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Photodiagnosis and Photodynamic Therapy 18 (2017) 284294

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Photodiagnosis and Photodynamic Therapy


journal homepage: www.elsevier.com/locate/pdpdt

Methylene blue, curcumin and ion pairing nanoparticles effects on


photodynamic therapy of MDA-MB-231 breast cancer cell
Reza Hosseinzadeh a, , Khatereh Khorsandi a,b
a
Medical Laser Research Center, ACECR, Tehran, Iran
b
Faculty of Biological Sciences, Islamic Azad University, North Tehran Branch, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Purpose: The aim of current study was to use methylene blue-curcumin ion pair nanoparticles and single
Received 29 August 2016 dyes as photosensitizer for comparison of photodynamic therapy (PDT) efcacy on MDA-MB-231 cancer
Received in revised form 4 February 2017 cells, also various light sources effect on activation of photosensitizer (PS) was considered.
Accepted 9 March 2017
Method: Ion pair nanoparticles were synthesized using opposite charge ions precipitation and lyophilized.
Available online 12 March 2017
The PDT experiments were designed and the effect of PSs and light sources (Red LED (630 nm; power
density: 30 mW cm2 ) and blue LED (465 nm; power density: 34 mW cm2 )) on the human breast cancer
Keywords:
cell line were examined. The effect of PS concentration (075 g. mL1 ), incubation time, irradiation time
Photodynamic therapy
Nanoparticle
and light sources, and priority in irradiation of blue or red lights were determined.
Methylene blue Results: The results show that the ion pairing of methylene blue and curcumin enhance the photody-
Curcumin namic activity of both dyes and the cytotoxicity of ion pair nanoparticles on the MDA-231 breast cancer
Ion pairing cell line. Blue and red LED light sources were used for photo activation of photosensitizers. The results
Singlet oxygen demonstrated that both dyes can activate using red light LED better than blue light LED for singlet oxygen
Photosensitizer producing.
Conclusion: Nano scale ion pair precipitating of methylene blue-curcumin enhanced the cell penetrating
and subsequently cytotoxicity of both dyes together.
2017 Elsevier B.V. All rights reserved.

1. Introduction tumor cells which consequently cause a signicant decrease in PDT


efcacy [6]. Hence, various methods have been employed for the
Photodynamic therapy is a form of radiation therapy that is transport of water-insoluble PSs such as curcumin to cells [714].
composed of three nontoxic elements producing reactive oxygen On the other hand, some problems concerned with water soluble PS
species (ROS), particularly singlet oxygen as a cytotoxic species such as methylene blue, due to simple reduction by reducing agents
[13]. These three important elements are: photosensitizer, molec- (such as ascorbic acid) in cells and organs. The reduced form of
ular oxygen and light in specied wavelength [1]. Photosensitizers methylene blue (MB), known as leucomethylene blue that is photo
are the molecular compounds that can absorb light at the spe- inactive molecule [15,16]. Also concentration based dimerization
cic wavelength and can be excited to triplet state [35]. PDT is can affect the PDT efcacy of MB [1618]. Microenvironment of
a noninvasive therapy method with fast healing process and with- photosensitizer have great role in PDT yield. For example, pH of cell
out scarring. According to the fact that many of photosensitizers microenvironment can switch the two type of reaction with MB.,
are hydrophobic compounds so tend to aggregate in aqueous solu- The 1 O2 production yield in aqueous solution of methylene blue is
tion. This limitation leads to poor uptake of photosensitizer (PS) in dependent on the pH. The excited triplet state of methylene blue is
an excited base. The kinetic constant related to protonation reaction
of triplet state of MB (TSMB) (at acidic solutions, pH 4) is higher
than the kinetic constant of TSMB reaction with molecular oxygen.
Abbreviations: PDT, photodynamic therapy; PSs, photosensitizers; LED, light
emitting diode; ROS, reactive oxygen species; MB, methylene blue; IP, ion pair; So in acidic conditions protonation of TSMB is favorable reaction.
DMSO, dimethyl sulfoxide; FBS, fetal bovine serum. Protonated TSMBH2+ has a longer life time and higher state elec-
Corresponding author. tron energy level. So the protonated MB react with oxygen with a
E-mail addresses: r.hoseinzadeh@ut.ac.ir, chem.reza@gmail.com
(R. Hosseinzadeh).

http://dx.doi.org/10.1016/j.pdpdt.2017.03.005
1572-1000/ 2017 Elsevier B.V. All rights reserved.
R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294 285

smaller rate constant that resulted in lowering PDT quantum yield stored at 4 C and solutions were prepared and used freshly. The
[16,19]. characteristics of ion pair were determined using FE-SEM images
This is a fact must be considering for treating of cancer cells and dynamic light scattering.
and tissues that have a lower pH value in surrounding cellular
microenvironment due to the anaerobic metabolism in cancerous 2.2.2. UVvis spectroscopic properties of dyes and FE-SEM ion
cells. Due to these limitations various researches have been done pair nanoparticle characterization
for overcoming problems and increasing PDT quantum yield. The UVvis absorption spectra were recorded using Cary 100
Ion-pair (IP) formed between both of oppositely charged ions UV/Vis spectrophotometer, equipped with quartz cells. The absorp-
by Coulombic force as physical bond. Both of ions together in ion tion spectra related to single dyes (curcumin and methylene blue)
pair complex behave like a single unit. IP reported to improve and ion paired dyes were recorded and compared with together. It
the hydrophobicity of polar drugs and subsequently accelerate must be mentioned that polyethylene glycol (PEG 400) was used
the transport rate of them across cell membranes. Decreasing the as co-solvent in the preparation of experimental solutions and for
hydrophilicity of ionic drugs, such as methylene blue, by ion- examination of the effect of PEG on cell viability, a set of exper-
pairing with suitable opposite charge ions have proven to be iments were considered in the presence of PEG 400 as reference
promising for several drug delivery and bioavailability applications. experiments. In all of them the PEG didnt show any effect and make
Also ion-pairing can be a valuable tool for enhancing stability and the inert environment in all PDT experiments.
bioavailability of drugs in body system. By using new approaches in For considering the other characteristic experiment in nanopar-
combination with ion pairing, the drugs efcacy and bioavailability ticle characterization, SEM image of nanoparticles was recorded.
can be improved. Nano-carriers or nano-drug systems for photo- Field Emission Scanning Electron Microscopy (FE-SEM, Hitachi S-
sensitizer delivery to tumor cells are one of preventative methods 4160, Japan) was used for nanoparticle characterization.
in drug delivery. Nanometer size of PS or PS carrying nanoparticles,
make good advantages for PS delivery to cancer cells and tissues.
2.2.3. Cell culture
Due to the sub-cellular size, nanoparticles can penetrate deep into
Human breast cancer cell line, MDA-MB-231, was supplied by
tissues and cells [2023].
the Institute of Pasture, Tehran, Iran. The cells were grown in DMEM
In the current study we design a series of photodynamic ther-
medium supplemented with 10% FBS, 100 IU/mL penicillin and
apy experiments using single dye photosensitizers (curcumin and
100 g/mL of streptomycin then incubated in a humidied incuba-
methylene blue) and nanoparticle ion pair (Curcumin-MB) for
tor containing 5% CO2 at 37 C. For the experiments, the cells were
investigating PDT efcacy on MDA-MB-231 cancer cells.
removed by trypsinizing (trypsin 0.025%, EDTA 0.02%) and washed
with phosphate-buffered saline (PBS).
2. Materials and methods
2.2.4. Effect of different analytes concentrations on human breast
2.1. Materials cancer cells
For considering and comparison the effect of various concen-
Curcumin (with IUPAC name as: (1E,6E)-1,7-bis(4-hydroxy-3- trations of MB, curcumin and IP nanoparticles on photodynamic
methoxyphenyl)hepta-1,6-diene-3,5-dione) and tetrazolium dye, efcacy, a series of experiments were designed for dark,blue, red
MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- and red/blue irradiations. Briey, the MDA-MB-231 cells were
mide) were supplied by Sigma-Aldrich Co. Methylene blue seeded using fresh culture medium in petri dishes and incubated
(Systematic name: 3,7-bis(Dimethylamino)-phenothiazin-5-ium under 5% CO2 , at 37 C for 24 h. Then the cells were incubated using
chloride) was purchased from Merck Co. (Germany). Trypan blue fresh cell culture medium containing certain amounts of analytes
solution (0.4% w/v) and dimethyl sulfoxide (DMSO) were achieved (Methylene blue, Curcumin and IP nanoparticles). After certain
from Merck company (Germany). Fetal bovine serum (FBS) and incubation time, the cells were washed by PBS buffer solution. One
antibiotics were purchased from Gibco (Gibco BRL). DMEM medium of the treated plates considers as reference plate (no irradiation
(Dulbeccos Modied Eagle Medium) was purchased from Invitro- (dark)) and the other plates irradiated using red and blue light LED
gen (Invitrogen, Carlsbad, California, US). The entire buffer salts and sources. One plate irradiated only with blue light for 30 min. Sec-
other chemicals were supplied from Merck Co. (Germany). Dou- ond plate used under red light irradiation for 30 min. The third
ble distilled deionized water was used for all experiments and plate consider for combination irradiation experiments (15 min
solutions. Red (630 nm; power density: 30 mW cm2 ) and blue red light then 15 min blue light). The cell viability was measured
(465 nm; power density: 34 mW cm2 ) light emitting LEDs were after incubations. The colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,
used as light sources. The LED power-metery were done using suit- 5 diphenyltetrazolium bromide (MTT) assay was used to determine
able power metric devices by the Electronics Research Institute at the cells viability. Each experiment was repeated 3 times.
Sharif University of Technology (SUT), Optic laboratory.
2.2.5. Effect of different incubation and irradiation time on cancer
2.2. Methods cells
The effect of incubation and irradiation times was optimized
2.2.1. Dyes ion pair nanoparticle preparation by considering various incubation and irradiation periods in the
Ion pair of methylene blue and curcumin was prepared by presence and absence of analytes. After incubation or irradiation
preparing the appropriate concentration of MB and curcumin under specied times the cell viability was determined using MTT
solutions. The curcumin solution was prepared in slightly alka- assay. All experiments were repeated three times.
line solution to forming anionic form of curcumin. The prepared
solutions were ltered. The MB solution was added to curcumin 2.2.6. Effect of priority of blue or red light irradiation on cancer
solution dropwise at continuous stirring conditions. The result- cell viability
ing precipitate was transferred to Teon lined autoclave and aged For considering the effect of blue and red lights priority, the
at 120 C for 2 h and then cooled at room temperature. The solu- cells were seeded and then the cells irradiated by blue and red
tion freezes simultaneously using liquid nitrogen and then was lights as: experiment 1; 30 min blue light irradiation, experiment 2;
lyophilized using freeze-drying method. The obtained powder 30 min red light irradiation and experiment 3; 15 min red light plus
286 R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294

15 min blue light irradiation. The cell viability at various irradiation characteristics of dyes. Electron resonance in conjugated struc-
experiments was obtained using MTT assay. ture induced the spectra. By blocking the electron in the molecular
ions the resonance affected and changed. Also in cationic molecules
2.2.7. In vitro photodynamic assay by adding electrons due to electrostatic anion-cation interactions,
The MDA-MB-231 cancer cells were grown in medium culture the electron density on the cation change and affects the elec-
cell and after reaching 8090% conuence; the cells were washed tronic resonance of ionic molecular structure that make changes
with PBS, afterwards detached from the ask by addition of 1.0 mL in related spectra. Fig. 1(D) is a photographic image of PEG assisted
of 0.25% trypsin for 13 min at 37 #XPS##x0030A;#XPS#9617;C. solution of photosensitizers and nanoparticles. The uorescence
Cells (1 104 cells/well) were seeded into ve 96-well plates. The spectra related to dye photosensitizers and ion pairs excited using
cells were then treated with methylene blue, curcumin and IP 420 nm and 630 nm wavelengths are presented in Fig. 1(E).
nanoparticles at different concentrations. After a further incuba-
tion of 24 h, one plate was considered as dark control plate and the 3.3. Solubility enhancement using PEG-400
other plates were illuminated as described above. The MTT assay
was used to determine the cell viability. Surface active reagents have been widely used as effective
approach to increase the water solubility of many poor soluble
2.2.8. MTT assay experiment chemicals and pharmaceuticals for providing acceptable dosage
Thiazolyl blue tetrazolium bromide (MTT) was used in deter- of drugs and chemicals in various pharmaceutic and industrial
mination of cell survival as colorimetric MTT assay. Cell viability applications. Curcumin as an insoluble (poor soluble in water
can be measured as a function of cells redox potential. Living cells <0.1 mg/mL and 420 nm = 55000 M1 cm1 in ethanol) polypheno-
convert the MTT compound to an insoluble formazan. The result- lic compound that the poor water solubility makes limitation in
ing formazan solubilized using dimethyl sulphoxide (DMSO) and curcumin applications in food and drug formulations. In here the
its concentration determined using spectrophotometric methods. PEG-400 was used for enhancing the solubility of curcumin. For this
Briey, culture medium was removed and cells were incubated purpose, the excess amount of curcumin weighted (0.015 mg) and
in medium containing 0.5 mg/mL of 3-(4, 5-Dimethylthiazol-2-yl)- dispersed in 2 mL of double distilled water. The various concentra-
2,5-Diphenyltetrazolium Bromide for 4 h at 37 C. The resulting tions of PEG-400 (ranging from 0 to 70 mM) were added and mixed
purple formazan crystals were dissolved in 100 L DMSO and well and after centrifugation (8000 rpm for 5 min) the upper clear
shacked for 15 min. The absorbance of solutions was measured at solution used for determination of curcumin absorbance (Fig. 2(A)).
540 nm by an ELISA reader (Hyperion, Inc., FL, U.S.A.). The effect of PEG-400 on curcumin solubility enhancement can
be characterized based on methods reported previously [25,26].
3. Results The solubilization constant (Ks = 4.33) and water/PEG400 distribu-
tion constant (Kx = 240.43) were estimated (Fig. 2(B)). The related
3.1. Interaction of curcumin with methylene blue free energy of distribution was calculated for curcumin/PEG-400
(GX0 = 13.58KJ/mol).
Titration of methylene blue (36 mg/mL water solubility and
667 nm = 56621 M1 cm1 ). solution (1 106 mol L1 ) using cur- 3.4. FE-SEM images of ion-pairing particles
cumin stock solution (stock concentration of curcumin was
2 102 mol L1 ) makes bathochromic shift in MB maximum Scanning electron microscope, is one of the powerful instru-
absorption spectra and the decrease in absorbance with increasing ments in particles topography, morphology and size determination.
curcumin concentration. These spectral variations demonstrated The FE-SEM image of synthesized nanoparticles was taken and used
that methylene blue interact with curcumin. Fig. 1(A) show the for characterization of nanoparticles. Fig. 3 represents the obtained
spectral change of MB by addition of curcumin. In Fig. 1(B), the vari- image of synthesized ion-pairing nano structures.
ation of (A/(A0 -A)) versus 1/[C] was constructed based on Benesi
Hildebrand equation [24]. The graph contains two linear regions, 3.5. Viability studies and MTT assay
the rst region is in lower concentration of curcumin and the
second is in higher concentrations of curcumin. Based on Benesi- Cellular effects of various treatments were measured and deter-
Hildebrand equations the linearity demonstrated that the complex mined using MTT assay for determination of cell viability after and
formation is 1:1 equilibrated interaction. By calculating of related before of treatments and irradiations. Fig. 4, summarized the effect
Kb (binding constant) for each of regions the strength of interac- of used compounds on cells viability at dark and in absence of LED
tions can be estimated. The obtained Kb1 (the binding constant for irradiation, in the other words, the cellular toxicity of each used
the rst region (Fig. 1(B)) is 5016.17 and Kb2 (the binding con- material were testes and examined without LED irradiation (dark
stant for the second region (Fig. 1(B)) is 29.33. Also the binding experiment).
free energies can be calculated using obtained binding constants Fig. 5, represents the related treatments in Fig. 4 in the pres-
(Gb10 = 21.11KJ/molandG0 = 8.37KJ/mol). ence of red LED irradiation in the presence (Fig. 5(B)) and absence
b2
(Fig. 5(A)) of PEG-400. Results show that in the presence of red
3.2. UVvis and uorescence spectrophotometric characterization light, the cell viability in blanks contains PBS and PEG, didnt show
any signicant changes that approve the cellular safety of PBS and
Fig. 1(C), represents the UVvis spectra of methylene blue and PEG in the presence of light and also approve that the used dosage
curcumin in free form and interacted electrostatically induced com- of light at the irradiation time didnt show any photo-toxicity in
plex form. Spectrophotometric studies are common procedure in the absence of photosensitizers. According to the spectroscopic
understanding of structural changes in molecular level. Methylene data, the solubility enhancement of PEG on curcumin solubiliza-
blue spectra contains the two distinct region which can be seen in tion contribute in curcumin cellular uptake and penetration and
visible region of spectra and the other one in below of 300 nm as consequently the increasing of PDT efcacy of curcumin can be
in ultra violet region. The curcumin spectra also have these peaks achieved. Also the effect of PEG on cellular uptake of MB and IP
in related spectra but according to the molecular structure of com- can be seen. By comparison of Fig. 5(A) and (B), by addition of
pounds these peaks appear in different wavelengths. However, the PEG the photodynamical effect of photosensitizers increased due
dye photosensitizers spectra are a result of electronic molecular to solubility enhancement and balancing between hydrophobicity
R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294 287

and hydrophilicity of photosensitizers. According to the nano-level ing effect on IP for PDT effect that is in agreement with cell viability
structure of IP, the cellular uptake and cell penetration efciency of data under blue light irradiation.
IP is high and so the adding of PEG-400 cannot have a great effect In Fig. 7, the continuously irradiation with red and blue lights
on photodynamical efcacy. By considering these results we exam- effect were examined and the cell viability were obtained.
ined the effect of red and blue light effect on photodynamic therapy
of used analytes in the presence of PEG-400 and comparing the data
with together.
Fig. 6 represents the results related to blue light irradiations 3.6. Light invert microscopic images
effect on cells viability in the presence of used PSs and nanopar-
ticles. According to the uorescence spectra of IP, by excitation The morphological changes in cells were represented using
at 420 nm, the emission spectra shows a peak at 650750 nm microscopic images of treated cells. Fig. 8 shows the morpholog-
(Fig. 1(E)). According to this result, the blue light can have excit- ical changes in cells that incubated in dark at the presence of PBS
(A) and PEG (B) as a blank treatments and also in the presence of
curcumin (C), methylene blue (D) and IP nanoparticles (E).

Fig. 1. (A), The spectrophotometric binding of curcumin with methylene blue (B), Benesi-Hildebrand plot related to curcumin-MB interaction. (C), UVvis absorption spectra
of MB, Curcumin and Ion-pair. (D), Image of dissolved PSs and IP nanoparticles by PEG-400 in PBS solution. (E), Fluorescence spectra of curcumin (420 nm), methylene blue
(630 nm) and IP recorded using 420 and 630 nm as excitation wavelengths.
288 R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294

Fig. 1. (Continued).

The results show that the morphology of cells in the absence nanoparticles. The results demonstrated that the PBS and PEG
of LED irradiation didnt have any signicant changes. The Fig. 9 didnt show any photo-toxicity in the presence of red LED.
represents the morphological change of cells treated with PSs and Clear changes in the morphology of cells can be seen in the
presence of red LED irradiation that treated with IP-nanoparticles.
R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294 289

Fig. 2. (A), The effect of various concentration of PEG-400 (070 mM) on curcumin solubilization (B), The variation of absorbance difference by increasing of PEG-400
concentration.

Fig. 10 shows the effect of blue LED irradiation in the same condi- blue shift (810 nm) and indicate that the cationic MB interact with
tions of Fig. 9, on the cells morphology. The results show the clear negatively charged curcumin due to electrostatic forces and make
changes in cells treated with nanoparticles. the ion pairing complex. So ion pairing decreases the hydrophilic-
ity of MB and hydrophobicity of curcumin. The hydrophobicity
of curcumin make the limitation in curcumin applications and
4. Discussions
uptakes, also the higher hydrophilicity of MB introduce some dis-
advantages in MB applications especially in PDT applications [16].
4.1. Characteristics of dyes and ion pair nanoparticle
According to the obtained results, the curcumin interacts with
methylene blue in two distinct regions. At the rs regions that
The solutions of methylene blue, curcumin and ion pair
occurred in lower concentrations of curcumin, the binding con-
nanoparticles were prepared and considered for recording UVvis
stant related to curcumin-methylene blue interaction is high and is
absorption spectra. According to Fig. 1 the methylene blue and
due to electrostatic interaction between anionic and cationic dyes.
curcumin spectra are normal however by ion-pairing any shift
The related free energy is negative and higher. These data suggest
in maximum absorption wavelengths occurred for both dyes. The
that in this region the ion-pair formation between dyes occurred.
red shift (24 nm) in curcumin spectra indicate that the negative
At higher concentrations of curcumin, the excess amount of cur-
charge on the curcumin interacts electrostatically and decrease the
cumin attributed in hydrophobic interactions with produced IP due
hydrophilicity of compound. The methylene blue spectra show the
290 R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294

Fig. 3. FE-SEM image of methylene blue-curcumin ion pair nanoparticles.

Fig. 4. The viability of MDA MB 231 breast cancer cells treated with various concentrations of analytes at dark. The results are expressed as the mean SD (n = 3).

to hydrophobic structural properties of curcumin. The binding con- ent concentrations of MB and curcumin (24 h incubation at 0, 25
stant related to this region is lower than rst region. According to and 75 g/mL concentrations) were determined at dark. Accord-
the results the ions have suitable and enough potential for ion-pair ing to the obtained results (Fig. 4), it is clear that the cytotoxicity
forming in similar molar concentration of dyes. Further character- of examined compounds is negligible in absence of light (dark)
istics were made using FE-SEM images of ion pair nanoparticles experiments.
(Fig. 3). Nano size structure of complex can make better uptake
of both dyes ion pair together in cells. Nowadays, nanotechnology
4.3. Effect of red light LED irradiation on cell viability
help suitable drug delivery efcacy in drugs uptake in body, espe-
cially for hydrophobic drugs. SEM images of nanoparticles show
As we know the three important essential parameters in photo-
that the size of ion-paired nanoparticles is about 4060 nm.
dynamic therapy are photosensitizer (PS), molecular oxygen and
light with suitable wavelength. Photosensitizer excitation using
4.2. Determination of MB and curcumin cytotoxicity suitable light wavelength can have critical role in excitation and
singlet oxygen or reactive oxygen species production yield. Excita-
In order to compare the cytotoxicity of MB and curcumin in tion of PS can achieve using other wavelengths with lower quantum
molecular and ion-pairing forms, the effect of these compounds on yields. According to the fact that ion pair nanoparticles are com-
cell viability were determined. Viability of treated cells with differ- plex of two photosensitizers so the effect of both red and blue light
R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294 291

Fig. 5. The viability of breast cancer cells treated with various concentrations of analytes and red light LED irradiation for 30 min in the presence and absence of PEG-400.
The results are expressed as the mean SD (n = 3). (A), without PEG-400 (B), at 50 mM of PEG-400.

emitting diodes in photodynamic therapy efcacy of nanoparticles son with MB alone or curcumin alone solutions. So the PDT efcacy
considered for examination. Fig. 5 represents the effect of red light must be lower than each dye at related light excitation. The pro-
emitting LED light source on the viability of MDA-MB-231 breast duced radicals can react with other part of ion pair for producing
cancer cells treated by curcumin, methylene blue and IP nanopar- further radicals and also the charge transferring between both pho-
ticles. The higher PDT effect (24.25% viability) achieved using IP tosensitizers can induce singlet oxygen production using both PS in
nanoparticles (75 g/mL) under red light LED irradiation. As the ion pair that lead to higher concentration of ROS or singlet oxygen
synthesized ion-pair nanoparticle is the stoichiometry precipita- in PDT process.
tion of both dyes, so the molar solution of ion pair nanoparticle
contains half dosage of methylene blue or curcumin in compari-
292 R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294

Fig. 6. The viability of breast cancer cells treated with various concentrations of analytes and blue light LED irradiation for 30 min. The results are expressed as the mean SD
(n = 3).

Fig. 7. The viability of breast cancer cells treated with various concentrations of analytes and red and blue light LED irradiation each one for 15 min, consequently. The results
are expressed as the mean SD (n = 3). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

4.4. Effect of blue light LED irradiation on cell viability higher wavelengths have better PDT effects in comparison with
lower wavelengths.
In the other experiment we consider the blue LED source as exit-
ing light for determination of PDT efcacy. The results (Fig. 6) show
that the effectiveness in PDT yield increased for curcumin using
blue LED. MB didnt show any signicant difference with previous 4.5. Effect of continuously red and blue light LED irradiations on
results and slight decrease in viability can be seen. IP nanoparticles cell viability
induced 27.7% viability that is higher that obtained results for red
LED. By comparison the results obtained for both red and blue lights For this purpose, we consider both red and blue light irradiation
effects, it can be concluded that the red light affectivity is higher with two 15 min irradiation times (the rst 15 min irradiation with
that blue light. The lower wavelengths lights have greater interac- red LED and the second 15 min irradiated using blue LED light). The
tion with body system that caused further ltering effect of body results are presented in Fig. 7. As can be seen, the effect of IP on
and decreased the light penetration in body organs. By decreas- PDT efcacy is better than MB and curcumin at higher concentra-
ing the light dose due to the body ltering effect the PDT efcacy tions (75 g/mL and 25.64 cell viability%). By comparison of the
decrease and affect the PDT yield. By considering these factors, obtained data, the effect of red light on cancer cell viability using
IP nanoparticles is better than others.
R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294 293

Fig. 8. Invert microscopy images (40 X) of cancer cells treated with, PEG-400, Curcumin, MB and IP nanoparticles without irradiation: (A) blank, (B) PEG-400, (C) Curcumin,
(D) MB and (E) IP nanoparticles.

Fig. 9. Invert microscopy images (40 X) of cancer cells treated with, PEG-400, Curcumin, MB and IP nanoparticles and red light LED irradiation: (A) blank, (B) PEG-400, (C)
Curcumin, (D) MB and (E) IP nanoparticles.

4.6. Microscopic study 5. Conclusion

Images of cancer cells treated with PEG-400, curcumin, MB and The results indicate that the red light effect on cell viability in
IP nanoparticles, with and without irradiation of red and blue light the presence of IP nanoparticles is better than blue light in compar-
LEDs were studied under invert microscopy to visualization of the ison with dyes alone. The nanoparticles distribution and diffusion
photo-toxicity effects. As seen in Figs. 810, morphological differ- in cells due to the nano-structural characteristics and also balanc-
ence between cells treated with IP nanoparticles with other test ing of hydrophobicity and hydrophilicity properties of each dye
can be detect clearly. enhance the photochemical reaction yield in photodynamic ther-
apy on MDA-MB-231 breast cancer cell line.
294 R. Hosseinzadeh, K. Khorsandi / Photodiagnosis and Photodynamic Therapy 18 (2017) 284294

Fig. 10. Invert microscopy images (40 X) of cancer cells treated with, PEG-400, Curcumin, MB and IP nanoparticles and blue light LED irradiation: (A) blank, (B) PEG-400, (C)
Curcumin, (D) MB and (E) IP nanoparticles.

Acknowledgments [14] K. Khorsandi, R. Hosseinzadeh, M. Fateh, Curcumin intercalated layered


double hydroxide nanohybrid as a potential drug delivery system for effective
photodynamic therapy in human breast cancer cells, RSC Adv. 5 (2015)
The authors wish to acknowledge from all peoples that help us 9398793994, http://dx.doi.org/10.1039/C5RA15888E.
to do this project. [15] D. Gabrielli, E. Belisle, D. Severino, A.J. Kowaltowski, M.S. Baptista, Binding,
aggregation and photochemical properties of methylene blue in
mitochondrial suspensions, Photochem. Photobiol. 79 (2007) 227232, http://
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