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Effects of continuous wave and fractionated diode laser on human fibroblast cancer and dermal normal cells by zinc phthalocyanine in photodynamic therapy: A comparative study
Journal of Photochemistry & Photobiology, B: Biology 161 (2016) 456–462
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Effects of continuous wave and fractionated diode laser on human fibroblast cancer and dermal normal cells by zinc phthalocyanine in photodynamic therapy: A comparative study Farzaneh Navaeipour a,b, Hadi Afsharan b,c, Habib Tajalli d, Mahmood Mollabashi b, Farideh Ranjbari e, Azadeh Montaseri a,f, Mohammad-Reza Rashidi c,e,⁎ a
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Faculty of Physics, Iran University of Science and Technology, Tehran, Iran c Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran d Research Institute for Applied Physics and Astronomy, University of Tabriz, Tabriz, Iran e Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran f Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran b
a r t i c l e
i n f o
Article history: Received 17 March 2016 Accepted 10 June 2016 Available online 11 June 2016 Keywords: Photodynamic therapy Continuous wave laser Fractionated laser Zinc phthalocyanine Human fibroblast cancer cells Human dermal normal cells
a b s t r a c t In this experimental study, cancer and normal cells behavior during an in vitro photodynamic therapy (PDT) under exposure of continuous wave (CW) and fractionated mode of laser with different irradiation power and time intervals was compared and investigated. At the first, human fibroblast cancer cell line (SW 872) and human dermal normal (HFFF2) cell line were incubated with different concentrations of zinc phthalocyanine (ZnPc), as a PDT drug. The cells, then, were irradiated with a 675 nm diode laser and the cell viability was evaluated using MTT assay. Under optimized conditions, the viability of the cancer cells was eventually reduced to 3.23% and 13.17%, and that of normal cells was decreased to 20.83% and 36.23% using CW and fractionated diode lasers, respectively. In general, the ratio of ZnPc LD50 values for the normal cells to the cancer cells with CW laser was much higher than that of the fractionated laser. Subsequently, cancer cells in comparison with normal ones were found to be more sensitive toward the photodynamic damage induced by ZnPc. In addition, treatment with CW laser was found to be more effective against the cancer cells with a lower toxicity to the normal cells compared with the fractionated diode laser. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cancer is one of the leading causes of death worldwide and its effective treatment represents a global challenge to public health care. Although chemotherapy, surgery and radiation are still the most widely used treatment options to treat cancer, there is a great need for other treatments that can eradicate cancer cells with much less damage to the normal cells [1,2]. Photodynamic therapy is a promising treatment of cancer and nonmalignant diseases such as skin [3], lung, head and neck [4], liver [5], bladder [6], esophageal [7] and breast cancer [8], brain tumors [9] and sterilization of blood to eliminate viruses such as HIV and hepatitis for transfusion [6]. This medical treatment modality is based on the localization of a chemical photosensitizer in the target cells followed by ⁎ Corresponding author at: Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, 51664-14766 Tabriz, Iran. E-mail address: [email protected] (M.-R. Rashidi).
http://dx.doi.org/10.1016/j.jphotobiol.2016.06.017 1011-1344/© 2016 Elsevier B.V. All rights reserved.
light irradiation at certain wavelength that corresponds to the peak absorption of the photosensitizer into the target tissue. The light irradiation leads to the excitation of the photosensitizer to the triplet state from the singlet state through intersystem crossing and transferring the excited energy to ground state molecular oxygen generating cytotoxic reactive oxygen species (ROS) such as OH•, O2•− and excited singlet oxygen (1O2) that destroy target cells through apoptosis or necrosis [4,10,11]. However, they also concentrate in some extent in normal tissues and unintended destructions of normal cells beside cancerous ones is considered as the main drawback of the PDT. Therefore, obtaining optimal conditions such as light source parameters and PDT agents are important factors to achieve high treatment performance. On the other hand, finding these parameters can lead to minimum normal cell death with high elimination of cancer cells [12]. Although it is desired that the photosensitizer preferentially accumulates in cancer lesions, it can also concentrate in some extent in normal tissues and unintended destructions of normal cells beside cancer
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ones is considered as the main drawback of the PDT. Therefore, obtaining optimal conditions such as laser parameters, PDT agents, and the use of appropriate targeted drug delivery systems are important factors and strategies to achieve high treatment performance. On the other hand, finding these parameters may lead to minimum normal cell death with high elimination of cancer cells [12]. The light source is one of the three major components of PDT and its optimization has an important influence on the performance of PDT. Selection of light source as the heart of PDT is highly dependent on photosensitizer and its absorption peak, location and type of tumors and light dose delivery [13]. Although traditional lamps and LEDs are still used in PDT, they have been replaced recently with lasers due to unique and innovative properties of lasers such as such as excellent coherency, high intensity, good penetration range and monochromatic beam [14]. Among all types of lasers, diode lasers have been on the focus because of their low cost, light-weight, small size, easy installation, facile mobility, simple operation and also they do not need any light transferring tool. Thus, diode lasers have become the most prevalent light sources used in PDT [15,16]. However, laser, in and itself, has some defects in cells, such as destruction of cells, disruption in proliferation and causing unintended cell death. In this regard, many researches have been conducted to find the optimized condition for the laser application during PDT [17]. For example, in an in vitro study Kawauchi et al. indicated that the efficiency of PDT utilizing CW laser was more than that of pulsed laser [18]. While other studies reported controversial results. According to some studies, fractionated illuminations were more effective than CW lasers in PDT of some solid tumors [19,20]. In an in vitro experiment, it has been shown that the increasing singlet oxygen concentration in pulsed mode is higher than in CW one and the pulsed irradiation mode mainly leads to apoptosis while the CW mode results in necrosis. Equal number of dead cells was obtained for both modes in this study [21]. Alongside these controversial results with regards to the effect of different modes of laser application, the behavior of normal cells in comparison with that of cancer cells has been almost neglected. In the present comparative study, the effect of PDT on human fibroblast cancer (SW 872) and human dermal normal (HFFF2) cell lines using ZnPc as a photosensitizer and a 675 nm diode laser as a light source has been investigated. Two laser modes (CW and fractionated) with different output power and different time of irradiation were employed in this study. During the experiments, the performance of PDT was evaluated by employing different concentrations of the photosensitizer and also changing the parameters associated with laser such as irradiation mode, output power and irradiation time. Human dermal fibroblast cell lines were selected because skin cancer is the most common type of cancer all around the world. Fibroblast is the most important type of cells in the skin, and its main function is to synthesize and secrete the extracellular matrix to preserve the structural consistency of connective tissue [22]. To the best of our knowledge, this is the first time that the behavior of cancer and normal cells during PDT is investigated by using two modes of laser in different exposure time and various output power alongside the incubation of broad range of photosensitizer concentrations.
ZnPc has a weak absorption peak at 345 nm and a strong one at 675 nm, which was proved by UV–visible spectrum (Shimadzu UV1650PC, Japan). Characterization of ZnPc as the photosensitizer is highly recommended due to its prominent role in photodynamic therapy and direct relation to the production of singlet oxygen. ZnPc was also characterized to find its main absorption peak in order to select appropriate laser and proper excitation of the compound. The UV–visible spectrum of ZnPc in DMSO is shown in Fig. 1. In this spectrum, the broad absorption peak at 345 nm is associated with B-band. Furthermore, an intense peak near 675 nm was observed, which is assigned to Q-band (π-π*). These data are consistent with other literatures of this case [23,24]. The results indicate that the selected diode laser at 675 nm is coincident with the absorption peak of ZnPc. 2.2. Cell Culture The SW 872 and HFFF2 cell line were purchased from the Pasteur Institute (Tehran, Iran). SW 872 cell line which was used as a cancer fibroblast cell line in this study is reported as an undifferentiated malignant tumor consistent with liposarcoma. Liposarcoma is a malignant tumor of connective tissue which predominantly consists of fibroblasts. HFFF2 cell line was derived from the foreskin of a 14 to 18-week-old Caucasian fetus. Both cell lines were defrosted and grown in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Biowest, France). Cells were incubated at 37 °C in 5% CO2 and 95% humidity until they reached at least 70% confluency. 2.3. Light Source To match the laser light with the maximum absorption of ZnPc, a continuous wave (emits light continuously and has a constant and steady light emission during exposure process) and fractionated (also is a CW laser system that has a discrete output power which is created by utilizing special electronics and used to be modulated between an “on” state and “off” state.) diode laser (Shenzhen Taiyong Technology, China) with a wavelength of 675 nm was used. The continuous wave laser was modulated in 25, 50 and 80 mW output power and the fractionated laser was utilized in 30, 70 and 100 mW power. 3. Experimental Design For investigating the effects of PDT on the cell lines, both cell types were firstly harvested using trypsin/EDTA enzyme (Bioidea, Iran) and after reaching to almost 70% of confluency, they were centrifuged and counted. The Suspended cells were then seeded in 96-well plates with a cell density of 10,000 cells/well. Finally and before performing any experiments, they were incubated overnight at 37 °C in a humid
2. Materials and Methods 2.1. Photosensitizer Zinc phthalocyanine (ZnPc) was purchased from Sigma-Aldrich (USA). The stock solution (100 μg/mL) of ZnPc was prepared using dimethylsulfoxide (DMSO) and RPMI culture media (Gibco, USA) followed by sonication in a bath type sonicator (Elma transsonic T420, Germany). The final concentration of DMSO in the stock solution was 2% (v/v) in RPMI. Subsequently, the required concentrations were prepared by diluting the stock solution with RPMI. It must be noted that
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Fig. 1. UV–visible spectrum of 100 μg/mL of ZnPc in DMSO.
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environment and in the presence of 5% CO2. Then, they received different dosages of ZnPc or laser irradiation as described below: The cells were categorized in four groups according to how they were examined. The first group, which is defined as a control group, was not treated with neither of ZnPc nor laser. The second group was just irradiated with laser in the absence of ZnPc. The third group received only ZnPc without laser exposure, and finally the fourth group treated with both ZnPc and laser irradiation. The cells in the third and fourth groups were incubated in dark condition with various concentrations of ZnPc (0.5 to 100 μg/mL). After 24 h, they were washed by PBS (using 0.1 M Na2HPO4, 0.1 M KH2PO4, 0.1 M KCl) and then, irradiated with laser in a dark room at room temperature. In the first set of experiments, normal and cancer cells were irradiated by CW laser at different power and exposure time. For instance, after the laser power was set to 25 mW, the cells were irradiated for 20, 40 and 60 s separately. Similarly, for 50 and 80 mW laser power, the experiments were exactly repeated. The tests on the normal and cancer cells were performed simultaneously. In the second set of experiments, the seeded normal and cancer cells were irradiated by fractionated laser at different output power and number of irradiation. The reason of using the fractionated laser instead of the CW one is to evaluate the assertion of achieving the same efficiency, by increasing the intensity and simultaneously, shorten the laser irradiation time. The first experiment was performed with 30 mW output power with 1000, 2000 and 3000 irradiation. After that, the 70 and 100 mW laser power were employed and used to evaluate the photodynamic treatment effect. Subsequently, the laser exposed cells were incubated at 37 °C for a day. All experiments were repeated twice in a triplicate. Parameters of the utilized lasers are shown in Table 1. To compare the resulted data of the CW and fractionated laser, the irradiance of the emitted light was calculated using Eq. (1): Fluency J=cm2 ¼ Time ð secÞ Power ðWÞ=surface cm2
[25–27]. Cell viability ð%Þ ¼ ðabsorbance of sample=absorbance of controlÞ 100 ð2Þ
3.1. Statistical Analysis Each test was done six times (n = 6). The mean, standard deviation and standard error were calculated using Microsoft Excel 2013 software. Further statistical analysis was conducted using GraphPad prism 6 software by two-way analysis of Variance (ANOVA). Moreover, determination of significant changes between experimental groups and respective controls was obtained by utilizing Tukey–Kramer Multiple Comparisons Test. Significant differences were represented in the graphs as (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001. 4. Results and Discussion 4.1. Investigation of ZnPc Cytotoxicity (Dark Toxicity) and Laser Effects on Cells Initially, the effects of ZnPc concentrations (without irradiation of laser) and laser irradiation (without ZnPc) on viability of both cell types were evaluated. According to the obtained data displayed in Fig. 2A, the cytotoxic effect of inactive ZnPc (without irradiation of laser) at different concentrations on the viability of SW 872 cancer cells and HFFF2 normal cells (the control group) was very low and negligible. In particular, in the normal cells, at low concentrations of ZnPc (0.5 to 5 μg/mL), the toxicity was quite negligible and the cells death were almost 3%. While, by incubating the cells at higher concentrations (such as 100 μg/mL) of ZnPc, the viability decreased to about 94%. Similarly,
ð1Þ
The laser impact on the cells death after irradiation was evaluated by MTT assay According to the [25,26], first, the MTT solution was prepared by mixing fresh culture medium and 2 mg/mL methyl-thiazolyl-tetrazolium, MTT (Sigma-Aldrich, USA) with the ratio of 3:1 μL. Next, 200 μL of the prepared MTT solution was added to the cell medium in each well. The plates were then incubated for 4 h at 37 °C. During the incubation process, yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide is reduced to a dark purple formazan product by mitochondrial succinate dehydrogenase. As this product is insoluble in water, an organic solvent is added to the solution and the amount of the released formazan is measured spectrophotometrically. Since the reduction of MTT can only occur in metabolically active cells, the level of activity is considered as a measure of the cells viability. Therefore, 100 μL DMSO was added to each well and the plate was placed in a microplate reader (Bio Tek, Elx 808, USA) to measure the absorbance of each well. The percentage of the viable cells in each well was, then, calculated using Eq. (2)
Table 1 The parameters of lasers used in this study. Parameter
Continuous wave laser
Fractionated laser
Wavelength Wave emission Beam diameter Output power Duration of irradiation Interval between irradiation Number of irradiation Fluency
675 (nm) Continuous 7 (mm) 25, 50, 80 (mW) 20, 40, 60 (s) –
675 (nm) Fractionated 7 (mm) 30, 70, 100 (mW) 15 (ms) 10 (ms)
– 1, 2, 3, 4, 6, 6.4, 9.6 (J/cm2)
1000, 2000, 3000 1, 2, 3, 4, 6, 6.4, 9.6 (J/cm2)
Fig. 2. (A) The cell viability in the presence of 0, 0.5, 2, 5, 10, 25, 50 and 100 μg/mL of ZnPc for both HFFF2 and SW 872. (B) The effect of irradiation of 25, 50 and 80 mW laser on SW 872 cells without incubating any ZnPc. Data points represent the mean ± SD, n = 6.
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the variation of viabilities via ZnPc concentrations in cancer cells (SW 872) was almost the same as that of HFFF2. Moreover, in the absence of ZnPc, by exposing different laser power (25, 50 and 80 mW) to the cells (listed as the second group), even for 60 s, the viability of the cells was not affected greatly, demonstrating that the laser light alone does not have any toxic defects on the cells (Fig. 2B). By utilizing the fractionated laser, the same results were obtained. All of the above gathered data showed that the cell death in the next investigations can be attributed to the effects of the photodynamic treatment. 4.2. Effect of the CW Laser on Cells The collected data after irradiation of CW laser with 25 mW (20 s; 1 J/cm2, 40 s; 2 J/cm 2 , 60 s; 3 J/cm2), 50 mW (20 s; 2 J/cm 2, 40 s; 4 J/cm2, 60 s; 6 J/cm2) and 80 mW (40 s; 6.4 J/cm2, 60 s; 9.6 J/cm2) output power on the cancer and normal cells are presented in Fig. 3. By increasing the ZnPc concentration, viability of the normal and cancer cells reduced. It is obvious in Fig. 3A that at concentrations above 2 μg/mL, as laser (25 mW) exposure time increased, the cell viability decreased. For example, at 100 μg/mL concentration and 60 s irradiation, normal cells viability decreased to 49.35%, while the viability of cancer cells was 18%. Based on the results obtained from these concentration-response experiments, the LD50 (the dose of ZnPc that is required for 50% viability of the cells) values of ZnPc (25 mW, 60 s;
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3 J/cm2) for the cancer and normal cells were calculated as 20 and 100 μg/mL, respectively. This indicates that ZnPc is more than five times more toxic toward cancer cells than to the normal cell line. Fig. 3B shows that by raising the laser power of 50 mW, the results are similar to those of 25 mW laser. As a result, increasing the ZnPc concentration and the duration of the irradiation was led to reduction of cell viabilities. For example, at 100 μg/mL concentration of ZnPc under 60 s exposure, the viability of normal cells reduced to 38.01%, whereas in the case of cancer cell line, only 5.19% of the cells could survive. In addition, one can find that higher power of laser was able to induce the cytotoxicity at lower concentrations of ZnPc, in which the LD50 of ZnPc for cancer and normal cells (50 mW, 60 s; 6 J/cm2) were 10 and 27 μg/mL, respectively. Fig. 3C reveals that irradiation of laser for 60 s, at 100 μg/mL concentration of ZnPc, led to much more toxic effect on the cancer cells compared to the normal cells. Furthermore, the LD50 of cancer cells is 2 μg/mL and that of the normal cells is 15 μg/mL when the experiment is done by a CW laser (80 mW, 60 s; 9.6 J/cm2). Although it is expected that the efficiency of the photodynamic treatment depends on the ZnPc concentration and the light dosage, the marked difference between the behavior of the normal and cancer cells toward these parameters was interesting. Also, the viability of normal cells was more than cancer cells, demonstrating that the photodynamic treatment acts more efficiently on cancer cells than on normal cells. Subsequently, this information satisfies the desired expectations
Fig. 3. The cell viability of SW 872 cancer and HFFF2 normal cell lines after photosensitization with different concentrations of ZnPc (0 to 100 μg/mL) and photoirradiation with CW laser for 20, 40 and 60 s at (A) 25, (B) 50 and (C) 80 mW. Data points represent the mean ± SD, n = 6.
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Fig. 4. The effect of the fractionated laser exposure with (A) 30, (B) 70 and (C) 100 mW output power on cell viability of SW 872 cancerous and HFFF2 normal cell lines incubated with 0 to 100 μg/mL of ZnPc for 1000, 2000 and 3000 irradiation. Data points represent the mean ± SD, n = 6.
about PDT and its role in eliminating cancer cells rather than normal ones. There are also other previously reported articles that are consistent with the above results [28,29]. 4.3. Influence of the Fractionated Laser on Cells Viability The influence of 30 mW (1000 irradiation; 1 J/cm2, 2000 irradiation; 2 J/cm2, 3000 irradiation; 3 J/cm2), 70 mW (1000 irradiation; 2 J/cm2, 2000 irradiation; 4 J/cm2, 3000 irradiation; 6 J/cm2) and 100 mW (2000 irradiation; 6.4 J/cm2, 3000 irradiation; 9.6 J/cm2), fractionated laser on both cell types has been presented in Fig. 4. First, the modulated laser was adjusted to 30 mW and exposed (with 1000, 2000 and 3000 irradiation) to the cancer and normal cells incubated with different concentrations of ZnPc. Fig. 4A illustrates the collected data after MTT. According to the figure, 3000 irradiation were led to the highest number of death imposed to the cells, which had been incubated with 100 μg/mL ZnPc. Applied conditions (3000 irradiation, 30 mW and 100 μg/mL ZnPc) resulted in decreasing the viability of cancer and normal cells to 37.31% and 48.9%, respectively. Additionally, the LD50 of cancer and normal cells irradiated with fractionated laser (30 mW, 3000 irradiation; 3 J/cm2) were obtained 75 and N 100 μg/mL. By repeating the experiments for 70 and 100 mW modulated lasers, almost the same behavior was observed. The gathered data of these parts of the experiments are shown in Fig. 4B and 4C. By employing the 6 J/cm2 (70 mW, 3000 irradiation), the LD50 of cancer and normal cells were calculated 40 and 90 μg/mL, and the cell viabilities in the
presence of 100 μg/mL ZnPc, were decreased to 5.19% and 38.01%, respectively. The LD50 after exposure of laser (100 mW, 3000 irradiation; 9.6 J/cm2) on both cell types (cancer and normal) were also calculated as 30 and 50 μg/mL, respectively. In addition, the viability for cancer cells was measured 3.23% and that of normal cells 20.83% was measured by MTT assay. 4.4. Optimizing the Laser Exposure Time It is important to obtain an optimized condition in which the time of PDT experiment becomes minimum while achieving similar or better efficiency. As a result, cells are less exposed to laser and the probability Table 2 Obtained LD50 values during PDT for cancer and normal cells. Fluency (J/cm2)
1 2 3 4 6 6.4 9.6
Continuous wave laser
Fractionated laser
Cancer cells LD50 (μg/mL)
Normal cells LD50 (μg/mL)
Cancer cells LD50 (μg/mL)
Normal cells LD50 (μg/mL)
63 ± 7.8 35 ± 4.3 20 ± 5.1 15 ± 3.8 10 ± 4.2 5 ± 3.3 2 ± 1.4
N100 ± 10.7 N100 ± 12.1 100 ± 8.6 45 ± 6.0 27 ± 5.3 25 ± 5.6 15 ± 3.2
N100 ± 9.1 N100 ± 13.0 75 ± 9.7 75 ± 6.8 40 ± 3.2 50 ± 5.7 35 ± 6.4
N100 ± 7.8 N100 ± 11.3 N100 ± 9.4 100 ± 5.8 90 ± 7.2 75 ± 5.5 50 ± 6.0
LD50: the dose required for 50% cell viability.
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Table 3 The LD50 obtained values of PDT using CW laser in this work in comparison with those reported by others. Cell type
PDT drug
Light source
Fluency (J/cm2)
LD50 value (μg/mL)
Reference
NIH3T3 B16-F0 MCF7 G361 THP-1 HepG2 A549 HepG2 HT29 HFFF2 SW 872
Phthalocyanine ClAlPcS2 Phthalocyanine ClAlPcS2 Phthalocyanine ClAlPcS2 Phthalocyanine ClAlPcS2 ZnPc-TiO2 ZnPc-TiO2 Dendrimer phthalocyanine Silicon(IV) phthalocyanines Silicon(IV) phthalocyanines ZnPc ZnPc
675 nm semiconductor laser (CW) 675 nm semiconductor laser (CW) 675 nm semiconductor laser (CW) 675 nm semiconductor laser (CW) 670 nm non-ionic laser light (CW) 670 nm non-ionic laser light (CW) Halogen lamp 675 nm laser (CW) 675 nm laser (CW) 675 nm diode laser (CW) 675 nm diode laser (CW)
30 30 30 30 10 10 10.8 30 30 9.6 9.6
5.6 N10 1.4 1.6 2.0 5.5 8.0 1.32 0.91 15 2
[31] [31] [31] [31] [25] [25] [32] [30] [30] This work This work
NIH3T3: mouse normal fibroblasts, B16-F0: mouse cancer melanoma, MCF7: human breast adenocarcinoma, G361: human melanoma, THP-1: human macrophage, HepG2: human hepatocellular carcinoma, A549: human lung adenocarcinoma, HT29: human colon adenocarcinoma, HFFF2: human fibroblast skin normal cells, SW 872: human fibroblast skin cancer cells, ZnPc: zinc phthalocyanine.
of laser defects on cells will be decreased. So, at the constant ZnPc concentration, by increasing the output power of laser and reducing the exposure time, in comparison to lower output power and longer exposure time, we can achieve almost the same transferred energy. For example, for cancer cells, at 50 μg/mL ZnPc concentration and using the CW laser with 2 J/cm2 energy, 60 s irradiation of 25 mW laser led to 38.5% of cell viability while 20 s irradiation of 50 mW laser resulted in 39.53% cell viability. In addition, by employing the same conditions, the viability of normal cells were obtained 60.6% and 61.78%, respectively. The same effects stood for the fractionated laser. Therefore, it can be concluded that by using the shorter exposure time, not only approximately equivalent PDT efficiency can be achieved, but also in some cases it could become even better. 4.5. Comparison of the Generated Effects by CW and Fractionated Lasers on Cancer and Normal Cells As we said before, to compare the resulted data of CW and fractionated laser, we have calculated the fluency of emitted light (according to Eq. (1)). By comparing the results it can be seen that the CW laser is more effective. On the other hand, the death rate of cells resulted by CW laser is more than that of fractionated laser. The intervals between irradiation of fractionated laser which prevent the steady activation process of ZnPc is the reason of this behavior. For instance, irradiation of 9.6 J/cm2 energy using a CW laser has reduced the viability of cancer cells to 2.23%, while, the fractionated laser at the same transferred energy (9.6 J/cm2) has reduced it to 13.17%. The concluded fact stands for the normal cells, too. Particularly, when the CW laser is exposed to normal cells carrying 9.6 J/cm2 radiation energy, the cell viabilities reduced 20.83%. In the same conditions, fractionated laser reduced the viability to 36.23%. For further clarification, the obtained LD50 values for normal and cancer cells treated by PDT have been listed in Table 2. Also, evaluating the data represented in Table 2, demonstrated the superior impact of CW laser on cancer cells over the normal cells which is consistent with previous obtained information. In addition, in Table 3, the LD50 obtained in the present study using CW laser have been compared with those have been reported by others. According to this table, the efficiency of PDT in this study, considering the fluencies of the conducted experiments is better or comparable with the other ones. Particularly, in a study [30], the research group evaluated the PDT effects on HT29 and HepG2 cells. They calculated the LD50 for these cells as 0.91 and 1.32 μg/mL, respectively, with using Silicon(IV)-phthalocyanine as photosensitizer and a 675 nm CW laser as a light source. Comparison of our obtained LD50 value (for cancer cells = 2 μg/mL) and those reported in [30] indicates the efficiency of the present study, by have this in mind that the fluency of our works is less than one third. As another example [31], Kolarova et al. have investigated PDT using a 675 nm CW semiconductor laser irradiated on
4 types of cell lines (NIH3T3, B16-F0, MCF7 and G361) which were incubated with phthalocyanine ClAlPcS2. By comparing the results with those of ours and again considering the fluency of each study, it can be seen that not only LD50 of our represented is better, but also performance of our experimental study is higher. Due to the fact that during PDT of the cancer cells, normal tissues or cells are also either damaged or destroyed, the performance of a PDT is highly dependent on minimum death of normal cells alongside the maximum death of cancer ones. In other words, the effect of PDT on normal cells should be lesser than that on cancer cells. In the mentioned study, NIH3T3 is a normal fibroblast mouse cell line and the B16-F0 is a cancer melanoma mouse cell line. It is obvious that the normal cells have lower LD50 in comparison with cancer ones (5.6 vs. N10 μg/mL). However, the LD50s obtained in the same conditions for the cancer and normal cell lines were 2 and 15 μg/mL, respectively. In general, the ratio of LD50 values for the normal cells to the cancer cells with CW laser was much higher than that of the fractioned laser (Table 2) indicating the effectiveness alongside relatively more safety of the former laser mode compared to the later one. 5. Conclusion In this study, the effects of laser irradiation in various conditions including power, time of exposure and the irradiation type on the cancer and normal skin cells in the presence of different concentrations of ZnPc were investigated. The data obtained indicate that not only the performance of the CW laser is better than the fractionated laser, but also, at the same conditions, the death rate of SW 872 (cancer cells) is more than that of HFFF2 (normal cells) which meets the desired expectation to treat cancer tumors. This is also reflected in the ratio of LD50 values for the HFFF2 to the SW 872 with CW and fractionated lasers. In addition, the accumulated data also show that by irradiating more powerful bursts of laser in shorter time, the same quite results of cell death are obtained in comparison with exposing longer and low power bursts. Acknowledgment This project was financially supported by Iran University of Science and Technology (IUST) and Stem Cell Research Center (SCRC) at Tabriz University of Medical Sciences. References [1] A.M. Kramer, M. Yan, K.S. Peggs, J. Anderson, K. Gustafsson, Tumor-associated antigen presentation by γδ T-cells in cancer immunotherapy, Blood 124 (21) (2014) 1411. [2] N.S. Iyer, L.M. Balsamo, M.B. Bracken, N.S. Kadan-Lottick, Chemotherapy-only treatment effects on long-term neurocognitive functioning in childhood ALL survivors: a review and meta-analysis, Blood 126 (3) (2015) 346–353.
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[3] H. Kolarova, P. Nevrelova, R. Bajgar, D. Jirova, K. Kejlova, M. Strnad, In vitro photodynamic therapy on melanoma cell lines with phthalocyanine, Toxicol. In Vitro. 21 (2) (2007) 249–253. [4] A. Gupta, P. Avci, M. Sadasivam, R. Chandran, N. Parizotto, D. Vecchio, et al., Shining light on nanotechnology to help repair and regeneration, Biotechnol. Adv. 31 (5) (2013) 607–631. [5] C.-Y. Wang, X. Wang, Y. Wang, T. Zhou, Y. Bai, Li Y-C, et al., Photosensitization of phycocyanin extracted from microcystis in human hepatocellular carcinoma cells: implication of mitochondria-dependent apoptosis, J. Photochem. Photobiol. B Biol. 117 (2012) 70–79. [6] L. Howe, J.Z. Zhang, The effect of biological substrates on the ultrafast excited-state dynamics of zinc phthalocyanine tetrasulfonate in solution, Photochem. Photobiol. 67 (1) (1998) 90–96. [7] L. Corti, J. Skarlatos, C. Boso, F. Cardin, L. Kosma, M.I. Koukourakis, et al., Outcome of patients receiving photodynamic therapy for early esophageal cancer, Int. J. Radiat. Oncol. Biol. Phys. 47 (2) (2000) 419–424. [8] L.-y. Xue, Chiu S-m, N.L. Oleinick, Atg7 deficiency increases resistance of MCF-7 human breast cancer cells to photodynamic therapy, Autophagy 6 (2) (2010) 248–255. [9] D. Bechet, S.R. Mordon, F. Guillemin, M.A. Barberi-Heyob, Photodynamic therapy of malignant brain tumours: a complementary approach to conventional therapies, Cancer Treat. Rev. 40 (2) (2014) 229–241. [10] A.M. Lima, C.D. Pizzol, F.B.F. Monteiro, T.B. Creczynski-Pasa, G.P. Andrade, A.O. Ribeiro, et al., Hypericin encapsulated in solid lipid nanoparticles: phototoxicity and photodynamic efficiency, J. Photochem. Photobiol. B Biol. 125 (2013) 146–154. [11] S. Choudhary, K. Nouri, M. Elsaie, Photodynamic therapy in dermatology: a review, Lasers Med. Sci. 24 (6) (2009) 971–980. [12] S. Yano, S. Hirohara, M. Obata, Y. Hagiya, Ogura S-I, A. Ikeda, et al., Current states and future views in photodynamic therapy, J. Photochem. Photobiol. C Photochem. Rev. 12 (1) (2011) 46–67. [13] L. Brancaleon, H. Moseley, Laser and non-laser light sources for photodynamic therapy, Lasers Med. Sci. 17 (3) (2002) 173–186. [14] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick, et al., Photodynamic therapy of cancer: an update, CA Cancer J. Clin. 61 (4) (2011) 250–281. [15] T.S. Mang, Lasers and light sources for PDT: past, present and future, Photodiagnosis Photodynamic Ther. 1 (1) (2004) 43–48. [16] J. Jankun, R.W. Keck, E. Skrzypczak-Jankun, L. Lilge, S.H. Selman, Diverse optical characteristic of the prostate and light delivery system: implications for computer modelling of prostatic photodynamic therapy, BJU Int. 95 (9) (2005) 1237–1244. [17] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick, et al., Photodynamic therapy of cancer: an update, CA Cancer J. Clin. 61 (4) (2011) 250–281. [18] S. Kawauchi, Y. Morimoto, S. Sato, T. Arai, K. Seguchi, H. Asanuma, et al., Differences between cytotoxicity in photodynamic therapy using a pulsed laser and a continuous wave laser: study of oxygen consumption and photobleaching, Lasers Med. Sci. 18 (4) (2004) 179–183.
[19] Z. Xiao, S. Halls, D. Dickey, J. Tulip, R.B. Moore, Fractionated versus standard continuous light delivery in interstitial photodynamic therapy of dunning prostate carcinomas, Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 13 (24) (2007) 7496–7505. [20] J.P. Estevez, M. Ascencio, P. Colin, M.O. Farine, P. Collinet, S. Mordon, Continuous or fractionated photodynamic therapy? Comparison of three PDT schemes for ovarian peritoneal micrometastasis treatment in a rat model, Photodiagnosis Photodynamic Ther. 7 (4) (2010) 251–257. [21] V.V. Klimenko, A.A. Bogdanov, N.A. Knyazev, A.A. Rusanov, M.V. Dubina, Different photodynamic effect between continuous wave and pulsed laser irradiation modes in k562 cells in vitro, J. Phys Conf. Series. 541 (1) (2014) 012040. [22] M. Caiazzo, M.T. Dell'Anno, E. Dvoretskova, D. Lazarevic, S. Taverna, D. Leo, et al., Direct generation of functional dopaminergic neurons from mouse and human fibroblasts, Nature 476 (7359) (2011) 224–227. [23] M. van Leeuwen, A. Beeby, S.H. Ashworth, The photochemistry and photophysics of a series of non-peripherally substituted zinc phthalocyanines, Photochem. Photobiol. Sci. 9 (3) (2010) 370–375. [24] A.R. Barron, J. Humphrey, Nanomaterials for alternative energy sources, Dalton Trans. (40) (2008) 5399. [25] T. Lopez, E. Ortiz, M. Alvarez, J. Navarrete, J.A. Odriozola, F. Martinez-Ortega, et al., Study of the stabilization of zinc phthalocyanine in sol-gel TiO2 for photodynamic therapy applications, Nanomed. Nanotechnol. Biol. Med. 6 (6) (2010) 777–785. [26] S.M. El-Daly, A.M. Gamal-Eldeen, M.A.M. Abo-Zeid, I.H. Borai, H.A. Wafay, A.-R.B. Abdel-Ghaffar, Photodynamic therapeutic activity of indocyanine green entrapped in polymeric nanoparticles, Photodiagnosis Photodynamic Ther. 10 (2) (2013) 173–185. [27] T.G. Sutedja, P.E. Postmus, Photodynamic therapy in lung cancer. a review, J. Photochem. Photobiol. B Biol. 36 (2) (1996) 199–204. [28] N.B.R. Vittar, C.G. Prucca, C. Strassert, J. Awruch, V.A. Rivarola, Cellular inactivation and antitumor efficacy of a new zinc phthalocyanine with potential use in photodynamic therapy, Int. J. Biochem. Cell Biol. 40 (10) (2008) 2192–2205. [29] K. Maduray, B. Odhav, T. Nyokong, In vitro photodynamic effect of aluminum tetrasulfophthalocyanines on melanoma skin cancer and healthy normal skin cells, Photodiagnosis Photodynamic Ther. 9 (1) (2012) 32–39. [30] J.T. Lau, P.C. Lo, W.P. Fong, D.K. Ng, Preparation and photodynamic activities of silicon (IV) phthalocyanines substituted with permethylated β-cyclodextrins, Chem. Eur. J. 17 (27) (2011) 7569–7577. [31] H. Kolarova, R. Lenobel, P. Kolar, M. Strnad, Sensitivity of different cell lines to phototoxic effect of disulfonated chloroaluminium phthalocyanine, Toxicol. In Vitro. 21 (7) (2007) 1304–1306. [32] S. Herlambang, M. Kumagai, T. Nomoto, S. Horie, S. Fukushima, M. Oba, et al., Disulfide crosslinked polyion complex micelles encapsulating dendrimer phthalocyanine directed to improved efficiency of photodynamic therapy, J. Control. Release. 155 (3) (2011) 449–457.
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Effects of continuous wave and fractionated diode laser on human fibroblast cancer and dermal normal cells by zinc phthalocyanine in photodynamic therapy: A comparative study Farzaneh Navaeipour a,b, Hadi Afsharan b,c, Habib Tajalli d, Mahmood Mollabashi b, Farideh Ranjbari e, Azadeh Montaseri a,f, Mohammad-Reza Rashidi c,e,⁎ a
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Faculty of Physics, Iran University of Science and Technology, Tehran, Iran c Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran d Research Institute for Applied Physics and Astronomy, University of Tabriz, Tabriz, Iran e Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran f Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran b
a r t i c l e
i n f o
Article history: Received 17 March 2016 Accepted 10 June 2016 Available online 11 June 2016 Keywords: Photodynamic therapy Continuous wave laser Fractionated laser Zinc phthalocyanine Human fibroblast cancer cells Human dermal normal cells
a b s t r a c t In this experimental study, cancer and normal cells behavior during an in vitro photodynamic therapy (PDT) under exposure of continuous wave (CW) and fractionated mode of laser with different irradiation power and time intervals was compared and investigated. At the first, human fibroblast cancer cell line (SW 872) and human dermal normal (HFFF2) cell line were incubated with different concentrations of zinc phthalocyanine (ZnPc), as a PDT drug. The cells, then, were irradiated with a 675 nm diode laser and the cell viability was evaluated using MTT assay. Under optimized conditions, the viability of the cancer cells was eventually reduced to 3.23% and 13.17%, and that of normal cells was decreased to 20.83% and 36.23% using CW and fractionated diode lasers, respectively. In general, the ratio of ZnPc LD50 values for the normal cells to the cancer cells with CW laser was much higher than that of the fractionated laser. Subsequently, cancer cells in comparison with normal ones were found to be more sensitive toward the photodynamic damage induced by ZnPc. In addition, treatment with CW laser was found to be more effective against the cancer cells with a lower toxicity to the normal cells compared with the fractionated diode laser. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cancer is one of the leading causes of death worldwide and its effective treatment represents a global challenge to public health care. Although chemotherapy, surgery and radiation are still the most widely used treatment options to treat cancer, there is a great need for other treatments that can eradicate cancer cells with much less damage to the normal cells [1,2]. Photodynamic therapy is a promising treatment of cancer and nonmalignant diseases such as skin [3], lung, head and neck [4], liver [5], bladder [6], esophageal [7] and breast cancer [8], brain tumors [9] and sterilization of blood to eliminate viruses such as HIV and hepatitis for transfusion [6]. This medical treatment modality is based on the localization of a chemical photosensitizer in the target cells followed by ⁎ Corresponding author at: Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, 51664-14766 Tabriz, Iran. E-mail address: [email protected] (M.-R. Rashidi).
http://dx.doi.org/10.1016/j.jphotobiol.2016.06.017 1011-1344/© 2016 Elsevier B.V. All rights reserved.
light irradiation at certain wavelength that corresponds to the peak absorption of the photosensitizer into the target tissue. The light irradiation leads to the excitation of the photosensitizer to the triplet state from the singlet state through intersystem crossing and transferring the excited energy to ground state molecular oxygen generating cytotoxic reactive oxygen species (ROS) such as OH•, O2•− and excited singlet oxygen (1O2) that destroy target cells through apoptosis or necrosis [4,10,11]. However, they also concentrate in some extent in normal tissues and unintended destructions of normal cells beside cancerous ones is considered as the main drawback of the PDT. Therefore, obtaining optimal conditions such as light source parameters and PDT agents are important factors to achieve high treatment performance. On the other hand, finding these parameters can lead to minimum normal cell death with high elimination of cancer cells [12]. Although it is desired that the photosensitizer preferentially accumulates in cancer lesions, it can also concentrate in some extent in normal tissues and unintended destructions of normal cells beside cancer
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ones is considered as the main drawback of the PDT. Therefore, obtaining optimal conditions such as laser parameters, PDT agents, and the use of appropriate targeted drug delivery systems are important factors and strategies to achieve high treatment performance. On the other hand, finding these parameters may lead to minimum normal cell death with high elimination of cancer cells [12]. The light source is one of the three major components of PDT and its optimization has an important influence on the performance of PDT. Selection of light source as the heart of PDT is highly dependent on photosensitizer and its absorption peak, location and type of tumors and light dose delivery [13]. Although traditional lamps and LEDs are still used in PDT, they have been replaced recently with lasers due to unique and innovative properties of lasers such as such as excellent coherency, high intensity, good penetration range and monochromatic beam [14]. Among all types of lasers, diode lasers have been on the focus because of their low cost, light-weight, small size, easy installation, facile mobility, simple operation and also they do not need any light transferring tool. Thus, diode lasers have become the most prevalent light sources used in PDT [15,16]. However, laser, in and itself, has some defects in cells, such as destruction of cells, disruption in proliferation and causing unintended cell death. In this regard, many researches have been conducted to find the optimized condition for the laser application during PDT [17]. For example, in an in vitro study Kawauchi et al. indicated that the efficiency of PDT utilizing CW laser was more than that of pulsed laser [18]. While other studies reported controversial results. According to some studies, fractionated illuminations were more effective than CW lasers in PDT of some solid tumors [19,20]. In an in vitro experiment, it has been shown that the increasing singlet oxygen concentration in pulsed mode is higher than in CW one and the pulsed irradiation mode mainly leads to apoptosis while the CW mode results in necrosis. Equal number of dead cells was obtained for both modes in this study [21]. Alongside these controversial results with regards to the effect of different modes of laser application, the behavior of normal cells in comparison with that of cancer cells has been almost neglected. In the present comparative study, the effect of PDT on human fibroblast cancer (SW 872) and human dermal normal (HFFF2) cell lines using ZnPc as a photosensitizer and a 675 nm diode laser as a light source has been investigated. Two laser modes (CW and fractionated) with different output power and different time of irradiation were employed in this study. During the experiments, the performance of PDT was evaluated by employing different concentrations of the photosensitizer and also changing the parameters associated with laser such as irradiation mode, output power and irradiation time. Human dermal fibroblast cell lines were selected because skin cancer is the most common type of cancer all around the world. Fibroblast is the most important type of cells in the skin, and its main function is to synthesize and secrete the extracellular matrix to preserve the structural consistency of connective tissue [22]. To the best of our knowledge, this is the first time that the behavior of cancer and normal cells during PDT is investigated by using two modes of laser in different exposure time and various output power alongside the incubation of broad range of photosensitizer concentrations.
ZnPc has a weak absorption peak at 345 nm and a strong one at 675 nm, which was proved by UV–visible spectrum (Shimadzu UV1650PC, Japan). Characterization of ZnPc as the photosensitizer is highly recommended due to its prominent role in photodynamic therapy and direct relation to the production of singlet oxygen. ZnPc was also characterized to find its main absorption peak in order to select appropriate laser and proper excitation of the compound. The UV–visible spectrum of ZnPc in DMSO is shown in Fig. 1. In this spectrum, the broad absorption peak at 345 nm is associated with B-band. Furthermore, an intense peak near 675 nm was observed, which is assigned to Q-band (π-π*). These data are consistent with other literatures of this case [23,24]. The results indicate that the selected diode laser at 675 nm is coincident with the absorption peak of ZnPc. 2.2. Cell Culture The SW 872 and HFFF2 cell line were purchased from the Pasteur Institute (Tehran, Iran). SW 872 cell line which was used as a cancer fibroblast cell line in this study is reported as an undifferentiated malignant tumor consistent with liposarcoma. Liposarcoma is a malignant tumor of connective tissue which predominantly consists of fibroblasts. HFFF2 cell line was derived from the foreskin of a 14 to 18-week-old Caucasian fetus. Both cell lines were defrosted and grown in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Biowest, France). Cells were incubated at 37 °C in 5% CO2 and 95% humidity until they reached at least 70% confluency. 2.3. Light Source To match the laser light with the maximum absorption of ZnPc, a continuous wave (emits light continuously and has a constant and steady light emission during exposure process) and fractionated (also is a CW laser system that has a discrete output power which is created by utilizing special electronics and used to be modulated between an “on” state and “off” state.) diode laser (Shenzhen Taiyong Technology, China) with a wavelength of 675 nm was used. The continuous wave laser was modulated in 25, 50 and 80 mW output power and the fractionated laser was utilized in 30, 70 and 100 mW power. 3. Experimental Design For investigating the effects of PDT on the cell lines, both cell types were firstly harvested using trypsin/EDTA enzyme (Bioidea, Iran) and after reaching to almost 70% of confluency, they were centrifuged and counted. The Suspended cells were then seeded in 96-well plates with a cell density of 10,000 cells/well. Finally and before performing any experiments, they were incubated overnight at 37 °C in a humid
2. Materials and Methods 2.1. Photosensitizer Zinc phthalocyanine (ZnPc) was purchased from Sigma-Aldrich (USA). The stock solution (100 μg/mL) of ZnPc was prepared using dimethylsulfoxide (DMSO) and RPMI culture media (Gibco, USA) followed by sonication in a bath type sonicator (Elma transsonic T420, Germany). The final concentration of DMSO in the stock solution was 2% (v/v) in RPMI. Subsequently, the required concentrations were prepared by diluting the stock solution with RPMI. It must be noted that
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Fig. 1. UV–visible spectrum of 100 μg/mL of ZnPc in DMSO.
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environment and in the presence of 5% CO2. Then, they received different dosages of ZnPc or laser irradiation as described below: The cells were categorized in four groups according to how they were examined. The first group, which is defined as a control group, was not treated with neither of ZnPc nor laser. The second group was just irradiated with laser in the absence of ZnPc. The third group received only ZnPc without laser exposure, and finally the fourth group treated with both ZnPc and laser irradiation. The cells in the third and fourth groups were incubated in dark condition with various concentrations of ZnPc (0.5 to 100 μg/mL). After 24 h, they were washed by PBS (using 0.1 M Na2HPO4, 0.1 M KH2PO4, 0.1 M KCl) and then, irradiated with laser in a dark room at room temperature. In the first set of experiments, normal and cancer cells were irradiated by CW laser at different power and exposure time. For instance, after the laser power was set to 25 mW, the cells were irradiated for 20, 40 and 60 s separately. Similarly, for 50 and 80 mW laser power, the experiments were exactly repeated. The tests on the normal and cancer cells were performed simultaneously. In the second set of experiments, the seeded normal and cancer cells were irradiated by fractionated laser at different output power and number of irradiation. The reason of using the fractionated laser instead of the CW one is to evaluate the assertion of achieving the same efficiency, by increasing the intensity and simultaneously, shorten the laser irradiation time. The first experiment was performed with 30 mW output power with 1000, 2000 and 3000 irradiation. After that, the 70 and 100 mW laser power were employed and used to evaluate the photodynamic treatment effect. Subsequently, the laser exposed cells were incubated at 37 °C for a day. All experiments were repeated twice in a triplicate. Parameters of the utilized lasers are shown in Table 1. To compare the resulted data of the CW and fractionated laser, the irradiance of the emitted light was calculated using Eq. (1): Fluency J=cm2 ¼ Time ð secÞ Power ðWÞ=surface cm2
[25–27]. Cell viability ð%Þ ¼ ðabsorbance of sample=absorbance of controlÞ 100 ð2Þ
3.1. Statistical Analysis Each test was done six times (n = 6). The mean, standard deviation and standard error were calculated using Microsoft Excel 2013 software. Further statistical analysis was conducted using GraphPad prism 6 software by two-way analysis of Variance (ANOVA). Moreover, determination of significant changes between experimental groups and respective controls was obtained by utilizing Tukey–Kramer Multiple Comparisons Test. Significant differences were represented in the graphs as (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001. 4. Results and Discussion 4.1. Investigation of ZnPc Cytotoxicity (Dark Toxicity) and Laser Effects on Cells Initially, the effects of ZnPc concentrations (without irradiation of laser) and laser irradiation (without ZnPc) on viability of both cell types were evaluated. According to the obtained data displayed in Fig. 2A, the cytotoxic effect of inactive ZnPc (without irradiation of laser) at different concentrations on the viability of SW 872 cancer cells and HFFF2 normal cells (the control group) was very low and negligible. In particular, in the normal cells, at low concentrations of ZnPc (0.5 to 5 μg/mL), the toxicity was quite negligible and the cells death were almost 3%. While, by incubating the cells at higher concentrations (such as 100 μg/mL) of ZnPc, the viability decreased to about 94%. Similarly,
ð1Þ
The laser impact on the cells death after irradiation was evaluated by MTT assay According to the [25,26], first, the MTT solution was prepared by mixing fresh culture medium and 2 mg/mL methyl-thiazolyl-tetrazolium, MTT (Sigma-Aldrich, USA) with the ratio of 3:1 μL. Next, 200 μL of the prepared MTT solution was added to the cell medium in each well. The plates were then incubated for 4 h at 37 °C. During the incubation process, yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide is reduced to a dark purple formazan product by mitochondrial succinate dehydrogenase. As this product is insoluble in water, an organic solvent is added to the solution and the amount of the released formazan is measured spectrophotometrically. Since the reduction of MTT can only occur in metabolically active cells, the level of activity is considered as a measure of the cells viability. Therefore, 100 μL DMSO was added to each well and the plate was placed in a microplate reader (Bio Tek, Elx 808, USA) to measure the absorbance of each well. The percentage of the viable cells in each well was, then, calculated using Eq. (2)
Table 1 The parameters of lasers used in this study. Parameter
Continuous wave laser
Fractionated laser
Wavelength Wave emission Beam diameter Output power Duration of irradiation Interval between irradiation Number of irradiation Fluency
675 (nm) Continuous 7 (mm) 25, 50, 80 (mW) 20, 40, 60 (s) –
675 (nm) Fractionated 7 (mm) 30, 70, 100 (mW) 15 (ms) 10 (ms)
– 1, 2, 3, 4, 6, 6.4, 9.6 (J/cm2)
1000, 2000, 3000 1, 2, 3, 4, 6, 6.4, 9.6 (J/cm2)
Fig. 2. (A) The cell viability in the presence of 0, 0.5, 2, 5, 10, 25, 50 and 100 μg/mL of ZnPc for both HFFF2 and SW 872. (B) The effect of irradiation of 25, 50 and 80 mW laser on SW 872 cells without incubating any ZnPc. Data points represent the mean ± SD, n = 6.
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the variation of viabilities via ZnPc concentrations in cancer cells (SW 872) was almost the same as that of HFFF2. Moreover, in the absence of ZnPc, by exposing different laser power (25, 50 and 80 mW) to the cells (listed as the second group), even for 60 s, the viability of the cells was not affected greatly, demonstrating that the laser light alone does not have any toxic defects on the cells (Fig. 2B). By utilizing the fractionated laser, the same results were obtained. All of the above gathered data showed that the cell death in the next investigations can be attributed to the effects of the photodynamic treatment. 4.2. Effect of the CW Laser on Cells The collected data after irradiation of CW laser with 25 mW (20 s; 1 J/cm2, 40 s; 2 J/cm 2 , 60 s; 3 J/cm2), 50 mW (20 s; 2 J/cm 2, 40 s; 4 J/cm2, 60 s; 6 J/cm2) and 80 mW (40 s; 6.4 J/cm2, 60 s; 9.6 J/cm2) output power on the cancer and normal cells are presented in Fig. 3. By increasing the ZnPc concentration, viability of the normal and cancer cells reduced. It is obvious in Fig. 3A that at concentrations above 2 μg/mL, as laser (25 mW) exposure time increased, the cell viability decreased. For example, at 100 μg/mL concentration and 60 s irradiation, normal cells viability decreased to 49.35%, while the viability of cancer cells was 18%. Based on the results obtained from these concentration-response experiments, the LD50 (the dose of ZnPc that is required for 50% viability of the cells) values of ZnPc (25 mW, 60 s;
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3 J/cm2) for the cancer and normal cells were calculated as 20 and 100 μg/mL, respectively. This indicates that ZnPc is more than five times more toxic toward cancer cells than to the normal cell line. Fig. 3B shows that by raising the laser power of 50 mW, the results are similar to those of 25 mW laser. As a result, increasing the ZnPc concentration and the duration of the irradiation was led to reduction of cell viabilities. For example, at 100 μg/mL concentration of ZnPc under 60 s exposure, the viability of normal cells reduced to 38.01%, whereas in the case of cancer cell line, only 5.19% of the cells could survive. In addition, one can find that higher power of laser was able to induce the cytotoxicity at lower concentrations of ZnPc, in which the LD50 of ZnPc for cancer and normal cells (50 mW, 60 s; 6 J/cm2) were 10 and 27 μg/mL, respectively. Fig. 3C reveals that irradiation of laser for 60 s, at 100 μg/mL concentration of ZnPc, led to much more toxic effect on the cancer cells compared to the normal cells. Furthermore, the LD50 of cancer cells is 2 μg/mL and that of the normal cells is 15 μg/mL when the experiment is done by a CW laser (80 mW, 60 s; 9.6 J/cm2). Although it is expected that the efficiency of the photodynamic treatment depends on the ZnPc concentration and the light dosage, the marked difference between the behavior of the normal and cancer cells toward these parameters was interesting. Also, the viability of normal cells was more than cancer cells, demonstrating that the photodynamic treatment acts more efficiently on cancer cells than on normal cells. Subsequently, this information satisfies the desired expectations
Fig. 3. The cell viability of SW 872 cancer and HFFF2 normal cell lines after photosensitization with different concentrations of ZnPc (0 to 100 μg/mL) and photoirradiation with CW laser for 20, 40 and 60 s at (A) 25, (B) 50 and (C) 80 mW. Data points represent the mean ± SD, n = 6.
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Fig. 4. The effect of the fractionated laser exposure with (A) 30, (B) 70 and (C) 100 mW output power on cell viability of SW 872 cancerous and HFFF2 normal cell lines incubated with 0 to 100 μg/mL of ZnPc for 1000, 2000 and 3000 irradiation. Data points represent the mean ± SD, n = 6.
about PDT and its role in eliminating cancer cells rather than normal ones. There are also other previously reported articles that are consistent with the above results [28,29]. 4.3. Influence of the Fractionated Laser on Cells Viability The influence of 30 mW (1000 irradiation; 1 J/cm2, 2000 irradiation; 2 J/cm2, 3000 irradiation; 3 J/cm2), 70 mW (1000 irradiation; 2 J/cm2, 2000 irradiation; 4 J/cm2, 3000 irradiation; 6 J/cm2) and 100 mW (2000 irradiation; 6.4 J/cm2, 3000 irradiation; 9.6 J/cm2), fractionated laser on both cell types has been presented in Fig. 4. First, the modulated laser was adjusted to 30 mW and exposed (with 1000, 2000 and 3000 irradiation) to the cancer and normal cells incubated with different concentrations of ZnPc. Fig. 4A illustrates the collected data after MTT. According to the figure, 3000 irradiation were led to the highest number of death imposed to the cells, which had been incubated with 100 μg/mL ZnPc. Applied conditions (3000 irradiation, 30 mW and 100 μg/mL ZnPc) resulted in decreasing the viability of cancer and normal cells to 37.31% and 48.9%, respectively. Additionally, the LD50 of cancer and normal cells irradiated with fractionated laser (30 mW, 3000 irradiation; 3 J/cm2) were obtained 75 and N 100 μg/mL. By repeating the experiments for 70 and 100 mW modulated lasers, almost the same behavior was observed. The gathered data of these parts of the experiments are shown in Fig. 4B and 4C. By employing the 6 J/cm2 (70 mW, 3000 irradiation), the LD50 of cancer and normal cells were calculated 40 and 90 μg/mL, and the cell viabilities in the
presence of 100 μg/mL ZnPc, were decreased to 5.19% and 38.01%, respectively. The LD50 after exposure of laser (100 mW, 3000 irradiation; 9.6 J/cm2) on both cell types (cancer and normal) were also calculated as 30 and 50 μg/mL, respectively. In addition, the viability for cancer cells was measured 3.23% and that of normal cells 20.83% was measured by MTT assay. 4.4. Optimizing the Laser Exposure Time It is important to obtain an optimized condition in which the time of PDT experiment becomes minimum while achieving similar or better efficiency. As a result, cells are less exposed to laser and the probability Table 2 Obtained LD50 values during PDT for cancer and normal cells. Fluency (J/cm2)
1 2 3 4 6 6.4 9.6
Continuous wave laser
Fractionated laser
Cancer cells LD50 (μg/mL)
Normal cells LD50 (μg/mL)
Cancer cells LD50 (μg/mL)
Normal cells LD50 (μg/mL)
63 ± 7.8 35 ± 4.3 20 ± 5.1 15 ± 3.8 10 ± 4.2 5 ± 3.3 2 ± 1.4
N100 ± 10.7 N100 ± 12.1 100 ± 8.6 45 ± 6.0 27 ± 5.3 25 ± 5.6 15 ± 3.2
N100 ± 9.1 N100 ± 13.0 75 ± 9.7 75 ± 6.8 40 ± 3.2 50 ± 5.7 35 ± 6.4
N100 ± 7.8 N100 ± 11.3 N100 ± 9.4 100 ± 5.8 90 ± 7.2 75 ± 5.5 50 ± 6.0
LD50: the dose required for 50% cell viability.
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Table 3 The LD50 obtained values of PDT using CW laser in this work in comparison with those reported by others. Cell type
PDT drug
Light source
Fluency (J/cm2)
LD50 value (μg/mL)
Reference
NIH3T3 B16-F0 MCF7 G361 THP-1 HepG2 A549 HepG2 HT29 HFFF2 SW 872
Phthalocyanine ClAlPcS2 Phthalocyanine ClAlPcS2 Phthalocyanine ClAlPcS2 Phthalocyanine ClAlPcS2 ZnPc-TiO2 ZnPc-TiO2 Dendrimer phthalocyanine Silicon(IV) phthalocyanines Silicon(IV) phthalocyanines ZnPc ZnPc
675 nm semiconductor laser (CW) 675 nm semiconductor laser (CW) 675 nm semiconductor laser (CW) 675 nm semiconductor laser (CW) 670 nm non-ionic laser light (CW) 670 nm non-ionic laser light (CW) Halogen lamp 675 nm laser (CW) 675 nm laser (CW) 675 nm diode laser (CW) 675 nm diode laser (CW)
30 30 30 30 10 10 10.8 30 30 9.6 9.6
5.6 N10 1.4 1.6 2.0 5.5 8.0 1.32 0.91 15 2
[31] [31] [31] [31] [25] [25] [32] [30] [30] This work This work
NIH3T3: mouse normal fibroblasts, B16-F0: mouse cancer melanoma, MCF7: human breast adenocarcinoma, G361: human melanoma, THP-1: human macrophage, HepG2: human hepatocellular carcinoma, A549: human lung adenocarcinoma, HT29: human colon adenocarcinoma, HFFF2: human fibroblast skin normal cells, SW 872: human fibroblast skin cancer cells, ZnPc: zinc phthalocyanine.
of laser defects on cells will be decreased. So, at the constant ZnPc concentration, by increasing the output power of laser and reducing the exposure time, in comparison to lower output power and longer exposure time, we can achieve almost the same transferred energy. For example, for cancer cells, at 50 μg/mL ZnPc concentration and using the CW laser with 2 J/cm2 energy, 60 s irradiation of 25 mW laser led to 38.5% of cell viability while 20 s irradiation of 50 mW laser resulted in 39.53% cell viability. In addition, by employing the same conditions, the viability of normal cells were obtained 60.6% and 61.78%, respectively. The same effects stood for the fractionated laser. Therefore, it can be concluded that by using the shorter exposure time, not only approximately equivalent PDT efficiency can be achieved, but also in some cases it could become even better. 4.5. Comparison of the Generated Effects by CW and Fractionated Lasers on Cancer and Normal Cells As we said before, to compare the resulted data of CW and fractionated laser, we have calculated the fluency of emitted light (according to Eq. (1)). By comparing the results it can be seen that the CW laser is more effective. On the other hand, the death rate of cells resulted by CW laser is more than that of fractionated laser. The intervals between irradiation of fractionated laser which prevent the steady activation process of ZnPc is the reason of this behavior. For instance, irradiation of 9.6 J/cm2 energy using a CW laser has reduced the viability of cancer cells to 2.23%, while, the fractionated laser at the same transferred energy (9.6 J/cm2) has reduced it to 13.17%. The concluded fact stands for the normal cells, too. Particularly, when the CW laser is exposed to normal cells carrying 9.6 J/cm2 radiation energy, the cell viabilities reduced 20.83%. In the same conditions, fractionated laser reduced the viability to 36.23%. For further clarification, the obtained LD50 values for normal and cancer cells treated by PDT have been listed in Table 2. Also, evaluating the data represented in Table 2, demonstrated the superior impact of CW laser on cancer cells over the normal cells which is consistent with previous obtained information. In addition, in Table 3, the LD50 obtained in the present study using CW laser have been compared with those have been reported by others. According to this table, the efficiency of PDT in this study, considering the fluencies of the conducted experiments is better or comparable with the other ones. Particularly, in a study [30], the research group evaluated the PDT effects on HT29 and HepG2 cells. They calculated the LD50 for these cells as 0.91 and 1.32 μg/mL, respectively, with using Silicon(IV)-phthalocyanine as photosensitizer and a 675 nm CW laser as a light source. Comparison of our obtained LD50 value (for cancer cells = 2 μg/mL) and those reported in [30] indicates the efficiency of the present study, by have this in mind that the fluency of our works is less than one third. As another example [31], Kolarova et al. have investigated PDT using a 675 nm CW semiconductor laser irradiated on
4 types of cell lines (NIH3T3, B16-F0, MCF7 and G361) which were incubated with phthalocyanine ClAlPcS2. By comparing the results with those of ours and again considering the fluency of each study, it can be seen that not only LD50 of our represented is better, but also performance of our experimental study is higher. Due to the fact that during PDT of the cancer cells, normal tissues or cells are also either damaged or destroyed, the performance of a PDT is highly dependent on minimum death of normal cells alongside the maximum death of cancer ones. In other words, the effect of PDT on normal cells should be lesser than that on cancer cells. In the mentioned study, NIH3T3 is a normal fibroblast mouse cell line and the B16-F0 is a cancer melanoma mouse cell line. It is obvious that the normal cells have lower LD50 in comparison with cancer ones (5.6 vs. N10 μg/mL). However, the LD50s obtained in the same conditions for the cancer and normal cell lines were 2 and 15 μg/mL, respectively. In general, the ratio of LD50 values for the normal cells to the cancer cells with CW laser was much higher than that of the fractioned laser (Table 2) indicating the effectiveness alongside relatively more safety of the former laser mode compared to the later one. 5. Conclusion In this study, the effects of laser irradiation in various conditions including power, time of exposure and the irradiation type on the cancer and normal skin cells in the presence of different concentrations of ZnPc were investigated. The data obtained indicate that not only the performance of the CW laser is better than the fractionated laser, but also, at the same conditions, the death rate of SW 872 (cancer cells) is more than that of HFFF2 (normal cells) which meets the desired expectation to treat cancer tumors. This is also reflected in the ratio of LD50 values for the HFFF2 to the SW 872 with CW and fractionated lasers. In addition, the accumulated data also show that by irradiating more powerful bursts of laser in shorter time, the same quite results of cell death are obtained in comparison with exposing longer and low power bursts. Acknowledgment This project was financially supported by Iran University of Science and Technology (IUST) and Stem Cell Research Center (SCRC) at Tabriz University of Medical Sciences. References [1] A.M. Kramer, M. Yan, K.S. Peggs, J. Anderson, K. Gustafsson, Tumor-associated antigen presentation by γδ T-cells in cancer immunotherapy, Blood 124 (21) (2014) 1411. [2] N.S. Iyer, L.M. Balsamo, M.B. Bracken, N.S. Kadan-Lottick, Chemotherapy-only treatment effects on long-term neurocognitive functioning in childhood ALL survivors: a review and meta-analysis, Blood 126 (3) (2015) 346–353.
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