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Combination photodynamic therapy of human breast cancer using salicylic acid and methylene blue
Accepted Manuscript Combination photodynamic therapy of human breast cancer using salicylic acid and methylene blue
Reza Hosseinzadeh, Khatereh Khorsandi, Maryam Jahanshiri PII: DOI: Reference:
S1386-1425(17)30375-X doi: 10.1016/j.saa.2017.05.008 SAA 15150
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received date: Revised date: Accepted date:
1 November 2016 29 April 2017 4 May 2017
Please cite this article as: Reza Hosseinzadeh, Khatereh Khorsandi, Maryam Jahanshiri , Combination photodynamic therapy of human breast cancer using salicylic acid and methylene blue, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.05.008
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Revised manuscript: SAA-D-16-02377-R1
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Combination photodynamic therapy of human breast cancer using salicylic acid and methylene blue
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Reza Hosseinzadeh*, Khatereh Khorsandi, Maryam Jahanshiri
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*Corresponding Authors:
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Medical Laser Research Center, ACECR, Tehran, Iran
Email: [email protected] ,
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[email protected]
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Abstract The objective of this study was to evaluate the effects of combination therapy with
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methylene blue (MB) assisted photodynamic therapy (PDT) and salicylic acid (SA) as chemo-therapy anticancer agent. The binding of salicylic acid to methylene blue was studied
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using spectrophotometric method. The results show the 1:2 complex formation between SA
=12.92 kJ.mol-1,
=9.02 kJ.mol-1). The spectrosphotometric results
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Kb2=38.13 and
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and MB. The binding constants and related Gibbs free energies o are obtained (Kb1=183.74,
show the improvement in solubilization and reduction prevention for SA and MB in the
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complex form. These results are in agreements with cellular experiments. The dark toxicity measurements represent the improve efficacy of chemotherapy using combination of SA and
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MB. The photodynamic therapy results (using red LED as light source (630 nm; power
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density: 30 mW cm-2)) show that the cancer cell killing efficiency of MB increases in the combination with SA due to reduction prevention and stabilization of monomeric form of
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MB.
Keywords: Photodynamic therapy, Salicylic Acid, Methylene blue, Molecular Interaction, Singlet oxygen, Combination Therapy
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ACCEPTED MANUSCRIPT Introduction According to the reports of world health organization (WHO), over 508 000 women died in 2011 due to breast cancer in worldwide (Global Health Estimates, WHO 2013). Breast cancer is a disease that make challenge for the all of countries in the world in both of the developed and less developed countries [1,2]. Breast cancer patients in Iran is 40 thousand and more
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than seven thousand patients are added to this number, yearly. According to the emotional
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status of women in family and the importance of the role of women in human society, diseases such as breast cancer, can induced high psychological stress in their families and
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communities [3]. So control and therapy of breast cancer is one of the essential challenges in
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all the world. Nowadays, there are many types of cancer therapy methods. The types of cancer treatment depend on the type of cancer and how advanced of cancer. Some patients
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give only one treatment. But most people have a combination of two or further cancer therapies that known as combination therapy. The combination therapy is one of new and
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interesting methods in cancer therapy researches. Combinations of multiple drugs in cancer
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treatments are proving more effective than one drug. Combination therapy not only provide a potential solution to addressing the heterogenic tumors but also can solve the drug resistance
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issues in cancer treatments[4–7]. Combination of photodynamic therapy (PDT) with chemotherapy is interesting method in this field of researches[8–10]. Photodynamic therapy
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is a new and effective therapy method that can be used for the treatment of various types of cancers. PDT is a form of radiation therapy that composed of three nontoxic elements. By illumination of photosensitizer in the presence of molecular oxygen, reactive oxygen species (ROS), particularly singlet oxygen as a cytotoxic species can be induced in tumor cells. The cancer cells would be destroyed by apoptosis or necrosis that switched by ROS, achieving, ultimately, the goal of local treatment with minimum invasion [11–19]. Combination of photodynamic therapy with salicylic acid based chemo therapy, is goal of our study.
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ACCEPTED MANUSCRIPT Salicylic acid is a mono-hydroxybenzoic acid, a type of phenolic compounds and commonly used as plant hormones in agricultural applications and it is probably best known for use as a key agent in topical anti-acne pharmaceutical products and is used as an anti-inflammatory drug. Salicylic acid is also known for its ability to ease aches and pains and reduce fevers. Anti-inflammatory drugs, such as salicylic acid, have been singled out as apoptosis inducing
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agents with anti-cancer activity. Kutlu and coworkers reported of anti-tumor activity the
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salicylic acid as chemotherapeutic drug, on A549 human lung adenocarcinoma cells [20].
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In here, the interaction of salicylic acid with methylene blue was investigated using spectrophotometric measurements and a series of combinatorial therapy were examined by
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considering effectiveness of photodynamic therapy of methylene blue in the presence of
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salicylic acid as chemotherapeutic anti-cancer drug on MDA-MB-231 breast cancer cells.
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Materials and instruments:
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Materials and Methods
Salicylic acid (chemical name; 2-Hydroxybenzoic acid) was purchased from Merck Co.
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(Germany) and tetrazolium dye, MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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bromide) was supplied by Sigma-Aldrich Co. (USA). Methylene blue (Systematic name: 3,7bis(Dimethylamino)-phenothiazin-5-ium chloride) was purchased from Merck Co. Trypan blue solution (0.4% w/v) and dimethyl sulfoxide (DMSO) were achieved from Merck company. Fetal bovine serum (FBS) and antibiotics were purchased from Gibco (Gibco BRL). DMEM medium (Dulbecco's Modified Eagle Medium) was purchased from Invitrogen (Invitrogen, Carlsbad, California, US). The entire buffer salts and other chemicals were supplied from Merck Co. Double distilled deionized water was used for all experiments and solutions.
C, using Metrohm 744 (Switzerland). The 4
ACCEPTED MANUSCRIPT UV-Vis absorption spectra were recorded using Cary 60 UV/Vis spectrophotometer, equipped with quartz cells. Red light emitting LED (630 nm; power density: 30 mW cm-2) was used as light source. The LED power-metery was done at the Electronics Research Institute at Sharif University of Technology (SUT), Optic laboratory, using suitable power
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metric devices. Methods:
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UV-Vis spectroscopic study of Salicylic acid interaction with methylene blue;
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Stock solutions of 5×10−6 mol.L-1 of methylene blue, as photosensitizer, was prepared by
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dissolving the certain amount of dye in distilled water. Stock solution of 2×10−2 mol.L-1 of SA was prepared by dissolving an appropriate amount of the SA in double distilled water.
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The variation of absorbance spectra of MB solution by increasing of SA concentrations was recorded at 200-800 nm wavelength using water as a blank. Methylene blue-salicylic acid
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binding constant was determined from the b o b c ’ of a series of solutions containing a
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fixed concentration of photosensitizer (CMB =1×10−6 mol.L-1) and increasing concentration
Cell culture
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of salicylic acid.
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Human breast cancer cell line, MDA-MB-231, was supplied by the Institute of Pasture, Tehran, Iran. The cells were grown in DMEM medium supplemented with 10% FBS, 100 IU/mL penicillin and 100 µg/mL of streptomycin then incubated in a humidified incubator containing 5% CO2 at 37 ˚C. For the experiments, the cells were removed by trypsinizing (trypsin 0.025%, EDTA 0.02%) and washed with phosphate-buffered saline (PBS). Effect of different analytes concentrations on human breast cancer Cells
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ACCEPTED MANUSCRIPT For considering and comparison the effect of various concentrations of methylene blue, salicylic acid on photodynamic efficacy, a series of experiments were designed for dark and red LED irradiations. Briefly, the MDA-MB-231 cells were seeded using fresh culture medium in petri dishes and incubated under 5% CO2,
37 ˚C for 24 h. Then the cells were
incubated using fresh cell culture medium containing certain amounts of analytes (Methylene
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blue, SA). After certain incubation time, the cells were washed by PBS buffer solution. One
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of the treated plates considers as reference plate (no irradiation (dark)) and the other plates
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irradiated using red LED for 30 min. The cell viability was measured after incubations. The colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay was
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used to determine the cells viability. Each experiment was repeated 3 times.
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Effect of different incubation and irradiation time on cancer cells The effects of incubation and irradiation times were optimized by considering various
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incubation and irradiation periods in the presence and absence of analytes. After incubation
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or irradiation under specified times the cell viability was determined using MTT assay. All experiments were repeated three times.
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In vitro photodynamic assay
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The MDA-MB-231 cancer cells were grown in medium culture cell and after reaching 80∼90% confluence; the cells were washed with PBS, afterwards detached from the flask by addition of 1.0
L of 0.
% y i fo 1−3
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37 ˚C. Cells (1×104 cells/well) were
seeded into five 96-well plates. The cells were then treated with methylene blue, Salicylic acid and SA-MB with together, at different concentrations. After a further incubation of 24 h, one plate was considered as dark control plate and the other plates illuminated as described above. The MTT assay was used to determine the cell viability.
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ACCEPTED MANUSCRIPT MTT assay experiment Thiazolyl blue tetrazolium bromide (MTT) was used in determination of cell survival as colorimetric MTT assay. Cell viability can be measured as a function of cells redox potential. Living cells convert the MTT compound to an insoluble formazan. The resulting formazan
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solubilized using dimethyl sulphoxide (DMSO) and its concentration determined using spectrophotometric methods. Briefly, culture medium was removed and cells were incubated
37 ˚C. Th
i g purple formazan crystals dissolved in 100 µL DMSO
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bromide for 4 h
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in medium containing 0.5 mg/mL of 3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
and shacked for 15 min. The absorbance of solutions was measured at 540 nm by an ELISA
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reader (Hyperion, Inc., FL ,U.S.A.).
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Results and Discussions
Interaction of salicylic acid with methylene blue:
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Addition of salicylic acid (stock concentration of SA was 2×10−2 mol.L-1) on MB solution (1×10−6 mol.L-1) makes bathochromic shift in MB maximum absorption spectra and the
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decrease in absorbance with increasing SA concentration. The results demonstrated that MB interact with SA. Figure 1 show the spectral change of MB by addition of SA concentration.
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In figure 2, the variation of 1/Abs versus 1/[C] was constructed based on Benesi Hidebrand equation [21–23]. The graph demonstrated that the variations is linear and complex formation is 1:1 equilibrated interaction. The related binding constant can be estimated.
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Figure 1, UV-Vis absorption spectra of methylene blue ([MB] = 5×10−6 mol.L-1)
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Determination of binding constant:
The value of the binding constant (Kb) can be obtained according to the methods described
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previously [21]. Due to the fact that binding of SA to MB is an equilibrium interaction, so
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equations (1) and (2) can be constructed as: (1)
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MB+SA⇌[Complex]
(2)
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We can rearrange the Eq. 2 as Eq 3:
Equation 3 can be constructed based on spectrophotometric data as Eq. 4;
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ACCEPTED MANUSCRIPT Kb can be estimated from the ratio of the intercept to the slope of the line constructed in figure 2. As can be seen in figure 2, the obtained plot can be divided to two distinct linear parts. According to the Zhi-Wu reports[24], the interactions with two binding interactions, if the binding strengths in both interactions have clear difference, the Benesi-Hildebrand plot is not linear and its due to the differing binding strengths in two binding interactions. By
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considering this fact, the plot can be divided to two linear regions that demonstrated the 1:2
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binding interaction between methylene blue and salicylic acid. The binding constant related to the first and second binding sites were obtained as Kb1=183.74 (slop=0.0923,
icy ic ci i
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hy
b
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C. According to the
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intercept=16.959 and R2=0.99) and Kb2=38.13 (slop=0.0422, intercept=1.609 and R2=0.99)
obtained results it is clear that the interaction of salicylic acid with methylene blue is based
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on molecular interactions and the electrostatic interactions are the initial forces in salicylic acid binding to methylene blue. At higher concentrations of salicylic acid, the hydrophobic
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interactions are the major forces in binding phenomena. In the other words, by further
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increasing in salicylic acid concentration, the hydrophobic interactions come important in binding phenomena. Due to the structural properties of salicylic acid, it behaves as an ionic
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surfactant. In the presence of methylene blue the electrostatic interaction of methylene blue and salicylic acid make complex of dye-drug and by increasing of SA concentration mixed
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micelle of dye-drug can produce. At these conditions, the MB can prevent from redox reactions in the cellular penetrations and also the salicylic acid solubilization can improved in mixed form with methylene blue[25]. Red shift in UV-Vis spectra approve this statement due to increasing of hydrophobicity around the microenvironment of methylene blue in the presence of salicylic acid [21]. Also by increasing of SA concentration, the monomeric form of MB is the stable form and dimerization of MB decreased. As reported previously, the lower wavelength shoulder in MB spectra is related to dimeric form of MB. By increasing of
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ACCEPTED MANUSCRIPT SA concentration, A620 decreased with higher slope in comparison with A665 (slope620=0.0764 and slope665=0.0519). Dimerization pf MB caused higher decreasing in photodynamic therapy efficiency of MB [25]. The Gibbs free energy of binding of SA with MB can be
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obtained using the following thermodynamic equation:
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The estimated value of Gibbs free energy for SA interaction with MB at first binding site is 12.92 kJ/mol and for second binding site is 9.02 kJ/mol that is related to molecular
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interactions. complex formation between SA and MB prevents the MB from environmental
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reduction to leuco-form of methylene blue. The leuco-form is not PDT-active form of MB.
Figure 2, Benesi-Hildebrand plot for interaction of methylene blue (5×10 −6 C [24].
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icy ic ci
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ACCEPTED MANUSCRIPT Viability studies and MTT assay: Cellular effects of various treatments were measured and determined using MTT assay for determination of cell viability after and before of treatments and irradiations. Figure 3, summarized the effect of used compounds on cells viability at dark and in absence of LED irradiation, in the other words, the cellular toxicity of each used material were tested and
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examined without LED irradiation (dark experiment).
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Determination of dark cytotoxicity used MB and SA compounds:
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In order to compare the cytotoxicity effect of MB and SA in the absence of irradiation, the
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effect of these compounds on cell viability were determined. Viability of treated cells with different concentrations of MB and SA (24 h incubation at 0, 10, 25, 50 and 75 µg/mL) were
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determined at dark. According to the obtained results (Fig. 3), it is clear that the cytotoxicity of salicylic acid is higher than MB in the absence of LED irradiation. The cytotoxicity of MB
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at used concentration range in the absence of irradiation is negligible while the cytotoxicity of
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combinational experiment is higher than each other. According to the obtained results in spectrophotometric studies, it is due to the equilibration between electrostatic and
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hydrophobic forces of each anticancer reactive agent. SA solubilization increased by complexation with MB also SA have preventative effect on MB reduction with
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environmental reductants such as NAD(P)H. By considering these effects, the anticancer effectiveness of both of reagents, SA and MB, increase. Dark toxicity experiments are in good agreement with spectroscopic results. Our experiments show that the SA have anticancer effect and can decreases the cell viability (80 %). The anticancer effect of salicylic acid has been reported previously by Kutlu and coworkers[20]. The concentration and dose affect the effectiveness of drugs. The anticancer effect of salicylic acid is dose dependent and can be better in higher concentrations but the side effects of chemotherapy anticancer drugs in higher concentrations make limitation in using higher concentrations. In here we used the 11
ACCEPTED MANUSCRIPT low concentrations of salicylic acid by combination with PDT for improving cell up taking and cellular penetration of SA for better anticancer effect in low concentrations. In the other hand, the laser and LED treatments can improve cell permeability of drugs due to the light
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effect on cell channel pumps and so affect the drugs cellular penetration [26].
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Figure 3, the viability of MDA MB 231 breast cancer cells treated with various concentrations of used compounds without irradiation. The results are expressed as the mean ± SD (n = 3).
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Determination of red light illuminated cytotoxicity of MB and SA: Figure 4, represents the related treatments in figure 3 in the presence of red LED irradiation. Results show that in the presence of red light, the cell viability in blanks contains PBS, i
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show any significant changes approve that the used dosage of light at the irradiation time i
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y ho o-toxicity in the absence of photosensitizers. In the presence of red light
irradiation, phototoxic reactions make further cell killing potential and reduced the cell viability in which photosensitizer are present. The cell killing potential for combinational 12
ACCEPTED MANUSCRIPT treatments by MB and SA, are higher than other experiments. According to the results it is clear that the reduction prevention effect of salicylic acid on methylene blue reduction in the cells is higher than other effects. In the presence of SA, the MB have good cellular penetration and also the reduction of MB in the cells was decreased effectively. Laser and LED lights can improve cellular penetration of drugs and in the other words, low level lasers
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and LEDs can affect the cellular permeability and cell and mitochondria membrane
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potentials. In the presence of light and under LED irradiation cellular penetration of SA
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increased and so the anticancer effect of SA increased due to cellular up taking [27].
Figure 4, the viability of MDA MB 231 breast cancer cells treated with various concentrations of used compounds under red LED irradiation. The results are expressed as the mean ± SD (n = 3).
Light invert microscopy: The morphological changes in cells were represented using microscopic images of treated cells. Figure 5 shows the morphological changes in cells that incubated in dark at the presence of (A) PBS (blank), (B) Salicylic acid, (C) Methylene blue and (D) Salicylic acid13
ACCEPTED MANUSCRIPT Methylene blue (mixed). Figure 6, represents the red LED irradiation on cell morphology of related to experiments considered in figure 5. Clear changes in the morphology of cells can be seen in the presence of red LED irradiation that treated with salicylic acid-methylen blue. B
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Figure 5, Invert microscopy images (40 X) of cancer cells treated with, PBS, SA, MB and SA-MB without irradiation: (A) blank (PBS), (B) SA, (C) MB, (D) SA-MB.
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Figure 6, Invert microscopy images (40 X) of cancer cells treated with, PBS, SA, MB, SA-MB and red light LED irradiation: (A) blank, (B) SA, (C) MB, (D) SA-MB.
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ACCEPTED MANUSCRIPT Images of cancer cells treated with PBS, SA, MB and SA-MB, with and without irradiation of red LED were studied under invert microscopy to visualization of the dark toxicity and photo-toxicity effects. As seen in figures 5 and 6, morphological difference between cells
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treated with SA-MB combination experiment with other test can be detect clearly.
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Conclusion
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The combination cancer therapy effect of Salicylic acid in the presence of methylene blue as a FDA approved PDT photosensitizer were considered. The results show that the anticancer
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potential of SA increased in the presence of MB due to solubilization performance and balancing between hydrophobic and electrostatic forces. Due to the reduction preventing
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effect of SA on MB, the photodynamic therapy killing potential of MB increases in the presence of SA. Also SA interaction with MB stabilize the monomer form of MB and
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dimerization of dye decreases. Due to the fact that monomer form of MB is active
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photosensitizer, so photodynamic therapy killing potential of MB increases in the presence of SA. The results demonstrated that combination photodynamic therapy of SA-MB can
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improve the cell killing effectiveness of MB in PDT assay on the human breast cancer cells.
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Acknowledgment
The authors wish to acknowledge from all peoples that help us to doing this project.
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ACCEPTED MANUSCRIPT Graphical Abstract
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ACCEPTED MANUSCRIPT Highlights
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Combination therapy using salicylic acid and methylene blub as photosensitizer Spectroscopic and thermodynamic characterization of methylene blue-salicylic acid complex Red light can lead to permeability of salicylic acid into cells photodynamic therapy killing potential of methylene blue increases in the presence of salicylic acid
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Reza Hosseinzadeh, Khatereh Khorsandi, Maryam Jahanshiri PII: DOI: Reference:
S1386-1425(17)30375-X doi: 10.1016/j.saa.2017.05.008 SAA 15150
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received date: Revised date: Accepted date:
1 November 2016 29 April 2017 4 May 2017
Please cite this article as: Reza Hosseinzadeh, Khatereh Khorsandi, Maryam Jahanshiri , Combination photodynamic therapy of human breast cancer using salicylic acid and methylene blue, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.05.008
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Revised manuscript: SAA-D-16-02377-R1
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Combination photodynamic therapy of human breast cancer using salicylic acid and methylene blue
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Reza Hosseinzadeh*, Khatereh Khorsandi, Maryam Jahanshiri
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*Corresponding Authors:
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Medical Laser Research Center, ACECR, Tehran, Iran
Email: [email protected] ,
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[email protected]
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Abstract The objective of this study was to evaluate the effects of combination therapy with
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methylene blue (MB) assisted photodynamic therapy (PDT) and salicylic acid (SA) as chemo-therapy anticancer agent. The binding of salicylic acid to methylene blue was studied
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using spectrophotometric method. The results show the 1:2 complex formation between SA
=12.92 kJ.mol-1,
=9.02 kJ.mol-1). The spectrosphotometric results
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Kb2=38.13 and
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and MB. The binding constants and related Gibbs free energies o are obtained (Kb1=183.74,
show the improvement in solubilization and reduction prevention for SA and MB in the
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complex form. These results are in agreements with cellular experiments. The dark toxicity measurements represent the improve efficacy of chemotherapy using combination of SA and
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MB. The photodynamic therapy results (using red LED as light source (630 nm; power
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density: 30 mW cm-2)) show that the cancer cell killing efficiency of MB increases in the combination with SA due to reduction prevention and stabilization of monomeric form of
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MB.
Keywords: Photodynamic therapy, Salicylic Acid, Methylene blue, Molecular Interaction, Singlet oxygen, Combination Therapy
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ACCEPTED MANUSCRIPT Introduction According to the reports of world health organization (WHO), over 508 000 women died in 2011 due to breast cancer in worldwide (Global Health Estimates, WHO 2013). Breast cancer is a disease that make challenge for the all of countries in the world in both of the developed and less developed countries [1,2]. Breast cancer patients in Iran is 40 thousand and more
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than seven thousand patients are added to this number, yearly. According to the emotional
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status of women in family and the importance of the role of women in human society, diseases such as breast cancer, can induced high psychological stress in their families and
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communities [3]. So control and therapy of breast cancer is one of the essential challenges in
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all the world. Nowadays, there are many types of cancer therapy methods. The types of cancer treatment depend on the type of cancer and how advanced of cancer. Some patients
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give only one treatment. But most people have a combination of two or further cancer therapies that known as combination therapy. The combination therapy is one of new and
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interesting methods in cancer therapy researches. Combinations of multiple drugs in cancer
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treatments are proving more effective than one drug. Combination therapy not only provide a potential solution to addressing the heterogenic tumors but also can solve the drug resistance
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issues in cancer treatments[4–7]. Combination of photodynamic therapy (PDT) with chemotherapy is interesting method in this field of researches[8–10]. Photodynamic therapy
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is a new and effective therapy method that can be used for the treatment of various types of cancers. PDT is a form of radiation therapy that composed of three nontoxic elements. By illumination of photosensitizer in the presence of molecular oxygen, reactive oxygen species (ROS), particularly singlet oxygen as a cytotoxic species can be induced in tumor cells. The cancer cells would be destroyed by apoptosis or necrosis that switched by ROS, achieving, ultimately, the goal of local treatment with minimum invasion [11–19]. Combination of photodynamic therapy with salicylic acid based chemo therapy, is goal of our study.
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ACCEPTED MANUSCRIPT Salicylic acid is a mono-hydroxybenzoic acid, a type of phenolic compounds and commonly used as plant hormones in agricultural applications and it is probably best known for use as a key agent in topical anti-acne pharmaceutical products and is used as an anti-inflammatory drug. Salicylic acid is also known for its ability to ease aches and pains and reduce fevers. Anti-inflammatory drugs, such as salicylic acid, have been singled out as apoptosis inducing
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agents with anti-cancer activity. Kutlu and coworkers reported of anti-tumor activity the
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salicylic acid as chemotherapeutic drug, on A549 human lung adenocarcinoma cells [20].
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In here, the interaction of salicylic acid with methylene blue was investigated using spectrophotometric measurements and a series of combinatorial therapy were examined by
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considering effectiveness of photodynamic therapy of methylene blue in the presence of
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salicylic acid as chemotherapeutic anti-cancer drug on MDA-MB-231 breast cancer cells.
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Materials and instruments:
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Materials and Methods
Salicylic acid (chemical name; 2-Hydroxybenzoic acid) was purchased from Merck Co.
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(Germany) and tetrazolium dye, MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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bromide) was supplied by Sigma-Aldrich Co. (USA). Methylene blue (Systematic name: 3,7bis(Dimethylamino)-phenothiazin-5-ium chloride) was purchased from Merck Co. Trypan blue solution (0.4% w/v) and dimethyl sulfoxide (DMSO) were achieved from Merck company. Fetal bovine serum (FBS) and antibiotics were purchased from Gibco (Gibco BRL). DMEM medium (Dulbecco's Modified Eagle Medium) was purchased from Invitrogen (Invitrogen, Carlsbad, California, US). The entire buffer salts and other chemicals were supplied from Merck Co. Double distilled deionized water was used for all experiments and solutions.
C, using Metrohm 744 (Switzerland). The 4
ACCEPTED MANUSCRIPT UV-Vis absorption spectra were recorded using Cary 60 UV/Vis spectrophotometer, equipped with quartz cells. Red light emitting LED (630 nm; power density: 30 mW cm-2) was used as light source. The LED power-metery was done at the Electronics Research Institute at Sharif University of Technology (SUT), Optic laboratory, using suitable power
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metric devices. Methods:
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UV-Vis spectroscopic study of Salicylic acid interaction with methylene blue;
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Stock solutions of 5×10−6 mol.L-1 of methylene blue, as photosensitizer, was prepared by
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dissolving the certain amount of dye in distilled water. Stock solution of 2×10−2 mol.L-1 of SA was prepared by dissolving an appropriate amount of the SA in double distilled water.
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The variation of absorbance spectra of MB solution by increasing of SA concentrations was recorded at 200-800 nm wavelength using water as a blank. Methylene blue-salicylic acid
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binding constant was determined from the b o b c ’ of a series of solutions containing a
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fixed concentration of photosensitizer (CMB =1×10−6 mol.L-1) and increasing concentration
Cell culture
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of salicylic acid.
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Human breast cancer cell line, MDA-MB-231, was supplied by the Institute of Pasture, Tehran, Iran. The cells were grown in DMEM medium supplemented with 10% FBS, 100 IU/mL penicillin and 100 µg/mL of streptomycin then incubated in a humidified incubator containing 5% CO2 at 37 ˚C. For the experiments, the cells were removed by trypsinizing (trypsin 0.025%, EDTA 0.02%) and washed with phosphate-buffered saline (PBS). Effect of different analytes concentrations on human breast cancer Cells
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ACCEPTED MANUSCRIPT For considering and comparison the effect of various concentrations of methylene blue, salicylic acid on photodynamic efficacy, a series of experiments were designed for dark and red LED irradiations. Briefly, the MDA-MB-231 cells were seeded using fresh culture medium in petri dishes and incubated under 5% CO2,
37 ˚C for 24 h. Then the cells were
incubated using fresh cell culture medium containing certain amounts of analytes (Methylene
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blue, SA). After certain incubation time, the cells were washed by PBS buffer solution. One
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of the treated plates considers as reference plate (no irradiation (dark)) and the other plates
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irradiated using red LED for 30 min. The cell viability was measured after incubations. The colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay was
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used to determine the cells viability. Each experiment was repeated 3 times.
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Effect of different incubation and irradiation time on cancer cells The effects of incubation and irradiation times were optimized by considering various
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incubation and irradiation periods in the presence and absence of analytes. After incubation
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or irradiation under specified times the cell viability was determined using MTT assay. All experiments were repeated three times.
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In vitro photodynamic assay
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The MDA-MB-231 cancer cells were grown in medium culture cell and after reaching 80∼90% confluence; the cells were washed with PBS, afterwards detached from the flask by addition of 1.0
L of 0.
% y i fo 1−3
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37 ˚C. Cells (1×104 cells/well) were
seeded into five 96-well plates. The cells were then treated with methylene blue, Salicylic acid and SA-MB with together, at different concentrations. After a further incubation of 24 h, one plate was considered as dark control plate and the other plates illuminated as described above. The MTT assay was used to determine the cell viability.
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ACCEPTED MANUSCRIPT MTT assay experiment Thiazolyl blue tetrazolium bromide (MTT) was used in determination of cell survival as colorimetric MTT assay. Cell viability can be measured as a function of cells redox potential. Living cells convert the MTT compound to an insoluble formazan. The resulting formazan
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solubilized using dimethyl sulphoxide (DMSO) and its concentration determined using spectrophotometric methods. Briefly, culture medium was removed and cells were incubated
37 ˚C. Th
i g purple formazan crystals dissolved in 100 µL DMSO
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bromide for 4 h
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in medium containing 0.5 mg/mL of 3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
and shacked for 15 min. The absorbance of solutions was measured at 540 nm by an ELISA
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reader (Hyperion, Inc., FL ,U.S.A.).
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Results and Discussions
Interaction of salicylic acid with methylene blue:
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Addition of salicylic acid (stock concentration of SA was 2×10−2 mol.L-1) on MB solution (1×10−6 mol.L-1) makes bathochromic shift in MB maximum absorption spectra and the
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decrease in absorbance with increasing SA concentration. The results demonstrated that MB interact with SA. Figure 1 show the spectral change of MB by addition of SA concentration.
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In figure 2, the variation of 1/Abs versus 1/[C] was constructed based on Benesi Hidebrand equation [21–23]. The graph demonstrated that the variations is linear and complex formation is 1:1 equilibrated interaction. The related binding constant can be estimated.
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Figure 1, UV-Vis absorption spectra of methylene blue ([MB] = 5×10−6 mol.L-1)
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Determination of binding constant:
The value of the binding constant (Kb) can be obtained according to the methods described
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previously [21]. Due to the fact that binding of SA to MB is an equilibrium interaction, so
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equations (1) and (2) can be constructed as: (1)
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MB+SA⇌[Complex]
(2)
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We can rearrange the Eq. 2 as Eq 3:
Equation 3 can be constructed based on spectrophotometric data as Eq. 4;
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ACCEPTED MANUSCRIPT Kb can be estimated from the ratio of the intercept to the slope of the line constructed in figure 2. As can be seen in figure 2, the obtained plot can be divided to two distinct linear parts. According to the Zhi-Wu reports[24], the interactions with two binding interactions, if the binding strengths in both interactions have clear difference, the Benesi-Hildebrand plot is not linear and its due to the differing binding strengths in two binding interactions. By
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considering this fact, the plot can be divided to two linear regions that demonstrated the 1:2
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binding interaction between methylene blue and salicylic acid. The binding constant related to the first and second binding sites were obtained as Kb1=183.74 (slop=0.0923,
icy ic ci i
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hy
b
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C. According to the
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fo
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intercept=16.959 and R2=0.99) and Kb2=38.13 (slop=0.0422, intercept=1.609 and R2=0.99)
obtained results it is clear that the interaction of salicylic acid with methylene blue is based
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on molecular interactions and the electrostatic interactions are the initial forces in salicylic acid binding to methylene blue. At higher concentrations of salicylic acid, the hydrophobic
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interactions are the major forces in binding phenomena. In the other words, by further
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increasing in salicylic acid concentration, the hydrophobic interactions come important in binding phenomena. Due to the structural properties of salicylic acid, it behaves as an ionic
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surfactant. In the presence of methylene blue the electrostatic interaction of methylene blue and salicylic acid make complex of dye-drug and by increasing of SA concentration mixed
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micelle of dye-drug can produce. At these conditions, the MB can prevent from redox reactions in the cellular penetrations and also the salicylic acid solubilization can improved in mixed form with methylene blue[25]. Red shift in UV-Vis spectra approve this statement due to increasing of hydrophobicity around the microenvironment of methylene blue in the presence of salicylic acid [21]. Also by increasing of SA concentration, the monomeric form of MB is the stable form and dimerization of MB decreased. As reported previously, the lower wavelength shoulder in MB spectra is related to dimeric form of MB. By increasing of
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ACCEPTED MANUSCRIPT SA concentration, A620 decreased with higher slope in comparison with A665 (slope620=0.0764 and slope665=0.0519). Dimerization pf MB caused higher decreasing in photodynamic therapy efficiency of MB [25]. The Gibbs free energy of binding of SA with MB can be
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obtained using the following thermodynamic equation:
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The estimated value of Gibbs free energy for SA interaction with MB at first binding site is 12.92 kJ/mol and for second binding site is 9.02 kJ/mol that is related to molecular
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interactions. complex formation between SA and MB prevents the MB from environmental
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reduction to leuco-form of methylene blue. The leuco-form is not PDT-active form of MB.
Figure 2, Benesi-Hildebrand plot for interaction of methylene blue (5×10 −6 C [24].
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icy ic ci
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ACCEPTED MANUSCRIPT Viability studies and MTT assay: Cellular effects of various treatments were measured and determined using MTT assay for determination of cell viability after and before of treatments and irradiations. Figure 3, summarized the effect of used compounds on cells viability at dark and in absence of LED irradiation, in the other words, the cellular toxicity of each used material were tested and
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examined without LED irradiation (dark experiment).
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Determination of dark cytotoxicity used MB and SA compounds:
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In order to compare the cytotoxicity effect of MB and SA in the absence of irradiation, the
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effect of these compounds on cell viability were determined. Viability of treated cells with different concentrations of MB and SA (24 h incubation at 0, 10, 25, 50 and 75 µg/mL) were
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determined at dark. According to the obtained results (Fig. 3), it is clear that the cytotoxicity of salicylic acid is higher than MB in the absence of LED irradiation. The cytotoxicity of MB
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at used concentration range in the absence of irradiation is negligible while the cytotoxicity of
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combinational experiment is higher than each other. According to the obtained results in spectrophotometric studies, it is due to the equilibration between electrostatic and
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hydrophobic forces of each anticancer reactive agent. SA solubilization increased by complexation with MB also SA have preventative effect on MB reduction with
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environmental reductants such as NAD(P)H. By considering these effects, the anticancer effectiveness of both of reagents, SA and MB, increase. Dark toxicity experiments are in good agreement with spectroscopic results. Our experiments show that the SA have anticancer effect and can decreases the cell viability (80 %). The anticancer effect of salicylic acid has been reported previously by Kutlu and coworkers[20]. The concentration and dose affect the effectiveness of drugs. The anticancer effect of salicylic acid is dose dependent and can be better in higher concentrations but the side effects of chemotherapy anticancer drugs in higher concentrations make limitation in using higher concentrations. In here we used the 11
ACCEPTED MANUSCRIPT low concentrations of salicylic acid by combination with PDT for improving cell up taking and cellular penetration of SA for better anticancer effect in low concentrations. In the other hand, the laser and LED treatments can improve cell permeability of drugs due to the light
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effect on cell channel pumps and so affect the drugs cellular penetration [26].
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Figure 3, the viability of MDA MB 231 breast cancer cells treated with various concentrations of used compounds without irradiation. The results are expressed as the mean ± SD (n = 3).
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Determination of red light illuminated cytotoxicity of MB and SA: Figure 4, represents the related treatments in figure 3 in the presence of red LED irradiation. Results show that in the presence of red light, the cell viability in blanks contains PBS, i
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show any significant changes approve that the used dosage of light at the irradiation time i
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y ho o-toxicity in the absence of photosensitizers. In the presence of red light
irradiation, phototoxic reactions make further cell killing potential and reduced the cell viability in which photosensitizer are present. The cell killing potential for combinational 12
ACCEPTED MANUSCRIPT treatments by MB and SA, are higher than other experiments. According to the results it is clear that the reduction prevention effect of salicylic acid on methylene blue reduction in the cells is higher than other effects. In the presence of SA, the MB have good cellular penetration and also the reduction of MB in the cells was decreased effectively. Laser and LED lights can improve cellular penetration of drugs and in the other words, low level lasers
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and LEDs can affect the cellular permeability and cell and mitochondria membrane
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potentials. In the presence of light and under LED irradiation cellular penetration of SA
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increased and so the anticancer effect of SA increased due to cellular up taking [27].
Figure 4, the viability of MDA MB 231 breast cancer cells treated with various concentrations of used compounds under red LED irradiation. The results are expressed as the mean ± SD (n = 3).
Light invert microscopy: The morphological changes in cells were represented using microscopic images of treated cells. Figure 5 shows the morphological changes in cells that incubated in dark at the presence of (A) PBS (blank), (B) Salicylic acid, (C) Methylene blue and (D) Salicylic acid13
ACCEPTED MANUSCRIPT Methylene blue (mixed). Figure 6, represents the red LED irradiation on cell morphology of related to experiments considered in figure 5. Clear changes in the morphology of cells can be seen in the presence of red LED irradiation that treated with salicylic acid-methylen blue. B
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Figure 5, Invert microscopy images (40 X) of cancer cells treated with, PBS, SA, MB and SA-MB without irradiation: (A) blank (PBS), (B) SA, (C) MB, (D) SA-MB.
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Figure 6, Invert microscopy images (40 X) of cancer cells treated with, PBS, SA, MB, SA-MB and red light LED irradiation: (A) blank, (B) SA, (C) MB, (D) SA-MB.
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ACCEPTED MANUSCRIPT Images of cancer cells treated with PBS, SA, MB and SA-MB, with and without irradiation of red LED were studied under invert microscopy to visualization of the dark toxicity and photo-toxicity effects. As seen in figures 5 and 6, morphological difference between cells
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treated with SA-MB combination experiment with other test can be detect clearly.
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Conclusion
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The combination cancer therapy effect of Salicylic acid in the presence of methylene blue as a FDA approved PDT photosensitizer were considered. The results show that the anticancer
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potential of SA increased in the presence of MB due to solubilization performance and balancing between hydrophobic and electrostatic forces. Due to the reduction preventing
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effect of SA on MB, the photodynamic therapy killing potential of MB increases in the presence of SA. Also SA interaction with MB stabilize the monomer form of MB and
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dimerization of dye decreases. Due to the fact that monomer form of MB is active
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photosensitizer, so photodynamic therapy killing potential of MB increases in the presence of SA. The results demonstrated that combination photodynamic therapy of SA-MB can
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improve the cell killing effectiveness of MB in PDT assay on the human breast cancer cells.
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Acknowledgment
The authors wish to acknowledge from all peoples that help us to doing this project.
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ACCEPTED MANUSCRIPT Graphical Abstract
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SA-MB
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ACCEPTED MANUSCRIPT Highlights
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Combination therapy using salicylic acid and methylene blub as photosensitizer Spectroscopic and thermodynamic characterization of methylene blue-salicylic acid complex Red light can lead to permeability of salicylic acid into cells photodynamic therapy killing potential of methylene blue increases in the presence of salicylic acid
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