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Ru(III) anticancer agents with aromatic and non-aromatic dithiocarbamates asligands: Loading into nanocarriers and preliminary biological studies
Ru(III) anticancer agents with aromatic and non-aromatic dithiocarbamates as
ligands: Loading into PF127 micelles and preliminary biological studies
S. Scintilla, L. Brustolin, A. Gambalunga, F. Chiara, A. Trevisan, C.
Nardon, D. Fregona
PII:
DOI:
Reference:
S0162-0134(16)30245-8
doi:10.1016/j.jinorgbio.2016.09.009
JIB 10081
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
8 March 2016
8 September 2016
13 September 2016
Please cite this article as: S. Scintilla, L. Brustolin, A. Gambalunga, F. Chiara, A. Trevisan, C. Nardon, D. Fregona, Ru(III) anticancer agents with aromatic and non-aromatic
dithiocarbamates as ligands: Loading into PF127 micelles and preliminary biological
studies, Journal of Inorganic Biochemistry (2016), doi:10.1016/j.jinorgbio.2016.09.009
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Ru(III) anticancer agents with aromatic and non-aromatic dithiocarbamates as
ligands: loading into PF127 micelles and preliminary biological studies.
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Scintilla S. a,#, Brustolin L. a,#, Gambalunga A.b, Chiara F. b, Trevisan A. b, Nardon C.
, Fregona D. a,*
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a,*
Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131 Padova, Italy
b
Department of Cardiac, Thoracic and Vascular Sciences, University of Padova, Via Giustiniani, 2,
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a
35128 Padova, Italy
corresponding authors
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these authors contributed equally to this work
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*
In memory of Professor Alessandro Bagno (Dept. of Chemical Sciences, University of Padova), for
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his contribution to NMR spectroscopy of species containing paramagnetic centers.
Abstract
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Since the discovery of cisplatin in the 1960s, other metal complexes have been investigated as
potential antitumor agents to overcome the side-effects associated with the administration of the Ptbased drug. In line with our previous research, in this work we report the synthesis and
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characterization of mono- and dinuclear Ru(III) complexes with the pyrrolidinedithiocarbamate
(PDT) ligand and the more sterically-hindered carbazole-dithiocarbamato ligand (CDT), to compare
their properties (both at the chemical and antiproliferative level), in an attempt to assess a structureactivity rationale. Moreover, to overcome the scarce solubility under physiological conditions of the
Ru(III)-dithiocarbamato compounds, the biocompatible copolymer Pluronic® F127 has been used,
to encapsulate the metal derivatives in water-soluble micellar carriers. Finally, preliminary
biological evaluations on CDT and PDT compounds along with their nanoformulations, open
intriguing perspectives in anticancer chemotherapy. In particular, comparing the structure of the
Ru(III) derivatives, the ionic dinuclear PDT complex shows an important cytotoxic action in
comparison to its neutral counterparts. Moreover, the micellar carrier improves the overall activity
of the encapsulated Ru(III)-PDT chemotherapeutics. On the other hand, the nanoformulation of the
CDT derivatives allows us to solubilize both the 1:3 and the 2:5 complexes and to state their
inactivity .
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Keywords: Ru(III) complexes, dithiocarbamate, Pluronic F127, cancer, micelle, nanoformulation.
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1. Introduction
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Cancer is now the leading cause of death worldwide [1] and intense efforts are still devoted to
develop new and more effective therapies [2–4]. Since the successful application of cisplatin against
a variety of solid tumors [5], a large number of metal-based complexes - including both platinum
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and non-platinum (Au, Cu, Os, Pd, Ru, Rh and Ir) centers, combined with an even larger variety of
ligands- have been extensively investigated to increase the efficiency and overcome the drawbacks
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exhibited by their parent compound [6–9].
The side interactions of metallodrugs with biomolecules (mainly those involving sulfur aminoacids,
such as glutathione or cysteine) are crucial in their pharmacology and clearance.[10]In the light of
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this consideration, in the early stages of clinical use, sulfur-containing nucleophiles were
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administered after treatment with Pt(II)-based drugs in the attempt to modulate the related patientdisabling side-effects. The results, obtained by using sodium diethyldithiocarbamate (DEDTNa) as
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a chemoprotectant for cisplatin [11], inspired us to develop compounds which combine - in a single
molecule - the chemoprotective properties of this type of organic moiety with the cytotoxic activity
of a chosen metal center [12]. Besides platinum, our research group has been studying also Ru(III)
derivatives, inspired by the promising activity of the three complexes NAMI-A, KP1019 and NKP-
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1339 (which have reached human clinical trials [14–16]) and other ruthenium-based complexes
exhibiting activity against cisplatin-resistant tumors with less severe side-effects compared to
platinum drugs [13]. Starting from the works of Pignolet and Hendrickson [17–20], our research
group has developed novel Ru(III) dithiocarbamato (dtc) complexes (Fig. 1, panel I) with the
ligands pyrrolidyldithiocarbamate (PDT), N,N-dimethyldithiocarbamate (DMDT) and alkylsarcosyldithiocarbamate (RSDT, R = Me, Et, tBu) (Fig. 1, panel II) [21–24].
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Fig. 1. I, Chemical structure of our Ru(III)-dithiocarbamato complexes [Ru(dtc)3] (a), α-[Ru2(dtc)5]Cl (b) and β[Ru2(dtc)5]Cl (c). II, Chemical structure of the investigated dithiocarbamato ligands. N,N-Dimethyl dithiocarbamate
(DMDT), Pyrrolidyl dithiocarbamate (PDT), Sarcosyl-alkyl-ester dithiocarbamate (RSDT) and Carbazolyl
dithiocarbamate (CDT).
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In vitro cytotoxicity assays performed on different human tumor cell lines indicated that the ionic
dinuclear complexes (in particular in the case of α-[Ru2(DMDT)5]Cl and α-[Ru2(TSDT)5]Cl (TSDT
= tert-butyl sarcosine dithiocarbamate), Fig. 1, panel II) had superior anticancer activity than
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cisplatin against solid tumors (IC50 < 1 μM). On the contrary, the monomeric species were more
selective towards leukemic cells. Recently, it has been reported that [Ru2(PDT)5]Cl is ten-fold more
active than cisplatin against NCI-H1975 line (non-small cell lung cancer). Very attractively, the
antiproliferative activity is strongly affected by the chemical nature of the coordinated
dithiocarbamato-ligand. Ru(III) complexes containing PDT ligands are much more active than the
DMDT counterparts, underlining the crucial role of the ligand moiety for the efficacy of the drug
[25].
In this work we report the synthesis and physico-chemical characterization (elemental analysis,
ESI-MS, 1H-NMR spectroscopy, UV-Vis and FT-IR spectrophotometries) of novel Ru(III)carbazolyl-dithiocarbamato-derivatives (CDT, Fig. 1, panel II). Compared to pyrrolidine, carbazole
possesses two aromatic rings, condensed on the central heterocycle, thus conferring a pronounced
hydrophobic character on the complex, which may favor interactions with the cell membrane and
specific regions of proteins and organelles, possibly enhancing its cytotoxicity. Both Ru(III)-PDT
and -CDT derivatives have been tested as anticancer chemotherapeutics towards two human tumor
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epithelial cell lines, HeLa (cervix adenocarcinoma) and HCT 116 (colon carcinoma), with the aim
to get structure/activity relationships. For the in vitro tests, Pluronic® F127, a non-ionic surfactant,
has been used to increase the water solubility and, hence, the bioavailability of the investigated
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Ru(III) complexes. The choice of Pluronic® F127 was not accidental. The cytotoxicity exhibited by
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Pluronic® micelles towards non-cancerous cells was significantly lower than that observed for
cancerous cells [26], pointing out intriguing selectivity properties. Its combination with Pluronic®
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L61 has been used to obtain doxorubicin-loaded mixed micelles, which have already reached Phase
III stage in human clinical trials on patients with advanced adenocarcinoma of the esophagus and
gastroesophageal junction [27,28]. These interesting properties have recently led our research group
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to investigate PF127 as a carrier for Au(III)-dithiocarbamato complexes, thus providing a smart
solution for the implementation of in vivo tests of metallodrugs [29,30]. In addition, literature
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reports of attempts to incorporate Ru(III)-based drugs into other nanosystems [31,32].
The results collected with PF127 micelles loaded with Ru(III) compounds are discussed in
comparison with those obtained via the common methodology, involving DMSO as a solubilizing
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agent [24].
2.1. Materials
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2. Experimental
All reagents and solvents were used as supplied, unless otherwise stated, from Sigma Aldrich
without any further purification: ruthenium(III) chloride hydrate (RuCl3·3H2O), carbazole, sodium
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hydride (NaH) 60% - dispersion in mineral oil -, carbon disulfide (CS2), sodium pyrrolidinedithiocarbamate (NaPDT), cisplatin, n-octanol, saline solution, sodium sulfide (Na2S), sulfuric acid
(H2SO4) 98%, dichloromethane (CH2Cl2), diethyl ether (Et2O), ethanol (EtOH), methanol (MeOH),
acetone, acetonitrile, dimethylsulfoxide (DMSO), tetrahydrofuran (THF), chloroform (CHCl3),
pentane, hexane, deuterated chloroform (CDCl3), dimethylsulfoxide-d6 (DMSO-d6), deuterium
oxide (D2O). Tetrahydrofuran (THF) was dried using standard distillation procedures. The synthetic
procedures to obtain the carbazolyl dithiocarbamate and Ru(III) derivatives were performed under
controlled nitrogen atmosphere with a Schlenk line and Schlenk glassware. All the aqueous
reactions were conducted in distilled water, purified by means of ionic exchange membrane filters.
A dialysis tubing cellulose membrane (avg. flat width 25 mm, MW cut-off 11,181 Da, SigmaAldrich) was used for release assays and prepared as reported in section 2.12.
For in vitro cytotoxicity studies, the following products were purchased and used as provided by
suppliers: HeLa cells, HCT 116 cells (American Type Culture Collection, ATCC); Dulbecco
Modified Eagle’s Medium (D-MEM), L-glutamine, penicillin, streptomycin, fetal bovine serum
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(FBS), trypsin (0.05%, EDTA 0.02% in PBS) (Euro Clone); Pluronic® F127, DMSO (>99.9%, for
biological treatments), in vitro toxicology assay kit (resazurin based) (Aldrich).
2.2. FT-IR spectroscopy
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Near-FT-IR spectra (4000-400 cm-1) were recordered at room temperature (32 scans, resolution 2
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cm-1) by Nicolet Nexus 5SXC spectrophotometer. KBr pellets of samples were prepared according
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to standard procedures. Far-FT-IR spectra (600-50 cm-1) were registered at room temperature with a
Nicolet Nexus 870 spectrophotometer. For the analysis, films of sample dispersed in nujol were
loaded on polyethylene discs (250 scans, resolution 4 cm-1). Spectra were processed with OMNIC
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5.2 (Nicolet Instrument Corporation).
2.3. NMR spectroscopy
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NMR spectra were recorded from samples with a typical concentration of 5mM at 298 K on a
Bruker Avance DRX300 spectrometer equipped with a BBI [1H, X] probe-head and on a Bruker
Avance DRX400 equipped with a BBI-5 mm z-field gradient probe-head. Typical one-dimensional
H-NMR spectra were acquired with 128 scans, recycle delay of 1-4 s, spectral window 0÷14 ppm.
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H spectra of paramagnetic samples were acquired with 256 scans, recycle delay of 40 ms, with -
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50÷50 ppm spectral window. The 1H-NMR chemical shifts (δ) of the signals are given in ppm and
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referenced to residual protons in the deuterated solvents: chloroform-d (CDCl3, 7.26 ppm),
dimethylsulfoxide-d6 (DMSO-d6, 2.50 ppm), deuterium oxide (D2O, 4.80 ppm). Data processing
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was carried out by means of MestReNova version 6.2.0 (Mestrelab Research S.L.).
2.4. UV-Vis spectroscopy
Absorption spectra of freshly prepared solutions of the investigated complexes were acquired at 298
K or 310 K in the range 200-800 nm by an Agilent Cary 100 UV-Vis double beam
spectrophotometer. Samples were dissolved in the appropriate solvents and the resulting solutions
were placed in QS quartz cuvette (path length 1 cm). According to the Lambert-Beer law, molar
extinction coefficients for the investigated complexes were extrapolated by calibration curves,
assessed by recording spectra at different concentrations (7, 5, 4, 3 and 2 ×10-5 M).
2.5. ESI-MS spectrometry
ESI-MS spectra were recorded on a Mariner Perspective Biosystem instrument, setting a 5 kV
ionization potential and a 20 μL/min flow rate. A mixture of coumarin and 6-methyl-triptophan was
used as a standard. Samples were dissolved in methanol or acetonitrile, whereas methanol with 1%
formic acid was used as eluent. ESI-MS spectra have been processed by the software Data Explorer.
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2.6. Silica gel and thin layer chromatography
Analytical TLC and preparative TLC were performed on Kiesegel F254 and Kiesegel 60 (thickness
2 mm) (Fluka), respectively. UV light (λ= 254 nm) allowed spot after TLC runs. Gravity column
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loaded compound was obtained by using the proper eluent mixture.
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chromatography was carried out on silica gel Kiesegel 60 (40-63 μm) (Fluka); the elution of the
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2.7. Elemental analysis
Elemental analyses were carried out at the Microanalysis Laboratory of the Department of
Chemical Sciences, University of Padua by using a microanalyzer Fisons EA-1108 CHNS-O and a
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microanalyzer Carlo Erba 1108 CHNS-O.
2.8. Synthesis of Ru(III) precursors
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2.8.1. Synthesis of [(DMSO)2H][trans-Ru(DMSO)2Cl4]. The synthetic procedure was carried out as
described elsewhere [33]. After precipitation of red-orange crystals the supernatant was removed
and the crystals washed with acetone, Et2O and dried under vacuum in presence of P2O5.
H-NMR (D2O, 300.13 MHz, 298 K, δ/ppm): 2.7 (br, 6H, S-CH3 [DMSO2H]+, cationic moiety), -
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Yield: 35%
17.0 (br, 6H, S-CH3, coordinated DMSO).
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2.8.2. Synthesis of Na[trans-Ru(DMSO)2Cl4]. The synthetic procedure was carried out as described
elsewhere [33]. The product rapidly precipitated from the mixture as light orange microcrystals,
which were filtered off, washed with cold EtOH, Et2O and dried under vacuum dried in presence of
Yield: 75%
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P2O5.
H-NMR (D2O, 300.13 MHz, 298 K, δ/ppm): -16.8 (br, 6H, S-CH3).
Anal. Calc. for C4H12Cl4O2S2RuNa (M.W.= 422.14) H 2.87; C 11.38; S 15.19. Found H 2.89; C
11.25; S 15.32.
2.9. Ru-based PDT complexes
2.9.1. Synthesis of [Ru(PDT)3]. The synthetic procedure was carried out as described previously
[21]. To a solution of Na[trans-Ru(DMSO)2Cl4] (42.2 mg, 0.10 mmol) in EtOH (4 mL), a solution
of NaPDT (64.6 mg, 0.38) in EtOH (4 mL) was added. The mixture was stirred for 20 minutes at
room temperature, leading to the formation of a dark brown solid which was filtered off and washed
with cold EtOH (2 x 2.0 mL). The solid was re-dissolved in CH2Cl2 and purified by silica gel
chromatography, using CH2Cl2 to elute the mononuclear complex first. Successively, the mixture
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was switched to the more polar eluent CH2Cl2/MeOH 95:5 to obtain dinuclear complexes (as
byproducts).
Yield: 14%, dark green solid
H-NMR (CDCl3, 300.13 MHz, 298 K, δ/ppm): 44.4 (br, 2H, (N)CH2), 36.0 (br, 4H, CH2-N-CH2),
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R.f. (on silica gel, CH2Cl2): 0.85
0.3 (br, 4H,CH2-CH2).
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ESI-MS [M]+ 539.4 m/z (calc. = 539.8).
Anal. Calc. for C15H24N3S6Ru (M.W.= 539.84) H 4.48; C 33.37; N 7.78; S 35.64. Found H 4.58; C
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33.45; N 7.56; S 36.02.
2.9.2. Synthesis of [Ru2(PDT)5]Cl ( and mixture). According to the literature [21], to a solution
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of RuCl3·3H2O (0.40 g, 1.50 mmol) in water (4 mL), 10 mL of a NaPDT aqueous solution (0.78 g,
4.60 mmol in 10 mL) were added dropwise. A dark brown solid precipitated instantaneously. The
mixture was stirred for 1 h, then the solid was filtrated and washed with cold water (3 x 3.0 mL) and
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cold Et2O (2 x 2.5 mL). The isolated product was re-dissolved in CH2Cl2 and purified by silica gel
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chromatography, by using CH2Cl2 to elute the mononuclear byproduct and, afterwards, a mixture
CH2Cl2/MeOH 95:5 to elute the desired isomer mixture complexes.
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Yield: 19%, dark red solid
R.f. (on silica gel, CH2Cl2/MeOH 92:8): 0.23
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H-NMR (CDCl3, 300.13 MHz, 298 K, δ/ppm): 4.25-3.15 (m, 20H, CH2-N-CH2), 2.15-1.04 (m,
20H, CH2-CH2).
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ESI-MS [Ru2(PDT)5]+ 933.1 m/z (calc. = 933.5).
Anal. Calc. for C25H40N5S10Ru2Cl (M.W.= 968.87) H 4.16; C 30.99; N 7.23; S 33.10. Found H
4.01; C 31.12; N 7.42; S 33.02.
2.9.3. Isomerisation of the /β mixture to β-[Ru2(PDT)5]Cl. The isomerization from αRu2(PDT)]5Cl to -[Ru2(PDT)]5Cl was carried out according to a method described in literature
[20], in order to achieve the most thermodynamically stable isomer. 0.14 g (0.14 mmol) of α,[Ru2(PDT)]5Cl mixture was heated under reflux into a minimum volume of MeOH (3 mL) for 8 h.
Successively, the mixture was allowed to cool down and a dark red solid was then separated and
dried under vacuum.
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H-NMR (CDCl3, 300.13 MHz, 298 K, δ/ppm): 3.98-3.40 (m, 20H, CH2-N-CH2), 2.18-1.80 (m,
20H, CH2-CH2).
ESI-MS [Ru2(PDT)5]+ 933.1 m/z (calc. = 933.5).
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Anal. Calc. for C25H40ClN5S10Ru2 (M.W.= 968.87) H 4.16; C 30.99; N 7.23; S 33.10. Found H
4.20; C 31.15; N 7.22; S 32.97.
2.10. Synthesis of Ru-based CDT complexes
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2.10.1. Synthesis of Na(CDT). The sodium salt of carbazole-dithiocarbamate (NaCDT) was
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synthetized by following a synthetic procedure reported in literature properly modified [34].
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According to this procedure, to 0.24 g (1.44 mmol) of carbazole previously dissolved in freshly
distilled THF under inert N2 atmosphere, 0.12 g (2.88 mmol) of NaH 60% suspension were added at
0 °C. After the reaction was kept under vigorous stirring for 10 minutes, the mixture was filtered
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under nitrogen to remove NaH residues. Then, 173 μL (2.88 mmol) of dry CS2 were added to the
resulting solution under stirring at 0 °C. After 2 h the solvent was removed under reduced pressure
and a bright yellow-orange solid was obtained. The product is conserved under inert atmosphere, as
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it resulted highly hygroscopic and air sensitive, decomposing within 30 s into a black tar. The
product is used without any further purification (Fig. 1, panel I).
Yield: 80%
H-NMR (CDCl3, 300.13 MHz, 298 K, δ/ppm): 8.82-8.75 (m, 1H, H5), 8.05-8.12 (m, 1H, H2), 7.93-
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7.87 (m, 1H, H3), 7.46-7.41 (m, 1H, H4).
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2.10.2. Synthesis of [Ru(CDT)3] and α-[Ru2(CDT)5]Cl. By using a Schlenk-line apparatus under N2
atmosphere, 0.31 g (1.15 mmol) of synthesized Na(CDT) and 0.24 g (0.92 mmol) of RuCl3·3H2O
were dissolved in 18 mL of freshly distilled THF. The mixture was left under stirring for 15 h at
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room temperature. The solvent was successively removed under reduced pressure, leaving a black
solid that was washed with cold pentane (5 x 5.0 mL). The dinuclear complex was isolated by
several precipitation cycles in THF/pentane mixture (1:1 v/v). The THF/pentane solution is dried
leaving a dark green solid that was dried under vacuum and purified by silica gel chromatography
using an eluting mixture CH2Cl2/hexane 4:6 to isolate the mononuclear complex.
[Ru(CDT)3]: Yield: 11%, aspect: dark green
R.f. (on silica gel, CH2Cl2/hexane 1:1): 0.88
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H-NMR (CDCl3, 400 MHz, 298 K, δ/ppm): 9.03-9.01 (d, 1H, H3), 8.91-8.88 (t, 1H, H4), 7.73-7.71
(d, 1H, H5).
ESI-MS [M]+ 828.0 m/z (calc. = 827.9).
Anal. Calc. for C39H24N3S6Ru (M.W.= 828.09) H 2.92; C 56.57; N 5.07; S 23.23. Found H 3.28; C
56.52; N 4.86; S 23.57.
α,β-[Ru2(CDT)5]Cl: Yield: 17%, aspect: dark orange
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R.f. (on silica gel, CH2Cl2/hexane 1:1): 0.80
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H-NMR (CDCl3, 400 MHz, 298 K, δ/ppm): 9.19-9.17 (d, 1H, H2), 8.00-7.98 (d, 1H, H5), 7.53-7.46
(m, 1H, H3+H4).
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ESI-MS [M+CH3CN]+ 1490.5 m/z (calc. = 1490.9).
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Anal. Calc. for C65H40ClN5S10Ru2 (M.W.= 1449.30) H 2.87; C 53.87; N 4.83; S 22.12. Found H
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2.70; C 53.79; N 4.67; S 22.18.
2.10.3. Isomerisation of /β mixture to β-[Ru2(CDT)5]Cl. The reaction of isomerization was carried
out by following the methodology described in Section 2.9.3.
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Aspect: dark orange
H-NMR (CDCl3, 400 MHz, 298 K, δ/ppm): 9.19-9.17 (d, 1H, H2), 8.00-7.98 (d, 1H, H5), 7.53-7.46
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(m, 1H, H3+H4).
ESI-MS [M+CH3CN]+ at 1490.5 m/z (calc. = 1490.9)
Anal. Calc. for C65H40ClN5S10Ru2 (M.W.= 1449.30) H 2.87; C 53.87; N 4.83; S 22.12. Found H
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2.11. In vitro cytotoxicity studies
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2.91; C 53.50; N 4.51 ; S 22.49.
2.11.1. Preparation of samples for cytotoxicity assays. Tests were carried out using Ru(III)-
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pyrrolidinedithiocarbamato and Ru(III)-carbazoledithiocarbamato complexes. Due to their low
solubility in water, Pluronic® F127 surfactant was used to facilitate solubilization of the compounds
in physiological media. The samples were prepared by dissolving both the polymer (a weighted
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amount to yield a final concentration of 40 mg/mL in saline solution – see below) and the complex
in CH2Cl2 and successively evaporating the solvent so to obtain a homogeneous thin layer. The
subsequent addition of saline solution (0.9% NaCl w/v) and sonication for 2 minutes led to the
formation of a mixture 140 M in Ru complex ready for cell treatment, upon proper dilution with
cell culture medium (D-MEM). DMSO (sterile) solutions of carbazole, [Ru(PDT)3] and β[Ru2(PDT)5]Cl were also prepared and, after ad hoc dilution with D-MEM medium, tested on the
cell cultures to compare the polymer-mediated release of the investigated compounds with the
activity found in DMSO vehicle. The notation PL{compound} stands for the use of micellar
systems.
2.11.2. Cell lines and culture conditions. Cell cultures have been incubated at 37 °C in a 5% carbon
dioxide controlled atmosphere of a Hera Cell 150i CO2 incubator (Thermo Scientific). Cellular
vitality was determined by absorbance measurements at 595 nm, using a plate reader ELISA
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Microplate Reader Model 550 (Bio-Rad). Cell counting was performed with a Bruker camera
emocytometer. Data were obtained and processed by Microplate Manager 4.0 and Origin 8.0
software.
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For this work, HeLa and HCT 116 cells were grown in a culture flask (surface 75 cm2) in
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Dulbecco’s modified Eagle’s medium (D-MEM), with addition of FBS (10%), L-glutamine (5 mM)
and antibiotics (streptomycin, penicillin) and incubated at 37 °C in a 5% carbon dioxide controlled
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atmosphere. After the removal of the medium, cells were washed with 6 mL of PBS (phosphate
buffered saline) solution and 1 mL of trypsin was added. The culture flask was incubated for 3 min
and then shaken to remove cells from the flask surface. 3 mL of D-MEM were successively added
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to the flask, to block the action of trypsin. After that, cellswere counted, transferred to the test plates
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(polystyrene, 96-well) and incubated for 24 h before treating with complexes.
2.11.3. Cell growth inhibition assay. The treatments were carried out for either 24 or 72 hours
starting from the following cell densities: 20,000 and 25,000 cells/mL in 24 hours-experiments for
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HeLa and HCT 116 lines, respectively; 10,000 and 15,000 cells/mL in 72 hours-experiments for
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HeLa and HCT 116 line, respectively. Briefly, when confluence was about 70-80%, D-MEM was
removed from the cell cultures. The previously described saline solutions (NaCl 0.9% w/v) of the
micellar samples were diluted with D-MEM (saline < 29% v/v) to yield the final tested
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concentrations (typically in the range 0.1 - 20 M) and hence added to the corresponding wells (200
μL/well). Similarly, DMSO solutions were properly diluted with medium at a safe DMSO
concentration of 0.1% v/v. At least three independent experiments were carried out under
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quadruplicate conditions for every tested μM concentration. Cisplatin (in saline solution, NaCl
0.9% w/v) has been used as a reference drug.
Growth inhibition was evaluated by Resazurin-test [35,36] , by removing the cell treatment medium
and adding 100 μL of a 10% Resazurin solution in D-MEM with an incubation time of 2 hours at 37
°C. Cellular viability was determined by absorbance measurements at 595 nm. Cytotoxicity data
were expressed as IC50 values, i.e. the concentration of the test complex inducing 50% reduction in
cell number compared with control cultures.
2.12. Release tests on PL{β-[Ru2(CDT)5]Cl} and PL{β-[Ru2(PDT)5]Cl}
2.12.1. Preparation of the dialysis tubing cellulose membrane. The dialysis tubing cellulose
membrane (Sigma Aldrich, avg. flat width 25 mm, MW cut-off 11,181 Da) was washed and
activated as follows: i) glycerol included as humectant was removed by washing with running water
for 3-4 hours; ii) removal of sulfur compounds was obtained by a treatment with a 0.3% (w/v)
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solution of sodium sulfide at 80 °C for 1 minute; iii) the tubing was washed with water at 60 °C for
2 minutes; iv) 0.2% (v/v) solution of sulfuric acid was added and a final rinse with hot water
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allowed the removal of the acid.
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2.12.2. Release test on PL{β-[Ru2(CDT)5]Cl} and PL{β-[Ru2(PDT)5]Cl}. A saline solution (NaCl
0.9% w/v) of each complex (1.4·10-4 M) was obtained by re-suspending PF127/complex thin layer
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(see section 2.11.1). 2 mL of each solution was injected in the previously described dialysis
membrane, with a final PF127 concentration of 40 mg/mL (the ideal dissolving concentration for
[Ru2(dtc)5]Cl complexes). Then, the membrane was opportunely sealed with clips and put in a
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container with 80 mL of saline solution and incubated at 37 °C under stirring. UV-Vis
measurements were recorded overtime up to 96 hours. For each measure, an aliquot of 1 mL was
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withdrawn from the bulk, immediately replenished with 1 mL of saline solution to keep the total
volume constant.
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2.13. Log P evaluation
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For the evaluation the of partition coefficient P, n-octanol was pre-saturated with deionized water
for 24 hours under vigorous stirring, then let to equilibrate for 6 hours at 25 °C. After that, weighted
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P
amounts of all Ru-dtc complexes were dissolved in a defined aliquot of the organic phase and
shaken for 2 hours in the presence of deionized water at 25 °C. Subsequently, the mixture was left
to equilibrate for 30 minutes. The concentration of every Ru-dtc derivative in the organic phase
before (C0) and after partitioning (C1) was measured by UV-Vis spectrophotometry, followed by the
[37].
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evaluation of the corresponding n-octanol/water partition coefficient (P ) as log P = log (C1)/(C0-C1)
11
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3. Results and discussion
3.1. The Ru(CDT) complexes
Scheme 1 reports the synthetic pathway to the mono- and dinuclear Ru(III) carbazole-
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dithiocarbamato (CDT) complexes. Bereman and Nalewajek reported in 1978 the synthesis of Cu,
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Ni, and Zn-CDT derivatives but, at the best of our knowledge, the synthesis reported here represents
the first attempt to obtain Ru(III)-CDT complexes [34]. The dithiocarbamic salt of carbazole
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(NaCDT, Scheme 1, I) is very hygroscopic and extremely air sensitive and was used as above
indicated (Scheme 1, II) to obtain the Ru(III)-CDT derivatives. In the following sections, FT-IR and
NMR characterizations are reported. ESI-MS spectra confirmed the nature of the compounds and
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P
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are reported in the SI (Fig. S1 and S2).
Scheme 1. Reaction scheme for the synthesis of Ru(CDT) compounds. (I); synthesis of NaCDT (II): synthesis of the
mononuclear complex [Ru(CDT)3] and the α-dinuclear complex [Ru2(CDT)5]Cl; the α-dinuclear complex is then
converted to its β-isomer by refluxing the mixture in MeOH.
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3.1.1. FT-IR characterization of Ru(CDT) complexes. Dithiocarbamato salts possess the ability to
be, at the same time, strong- and weak-field ligands. The dithiocarbamate can be considered as a
strong-field ligand when the dithiocarbamic mononegative form (hereinafter I) is dominating (Fig.
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2, left). On the other hand, a significant contribution of the thioureide resonance hybrid (hereinafter
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II, Fig. 2, right) is associated with a weak-field ligand behavior [38–40]. The contribution of the
latter (presenting a positive charge on the nitrogen with each sulfur atom negatively charged) makes
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these ligands capable to stabilize metal ions in a wide range of oxidation states [38]. Carbazoledithiocarbamato (CDT) ligand exhibits only the resonance form (I), as the resonance structure (II)
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previously described would hamper the aromaticity of the molecule (Fig. 2) [34].
Fig. 2. Binding of the ligands CDT and PDT to Ru(III) ion. In the case of CDT (left), the dithiocarbamic resonance
form (I) mainly contributes to the final structure. For ligands such as PDT (right), the thioureide form (II) is the most
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influent resonance, thus endowing the ligand with the ability to stabilize metal ions in a wide range of oxidation states.
These important features can be proved by comparing FT-IR analysis of both the Ru(III)-CDT
complexes and their PDT [24] analogues (Table 1, Fig. S3-S6).
Table 1. FT-IR data of Ru(III) complexes with CDT and PDT ligands in the region 4000-400 cm-1.
Assignment
[Ru(CDT)3]
[Ru(PDT)3]
[Ru2(CDT)5]Cl
[Ru2(PDT)5]Cl
ν(SSC-N)
1363s; 1324s;
1487s; 1469s;
1364s; 1326s;
1498s; 1472s;
1294s cm-1
1444s cm-1
1299s cm-1
νasym(S-C-S)
1036m cm-1
941m cm-1
1038 m cm-1
947m cm-1
νsym(S-C-S)
585 cm-1
571 cm-1
585 cm-1
569 cm-1
νasym(Ru-S)
459 cm-1
438 cm-1
467, 440 cm-1
438, 429 cm-1
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1443s cm-1
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413 cm-1
νsym(Ru-S)
342 cm-1
416 cm-1
340 cm-1
(asym= asymmetric, sim= symmetric; s= strong, m= medium, w= weak)
The FT-IR spectra present, for both mono- and dinuclear complexes, three strong and well defined
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ν(SSC-N) bands (Fig. S3 and S5). In the case of mononuclear complexes, such a pattern is due to
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their distorted octahedral geometry, as similarly reported for [Fe(PDT)3][41], resulting in three not
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equivalent dtc ligands [22]. This is visible in the X-ray crystal structures obtained also for dinuclar
derivatives [24]. A shift to lower energies of the ν(SSC-N) bands is detected for Ru(III)-CDT
complexes with respect to their Ru(III)-PDT analogues. This shift is ascribable to the partial double
character of SSC-N bond, caused by the predominant contribution of the resonance form (II) for
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coordinated PDT, contrarily to Ru(III)-CDT complexes, for which the limiting form (I) is
dominating (single SSC-N bond). At the same time the ν(S-C-S) as well as the ν(Ru-S) bands, are
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shifted to higher energies in CDT-complexes than in PDT derivatives. This energy increase
suggests the presence of a metal-to-ligand back-donation, leading to a multiple bond character for
the RuSCS four-membered ring of Ru(III)-CDT complexes (Table 1, Fig. S4 and S6). In addition,
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the detection of a single band at ca. 1000 cm-1 in all the complexes clearly indicates a symmetrical
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bidentate coordination of the -NCSS moiety to the metal ion, as confirmed by the Bonati-Ugo
criterion (Fig. S3 and S5) [42].
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Interestingly, the dinuclear species show a splitting of the band attributable to the asymmetric ν(RuS) (467 and 440 cm-1, 438 and 429 cm-1, respectively, Fig. S4 and S6, Table 1), owing to the
simultaneous presence of differently coordinated (bidentate or bridged) dithiocarbamato ligands.
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Similarly to mononuclear complexes, for [Ru2(CDT)5]Cl the ν(S-C-S) and ν(Ru-S) occur at higher
wavenumbers with respect to [Ru2(PDT)5]Cl, because of the presence of metal-to-ligand backbonding, and hence of a multiple bond character for the RuSCS ring (Fig. 2).
Table S1 shows the bands related to the aromatic moiety of Ru(III)-CDT complexes (Fig. S3 and
S5) [43].
3.1.2 1H-NMR characterization of Ru(III)-CDT complexes. 1H-NMR data for the Ru-CDT
complexes, together with the sodium carbazoledithiocarbamate and the carbazole precursor is
shown in Table 2.
Table 2. 1H-NMR data in CDCl3 found for Ru(III)-CDT complexes, Na(CDT) and carbazole (400 MHz, 298 K).
NH δ/ppm
H5 δ/ppm
H4 δ/ppm
H3 δ/ppm
H2 δ/ppm
Carbazole
8.03 (s)
8.08 (d)
7.24 (t)
7.42 (m)
7.42 (m)
Na(CDT)
-
8.82-8.75 (m)
7.46-7.41 (m)
7.93-7.87 (m)
8.12-8.05 (m)
Compound
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[Ru(CDT)3]
-
7.73-7.71 (d)
8.91-8.88 (t)
9.03-9.01 (d)
-
α-[Ru2(CDT)5]Cl
-
8.00-7.98 (d)
7.53-7.46 (m)
7.53-7.46 (m)
9.19-9.17 (d)
β-[Ru2(CDT)5]Cl
-
8.00-7.98 (d)
7.53-7.46 (m)
7.53-7.46 (m)
9.19-9.17 (d)
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m= multiplet, s= singlet, d= doublet, t= triplet, br= broad.
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The signals related to H2 nuclei of [Ru(CDT)3] (see Fig. 1, Fig. S7) are broadened under the limit of
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detection. Generally, both the coordination to a paramagnetic metal ion and the presence of an
aromatic ring causes a broadening of the proton signals [44]. Nevertheless, no difference was found
in the recorded spectra by increasing the relaxation time up to 10 seconds, suggesting that the
paramagnetic influence of Ru(III) ion is the main cause of such behavior.
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In the case of [Ru(PDT)3] the paramagnetic effect of the metal center not only affects the
broadening of the signals but also determines a large downfield shift of the resonances (up to 40
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ppm) [24]. Remarkably, in the spectra of the mixture of the two Ru(III)-CDT dinuclear isomers, the
signals related to H2 (Fig. 1) are detected. In fact, the two Ru(III) nuclei are antiferromagnetically
coupled [21], thus quenching the paramagnetic effect of both metal centers (Fig. S8). The spectra
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recorded for the two Ru(III)-CDT dinuclear isomers are basically identical, making it impossible to
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assess the degree of the / conversion (Scheme 1, Fig. S8), contrarily to what found for /[Ru2(PDT)5]Cl [24]. In the light of this evidence, 1H-NMR spectroscopy on Ru(dtc) complexes
isolated species.
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does not allow a deep structural characterization although being useful to assess the purity of the
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3.1.3. Ru(III)-CDT and Ru(III)-PDT: UV-Visible characterization and stability studies in solution.
UV-Vis spectra (spectral range 240-800 nm) of the ruthenium(III) complexes with PDT and CDT
ligands (hereinafter L) were recorded in CH2Cl2 over time to assess their stability in solution
(neutral monomers Fig. S9 and S10; dinuclear analogues Fig. S11 and S12). The most significant
spectroscopic features found for the compounds [RuL3] and [Ru2L5]Cl are summarized in Table 3.
Table 3. UV-Vis spectral data recorded in CH2Cl2 at 25 °C for the monomeric species [RuL3] and the dinuclear
complexes [Ru2L5]Cl (L = PDT, CDT).
λmax/nm (ε / M -1 cm-1)
Compound
241
255sh
280sh
362
467
565
(21400)
(19762)
(13092)
(5334)
(1468)
(963)
[Ru(CDT)3]
-a
-a
279
341
445
626
(18552)
(15559)
(9687)
(1441)
[Ru2(PDT)5]Cl
247
268
288
333sh
468
[Ru(PDT)3]
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(23419)
[Ru2(CDT) 5]Cl
-
a
(24516)
-
(24223)
278
a
sh
(36794)
(9401)
(1031)
361
421sh
(28260)
(16943)
sh= shoulder; a= band occurring at wavelength lower than solvent cut-off (240 nm)
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All the analyzed complexes have shown high stability in CH2Cl2 solution, as no significant spectral
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change was observed even after days. The first band at ca. 240 nm for PDT complexes (Fig. S9 and
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S11) is not detectable for CDT derivatives (Fig. S10 and S12) in dichloromethane but is found at
about 210 nm for the latter in saline solution (Fig. S14 and S16). The corresponding transition
cannot be attributable unambiguously as it is still subject of debate in literature and in most cases it
has been not even ascribed. Anyway, it could be due either to an intraligand π*←π transition
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located in the –NCSS moiety or to an intraligand p←d transition between levels originated by
sulfur atoms [45]. The bands at around 260 nm and 280-290 nm have been attributed to the
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intraligand π*←π transition located on the N-C-S and S-C-S moieties of the dithiocarbamato
ligands, respectively [46,47]. In the range 300-700 nm, the spectra of the two mononuclear species
(Fig. S9 and S10) consist of three absorption bands, significantly differing from each other in terms
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of intensity and spectral shape. In fact, based on the high value of the molar extinction coefficient (ε
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> 103 M-1 cm-1), the absorption band at about 330-360 nm can be assigned to a charge-transfer (CT)
transition rather than to d-d transitions. As the dithiocarbamato ligand is characterized by both filled
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π (bonding) and empty π* (antibonding) orbitals localized on the sulfur atoms, an interaction with
the orbitals t2g of the metal ion is conceivable. Although both the transitions of the type ligand-tometal (d←π, LMCT) and metal-to-ligand (π←d, MLCT) charge transfer are theoretically allowed,
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the first one is the most favored in this case, as it can easily occur when the metal is in a high
oxidation state. On the other hand, the bands at 440-460 nm and at ca. 600 nm may be assigned to
d-d transitions. According to the Tanabe-Sugano diagrams, the crystal field ground state of low-spin
Ru(III) centers is 2T2g arising from the t2g5eg0 configuration in an Oh environment. All d-d
transitions are spin-allowed and the absorption spectrum should show four bands, as the excited
states are 2Eg, 2T2g, 2A1g and 2A2g,2T1g (with the last two being degenerate) [40]. It is known that the
d-d transitions have small molar extinction coefficients (ca. 1-100 M-1 cm-1) since they are Laporteforbidden. Nevertheless, the X-ray structure of [Ru(PDT)3] shows a slightly distorted octahedral
geometry of the complex, therefore, upon loss of symmetry, it shows more intense d-d transitions (ε
ca. 1000 M-1 cm-1) [22]. The high molar extinction coefficients observed for the bands at 440-460
nm can be explained by considering also the contribution of charge-transfer transitions.
Interestingly, in the case of [Ru(CDT)3], the aromatic nature of the ligand emphasizes this aspect,
giving rise to a ε value of ca. 9000 M-1 cm-1. Finally, for these Ru(III) complexes, it is worth noting
16
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that the absorption bands observed in the visible region are very broad, supporting the hypothesis
that some transitions are overlapped.
The absorption spectra of the [Ru2L5]Cl derivatives show an intense band at 330-360 nm and a
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broad weak band at 420-470 nm (Fig. S10 and S12). Due to their quite dissimilar structures, any
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correlation between the spectra found for mono- and di-nuclear complexes cannot be established.
Dinuclear complexes involve two ruthenium(III) ions antiferromagnetically coupled, overall
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annihilating the paramagnetic effect of each metal center [21]. Therefore, since a strong metal-metal
interaction may take place, the absorption band at 330-360 nm can be ascribed to a metal-metal to
ligand charge transfer transition (MMLCT), whereas the very broad band at 420-470 nm may be
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due to a metal-to-metal charge transfer (MMCT) (Fig. S10 and S12). In fact, their molar extinction
coefficient values are higher than those expected for d-d transitions. In particular, the former
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(MMLCT) involves a metal–metal bonding orbital as the donor, similarly to other ruthenium
dinuclear complexes with diverse bridging ligands[48,49]. On the other hand, the latter (MMCT) is
unlikely to be a d–d transition in light of the strong interaction between the two metal centers [21].
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On passing from Ru(III)-PDT dinuclear derivatives to CDT counterparts, about a 3-fold and 17-fold
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increase of the molar extinction coefficient is observed for MMLCT and MCT transitions,
respectively. In addition, a bathochromic and a hypsochromic shift is detected for the same bands.
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To elucidate these phenomena, DFT calculations are planned. However, the stronger the sulfurmetal bond, the shorter the Ru-Ru distance (see Section 3.1.1). This may account for a higher ε
value for the aromatic derivatives.
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3.2. Pluronic® F127 block copolymer as a micellar nanocarrier
3.2.1. Pluronic® F127. Generally, metal compounds with scarce solubility in aqueous solutions
such as cell culture media (i.e., D-MEM) or other aqueous media suitable for cell culture conditions
(e.g., saline solution or PBS), are first dissolved in an organic solvent (e.g., DMSO, EtOH) to
facilitate the dissolution. These organic solvents, if added in cell culture medium at low
concentrations (< 0.5% v/v), are well tolerated by cells; thus, their presence under this limit does
not affect the IC50 values found for the investigated cytotoxic species. Ru(III)-PDT derivatives are
soluble in DMSO. A previous work reported the stability of [Ru(PDT)3] and of the two
[Ru2(PDT)5]Cl isomers in such organic solvent for several days. Moreover, these compounds
proved stable also in D-MEM cell-culture medium by means of UV-Vis spectrophotometry [22].
Due to the low solubility of Ru(III)-CDT species in the aforementioned organic solvents, new
strategies are required to treat cells with these compounds in aqueous media.
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The copolymer Pluronic® F127, through a process of micelle formation (micellization), allows for
the solubilization of our Ru-complexes in aqueous media. Such block copolymer is a non-ionic
surfactant consisting of hydrophilic poly(ethylene oxide) (PEO) and hydrophobic poly(propylene
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oxide) (PPO), arranged in A-B-A tri-block structure (PEO106-PPO70-PEO106), with an average
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molecular weight of 12,600 and a critical micelle concentration (CMC) of 2.8·10-6 M [50,51]. The
micelle structure will comprise a hydrophobic PPO region covered by a hydrophilic shell, made up
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of PEO chains. Internalization of low-MW compounds into Pluronic® micelles can increase their
solubility and stability in aqueous media. In case of biologically active species, this would
significantly enhance their bioavailability [52].
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Pluronic® F127 is one of the least toxic copolymers among all those commercially available [53].
Remarkably, the cytotoxicity exhibited by Pluronic® micelles towards non-cancerous cells was
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significantly lower than that observed in case of cancerous cells [26], pointing out inherent
selectivity properties. In fact, drug encapsulation in micelles reduces extravasation into normal
tissues. The diffusion is indeed increased in tumor tissues by means of the enhanced permeability
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and retention (EPR) effect, which is related to the abnormal high permeability of tumor blood
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vessels, thus resulting in a passive drug targeting to tumors. Because of these interesting properties,
Pluronic® F127 has been used in combination with Pluronic® L61 to obtain doxorubicin-loaded
mixed micelles, which have already reached Phase III stage in human clinical trials [53,28]. Our
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research group has recently studied a Pluronic® F127-based supramolecular system encapsulating
Au(III)-based compounds to yield a new strategy potentially exploitable in anticancer
chemotherapy [29,30]. Successively, there have been attempts to load Ru(III)-based complexes into
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similar systems [31,32].
In this work, the loaded compound/copolymer ratio (w/w) has been optimized to obtain full
solubilization of the Ru(III) complex and to avoid gelification processes in aqueous media. In order
to promote the micellization process, each sample was sonicated for 5 min to favor hydrophobic
interactions of the compounds with the poly(propylene oxide) domains (Fig. 3).
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Fig. 3. Scheme of the micelle formation and Ru(III) complex internalization process by Pluronic ® F127 block
copolymer.
3.2.2. PF127 micelle-loaded Ru(III)-CDT and Ru(III)-PDT complexes: UV-Vis stability studies.
T
Generally DMSO has been used so far as a vehicle for in vitro testing of our poor water soluble
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complexes. In this work, supramolecular carriers have been taken into account to enhance the
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bioavailabilty of our hydrophobic Ru(III)-dtc complexes. In particular, Pluronic® F127 micelles
were loaded with mono and dinuclear Ru complexes of CDT and PDT and tested for their stability
in saline solution (NaCl 0.9% w/v) over 72 hours at 37°C by UV-Vis spectrophotometry.
No significant change was observed over time. The absence of any modification of Ru(III)
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complexes after encapsulation (Fig. S13-S16) points out Pluronic® F127 as a suitable solubilizing
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agent for in vitro cytotoxicity studies on the investigated complexes.
3.3. In vitro cytotoxicity test on HeLa and HCT 116 cell lines
The in vitro antiproliferative activity of Ru(III)-CDT and Ru(III)-PDT, both mononuclear and
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dinuclear species, has been evaluated against HeLa and HCT 116 human tumor cell lines, over 24-
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and 72-h treatment. HeLa (cervix adenocarcinoma) cells represent a starting point for preliminary
testing in light of their widespread use in the last sixty-five years [55]. HCT 116 colon carcinoma
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cells were chosen as this tumor represents the third most commonly diagnosed cancer in males and
the second in females [1,55].
All the complexes were tested in saline solution upon micellization with PF127 or, when possible
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(Ru(III)-PDT complexes), upon previous dissolution in DMSO. Cisplatin was instead dissolved in
saline solution, to avoid the formation of the less cytotoxic species cis-[PtCl(NH3)2(DMSO)]+ [57].
Remarkably, the dilution of the cell culture medium with the saline solution (up to 29% v/v) did not
affect the growth conditions. In fact, for each test, the amount of grown cells was comparable in the
presence of either pure or diluted medium (both over 24 h and 72 h). Over 24 hours, none of the
compounds reached the IC50 value, including the reference drug. This is in agreement with the slow
ligand-substitution kinetics of both Ru(III) and Pt(II) complexes [58]. Therefore, the attention was
addressed to the 72-h treatment, which is a commonly used incubation time in preliminary
screening tests, involving also cisplatin for comparison purposes. The calculated IC50 values after
72-h treatment are summarized in Table 4.
Table 4. IC50 values (μM) evaluated after 72-h treatment with carbazole and Ru(III)-dtc (CDT and PDT) complexes in
Pluronic® F127 micelles and DMSO (reference drug: cisplatin).
Compound
HeLa
19
HCT 116
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>20
>20
PL{[Ru(PDT)3]}
5.9 ± 0.9
4.9 ± 0.2
PL{α-[Ru2(CDT)5]Cl}
>20
>20
PL{β-[Ru2(CDT)5]Cl}
>20
>20
PL{β-[Ru2(PDT)5]Cl}
0.28 ± 0.04
PL{carbazole}
>20
[Ru(PDT)3]a
>20
β-[Ru2(PDT)5]Cla
0.63 ± 0.06
0.90 ± 0.03
Carbazolea
>20
>20
6.6 ± 0.3
IP
>20
>20
15.96 ± 0.08
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Cisplatin
0.56 ± 0.08
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b
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PL{[Ru(CDT)3]}
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PL{compound} = micellar derivatives; a = solubilized in DMSO; b = solubilized in saline solution.
(The error was evaluated as standard deviation of the average IC50 value deriving from three or more independent
experiments)
Among the investigated formulations, three showed promising antiproliferative activity against both
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the human tumor cell lines with IC50 values comparable or lower than cisplatin, which was found
more active towards HeLa cells than HCT ones. As an example, Fig. 4 reports a plot related to the
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results obtained by treating HeLa and HCT116 cells with the micelle formulation PL{β-
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[Ru2(PDT)5]Cl}.
Fig. 4. Sensitivity profile of HeLa (continuous line) and HCT166 (dotted line) cell lines to PL{β-[Ru2(PDT)5]Cl} as a
function of compound concentration. The plots come from the average of three independent experiments, each one
being carried out under quadruplicate conditions.
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Interestingly, the Ru(III)-PDT dinuclear complex displayed a significantly higher activity when
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loaded into Pluronic® F-127 micelles if compared to the standard administration via DMSO. This
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phenomenon occurs also for the mononuclear PDT complex, associated with at least 3-fold increase
of anticancer activity.. The intrinsic nature of Pluronic® F-127 may account for such a behavior. In
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fact, Batrakova and Kabanov already proposed that this class of copolymers may act as a biological
response modifier [52,59]. Pluronic® surfactants sensitize multi-drug resistant (MDR) cancer cells
since they can (i) be incorporated into membranes, changing their microviscosity; (ii) induce a
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dramatic reduction in ATP levels in cancer and barrier cells; (iii) inhibit drug efflux transporters;
(iv) induce release of cytochrome c and increase reactive oxygen species (ROS) levels in the
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cytoplasm, thus favoring cell death; (v) enhance pro-apoptotic signaling and decrease anti-apoptotic
defense; (vi) inhibit the glutathione/glutathione S-transferasedetoxification system and (vii) hamper
drug sequestration within cytoplasmic vesicles. Therefore, in order to assess the PF127 vehicle
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cytotoxic effects we have also treated cells with empty Pluronic® F-127 micelles at different
observing any relevant effect.
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concentrations of copolymer (comparable to those used for solubilizing Ru(III) complexes), without
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In the light of the obtained results, the presence of the rigid PDT ligand has been associated with a
great anticancer activity in vitro. This result has been observed not only with ruthenium, but also
with gold and copper complexes [24,60,61].
Ru(III)-CDT complexes encapsulated in Pluronic® F127 micelles did not result active and, because
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of their poor solubility, they could not be tested with DMSO or EtOH as vehicles.
3.4. Release tests and log P comparison
The ability of a molecule to pass through the biological barriers (e.g., cell membrane,
gastrointestinal barrier) is often described by the n-octanol-water partition coefficient (log P) [62].
Table 5 summarizes the log P values obtained for the investigated mono- and dinuclear Ru(dtc)
complexes.
Table 5. Log P values of the evaluated Ru(III) derivatives as partition coefficient n-octanol/water.
Compound
log P (pH 7; 25 °C)
Ru(PDT)3
>4
Ru(CDT)3
>4
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0.92 ± 0.07
β-[Ru2(CDT)5]Cl
1.56 ± 0.09
T
β-[Ru2(PDT)5]Cl
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All the Ru(III) derivatives showed a log P value > 0, pointing out a great affinity to lipophilic
systems, including the phospholipidic layer of cell membrane and, on the other hand, a great affinity
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for the hydrophobic core of supramolecular carriers, both features being essential for biological
applications [63]. The results well correlate with the neutral and hydrophobic nature of
mononuclear complexes (log P > 4) as well as with the ionic character of their dinuclear
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counterparts, the latter being associated with a lower log P value than the former. Moreover,
concerning the dinuclear derivatives, it should be underlined the presence of the aromatic moiety in
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the Ru-CDT derivative, bearing five hydrophobic ligands, results in a higher log P value than the
PDT counterpart.
Only the ionic derivatives were chosen for dialysis studies as (i) they are endowed with a
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pharmacologically-relevant partition coefficient [64] and (ii) upon release, they possess more
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chances to be spectrophotometrically detectable, due to the higher solubility in saline solution and
higher molar extinction coefficient. UV-Vis measurements were carried out to monitor the micelle-
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unloading of the encapsulated Ru-PDT and Ru-CDT dinuclear complexes, through a dialysis
membrane in saline solution (as described in section 2.13). The samples collected for both the
complexes displayed a similar spectral trend, with a band at 204 nm increasing in intensity over
time and progressively undergoing a weak red-shift (Fig. S17, panel a and b). By considering both
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the membrane cut-off and the equilibrium of micellar aggregates with their monomeric components
at concentrations higher than the CMC (2.8·10-6 M), this behavior is ascribable to the presence of
free PF127 monomer (MW 12,600) in the saline solution (Fig. S17, panel c) [50,51]. On the other
hand, the total lack of spectral features attributable to the Ru(III) complexes (see Fig. S10 and S12)
confirms the high stability of the investigated supramolecular formulations (previously discussed in
section 3.2.2), thus hampering compound release over 96 hours. On the contrary, even if a defined
amount of compound was released, this was not detected because of a very poor solubility of the
tested complexes in saline solution. Precipitation phenomena were indeed observed inside the
dialysis bag after eight days as a brown mud for both complexes.
These findings could correlate with the higher in vitro activity of PF127-encapsulated Ru-PDT
dinuclear complex, compared to its DMSO counterpart. In fact, a clathrin-mediated internalization
process could be involved in the uptake and hence activity of the studied compounds [65]. In other
words, we hypothesize the interaction between the carrier and the cell membrane plays a key role in
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a passive and huge delivery of ruthenium-containing molecules with PDT ligand. On the contrary,
the aromatic nanoformulation does not trigger cancer cell death, likely because of a non-proper
release of its Ru-CDT cargo. In fact, the strong hydrophobic character of the CDT moiety, as well
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as its planarity, may prevent the complex release as a consequence of a too long-lasting
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supramolecular interaction (longer than 72 hours) either in the cytoplasm upon cell-uptake or in the
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cell culture medium.
4. Concluding remarks
In this work, we have reported the synthesis and characterization of mono- and dinuclear Ru(III)
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complexes in which the metal ion coordinates to dithiocarbamato derivatives of cyclic aromatic or
aliphatic amines, forming homoleptic compounds. The structural features of the novel derivatives
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[Ru(CDT)3] and [Ru2(CDT)5]Cl have been compared with their Ru-pyrrolidine dithiocarbamato
(PDT) analogues, with the main goal of obtaining structure-activity relationships. In contrast with
PDT complexes, the aromatic heterocyclic moiety of CDT leads to a resonance limiting form in
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which the C-N bond possesses mainly a single bond character due to the hampered nitrogen lone
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pair donation. Consequently, sulfur atoms have empty low-energy orbitals which can accept πback-donation from metal d orbitals. These features have been fully characterized by FT-IR
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spectrophotometry. The thorough characterization has pointed out a stronger Ru-S bond for the
aromatic derivatives compared to the PDT ones, thus explaining the lower reactivity, under
biological conditions, observed for the former. Moreover, Pluronic® copolymer was used to
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encapsulate the Ru(III) derivatives, ultimately improving their water solubility, stability and
bioavailability. The use of supramolecular architectures (e.g., micelles, liposomes) for poor watersoluble metal derivatives is a new horizon in cancer treatment [29,32] and some platinum-based
formulations have already reached clinical trials [28]. Our results on HeLa and HCT 116 cancer cell
lines highlight the activity of the [Ru2(PDT)5]Cl derivative, which increases when delivered via the
PF127 carrier. These promising findings are the base for future in vivo investigations. On the other
hand, the formulations with Ru(III)-CDT have shown no significant activity against the tested
human cancer cell lines. Log P evaluations have underlined the pronounced hydrophobic nature of
the complexes, entailing a strong interaction with the PPO units of the Pluronic® F127 micelles, and
hence preventing the release of the loaded compounds. A number of studies are planned to elucidate
the mechanism of action of micelles loaded with the Ru(III)-PDT complexes. Concluding, this work
paves the way to the use of micellar formulations for the solubilization of our hydrophobic
complexes overcoming the use of DMSO in future in vivo experiments.
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Acknowledgements
We thank the National Institutes of Health and National Cancer Institute for grant 1R13CA200223-01A1 (Conference
Organization support, 1st International Symposium on Clinical and Experimental Metallodrugs in Medicine: Cancer
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Chemotherapy, CEMM).
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We are grateful to Dr. L. Calore (Dept. of Chemical Sciences, University of Padova, Italy) and Dr. D. Dalzoppo (Dept.
of Pharmaceutical and Pharmacological Sciences, University of Padova, Italy) for technical support, and to
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A.R.TE.M.O. Association and T.R.N. IMBALLAGGI– logistic services (www.trnimballaggi.it/en/), for financial
support.
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Synopsis
This work presents the synthesis and characterization of Ru(III) complexes with the
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aromatic carbazole-dithiocarbamato ligand, to compare their anti-proliferative
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properties with those of the aliphatic pyrrolidine-dithiocarbamato analogues
(structure-activity relationships). The nonionic surfactant PF127 was used in in vitro
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cytotoxicity experiments, thus overcoming the poor water solubility of the
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compounds.
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Highlights
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Investigated four Ru(III) complexes with aromatic and aliphatic dithiocarbamato ligands
Their peculiar electronic structure and hydrophobicity result in different cytotoxicity
Their encapsulation in micellar nanocarriers improves water solubility
Dinuclear ionic compounds show higher activity than their mononuclear counterparts
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