Protein-stabilized gold nanoclusters for PDT: ROS and singlet oxygen generation

Journal of Photochemistry & Photobiology, B: Biology 204 (2020) 111802

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Protein-stabilized gold nanoclusters for PDT: ROS and singlet oxygen generation

T

Vilius Poderysa, Greta Jarockytea, Saulius Bagdonasb, Vitalijus Karabanovasa,c, , Ricardas Rotomskisa,b ⁎

a

Laboratory of Biomedical Physics, National Cancer Institute, Baublio 3b, LT-08406 Vilnius, Lithuania Biophotonics group of Laser Research Center, Faculty of Physics of Vilnius University, Sauletekio 9, bldg. 3, LT-10222 Vilnius, Lithuania c Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Sauletekio 11, LT-10223 Vilnius, Lithuania b

ARTICLE INFO

ABSTRACT

Keywords: Protein-stabilized gold nanoclusters Luminescent nanoparticles Bovine serum albumin Photodynamic therapy Singlet oxygen ROS SOSG Dihydrorhodamine Cancerous cells Viability tests

Suitable properties as well as eco-friendly synthesis of photoluminescent Au nanoclusters (NCs) make them promising compounds for biomedical diagnostics and visualization applications. However, the potential photochemical activity of such agents on cancerous cells is largely unknown. The nanoclusters (BSA-Au NCs) were synthetized in the presence of BSA (an average hydrodynamic diameter was about 9.4 nm, while the size of the metal cluster was < 1.3 nm according to atomic force microscopy measurements) and possessed a broad photoluminescence band at 680 nm in buffered (pH 7.2) aqueous medium. The photochemical activity was studied by adding two fluorescent probes (dihydrorhodamine or Singlet Oxygen Sensor Green) for detection of reactive oxygen species in samples irradiated at 405 nm to minimize direct excitation of the probes. The photoluminescence measurements evidenced the capability of BSA-Au NCs to generate reactive oxygen species upon light exposure, while the observed sensitivity of the photoluminescence properties might be used to indicate photooxidative processes in the medium. The viability test performed on breast cancer cells after incubation with BSA-Au NCs and subsequent irradiation revealed notable difference in induced phototoxicity between two cell lines, which was not the case after the corresponding treatment using the photosensitizer chlorin e6.

1. Introduction Due to unique chemical and physical properties noble metal nanomaterials have been extensively studied in recent years aiming to use them in a rapidly evolving environment of new technologies as multifunctional luminescent probes for biomedical imaging, detection, and therapy [1,2]. Unlike bulk gold, conventional gold nanoparticles (bigger than 2 nm) possess size-depending electronic properties and trigger distinctive physical phenomena, such as surface plasmon resonance, leading to a photothermal effect and promising biomedical applications [3,4]. As the size reduces to the subnanometer scale, metal clusters are too small to support plasmons, and a transition from metal to molecule ensues [5]. Gold nanoclusters (Au NCs), composed by tens or hundreds of Au atoms, are highly stable nanoparticles < 2 nm in diameter possessing molecular-like optical features [6]. The dimension of gold nanoclusters is comparable to the Fermi wavelength of electrons (~0.7 nm) and induces a strong quantum confinement effect of free electrons in the particles. Consequently, the continuous density turns into discrete energy levels, which leads to size-dependent luminescence

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and other unique molecule-like properties [2,7–9]. Recent advances in synthesis of Au NCs fostered easier production of water-soluble and biocompatible Au NCs with a smaller size and distinct emission colour [10]. Nevertheless, biocompatible stabilizing agents have to be used in order to synthesize Au NCs for biological applications [2,7,8]. Besides commonly used small thiol molecules, large and relatively more complex ligand types such as proteins and other biomolecules have been implemented to stabilize Au nanoclusters at various sizes, which further improves their integration into biomedicine-related applications [10]. One of the most common proteins being investigated for the synthesis of gold nanoclusters has been bovine serum albumin (BSA) [10]. The first protein-directed synthesis of luminescent Au NCs possessing a high quantum yield of photoluminescence (with bovine serum albumin as a stabilizing agent) was reported by Xie et al. in 2009 [11]. Notably, BSA functions both as a stabilizing agent and a reductant in this approach. As a matter of fact, the process is similar to the biomineralization occurring in nature [5]. Low toxicity, stability, possibility of surface modification providing controllable bioconjugation as well as eco-friendly synthesis make

Corresponding author at: Laboratory of Biomedical Physics, National Cancer Institute, Baublio 3b, LT-08406 Vilnius, Lithuania. E-mail addresses: [email protected] (V. Karabanovas), [email protected] (R. Rotomskis).

https://doi.org/10.1016/j.jphotobiol.2020.111802 Received 12 August 2019; Received in revised form 10 October 2019; Accepted 18 January 2020 Available online 20 January 2020 1011-1344/ © 2020 Elsevier B.V. All rights reserved.

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Fig. 1. (A) Topography of BSA-Au NCs spread on mica surface and being measured using an atomic force microscope; a scale bar indicates 200 nm; (B) Distribution of hydrodynamic sizes of BSA and BSA-Au NCs measured using a dynamic light scattering technique; (C) MALDI MS spectra of BSA (black trace) and BSA-Au NCs (blue trace). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

photoluminescent Au NCs attractive compounds in comparison with semiconductor nanoparticles. The promising luminescence properties of protein-stabilized Au NCs have been reported [5], their distinct structural features and possible applications were briefly reviewed by Chevrier et al. [12]. Currently, Au NCs are considered for usage in variety of applications such as material sensing, dual-modality imaging and biological labeling [2,7,8,13,14]. Other possibilities were also explored, such as to use BSA-Au25 for radiotherapy [15] and microwavesinduced hyperthermia [16]. In addition, BSA-Au NCs exhibit a long photoluminescence lifetime suggesting that the NCs can be capable of singlet oxygen generation [17]. Reactive oxygen species (ROS), including singlet oxygen, play a key role in the photodynamic therapy as damaging cellular factors [18]. However, publications on photochemical properties of Au NCs form an equivocal opinion about the generation effectiveness. Yu et al. [19] showed poor efficiency of singlet oxygen generation for BSA-Au25 NC with the quantum yield value being ≈ 0.07. This result suggests that triplet states being ready to generate 1 O2 are actually scarce in the excited BSA-Au25 [17]. Contrary to that, Kawasaki et al. [20] demonstrated that 1O2 can be efficiently produced through the direct photosensitization of cancer cells by Au25(SR)18− clusters (H − SR = phenylethanethiol or captopril) without using conventional organic photosensitizers under visible/near-IR (532, 650 and 808 nm) irradiation. Moreover, Yamamoto et al. [21] showed that the 1O2-generation efficiency of BSA– Au25 NCs was higher than that of Au25(SG)18 NCs (SG = glutathione), the latter also being dependent on the size of thiolate protected Au nanoclusters. The long lifetime of the electronic excitations and the high photostability were noted as key factors facilitating the formation of 1O2 in BSA–Au25 NCs. Conjugation of BSA-Au NCs and photosensitizers has been developed as yet another approach to improve 1O2 generation of nanoclusters for the photodynamic therapy (PDT) [22–24]. While singlet oxygen generation by BSA-Au NCs was addressed in several studies, there is still no consensus concerning both the toxicity and the photosensitizing capacity of those nanoclusters. Some studies demonstrated neither morphological damage and cytotoxicity nor modifications to the cell cycle and doubling time after the cellular contact with BSA-Au NCs [25,26]. In contrast, it was reported that BSAAu NCs could increase intracellular production of ROS, further causing cell apoptosis in different cell lines in a dose- and time-dependent manner [27]. The present study aimed to assess the generation of reactive oxygen species and singlet oxygen (1O2) by synthetized BSA-Au NCs under light exposure by using two different fluorescing indicators as well as to determine the toxicity and the PDT effect of the nanoclusters on two selected breast tumor cell lines.

2. Materials and Methods 2.1. Chemicals In order to investigate the capacity of BSA-Au NCs to generate reactive oxygen species under exposure to light, dihydrorhodamine 123 (DHR123) was chosen as a fluorescent probe, since DHR123 is readily oxidized back to the parent fluorescent dye Rhodamine 123 (Rhod123) by reactive oxygen species such as hydrogen peroxide [28,29]. Another commercially available fluorescent sensor named Singlet Oxygen Sensor Green (SOSG) was chosen as a highly selective probe for singlet oxygen without any appreciable response to hydroxyl radicals or superoxide [30,31]. In the presence of 1O2, SOSG is oxidized turning into SOSG endoperoxides (SOSG-EP) that emit strong green fluorescence with the maximum at 531 nm [32]. 2.2. Preparation and Characterization of BSA-Au NCs BSA-Au NCs were prepared according to a previously reported “green” synthesis route [11] with slight modifications. Typically, aqueous HAuCl4 (52% Au basis, M = 339.79 g/mol, Sigma-Aldrich, Germany) solution (5 mL, 37 °C, c = 5.27 × 10−3 M) was added into a BSA (V fraction, M ~ 66 kDa, Sigma-Aldrich, Germany) solution (5 mL, 37 °C, c = 7.53 × 10−4 M) under continuous stirring. Two minutes later, a NaOH (Sigma-Aldrich, Germany) solution (0.5 mL, 1.0 M) was added. The reaction was allowed to proceed under vigorous stirring for 12 h at the constant temperature of 37 °C yielding the BSA-Au NCs. After synthesis protein-stabilized luminescent gold nanoclusters were produced. The solution was then filtered using Whatman 25 mm GD/X syringe filter with CA (Cellulose Acetate) membrane, pore size 0.22 μm. A hydrodynamic diameter of particles was measured using an instrument Zeta Plus PALS (Brookhaven Inc., USA), and an average value for BSA-Au NCs was around 9.4 nm (Fig. 1(B)). The corresponding measurements of the solutions containing only BSA yielded an average value of about 6.4 nm, the largest values reaching up to 8 nm. In atomic force microscopy images of BSA-Au NCs being measured on mica surface using AFM diInnova (Veeco Inc.), the disc shape objects of approximately 1.3 nm in height were detected (Fig. 1(A)). As seen for NCs being dispersed on mica, BSA has lost its ellipsoidal structure and flattened. However, dried films of BSA-Au NCs still preserved PL capacity indicating that the gold nanoclusters had not degraded. Thus, the measured height of BSA-Au NCs on mica shows that the sizes of gold nanoclusters formed inside protein are smaller than 1.3 nm. These results are in accordance with the data reported in [33]. In addition to AFM measurements BSA and BSA-Au NCs samples were analysed by 2

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(Varian Inc., Australia). The spectra of both control and irradiated samples were measured after each period of irradiation. 2.4. The Photodynamic Effect of BSA-Au NCs in vitro Human breast cancer cell lines MCF-7 and MDA-MB-231 were used as experimental models to study the photosensitizing efficacy of NCs. MCF-7 cell line was purchased from The European Collection of Cell Cultures and MDA-MB-231 cell line – from American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle's Medium (Corning, US) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, USA), 100 U/mL penicillin, 100 μg/L streptomycin (Gibco, USA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% of CO2. For the in vitro experiments the solution of synthesized BSA-Au NCs was filtered using a 0.02 mm syringe filter (polyethersulfone membrane, TPP, Switzerland). To determine the PDT effect of the BSA-Au NCs in cell cultures, MCF-7 and MDA-MB-231 cells were seeded into 8chambered cover glass plates (Nalge Nunc International, USA) with a density of 3 × 104 cells/chamber and incubated at 37 °C in a humidified atmosphere containing 5% of CO2 for 24 h. After that the cells were treated with 56 mg/mL of BSA-Au NCs, 56 mg/mL BSA or 10−5 M chlorin e6 for comparative purposes and then incubated under the same conditions for the next 24 h. The cells being incubated with medium alone were taken as control. After 24 h the initial incubation medium with BSA-Au NCs, BSA or Ce6 was carefully aspirated and replaced with fresh one. Then the cells were irradiated with a xenon light source MAX-302 (Asahi Spectra, Japan) through a 400 / 10 nm bandpass filter (power density of 55 mW/cm2) up to 35.7 J/cm2 dose. The other group of cell samples was kept in the dark. After irradiation, the samples were put in the incubator for additional 24 h. A LIVE/DEAD viability/cytotoxicity kit (Thermo Fisher, USA) was used to stain both control and exposed cells. There are two dyes in this kit to mark live and dead cells: green-fluorescent calcein-AM for indication of the intracellular esterase activity, and red-fluorescent ethidium homodimer-1 for indication of the loss of plasma membrane integrity. The survival of exposed and control cells was assessed using the Nikon Eclipse Te2000eU microscope (Nikon, Japan) with the confocal laser scanning system C1si. Imaging was performed using a 20×/0.5 NA dry objective (Plan Apo VC, Nikon, Japan). Calcein-AM was excited at 488 nm with an argon ion laser, and ethidium homodimer-1 was excited at 543 nm with a HeeNe laser. The cells were kept/maintained at 37 °C in the Microscope Stage Incubation System (OkoLab, Italy) in a humidified atmosphere containing 5% of CO2 (0.80 Nl/min O2 and 0.04 Nl/min CO2). Image processing was performed using the Nikon EZ-C1 Bronze version 3.80 and ImageJ 1.46 software. The PDT effect on both cell lines was additionally quantified by means of the Adam-MC Automatic Cell Counter (Digital Bio, Seoul, Korea). The cells were seeded into a 24-well plate at a density of 5 × 104 cells/well, then incubated for 24 h and treated as described earlier. The same exposure dose of 35.7 J/cm2 was applied on the samples through a 400 / 10 nm bandpass filter using power density of 22 mW/cm2. The control samples were kept in the dark. After exposure, all samples were put into the incubator for 24 h. Then cells were trypsinized and harvested in old medium. 20 μL of the cell suspension was mixed with 20 μL of Accustain T solution (Digital Bio, Seoul, Korea) and 20 μL of Accustain N solution (Digital Bio, Seoul, Korea) for calculations of total and non-viable cells. The viability was automatically calculated by means of the ADAM-MC software after each quantitative measurement of the total cells and the non-viable cells. The data of cell viability were shown as the representative images or as the mean values of at least three independent experiments ± standard deviation (SD). Statistical analysis was performed using the two-tailed Student's t-test; differences were considered significant at p ≤ .05.

Fig. 2. Photoluminescence spectra acquired using a fiber optic dip probe at 1 h intervals during synthesis of BSA-Au NCs (λex = 405 nm). An insert presents changes in integrated intensity of the PL band (from 590 nm to 800 nm).

MALDI TOF/TOF mass spectrometer ABI 4800 (Applied biosystems, USA) (Fig. 1). The MALDI-MS spectra of the BSA showed peak at 66.3 kDa whereas BSA-Au NCs had a peak at 70.9 kDa. Mass difference between these peaks is approximately equal to mass of 24 gold atoms. This indicates, that Au NCs that formed in BSA template consists of around 24 atoms. These findings are in a good agreement with data previously reported data by other authors [11]. 2.3. ROS and Singlet Oxygen Generation DHR123 (6 μL, 5 mM, M = 346.38 g/mol, Invitrogen, USA) was diluted with PBS (909 μL, pH = 7.2) (Merck KGaA, Germany) to prepare a DHR123 stock solution (33 μM). Freshly made, filtered BSA-Au NCs solution (0.9 mL, c = 7.53 × 10−4 M) was diluted with PBS (9.1 mL) to prepare a BSA-Au NCs stock solution. Three types of samples were prepared. The first and second samples were made by diluting BSA-Au NCs stock solution (3.6 mL) with PBS (0.4 mL) or a DHR123 stock solution (0.4 mL). The third sample was made by diluting a DHR123 stock solution (0.4 mL) with PBS (3.6 mL). Final concentrations of BSA-Au NCs and DHR123 in studied solutions were c = 6.77 × 10−5 M and c = 3.30 × 10−6 M, respectively. SOSG (100 μg, Invitrogen, USA) was dissolved in methanol (33 μL), and then the 12 μL of this solution (5 mM) was diluted with a phosphate buffer (final volume 1.2 mL, 50 μM, pH = 7.4) to prepare a SOSG stock solution. Three types of samples were prepared in a similar way as described above: by mixing a BSA-Au NCs stock solution (3.6 mL) with 0.4 mL of PBS or a SOSG stock solution or by diluting a SOSG stock solution (0.4 mL) with PBS (3.6 mL). Final concentrations of BSA-Au NCs and SOSG in studied solutions were c = 6.77 × 10−5 M and c = 5 × 10−6 M, respectively. A photosensitizer chlorin e6 (Ce6, Frontier Scientific Inc., USA) was diluted in PBS to make a stock solution (1 mM). Further dilution was done just before in vitro PDT experiments. The impact of applied light exposure on the samples was studied using a continuous wave diode laser (λ = 405 nm, I = 66 mW/cm2) (Roithner Lasertechnik GmbH, Germany). Each type of sample was split into two (each of 2 mL, for control and experiment), and poured into 1 cm polystyrene cuvettes, which were tightly sealed afterwards. The experiment samples were irradiated with 405 nm light under constant stirring, giving the total dose of 35.8 J/cm2. The steady state absorption and photoluminescence spectra of the Au-BSA NCs and other solutions were measured using a UV–visible absorption spectrometer Carry 50 (Varian Inc., Australia) and a fluorescence spectrometer Cary Eclipse 3

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3. Results and Discussion 3.1. Optical characterization of BSA-Au NCs Photoluminescence spectra registered during the BSA-Au NCs synthesis procedure are presented in Fig. 2. Occurrence and growth of a PL band at 680 nm indicates formation of BSA-Au NCs. At first the PL band appeared in a red spectral region, peaking at 665 nm approximately 45 min after the beginning of synthesis. During next 2 h the peak of this PL band was constantly shifting to longer wavelengths till ~680 nm and afterwards remained in a fixed position till the end of the synthesis. The integrated intensity of the PL band was constantly growing during 2.5 h from the beginning of the synthesis (Fig. 2, an insert). Then it slightly decreased (approximately by 5%) during next hour, but afterwards continued to increase. It should be noted that the PL intensity did not reach a stable (maximum) value at the end. Extension of the synthesis time from 12 h to 60 h led to further formation of the PL band of BSA-Au NCs, the integrated intensity of which became approximately 2.2 times higher (Fig. S1 in the Supporting Information). Absorption, photoluminescence and photoluminescence excitation spectra of freshly synthesized BSA-Au nanoclusters (c = 7.53 × 10−5 M, concentration estimated assuming that NC was formed in every BSA molecule) are presented in Fig. 3. Absorbance of BSA-Au NCs increased

Fig. 3. Absorption (black), PL excitation (λem = 680 nm) (blue), PL emission (λex = 405 nm) (red) spectra of BSA-Au NCs, and an absorption spectrum of BSA (a purple dashed line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. (A) The photoluminescence spectra of the BSA-Au NCs sample, the DHR123 sample and the mixed BSA-Au NCs and DHR123 sample, normalized to the intensity of the BSA-Au NCs band; (B) the spectra of the irradiated DHR123 sample; (C) the spectra of the irradiated mixed BSA-Au NCs and DHR123 sample; (D) the spectra of the control (non-irradiated) mixed BSA-Au NCs and DHR123 sample. The spectra in (B)-(D) were normalized to initial intensity of Rhod123. Inserts in (C) and (D) show dynamics of the PL band of BSA-Au NCs during experiment time in irradiated and control samples, respectively. PL registration settings: for (A)-(D) and inserts, λex = 480 nm, Δλex = 1 nm, Δλem = 5 nm. 4

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Fig. 5. Normalized photoluminescence spectra of: (A) samples of BSA-Au NCs, SOSG and the mixture; (B) the irradiated sample of SOSG; (C, D) the irradiated sample and the control (non-irradiated) sample of BSA-Au NCs and SOSG mixture, respectively. Inserts in (C) and (D) show dynamics of the PL band of BSA-Au NCs during experiment time in irradiated and control samples, respectively. PL registration settings: for (A)-(D) and inserts, λex = 480 nm, Δλex = 1 nm, Δλem = 5 nm.

in a region of shorter wavelengths, and the spectrum had an absorption band with a peak at around 278 nm, which corresponds to the absorption band of BSA solution with the same concentration of protein (Fig. 3). Photoluminescence spectrum of a BSA-Au NCs solution possessed two bands with peak positions at 488 nm and 680 nm in a visible region. As it has been shown in an earlier study, the PL band at 488 nm is not related to the BSA-Au NCs photoluminescence [33]. The appearance of a similar band in the fluorescence spectra of proteins was explained as a result of electron delocalization through the peptide chain [34]. However, neither components used in BSA-Au NCs synthesis (HAuCl4, BSA), nor the mixture of those two materials have a photoluminescence band in a red spectral region (600–700 nm). The photoluminescence excitation spectrum of a BSA-Au NCs solution (λem = 680 nm) had a band at about 505 nm and a gradual slope towards a region of longer wavelengths. However, only a negligible shoulder was detected around 505 nm in the absorption spectrum implying that the NCs sample contained nonfluorescent species, which were also formed during the synthesis process.

Fig. 4(A). A fluorescence (FL) spectrum of DHR123 solution had a weak band at 525 nm, which can be attributed to the Rhod123 formed from DHR123. A FL spectrum of BSA-Au NCs and DHR123 mixture had two bands at 536 nm and 686 nm. It should be noted that in the presence of BSA-Au NCs the former band was decreased and red-shifted by 11 nm. Wang et al. showed that rhodamine in solutions readily interacts with BSA [35]. This interaction could be responsible for the observed shift of the Rhod123 FL band. When a mixture of BSA-Au NCs and DHR123 was irradiated with 405 nm light, DHR123 was readily oxidized back to the parent dye rhodamine 123 by generated reactive oxygen species and thus served as a fluorescent probe for ROS [28,29]. The photoinduced changes in photoluminescence spectra of the samples are shown in Fig. 4(B,C). The intensity of the FL band of Rhod123 increased approximately 2 times relatively to the initial intensity in the sample of DHR123 after the final irradiation dose, while the position of the peak didn't change (Fig. 4(B)). The corresponding increase in intensity of the Rhod123 FL band up to 25 times was measured in the mixture of BSAAu NCs and DHR123 (Fig. 4(C)). In addition, a PL peak intensity of BSAAu NCs decreased by 48%, and its position underwent a hypsochromic shift from 686 nm to 677 nm (Fig. 4(C), an insert). Observation of the control non-irradiated sample of BSA-Au NCs and DHR123 mixture has revealed that the intensities of the Rhod123 FL and the BSA-Au NCs PL bands increased up to 160% and 105%, respectively, while the peak positions remained constant (Fig. 4(D) and an insert). These data indicate that several processes were possibly responsible for such

3.2. Detection of ROS and singlet oxygen In order to test ROS generation capability of BSA-Au NCs by means of fluorescence spectroscopy, two different probes were added into aqueous solutions of nanoparticles. Photoluminescence spectra of DHR123 solution, BSA-Au NCs solution and solution containing DHR123 mixed with BSA-Au NCs before exposure are presented in 5

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Fig. 6. The photoinduced and control (time-related) changes in the peak fluorescence intensity of the ROS indicators as well as the in the photoluminescence intensity of BSA-Au NCs being measured in samples of: (A) DHR123 and its mixture with NCs; (B) SOSG and its mixture with NCs; (C) NCs and its both mixtures.

intensity gain of the Rhodamine 123 FL band in the irradiated mixed samples, albeit to different extent. On the one hand, the DHR123 in solution is slowly oxidized back to Rhod123 by dissolved oxygen and, furthermore, this process is accelerated by the light being absorbed both by DHR123 and Rhod123. On the other hand, the strongly accelerated oxidation of DHR123 in the irradiated mixed samples as well as the extent of the observed increase in FL intensity indicate the formation of ROS, the origin of which cannot be explained otherwise than being a result of the photoexcitation of BSA-Au NCs. The decrease of the PL band of BSA-Au NCs as well as its hypsochromic shift detected after the irradiation imply the photoinduced degradation of nanoclusters. Furthermore, in contrast to the irradiated samples, the PL band of BSAAu NCs in the control samples has increased without any changes in its spectral position (Fig. 4(D), an insert). These findings are very similar to those reported in our previous study on photostability of BSA-Au NCs [33]. Subsequent experiment was performed in order to address a question whether BSA-Au NCs can produce a specific ROS – singlet oxygen. To detect singlet oxygen, the commercially available fluorescent probe named SOSG was selected. In the presence of 1O2, SOSG turns into endoperoxide (SOSG-EP) that emits an intense green fluorescence band peaking at 531 nm [32]. An intensity of the SOSG-EP FL band is mainly dependent on the initial concentration of the source producing 1O2, concentration of SOSG and the irradiation dose used for 1O2 generation [32]. The wavelength (405 nm) of a light source was chosen so that to minimize the direct excitation of the oxygen sensor, because absorption

of SOSG at this wavelength is considerably lower than that of BSA-Au NCs (see Fig. S2). Photoluminescence spectra of samples containing SOSG, BSA-Au NCs and BSA-Au NCs mixed with SOSG are presented in Fig. 5(A). Similarly as in the case of DHR123, the sample of SOSG initially exhibited a relatively weak fluorescence around 530 nm indicating slow auto-oxidation of the dye, which is consistent with findings in the literature [39]. A FL spectrum of the SOSG and BSA-Au NCs mixed solution had two bands at 537 nm and 682 nm (Fig. 5(A)). The band at 537 nm can be attributed to the formed SOSG-EP interacting with BSA. It had been shown that upon addition of BSA to a solution of SOSG, the FL intensity increased considerably and underwent a bathochromic shift [30]. These observations led to the conclusion that in the presence of the protein the quantum efficiency of the SOSG-EP fluorescence increases. Nevertheless, the appreciable irradiation-dependent changes had been recorded measuring both absorbance and fluorescence intensity of the samples with SOSG [30]. The dependencies of changes in photoluminescence spectra of the samples on irradiation doses are shown in Fig. 5(B,C). A slight variation in intensity of the FL band was detected in samples of SOSG after the last irradiation dose (Fig. 5(B)). An intensity of the SOSG-EP FL band increased by 170%, and the PL band of BSA-Au NCs decreased by 51% in the mixed sample of SOSG and BSA-Au NCs during irradiation (Fig. 5(C)). In addition, the peak position of the BSA-Au NCs PL band underwent a hypsochromic shift for 14 nm (Fig. 5(C), an insert). The intensity of the SOSG-EP FL band in the control unexposed sample of SOSG and BSA-Au NCs mixture increased slightly (2%, Fig. 5(D)), while 6

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Fig. 8. Viability assessment of MCF-7 and MDA-MB-231 cells: control, incubated with BSA, with BSA-Au NCs and with Ce6. One group of samples was kept in the dark (left), the other group was irradiated with a xenon lamp (400 nm, 22 mW/cm2; 37.5 J/cm2). Error bars show standard deviations. Statistically significant differences compared to the control cells are shown (*, p ≤ .05; **, p ≤ .01); # indicates significant differences between the MCF-7 and MDA-MB-231cell lines (p ≤ .01).

conditions. BSA was used to test whether it alone (without Au NCs) could induce any harm for cells when exposed to violet radiation. Ce6 was chosen for positive control as a well-known photosensitizer used for PDT. As demonstrated in Fig. 7, no negative effects on viability or morphological features were detected in both control MCF-7 and MDAMB-231 cells as well as in cells incubated with BSA solution before exposure and afterwards. However, some minor damage was observed in non-irradiated cells, which were incubated with BSA-Au NCs or Ce6. Viability of irradiated cells, which were incubated with BSA-Au NCs, decreased, and their natural morphological appearance underwent certain changes. The cells become more spherical in shape, which could indicate occurring apoptosis process [37]. Incubation with Ce6 and irradiation induced the highest damage – majority of cells were stained with red fluorescence of ethidium homodimer-1 indicating that cells were non-viable. Quantification data of cell viability for the samples kept in the dark and those after the irradiation are presented in Fig. 8. Addition of BSA in the medium stimulated growing of both MCF-7 and MDA-MB-231 cell lines, which resulted in 5% more MCF-7 and 15% MDA-MB-231 cells at the end of the experiment. However, the comparison between the samples of MCF-7 and MDA-MB-231 cells, which were incubated with BSA-Au NCs in the dark, and the control samples showed that viability of the former decreased to about 78% and 76%, respectively. The similar reduction, though, has been detected for MCF-7 cells after incubation with Ce6. There were no noticeable toxic effects in the control group of cells and those incubated with BSA after irradiation, but a slightly reduced growth rate of MDA-MB-231 cells in the BSA group. At the same time the viability of MCF-7 and MDA-MB-231 cells incubated with BSA-Au NCs decreased to 13% and 50% after the exposure, whereas viability of those cells, which were incubated with Ce6, decreased to 11% and 4%, respectively. Interestingly, there was a considerable difference in phototoxicity that was induced in the two cell lines incubated with nanoclusters after the same applied exposure: the cells of MDA-MB-231 line were more resistant than MCF-7 cells. On the other hand, MDA-MB-231 cells were much more sensitive to the treatment with Ce6, demonstrating viability at an even slightly lower level than that detected for MCF-7 cells. The observed differences in phototoxicity of the BSA-Au nanoclusters might be due to some cytological variations between the two cancer cells. Thus, MDA-MB-231 cell line represents a model for CD44+/CD24−/EpCAM+ breast cancer cells, which are known for poor prognosis and are linked with cancer stem-like cells, whereas MCF-7 cell line serves as a model for CD44low/ − /CD24+/EpCAM+ breast cancer cells, which are less malignant, more sensitive to chemotherapy treatment and known for good prognosis after treatment [41]. It has also to be noted that the applied method for

Fig. 7. PDT effect of BSA-Au NCs in MCF-7 and MDA-MB-231 cell cultures. Confocal fluorescence microscopy images: control (untreated), incubated with BSA (6.77 × 10–5 M), BSA-Au NCs (6.77 × 10–5 M) and Ce6 (10−5 M). After 24 h of incubation the selected part of cells was irradiated (55 mW/cm2; 37.5 J/ cm2) and afterwards all the cells were treated with LIVE/DEAD cell viability/ cytotoxicity dyes (green-fluorescing calcein-AM stains alive cells, red-fluorescing ethidium homodimer-1 stains dead cells). A scale bar is 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the PL band of BSA-Au NCs remained unchanged (Fig. 5(D), an insert). It is known that SOSG can generate 1O2 (yet, very ineffectively) [40], and this leads to an increase of the SOSG-EP fluorescence band in the dark. However, a negligible increase in the FL intensity being detected in the control non-irradiated sample (Fig. 5(D)) implies that SOSG itself could not contribute with such amount of 1O2 that could explain the observed increase of SOSG-EP fluorescence in the presence of BSA-Au NCs (70%) at chosen exposure conditions. The normalized changes in the peak FL intensities of both ROS probes and the corresponding PL changes of BSA-Au NCs being measured in the exposed samples as well as those in the control samples are shown in Fig. 6(A,B). The presence of either DHR or SOSG revealed the clear enhancement of the production of ROS in irradiated solutions of NCs, which was monitored spectroscopically under excitation at the selected spectral region. The changes in intensity of the PL band of BSAAu NCs being measured in different samples are compared in Fig. 6(C). The natural explanation for the reduction in the PL intensity of BSA-Au NCs among all the exposed samples (Fig. 6 (C)) is the autooxidation that damages the structure of the fluorophore. The time-resolved photoluminescence measurements that were performed on control and irradiated samples demonstrated the three-exponential PL decay pattern in both cases, which showed a tendency to accelerate slightly with increasing irradiation dose (see Fig. S3 for details). 3.3. Photocytotoxicity of BSA-Au NCs Previous investigation on the intracellular uptake of BSA-Au NCs has shown that BSA-Au NCs accumulate in MCF-7 and MDA-MB-231 cells: 73.5% of the MCF-7 and 74.6% of MDA-MB-231 cells had internalized BSA-Au NCs after 24 h of incubation [36]. Thus, 24 h incubation was chosen for sensitization of cells with BSA-Au NCs. The PDT effects of added BSA and Ce6 were tested under the same exposure 7

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Fig. 9. A tentative model for the interactions of ROS produced by BSA-Au NCs with sensors (A) SOSG and (B) DHR.

Appendix A. Supplementary data

quantitative assessment of inactive cells was based on direct sensitivity for the membrane damage, which is not always occurring in the case of apoptotic processes at an early stage. The tentative interactions between ROS and the molecular sensors adsorbed to NCs are schematically depicted in Fig. 9. The photoactivation of ROS in the vicinity of NCs presumably can result in oxidative reactions with protein itself, thus affecting the frame stability of the Au cluster, or targeting the Au fluorophore directly. The presence of probes within the protein moiety makes it possible to observe their slight protective effect on the PL intensity with respect to the exposed samples containing only NCs, which appeared at higher exposure doses. Thus, the extent of the photochemical activity being detected in cancer cells as well as low toxicity comparable with that of a conventional photosensitizer in the absence of light, demonstrate the limited potential of BSA-Au nanoclusters as biocompatible photosensitizing agents, while the concomitant changes in the PL intensity could be used for observation of local oxidative processes occurring in biological systems.

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4. Conclusions The exposure of gold nanoclusters synthetized in bovine serum albumin moiety to light in the presence of two fluorescing probes revealed diverse photochemical processes in aqueous medium involving not only singlet oxygen but also other reactive oxygen species. The photoinduced cytotoxicity of nanoclusters was found to be notable in the two studied breast cancer cell lines, but also showed considerable variation between them. This confirms that such gold nanoclusters can be considered as photosensitizing agents, albeit, not for the general usage in the photodynamic therapy of cancer. The sensitivity of photoluminescence properties in the red spectral region to the applied exposure in addition to the relative biocompatibility might be also exploited for the optical monitoring of the photoinduced oxidative processes in biological systems. Declaration of Competing Interest 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. Acknowledgements This research was partially funded by the project “Programming cells and management of tumor microenvironment for personal therapy in oncology – LASTER”, VP1-3.1-ŠMM-10-V-02-027. 8

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