY) techniques. The complexes were assayed in vitro for cell proliferation inhibitory activity in human colon and ovarian tumors. The cell uptake studies were monitored by fluorescent imaging of fixed HCT116 colon tumor cell lines with complexes. The cellular mechanism of action was investigated by assaying the proapoptotic p53, caspase-3, and PARP by western blot and flow cytometry. The results indicate an interesting profile of biological activity with weak binding to HSA, indications that both cytotoxic and cytostatic mechanisms contribute to proliferation inhibition while the lead compound cis-[Ru(phen)2(ImH)2]2+ maintains luminescence in vitro allowing cellular localization and distribution studies.

Figure 1. Structures of NAMI-A, KP1019, and the Ru(II) complexes studied in this work.

RESULTS AND DISCUSSION Synthesis and Characterization. The complexes were synthesized by reacting cis-[Ru(L-L)2Cl2]60 with 2 equiv of L, or 1 equiv of L-L in the case of histamine, in H2O/EtOH (1:1). All complexes were isolated as hexafluorophosphate salts, and their composition and structure were confirmed from CHN analysis and NMR spectroscopy. We prepared the complex as PF6 counterion for characterization purposes. The counterion was replaced by chloride to increases the solubility of complexes in aqueous solution. A sample of complex was dissolved in acetone, and an excess of tetrabutylammonium chloride (N(n-Bu)4Cl) was added. The precipitate formed immediately was filtered, rinsed with ether to remove the excess of N(n-Bu)4Cl, and dried. Preliminary cytotoxicity studies (see below) indicated the pair of Ru−phen complexes to be of most interest; therefore, detailed studies were carried out on these compounds. The geometries of the phenanthroline complexes were density functional theory (DFT) optimized using B3LYP/ LANL2DZ including solvent using the PCM model and acetonitrile. Selected bond lengths and angles for the optimized geometry in CH3CN are given in Table S1 (Supporting Information) and Figure S1 (Supporting Information). They reproduce the X-ray crystal structural data of related ruthenium α-diimine complexes quite closely. The planar configuration of the phen ligand and the dihedral angles between the plane of the phen and imidazole ligand provide hydrogen-bonding and π−π stacking interactions that may stabilize the complex with proteins, nucleic acids, and DNA.

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Figure 2. (A) UV−vis absorption spectrum of RuphenImH in aqueous solution in the visible region; insert: UV−vis in the region 200−600 nm. (B) (right) Absorption and (left) emission (normalized) (λexc = 490 nm) spectra.

Figure 3. (A) RuphenImH binding to HSA determined by ITC. The upper panel shows the raw data of binding and the bottom panel shows the integrated interaction heat. (B) CD spectra of HSA + RuphenImH at varying concentrations. 1

H NMR Spectroscopy. The 1H NMR spectra in D2O for the complexes RuphenImH and Ruphen1MeIm exhibit eight different signals for the asymmetric phenanthroline in the region 7.40−9.40 ppm and three signals for the imidazole ligand in the region 6.80−7.70 ppm, Figure S2 (Supporting Information). Complex RuphenImH exhibits signals at 7.70, 6.90, and 7.00 ppm corresponding to the protons Hγ,γ′, Hβ,β′, and Hα,α′ of the imidazole ring, respectively. The 1H NMR spectrum of complex Ruphen1MeIm exhibits three signals at 7.60, 6.90, and 6.85 ppm corresponding to the protons Hγ,γ′, Hβ,β′, and Hα,α′ of the imidazole ring and one signal at 3.45 ppm (Hδ,δ′) assigned to the methyl group. Electrochemistry. Figure S3 (Supporting Information) shows the cyclic voltammograms of the two RuIIphen complexes in DMF at a platinum electrode versus Fc+/Fc (100 mV s−1), which exhibit a quasireversible metal-based redox RuII/III couple at E1/2 = 1.19 V for RuphenImH and E1/2 = 1.15 V for Ruphen1MeIm. In accord, Figure S4 (Supporting Information) shows that the anodic peak current (ipa)

(Supporting Information), the numbers of electrons involved in the electrochemical reaction were found to be 1.3 and 0.92 for RuphenImH and Ruphen1MeIm, respectively. Absorption and Emission Spectrum. The UV−visible (UV−vis) absorption spectrum of complexes in aqueous solution are characterized by intense UV absorption bands mostly π−π* in origin and a broad and intense absorption with maximum at 490 nm (ε = 7550 mol−1 L cm−1) in the visible region, Figure 2A. The emission spectra of the complexes are similar in both spectral structure and position suggesting a 3MLCT emitting state, Figure 2B. For both complexes, the Stokes shift was large, ∼5500 cm−1, and the phosphorescence at 660 nm was fit as a single exponential decay with lifetimes of 239 and 211 ns for complexes Ruphen1MeIm and RuphenImH, respectively. The UV−vis and emissive characteristics of complexes are retained in an aqueous buffer solution (Tris/HCl, pH 7.4) in the absence and in the presence of HSA. Stability of Complexes. The complexes are stable in the dark both in the solid state and in aqueous solution. The 1H NMR spectra of complexes RuphenImH and Ruphen1MeIm in D2O at pD 7.8 (phosphate buffer) remain unchanged for at

1

increases with the square root of the scan rate (ν /2), but it is not quite linear. By using the Tafel plot,61 Figure S5 4908



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Figure 4. of f resonance and STD spectra of RuphenImH in the presence of HSA.

least three days, Figure S6A (Supporting Information). The chemical stability of the complex as a function of the pD of solution was also investigated by 1H NMR. D2O solutions of the complex at pD 2.6, 4.6, 7.5, 9.7, and 12.6 were prepared, and the spectrum was taken at appropriate time intervals. Figure S6B (Supporting Information) indicates that the complexes are quite stable toward hydrolysis and that the imidazole ligand remains coordinated to the metal center at pD values from 2.6 to 12.6. Only at pD 12.6, formation of the imidazolate species is observed where the extent of deprotonation is 15%. HSA−Complex Interaction. The interaction of RuphenImH and Ruphen1MeIm with HSA was studied by ITC, NMR-STD, NMR-DOSY, and CD. Figure 3A shows the calorimetric data for the titration of 523 μmol L−1 of the RuphenImH into 30 μmol L−1 of HSA at pH 7.4 buffer solution at 37 °C. The binding isotherms (heat change versus RuphenImH/HSA molar ratio) were obtained from the integration of raw data, and the best fit was obtained using a single binding site model, with Kb = 9.8 × 104 mol−1 L. The analysis of data indicates that the interaction between RuphenImH and HSA is enthalpy driven, exothermic, ΔH = −11.5 ± 0.1 kcalmol−1, and shows an unfavorable binding entropy, TΔS = −4.46 ± 0.3 kcal mol−1. The weak binding affinity for RuphenImH to HSA suggests the formation of noncovalent interactions, observed by other methods such as CD, STD-NMR, and DOSY-NMR analyses. Given that the ITC signal for the Ruphen1MeIm complex binding to the HSA shows no interaction, it is likely that hydrogen-bond and electrostatic interactions contribute to the RuphenImH−HSA binding reaction. The unfavorable entropy contribution indicates a less pronounced effect of release of water of hydration and counterions during complex binding (hydrophobic effect). The CD spectrum monitored in the region of HSA absorption, Figure 3B, also supports this conclusion. No changes in the secondary structure of HSA were observed for concentrations as high as 25 μM of complex, Figure 3B.

Figure 4 shows the STD-NMR spectrum obtained for the interaction of RuphenImH with HSA. The signals exhibited in the STD-NMR spectrum come from the interaction of the protons of the complex, which are interacting with protein sites less than 5 Å distant.62 The intensities of the signals increase as the distance reduces, and this behavior is related to the strength of the interaction, being possible to obtain an epitope map, as pointed in the structures of the complexes. The epitope map demonstrates that all the protons from the complex interact with the protein, with the imidazole protons exhibiting a greater interaction with HSA compared with those on phenanthrolines. Presumably, a hydrogen bond from the imidazole may interact with the protein allowing closer approach of the vicinal hydrogens. This hydrogen bond is itself difficult to observe because of the exchange with deuterium from solvent (D2O), where the signal vanishes as expected. The STD-NMR experiments therefore confirm the demonstration from the ITC experiments that the complex−protein interaction presents a polar character where the imidazole ligand exhibits a higher polarity when compared with phenanthroline ligand. In this context, DOSY-NMR experiments were also performed, and a small shift in the diffusion coefficient of the complex in the presence of protein (average D = 4.36 × 10−10 m2 s−1) was obtained when compared with free complex (D = 4.78 × 10−10 m2 s−1), and free protein D = 7.22 × 10−11 m2 s−1, Figure S7A, B (Supporting Information). It is interesting to note that the free complex exhibits the same D value for all the signals (D = 4.78 × 10−10 m2 s−1). However, when the protein is present, two different diffusion coefficients are observed. According to the STD-NMR data, the protons with weaker interaction had a diffusion coefficient of 4.57 × 10−10 m2 s−1, whereas those ones with a stronger interaction (H2, H2′, α, α′, and β, β′) are less mobile and thus exhibit slow diffusion (D = 4.16 × 10−10 m2 s−1). These results are in agreement with those obtained by STDNMR, which showed that the protons from imidazole (α, α′ and β, β′) and also the (H2 and H2′) from phenanthroline are 4909



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the ones that present the highest STD-effect (66.66%, 100.00%, and 51.11%). The studies on the interaction between the complex Ruphen1MeIm and HSA were also evaluated by STDNMR, but due to the weaker interaction, the signals of the STD experiment were too small to determine the percentages (Figure S8, Supporting Information). In summary, the results indicate that the weak HSA− RuphenImH interaction involves hydrogen bonding from the imidazole ligand and π−π interactions from phen to the HSA protein. Biological Studies. Cell Proliferation Assay. The inhibition of cell proliferation in ovarian tumor A2780 and the isogenic HCT116 p53+/+ and HCT116 p53−/− human colon cancer cell lines by the complexes was evaluated using the MTT assay, Table 1. For comparison purposes, cisplatin was also Table 1. IC50 Values for Ruthenium Complexes and Cisplatin in Cell Lines HCT116 p53+/+ and HCT116 p53−/− IC50 (μmol L−1) complex RuphenImH Ruphen1MeIm RubpyImH Rubpy1MeIm RuphenBiHim cisplatin

HCT116 (p53 0.72 4.00 1.02 6.52 1.65 9.15

± ± ± ± ± ±

+/+

0.18 0.76 0.22 0.72 0.87 1.18

)

HCT116 (p53−/−) 0.10 5.13 0.53 0.70 1.04 6.00

± ± ± ± ± ±

0.03 0.45 0.16 0.19 0.12 0.78

Figure 5. Cell cycle analysis of p53+/+ and p53−/− cells after 24 and 48 h of exposure to complex RuphenImH.

A2780 1.30 6.92 8.24 6.52 1.65 1.30

± ± ± ± ± ±

1.0 0.79 1.09 0.84 0.69 0.17

assayed under the same experimental conditions. The IC50 values for all complexes were comparable to that of cisplatin. Among the Ru(II) complexes, complex RuphenImH is the most interesting compound, and the higher activity in p53−/− than p53+/+ is of special interest. The greater activity of both RuphenImH and RubpyImH complexes compared to their Ruphen1MeIm and Rubpy1MeIm analogues confirms our proposal that the hydrogen bond on the imidazole ligand has an important impact on the cytotoxicity of these complexes. Given this initial survey and the responses of RuphenImH, we focused on the mechanism of action studies on this complex, described in the next section. Cell Cycle Arrest and Apoptosis Assays with Complex RuphenImH. Figure 5 shows the flow cytometry results for cell cycle analysis of HCT116 p53+/+ and HCT116 p53−/− cells treated with 20 μmol L−1 complex RuphenImH and incubated for 24 and 48 h. The complex induces a robust G1 arrest, as the majority of the cell population accumulates in the G1, or growth phase. This effect coincides with a reduction in the number of cells in the S phase, or synthesis phase, when the DNA is copied. The cell cycle arrest shows to be p53−/− dependent, as p53+/+ cells arrest upon treatment with the complex, while p53−/− cells do not. We investigated the effect of RuphenImH on the cleavage and activation of the proapoptotic proteins p53, PARP, and caspase 3. The samples were prepared from cells treated with or without RuphenImH (20 μmol L−1) and subject to western blot analysis. The blot was probed with β-actin to control for equal gel loading and cisplatin as a positive control. The cells were lysed after 48 and 72 h of treatment. Western blot analysis revealed that treatment of HCT116 with the complex leads to induction of p53 in HCT116 p53+/+ cells, Figure 6. p53 induction classically leads to cell cycle arrest and is consistent with the G1 arrest observed during cell cycle analysis. Caspase-3 and Parp-1 cleavage are not observed in

Figure 6. Western blot analysis for PARP, caspase 3, P53, and β-actin with incubation of RuphenImH complex and cisplatin for 48 and 70 h.

HCT116 p53+/+ cells, indicating the apoptosis may not be causative of the observed cell inhibitory effects. Interestingly, Parp-1 cleavage is observed in HCT116 p53−/− cells. This difference may be a result of the inability of p53−/− cells to elicit an arrest allowing for evaluation and repair, the resulting damage thereby inducing an apoptotic cascade. In light of these results, it is probable that the primary mechanism of action involves a cytostatic pathway. Indeed, as can be seen in Table 2, the number of viable HCT116 cells does not change significantly in both p53+/+ and p53−/− cells over time when compared to the control, confirming a cytostatic effect of RuphenImH. Table 2. Proliferation of HCT116 Cells with Incubation Time of RuphenImH (20 μmol L−1) time (h) 30 control RuphenImH control RuphenImH 4910

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HCT116 p53+/+ (104 cells/mL) 120 190 37 42 HCT116 p53−/− (104 cells/mL) 96 120 30 32

77 320 45 160 35



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Synthesis of cis-[Ru(phen)2(ImH)2](PF6)2 (RuphenImH) and cis-[Ru(phen)2(1MeIm)2](PF6)2 (Ruphen1MeIm). cis-RuCl2(phen)2 (0.187 mmol) was dissolved in a 1:1 EtOH/H2O mixture (10 mL), and an amount of ImH/CH3−Im (0.375 mmol) ligand was added. The solution was stirred under nitrogen atmosphere for 8 h under reflux. A stoichiometric amount of NH4PF6 was added to precipitate the complex. The dark red precipitate was filtered, recrystallized with a 1:1 EtOH/H2O mixture, and washed with water and ethanol under vacuum. RuphenImH, Yield: 79%. Anal. Calcd for C30H24F12N8P2Ru: C, 40.59; H, 2.72; N, 12.62. Found: C, 39.21; H, 2.88; N, 12.03. Ruphen1MeIm, Yield: 69%. Anal. Calcd for C32H28F12N8P2Ru: C, 41.97; H, 3.08; N, 12.23. Found: C, 44.02; H, 3.21; N, 11.81. Synthesis of cis-[Ru(bpy)2(ImH)2](PF6)2 (RubpyImH) and cis[Ru(bpy)2(1MeIm)2](PF6)2 (Rubpy1MeIm). cis-RuCl2(bpy)2 (0.206 mmol) was dissolved in a 1:1 EtOH/H2O mixture (10 mL), and an amount of ImH/CH3−Im (0.412 mmol) ligand was added. The solution was stirred under nitrogen atmosphere for 8 h under reflux. A stoichiometric amount of NH4PF6 was added to precipitate the complex. The dark red precipitate was filtered, recrystallized with a 1:1 EtOH/H2O mixture, and washed with water and ethanol under vacuum. RubpyImH, Yield: 70%. 1H NMR (D2O): δ 8.91 (H1,H1′ d), 8.24 (H8,H8′, d), 8.16 (H7,H7′, d), 7.95 (H4,H4′, d),7.90 (H5,H5′, d), 7.73 (H3,H3′, t), 7.54 (H6,H6′,Hγ,Hγ′, m), 7.14 (H2,H2′, t), 6.97 (Hβ,Hβ′, s), 6.74 (Hα,Hα′, s) ppm. Anal. Calcd for C26H24F12N8P2Ru: C, 37.10; H, 2.88; N, 13.34. Found: C, 36.32; H, 3.10; N, 13.21. Rubpy1MeIm, Yield: 65%. 1H NMR (D2O): δ 8.90 (H1, H1′, d), 8.26 (H8, H8′, d), 8.18 (H7, H7′, d), 7.95 (H4, H4′, t),7.87 (H5, H5′, d), 7.73 (H3, H3′, t), 7.56 (H6, H6′, t), 7.44 (Hγ, Hγ′, s), 7.13 (H2, H2′, t), 6.89 (Hβ, Hβ′, s), 6.69 (Hα, Hα′, s), 3.47 (Hδ, Hδ′, s) ppm. Anal. Calcd for C28H28F12N8P2Ru: C, 38.76; H, 3.25; N, 12.96. Found: C, 38.07; H, 3.41; N, 13.00. Synthesis of cis-[Ru(phen)2(BiHim)](PF6)2 (RuphenBiHim). This complex was prepared according to the literature procedure.63 ITC Studies. Titration experiments were carried out on an ITC instrument (VP-ITC, MicroCal).64 Reaction cells (1.43 mL) were filled with solutions and equilibrated at 37 °C. After this equilibration, an additional delay period was allowed to generate the baseline used in the subsequent data analyses. Solutions were degassed by use of a vacuum degasser (ThermoVac, MicroCal) for 5 min prior to any experimental run. Stirring speed was maintained at 307 rpm, and heat flow (μcal s−1) was recorded as a function of time. The ligand RuphenImH (523 μmol L−1) was titrated into a cell containing the protein HSA (30 μmol L−1). Protein and ligand containing solutions were prepared in the same buffer (0.1 mol L−1 Na2HPO4, 0.1 mol L−1 NaH2PO4, 2% DMSO, pH 7.40). The experiment was followed by 30 injections of 8 μL at 180 s intervals. The heat of dilution, measured by the injection RuphenImH into the same buffer assay solution, was subtracted from each titration to obtain the net reaction heat value. The heat of RuphenImH dilution in buffer is less than 0.8 μcal/s. Data were recorded and analyzed using Origin (version 7, OriginLab) software. ITC curves were fitted using a one-site independent model for measuring thermodynamics parameters. The shape of the binding isotherm changed according to the product of the binding constant and the target concentration through the so-called c value, which is defined as Kb[M]n (or [M]n/Kd), where Kb is the binding constant, [M] is the macromolecule concentration, and n the number of interaction sites.65 For accurate determination of binding constants, a c value between 1 and 1000 is recommended.64,66 In conformity with this recommendation, the c value obtained was 3. CD Spectroscopy. The interaction was monitored by far-UV CD spectroscopy over a wavelength range 200−250 nm, using a J-715 Jasco spectropolarimeter. CD spectra were measured from samples in 0.1 cm quartz cuvettes and were the average of 16 accumulations. The protein concentration was 3 μmol L−1 in phosphate buffer (0.1 mol L−1 Na2HPO4 and 0.1 mol L−1 NaH2PO4, pH 7.40). The complex was incubated for 24 h with HSA before measurement. The concentrations of the complex were 3 and 25 μmol L−1.

Cell Uptake. Figure 7 shows the luminescent microscopy images for RuphenImH and Ruphen1MeIm in HCT116 p53+/+

Figure 7. Fluorescent confocal imaging for complexes RuphenImH and Ruphen1MeIm in the HCT116 p53+/+ cell line.

cells treated for 24 h with 20 μmol L−1 concentration of complex, which is the value of IC70. The cells were marked with DAPI as a nuclear stain. As shown in Figure 7, the complexes were transported into the interior of the cell and located both in the cytoplasm and in the nucleus. We note that, in principle, substitution and/or redox processes could occur during the uptake experiments, in which case the absorption spectrum would correspond with the spectrum of the aquo/hydroxo or Ru(III) product. Since these species are not emissive, it is concluded that they are not being formed during the uptake experiment.

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CONCLUSIONS The series cis-[Ru(phen/bipy)2(L)2]2+ adds to the diversity of Ru(II) chemotypes available for comparative study and development. These features include feasibility of synthesis, water solubility, and luminescence in the near IR region that is maintained in vitro allowing cellular localization and distribution studies. The high inhibition of HCT116 p53−/− related to p53+/+ by cis-[Ru(phen)2(ImH)2]2+ is of significance, given that almost 70% of colon rectal tumor cells and 50% of all tumor cells present the mutations in the p53 gene. The interesting profile of biological activity with weak binding to HSA allowing free perfusion to tissue while the inhibition of cellular proliferation probably involves a combination of both cytostatic and cytotoxic mechanisms.

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EXPERIMENTAL SECTION

General Methods. Reagents were purchased from Aldrich and used without further purification. The elemental analysis shows that the complexes are greater than 95% pure. 1H NMR analyses were used to confirm the purity of all compounds. Materials. HSA was purchased from Sigma−Aldrich. The HSA solutions were prepared in 0.1 mmol L−1 phosphate buffer of pH 7.4 considering the molecular weight of 66.5 kDa. The primary antibodies used were p53 (cell signaling, no. 9282), total caspase 3 (cell-signaling, 9662), total Parp (cell signaling, no. 9541), and β-actin (Abcam, ab8226). 4911



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NMR Experiments. All the NMR experiments were conducted in a Bruker Avance III 600 MHz spectrometer, by employing a 1H {13C, 15 N} TCI triple resonance cryogenic cooled probehead, equipped with a z-gradient coil producing a nominal maximum gradient of 53.5 G cm−1. To simulate the biological medium, all the experiments were run at 37 °C. STD-NMR. Sample for STD-NMR experiments were prepared with a protein HSA concentration of 9 × 10−6 mol L−1 and the complexes RuphenImH and Ruphen1MeIm concentration of 1 × 10−3 mol L−1 dissolved in a solution of 297 μL of buffer (2.03 mmol L−1 Na2HPO4 and 0.437 mmol L−1 NaH2PO4, pD 7.8) in D2O (99%) and 3 μL DMSO (1%). A recovery time of 4 s of acquisition time by collecting 64 k points were used. 256 scans were averaged for both on- and offresonance experiments, which were run by irradiating at 20 000 and 385 Hz, respectively. A train of 2000 Gaussian pulses of 1 ms each with a 4 μs delay between pulses was used, with a total saturation time of 2 s. The STD effect, ASTD, can be calculated using the equation below: A STD =

propidium iodide, 0.1% Triton-X in PBS) containing RNase A (200 U mL−1). Cell cycle analysis was done after 24 h incubation on a FACSort flow cytometer (Becton Dickinson). Western Blot Analysis. Following PPC treatments, both floating and adherent cells were harvested, washed once with ice-cold PBS, and pelleted (10 000 rpm, 5 min, 4 °C). The pellets were then lysed in SDS lysis buffer (62.5 mmol L−1 Tris−HCl, pH 7.5, 5% glycerol, 4% SDS, 4% complete protease inhibitor (Roche) and 5% BME). Protein concentrations were determined by the Bradford assay and transferred to PVDF membrane (350 mA, 2h, 4 °C). The membrane was blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween20 for 30 min. The membranes were then probed with the primary antibodies in blocking buffer overnight at 4 °C, followed by a secondary antibody for 1 h at room temperature. Chemiluminescent protein bands were visualized on X-ray films. Computational Details. Geometry-optimized structure was obtained using Gaussian 09, revision B.01, C.0167 employing DFT calculations, using the hybrid B3LYP exchange-correlation functional68 and the LanL2DZ basis.69−71 The solvent (acetonitrile) was included using the polarized continuum model (PCM).72,73 A tight convergence (10−8 au) was used for all DFT calculations.

ISTD [L]T × I0 [P]

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Where [L]T is the total ligand concentration, [P] is the protein concentration, ISTD is the peak intensity of the STD NMR spectra, and I0 is the intensity of the peaks in the 1H off-resonance spectra. DOSY-NMR. The pulsed field gradient (PFG) stimulated spin−echo (STE) experiment was used with a pulse field gradient length of 1.3 ms (little delta) and a diffusion delay (big delta) of 60 ms. An acquisition time of 1.8 s (32 k points) were used for acquiring a sweep window of 7211.54 Hz. Sixteen experiments were recorded with gradient intensity linearly sampled from 2% to 98%, and 16 scans were averaged for each experiment; the recovering time used was 2 s. For the DOSY experiment, the sample preparation was the same as in the STD experiment but here using a protein concentration of 5 × 10−5 mol L−1 in order to visualize not only the protein signals and its diffusion coefficient but also an effective interaction between complex and protein. Cell Lines. HCT116 p53+/+ and HCT116 53−/− cells lines were cultured in RPMI 1640 (Invitrogen), supplemented with 10% calf serum (Atlanta Biologicals) and 1% penicillin/streptomycin (Invitrogen). Cells were maintained in logarithmic growth as a monolayer in T75 culture flasks at 37 °C in a humidified atmosphere containing 5% CO2. Cellular Growth Inhibition. To measure growth inhibition using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cells were seeded in 96-well plates at 5 × 103 cells/well and allowed to attach overnight. RPMI 1640 media was removed, and 100 μL of each compound was added after serial dilution to treatment concentrations of 200−0.0002 μmol L−1 in quadruplicate wells and exposed to cells for 72 h. Plates were then washed with PBS, and 100 μL of 5 mg/mL MTT solution was added. MTT solution was incubated with cells for 3 h at 37 °C. After incubation, MTT solution was removed, and 100 μL of DMSO was added. Quantification of cell growth in treated and control wells was then assessed by measurement of absorbance at 595 nm. IC50 values were determined by using the standard curve analysis of Origin. Confocal Laser Scanning Microscopy. HCT116 p53+/+ cells were seeded in 8-well chamber slides (Lab-Tekll Chamber slide) for 24 h. The cells were treated with complex (20 μmol L−1) for 24 h. After treatment, slides were washed 3 times with ice-cold PBS and fixed with 3% paraformaldehyde. Paraformaldehyde was removed, and cells were washed again 3 times with ice-cold PBS and allowed to dry. Slides were then mounted with Vectashield mounting media containing DAPI. Fluorescence was observed by confocal laser scanning microscopy (Zeiss LSM 510). Cell Cycle Arrest. HCT116 p53+/+ and HCT116 p53−/− cells were seeded in Petri dishes for 24 h. The cells were treated with complex (20 μmol L−1) for 24 and 48 h. After treatment, the cells were detached using trypsin (Gibco), washed twice with PBS, and suspended in propidium iodide staining solution (50 μg mL−1

ASSOCIATED CONTENT

S Supporting Information *

Optimized structure of the complex RuphenImH; bond lengths and angles for the optimized structure of RuphenImH; 1H NMR spectra in D2O of complexes RuphenImH and Ruphen1MeIm; cyclic voltammetry to complex RuphenImH and Ruphen1MeIm in water solution; plots of anodic peak 1 current as a function of ν /2 and log j versus E for RuphenImH and Ruphen1MeIm; 1H NMR spectra in different time and pH for the complex RuphenImH; DOSY-NMR for the complex RuphenImH and RuphenImH in the presence of HSA; and STD-NMR spectrum for Ruphen1MeIm in the presence of HSA. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(N.P.F.) Phone: (804) 828-6320; e-mail: [email protected]. *(R.M.C.) Phone: (+55) 16-3351-8780; e-mail: rosem@ufscar. br. Author Contributions

All authors contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Luciana Vizotto from UFSCar for NMR measurements, Prof. Ana Paula Araújo and Dr. Julio Cesar Pissuti Damalio from USP-São Carlos for CD measurements, and Prof. Amando S. Ito from USP-RP for emission lifetime measurements. FAPESP (proc. 2009/08218-0) and CAPES for the grants and fellowships. Supported in part by RO1CA-78754 to N.F.

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ABBREVIATIONS USED ITC, isothermal titration calorimetry; CD, circular dichroism; STD, saturation transfer difference; DOSY, diffusion ordered spectroscopy; DFT, density functional theory

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REFERENCES

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