Unveiling Ga(III) phthalocyanine—a different photosensitizer in neuroblastoma cellular model

Abstract Phthalocyanines (Pc) and their metallated derivatives are strongly considered for photodynamic therapy (PDT) possessing unique properties as possible new photosensitizers (PS). We have used toxicological assessments, real‐time monitoring of cellular impedance, and imagistic measurements for assessing the in vitro dark toxicity and PDT efficacy of Ga(III)‐Pc in SHSy5Y neuroblastoma cells. We have established the non‐toxic concentration range of Ga(III)‐Pc, a compound which shows a high intracellular accumulation, with perinuclear distribution in confocal microscopy. By choosing Ga(III)Pc non‐toxic dose, we performed in vitro experimental PDT hampering cellular proliferation. Our proposed Ga(III)‐Pc could complete a future PS panel for neuroblastoma alternate therapy.


| INTRODUCTION
The current therapeutic lines for cancer treatment such as surgery, chemotherapy, and/or radiotherapy can cause significant trauma, systemic toxicity, and tissue injury, especially if repetitive treatment is necessary due to a refractory tumour. Therefore, the clinical interest in additional treatment strategies like photodynamic therapy (PDT) is steadily increasing in the last years. PDT is continuously expanding due to the efficient and selective photosensitizers (PS) accumulation that upon activation induces an increased tumour eradication. Moreover, this therapy has the advantage of being safely repeated when required. 1 In PDT, a compound that has PS properties can be delivered by different routes (intravenously, intraperitoneally, or topically). accumulation in 3-96 hours, depending on the PS nature and tumour type. 2,3 Conversely, sensitizer fluorescence could be used to diagnose and detect a certain tumour type. Alternatively, by irradiation at a specific wavelength, PS accumulated in the cell will generate reactive oxygen species responsible for apoptotic processes in tumour cells. 4 Among different classes of PS, phthalocyanines (Pc) are currently intensely considered for PDT applications. Their metal derivatives are the most investigated chemical forms, taking into account both their photosensitizing/PS properties and imagistic applications. Pc and Pc derivatives are macrocyclic compounds easily activated by light at a specific wavelength. 5 There are combined efforts focused to develop Pc forms for alternative cancer therapies, to decipher the mechanisms of action of Pc through PDT, its pharmaceutical development in order to enhance its "drugability," and to improve its targeted intracellular distribution. 6 Although gallium (Ga) has been discovered more than 100 years ago, its antitumour properties do not have a long historical record.
Nevertheless, its potential to bind and modify some intracellular proteins, especially enzymes, has been exploited for tumour imaging for instance as 67 Ga citrate. 7,8 Compounds with Ga and different chemical substituents (e.g., phenolate ligands or halogenated compounds) could induce apoptosis in C4-2B prostate tumour cells or inhibit tumour development in mice-bearing prostate cancer xenografts. 9 Ga compounds may hold promise for antitumour treatment; however meanwhile, there is an utmost need to elucidate the mechanisms of Ga antitumor activity, type, and nature of its molecular targets in tumour cells. 10 Furthermore, the cytotoxicity of Ga compounds and potential drug resistance represent significant challenges for biomedical scientists transposed in a continuous search for new Ga(III) formulas as therapeutic agents. 8 Ga(III) salts are envisioned as therapeutical compounds because Ga(III) salts can be administrated orally being less toxic and allowing a chronic therapy. Moreover, this administration was shown that has an improved bioavailability for tumours compared to the parenteral use. 7 In view of the anticancer potential of Ga(III) compounds, Pc-containing Ga(III) could be a new type of PDT antineoplastic agent, expanding the Ga-based therapeutic compounds list. 8 Neuroblastoma is a solid tumour of the peripheral nervous system accounting for 8%-10% of childhood tumours. 11 Neuroblastoma is a tumour type in which Ga(III) compounds are used as possible imaging and therapy complexes. 12 The current treatment for neuroblastoma involves a combination of surgery, radiation therapy, and chemotherapy (cisplatin and carboplatin) which unfortunately are prone to drug resistance development and toxic side effects. One way of overcoming chemotherapy side effects is to directly target the drug into the tumour and more so to track its delivery by in vivo imaging. Ga(III) could act as tumour imaging compound in radionuclide form or as part of chemical complexes for tumour therapy, having thus theranostic features. 13,14 A series of Ga(III) complexes comprising pyridine and phenol moieties encompassing the methoxy and halogenated groups was tested on a cisplatin-resistant BE(2)-C neuroblastoma cell line. The Ga(III)-halogenated compounds induced apoptosis in BE(2)-C neuroblastoma cells more efficiently than cisplatin and even superior to the nitro-Ga(III) forms. 10,12 For PDT applications, Ga(III) fits better as PS in the form of Ga (III)Pc. It should be emphasized that Pc are of great interest as PS, by keeping the fine balance between the hydrophobic and hydrophilic characteristics of a qualified PS. Pc skeletons are mostly hydrophobic, but their solubility in the physiological milieu can be improved by chemical substitution, which increases its π-electron density thus allowing solubilization in aqueous media. The chemistry of Pc is a promising area because there is a tremendous effort to search for water-soluble forms (with inserted chemical groups such as sulphonates, carboxylates, phosphonates, and quaternary amino) and for controlling their tendency of aggregation in polar solvents.

| MATERIALS AND METHODS
Ga(III)-Pc-chloride was prepared according to a previously published methodology. 16 Furthermore, the Ga-compound was solubilized in dimethylsulphoxide (DMSO) in order to obtain a 1 mmol/L stock solution that was further diluted in cell medium to prepare all working solutions for cellular testing systems. The DMSO final concentration in the medium was <0.05% non-toxic for cells. 17 For irradiation PDT experiments, quartz cuvettes with 0.5-2 cm optical path lengths containing 1 mL of SHSy5Y cell suspension each were used.

| Cellular viability
Lactate dehydrogenase (LDH) release assay was used for cellular viability and membrane integrity evaluation upon cells incubated with various doses of Ga(III)Pc. Cells were seeded at 2 × 10 3 per well in 96-well flat bottom plates and maintained at 37°C in humidified atmosphere with 5% CO 2 . After 24 hours, cells were treated with 0.4, 2, 10, and 20 μg/mL Ga(III)Pc.
Cytotox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI, USA) was used for assessing the released LDH from cells loaded with different doses of Ga(III)Pc as a viability test for both dark toxicity tests and PDT. For each type of sample, experiments were performed in triplicate and results were recorded spectrophotometrically and expressed as OD490 nm value compared to control.

| Cellular proliferation
Cells were seeded at 2 × 10 3 per well in 96 wells, maintained at 37°C in humidified atmosphere with 5% CO 2 , and after 24 hours, cells were treated with 0.4, 2, 10, and 20 μg/mL respectively Ga reduction to a quantifiable formazan compound, reaction developed only by metabolically active cells. 18 For each type of sample, three experiments were performed in triplicate. Results were expressed as OD490 nm value compared to untreated control cells.

| Intracellular staining for confocal microscopy assessment
SHSy5Y cells were seeded at 5 × 10 4 per cm 2 cell density on 18 × 18 mm coverslips and cultured at 37°C in a humidified atmosphere with 5% CO 2 , prior to loading with Ga(III)Pc. After 24 hours, cells were exposed for 24 hours to 10 μg/mL Ga(III)Pc before dsDNA staining. Cells untreated with Ga(III)Pc were used as control. After which was diluted 1:40 (BSA 1%) and applied to coverslips for 30 minutes; the DAPI solution was used for nuclei staining (10 minutes) and samples were mounted with Prolong Gold antifade reagent (Molecular Probes, P36934). All the solutions were prepared in PBS and the staining protocol was done at room temperature.

| Confocal microscopy of cells loaded with Ga (III) compound
Confocal images were acquired using a Leica TCS SP8 system (Leica

| Statistics
All the experiments were performed three times and for all the indi-

| RESULTS AND DISCUSSION
We aim to establish a toxicology pattern of a Ga(III)-Pc as a poten-

| Intracellular localization
In order to investigate the intracellular compartments in which PS accumulated and further reveal a possible apoptotic pathway, we used confocal microscopy to analyse cells loaded with Ga(III)Pc.
There were no significant changes in morphology between control and treated cells, as revealed by phalloidin staining of F-actin. PS showed a perinuclear/cytosolic distribution ( Figure 4) and seemingly did not associate with cytoskeletal components. To confirm this distribution, in 3D, we generated an image stack which showed uniform accumulation of PS in the perinuclear cytoplasmic regions (supplementary file 3D rendering of image stack). tissue penetrability. 25 The Ga nitrate is clinically approved by FDA for the treatment of cancers such as lymphoma and bladder cancer.

| Experimental PDT
It is also approved for use in microbial infections, but its administration procedure is cumbersome and somewhat invasive. 8 28 There is a fraction of PDT-resistant cells that are not immediately killed by the photoactivated Pcs. 29 We have shown that even this cell fraction can be affected by the PDT late action. Thus, we do not exclude two types of mechanisms, mutually probable. One is that cells that were not potential PS for use in PDT of cancer. 30 The photophysical (fluorescence quantum yields and life-times) and photochemical (singlet oxygen and photodegradation quantum yields) properties of these novel Pcs were exploited in light-dependent photodamage in HeLa, HuH-7, 31 and K562 cancer cells. 32 Using an established improved toxicological testing workflow, 33 we found that the photosensitivity and the intensity of damage are directly related to the concentration of the PS. As brain tumours remain a major health concern with limited therapeutic options, 34 our study has shown that neuroblastoma tumour cells can be damaged by PS, and in the future, these compounds may be attractive for clinical exploitation in both PDT and PDD.
In conclusion, in solid cancers, alternative treatment approaches like PDT are evolving in at least two directions, one by enlarging the tumour types where this can be applicable and second by designing new PS. Hence, our study has evaluated a new compound, Ga(III) Pc as a potential PS in neuroblastoma therapy. Our results have shown that Ga(III) Pc has a good toxicological profile and PS characteristics for applicable PDT in neuroblastoma cell line.
In addition, our results point out new mechanisms that should be further investigated as the PS localization and the long-term delayed decline effect post-PDT indicate complex intracellular pathways triggering tumour cell death.