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Metal dipyrrin complexes as potential photosensitizers for photodynamic therapy
Journal Pre-proofs Research paper Metal Dipyrrin Complexes as Potential Photosensitizers for Photodynamic Therapy Johannes Karges, Olivier Blacque, Gilles Gasser PII: DOI: Reference:
S0020-1693(19)32004-3 https://doi.org/10.1016/j.ica.2020.119482 ICA 119482
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
11 January 2020 27 January 2020 27 January 2020
Please cite this article as: J. Karges, O. Blacque, G. Gasser, Metal Dipyrrin Complexes as Potential Photosensitizers for Photodynamic Therapy, Inorganica Chimica Acta (2020), doi: https://doi.org/10.1016/j.ica.2020.119482
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© 2020 Published by Elsevier B.V.
Metal Dipyrrin Complexes as Potential Photosensitizers for Photodynamic Therapy Johannes Kargesa, Olivier Blacqueb and Gilles Gassera,*
a
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology, 75005 Paris, France.
b
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
*Email: [email protected]; Tel. +33 1 44 27 56 02; WWW: www.gassergroup.com.
ORCID-ID: Johannes Karges: 0000-0001-5258-0260 Olivier Blacque: 0000-0001-9857-4042 Gilles Gasser: 0000-0002-4244-5097
Keywords: Anticancer, Medicinal Inorganic Chemistry, Metals in Medicine, Photodynamic Therapy.
ABSTRACT Over the last decades, the use of photodynamic therapy (PDT) for the treatment of various types of cancer as well fungal, viral and bacterial infections has received increasing attention. Despite its clinical success, the currently approved photosensitizers (PSs) are associated with poor water solubility, aggregation, low photostability and a long excretion time. To overcome these limitations, much research is devoted towards the development of PSs based on transition metal complexes. However, the majority of metals used for this purpose are rare and expensive. Therefore, it would be of high interest to develop effective PDT PSs based on cheap and abundant metals. In this article, the use of Cu(II) and Ni(II) dipyrrin complexes as potential PDT PSs against cancer is presented. As required for PDT applications, these complexes were found to have a strong absorption in the green spectrum and to be stable in an aqueous solution in the dark as well as upon light irradiation. Biological studies revealed that the complexes have a very low cytotoxic effect in the dark with a slight effect upon irradiation at 510 nm in human cervical carcinoma (HeLa) cells.
Highlights: -
Synthesis and characterisation of two Cu(II) and Ni(II) dipyrrin complexes.
-
Investigations of the use of these compounds as chemotherapeutic and photodynamic therapy agents.
-
Photophysical studies studied revealed absorption in the biological spectral window.
-
(Photo-)cytotoxicity of the metal complexes in cancerous human cervical carcinoma cells was assessed.
Graphical Abstract:
1. Introduction Dipyrrins are small organic molecules consisting of two pyrroles moieties, which are linked together over a methionine group. Upon deprotonation, the ligand scaffold benefits from resonance stabilisation, which enables it to act as a bidentate ligand and to coordinate to metal ions. As the predominant class of compounds, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) complexes and its derivatives are highly investigated due to their attractive photophysical properties, which include high stability in a biological environment and sharp fluorescence with very high quantum yields. These photophysical properties initiated extensive research for the use of these compounds as biomedical fluorescence probes or chemical sensors.1-4 Apart from boron, the dipyrrin ligand scaffold is also able to coordinate to many other metal ions like, for example, Ni(II), Zn(II), Cd(II), Cu(II), Fe(III), Ga(III), Co(III), In(III) and Rh(III). Due to the possibility to generate nanoarchitectures, these compounds have found application as one-dimensional nanowires, two-dimensional nanosheets or metalorganic frameworks (MOFs).5-14 Recently, research has shifted to employ the great photophysical properties of bis(dipyrrinato)metal(II) and tris(dipyrrinato)metal(III) complexes in other fields of research.15-18 Exemplary, the use of bis(dipyrrinato)Zn(II) complexes as fluorescent probes has gained much interest. Despite much progress in the tuning of the photophysical properties of these complexes, the corresponding BODIPY complexes remain the best of the class.19-24 Next to applications as fluorescent probes, dipyrrin complexes and especially BODIPY complexes have also recently received attention for their utilisation as photosensitizers (PSs) for photodynamic therapy (PDT) for the treatment of bacterial, fungal or viral infections as well as various types of cancer (e.g., lung, bladder, oesophageal, and brain cancer).25-30 During a PDT treatment, a PS is injected, either locally or systemically. After a certain circulation time to allow for accumulation of the PS in the targeted region, the target is exposed to light at a specific wavelength, enabling activation of the PS. The latter then generates photocatalytically reactive oxygen species (ROS). Although different kinds of radials can be involved in this process, the majority of PSs are generating singlet oxygen (1O2). As ROS and 1O2 are highly reactive, oxidative stress is caused and cell death triggered.31-35 To date, the majority of PSs are based on a tetrapyrrolic scaffold (i.e., porphyrin, chlorine). Despite their undeniable clinical success, the status of these PDT agents generally remains unsatisfactory. These compounds are generally associated with poor water solubility,
aggregation, low photostability and a long excretion time of the body, leading to photosensitivity.36, 37 To overcome these limitations, much research is devoted towards the development of new classes of PDT PSs or the improvement of the currently approved PDT PSs. Among other concepts, the introduction of a metal ion into a PDT PS was found to sometimes overcome these drawbacks. For example, the tetrapyrrolic compounds complexed with Sn(IV) (Purlytin®), Lu(III) (Lutrin®/Antrin®), Al(III) (Photosens®) and Pd(II) (TOOKAD®/TOOKAD soluble®) have been or are currently investigated in clinical trials or have even been approved.38,
39
In addition, the Ru(II) polypyridyl complex TLD-1433 is
currently investigated in Phase II clinical trials to treat certain types of bladder cancer. Therefore, much efforts are devoted towards the development of transition metal complexes as PDT PSs.40 The majority of complexes studied are based on Ru(II),41-51 Os(II),52-54 Ir(III),55-61 Rh(III)62-64 and Re(I).65-69 As these metals are expensive, it would be of high interest to develop PSs based on cheap, abundant metals. With this aim in mind, we have recently investigated the use of Fe(II) polypyridine complexes as potential PDT PSs.70, 71 Based on the clinical success of tetrapyrrolic compounds and the high potential of transition metal complexes, it would be of great interest to combine both concepts by preparing bis(dipyrrinato)metal(II) complexes based on cheap, abundant metals. In this context, we have recently described bis(dipyrrinato)Zn(II) complexes with exceptionally long excited state lifetimes. By placing iodine atoms on the ligand scaffold, the heavy atom effect was able to promote the intersystem crossing process, generating PDT active compounds.72 Capitalising on these results, we were interested in determining if the scope of metals used for this purpose could be extended. In this article, we report our results on the use of Cu(II) and Ni(II) dipyrrin complexes. This class of complexes has been previously studied in the literature primarily due to their electrochemical properties, their ability to form nanoarchitectures but also their catalytic properties like the generation of hydroxyl radials.73-79 Although the majority of Ni(II) and Cu(II) complexes are known to be non-luminescent, some compounds including the structurally
related
Cu(II)(5-phenyldipyrromethene)2
and
Cu(II)(1,5,9-
triphenyldipyrromethene)2 complexes were previously found to be emitting.80-82 Capitalising on these studies, two new compounds (complexes 1-2) were synthesised and characterised in depth, including by single crystal X-ray diffraction. Their potential as PDT PSs is reported.
2. Experimental Section
2.1. Instrumentation and methods 1H
and 13C NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer. Chemical
shifts (δ) are reported in parts per million (ppm) referenced to tetramethylsilane (δ 0.00) ppm using the residual proton solvent peaks as internal standards. Coupling constants (J) are reported in Hertz (Hz) and the multiplicity is abbreviated as follows: s (singlet). ESI-MS experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientific (Thermo Fisher Scientific) and operated in positive ionization mode. Elemental microanalyses were performed on a Thermo Flash 2000 elemental analyzer.
2.2. Materials All chemicals were obtained from commercial sources and were used without further purification. Dulbecco’s Modified Eagles Medium (DMEM), Dulbecco’s Modified Eagles Medium supplemented with nutrient mixture F-12 (DMEM/F-12), Fetal Bovine Serum (FBS), Gibco Penicillin-Streptomycin-Glutamine (Penstrep), Dulbecco’s Phosphate-Buffered Saline (PBS) were purchased from Fisher Scientific and Resazurin from ACROS Organics.
2.3. Synthesis (Z)-2-((3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-3,5-dimethyl-1H-pyrrole: 2,4,6-Trimethylbenzaldehyde (200 mg, 1.35 mmol) and 2,4-dimethylpyrrole (322 mg, 3.38 mmol) were dissolved in dry dichloromethane (30 mL) under N2 atmosphere. Trifluoroacetic acid (100 L) was added and the reaction mixture was stirred at room temperature for 24 h. After this time, tetrachloro-p-benzoquinone (332 mg, 1.35 mmol) was added and the reaction mixture stirred additional 4 h. Afterwards the solution was filtered and the solvent evaporated. The crude product was purified by column chromatography on neutral alumina with a gradient of dichloromethane/hexane. The fractions containing the product were united and the solvent was removed. The solid was washed with Et2O and dried. The compound was used for the next synthetic step without further purification. (Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4-dimethyl1H-pyrrole:
(Z)-2-((3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-3,5-dimethyl-1H-pyrrole (318 mg, 1 mmol) was dissolved in a 1:1 chloroform/ethanol (150 mL) mixture to which iodine (508 mg, 2 mmol) and iodic acid (352 mg, 2 mmol) was added portion wise. The mixture was stirred at room temperature for 4 h under N2 atmosphere. After this time, the mixture was diluted with chloroform (500 mL) and the organic layer was washed three-times with 5% sodium sulphite solution (200 mL). The product was dried over anhydrous sodium sulphate and the solvent was evaporated. The compound was used for the next synthetic step without further purification. Bis((Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4dimethyl-1H-pyrrole)nickel(II) (1): (Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4-dimethyl-1Hpyrrole (200 mg, 0.35 mmol) was dissolved in a mixture of 3:2:3 butanol/ethanol/H2O (250 mL). Ni(OAc)2 . 4H2O (35 mg, 0.14 mmol) and Et3N (142 mg, 1.4 mmol) were added and the reaction mixture heated at reflux for 24 h under nitrogen atmosphere. After completion of the reaction, the solvent was removed. The crude product was purified by column chromatography on silica with a gradient of dichloromethane/pentane. The fractions containing the product were united and the solvent was removed. The solid was washed with Et2O and dried. Yield: 53%. 1H-NMR
(CD2Cl2, 400 MHz): 6.98 (s, 4H), 2.35 (s, 6H), 2.09 (s, 12H), 2.05 (s, 12H), 1.53 (s,
12H). 13C-NMR (CD2Cl2, 100 MHz): 157.8, 145.6, 144.4, 139.3, 136.2, 135.1, 129.2, 129.0, 84.3, 23.2, 19.6, 17.8, 17.5. ESI-HRMS (pos. detection mode): calcd for C44H47I4N4Ni [M+H]+ m/z 1196.9333; found: 1196.9327, UV/VIS (CH2Cl2) λmax (ε x 103): 526 (117.7), 392 (17.5) nm, Elemental analysis calcd for C44H46I4N4Ni (%): C 44.14, H 3.87, N 4.69; found: C 44.36, H 3.95, N 4.31. Bis((Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4dimethyl-1H-pyrrole)copper(II) (2): (Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4-dimethyl-1Hpyrrole (200 mg, 0.35 mmol) was dissolved in a mixture of 3:2:3 butanol/ethanol/H2O (250 mL). Cu(OAc)2 (25 mg, 0.14 mmol) and Et3N (142 mg, 1.4 mmol) were added and the reaction mixture heated at reflux for 24 h under nitrogen atmosphere. After completion of the reaction, the solvent was removed. The crude product was purified by column chromatography on silica with a gradient of dichloromethane/pentane. The fractions containing the product were united and the solvent was removed. The solid was washed with Et2O and dried. Yield: 74%. ESI-
HRMS (pos. detection mode): calcd for C44H47I4N4Cu [M+H]+ m/z 1201.9275; found: 1201.9278, UV/VIS (CH2Cl2) λmax (ε x 103): 526 (96.2), 392 (14.0) nm, Elemental analysis calcd for C44H46I4N4Cu (%): C 43.97, H 3.86, N 4.66; found: C 43.72, H 4.09, N 4.49.
2.4. X-ray crystallography Single crystal X-ray diffraction data were collected at 183(1) K on a Rigaku OD Synergy (Pilatus 200K detector) diffractometer for 1 and on a Rigaku OD Supernova (Atlas CCD detector) diffractometer for 2 equipped with Oxford liquid-nitrogen Cryostream coolers and using a single wavelength X-ray source from a micro-focus sealed X-ray tube with the Cu Kα radiation (λ = 1.54184 Å). The selected single crystals were mounted using polybutene oil on a flexible loop fixed on a goniometer head and transferred to the diffractometer. Preexperiments, data collections, data reductions and analytical absorption corrections83 were performed with the program suite CrysAlisPro.84 Using Olex2,85 the structures were solved with the SHELXT86 small molecule structure solution program and refined with the SHELXL2018/3 program package87 by full-matrix least-squares minimization on F2. The crystal data collections and structure refinement parameters are summarized in Table 1. CCDC 1976222 (for 1) and 1976221 (for 2) contain the supplementary crystallographic data for these compounds, and can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. In the crystal structure of 1, the Ni complex lies on a two-fold axis, only half of the molecule had to be refined the second half being reproduced by a symmetry operation. Solvent molecules of methanol cocrystallized with the main species but a reasonable modelling of the disordered positions could not be obtained. The PLATON SQUEEZE tool88 was used to calculate the number of electrons attributed to solvent molecules in the primitive cell and to take this solvent contribution into account in the calculated structure factors. A ratio of three molecules of methanol for one molecule of the Ni complex was considered. In the crystal structure of 2, two crystallographically independent molecules of the Cu complex are present in the asymmetric unit. Solvent molecules of hexane cocrystallized with the main species but like for 1 a reasonable modelling of the disordered positions could not be obtained. The PLATON SQUEEZE tool was and a ratio of four molecules of the Cu complex for one molecule of solvent was considered. For both structures the solvent molecules were included in the CIF files (formula sum and formula moiety) causing many alerts in the IUCr checkCIF reports.
2.5. Spectroscopic measurements The absorption and emission of the samples has been measured with a SpectraMax M2 Spectrometer (Molecular Devices). For the determination of the luminescence quantum yield, the samples were prepared in degassed CH2Cl2 solution with an absorbance of 0.1 at 520 nm. The luminescence quantum yields were calculated by comparison with the reference Rhodamine B in ethanol (Φem=0.50)89 applying the following formula:
Φem,
sample
= Φem, reference ∗
𝐹reference Fsample
∗
Isample Ireference
∗
(
nsample nreference
2
)
F = 1 ― 10 ―𝐴 Φem = luminescence quantum yield, F = fraction of light absorbed, I = integrated emission intensities, n = refractive index, A = absorbance of the sample at irradiation wavelength
2.6. Singlet oxygen measurements -
direct evaluation
The samples were prepared in an air saturated CH3CN or D2O solution with an absorbance of 0.2 at 450 nm. This solution was irradiated in fluorescence quartz cuvettes (width 1 cm) using a mounted M450LP1 LED (Thorlabs) whose irradiation, centred at 450 nm, has been focused with aspheric condenser lenses. The intensity of the irradiation has been varied using a T-Cube LED Driver (Thorlabs) and measured with an optical power and energy meter. The emission signal was focused and collected at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator. A longpass glass filter was placed in front of the monochromator entrance slit to cut off light at wavelengths shorter than 850 nm. The slits for detection were fully open. As a detector an EO-817L IR-sensitive liquid nitrogen cooled germanium diode detector (North Coast Scientific Corp.) has been used. The singlet oxygen phosphorescence at 1270 nm was measured by recording spectra from 1100 to 1400 nm. For the data analysis, the singlet oxygen luminescence peaks at different irradiation intensities were integrated. The resulting areas were plotted against the percentage of the irradiation intensity and the slope of the linear regression calculated. The absorbance of the sample was
corrected with an absorbance correction factor. As reference for the measurement in a D2O solution [Ru(bipy)3]Cl2 (ΦRu(bipy)₃Cl₂ = 0.22)90 was used and the singlet oxygen quantum yields were calculated using the following formula: 𝛷sample = 𝛷reference ×
Ssample Sreference
×
Ireference Isample
I = I0 × (1 ― 10 ―𝐴)
Φ = singlet oxygen quantum yield, S = slope of the linear regression of the plot of the areas of the singlet oxygen luminescence peaks against the irradiation intensity, I = absorbance correction factor, I0 = light intensity of the irradiation source, A = absorbance of the sample at irradiation wavelength.
-
indirect evaluation
For the measurement in CH3CN: The samples were prepared in an air-saturated CH3CN solution containing the complex with an absorbance of 0.1 at the irradiation wavelength, N,Ndimethyl-4-nitrosoaniline aniline (RNO, 24 µM) and imidazole (12 mM). For the measurement in PBS buffer: The samples were prepared in an air-saturated PBS solution containing the complex with an absorbance of 0.1 at the irradiation wavelength, N,N-dimethyl-4nitrosoaniline aniline (RNO, 20 µM) and histidine (10 mM). The samples were irradiated on 96 well plates with an Atlas Photonics LUMOS BIO irradiator for different times. The absorbance of the samples was measured during these time intervals with a SpectraMax M2 Microplate Reader (Molecular Devices). The difference in absorbance (A₀-A) at 420 nm for the CH3CN solution or at 440 nm a PBS buffer solution was calculated and plotted against the irradiation times. From the plot the slope of the linear regression was calculated as well as the absorbance correction factor determined. The singlet oxygen quantum yields were calculated using the same formulas as used for the direct evaluation.
2.7. Stability in CH2Cl2/H2O The stability of the complex in CH2Cl2 or H2O (2% DMSO, v%) was investigated by UV/Vis spectroscopy. The complex was dissolved with about 0.5 absorption at its absorption maximum
and stored at room temperature in the dark. The absorption spectrum from 300-600 nm was recorded with a SpectraMax M2 Microplate Reader (Molecular Devices) after each time interval (0, 1, 4, 12, 24, 48 h) and compared.
2.8. Photostability The photostability of the complex in CH2Cl2 was investigated by UV/Vis spectroscopy. The complex was dissolved with about 0.5 absorption at its absorption maximum and exposed to a constant LED irradiation 510 nm (4.2 mW/cm2) with an Atlas Photonics LUMOS BIO irradiator. The absorption spectrum from 300-600 nm was recorded with a SpectraMax M2 Microplate Reader (Molecular Devices) after each time interval (0, 5, 10, 20, 30 min) and compared.
2.9. (Photo-)cytotoxicity The cytotoxicity of the complexes was assessed by measuring cell viability using a fluorometric resazurin assay. The cultivated cells were seeded in triplicates in 96 well plates with a density of 4000 cells per well in 100 μL of media. After 24 h, the medium was removed and the cells were treated with increasing concentrations of the complex diluted in cell media achieving a total volume of 200 μL. The cells were incubated with the complex for 4 h. After this time, the media was removed and replaced with 200 μL of fresh medium. For the phototoxicity studies, the cells were exposed to light with an Atlas Photonics LUMOS BIO irradiator. Each well was constantly illuminated with either a 510 nm irradiation. During this time, the temperature was maintained constantly at 37 °C. The cells were grown in the incubator for additional 44 h. For the determination of the dark cytotoxicity, the cells were not irradiated and after the medium exchange directly incubated for 44 h. After this time, the medium was replaced with fresh medium containing resazurin with a final concentration of 0.2 mg/mL. After 4 h incubation, the amount of the fluorescent product resorufin was determined upon excitation at 540 nm and measurement its emission at 590 nm using a SpectraMax M2 Microplate Reader (Molecular Devices). The obtained data was analyzed with the GraphPad Prism software.
3. Results and Discussion 3.1. Synthesis The Ni(II) (1) and Cu(II) (2) complexes were synthesised as visualised in Scheme 1. In the first step,
the
ligand
scaffold
was
generated
by
reacting
2,4,6-trimethyl-2-
mesitylenecarboxaldehyde with 2, 4-dimethylmethylpyrrole using catalytic amounts of trifluoracetic acid. The ligand was then oxidised using tetrachloro-p-benzoquinone, achieving a complete conjugation of the aromatic system. Following this, the peripheral positions were iodinated using a mixture of iodine and iodic acid. At last, the metal ion was coordinated using the corresponding acetate salt. While the Cu(II) complex (2) is paramagnetic, the analogous Ni(II) complex (1) was found to be diamagnetic and was therefore characterised by 1H- and 13C-NMR
(Figures S1-S2). Worthy of note, structurally related Ni(II) complexes were also
found to be diamagnetic.73-75,77
I
I H
a) NH
HN
N
N
O c)
b) NH
HN
I
I NH
N
NH
d)
M
N N
N
I
I
M = Ni Cu
1 2
Scheme 1. Synthetic procedures for the synthesis of 1-2. a) TFA, CH2Cl2; b) Tetrachloro-pbenzoquinone; c) CHCl3:EtOH (1:1), I2, HIO3; d) butanol/ethanol/H2O (3:2:3), Et3N, 1: Cu(OAc)2, 2: Ni(OAc)2 . 4H2O.
3.2. X-ray crystallography The nickel and copper centers in 1 and 2 (Figure 1, Table S1) adopt a distorted tetrahedral geometry coordinated by the four nitrogen atoms of the two substituted dipyrrinato ligands. The Ni–N bond lengths are 1.930(7) and 1.937(5) Å while the Cu–N bond lengths fall in the similar range 1.931(3) – 1.950(3) Å (from two crystallographically independent molecules). The N–M–N bond angles are 92.8(2)° in 1 (Ni) and 92.89(13) – 93.67(13)° in 2 (Cu), and the
dihedral angles between the two N-M-N planes are 80.99° in 1 (Ni) and 65.11 – 69.00° in 2 (Cu). In the Cambridge Structural Database,91 reported α,α′‐substituted analogues show dihedral angles in the range of 83.6 – 86.2° for Ni complexes77, 92, 93 and 62.9 – 66.9° for Cu complexes.77, 81, 92, 93 Consequently, the geometry of our two new molecules seems to be typical for such metal complexes. Although Ni(II) and Cu(II) complexes favor square planar coordinations, steric repulsion between the methyl groups at the α position of the dipyrrinato ligand prevent planar structures in these complexes. The same feature is observed in our previously reported bis(dipyrrinato)zinc(II) complex.72
Figure 1. a) Molecular structure of 1 (all H atoms are omitted for clarity). The thermal ellipsoids are drawn at the 20% probability level. b) Molecular structure of 2 (one of the two independent molecules; all H atoms are omitted for clarity). The thermal ellipsoids are drawn at the 50% probability level.
3.3. Photophysical Properties After chemical characterisation of the complexes, their photophysical properties were evaluated in view of applications as PDT PSs. An important factor during a PDT treatment is the wavelengths employed as this directly correlates with the light penetration depth inside tissue. Worthy of note, there is no ideal wavelength for a treatment as this depends, among others, on the tumour size and localisation. Very importantly, it has to be ensured that only tumour tissue and not underlying healthy tissue is damaged. However, with the aim to treat deep seated or large tumors, much research is devoted towards the development of PSs with an
absorption in or near the biological spectral window (600-900 nm). This would allow light to penetrate significantly deeper inside tissue/tumour. Having this in mind, the absorption profile of the complexes 1-2 were measured (Figure 2). Both complexes were found to have a strong absorption at 526 nm, which is significantly more red-shifted than many transition metal complexes investigated for PDT applications. Interestingly, both complexes showed a highly similar absorption profile, indicating that the coordinated metal does not drastically influence this property. Worthy of note, previously reported Ni(II) and Cu(II) dipyrrin complexes were found to have similar absorption profiles.94-96 Following this, the luminescence of the complexes upon irradiation at 520 nm was investigated. While no signal for the Ni(II) complex 1 was detected, a low emission signal with a luminescence quantum yield of >0.1% and a maximum at 551 nm for the Cu(II) complex 2 was detected (Figure S3). This result is in agreement with the luminescence study of structurally related Cu(II)(dipyrrin)2 complexes.81
Figure 2. Absorption spectra of 1-2 in CH2Cl2.
Subsequently, the ability of complexes 1 and 2 to generate singlet oxygen (1O2), which is considered the predominant toxic species in PDT upon irradiation at 450 and 510 nm was investigated by two complementary methods: 1) direct by measurement of the phosphorescence of 1O2, 2) indirect by following the change in absorption of a 1O2 scavenger. The experiment was performed in the organic solvent CH3CN and in an aqueous solution. The results showed
that both complexes are poorly generating 1O2 at the detection limit of our used apparatus with quantum yields >1% in CH3CN and in an aqueous solution.
3.4. Stability As a crucial parameter for PDT applications, the stability of a compound needs to be investigated as previous studies have shown that this could be problematic.97-99 To do so, complexes 1-2 were incubated in the dark for varying time points (0 h, 1 h, 4 h, 12 h, 24 h and 48 h) in CH2Cl2 and H2O and the corresponding absorption spectra monitored (Figure S4-S7). As no significant difference were observed, the stability of both metal complexes is indicated. Beside the stability in a chemical environment, the stability upon irradiation remains a different issue. Therefore, the complexes were exposed to light at 510 nm and their absorption spectra monitored for varying time points (0, 5, 10 20 and 30 min). Only minimal changes were observed (Figure S8-S9), suggesting that the complexes are stable upon light irradiation.
3.5. (Photo-)cytotoxicity After an analysis of the chemical and photophysical properties of the compounds, their cytotoxicity on non-cancerous retinal pigment epithelium (RPE-1) and human cervical carcinoma (HeLa) cells in the dark as well as upon irradiation at 510 nm (20 min, 5.0 J/cm2) was investigated using a fluorometric resazurin assay. The results (Table 1) show that the compounds have a very low cytotoxic effect in the dark. Interestingly, the obtained IC50 values of both compounds were found in the same range, indicating that the central metal does not significantly influence this property. Unfortunately, light irradiation was found to have only a negligible effect on the cell viability. This is most likely caused by the poor photophysical properties (i.e., 1O2 generation) of complexes 1-2. In comparison, the PS Protoporphyrin IX (PpIX) and the anticancer drug cisplatin were found to have drastically lower IC50 values.
Table 1. IC50 value (μM) in the dark and upon irradiation at 510 nm (20 min, 5.0 J/cm2) for complexes 1-2, the PDT PS Protoporphyrin IX (PpIX) and the anticancer drug cisplatin in noncancerous retinal pigment epithelium (RPE-1) and human cervical carcinoma (HeLa) cells. Average of three independent measurements.
HeLa
RPE-1
dark
510 nm
dark
510 nm
1
157.1 ± 8.9
159.3 ± 9.1
184.3 ± 6.3
179.5 ± 7.0
2
148.3 ± 7.4
133.9 ± 8.5
172.4 ± 8.6
163.1 ± 8.9
PpIX
>100
2.8 ± 0.2
>100
4.1 ± 0.2
cisplatin
10.5 ± 0.8
-
23.9 ± 1.4
-
4. Conclusions In this study, the role of the metal ions Ni(II) and Cu(II) on the photophysical and biological properties of homoleptic dipyrrin complexes was investigated. Both metal complexes prepared were characterised in-depth, including by single crystal X-ray diffraction. The complexes were found to have a strong absorption in the green area of the electromagnetic spectrum, which is a desired property for PDT agent. Photophysical studies demonstrated that these complexes were poorly emissive and barely produced 1O2 upon irradiation. Stability studies indicated that both compounds were stable in a biological environment and upon light irradiation. Biological evaluation of the compounds in non-cancerous retinal pigment epithelium and human cervical carcinoma cells revealed a very low cytotoxicity in the dark, as requested for a PDT agent. However, only a negligible effect on cell viability was observed upon light irradiation. This is most likely caused by the poor photophysical properties (i.e. 1O2 generation) of the complexes. We are currently pursuing other options to design PDT PSs based on cheap, abundant metals.
Acknowledgements We thank Dr. Philippe Goldner for access to state-of-the-art laser apparatus. This work was financially supported by an ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679), has received support under the program “Investissements d’Avenir” launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.).
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All authors were involved with the design and interpretation of experiments and with the writing of the manuscript. Chemical, photophysical and biological experiments were carried out by J.K. X-ray crystallography was carried out by J.K. and O.B. All authors have given approval to the final version of the manuscript
Highlights: -
Synthesis and characterisation of two Cu(II) and Ni(II) dipyrrin complexes.
-
Investigations of the use of these compounds as chemotherapeutic and photodynamic therapy agents.
-
Photophysical studies studied revealed absorption in the biological spectral window.
-
(Photo-)cytotoxicity of the metal complexes in cancerous human cervical carcinoma cells was assessed.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0020-1693(19)32004-3 https://doi.org/10.1016/j.ica.2020.119482 ICA 119482
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
11 January 2020 27 January 2020 27 January 2020
Please cite this article as: J. Karges, O. Blacque, G. Gasser, Metal Dipyrrin Complexes as Potential Photosensitizers for Photodynamic Therapy, Inorganica Chimica Acta (2020), doi: https://doi.org/10.1016/j.ica.2020.119482
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© 2020 Published by Elsevier B.V.
Metal Dipyrrin Complexes as Potential Photosensitizers for Photodynamic Therapy Johannes Kargesa, Olivier Blacqueb and Gilles Gassera,*
a
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology, 75005 Paris, France.
b
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
*Email: [email protected]; Tel. +33 1 44 27 56 02; WWW: www.gassergroup.com.
ORCID-ID: Johannes Karges: 0000-0001-5258-0260 Olivier Blacque: 0000-0001-9857-4042 Gilles Gasser: 0000-0002-4244-5097
Keywords: Anticancer, Medicinal Inorganic Chemistry, Metals in Medicine, Photodynamic Therapy.
ABSTRACT Over the last decades, the use of photodynamic therapy (PDT) for the treatment of various types of cancer as well fungal, viral and bacterial infections has received increasing attention. Despite its clinical success, the currently approved photosensitizers (PSs) are associated with poor water solubility, aggregation, low photostability and a long excretion time. To overcome these limitations, much research is devoted towards the development of PSs based on transition metal complexes. However, the majority of metals used for this purpose are rare and expensive. Therefore, it would be of high interest to develop effective PDT PSs based on cheap and abundant metals. In this article, the use of Cu(II) and Ni(II) dipyrrin complexes as potential PDT PSs against cancer is presented. As required for PDT applications, these complexes were found to have a strong absorption in the green spectrum and to be stable in an aqueous solution in the dark as well as upon light irradiation. Biological studies revealed that the complexes have a very low cytotoxic effect in the dark with a slight effect upon irradiation at 510 nm in human cervical carcinoma (HeLa) cells.
Highlights: -
Synthesis and characterisation of two Cu(II) and Ni(II) dipyrrin complexes.
-
Investigations of the use of these compounds as chemotherapeutic and photodynamic therapy agents.
-
Photophysical studies studied revealed absorption in the biological spectral window.
-
(Photo-)cytotoxicity of the metal complexes in cancerous human cervical carcinoma cells was assessed.
Graphical Abstract:
1. Introduction Dipyrrins are small organic molecules consisting of two pyrroles moieties, which are linked together over a methionine group. Upon deprotonation, the ligand scaffold benefits from resonance stabilisation, which enables it to act as a bidentate ligand and to coordinate to metal ions. As the predominant class of compounds, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) complexes and its derivatives are highly investigated due to their attractive photophysical properties, which include high stability in a biological environment and sharp fluorescence with very high quantum yields. These photophysical properties initiated extensive research for the use of these compounds as biomedical fluorescence probes or chemical sensors.1-4 Apart from boron, the dipyrrin ligand scaffold is also able to coordinate to many other metal ions like, for example, Ni(II), Zn(II), Cd(II), Cu(II), Fe(III), Ga(III), Co(III), In(III) and Rh(III). Due to the possibility to generate nanoarchitectures, these compounds have found application as one-dimensional nanowires, two-dimensional nanosheets or metalorganic frameworks (MOFs).5-14 Recently, research has shifted to employ the great photophysical properties of bis(dipyrrinato)metal(II) and tris(dipyrrinato)metal(III) complexes in other fields of research.15-18 Exemplary, the use of bis(dipyrrinato)Zn(II) complexes as fluorescent probes has gained much interest. Despite much progress in the tuning of the photophysical properties of these complexes, the corresponding BODIPY complexes remain the best of the class.19-24 Next to applications as fluorescent probes, dipyrrin complexes and especially BODIPY complexes have also recently received attention for their utilisation as photosensitizers (PSs) for photodynamic therapy (PDT) for the treatment of bacterial, fungal or viral infections as well as various types of cancer (e.g., lung, bladder, oesophageal, and brain cancer).25-30 During a PDT treatment, a PS is injected, either locally or systemically. After a certain circulation time to allow for accumulation of the PS in the targeted region, the target is exposed to light at a specific wavelength, enabling activation of the PS. The latter then generates photocatalytically reactive oxygen species (ROS). Although different kinds of radials can be involved in this process, the majority of PSs are generating singlet oxygen (1O2). As ROS and 1O2 are highly reactive, oxidative stress is caused and cell death triggered.31-35 To date, the majority of PSs are based on a tetrapyrrolic scaffold (i.e., porphyrin, chlorine). Despite their undeniable clinical success, the status of these PDT agents generally remains unsatisfactory. These compounds are generally associated with poor water solubility,
aggregation, low photostability and a long excretion time of the body, leading to photosensitivity.36, 37 To overcome these limitations, much research is devoted towards the development of new classes of PDT PSs or the improvement of the currently approved PDT PSs. Among other concepts, the introduction of a metal ion into a PDT PS was found to sometimes overcome these drawbacks. For example, the tetrapyrrolic compounds complexed with Sn(IV) (Purlytin®), Lu(III) (Lutrin®/Antrin®), Al(III) (Photosens®) and Pd(II) (TOOKAD®/TOOKAD soluble®) have been or are currently investigated in clinical trials or have even been approved.38,
39
In addition, the Ru(II) polypyridyl complex TLD-1433 is
currently investigated in Phase II clinical trials to treat certain types of bladder cancer. Therefore, much efforts are devoted towards the development of transition metal complexes as PDT PSs.40 The majority of complexes studied are based on Ru(II),41-51 Os(II),52-54 Ir(III),55-61 Rh(III)62-64 and Re(I).65-69 As these metals are expensive, it would be of high interest to develop PSs based on cheap, abundant metals. With this aim in mind, we have recently investigated the use of Fe(II) polypyridine complexes as potential PDT PSs.70, 71 Based on the clinical success of tetrapyrrolic compounds and the high potential of transition metal complexes, it would be of great interest to combine both concepts by preparing bis(dipyrrinato)metal(II) complexes based on cheap, abundant metals. In this context, we have recently described bis(dipyrrinato)Zn(II) complexes with exceptionally long excited state lifetimes. By placing iodine atoms on the ligand scaffold, the heavy atom effect was able to promote the intersystem crossing process, generating PDT active compounds.72 Capitalising on these results, we were interested in determining if the scope of metals used for this purpose could be extended. In this article, we report our results on the use of Cu(II) and Ni(II) dipyrrin complexes. This class of complexes has been previously studied in the literature primarily due to their electrochemical properties, their ability to form nanoarchitectures but also their catalytic properties like the generation of hydroxyl radials.73-79 Although the majority of Ni(II) and Cu(II) complexes are known to be non-luminescent, some compounds including the structurally
related
Cu(II)(5-phenyldipyrromethene)2
and
Cu(II)(1,5,9-
triphenyldipyrromethene)2 complexes were previously found to be emitting.80-82 Capitalising on these studies, two new compounds (complexes 1-2) were synthesised and characterised in depth, including by single crystal X-ray diffraction. Their potential as PDT PSs is reported.
2. Experimental Section
2.1. Instrumentation and methods 1H
and 13C NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer. Chemical
shifts (δ) are reported in parts per million (ppm) referenced to tetramethylsilane (δ 0.00) ppm using the residual proton solvent peaks as internal standards. Coupling constants (J) are reported in Hertz (Hz) and the multiplicity is abbreviated as follows: s (singlet). ESI-MS experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientific (Thermo Fisher Scientific) and operated in positive ionization mode. Elemental microanalyses were performed on a Thermo Flash 2000 elemental analyzer.
2.2. Materials All chemicals were obtained from commercial sources and were used without further purification. Dulbecco’s Modified Eagles Medium (DMEM), Dulbecco’s Modified Eagles Medium supplemented with nutrient mixture F-12 (DMEM/F-12), Fetal Bovine Serum (FBS), Gibco Penicillin-Streptomycin-Glutamine (Penstrep), Dulbecco’s Phosphate-Buffered Saline (PBS) were purchased from Fisher Scientific and Resazurin from ACROS Organics.
2.3. Synthesis (Z)-2-((3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-3,5-dimethyl-1H-pyrrole: 2,4,6-Trimethylbenzaldehyde (200 mg, 1.35 mmol) and 2,4-dimethylpyrrole (322 mg, 3.38 mmol) were dissolved in dry dichloromethane (30 mL) under N2 atmosphere. Trifluoroacetic acid (100 L) was added and the reaction mixture was stirred at room temperature for 24 h. After this time, tetrachloro-p-benzoquinone (332 mg, 1.35 mmol) was added and the reaction mixture stirred additional 4 h. Afterwards the solution was filtered and the solvent evaporated. The crude product was purified by column chromatography on neutral alumina with a gradient of dichloromethane/hexane. The fractions containing the product were united and the solvent was removed. The solid was washed with Et2O and dried. The compound was used for the next synthetic step without further purification. (Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4-dimethyl1H-pyrrole:
(Z)-2-((3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-3,5-dimethyl-1H-pyrrole (318 mg, 1 mmol) was dissolved in a 1:1 chloroform/ethanol (150 mL) mixture to which iodine (508 mg, 2 mmol) and iodic acid (352 mg, 2 mmol) was added portion wise. The mixture was stirred at room temperature for 4 h under N2 atmosphere. After this time, the mixture was diluted with chloroform (500 mL) and the organic layer was washed three-times with 5% sodium sulphite solution (200 mL). The product was dried over anhydrous sodium sulphate and the solvent was evaporated. The compound was used for the next synthetic step without further purification. Bis((Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4dimethyl-1H-pyrrole)nickel(II) (1): (Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4-dimethyl-1Hpyrrole (200 mg, 0.35 mmol) was dissolved in a mixture of 3:2:3 butanol/ethanol/H2O (250 mL). Ni(OAc)2 . 4H2O (35 mg, 0.14 mmol) and Et3N (142 mg, 1.4 mmol) were added and the reaction mixture heated at reflux for 24 h under nitrogen atmosphere. After completion of the reaction, the solvent was removed. The crude product was purified by column chromatography on silica with a gradient of dichloromethane/pentane. The fractions containing the product were united and the solvent was removed. The solid was washed with Et2O and dried. Yield: 53%. 1H-NMR
(CD2Cl2, 400 MHz): 6.98 (s, 4H), 2.35 (s, 6H), 2.09 (s, 12H), 2.05 (s, 12H), 1.53 (s,
12H). 13C-NMR (CD2Cl2, 100 MHz): 157.8, 145.6, 144.4, 139.3, 136.2, 135.1, 129.2, 129.0, 84.3, 23.2, 19.6, 17.8, 17.5. ESI-HRMS (pos. detection mode): calcd for C44H47I4N4Ni [M+H]+ m/z 1196.9333; found: 1196.9327, UV/VIS (CH2Cl2) λmax (ε x 103): 526 (117.7), 392 (17.5) nm, Elemental analysis calcd for C44H46I4N4Ni (%): C 44.14, H 3.87, N 4.69; found: C 44.36, H 3.95, N 4.31. Bis((Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4dimethyl-1H-pyrrole)copper(II) (2): (Z)-3-iodo-5-((4-iodo-3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-2,4-dimethyl-1Hpyrrole (200 mg, 0.35 mmol) was dissolved in a mixture of 3:2:3 butanol/ethanol/H2O (250 mL). Cu(OAc)2 (25 mg, 0.14 mmol) and Et3N (142 mg, 1.4 mmol) were added and the reaction mixture heated at reflux for 24 h under nitrogen atmosphere. After completion of the reaction, the solvent was removed. The crude product was purified by column chromatography on silica with a gradient of dichloromethane/pentane. The fractions containing the product were united and the solvent was removed. The solid was washed with Et2O and dried. Yield: 74%. ESI-
HRMS (pos. detection mode): calcd for C44H47I4N4Cu [M+H]+ m/z 1201.9275; found: 1201.9278, UV/VIS (CH2Cl2) λmax (ε x 103): 526 (96.2), 392 (14.0) nm, Elemental analysis calcd for C44H46I4N4Cu (%): C 43.97, H 3.86, N 4.66; found: C 43.72, H 4.09, N 4.49.
2.4. X-ray crystallography Single crystal X-ray diffraction data were collected at 183(1) K on a Rigaku OD Synergy (Pilatus 200K detector) diffractometer for 1 and on a Rigaku OD Supernova (Atlas CCD detector) diffractometer for 2 equipped with Oxford liquid-nitrogen Cryostream coolers and using a single wavelength X-ray source from a micro-focus sealed X-ray tube with the Cu Kα radiation (λ = 1.54184 Å). The selected single crystals were mounted using polybutene oil on a flexible loop fixed on a goniometer head and transferred to the diffractometer. Preexperiments, data collections, data reductions and analytical absorption corrections83 were performed with the program suite CrysAlisPro.84 Using Olex2,85 the structures were solved with the SHELXT86 small molecule structure solution program and refined with the SHELXL2018/3 program package87 by full-matrix least-squares minimization on F2. The crystal data collections and structure refinement parameters are summarized in Table 1. CCDC 1976222 (for 1) and 1976221 (for 2) contain the supplementary crystallographic data for these compounds, and can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. In the crystal structure of 1, the Ni complex lies on a two-fold axis, only half of the molecule had to be refined the second half being reproduced by a symmetry operation. Solvent molecules of methanol cocrystallized with the main species but a reasonable modelling of the disordered positions could not be obtained. The PLATON SQUEEZE tool88 was used to calculate the number of electrons attributed to solvent molecules in the primitive cell and to take this solvent contribution into account in the calculated structure factors. A ratio of three molecules of methanol for one molecule of the Ni complex was considered. In the crystal structure of 2, two crystallographically independent molecules of the Cu complex are present in the asymmetric unit. Solvent molecules of hexane cocrystallized with the main species but like for 1 a reasonable modelling of the disordered positions could not be obtained. The PLATON SQUEEZE tool was and a ratio of four molecules of the Cu complex for one molecule of solvent was considered. For both structures the solvent molecules were included in the CIF files (formula sum and formula moiety) causing many alerts in the IUCr checkCIF reports.
2.5. Spectroscopic measurements The absorption and emission of the samples has been measured with a SpectraMax M2 Spectrometer (Molecular Devices). For the determination of the luminescence quantum yield, the samples were prepared in degassed CH2Cl2 solution with an absorbance of 0.1 at 520 nm. The luminescence quantum yields were calculated by comparison with the reference Rhodamine B in ethanol (Φem=0.50)89 applying the following formula:
Φem,
sample
= Φem, reference ∗
𝐹reference Fsample
∗
Isample Ireference
∗
(
nsample nreference
2
)
F = 1 ― 10 ―𝐴 Φem = luminescence quantum yield, F = fraction of light absorbed, I = integrated emission intensities, n = refractive index, A = absorbance of the sample at irradiation wavelength
2.6. Singlet oxygen measurements -
direct evaluation
The samples were prepared in an air saturated CH3CN or D2O solution with an absorbance of 0.2 at 450 nm. This solution was irradiated in fluorescence quartz cuvettes (width 1 cm) using a mounted M450LP1 LED (Thorlabs) whose irradiation, centred at 450 nm, has been focused with aspheric condenser lenses. The intensity of the irradiation has been varied using a T-Cube LED Driver (Thorlabs) and measured with an optical power and energy meter. The emission signal was focused and collected at right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i monochromator. A longpass glass filter was placed in front of the monochromator entrance slit to cut off light at wavelengths shorter than 850 nm. The slits for detection were fully open. As a detector an EO-817L IR-sensitive liquid nitrogen cooled germanium diode detector (North Coast Scientific Corp.) has been used. The singlet oxygen phosphorescence at 1270 nm was measured by recording spectra from 1100 to 1400 nm. For the data analysis, the singlet oxygen luminescence peaks at different irradiation intensities were integrated. The resulting areas were plotted against the percentage of the irradiation intensity and the slope of the linear regression calculated. The absorbance of the sample was
corrected with an absorbance correction factor. As reference for the measurement in a D2O solution [Ru(bipy)3]Cl2 (ΦRu(bipy)₃Cl₂ = 0.22)90 was used and the singlet oxygen quantum yields were calculated using the following formula: 𝛷sample = 𝛷reference ×
Ssample Sreference
×
Ireference Isample
I = I0 × (1 ― 10 ―𝐴)
Φ = singlet oxygen quantum yield, S = slope of the linear regression of the plot of the areas of the singlet oxygen luminescence peaks against the irradiation intensity, I = absorbance correction factor, I0 = light intensity of the irradiation source, A = absorbance of the sample at irradiation wavelength.
-
indirect evaluation
For the measurement in CH3CN: The samples were prepared in an air-saturated CH3CN solution containing the complex with an absorbance of 0.1 at the irradiation wavelength, N,Ndimethyl-4-nitrosoaniline aniline (RNO, 24 µM) and imidazole (12 mM). For the measurement in PBS buffer: The samples were prepared in an air-saturated PBS solution containing the complex with an absorbance of 0.1 at the irradiation wavelength, N,N-dimethyl-4nitrosoaniline aniline (RNO, 20 µM) and histidine (10 mM). The samples were irradiated on 96 well plates with an Atlas Photonics LUMOS BIO irradiator for different times. The absorbance of the samples was measured during these time intervals with a SpectraMax M2 Microplate Reader (Molecular Devices). The difference in absorbance (A₀-A) at 420 nm for the CH3CN solution or at 440 nm a PBS buffer solution was calculated and plotted against the irradiation times. From the plot the slope of the linear regression was calculated as well as the absorbance correction factor determined. The singlet oxygen quantum yields were calculated using the same formulas as used for the direct evaluation.
2.7. Stability in CH2Cl2/H2O The stability of the complex in CH2Cl2 or H2O (2% DMSO, v%) was investigated by UV/Vis spectroscopy. The complex was dissolved with about 0.5 absorption at its absorption maximum
and stored at room temperature in the dark. The absorption spectrum from 300-600 nm was recorded with a SpectraMax M2 Microplate Reader (Molecular Devices) after each time interval (0, 1, 4, 12, 24, 48 h) and compared.
2.8. Photostability The photostability of the complex in CH2Cl2 was investigated by UV/Vis spectroscopy. The complex was dissolved with about 0.5 absorption at its absorption maximum and exposed to a constant LED irradiation 510 nm (4.2 mW/cm2) with an Atlas Photonics LUMOS BIO irradiator. The absorption spectrum from 300-600 nm was recorded with a SpectraMax M2 Microplate Reader (Molecular Devices) after each time interval (0, 5, 10, 20, 30 min) and compared.
2.9. (Photo-)cytotoxicity The cytotoxicity of the complexes was assessed by measuring cell viability using a fluorometric resazurin assay. The cultivated cells were seeded in triplicates in 96 well plates with a density of 4000 cells per well in 100 μL of media. After 24 h, the medium was removed and the cells were treated with increasing concentrations of the complex diluted in cell media achieving a total volume of 200 μL. The cells were incubated with the complex for 4 h. After this time, the media was removed and replaced with 200 μL of fresh medium. For the phototoxicity studies, the cells were exposed to light with an Atlas Photonics LUMOS BIO irradiator. Each well was constantly illuminated with either a 510 nm irradiation. During this time, the temperature was maintained constantly at 37 °C. The cells were grown in the incubator for additional 44 h. For the determination of the dark cytotoxicity, the cells were not irradiated and after the medium exchange directly incubated for 44 h. After this time, the medium was replaced with fresh medium containing resazurin with a final concentration of 0.2 mg/mL. After 4 h incubation, the amount of the fluorescent product resorufin was determined upon excitation at 540 nm and measurement its emission at 590 nm using a SpectraMax M2 Microplate Reader (Molecular Devices). The obtained data was analyzed with the GraphPad Prism software.
3. Results and Discussion 3.1. Synthesis The Ni(II) (1) and Cu(II) (2) complexes were synthesised as visualised in Scheme 1. In the first step,
the
ligand
scaffold
was
generated
by
reacting
2,4,6-trimethyl-2-
mesitylenecarboxaldehyde with 2, 4-dimethylmethylpyrrole using catalytic amounts of trifluoracetic acid. The ligand was then oxidised using tetrachloro-p-benzoquinone, achieving a complete conjugation of the aromatic system. Following this, the peripheral positions were iodinated using a mixture of iodine and iodic acid. At last, the metal ion was coordinated using the corresponding acetate salt. While the Cu(II) complex (2) is paramagnetic, the analogous Ni(II) complex (1) was found to be diamagnetic and was therefore characterised by 1H- and 13C-NMR
(Figures S1-S2). Worthy of note, structurally related Ni(II) complexes were also
found to be diamagnetic.73-75,77
I
I H
a) NH
HN
N
N
O c)
b) NH
HN
I
I NH
N
NH
d)
M
N N
N
I
I
M = Ni Cu
1 2
Scheme 1. Synthetic procedures for the synthesis of 1-2. a) TFA, CH2Cl2; b) Tetrachloro-pbenzoquinone; c) CHCl3:EtOH (1:1), I2, HIO3; d) butanol/ethanol/H2O (3:2:3), Et3N, 1: Cu(OAc)2, 2: Ni(OAc)2 . 4H2O.
3.2. X-ray crystallography The nickel and copper centers in 1 and 2 (Figure 1, Table S1) adopt a distorted tetrahedral geometry coordinated by the four nitrogen atoms of the two substituted dipyrrinato ligands. The Ni–N bond lengths are 1.930(7) and 1.937(5) Å while the Cu–N bond lengths fall in the similar range 1.931(3) – 1.950(3) Å (from two crystallographically independent molecules). The N–M–N bond angles are 92.8(2)° in 1 (Ni) and 92.89(13) – 93.67(13)° in 2 (Cu), and the
dihedral angles between the two N-M-N planes are 80.99° in 1 (Ni) and 65.11 – 69.00° in 2 (Cu). In the Cambridge Structural Database,91 reported α,α′‐substituted analogues show dihedral angles in the range of 83.6 – 86.2° for Ni complexes77, 92, 93 and 62.9 – 66.9° for Cu complexes.77, 81, 92, 93 Consequently, the geometry of our two new molecules seems to be typical for such metal complexes. Although Ni(II) and Cu(II) complexes favor square planar coordinations, steric repulsion between the methyl groups at the α position of the dipyrrinato ligand prevent planar structures in these complexes. The same feature is observed in our previously reported bis(dipyrrinato)zinc(II) complex.72
Figure 1. a) Molecular structure of 1 (all H atoms are omitted for clarity). The thermal ellipsoids are drawn at the 20% probability level. b) Molecular structure of 2 (one of the two independent molecules; all H atoms are omitted for clarity). The thermal ellipsoids are drawn at the 50% probability level.
3.3. Photophysical Properties After chemical characterisation of the complexes, their photophysical properties were evaluated in view of applications as PDT PSs. An important factor during a PDT treatment is the wavelengths employed as this directly correlates with the light penetration depth inside tissue. Worthy of note, there is no ideal wavelength for a treatment as this depends, among others, on the tumour size and localisation. Very importantly, it has to be ensured that only tumour tissue and not underlying healthy tissue is damaged. However, with the aim to treat deep seated or large tumors, much research is devoted towards the development of PSs with an
absorption in or near the biological spectral window (600-900 nm). This would allow light to penetrate significantly deeper inside tissue/tumour. Having this in mind, the absorption profile of the complexes 1-2 were measured (Figure 2). Both complexes were found to have a strong absorption at 526 nm, which is significantly more red-shifted than many transition metal complexes investigated for PDT applications. Interestingly, both complexes showed a highly similar absorption profile, indicating that the coordinated metal does not drastically influence this property. Worthy of note, previously reported Ni(II) and Cu(II) dipyrrin complexes were found to have similar absorption profiles.94-96 Following this, the luminescence of the complexes upon irradiation at 520 nm was investigated. While no signal for the Ni(II) complex 1 was detected, a low emission signal with a luminescence quantum yield of >0.1% and a maximum at 551 nm for the Cu(II) complex 2 was detected (Figure S3). This result is in agreement with the luminescence study of structurally related Cu(II)(dipyrrin)2 complexes.81
Figure 2. Absorption spectra of 1-2 in CH2Cl2.
Subsequently, the ability of complexes 1 and 2 to generate singlet oxygen (1O2), which is considered the predominant toxic species in PDT upon irradiation at 450 and 510 nm was investigated by two complementary methods: 1) direct by measurement of the phosphorescence of 1O2, 2) indirect by following the change in absorption of a 1O2 scavenger. The experiment was performed in the organic solvent CH3CN and in an aqueous solution. The results showed
that both complexes are poorly generating 1O2 at the detection limit of our used apparatus with quantum yields >1% in CH3CN and in an aqueous solution.
3.4. Stability As a crucial parameter for PDT applications, the stability of a compound needs to be investigated as previous studies have shown that this could be problematic.97-99 To do so, complexes 1-2 were incubated in the dark for varying time points (0 h, 1 h, 4 h, 12 h, 24 h and 48 h) in CH2Cl2 and H2O and the corresponding absorption spectra monitored (Figure S4-S7). As no significant difference were observed, the stability of both metal complexes is indicated. Beside the stability in a chemical environment, the stability upon irradiation remains a different issue. Therefore, the complexes were exposed to light at 510 nm and their absorption spectra monitored for varying time points (0, 5, 10 20 and 30 min). Only minimal changes were observed (Figure S8-S9), suggesting that the complexes are stable upon light irradiation.
3.5. (Photo-)cytotoxicity After an analysis of the chemical and photophysical properties of the compounds, their cytotoxicity on non-cancerous retinal pigment epithelium (RPE-1) and human cervical carcinoma (HeLa) cells in the dark as well as upon irradiation at 510 nm (20 min, 5.0 J/cm2) was investigated using a fluorometric resazurin assay. The results (Table 1) show that the compounds have a very low cytotoxic effect in the dark. Interestingly, the obtained IC50 values of both compounds were found in the same range, indicating that the central metal does not significantly influence this property. Unfortunately, light irradiation was found to have only a negligible effect on the cell viability. This is most likely caused by the poor photophysical properties (i.e., 1O2 generation) of complexes 1-2. In comparison, the PS Protoporphyrin IX (PpIX) and the anticancer drug cisplatin were found to have drastically lower IC50 values.
Table 1. IC50 value (μM) in the dark and upon irradiation at 510 nm (20 min, 5.0 J/cm2) for complexes 1-2, the PDT PS Protoporphyrin IX (PpIX) and the anticancer drug cisplatin in noncancerous retinal pigment epithelium (RPE-1) and human cervical carcinoma (HeLa) cells. Average of three independent measurements.
HeLa
RPE-1
dark
510 nm
dark
510 nm
1
157.1 ± 8.9
159.3 ± 9.1
184.3 ± 6.3
179.5 ± 7.0
2
148.3 ± 7.4
133.9 ± 8.5
172.4 ± 8.6
163.1 ± 8.9
PpIX
>100
2.8 ± 0.2
>100
4.1 ± 0.2
cisplatin
10.5 ± 0.8
-
23.9 ± 1.4
-
4. Conclusions In this study, the role of the metal ions Ni(II) and Cu(II) on the photophysical and biological properties of homoleptic dipyrrin complexes was investigated. Both metal complexes prepared were characterised in-depth, including by single crystal X-ray diffraction. The complexes were found to have a strong absorption in the green area of the electromagnetic spectrum, which is a desired property for PDT agent. Photophysical studies demonstrated that these complexes were poorly emissive and barely produced 1O2 upon irradiation. Stability studies indicated that both compounds were stable in a biological environment and upon light irradiation. Biological evaluation of the compounds in non-cancerous retinal pigment epithelium and human cervical carcinoma cells revealed a very low cytotoxicity in the dark, as requested for a PDT agent. However, only a negligible effect on cell viability was observed upon light irradiation. This is most likely caused by the poor photophysical properties (i.e. 1O2 generation) of the complexes. We are currently pursuing other options to design PDT PSs based on cheap, abundant metals.
Acknowledgements We thank Dr. Philippe Goldner for access to state-of-the-art laser apparatus. This work was financially supported by an ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679), has received support under the program “Investissements d’Avenir” launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.).
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All authors were involved with the design and interpretation of experiments and with the writing of the manuscript. Chemical, photophysical and biological experiments were carried out by J.K. X-ray crystallography was carried out by J.K. and O.B. All authors have given approval to the final version of the manuscript
Highlights: -
Synthesis and characterisation of two Cu(II) and Ni(II) dipyrrin complexes.
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Investigations of the use of these compounds as chemotherapeutic and photodynamic therapy agents.
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Photophysical studies studied revealed absorption in the biological spectral window.
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(Photo-)cytotoxicity of the metal complexes in cancerous human cervical carcinoma cells was assessed.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: