Optical modulation of cell nucleus penetration and singlet oxygen release of a switchable platinum complex

Summary

The activation of anticancer molecules with visible light constitutes an elegant strategy to target tumors and to improve the selectivity of treatments. In this context, we report here a visible-light activatable bis-platinum complex (DHP-Pt₂) incorporating an organic photo-switchable ligand based on the dimethyldihydropyrene moiety. Illumination of this metal complex with red light (660 nm) under air readily produces the corresponding endoperoxide form (CPDO₂-Pt₂). These two metal complexes exhibit different DNA binding properties and, more importantly, we show that only the photogenerated CPDO₂-Pt₂ is able to penetrate into cancer cell nuclei, where it is then capable of releasing cytotoxic singlet oxygen. This study represents the first proof-of-concept showing that dimethyldihydropyrene derivatives can be used to transport and deliver singlet oxygen into cancer cell nuclei upon visible-light activation.

Graphical abstract

Highlights

• •A bis-platinum complex incorporating a photochromic unit was investigated
• •Red-light illumination of the complex under air generates an endoperoxide form
• •Interaction with DNA and penetration efficiency in cells is photo-modulated
• •The photogenerated system is able to transport and release ¹O₂ inside the nucleus

Chemistry; Physics; Biological sciences

Introduction

Nowadays, several efficient chemotherapies and therapeutic agents are available.^1,2,3 However, traditional anti-cancer drugs such as cisplatin and its derivatives suffer from a lack of selectivity that generates many important side effects.^4,5,6,7,8 In this context, the use of photopharmacology^9,10,11,12 and photoactivated chemotherapy (PACT)^13,14,15 constitutes efficient solutions to target cancerous cells and to reach a better specificity since the photochemical activation of the drugs allows a precise spatial and temporal activation of the system.^13,14

In particular, light-responsive molecular systems and therapies have been developed using coordination complexes. In these systems, the photoactivation is usually metal-centered and the formation of the excited state is followed by a variety of effects and mechanisms that allow the therapeutic effects.^16,17,18 In 2015, Pérez-Tomás and Gamez proposed an innovative approach in which the PACT technique relies on the optical activation of a coordinated switchable ligand.¹⁹ In their systems, the properties of the metal complexes have been modulated through the isomerization of an organic dithienylethene unit which have been triggered by light irradiation at precise wavelengths. These photochromic complexes were shown to exhibit two photo-interconvertible isomers that display distinct DNA-interacting properties and cytotoxic behaviors against cancer cell lines.^19,20

The major drawback of photochromic systems to extend their use to biological and medical applications is that their excited state is usually reached by UV light irradiation. Indeed, such light source must be avoided because of its short penetration length into the tissue and because it also causes severe damages.^12,21,22,23 A strategy to solve this problem is to use a negative photochrom, i.e., a system in which the initial and thermodynamically stable state corresponds to the colored one that can thus be activated by visible light.²⁴

In this context, we report herein the study of a bimetallic-platinum(II) complex (DHP-Pt₂, Figure 1) in which the two metal centers are bridged through a photoactive bis-pyridine-dimethyldihydropyrene ligand. The dimethyldihydropyrene (DHP, closed isomer) unit is an organic and negative photochrom that can be excited with visible light to yield the corresponding cyclophanediene (CPD, open form) isomer following the opening of its central carbon-carbon bond.^{25,26,27,28,29,30,31} In addition to this photochromic behavior that can be exploited for many domains,^{21,32,33,34,35,36,37,38,39} we previously reported that some DHP derivatives, upon suitable chemical functionalization, may readily lead to an endoperoxide-cyclophanediene (CPDO₂) form when irradiated with visible (red) light under aerobic conditions.^40,41,42,43 In this reaction, the DHP form is isomerized into its corresponding cyclophanediene isomer (CPD) and, in parallel, it can also act as a photosensitizer and produce singlet oxygen (¹O₂) when illuminated under air. The photogenerated CPD isomer and ¹O₂ then react together to yield the corresponding endoperoxide (CPDO₂). Finally, this compound is able to thermally release a cytotoxic singlet oxygen molecule (typically at 37°C).^40,41 These photoactivable systems are thus able to store, transport and deliver singlet oxygen whose toxicity can be explored in particular for life sciences.

Figure 1.

The DHP-Pt₂/CPDO₂-Pt₂ system

Top: reversible conversion of the DHP-Pt₂/CPDO₂-Pt₂ couple. Down: molecular structure of DHP-Pt₂ (ORTEP view). Thermal ellipsoids are scaled to a 50% probability level. Hydrogen atoms have been omitted for clarity.

Based on these peculiar properties of DHP derivatives, we report herein the preparation and characterization of the bis-platinum(II) DHP-Pt₂ complex as well as its corresponding endoperoxide form CPDO₂-Pt₂ (Figure 1) and we investigate the DNA-binding properties and cytotoxic features of these two forms in cancer cells.

Results and discussion

Preparation and characterization of DHP-Pt₂ and CPDO₂-Pt₂

The starting bis-pyridine-DHP ligand (2,7-di-tert-butyl-4,9-di-(4-pyridyl)-trans-10b,10c-dimethyl-10b,10c-dihydropyrene; DHP-Py₂) was synthesized following our previously reported procedure, using a Suzuki coupling reaction (See supplemental information).^40,44 The DHP-Pt₂ complex was then prepared by dissolving 2 equivalents of (cis-[Pt(DMSO)₂Cl₂]⁴⁵ in MeOH, followed by the addition of 1 M equivalent of the organic DHP-Py₂ ligand. Upon stirring, at room temperature (RT) during 12 h under dark conditions, a precipitate was formed and DHP-Pt₂ was isolated as a brown powder upon filtration. Mass spectrometry as well as ¹H and ¹³C NMR spectroscopy analysis were in full accordance with the proposed bimetallic structure. In particular, in addition to the signals of the aromatic part of the DHP core and of the pyridine groups, ¹H-NMR data (see Figure 2A) show the presence of a unique singlet at δ = −3.72 ppm (6H) corresponding to the signature of the two internal methyl groups of the symmetrical photochromic unit in its closed form. Crystals of DHP-Pt₂ were further obtained by slow diffusion of Et₂O in a CH₃CN solution and the structure of the complex was confirmed by single-crystal X-ray diffraction (Figure 1).⁴⁶ DHP-Pt₂ crystallizes in a monoclinic system. In this structure, the two metal centers exist in a slightly distorted square planar geometry, their coordination sites being ensured by one pyridine unit, one dimethyl sulfoxide (DMSO) molecule and 2 chloride units in trans positions. The distance between the two internal sp³ carbon (C₁-C₂) is 1.50 Å, which is in accordance with other DHP derivatives.^40,44 In addition, in the solid state, the pyridine units adopt a coplanar arrangement, with dihedral angles of 57° with respect to the main DHP plane.

Figure 2.

NMR and UV-vis spectroscopic characterization

Partial NMR spectra of a solution of DHP-Pt₂ (∼2 mg in 600 μL in CD₂Cl₂) before (A) and after (B) 1 h irradiation with visible light.

(C) UV/Vis spectra evolution of a solution of DHP-Pt₂ in CH₂Cl₂ under air during irradiation with red light (corresponding to the formation of CPDO₂-Pt₂; total time: 20 min). Irradiation conditions: λ > 630 nm, T = 20°C.

The absorption spectrum of a CH₂Cl₂ solution of DHP-Pt₂ (Figure 2C) displays several bands in the visible range attributed to π→π^∗ transitions that implies singlet electronic excited states of the chromophore core.²⁵ In particular, the lowest energy transition S₀→S₁ is located at 668 nm in DHP-Pt₂.

The reactivity of DHP-Pt₂ toward light was then tested. First, the photosensitizing properties were verified by measuring the production of singlet oxygen during illumination of the compound with red light and under aerobic conditions (air, p = 1 atm, 20°C). For this, the emission band of ¹O₂ at 1270 nm was measured (see supplemental information) and the results showed that ¹O₂ was readily produced with a quantum yield of φ_Δ = 38 ± 3%.

The preparation of the corresponding cyclophanediene-endoperoxide form (CPDO₂-Pt₂, Figure 1) could be then realized by illumination of DHP-Pt₂ in CH₂Cl₂ with red light (λ_exc = 660 nm) at RT and under aerobic conditions (air, p = 1 atm). This reaction was followed by UV/Vis spectroscopy (Figure 2C). In accordance with the photo-opening of the central carbon-carbon bond, a discoloration of the solution was observed (from orange to light yellow). This was accompanied by the progressive disappearance of the visible bands of the absorption spectrum and the growth of a new signal in the UV part.

The formation of CPDO₂-Pt₂ was confirmed by ¹H NMR spectroscopy (Figure 2B). Upon irradiation under air of the DHP-Pt₂ isomer in CD₂Cl₂, a new set of peaks were obtained and their analysis indicated a loss in symmetry, in accordance with the formation of the endoperoxide form. In particular, the signal of the internal methyl groups in the DHP state (singlet at δ ≈ −3.7 ppm, 6H) disappeared and two new singlets (2 × 3H) were observed at δ ∼ +1.5 ppm and 0 ppm, this latter being typical of the signal of the internal methyl group connected to the peroxide function in CPD-O₂ derivatives.⁴¹ In addition, mass spectrometry analysis corroborated the presence of two additional oxygen atoms in the compound upon illumination (See Figures S10 and S11). Finally, CPDO₂-Pt₂ was isolated by evaporation of the solvent under vacuum at low temperature (4°C).

At this stage, it is important to outline that the particular (photo)reactivity of dimethyldihydropyrenes toward dioxygen requires a chemical functionalization by suitable electron-withdrawing groups that modify their underlying photochemical mechanisms.^33,43,44 In particular, unlike the DHP-Pt₂ metal complex, the bis-pyridine DHP-Py₂ ligand does not form endoperoxides and cannot be isomerized by red light irradiation. Therefore, the presence of the platinum centers is clearly essential in this work.

The reversion process between CPDO₂-Pt₂ and DHP-Pt₂ forms and consequent release of ¹O₂ were thermally evaluated at 25°C using NMR studies. After 48 h, the endoperoxide form totally disappeared and a major part of DHP-Pt₂ was recovered. It should be noted that some undefined products were also formed, certainly due to some oxidation reactions occurring during the experiment.

The fluorescence properties of DHP-Pt₂ and CPDO₂-Pt₂ have been also examined. Whereas CPDO₂-Pt₂ does not exhibit any emission, the DHP-Pt₂ complex displays a narrow fluorescence signal at 687 nm upon excitation at 450 nm (Figure S1). The relatively small value of the Stokes Shift (−0.0514 eV) indicates that the geometry in the first excited state (S₁) and in the ground state (S₀) of DHP-Pt₂ are close. The radiative deexcitation from the first excited state was found to be mono-exponential with an associated lifetime τ = 2.2 ns and a fluorescence quantum yield of φ_f = 8.6.10⁻³. These differences in the fluorescence properties between DHP-Pt₂ and CPDO₂-Pt₂ can be utilized to identify the state of the system, in particular in biological media.

Biological investigations

Following the previous experiments, the biological properties of DHP-Pt₂ and CPDO₂-Pt₂ were investigated. The interaction of the two metallic complexes with DNA was first studied by measuring the electrophoretic mobility in an agarose gel of pBR322 DNA plasmid treated with increasing concentrations of each compound. DNA treated with cisplatin (cis-diamminedichloroplatinum(II)) was also used for comparison. Figure 3 shows the electrophoretic migration patterns of DNA plasmid for each condition.

Figure 3.

Interaction of DHP-Pt₂ and CPDO₂-Pt₂ with DNA

Agarose gel electrophoresis images of pBR322 DNA plasmid (13 μg mL⁻¹) incubated for 16 h at 37°C with increasing concentrations of (A) DHP-Pt₂, (B) CPDO₂-Pt₂ and (C) cisplatin. Lane 1: pure plasmid DNA; lanes 2–10: 0.09, 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25 μM of compound.

Before any treatment, DNA plasmid was essentially composed of supercoiled form I and some small fraction of relaxed circular form II. When incubated with increasing concentrations of platinum compounds, a proportional decrease in the electrophoretic mobility of the supercoiled DNA plasmid form I and increase in that of the relaxed circular form II were observed until both forms comigrates. The lower mobility of the DNA form I is a consequence of DNA unwinding and removal of negative supercoils due to the interaction of the metallocomplexes. When the concentration was further increased, a positively supercoiled DNA migrated faster into the agarose gel. The concentration for which the two bands (DNA forms I and II) are coalescent is currently used as an indicator of the DNA unwinding relative to the effectiveness of compounds to interact with DNA and to form adducts with nucleobases.^47,48,49 It has to be mentioned that no change in the electrophoretic mobility of the DNA (forms I and II) has been detected when plasmid was incubated with the bis-pyridine-DHP ligand (Figure S3).The point of coalescence for DNA treated with cisplatin was obtained at a concentration of 6.25 μM (Figure 3C, lane 8) while it was observed at 16 and 4 times lower concentrations for DNA treated with DHP-Pt₂ and CPDO₂-Pt₂ respectively (Figure 3A, lane 4; Figure 3B, lane 6), suggesting a better interaction of the bis-platinum compounds with DNA compared to cisplatin. More importantly, comigration of DNA forms I and II was observed at a DHP-Pt₂ concentration 4 times lower than CPDO₂-Pt₂ (0.39 μM and 1.56 μM respectively) indicating that these two compounds interact differently with DNA. This result may be attributed to the difference in geometry between the starting DHP complex and the photogenerated endoperoxide form. Indeed, the flat aromatic part of DHP-Pt₂ (Figure 1) may facilitate its interaction with DNA and thus promotes the formation of covalent bonds with nucleobases. In contrast, CPDO₂-Pt₂ is expected to exhibit a very different shape due to the photo-opening of the central C-C bond followed by the formation of the C-O-O-C bridge that leads to a change in the hybridization states of the carbon atoms, from sp² to sp³ and this structure is certainly less conducive to interaction with DNA.⁴¹

Cell viability was next investigated with four cancer cell lines, BT-549 (human breast ductal carcinoma), HeLa (human cervical carcinoma), A-549 (human lung adenocarcinoma) and A-375 (human melanoma). In a first assay, all cancer cell lines were incubated for 48 h with a single concentration of 50 μM of DHP-Pt₂ or CPDO₂-Pt₂ and their viabilities were measured using standard MTT assays. Results are depicted in Figure 4. A disparity in cell viability among the various lines treated with the bis-platinum complexes was clearly noticed. The lowest cell viabilities were measured with A-375 (DHP-Pt₂: 50.42%, CPDO2-Pt₂ 56.80%) and HeLa (DHP-Pt₂: 57.15% and CPDO₂-Pt₂: 63.69%), while very modest or even no effect on the cell viability was observed with A-549 and BT-549 (A-549 cell viability: 80.99% with DHP-Pt₂ and 76.94% with CPDO₂-Pt₂). For all cell lines, no significant difference in cell viability at 48 h was measured based on whether they were treated with 50 μM of DHP-Pt₂ or CPDO₂-Pt₂.

Figure 4.

Cancer cell viability studies in the presence of DHP-Pt₂ and CPDO₂-Pt₂

Cell-viability assay with DHP-Pt₂ or CPDO₂-Pt₂ performed with four human cancer cell lines, BT-549 (breast carcinoma), HeLa (cervical carcinoma), A-549 (lung adenocarcinoma) and A-375 (melanoma). Assays were conducted using a concentration in each bis-platinum complex of 50 μM and an incubation time of 48 h. Percentages in cell viability are reported.

IC₅₀ values at 24, 48 and 72 h of incubation with DHP-Pt₂, CPDO₂-Pt₂ and cisplatin were then determined for cells that showed a greater sensitivity toward the bis-platinum compounds in the first viability assay (A-375 and HeLa; results are reported in Table S2). Compounds cytotoxicity was also investigated at 48h with HFF-1 non-cancer cells (human foreskin fibroblasts).The data were consistent with those obtained with the first single point toxicity assay, showing very similar cytotoxicity properties for both DHP-Pt₂ and CPDO₂-Pt₂ after 24, 48 or 72 h of incubation with both cancer cell lines (IC₅₀ values at 48 h, for DHP-Pt₂/CPDO₂-Pt₂ 52.5 μM versus 56.2 μM with A-375 and 59.8 μM versus 61.9 μM with HeLa). It should be pointed out that these IC₅₀ values remain higher than those determined with cisplatin, certainly because of the lower ability of the compounds to cross the cell membrane and penetrate the nucleus compared to the small cisplatin molecule. However, an important point to mention is that DHP-Pt₂ and CPDO₂-Pt₂ were found to be more toxic on both cancer cell lines than on healthy HFF-1 cells (see Table S2).

It is important to note that the close cytotoxic properties measured for both compounds contrast with the lower ability for CPDO₂-Pt₂ to interact with DNA compared to DHP-Pt₂. This result suggests that different cytotoxic processes may be involved for cells treated with these two complexes. To corroborate this hypothesis, the potential of each of the two bis-platinum complexes to penetrate cells and reach the nucleus was examined. For this, the presence of DHP-Pt₂ was measured by confocal microscopy using its fluorescence properties. The cellular uptake of both bis-platinum compounds with A-375 cell line (for which the higher cytotoxicity was observed) and with BT-549 (that exhibits a low sensitivity toward the compounds) was detected. Analyze of cell fluorescence indicated that the penetration of DHP-Pt₂ and CPDO₂-Pt₂ into cell lines is clearly different (Figure 5).

Figure 5.

Penetration studies of DHP-Pt₂ and CPDO₂-Pt₂ into cell lines

Confocal fluorescence microscopic images of BT-549 and A-375 cells treated with 25 μM of DHP-Pt₂ or CPDO₂-Pt₂ for 24 h at 37°C. The merged images were obtained from Z-stacks of one of the platinum complexes (red) and nuclear marker Hoechst (blue).

Indeed, while most of the DHP-Pt₂ molecules appeared accumulated into the cell cytoplasm of A-375 cells, important amounts of CPDO₂-Pt₂ were capable to enter into the nucleus (and were then detected as their DHP-Pt₂ form upon their singlet O₂ release; vide infra). For BT-549 cells, used as negative control, both compounds penetrated the cell membrane but did not reach the nucleus. These observations could explain the low cytotoxicity of the compounds with this cell line. To confirm these observations, the fluorescence intensity profiles of the bis-platinum compounds and the nuclear marker (Hoechst 33342), in both cancer cell lines have been analyzed (see Figure S4). As clearly shown by the colocalization profiles, only CPDO₂-Pt₂ enters the A-375 cell nucleus demonstrating that the penetration of the bis-platinum complex in the nuclear compartment depends on the (photo)state of the system.

As written previously, a great asset of endoperoxide derivatives, photogenerated from DHP systems, deals with their ability to thermally release singlet oxygen.^40,41 The reactive oxygen species (ROS) cell delivery with this platinum complex was thus investigated by microscopy. For this, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as an ROS probe to evidence the intracellular delivery of ¹O₂.⁵⁰ A-375 cells pre-treated with DCFH-DA were incubated with 10 μM of CPDO₂-Pt₂ or DHP-Pt₂ (that was used as a negative control since it was not expected to release singlet oxygen) and the cell fluorescence was examined by confocal microscopy (Figure 6). Whereas significant cell fluorescence was observed for cells treated with CPDO₂-Pt₂, no emission was detected with DHP-Pt₂. This important result shows that it is possible to control the transportation and release of cytotoxic ¹O₂ in cell nuclei using the endoperoxide form of the platinum complex.

Figure 6.

Singlet oxygen release in tumor cells

Imaging of the ¹O₂ release from complexes inside cultured A-375 cells using DCFH-DA as intracellular ROS probe. (A) incubation with 10 μM DHP-Pt₂, (B) incubation with 10 μM CPDO₂-Pt₂, (C) control cells without platinum complex. Top row DIC, middle row fluorescence image, bottom, merged. Scale bar is 20 μm.

Limitations of the study

This study shows that DHP-Pt₂ and CPDO₂-Pt₂ exhibit different mechanisms of cytotoxicity. Indeed, DHP-Pt₂ has higher interactions with DNA while only CPDO₂-Pt₂ is able to enter in the nuclear compartment and to release toxic singlet dioxygen. However, the difference in the cytotoxicity of the two states, i.e., before and after illumination, must be improved in order to avoid side effects. This can be done by further chemical modification of the platinum complex. In addition, the selectivity of the system can be increased by introducing appropriate vectors targeting proteins overexpressed in tumor tissues, enabling accumulation of the platinum complex specifically in cancer cells.

Conclusion

In summary, we have synthetized and investigated a new photoswitchable bis-platinum complex, DHP-Pt₂, based on the organic dimethyldihydropyrene core. DHP-Pt₂ can be readily converted upon red-light illumination under aerobic conditions into its corresponding endoperoxide form CPDO₂-Pt₂. The biologic evaluation of DHP-Pt₂ and CPDO₂-Pt₂ revealed their different abilities to interact with DNA. In contrast, these two platinum complexes exhibited similar cytotoxic properties against A-375 cells line, suggesting that the lower ability of CPDO₂-Pt₂ to interact with DNA and form toxic adducts with nucleobases, is compensated by a different mechanism of cytotoxicity and in particular by the release of ¹O₂. This hypothesis was supported by further experiments that demonstrate a high penetration of the bis-platinum complex in the nuclear compartment only under its endoperoxide state. In addition, the CPDO₂-Pt₂ form is capable to thermally release toxic singlet dioxygen inside the nucleus. Several therapeutic effects (due to the platinum centers and to the production of singlet O₂) can be thus photo-triggered and accumulated in the same molecular system.

This work represents the first example of biological investigations of dimethyldihydropyrenes capable of singlet oxygen production. We believe that these results open new perspectives in the domain of photopharmacology, especially for therapeutics targeting hypoxic tumor cells and hypoxic microenvironments. Our next steps will be to improve the therapeutic effects by further chemical functionalization of the system and to investigate in vitro and in vivo studies with in situ illuminations.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
pBR322 DNA	Euromedex	cat#10-0441
Chemicals, peptides, and recombinant proteins
Acetonitrile	Sigma Aldrich	CAS No.: 75-05-8
Tetrahydrofurane	Sigma Aldrich	CAS No.: 109-99-9
Potassium tetrachloroplatinate(II)	Sigma Aldrich	CAS No.: 10025-99-7
Dimethylsulfoxyde	Sigma Aldrich	CAS No.: 67-68-5
Cisplatin	Sigma Aldrich	cat#1134357 CAS No.: 15663-27-1
Agarose	Euromedex	cat#BI-D0012 CAS No.: 9012-36-6
Boric acid	Euromedex	cat#BI-BB0044 CAS No.: 10043-35-3
Tris base	Euromedex	cat#26-128-3094-B CAS No.: 77-86-1
Urea	Euromedex	cat#EU0014-B CAS No.: 57-13-6
DCFHDA	Sigma Aldrich	cat#35845 CAS No.: 2044-85-1 ID: 57649438
bis Benzimide H 33342 trihydrchloride ( Hoeschst 33342)	Sigma Aldrich	cat#B2261 CAS No.: 875756-97-1 ID: 24891639
MTT	Sigma Aldrich	cat#475989 CAS No.: 298-93-1
Dulbecco's modified Eagle"s medium (DMEM)	Sigma Aldrich	cat#D0819
RPMI-1640 Medium	Sigma Aldrich	cat#R2405
Fetal Bovine Serum (FBS)	Sigma Aldrich	cat#F7524
penicillin-Streptomycin	Sigma Aldrich	cat#P4458
Phosphate Buffered Saline (PBS)	Sigma Aldrich	cat#806552
Trypsin-EDTA	Sigma Aldrich	cat#T2610
phenalenone	Sigma Aldrich	cat#P10801 CAS No.: 548-39-0
Experimental models: Cell lines
BT-549	ATCC	Cat#HTB-122
HeLa	ATCC	Cat#CCL-2
A-549	ATCC	Cat#CCL-185
A-375	ATCC	Cat#CRL-1619
Software and algorithms
ImageJ software	https://imagej.net/ij

Resource availability

Lead contact

Lead contact: Guy Royal (guy.royal@univ-grenoble-alpes.fr). Information and requests should be directed to Nathalie Berthet (nathalie.berthet@univ-grenoble-alpes.fr) for biological studies or Guy Royal for (photo)chemical studies.

Materials availability

This study did not generate new materials.

Data and code availability

Crystallographic structure of DHP-Pt₂ can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. CCDC-2240890 contains the supplementary crystallographic data for this paper. All other datasets, including experimental spectra and data are available in the supplemental information and are publicly available as of the date of publication. This study does not report original code.

Method details

Organic and inorganic reagents used in the synthetic procedures were purchased from Aldrich and were used without additional purification. Solvents were used as received except THF that was distilled over sodium/ benzophenone under argon. pBR322 plasmid DNA, agarose, boric acid, tris base and urea were purchased from Euromedex. 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), Hoechst 33342, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell culture relevant reagents such Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Institute (RPMI)-1640 medium, fetal bovine serum (FBS), antibiotics (penicillin, streptomycin), phosphate buffered saline (PBS), trypsin were all purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA.

All evaporations were carried out under reduced pressure with a rotatory evaporator, and all organic extract were washed with water and dried over MgSO₄. Merck Kieselgel silica gel, 40-63 μm was used for column chromatography. NMR spectra were performed on a Brucker Avanced 500 or 400 MHz spectrometer using deuterated solvents. Chemicals shifts were calibrated to residual solvent peaks. Coupling constant values (J) are given in hertz and chemical shifts (d) in ppm. High-resolution mass spectrometry analyses were conducted using the HRMS, Bruker maXis mass spectrometer and were performed in positive Electrospray Ionisation (ESI⁺) at the DCM mass facility.

Synthesis

2,7-di-tert-butyl-4,9-di-(4-pyridyl)-trans-10b,10c-dimethyl-10b,10c-dihydropyrene (bis-pyridine-DHP ligand) was synthetized following our previously reported procedure.^32,40 Cis-[PtCl₂(DMSO)₂] was prepared using the method previously reported by Wayland and coll.⁴⁵

Preparation of DHP-Pt₂

15.2 mg of cis-[PtCl₂(DMSO)₂] (36 μmol) were suspended in 2.5 mL of methanol and the mixture was refluxed until the complex was dissolved (∼1h). 9 mg (18 μmol) of bis-pyridine-DHP ligand was then added and the solution was stirred at room temperature under dark conditions. After 12 hours, the resulting green-brown mixture was filtrated and the solid was washed with MeOH and Et₂O and dried under vacuum. If needed, DHP-Pt₂ can be crystalized by dissolution in dichloromethane flowed by slow diffusion of Et₂O. yield: 72% (15.5 mg).

¹H NMR (400 MHz, 298 K, CD₂Cl₂) δ (ppm): -3.65 (s, 6H, int. CH₃), 1.68 (s, 18H, tBu), 3.54 (s, 12H, -CH₃ DMSO), 8.01 (m, 4H), 8.58 (s, 2H), 8.75 (m, 4H), 9.00 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm): 154.0, 151.6, 149.0, 137.4, 133.3, 130.1, 127.5, 124.8, 123.6, 120.2, 76.9, 51.1, 44.5, 36.6, 31.8, 30.5, 15.1. ESIMS: m/z: calcd for C₄₀H₅₀Cl₄N₂O₂Pt₂S₂: 1184.1 found: 1184.1.

Preparation of CPDO₂-Pt₂

CPDO₂-Pt₂ was prepared by in situ illumination of a solutions of DHP-Pt₂, as reported below (see section “Irradiation procedures”). The compound can be isolated as a yellow powder upon evaporation of the solvent at low temperature (<10°C) and can be stored for months at 4°C.

¹H NMR (400 MHz, CD₂Cl₂) δ (ppm): 0.00 (s, 3H), 1.09 (s, 9H), 1,28 (s, 9H), 2.10 (s, 3H), 3.47 (s, 6H), 3.49 (s, 6H), 6.79 (s, 1H); 6.98 (s, 1H), 7.06 (m, 1H), 7.29 (m, 2H), 7.38 (s, 1H), 7.52 (m, 2H), 7.63 (m, 2H), 8.70 (m, 2H), 8.77 (m, 2H). ESIMS: m/z: calcd for C₄₀H₅₀Cl₄N₂O₄Pt₂S₂Na: 1239.2 [M+Na⁺] found: 1239.1.

X-Ray diffraction

Data for single crystal X-Ray diffraction were collected at 200 K on a Bruker AXS Enraf-Nonius Kappa APEXII diffractometer using the Mo-Kα monochromated radiation. Intensity data were collected for Lorentz and polarization effects and absorption with the EVAL14 software. Structures solutions and refinements were performed with the SHELX softwares implemented by Olex2. All non-hydrogen were refined by full matrix least-squares with anisotropic thermal parameters. Hydrogen atoms were introduced at calculated positions as riding atoms. Crystallographic structure of DHP-Pt₂ was drawn with the Mercury 2022.3.0 software. CCDC-2240890 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Spectroscopy

Absorption spectra were recorded using a Varian Cary 60 Scan UV/visible spectrophotometer equipped with a temperature controller unit. Absorption measurements over the spectral range from 250 to 800 nm were carried out in Hellma quartz suprasil cells (QS) having an optical path length of 1 cm. Emission spectra were recorded in CH₂Cl₂ at room temperature on a Varian Cary Eclipse fluorescence spectrophotometer.

Irradiation procedures

The solutions of DHP-Pt₂ were irradiated in UV-visible quartz cells, NMR tubes or flasks under air (P = 1 atm.). Typically, concentrations used for UV-visible spectroscopy and NMR experiments were 2.10^-5 M and 2 mg.mL^-1 respectively. The visible irradiations for making the reaction from DHP-Pt₂ to its corresponding endoperoxide form CPDO₂-Pt₂ were carried out at room temperature either with a Xe-Hg lamp (500W) using a 630 nm cut-off filter or with mounted LEDs from Thorlab (M660L4, FWHM = 20 nm, 940-1050 mW), in combination of a Thorlab DC2200 led driver and an adjustable collimation adapter (SM2F32-A). The conversion reaction between the two forms was investigated from UV-visible and NMR experiments. Intermediate spectra were recorded at different times depending on the isomerization process rate.

Singlet oxygen production (Φ_Δ)

Singlet oxygen production quantum yields Φ_Δ were determined by the measure of the phosphorescence of singlet oxygen at λ = 1270 nm⁵¹ with an Edinburgh Instruments FLS1000 fluorimeter equipped with an InGaAs detector. Experimentations were achieved at room temperature, with an oxygen non-saturated atmosphere (P = 1 atm).^52,53 Typically, solutions at 10^-5 M in dichloromethane were prepared. Note that the reference and the sample have to be prepared in the same solvents in order to avoid singlet oxygen lifetimes deviation. Reference and sample were excited at a wavelength where they both have an optical density lower than 0.1. Excitation wavelength has to be preferentially in a zone where reference and sample have same absorbance tendency in order to reduce measurements uncertainty. Phenalenone photosensitizer was used as reference since it has high quantum yields near to the unity (${\mathrm{\Phi }}_{\Delta }\ge 0.94$) and it is soluble in a wide variety of solvents.⁵⁴ Φ_Δ was determined according to the following equation:

$${\Phi }_{\Delta }^s={\Phi }_{\Delta }^{ref}\times \frac{I_{{\lambda }^{ex}}^s{A}_{{\lambda }^{ex}}^{ref}{\eta }_s^2}{I_{{\lambda }^{ex}}^{ref}{A}_{{\lambda }^{ex}}^s{\eta }_{ref}^2}={\Phi }_{\Delta }^{ref}\times \frac{I_{{\lambda }^{ex}}^s{A}_{{\lambda }^{ex}}^{ref}}{I_{{\lambda }^{ex}}^{ref}{A}_{{\lambda }^{ex}}^s}={\Phi }_{\Delta }^{ref}\times \frac{S_{{\lambda }^{ex}}^s}{S_{{\lambda }^{ex}}^{ref}}$$

Where ${\mathrm{\Phi }}_{\Delta }^{\mathrm{s}}$ and ${\mathrm{\Phi }}_{\Delta }^{\text{ref}}$ are singlet oxygen production quantum yields for sample and reference respectively. Reported singlet oxygen production quantum yield for phenalenone in dichloromethane is ${\mathrm{\Phi }}_{{\Delta }_{\text{CH}2\text{Cl}2}}^{\text{ref}}=0.98\pm 0.08$. ${\eta }_{\mathrm{s}}$ and ${\eta }_{\text{ref}}$ are the refractive index of the solvents. ${\eta }_{\mathrm{s}}={\eta }_{\text{ref}}$ as we work in the same solvents. ${\mathrm{I}}_{{\lambda }^{\text{ex}}}^{\mathrm{s}}$ and ${\mathrm{I}}_{{\lambda }^{\text{ex}}}^{\text{ref}}$ are the integrated weak near-infrared phosphorescence of ¹O₂ at λ = 1270 nm for the sample and the reference. This emission is characteristic of the deexcitation of the metastable singlet oxygen to its triplet ground state. The integrals were computed using an absolute integration by mathematical trapezoid sum algorithm implemented in Origin 9.5.0 software package.⁵⁵ ${\mathrm{A}}_{{\lambda }^{\text{ex}}}^{\mathrm{s}}$ and ${\mathrm{A}}_{{\lambda }^{\text{ex}}}^{\text{ref}}$ denote for the absorbance of the sample and of the reference at the excitation wavelength ${\lambda }^{\text{ex}}$. ${\mathrm{S}}_{{\lambda }^{\text{ex}}}^{\mathrm{s}}$ and ${\mathrm{S}}_{{\lambda }^{\text{ex}}}^{\text{ref}}$ stand for the slope of the line of the function $\mathrm{f}\left({\mathrm{A}}_{{\lambda }^{\text{ex}}}\right)={\mathrm{I}}_{{\lambda }^{\text{ex}}}$ for the sample and the reference respectively meaning that ${\mathrm{S}}_{{\lambda }^{\text{ex}}}^{\mathrm{s}}={\mathrm{I}}_{{\lambda }^{\text{ex}}}^{\mathrm{s}}/{\mathrm{A}}_{{\lambda }^{\text{ex}}}^{\mathrm{s}}$ and ${\mathrm{S}}_{{\lambda }^{\text{ex}}}^{\text{ref}}={\mathrm{I}}_{{\lambda }^{\text{ex}}}^{\text{ref}}/{\mathrm{A}}_{{\lambda }^{\text{ex}}}^{\text{ref}}$. By plotting the function $\mathrm{f}\left({\mathrm{A}}_{{\lambda }^{\text{ex}}}\right)={\mathrm{I}}_{{\lambda }^{\text{ex}}}$ for at least 5 different concentrations, ${\mathrm{S}}_{{\lambda }^{\text{ex}}}^{\mathrm{s}}$ and ${\mathrm{S}}_{{\lambda }^{\text{ex}}}^{\text{ref}}$ can be determined as the slopes of the lines.

Confocal microscopy

The z-stack images of cell-cultures were captured using a TCS SP8 GSU Leica (HC PL APO CS2, 40X/1.30 OIL, zoom 3.2, with a z voxel size = 0.569 μm and an image resolution of 1024 x 1024 pixels. H₂DCFDA probe: Excitation wavelength was 488 nm and emission was collected in the 500 - 550 nm range. Hoechst 33342: Excitation: λ = 405 nm and fluorescence emission collected between λ = 450 and 500 nm. DHP-Pt₂ or CPDO₂-Pt₂: Excitation at λ = 405 nm and fluorescence emission collected between λ = 650 and 750 nm. Images were processed using ImageJ software.⁵⁶ The solution-absorbance in microplates was read on a POLARstar Omega plate reader (BMG labtech).

Cell lines and culture conditions

BT-549 (human breast ductal carcinoma), HeLa (human cervical carcinoma), A-549 (human lung adenocarcinoma) and A-375 (human melanoma) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). BT-549 , A-549 and A-375 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin and streptomycin in a humidified 5% CO₂ incubator at 37°C. HeLa and HFF-1 cells were grown in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin at 37°C under 5% CO₂ atmosphere.

Interaction with DNA plasmid

Gel-electrophoretic mobility shift assay is commonly used to study the interaction via intercalation or covalent crosslinking of metallic complexes with double helix DNA. Stock solutions of platinum (II) compounds were prepared in 10 mM phosphate buffer, pH 7.5, 10% DMSO. pBR322 plasmid DNA aliquots (13 μg.mL^-1 in 10 mM phosphate buffer, pH 7.5 containing 10% DMSO) were incubated with increasing concentrations of platinum complexes ranging from 0.09 to 25 μM at 37°C for 16h in the dark (CPDO₂-Pt₂ was freshly preformed before incubation with DNA by illumination of a solution of DHP-Pt₂ as previously described). DNA loading buffer was then added to the samples and the resulting mixtures containing complexes-DNA products were loaded onto 1% agarose gel in tris-Boric-EDTA buffer (pH 8.2) (0.5 x TBE). Electrophoresis was performed at 70 V for 2h. After DNA migration, the gels were stained in the same buffer containing ethidium bromide (0.5 mg.mL^-1) and visualised using Molecular Imager, Gel DocTM XR and image LabTM Software.

Intracellular singlet oxygen release

DCFH-DA was used as ¹O₂ probe. Cells (1.10⁵ cells.mL^-1) were seeded in Lab-Tek chamber slides and allowed to grow for 24h. Compounds (DHP-Pt₂ or CPDO₂-Pt₂) were directly added in the cell culture (10 μM final concentration) and the incubation was continued for 24h at 37°C, 5% CO₂. Culture medium was then removed and cells were treated with DCFH-DA (4 μM in PBS) for another 0.5 h at 37°C. In parallel, cells without compound treatment were incubated with DCFH-DA and used as control. Cells were then washed with PBS and confocal fluorescence imaging was used to image intracellular singlet oxygen delivery.

Cell viability assay

Cytotoxicity of platinum complexes (DHP-Pt₂ or CPDO₂-Pt₂) and cisplatin used as reference were measured using MTT assay as previously described.⁵⁷ Cells were seeded in 96-well plates (4.10³ cells.well^-1 for A-549 and A-375, 1.10⁴ cells.well^-1 for HeLa and 2.10⁴ cells.well^-1 for BT-549) and allowed to attach for overnight. Cells were then treated with 50 μM of each compound for 48h for single concentration assay or with a range concentration of 0.78 to 100 μM for 24h, 48h and 72h for dose-response curves. MTT solution (0.5 mg.mL^-1 in culture media) was added to each well and the cells were further incubated at 37°C for 4h. Subsequently, the media was removed and the blue formazan crystals were dissolved in 100 μL of DMSO. The absorbance value was measured at 570 nm with a microplate reader and the cell viability was calculated using the following formula:

$$Cellviability(\%)=\left(\frac{O{D}_{Sample}-O{D}_{Blank}}{O{D}_{Control}-O{D}_{Blank}}\right)\times 100$$

Confocal microscopy for cell-compound localization

Cells were seeded onto coverslips in 8-wells labtek with a cell density of 4.10⁴ cells.well^-1 and allowed to attach for 24h. Complexes (25 μM) or blank PBS were added and cells were incubated at 37°C in a humidified 5% CO₂ incubator for 24 h. The medium was then removed and cells were washed with PBS before to be fixed for 10 min at 4°C in 4% paraformaldehyde (PFA) solution. Cell nucleus were then stained with Hoechst 33342 (1 μM) for 20 min at room temperature. After washing with PBS, cells were imaged with confocal microscope.

Published: December 9, 2023