Synthesis, characterization and antitumor activity of

Inorganica Chimica Acta 480 (2018) 83–90

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Research paper

Synthesis, characterization and antitumor activity of two new dipyridinium ylide based lanthanide(III) complexes Andreea Cârâc a, Rica Boscencu a, Rodica Mihaela Dinica˘ b, Joana F. Guerreiro c, Francisco Silva c, Fernanda Marques c, Maria Paula Cabral Campello c, Ca˘lin Moise d, Oana Brîncoveanu d, Marius Ena˘chescu d, Geta Cârâc b, Aurel Ta˘ba˘caru b,⇑ a

Faculty of Pharmacy, ‘‘Carol Davila” University of Medicine and Pharmacy, 6 Traian Vuia Street, 020956 Bucharest, Romania Department of Chemistry, Physics and Environment, Faculty of Sciences and Environment, ‘‘Dunarea de Jos” University of Galati, 111 Domneasca Street, 800201 Galati, Romania c Centro de Ciências e Tecnologias Nucleares (C2TN), Instituto Superior Técnico, Universidade de Lisboa, Portugal d Center for Surface Science and Nanotechnology, Politehnica University of Bucharest (CSSNT-UPB), 313 Splaiul Independentei, 060042 Bucharest, Romania b

a r t i c l e

i n f o

Article history: Received 20 November 2017 Received in revised form 29 April 2018 Accepted 5 May 2018

Keywords: Trivalent lanthanide complexes Heterocyclic ligands Ylide Dipyridinium ylide Antitumor activity

a b s t r a c t Two new dinuclear lanthanide(III) complexes, La-DPY and Nd-DPY, with the stoichiometric formula [{Ln(Et3N)(SO4)}2(l-DPY)(l4-SO4)] (Ln = La, Nd; DPY = ylide form of DPB, Et3N = triethylamine), were obtained through the reaction of N-heterocylic diquaternary salt N,N’-diphenacyl-4,40 -dipyridinium dibromide (DPB) and lanthanide(III) sulfate in methanol, in the presence of triethylamine. The obtained complexes were characterized by Fourier transform infrared spectroscopy, proton nuclear magnetic resonance, elemental analysis, UV–vis spectroscopy, thermogravimetric analysis and powder X-ray diffraction. Scanning electron microscopy (SEM) was used to investigate the morphology and particles size of the complexes, confirming that their particles are quite homogenous and uniform. The antitumor activity of the complexes was evaluated in the human cancer cells MCF7 and A2780 and compared to cisplatin, the metal-based drug in cancer therapy. The complexes were found to induce apoptotic cell death and, to a lesser extent, production of ROS, although these are not the unique mechanisms of action. In conclusion, we anticipate that these types of Ln(III) complexes have potential and could be further explored for applications as antitumor agents. Ó 2018 Elsevier B.V. All rights reserved.

1. Introduction Lanthanide (Ln) chemistry is currently a very active area of research. Due to their electronic structure these elements have unusual properties that make them suitable for applications in catalysis, organometallic synthesis, electronic and luminescent materials [1–11]. In the biomedical field, lanthanide complexes have received considerable attention, particularly in bioanalysis, imaging and radioimmunotherapy [2]. Recently, some lanthanide complexes have shown photocytotoxic activities in tumoral cells and hence with potential use in photodynamic therapy (PDT) [12–16]. As far as the clinical applications are concerned, lanthanides cannot be administered in the form of simple salts or metal ions, due to their toxic effects, but they can be administered in the form of thermodynamically and kinetically stable complexes. Upon

⇑ Corresponding author. E-mail address: [email protected] (A. Ta˘ba˘caru). https://doi.org/10.1016/j.ica.2018.05.003 0020-1693/Ó 2018 Elsevier B.V. All rights reserved.

coordination, the ligands play a key role in tuning the properties of the corresponding complexes [17], which is particularly relevant to biological, biochemical and medical applications [18]. Due to their size, lanthanide ions form stable complexes with high geometric flexibility and high coordination number [15–19]. As strong Lewis acids, lanthanide ions coordinate with highly electronegative donor atoms such as nitrogen or oxygen. N-donor ligands such as quaternary pyridinium derivatives present a positive charge at the nitrogen atom and therefore are expected to be more suitable for the synthesis of very large complexes of 4f-elements than O-donor ligands [9,20]. Nevertheless, the synthesis of lanthanide complexes with these types of ligands present certain limitations, i.e., if anhydrous conditions are not met, the presence of a nitrogen donor ligand such as 4,40 -bipyridine can favor deprotonation of any coordinated water, with formation of undesired sub-products as hydroxo- or oxo-lanthanide derivatives [20,21]. Heterocyclic ligands present intrinsic versatility to form metal complexes with biomedical importance and, in this direction, a

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wide range of complexes with biological activities such as antibacterial, antifungal, antiviral, and antitumor activities have been reported [22–29]. Novel synthetic strategies with the advances made in coordination chemistry strongly influenced the design of prospective lanthanide-based drugs with higher selectivity and improved toxicological and pharmacokinetic profiles [30,31]. In this context, we describe here the synthesis and characterization of the sulfate complexes of La3+ and Nd3+ with the diquaternary N,N’-diphenacyl-4,40 -dipyridinium dibromide (DPB) proligand (Scheme 1). The antitumor activity of these complexes in cancer cell lines was performed and compared with cisplatin, the metalbased drug in clinical use, in order to allow their evaluation as chemotherapeutic agents. The mechanisms involved in cell death were also explored.

2. Experimental section 2.1. Materials and methods All the chemicals and reagents were purchased from SigmaAldrich Co and used without further purification. The N-heterocylic diquaternary salt N,N’-diphenacyl-4,40 -dipyridinium dibromide (DPB) was prepared by the previously published method [32]. Ln2(SO4)3 (Ln = La, Nd) was prepared by dissolving Ln2O3 in concentrated H2SO4. The solvents were used as supplied or distilled using standard methods. Elemental analyses (C, H, N, S) were performed in-house with Fisons Instruments 1108 CHNS-O Elemental Analyzer. Before performing the analytical characterization, all samples were dried in vacuo (50 °C, 104 bar) until a constant weight was reached. Melting points are uncorrected and were taken on an SMP3 Stuart instrument with a capillary apparatus. The IR spectra were recorded from 4000 to 450 cm1 with a Spectrum Two IR spectrometer from PerkinElmer. In the following, the IR bands are classified as very weak (vw), weak (w), medium (m), strong (s) and very strong (vs). The 1H NMR spectrum of La-DPY was acquired at room temperature in dimethyl sulfoxide (DMSO) with a VXR-300 Varian spectrometer operating at 400 MHz, using tetramethylsilane (TMS) as internal standard. In the following, the 1H NMR signals are classified as singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m). Due to the paramagnetism of Nd(III) ion, the 1H NMR spectrum of Nd-DPY was not acquired. UV–vis spectra were recorded in the 200–900 nm range for both the ligand and the two complexes at 105 M in ethanolic solutions using a LAMBDA 950 spectrophotometer from PerkinElmer. For the stability tests of the complexes, the UV–vis spectra, performed in 105 M DMSO solutions, were collected in a Shimadzu UV-1800 Spectrophotometer (range: 200–800 nm). Thermogravimetric analyses (TGA) were carried out under a N2 flow from room temperature to 1200 °C and with a heating rate of 10 °C/min using a PerkinElmer STA 6000 Simultaneous Thermal Analyzer. Powder X-ray diffraction (PXRD) analyses were carried out in the 5–90° 2h range on a Rigaku SmartLab X-ray diffractometer using Cu Ka radiation (k = 0.154060 nm) in the general (Bragg Brentano Focusing) type of measurements, operating at room temperature. The generator was set at 45 kV and 200 mA.

Scheme 1. Structure of N,N’-diphenacyl-4,40 -dipyridinium dibromide (DPB).

Scanning electron microscopy (SEM) measurements were carried out using a Hitachi SU 8230 Scanning Electron Microscope equipped with a detector able to achieve the low angle backscattering electrons (LA-BSE), for both morphological and compositional contrast information. 2.2. Synthesis of Ln(III) complexes: General procedure Complexes La-DPY and Nd-DPY were synthesized according to the following procedure: in two 50 mL round bottom flasks, 0.277 g of N,N’-diphenacyl-4,40 -dipyridinium dibromide (0.5 mmol) were dissolved in 20 mL of methanol under heating at 50–60 °C and stirring until complete dissolution. Lanthanum(III) sulfate (0.283 g, 0.5 mmol) or neodymium(III) sulfate (0.288 g, 0.5 mmol) were then added, and after several minutes of stirring, 1 mL of triethylamine was added. Then, deep violet precipitates started to appear, which were left under reflux and continuous stirring overnight. After cooling, the solid products were filtered off, washed three times with 5 mL of methanol and distilled water, and were finally dried under vacuo for 24 h. [{La(Et3N)(SO4)}2(l-DPY)(l4-SO4)] (La-DPY). Violet powder. Yield: 85%. M.p. 200–205 °C dec., weakly soluble in methanol, and dimethylformamide. Anal. Calc. for C38H50La2N4O14S3 (FW = 1160.83 g mol1): C, 39.32; H, 4.34; N, 4.83; S, 8.29%. Found: C, 39.15; H, 4.45; N, 4.62; S, 7.98%. IR m(cm1): 3100–3000 (vw), 3000–2800 (vw), 1580 (m), 1542 (m), 1487 (vs), 1454 (s), 1420 (vs), 1336 (s), 1281 (w), 1223 (w), 1190 (vs), 1086 (w), 1014 (m), 943 (vw), 889 (s), 845 (w), 818 (s), 785 (w), 694 (vs), 664 (w), 503 (m). 1H NMR (DMSO d6, 400 MHz): d, 1.56q, 3.28 t (30H, (CH3CH2)3N), 5.60 s (2H, CHmethine), 7.14–7.30 m (10H, CHphenyl), 7.60d (J = 8.4 Hz, 4H, CHbipyridyl), 8.65d (J = 8.4 Hz, 4H, CHbipyridyl). [{Nd(Et3N)(SO4)}2(l-DPY)(l4-SO4)] (Nd-DPY). Violet powder. Yield: 81%. M.p. 200–205 °C dec., weakly soluble in methanol, and dimethylformamide. Anal. Calc. for C38H50Nd2N4O14S3 (FW = 1171.50 g mol1): C, 38.96; H, 4.30; N, 4.78; S, 8.21%. Found: C, 38.75; H, 4.48; N, 4.57; S, 7.89%. IR m(cm1): 3100–3000 (vw), 3000–2800 (vw), 1580 (m), 1542 (m), 1487 (vs), 1454 (s), 1420 (vs), 1336 (s), 1281 (w), 1223 (w), 1190 (vs), 1086 (w), 1014 (m), 943 (vw), 889 (s), 845 (w), 818 (s), 785 (w), 694 (vs), 664 (w), 503 (m). 2.3. Biological assays 2.3.1. Cellular viability by the MTT assay Human tumor cell lines, breast MCF7 and ovarian (cisplatin sensitive) A2780, were cultured in RPMI (A2780) or DMEM culture media (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C, 5% CO2 in a humidified atmosphere (Heraeus, Germany). The cells were adherent in monolayers and upon confluence were harvested by digestion with trypsin-EDTA (Gibco). Cell viability was evaluated using the tetrazolium salt MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]), which is reduced by a mitochondrial succinate dehydrogenase in metabolically active cells to insoluble purple formazan crystals [33]. For the assays, cells were seeded in 96-well plates at a density that ensures exponential growth of controls (untreated cells) throughout the experiments. Cells (2-3 104 cells/200 lL medium) were allowed to settle overnight followed by the addition of dilution series of the compounds in fresh medium in aliquots of 200 lL per well. Ligand and complexes were first solubilized in DMSO and then in medium, and added to final concentrations from 1 mM to 50 mM. The final concentration of DMSO in cell culture medium did not exceed 1%. After continuous exposure to the compounds for 24 h and 48 h at 37 °C/5% CO2, the medium was removed, and the cells were incubated with 200 lL of MTT solution in phosphate buffer saline (PBS) (0.5 mg/mL). After 3 h at 37 °C/5%


CO2, the solution was discarded, and the purple formazan crystals formed inside the cells were dissolved in 200 lL DMSO. The cellular viability was evaluated by measuring the absorbance at 570 nm on a plate spectrophotometer (PowerWave Xs, Bio-Tek Instruments, Winooski, VT, USA). The cytotoxic effects of the compounds were quantified by calculating the compound concentration that inhibit tumor cell growth by 50% (IC50), based on non-linear regression analysis of dose response data (GraphPad Prism software vs 5.0). All compounds were tested in at least two independent experiments, each comprising six replicates per concentration. 2.3.2. Apoptosis evaluation by Hoechst staining A2780 cells were plated in a cell culture slide at a concentration of 2 105 cells/mL and incubated for 48 h in culture medium in the absence or presence of the compounds at their IC50. The Hoechst staining method (Hoechst 33342, Thermo Fisher Scientific) was used to detect apoptotic nuclei as described elsewhere [34], with minor adaptations. Briefly, the culture medium was removed, and the cells were washed thwice with PBS before fixation with 4% (v/v) paraformaldehyde (in PBS) for 15 min at room temperature. After fixation, cells were washed thwice with PBS and incubated with Hoechst dye 33342 at a 1:2000 dilution, according to the manufacturer’s instructions. Following three washing steps with PBS, the cells were mounted in anti-fade mounting media (Vectashield H-100, Vector Laboratories, Burlingame, Canada). The slides were photographed under 20 (for counting purposes) or 63 magnification in a Zeiss Axioplan2 fluorescence microscope. Several random microscopic fields per sample were analyzed, corresponding to at least 300 nuclei per sample, in two independent experiments. 2.3.3. Reactive oxygen species by the NBT assay A2780 cells were seeded in 96-well plates with a density of 2 104 cells/200 mL. After 24 h, the medium was replaced with fresh medium containing the compounds at selected concentrations. ROS generation was detected by the nitroblue tetrazolium (NBT) assay following a previously described method [35]. Briefly, after 1 h pretreatment with the compounds, 20 lL of a NBT solution (10 mg/mL in water) (NBT = 2,20 -bis(4-Nitrophenyl)-5,50 diphenyl-3,30 -(3,30 -dimethoxy-4,40 -diphenylene)ditetrazolium chloride) was added to the medium and incubation continued for another 1 h at 37 °C. The NBT solution was discarded and the formazan deposits were solubilized with 200 lL of 90% DMSO:10% 0.01 M NaOH plus 0.1% SDS. After careful homogenization of the wells deposits, the absorbance was measured at 550 nm. All compounds were tested in at least two independent experiments, each comprising 4 replicates per concentration. Results (mean ± SD) are expressed as % of controls (no treatment).

tions (Et3N) of DPB proligand (Scheme 2, A) into its reactive dipyridinium ylide intermediate DPY (Scheme 2, B) which was in resonance with its enolic form (Scheme 2, C) [37]. The resulting DPY species actually acted as the ligand towards the complexation with the two lanthanide(III) ions, making both of the complexes colored violet. A preliminary PXRD analysis of the two complexes revealed their isostructurality (Fig. 1). Attempts to obtain single crystals suitable for the X-ray structure determination of these complexes were so far unsuccessful, and therefore, their crystal structure remains presently unknown. However, based on several characterization techniques (IR, EA, 1H NMR, UV–vis, TGA), we were able to propose a possible structure for the obtained lanthanide(III) complexes. The elemental analysis was found to be consistent with the general stoichiometric formula [{Ln(Et3N)(SO4)}2(l-DPY)(l4SO4)] (Ln = La, Nd) for both complexes. The IR spectra of the complexes show medium and strong bands in the region 1543–1420 cm1 assigned to the stretching vibrations C@C and C@N double bonds from the aromatic rings. While the IR spectrum of the DPB proligand presents a strong band at 1694 cm1, specific to the carbonyl (C@O) group, the IR spectra of both complexes lack such a band, which indicates the transformation of DPB proligand into the ylide form DPY (Fig. 2). This transformation is also supported by the appearance of a medium band located at 1010 cm1 and another weak band at 1063 cm1, which were assigned to the bending mode of vibration of the methine AC@CH group and to the stretching vibration of the CAO group resulting from the carbonyl group [38]. The IR spectrum of the free groups, i.e. ionic, sulfate (having the Td symmetry) displayed very strong bands at 1104 cm1 and at 613 cm1, which were assigned to the m3 stretching [md(SO)] and respectively, m4 bending [dd(OSO)] modes [39]. On the other hand, for the IR spectrum of the two complexes, the bands observed in the range 1275–1090 cm1 were ascribed to the m1 and m3 vibrations modes of the sulfate ions in low symmetry, suggesting the occurrence within the same complex of both the tetradentate coordination of bridging sulfate group in a bis-chelate mode and the bidentate coordination of sulfate group in a mono-chelate mode [39]. This relatively rare occurrence was also observed, e.g., with the Sm(III) complex (H2prz)[Sm2(HIDC)2(SO4)2] (HIDC = imidazole-4,5-dicarboxylic monoacid, H2prz = piperazine) for the bridging bis-chelate mode [40], or the La(III) complexes (H2en)2[La2M (SO4)6(H2O)2] (M = Co, Ni) [41]. The intense absorption band

3. Results and discussion 3.1. Synthesis and characterization of complexes The synthesis of the two new lanthanide(III) complexes, La-DPY and Nd-DPY, has been conducted in methanol solution by the reaction of Ln(III) sulfates with the diquaternary salt of 4,40 -dipyridinium DPB, as proligand, in the presence of triethylamine, using a 1:1 molar ratio. Worthy of note, the absence of a base, either inorganic or organic, did not lead to the formation of any complex. On the other hand, attempts to prepare Ln(III) complexes in the 1:2 or 1:3 molar ratio were not successful, presumably due to the steric factors of both the Ln(III) ions and the ligands [36]. Both complexes were isolated in good yields as violet powders and showed to be stable at air and moisture. The violet color of the complexes was an indicator of the transformation in basic condi-

85

Scheme 2. Transformation route of the DPB proligand into the DPY ligand.


6000

Intensity (cps)

5000 4000 3000 DPB

2000

Nd-DPY

1000

La-DPY

0 10

20

30

40

50

60

70

80

90

2 theta (deg.) Fig. 1. PXRD patterns of DPB (black), La-DPY (red) and Nd-DPY (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

T (%)

Nd-DPY

La-DPY

DPB

4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm ) Fig. 2. IR spectra of DPB proligand (black), La-DPY (red) and Nd-DPY (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

located at 817 cm1 can be attributed to the Ln–O vibration [42], whereas the weak absorption band observed at 1225 cm1 was assigned to the C–N stretching from coordinated triethylamine molecules [39]. The 1H NMR spectrum of La-DPY also revealed the presence of a quartet and a triplet at 1.56 and 3.28 ppm, which were assigned to the protons from coordinated triethylamine molecules. Corroboration of the analytical data with the spectroscopic techniques so far described allowed to propose the structure of complexes La-DPY and Nd-DPY (Fig. 3), which contain one DPY ligand in the enolic form, two Ln(III) ions, three sulfate groups

and two Et3N molecules per formula unit. Each Ln(III) ion is coordinated by one oxygen atom from the DPY ligand, two oxygen atoms from one bidentate mono-chelate sulfate group, two oxygen atoms from one tetradentate bridging sulfate group and one nitrogen atom from Et3N, therefore assuming an octahedral geometry. The two Ln(III) ions, interconnected by the bridging tetradentate sulfate group, are closing a cycle with the exo-bidentate DPY ligand, and the resulting complexes are dinuclear. Additionally, the positive charges of the nitrogen atoms are compensated, within the same structure of the complexes, by the negative charges of the enolate oxygen atoms, as was also observed in a palladium(II)– quaternary 2,20 -bipyridylium ylide complex reported by Dinica et al. [43]. These six-coordinate Ln(III) complexes are thus coming to join the very scarce family of complexes containing species with CN = 6 [44–47]. The higher coordination requirements of Ln(III) ions are a probable cause for the occurrence of dinuclear complexes, for which the ‘large ionic radii – low coordination numbers’ relationship [45] could be strategically achieved by the use of the bulky DPY ligand. Thermogravimetric analysis (TGA) was employed to investigate the thermal stabilities of the obtained Ln(III) complexes compared to the DPB proligand, along with the evaluation of the main weight losses recorded upon heating from room temperature to 1200 °C under a nitrogen atmosphere. Moreover, this analysis was used as a complementary tool for the confirmation of the proposed structure of the obtained complexes. The TGA traces of both the DPB proligand and the two complexes are collectively gathered in Fig. 4. The free DPB proligand is stable up to 225 °C, temperature at which a rapid decomposition was registered until a black carbonaceous residue was found. The decomposition of DPB proligand at such a high temperature is likely due to its quaternary ammonium nature [48,49]. Both Ln(III) complexes showed a slightly lower stability with respect to the DPB proligand, their decomposition onset being established at about 205 °C. At this temperature, a rapid decomposition of the complexes began with a weight loss of ca. 18.5% for La-DPB (theoretical weight loss 17.4%) and ca. 16.5% for Nd-DPY (theoretical weight loss 17.3%), corresponding to the evaporation of the coordinated Et3N molecules in both complexes. A progressive decomposition is continued, until 1200 °C, with the complete evolution of three sulfate groups and one DPY ligand, amounting to ca. 59.5% for La-DPB (theoretical weight loss 58.6%) and ca. 54.9% for Nd-DPY (theoretical weight loss 55.7%). The final residues, which have remained at the end of the heating

100

Weight loss (%)

86

80

60

La-DPY

40

Nd-DPY 20

DPB 0 0

200

400

600

800

1000

1200

Temperature (oC) Fig. 3. Proposed structure of complexes [{Ln(Et3N)(SO4)}2(l-DPY)(l4-SO4)] (Ln = La, Nd).

Fig. 4. TGA traces of DPB proligand (blue), La-DPY (red) and Nd-DPY (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


process, were associated to the high melting point metallic lanthanum and neodymium. The UV–vis absorption spectra of DPB proligand and the two lanthanide(III) complexes in ethanol are shown in Fig. 5. The DPB proligand exhibits a single absorption band at 260 nm, which is assigned to intraligand (p–p⁄) charge-transfer transitions [50]. The related lanthanide(III) complexes exhibit more intense absorption bands located at the same wavelength as DPB, and a hyperchromic effect is therefore attained as a consequence of metal complexation. The UV–vis spectra of the complexes also show two weak and broad absorption bands in the visible region which are likely due to the ligand field effect and could be assigned to the metal-to-ligand charge transfer (MLCT) phenomenon [51]. For the biological assays, the lanthanide complexes were initially dissolved in DMSO. In order to assess the stability of the complexes in this medium, UV–vis absorption spectra were recorded at room temperature at different time intervals. In DMSO solution, the single absorption band of the DPB proligand is shifted to 351 nm (see Fig. S1). As shown in Fig. 6, the pattern of the spectra of both lanthanide complexes in DMSO is similar to that observed in ethanolic solution and reproducible over time, although the three absorption bands observed in DMSO are slightly shifted relatively to those observed in ethanolic solution. These findings led us to conclude that the compounds are stable in DMSO. Scanning electron microscopy (SEM) was used to investigate the morphology and particles size of the synthesized Ln(III) complexes. Both complexes possess multiform crystal structures and morphologies (elongated micro- and nanoparticles, aggregated micro- and nanoparticles. Fig. 7 shows the SEM images of DPB ligand, La-DPY and Nd-DPY complexes at different magnifications. A comparison of the SEM images of covalently bonded complexes and ligand shows that the former appears to be quite homogenous and uniform. The morphology of the La and Nd complexes reveals aggregates of nanoparticles with dimensions varying from 10 to 40 nm, and from 30 to 250 nm, respectively. These micro- and nanoparticles might be formed by the coordination of N-heterocyclic bipyridine to the lanthanide(III) ions [52]. Complexes with well-defined shape and dimensionality were achieved and their morphology is determined mainly by the interaction between lanthanide ions and the ligand [53]. The SEM images of these complexes also reveal information regarding their

1.0 La-DPY Nd-DPY DPB

Absorbance

0.8 0.6 0.4 0.2 0.0 200

400

600

800

Wavelength (nm) Fig. 5. Room temperature UV–vis spectra of DPB (black), La-DPY (red) and Nd-DPY (blue), recorded on 105 M ethanolic solutions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Room temperature UV–vis spectra of lanthanide complexes, recorded on 105 M DMSO solutions at different time intervals: Nd-DPY, t = 0 (blue); Nd-DPY, t = 48 h (pink), La-DPY, t = 0 (black); La-DPY, t = 48 h (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

texture. Moreover, quite a few spheroids, with diameter between 10 nm and 40 nm, have emerged at the fracture surface, and we suggest that the ligand triggered the formation of this attractive morphology. The first microstructure of microrods, with a length from 2 to 6 mm and width from 0.1 to 0.5 mm, can be distinguished in La-DPY and Nd-DPY, and it is associated with the crystal structure of Ln2(SO4)3 (Ln = La and Nd), whereas the second one corresponds to the secondary phase, agglomerates of the smaller size particles with no well-defined shape (Fig. 8). These particles might be formed by the coordination of the DPY ligand to the lanthanide ions under the experimental conditions. The chelating ligand was unable to coordinate to the same Ln3+ because of the limits of spatial geometry. The coordination interaction therefore occurs intermolecularly, but not intramolecularly. The results highlight that the confinement did not disturb the first coordination sphere of the lanthanide ions. 3.2. Biological activity 3.2.1. Cellular viability The cytotoxicity of the complexes was assessed in the human tumor cells A2780 (ovarian cisplatin sensitive) and MCF7 (breast) within the concentration range 1 mM–50 mM. The effects of the compounds on the cellular viability were evaluated by the MTT assay after continuous exposure for 24 h and 48 h. As can be observed in Fig. 9 (Table S1 and Fig. S2), the cytotoxic activity of DPB increased upon coordination to the two Ln(III) ions, especially in A2780 cells. The lanthanide sulfate salts were also included in this study and showed considerably lower activity than the corresponding complexes in both cell lines (IC50 > 100 mM). The cytotoxic effect observed was higher in ovarian cells compared to breast cells, increasing drastically for longer incubation times (48 h). The antitumor activity displayed by cisplatin in A2780 cells after 24 h incubation was of the same order of magnitude than the Nd-DPY complex (26.0 ± 5.0 vs 30 ± 11). In MCF7 cells, both complexes were even more active than cisplatin, in particular the

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Fig. 7. SEM images of DPB ligand (a), La-DPY (b), and Nd-DPY (c), at different magnifications (top 5kx, and bottom 25kx).

Fig. 8. SEM images of La-DPY (a) and Nd-DPY (b), showing the particle and rod dimensions.

Fig. 9. Cytotoxic activity measured as the half-inhibitory concentration (IC50) for the complexes, the ligand and the lanthanide salts against MCF7 and A2780 cells after 24 h and 48 h incubation.

3.2.2. Apoptosis evaluation In order to elucidate the mechanisms that might underlie the cytotoxicity of the complexes, the Hoechst nuclei staining assay was used to estimate the level of apoptosis occurring in a cell population of A2780 cells exposed, or not, to the complexes. This assay is based on the ability of Hoechst 33342 to permeate the cells and stain the DNA, allowing the detection of nuclei exhibiting apoptotic markers, such as condensation or fragmentation of chromatin and formation of apoptotic bodies [54]. The results obtained indicate that both complexes can induce apoptosis in A2780 cells, as evidenced by the slight increase in the number of cells displaying typical apoptotic markers (Fig. 10 and Fig. S3). However, this effect is not as marked as expected based on the fact that the IC50 concentration for the complexes was used. This suggests that apoptosis is not the predominant pathway of cell death at play, and raises the question whether the cytotoxic effects induced by the complexes are also dependent on other types of programmed cell death.

Nd-DPY complex. Compared with cisplatin, the widely used chemotherapy drug, and using the same experimental conditions, both complexes presented important cytotoxic properties, in particular in ovarian cancer cells.

3.2.3. ROS production The NBT assay was used to evaluate the induction of ROS (superoxide anion) in A2780 cells exposed to the compounds. This colorimetric assay is based on the reduction of the membrane

100

IC 50 (μM)

80 60

Nd2(SO4)3 La2(SO4)3

40 20 0

DPB NdDPY LaDPY

89


Control

10 μm

10 μm

10 μm

Nd-DPY

La-DPY

Fig. 10. Staining of A2780 cells with Hoechst 33342 in the absence (control) or upon exposure to the complexes for visualization of apoptotic nuclei. The images shown are representative of two independent experiments where at least 300 nuclei were counted per sample. The white arrows indicate the presence of chromatin condensation, nuclear fragmentation, or apoptotic bodies, typical of apoptosis.

permeable yellow-colored nitroblue tetrazolium to an insoluble blue-product formazan by O–2. NBT conversion occurs intracellularly through mitochondrial NAD(P)H oxidase [55]. As can be seen from Fig. 11, the production of ROS is not considerably different in

relation to controls (no treatment), slightly increasing for La-DPY and Nd-DPY only at 50 mM. In these experimental conditions, the oxidative burst i.e., the rapid release of ROS (O–2), may not be the exclusive mechanism responsible for cellular cytotoxicity.

4. Conclusions

Fig. 11. NBT reduction in A2780 cells in response to the compounds at selected concentrations: 10, 20 and 50 mM after 2 h incubation.

Two new lanthanide(III) complexes La-DPY and Nd-DPY were synthesized through the reaction between Ln2(SO4)3 (Ln = La, Nd) and the diquaternary bis(pyridinium) salt DPB, acting as proligand, in alcoholic medium. The analytical and spectroscopic data revealed the dinuclear structure of both complexes, also highlighting the conversion of the DPB proligand into its ylide reactive intermediate DPY ligand in basic conditions. The antitumor activity of the complexes was evaluated in both ovarian and breast cancer cells and was found to be superior to cisplatin, in particular for Nd-DPY. However, induction of apoptotic cell death and production of ROS do not seem to be the exclusive mechanisms responsible for cellular cytotoxicity, so these will be worthy of further clarification in the future. In brief, we anticipate that these types of Ln(III) complexes have potential and could be further explored as antitumor agents.

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Acknowledgements Andreea Cârâc gratefully acknowledge the support through POSDRU/159/1.5/S/138907-EXCELIS project. C2TN/IST authors gratefully acknowledge the FCT support through the UID/ Multi/04349/2013 project. The authors also thank the helpful assistance of Geanina Mihai, Cosmin Gheorghe, Raluca Mesterca, Oana Lazar and Aida Pantazi, PhD students from CSSNT-UPB. Professors Claudio Pettinari and Fabio Marchetti from the University of Camerino are also acknowledged for helpful experimental assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ica.2018.05.003. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]

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