Nanoparticles of methylene blue enhance photodynamic therapy

Nanoparticles of methylene blue enhance photodynamic therapy

Accepted Manuscript Title: Nanoparticles and Methylene Blue for Enhancement Photodynamic Therapy Authors: V.P.S. Jesus, L. Raniero, G.M. Lemes, T.T. B...

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Accepted Manuscript Title: Nanoparticles and Methylene Blue for Enhancement Photodynamic Therapy Authors: V.P.S. Jesus, L. Raniero, G.M. Lemes, T.T. Bhattacharjee, P.C. Caetano J´unior, M.L. Castilho PII: DOI: Reference:

S1572-1000(18)30044-9 https://doi.org/10.1016/j.pdpdt.2018.06.011 PDPDT 1187

To appear in:

Photodiagnosis and Photodynamic Therapy

Received date: Revised date: Accepted date:

20-2-2018 6-5-2018 13-6-2018

Please cite this article as: Jesus VPS, Raniero L, Lemes GM, Bhattacharjee TT, Caetano J´unior PC, Castilho ML, Nanoparticles and Methylene Blue for Enhancement Photodynamic Therapy, Photodiagnosis and Photodynamic Therapy (2018), https://doi.org/10.1016/j.pdpdt.2018.06.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nanoparticles and Methylene Blue for Enhancement Photodynamic Therapy V. P. S. Jesusa,b, L. Ranierob, G. M. Lemesa,b, T. T. Bhattacharjeeb, P. C. Caetano Júniorb, M. L. Castilhoa aLaboratório

de Bionanotecnologia, Instituto de Pesquisa & Desenvolvimento, Universidade do Vale do Paraíba, Av.

bLaboratório

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Shishima Hifumi, 2911, Urbanova, São José dos Campos, São Paulo 12244-000, Brazil. de Nanossensores, Instituto de Pesquisa & Desenvolvimento, Universidade do Vale do Paraíba,

Av. Shishima Hifumi, 2911, Urbanova, São José dos Campos, São Paulo 12244-000, Brazil.

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Graphical abstract

Graphical abstract: Schematic representation of the mechanism of action of PDT with nanoparticles in breast cancer cells. In particular, AgNPs induce mitochondrial and DNA damage by ROS.

Highlights   

Nanoparticles combined to methylene blue have great potential as an agent in PDT. The cellular uptake of MDA-MB-468 was investigated by fluorescence. The cytotoxicity effects of AgNPs are the release of Ag + ions from their surface.

Abstract Breast cancer is the most commonly diagnosed cancer and the second leading cause of death related to

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cancer among women worldwide. Screening and advancements in treatments have improved survival rate of women suffering from this ailment. Novel therapeutic techniques may further reduce cancer related mortality. One of the several emerging therapeutic options is Photodynamic Therapy (PDT) that

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uses light activated photosensitizer (PS) inducing cell death by apoptosis and/or necrosis. Nanotechnology has made contribution to improve photosensitizer for PDT, increasing the efficiency of therapy using gold and silver nanoparticles. Efforts have been done to develop better mechanism to improve PS and consequently PDT effects. In this study, we investigate the efficacy of the PDT using

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gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) when mixed to methylene blue (MB) in the treatment of the human breast adenocarcinoma cell line (MDA-MB-468). The MDA-MB-468 was

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treated in the presence of different MB concentrations with/without AuNPs or AgNPs. The colloidal

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solution of AgNPs showed a plasmon resonance band at 411 nm in UV-visible range and a bimodal size distribution. The results of viability analysis showed that cells treated with nanoparticles exhibited higher

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cytotoxicity than cells treated with only MB, improving the efficiency of the treatment in the tumor cells. The cytotoxicity effect of MB associated with AgNPs on MDA-MB-468 cell line could be related to

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increased reactive oxygen species production due to the release of Ag + ions from nanoparticles surface, suggesting that the association between FS and AgNPs has potential as a PDT agent.

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Introduction

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Keywords: Gold nanoparticles, Silver nanoparticles, Methylene blue, Photodynamic Therapy, Breast

Breast cancer remains the most commonly diagnosed and the second most frequent cause of women

death in the United States, according to the latest 2017 statistics [1]. Estimated 252,710 new cases and 40,610 deaths were expected that year. It has been shown that the overall cancer death rate has reduced

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by 39% from 1989 to 2015. Breast cancer survivorship statistics in 2016 showed that 3.5 American women with history of breast cancer alive, with 89, 83 and 78% relative survival rates for 5, 10 and 15 years, respectively [2]. This is primarily due to prevalence of breast cancer screening and emergence of novel therapeutic options. The triple-negative breast cancer (TNBC) is the most aggressive subgroup, which is characterized by the lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor 2. In TNBC, the epidermal growth factor receptor (EGFR) is frequently overexpressed and, when it is activated by a ligand triggers a process of differentiation and cellular proliferation leading to worse

prognosis. The MDA-MB-468 breast cancer cell line features overexpression of EGFR (1.9x106 EGFR/cell) characterizing a TNBC, which require advancements in breast cancer treatment [3-5]. One emerging treatment option is Photodynamic therapy (PDT). PDT involves activation of a photosensitizer (PS) by irradiation, which generates reactive oxygen species (ROS) from cellular oxygen and free radicals. ROS are highly reactive and result in cells membrane damage, inducing the cells death. This technique has been an option for TNBC treatment due to physical, chemical and biological effects, such as reproducibility without drug resistance, low toxic in the absence of irradiation, irradiation nonionizing and no cumulative toxic effects [6-9].

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Methylene blue (MB) is a PS belongs to the phenothiazine’s family, which is a heterocyclic aromatic dye soluble in alcohol or water. MB presents an excellent penetration in the cellular membrane due to the

capacity of its benzene ring to concentrate in the mitochondria, lysosomes and double-stranded DNA. It

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has photochemical properties that produce a good quantum yield, hydrophilicity and low costeffectiveness [10-13].

The limitations for in vivo PDT involve biocompatibility, kinetics, toxicity, pharmacokinetics, biodistribution, deposition, cross-interactions and others. In this context, the nanotechnology has contributed to increase the PS efficiency, improving the effects of PDT on cancer treatment using gold

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(AuNPs) and silver nanoparticles (AgNPs), which can be functionalized with the PS [12,13,14,15 ]. It is

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well known that nanostructured materials such as nanoparticles, have been increasingly applied in cancer treatments, due to their biocompatibility, permeation, and distribution in cells, as well as their capacity to

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be chemically inert and scatter light strongly [8, 16-18].

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Gold and silver nanoparticles have been attracting interest in the treatment of cancer, as result of their physical and chemical properties. These properties are size, charge, shape and surface characteristics, they are fundamental in biodistribution and cellular internalization [19]. The AuNPs applications become very

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favorable because of its unique properties and characteristics such as low toxicity, biocompatibility, easily can be chemically modified in its surface area, excellent plasmonic activity and act as an agent facilitator in the process of absorption of hydrophobic drugs [20-22]. The AgNPs have gained important attention

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as an anticancer agent via PDT, as they generate cytotoxic reactive oxygen species when irradiated with suitable light wavelengths. Studies show that the cytotoxicity effects of AgNPs can be attributed to Ag+

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ions releasing from their surface, induce higher levels of intracellular ROS, and leading to cancer cell death [15, 23-27].

The study of nanoparticle cytotoxicity has been promising since it involves many biological

applications, thereby contributing to the advance of the diagnosis, prevention and treatment of diseases [16]. The cell viability assays evaluates the interaction of cells with NPs determining their therapeutic

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applications. The nanoparticles can enhance the photosensitizers action increasing the oxygen species upon laser irradiation; causing damage in the DNA and chromosomes of the cancers cells [15 - 28]. In an effort to improve PDT effects, this work describes the syntheses and characterizes of nanoparticles by UV-visible Spectroscopy and Dynamic Light Scattering (DLS). The Fluorescence Spectroscopy and Confocal Fluorescence Microscopy were used to determine the cellular uptake of methylene blue in the MDA-MB-468 cells line. In vitro assays in MDA-MB-468 cells line will show the efficacy of nanoparticles compared to free MB, which was determined by the trypan blue exclusion method.

Material and methods Cell culture The human breast carcinoma cell line (MDA-MB-468) was obtained from the Banco de Célula Rio de Janeiro (BCRJ). They were cultured in 25 cm2 tissue culture flasks in L-15 medium (Leibovitz, Sigma Life Science, L4386) and supplemented with 10% Fetal Bovine Serum (FBS, Sigma Aldrich, F2561500ML). The cells were grown at 37◦C in a humidified 95% air atmosphere.

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Synthesis of gold and silver nanoparticles Gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) were synthetized using the conventional method described by Lee and Meisel [29]. Briefly, AuNPs were prepared using gold chlorate

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trihydrate (Sigma Aldrich, 520918) diluted in ultra-pure water to a final concentration of 0.04%, heated to

~98ºC, adding sodium citrate solution (S4641, Sigma Aldrich) at 1%, data previously showed [18]. The AgNPs were obtained by reducing the silver nitrate (Sigma Aldrich, 209139) (0.0182%) using a sodium citrate solution at 2% under constant stirring and boiling (~98ºC). The system was kept under heating and stirring for 30 min to nucleate nanoparticles. The AgNPs were characterized by UV-visible spectroscopy,

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DLS and Zeta Potential.

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Fluorescence Spectroscopy

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The fluorescence spectroscopy allows the monitoring of the incorporation time and quantification of photosensitizer methylene blue in the MDA-MB-468 cells line. The 105cells suspension were seeded in a 96-well plate for 24 h in order to attach. After this period, the culture medium was replaced with 2 μg/mL

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of MB and incubated for different periods (15, 30, 60, 120, 240, 360, 720, 1440 and 2880 min) in the dark at 37 °C. The plate was washed twice with PBS after each incubation time and the fluorescence intensity was measured using a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, USA) with

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excitation at 664 nm and emission at 690 nm.

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Confocal Fluorescence Microscopy The micrographs were registered by Confocal Laser Scanning Microscope (LSM 700, Zeiss) to

confirm the incorporation of photosensitizer in the MDA-MB-468 cells line. Thin borosilicate glasses with 105 cells were incubated in the dark with MB (2 μg/mL) for 2h and 24h in the 24-well plate. The group without MB was used as control. After incubation times the plate was washed twice with PBS and fixed in

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a paraformaldehyde solution at 4% for 30 min. The borosilicate glasses were washed with PBS again and Prolong® Antifade (Life Technologies, P36971) was added to highlight the cells cytoplasm. The images were processed in Zeiss software ZEN Black edition.

Photodynamic therapy assay The MDA-MB-468 suspension cells (105cells) were seeded onto two 96-well plates and allowed to attach for 24 h. After cells adhesion, the medium was replaced and the PDT was performed in the presence

of different methylene blue concentrations (2, 4, and 6 μg/mL) with/without AuNPs or AgNPs (9x1010 particles/105cells). Control cells received only culture medium. The cells were incubated for 2h in the dark environment at 37 °C and then washed with PBS, before medium replacement. Following these procedures, one plate kept in the dark and the other was irradiated with an Irrad-Led5 660 (660nm wavelength, Biopdi), using an irradiance of 25 mW/cm2, potency 70 mW and fluence 25 J/cm2. After 18 h PDT, the cells were kept under dark environmental at 37◦C. The cell viability was measured using the trypan blue exclusion test (Sigma Aldrich, T8154). A trypan blue solution at 0.4% was added to the 96 well plate, during 5 min, followed by two PBS washing. Five

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photographs were randomly collected from each well and the cells were counted using ImageJ software.

Intracellular ROS production

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Accumulation of ROS was quantified using 2’, 7’- dichlorodihydrofluorescein diacetate (H2DCF-DA) (Molecular Probes, Eugene, OR, USA) staining. MDA-MB-468 suspension cell (105 cells) was seeded, in

a 96-well plate and allowed to attach at 37ºC. After 24h, the MB (2 μg/mL) was incubated with/ without AuNPs or AgNPs for 2h in the dark environment at 37 °C. Following these procedures, the plate was

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irradiated using a fluence of 25 J/cm2 and kept 1 h under dark environment at 37 °C. After this period, the cells were washed with PBS and 4µl of H2DCF-DA (10 µM) was added and the cells were incubated for

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1h. The fluorescence intensity of the suspension was measured directly, in arbitrary units, using a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, USA) with excitation at 485 nm and emission

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at 530 nm.

Results and Discussions

Analyses of the nanoparticles performed by the UV-visible spectroscopy and DLS technique are

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shown in Fig. 1. The absorption spectrum of the colloidal solutions shows a characteristic plasmon resonance band centered at 411 nm for the AgNPs (Fig. 1a), a hydrodynamic diameter around 43 nm and a polydispersity index value of 0.676 (Fig. 1b). These results are in agreement to the observations of

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Paramelle et al. [30].The synthetized gold nanoparticles showed a UV-visible spectrum with a plasmon resonance band maximum at 524 nm and an average size around 21 nm (Data was not shown, published in

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Castilho et. al. 2017 [18]).

The DLS of AgNPs show a bimodal particle size distribution, which suggests that ‘large’ (43nm) and

‘small’ (2nm) particles coexist. In fact, these results are in better agreement with nucleation conditions described by Ostwald ripening process, which suggests that small particles tend to coalesce on the surface of the larger particles. However, many seeds are continuously being formed in this process and cannot grow

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because of the formation of depletion layers around, consequently generating two populations of particles [29, 30]. In addition, more than 85% of AgNPs are in the population centered at 43 nm (Figure 1b). The zeta potential value of -48mV is indicative of good stability [31].

Fig. 1: Characterization of the silver nanoparticle. a) UV–visible spectra of the as-synthesized AgNPs. b) DLS-derived histogram of hydrodynamic diameter of AgNPs.

These results are important to elucidate nanoparticles application in cancer cells. The colloidal solution showed an appropriated size distribution to cell uptake and the zeta potential indicated chemical stability against aggregation. The mixture of MB with nanoparticles did not change this stability and the cellular uptakes in MDA-MB-468 cancer cells, which was investigated by fluorescence spectroscopy, by elucidating the number of molecules of the photosensitizer internalized per cell. The treated cells show time-dependent increase of fluorescence signal (Fig. 2). This fact can be related to the characteristics of the PS from phenothiazine’s group, which present fast incorporation kinetics due to such characteristic as low molecular weight, hydrophobicity and positive charge, promoting its transition through the cell membrane

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[32]. Although PS passes easily through the cell membrane, the incubation time for treatment of tumor cell lines is not well defined in the literature, accomplishing PDT treatment difficult [12, 33-35]. The behavior of this PS is quite peculiar, since it is observed the successive increase of the MB within the cells is

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approximately 10-fold greater; according to the time of incorporation as shown in Fig. 2. However, the 2h period for the cellular uptake of this PS is effective for PDT applications, due to the number of internalized molecules (2.24 x 108).

Fig. 2: Cellular uptake and internalization of methylene blue was conducted on MDA-MB-468 cancer cells

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until 48h.

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In order to characterize the cellular uptake and localization of methylene blue, the confocal fluorescence microscopy analysis were performed on MDA-MB-468 cancer cells, as shown in Fig.3. The

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micrographs exhibit red fluorescent dots in the cytoplasm, which indicates intracellular uptake of the PS;

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while the blue fluorescence refers to the labeling of nuclear DNA by DAPI marker. Gradual increase in red fluorescence of MB treated cells was observed, according which corroborates the results of Fluorescence Spectroscopy analysis. Initially after 2h of incubation at 37ºC, the MDA-MB-468 cells exhibit very weak

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intracellular fluorescence intensity of MB, when compared to 24h. However, the number of MB molecules determined by fluorescence spectroscopy and the 3D images confocal fluorescence microscopy becomes

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sufficient for treatment via PDT for 2h, based on the presence of PS in the intracellular region.

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Fig. 3: Micrographs of MDA-MB-468 after 2h and 24h incubation with M.B and control group.

To determine the effects of PDT on cancer cells (MDA-MB-468) micrographs in bright field were

obtained by staining the cells with trypan blue dye and quantifying the number of viable cells (Figure 4). The survival rate of the cells was defined by staining, blue stained represent dead cells whereas live cells were represented by unstaining. In control group (Figure 4a) cells membranes are intact and trypan blue

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dye being unable to penetrate healthy cells. The Figure 4b and 4c shows the ability of PDT to induce cells death with similar results were observed, despite low efficiency. MB and AgNPs treated cells exhibited high efficiency by PDT in comparison to other groups, evidencing damage to cells membrane (Figure 4d).

Fig. 4: Trypan blue exclusion test of MDA-MB-468 cell viability after PDT. (A) Control group; (B) Cells treated with MB at 2 μg/ml; (C) Cells treated with MB at 2 μg/ml and 10 9 AuNPs /ml; and (D) Cells treated with MB at 2 μg/ml and 109 AgNPs /ml.

In the PDT, the efficacy of MB mixture to AuNPs or AgNPs was compared to free MB in MDA-MB468 cells and quantified by trypan blue exclusion test (Fig. 5). In the absence of irradiation, no significant cell toxicity was observed for all groups, confirming that the free MB, AuNPs mixture MB and AgNPs mixture MB are non-cytotoxic for the experimental conditions used (Fig. 5b). Upon irradiation at 660 nm and fluence 25 J/cm2, the MDA-MB-468 cells treated with 2, 4 and 6 µg/mL of free MB had 82.4%, 70.6% and 32.8% of viable cells, respectively (Fig. 5a). The cells treated with MB mixture to 9x1010 AuNPs did

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not show evidence of photodynamic therapy improvement, when compared with free MB. Yu et al. (2014), reported that gold nanoparticles that carried MB retained the PDT efficiency as pure MB in cervical cancer

cells, which are consistent with the observations of this work [12]. However, almost all cells treated with MB mixture to AgNPs die using 2 µg/mL of MB rather than that free MB obtains 17.6% cytotoxicity. These

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results can be explained by the cytotoxicity effects of AgNPs, when Ag+ ions are released from their surface, leading to higher levels of intracellular ROS [12,15, 24-25, 27].

Fig. 5: The viability of MDA-MB-468 measured by trypan blue exclusion test after 2h incubation time.

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a) Upon irradiation (660 nm, 25 mW/cm2, 70 mW and 25 J/cm2). b) Without laser irradiation. The final

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nanoparticles concentration was 9x1010 particles/105 cells. Data are presented as mean ± SE (n = 3).

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In order to study the mechanism by which PDT with AgNPs increased the effectiveness of treatment in TNBC subgroup cells, intracellular ROS production was determined by H2DCF-DA (Figure 6). It was

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observed that in control group (only cells) does not show modify ROS accumulation. Comparing to control, free MB and MB mixture AuNPs demonstrate an increase by three times of intracellular ROS production.

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However, the cells treated by MB mixture AgNPs exhibited a significant increase intracellular ROS production in six time when compared control group. This fact may be related by the oxidative stress damage of mitochondria induced by the presence of AgNPs and Ag +.

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In addition, the AgNPs and Ag+ ions exhibit a high affinity for thiol groups that are present in the cytoplasm, cell membrane, and inner membrane of mitochondria, concatenating in an oxidative damage to proteins, DNA and inducing a mitochondrial dysfunction [27]. Srinivasan et al. (2016), describe that the

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releasing of these metallic ions induces the formation of ROS and they depend on many factors such as capping agent, medium pH, incubation time, and incubation temperature [15]. Foldbjerg and co-autors (2011) showed that AgNPs could induce oxidative stress correlating with cytotoxicity and genotoxicity in the human lung cancer cell line [24]. Guo et al. (2015) evaluated the anticarcinogenic activity of the human

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Hepatoma cell (HepG2), showing that AgNPs into the cell inhibits the viability of the cell by apoptosis. Thus, the AgNPs are cytotoxic due to their interaction with mitochondria, triggering apoptosis and producing ROS [36]. Ishida (2017) reported that AgNPs had inhibitory effects on growth and angiogenesis in adenocarcinoma cells (A549) [37].

Fig. 6: Effect of PDT and nanoparticles on intracellular ROS production in MDA-MB-468. The ROS production was determined 1 h after PDT, using 2 µg/mL of MB and 9x10 10 particles/105 cells. Data are presented as mean ± SE (n = 3).

Nevertheless, the effects of AgNPs have not been fully elucidated yet and the release of Ag+ ions and the production of ROS still need to be more explored [37]. In summary, AgNPs show excellent biocompatibility as well as greater cytotoxicity upon irradiation, suggesting it has a potential to be used as a PDT agent with to methylene blue in breast cancer cells.

Conclusion

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The AgNPs showed good chemical stability and a bimodal distribution with hydrodynamic diameter of 43 nm, characterized by DLS and UV-Visible spectroscopy, which showed a resonant band centered at

410 nm. Fluorescence spectroscopy and confocal fluorescence microscopy suggested the 2-hour period best suited for MB incorporation into the MDA-MB-468, providing a high affinity to the cell membrane and

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may be of great relevance for PDT optimization. The cytotoxicity study showed that AgNPs were more

effective (99.3%) in the treated MDA-MB-468 cells, improving the efficiency of tumor cell treatment. In contrast, free MB with AuNPs did not show significant change in cell death, which was performed by trypan blue exclusion tests. MB mixture with AgNPs showed an increase in ROS production, which could

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be explained by the release of Ag+ ions. Thus, the in vitro test exhibited performance, indicating that the

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association between FS and nanoparticles has potential as a PDT agent.

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Acknowledgments

This research was financially supported by the FAPESP (Project 2013/17404-7), CNPq

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(302132/2015-5) FINEP (Conv. 01.13.0275.00) and CAPES for the scholarship. The authors would like to thank Ph.D. Juliana Ferreira Strixino, “Laboratório de Terapia Fotodinâmica” (FAPESP 2010/00488-5)

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from Universidade do Vale do Paraíba, for lending the PDT equipment.

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PT

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IP T

SC R

U

N

A

M

A ED

PT

CC E

IP T

SC R

U

N

A

M