Chemico-Biological Interactions 222 (2014) 44–49
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Symmetrical diiodinated squaraine as an efficient photosensitizer for PDT applications: Evidence from photodynamic and toxicological aspects M.S. Soumya a, K.M. Shafeekh b, Suresh Das b,⇑, Annie Abraham a,⇑ a
Department of Biochemistry, University of Kerala, Kariavattom Campus, Trivandrum 695581, Kerala, India Photosciences and Photonics Section, Chemical Sciences and Technology Division, National Institute for Interdisciplinary Science and Technology (NIIST CSIR), Industrial Estate P.O., Trivandrum 695019, India b
a r t i c l e
i n f o
Article history: Received 15 November 2013 Received in revised form 22 May 2014 Accepted 18 August 2014 Available online 26 August 2014 Keywords: Photodynamic therapy Squaraine dye Ehrlich’s Ascites Carcinoma (EAC) cells Photosensitizer
a b s t r a c t Photodynamic therapy (PDT) is emerging as a promising non-invasive treatment for cancers. It involves three key components; a photosensitizer, light and tissue oxygen. Even though several photosensitizers have been investigated for their use in PDT, they have several disadvantages and hence the search for more effective sensitizers has become important in recent years. The dye selected in our study – symmetrical diiodinated benzothiazolium squaraine (SQDI) – is one of the newly developed photosensitizers. The study aimed to evaluate the in vitro cytotoxicity of the dye on Ehrlich’s Ascites Carcinoma (EAC) cells and to assess the in vivo toxicity on Swiss Albino mice. The EAC cells were maintained in the peritoneum of mice and used to study the dark toxicity and phototoxicity by Trypan blue dye exclusion method, estimation of Reactive Oxygen Species (ROS), caspase activity and levels of thiobarbituric acid reactive substances (TBARS). The in vitro studies revealed that the dye induces toxicity in the presence of light and mediates cell death. The in vivo part of the study, which dealt with the toxicity evaluation in the body of Swiss Albino mice, was done by analyzing the parameters like serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), lactate dehydrogenase (LDH), creatine kinase (CK) and alkaline phosphatase (ALP). No significant change was observed in the above mentioned parameters in the dye administered group when compared to control. Altogether, this experiment indicates that the SQDI selected for our study may be used as an efficient photosensitizer for PDT applications and does not elicit acute toxicity to normal tissues in the absence of light. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Photodynamic therapy (PDT) involves administration of a tumor localizing photosensitizing agent, followed by the activation of the agent by light of a specific wavelength. This therapy results in a sequence of photochemical and photobiological processes that cause irreversible photo damage to tumor tissue. Photodynamic therapy (PDT) has emerged as an alternative strategy for treating cancer. PDT consists of three main components: a photosensitizer, light, and oxygen. PDT takes advantage of an appropriate wavelength of light that excites a photosensitizer to its singlet excited state and a subsequent intersystem crossing to reach the triplet
⇑ Corresponding authors. Tel.: +91 471 2308078 (O), +91 9447246692 (mobile) (A. Abraham). Tel.: +91 04712515226 (O) (S. Das). E-mail addresses: [email protected] (S. Das), [email protected] (A. Abraham). http://dx.doi.org/10.1016/j.cbi.2014.08.006 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
energy state [1–3]. In the presence of molecular oxygen, energy is transferred to relax the excited state of the photosensitizer. This energy transfer in turn excites molecular oxygen to form excited, singlet state oxygen. Singlet oxygen induces cell death via damaging oxidation or redox-sensitive cellular signaling pathways, thus mediating the effects of PDT [1,4,5]. Intriguingly, PDT has also been shown to regulate processes beyond tumor cell death including tumor angiogenesis and modulation of the immune system [1,4,6,7]. Each photosensitizer is activated by light of a specific wavelength [8]. This wavelength determines how far the light can travel into the body [9,10]. An ideal photosensitizer should meet the following criteria that are clinically relevant: a commercially available pure chemical, possessing low dark toxicity but strong photocytotoxicity, good selectivity towards target cells, long-wavelength absorbing, rapid removal from the body and ease of administration through various routes. These criteria provide a general guideline for comparison. Although some photosensitizers satisfy all of or some of these
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criteria, there are currently only a few photosensitizers that have received official approval around the world. However, PDT with the currently FDA-approved photosensitizers is not without adverse effects. For example, PhotofrinÒ, the first systemic drug approved, is well known for causing an intense inflammatory and necrotic reaction at the treated site and prolonged widespread photosensitivity for up to several weeks post-PDT, thereby imposing severe limitations on the patient’s lifestyle [11,12]. Because of this and other drawbacks of PhotofrinÒ, many additional photosensitizers have been synthesized, and a few of them have developed into FDA-approved drugs or are under clinical trial. The aim of this study was to determine the photodynamic efficacy of one of the newly developed photosensitizer – Symmetrical diiodinated squaraine (SQDI) [13] – on Ehrlich’s Ascites Carcinoma (EAC) cells and also to assess the in vivo toxicity in the absence of light on Swiss Albino mice. The in vitro PDT efficacy was done on EAC cells developed in the peritoneum of mice. The in vivo toxicity evaluation was done after the administration of the dye through intraperitoneal cavity of mice and compared with control mice which were not administered with the dye. 2. Experimental 2.1. Chemicals All reagents used for the study were of analytical grade. All biochemicals used were obtained from M/s. Sigma, St. Louis, MO, USA. A 1000 W halogen lamp (Philips PF 811) was used as the light source for the PDT studies. 2.2. Synthesis of SQDI A mixture of N-sulfobutyl-6-iodo-2-methyl-1, 3 benzothiazole (200 mg, 0.48 mmol) and squaric acid (28 mg, 0.24 mmol) were refluxed for 6 h in 2:1 butanol:benzene mixture in presence of 1 ml of quinoline fitted with a Dean stark condenser for the azeotropic removal of water. The reaction mixture was cooled, concentrated to 2 ml, and precipitated by adding 20 times excess of hexane. It was then filtered and was purified by column chromatography using neutral alumina with 12:7:1 chloroform:methanol:triethylamine as the eluent to give SQDI as pure product. Yield: 80 mg (37%); m.p. = 208–210° C (decomp.); IR (in KBr): 2939.5, 2678.2, 1562.3, 1474.6, 1270.1, 1202.6, 1040.6, 963.4, 811.0, 518.8 cm1; 1H NMR (500 MHz, CD3OD): 8.06 (s, 2H); 7.73–7.75 (d, J = 8.5 Hz, 2H); 7.34–7.35 (d, J = 8.4 Hz, 2H); 6.61 (s, olefinic, 2H); 4.25 (t, 4H); 2.89–2.92 (t, 4H); 1.97 (m, 8H) ppm; 13 C NMR (125 MHz, CD3OD): 172.61, 160.60, 142.38, 140.96, 139.22, 137.54, 136.83, 133.34, 115.33, 95.87, 62.55, 53.77, 30.15, 23.73 ppm. MALDI-TOF: Calcd: 900.02; Found: 900.94. 2.3. Animal models The animal models for the in vivo study (Swiss Albino miceMale, weighing 20–25 g) were from the departmental animal house. The animals were housed in polypropylene cages in rooms maintained at 25 ± 1 °C. Drinking water was given ad libitum. Mice were fed with standard laboratory diet supplied by Lipton India Ltd. For maintaining the experimental animals, the Institutional Ethical guidelines were absolutely followed as per CPCSEA rules [Sanction No: IAEC- KU-4/2010-11-BC-AA (15)]. 2.4. Maintenance of Ehrlich’s Ascites Carcinoma (EAC) cells on Swiss Albino mice To maintain the cells, the Ehrlich’s Ascites Carcinoma (EAC) cells were injected into the intraperitoneal cavity of mice and
maintained up to 3 weeks in aseptic conditions in the departmental animal house. The matured cells were aspirated from the peritoneum to apply for in vitro studies. 2.5. In vitro cytotoxicity assessment on cancer cell lines The EAC cells were aspirated from peritoneal cavity of tumor bearing mice, washed three times with ice cold PBS and checked the viability using Trypan blue dye exclusion method [14] (Cell viability should be above 98%). Serial dilutions were made of 101, 102 and 103. Counted the cells in the 103 dilution on hemocytometer and adjusted the cell number to 1 106 cells/ml. The cells were incubated at 37 °C with different concentrations of SQDI dye with 1 106 cells for different time intervals. Another set of cells, after treatment with different concentrations of the dye were illuminated in ice for 10 min with a 1000 W halogen lamp kept at a distance of 33 cm. A control was set with the cells, which received the illumination alone (without treatment with the dye). 0.1 ml of Trypan blue was added and the number of dead cells was determined using a hemocytometer. The percentage of dead cells was calculated by the formula:
Percentage of dead cells ¼
Number of dead cells 100 Total number of cells
2.6. In vitro photodynamic therapy effects of SQDI on Ehrlich’s Ascites Carcinoma (EAC) cells From the in vitro cytotoxicity and PDT studies, it was found that the cytotoxicity was produced by the synergestic effect of the dye and light. Neither the dye alone nor light alone induced cytotoxicity on EAC cells nor therefore to study the mechanism of cell death, the following parameters were analyzed both in the presence and absence of light after treatment of the cells with the dye. A control was set with the EAC cells alone which neither treated with dye nor subjected to light treatment. The second group of cells received the illumination alone (without treatment with the dye). The third group of cells was incubated at 37 °C with SQDI (0.2 mg/ml) with 1 106 cells for 3 h. Another set of cells, after treatment with SQDI (0.2 mg/ml) were illuminated for 10 min with a 1000 W halogen lamp kept at a distance of 33 cm. The total light dose was not delivered at a single stretch. Instead the method of fractionated light delivery with short term intervals (dark periods) was used. This allows re-oxygenation of tumor tissue during the treatment and thus enhances efficacy of PDT by also preventing thermal injury. 2.6.1. Estimation of levels of ROS after PDT treatment The dichlorofluorescein (DCF) assay was used to measure the levels of ROS in cells according to the procedure as described previously [15]. Direct evidence of intracellular oxidation in cells using the oxidant-sensitive probe 20 ,70 - dichlorofluorescein diacetate (DCFDA) was used to measure of ROS, which is expressed as fluorescence intensity/mg protein. A 10% (w/v) cells was prepared in phosphate buffer and aliquots were taken from this for ROS determination and protein estimation. 150 ll DCFDA in ethanol was added and incubated at 37 °C for 30 min. Supernatant obtained after centrifugation at 10,000 rpm for 15 min was analyzed for fluorescence at an excitation wavelength of 502 nm and emission wavelength of 523 nm using a spectrofluorimeter (Luminescence Spectrometer-45, Perkin Elmer). 2.6.2. Measurement of caspase-3 activity Caspase-3 was estimated by Caspase-3 Assay Kit (Sigma Aldrich). The process of measurement was according to the instructions in the reagent kit.
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2.6.3. Estimation of levels of lipid peroxidation products-TBARS TBARS was estimated by thiobarbituric acid assay. Reagents (a) 0.9% NaCl (b) TCA (c) 0.1 N NaOH
S O I
S
O
N HO3S HO 3S
Procedure EAC cells were photodynamically treated with the photosensitizer. The cells were washed with phosphate-buffered saline solution (PBS), trypsinized and pelleted. The pellets (106 cells) were suspended in 2 ml of 0.9% NaCl and vortexed vigorously, and an aliquot was taken from each sample for a Lowry protein assay. The remaining samples were added with 230 ll of trichloroacetic acid-saturated solution, vortexed and centrifuged at 3000g for 10 min to precipitate the proteins. The supernatants (1.6 ml) were placed in glass test tubes, and 200 ll of 14.4 mg/ml 2-thiobarbituric acid and 0.1 N NaOH solution were added. The samples were incubated for 45 min at 75 °C. Standards were prepared from the hydrolysis of 1,1,2,2-ethoxypropane in a 40% trichloroacetic acid solution. After the samples were cooled, the absorbance of the samples was read at 535 nm by spectrophotometer. thiobarbituric acid reactive substances (TBARS) values were calculated as nmol/mg of total protein.
I
N
SQDI
Fig. 1. Structure of SQDI.
the light treatment, the percentage of cell death was found to be increasing in a concentration and dark incubation dependant manner.100% cytotoxicity was found for the sample incubated with SQDI at a concentration of 0.6 mg/ml for 1 h, whereas when the samples were dark incubated for 3 h, 100% cytotoxicity was found at very lower concentrations (0.2 mg/ml) (Fig. 2). Cytotoxicity was not observed in the samples which were subjected to either incubation with the photosensitizer alone or light treatment alone (data not shown). Cell death occurs only after combined action of light and photosensitizer. From this result, it can be stated that cytotoxicity is produced by the synergistic effect of both the photosensitizer and light. 3.3. In vitro photodynamic therapy effects of SQDI on Ehrlich’s Ascites Carcinoma EAC cells
2.8. In vivo studies of SQDI To determine whether there is any toxicity produced by SQDI in the body, the following studies were done. SQDI was administered through the peritoneum of mice at a concentration of 12.5 mg/kg body weight. The toxicity parameters SGPT, SGOT, LDH, CK and ALP were assayed at 4 h and 24 h after the administration of the dye (since from the biodistribution study it was found that the dye is retained at maximum concentration at 4 h in the tissue of mice and completely cleared out from the body by 24 h) [16]. The enzyme activity was assayed for both the groups and compared with control mice which were not subjected to dye treatment. Number of animals used per group is 7. 2.9. Statistical analysis The data were statistically analyzed using analysis of variance (ANOVA) and significant difference of means was determined using Duncan’s multiple range test [17] at the level of p < 0.05.
Cytotoxicity of different concentrations of SQDI with and without light treatment on EAC cells showed that maximum cell death was found at a concentration of 0.2 mg/ml. Hence, further PDT studies were carried out with this concentration (0.2 mg/ml). 3.3.1. Estimation of ROS levels ROS may act as signaling molecules for the initiation and execution of the apoptotic cell death program [18]. The mechanism of action of most photosensitizers revealed that, in the presence of molecular oxygen, the light irradiation of photosensitizer and energy transfer can lead to a series of photochemical reactions and thereby generation of various cytotoxic species (e.g. singlet oxygen and ROS) which consequently induce cellular damage by apoptosis/necrosis. In the present study the generation of ROS was evaluated both in the presence and absence of light after the treatment with SQDI and the result showed a significant increase in ROS when the EAC cells were treated with photosensitizers in 120
3. Results and discussion
The dye was synthesized as described in Section 2.2. The structure of the dye is shown in Fig. 1. 3.2. In vitro cytotoxicity assessment by Trypan blue dye exclusion method The in vitro cytotoxicity of SQDI at different concentrations was analyzed on Ehrlich’s Ascites Carcinoma (EAC) cells to evaluate the cytotoxicity in the absence and presence of light (phototoxicity). The cells were incubated with different concentration of the dye in phosphate buffered saline (PBS) for different time intervals. The in vitro photodynamic effect of SQDI was checked by exposing the EAC cells to visible light from a 1000 W halogen lamp, kept at a distance of 33 cm and provided illumination for 10 min corresponding to the light dose of 46 J/cm2. The percentage of cell death was then analyzed using trypan blue exclusion method respectively. After
Percentage of cytotoxicity
3.1. Synthesis of SQDI
100 80 60 40
1h 2h 3h
20 0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Concentration of the dye (mg/ml) Fig. 2. In vitro cytotoxicity assessment of SQDI on EAC cells. The in vitro cytotoxicity study was done by Trypan blue dye exclusion method. When the cells were subjected to light treatment with the different concentrations of the dye, there was a significant induction of cytotoxicity in a concentration dependent manner (after dark incubation of 1, 2 and 3 h). Neither the dye alone nor the light alone induced toxicity to EAC cells. Values are mean of seven estimations ± SD.
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the presence of light whereas no significant ROS level was observed in the dye alone treated group without light when compared to control (Fig. 3).
3.3.3. Estimation of levels of TBARS Free radicals cause lipid peroxidation which result in cellular damage. Thiobarbituric acid reactive substance assay is most commonly used for the quantification of the end products of lipid peroxidation, specifically malondialdehyde (MDA) [20]. Malondialdehyde is a reactive species that occurs naturally as a product of lipid peroxidation. Reactive Oxygen Species degrade polyunsaturated lipids, forming malondialdehyde. This compound is a reactive aldehyde and is one of the many reactive electrophile species that cause toxic stress in cells and form advanced glycation end products. The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism [21]. In the present study, the activity of malondialdehyde (TBARS) was assessed on EAC cells both in the absence and presence of light and it was found that, administration of SQDI induced significant change in the concentration of malondialdehyde (Fig. 5). 3.4. In vivo studies of SQDI
Fluorescence intensity/ mg protein
Since the results of the in vitro part of the study were encouraging, we decided to check whether the dye induces any toxicity to normal tissues in the absence of light. Before analyzing the 350 300 250 200 150 100 50 0 1
2
3
4
Groups Fig. 3. Estimation of ROS. Group I: control (EAC alone), Group II: EAC + light, Group III: EAC + SQDI, Group IV: EAC + SQDI + light. The result shows that when the cells were subjected to squaraine PDT, there was a significant increase in the level of ROS when compared to control. Samples in Group 2 and 3 did not show significant increase. Values are mean of 7 estimations per group ± SD.
Fig. 4. Activity of caspase. Group I: control (EAC alone), Group II: EAC + light, Group III: EAC + SQDI, Group IV: EAC + SQDI + light. The result shows that when the cells were subjected to squaraine PDT, there was a significant increase in the activity of caspase when compared to control. Samples in Group 2 and 3 did not show significant increase. Values are mean of 7 estimations per group ± SD.
0.03 0.025
nM/mg protein
3.3.2. Measurement of caspase-3 activity Caspases are a family of cysteine proteases that are activated during the execution phase of apoptotic process [19]. They are activated at an early stage of apoptosis and are responsible for triggering most of the changes observed during cell death. Caspases accomplish this feat by cleaving a selected group of essential proteins like protein kinases, cytoskeletal proteins etc. Caspase-3 is a key executioner of apoptosis whose activation is mediated by the initiator caspases such as caspase-9. It is responsible either partially or wholly for the proteolytic cleavage of many key proteins involved in the Apoptotic signaling pathways. Generation of ROS can trigger the intrinsic pathway of apoptosis. Induction of apoptosis in cancer cells is considered as an effective anticancer strategy, since it leads to marked damage to the highly proliferating cancer cells. In the present study, in presence of light, caspase-3 is found to be markedly increased in SQDI PDT treated groups whereas no significant change is observed in photosensitizer alone and light alone treated groups (Fig. 4). Thus it can be inferred that, in EAC cells, PDT mediated oxidative stress result in apoptosis, which was induced by combined action of SQDI and light.
0.02 0.015 0.01 0.005 0 1
2
3
4
Groups Fig. 5. Estimation of levels of TBARS. Group I: control (EAC alone), Group II: EAC + light, Group III: EAC + SQDI, Group IV: EAC + SQDI + light. The result shows that when the cells were subjected to squaraine PDT, there was a significant increase in the levels of TBARS when compared to control. Samples in Group 2 and 3 did not show significant increase. Values are mean of 7 estimations per group ± SD.
therapeutic efficacy of the compound as a photosensitizer, it should be confirmed that it does not elicit any toxic manifestations in the body, when not illuminated. Thus we have checked whether SQDI is an ideal photosensitizer in this aspect. Swiss Albino mice were taken as experimental models to study this. SQDI was administered through the intraperitoneal cavity of the mice and sacrificed at 4 and 24 h after the treatment with the dye (since from the biodistribution study it was found that SQDI is retained at maximum concentration at 4 h in the body of mice and completely cleared out from the body by 24 h). For the acute toxicity assessment, the activity of the enzymes SGOT, SGPT, CK, LDH and ALP were analyzed. Aspartate transaminase (AST) also called serum glutamic oxaloacetic transaminase (SGOT) or aspartate aminotransferase (ASAT/ AAT) is similar to alanine transaminase (ALT) in that it is another enzyme associated with liver parenchymal cells. It is raised in acute liver damage. It is also present in red blood cells and cardiac muscle, skeletal muscle, kidney and brain tissue, and elevated due to damage to those sources as well. AST (SGOT) is commonly measured clinically as a part of diagnostic liver function tests, to determine liver health. In the present study, the activity of SGPT was assessed in serum and it was found that the administration of SQDI induced no significant effect in the activity of SGOT in both 4 and 24 h when compared to the control (Fig. 6).
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Fig. 6. Activity of SGOT. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of GOT in the serum after the administration of SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
Serum glutamic pyruvic transaminase (SGPT) or alanine aminotransferase (ALAT) is found in serum and in various bodily tissues, but is most commonly associated with the liver. ALT is commonly used as a way of screening for liver problems. When elevated ALT levels are found in the blood, the possible underlying causes can be further narrowed down by measuring other enzymes. For example, elevated ALT levels due to liver-cell damage can be distinguished from biliary duct problems by measuring alkaline phosphatase. Several drugs elevate the ALT levels. In the present study, the activity of SGPT was assessed in serum and it was found that the administration of SQDI induced no significant effect in the activity of SGPT in both 4 and 24 h when compared to the control (Fig. 7). Lactate dehydrogenase is a cytoplasmic enzyme found in organs like heart, liver, kidney and skeletal muscle. Elevated LDH levels in serum are observed in hemolytic, cardiac, skeletal muscle and renal diseases. Other uses are assessment of tissue breakdown in general; this is possible when there are no other indicators of hemolysis. The activity of LDH was assessed in all the groups and it was found that the administration of SQDI induced no significant effect in the activity of the enzyme among the groups (Fig. 8). Creatine kinase (CK), also known as creatine phosphokinase (CPK) or phospho-creatine kinase, is an enzyme expressed by various tissues and cell types. Clinically, creatine kinase is assayed in blood tests as a marker of myocardial infarction (heart attack), rhabdomyolysis (severe muscle breakdown), muscular dystrophy, and in acute renal failure. Elevation of CK is an indication of damage to muscle. In the present study, the activity of CK was assessed in serum and it was found that the administration of SQDI induced no significant effect in the activity of the enzyme in SQDI treated groups when compared to the control (Fig. 9).
Fig. 8. Activity of LDH in serum. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of LDH in the serum after the administration of SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
350 Activity of CK (units/ mg protein)
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345 340 335 330 325 320 315 310 305 300 1
2 Groups
3
Fig. 9. Activity of CK in serum. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of CK in the serum after the administration of the SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
Fig. 10. Activity of ALP in serum. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of ALP in the serum after the administration of the SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
Fig. 7. Activity of SGPT. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of GPT in the serum after the administration of the SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. Alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney and bone. Higher levels are seen in polycythemia vera (PV), essential thrombocytosis (ET), primary myelofibrosis (PM), and the leukemoid reaction. Lower levels are found in chronic myelogenous leukemia (CML) and paroxysmal nocturnal hemoglobinuria (PNH). In the present study, the activity of ALP was assessed in serum and it was found that the administration of SQDI induced no significant effect in the activity of the enzyme when compared to the control group (Fig. 10).
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4. Conclusions In the present study, we have assessed the in vitro and in vivo cytotoxicity effect of SQDI, a newly developed PDT sensitizer, on EAC Cells and Swiss Albino mice respectively. The in vitro cytotoxicity studies revealed that cytotoxicity is produced by the synergistic effect of SQDI and light on EAC cells. Neither the SQDI alone nor the light alone was found to be toxic. The in vivo part of the study dealt with the acute toxicity assessment of the SQDI on Swiss Albino mice and the results shown that the SQDI leaves no acute toxicity to the tissues in the absence of light. Thus, from the present study, it can be concluded that the dye selected for our study, SQDI, may be used as a promising agent for the application in photodynamic therapy, without any significant toxicity to the normal tissues as evident from the absence of clinical signs of toxicity in the mice after the administration of dye. Conflict of Interest None declared. Transparency Document The Transparency document associated with this article can be found in the online version.
Acknowledgements We gratefully acknowledge the financial assistance received in the form of CSIR-JRF, Govt. of India to the first and second authors of the paper. References [1] A. Juarranz, P. Jaen, F. Sanz-Rodriguez, J. Cuevas, S. Gonzalez, Photodynamic therapy of cancer. Basic principles and applications, Clin. Transl. Oncol. 10 (2008) 148–154. [2] B. Ortel, C.R. Shea, P. Calzavara-Pinton, Molecular mechanisms of photodynamic therapy, Front. Biosci. 14 (2009) 4157–4172.
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Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Symmetrical diiodinated squaraine as an efficient photosensitizer for PDT applications: Evidence from photodynamic and toxicological aspects M.S. Soumya a, K.M. Shafeekh b, Suresh Das b,⇑, Annie Abraham a,⇑ a
Department of Biochemistry, University of Kerala, Kariavattom Campus, Trivandrum 695581, Kerala, India Photosciences and Photonics Section, Chemical Sciences and Technology Division, National Institute for Interdisciplinary Science and Technology (NIIST CSIR), Industrial Estate P.O., Trivandrum 695019, India b
a r t i c l e
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Article history: Received 15 November 2013 Received in revised form 22 May 2014 Accepted 18 August 2014 Available online 26 August 2014 Keywords: Photodynamic therapy Squaraine dye Ehrlich’s Ascites Carcinoma (EAC) cells Photosensitizer
a b s t r a c t Photodynamic therapy (PDT) is emerging as a promising non-invasive treatment for cancers. It involves three key components; a photosensitizer, light and tissue oxygen. Even though several photosensitizers have been investigated for their use in PDT, they have several disadvantages and hence the search for more effective sensitizers has become important in recent years. The dye selected in our study – symmetrical diiodinated benzothiazolium squaraine (SQDI) – is one of the newly developed photosensitizers. The study aimed to evaluate the in vitro cytotoxicity of the dye on Ehrlich’s Ascites Carcinoma (EAC) cells and to assess the in vivo toxicity on Swiss Albino mice. The EAC cells were maintained in the peritoneum of mice and used to study the dark toxicity and phototoxicity by Trypan blue dye exclusion method, estimation of Reactive Oxygen Species (ROS), caspase activity and levels of thiobarbituric acid reactive substances (TBARS). The in vitro studies revealed that the dye induces toxicity in the presence of light and mediates cell death. The in vivo part of the study, which dealt with the toxicity evaluation in the body of Swiss Albino mice, was done by analyzing the parameters like serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), lactate dehydrogenase (LDH), creatine kinase (CK) and alkaline phosphatase (ALP). No significant change was observed in the above mentioned parameters in the dye administered group when compared to control. Altogether, this experiment indicates that the SQDI selected for our study may be used as an efficient photosensitizer for PDT applications and does not elicit acute toxicity to normal tissues in the absence of light. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Photodynamic therapy (PDT) involves administration of a tumor localizing photosensitizing agent, followed by the activation of the agent by light of a specific wavelength. This therapy results in a sequence of photochemical and photobiological processes that cause irreversible photo damage to tumor tissue. Photodynamic therapy (PDT) has emerged as an alternative strategy for treating cancer. PDT consists of three main components: a photosensitizer, light, and oxygen. PDT takes advantage of an appropriate wavelength of light that excites a photosensitizer to its singlet excited state and a subsequent intersystem crossing to reach the triplet
⇑ Corresponding authors. Tel.: +91 471 2308078 (O), +91 9447246692 (mobile) (A. Abraham). Tel.: +91 04712515226 (O) (S. Das). E-mail addresses: [email protected] (S. Das), [email protected] (A. Abraham). http://dx.doi.org/10.1016/j.cbi.2014.08.006 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
energy state [1–3]. In the presence of molecular oxygen, energy is transferred to relax the excited state of the photosensitizer. This energy transfer in turn excites molecular oxygen to form excited, singlet state oxygen. Singlet oxygen induces cell death via damaging oxidation or redox-sensitive cellular signaling pathways, thus mediating the effects of PDT [1,4,5]. Intriguingly, PDT has also been shown to regulate processes beyond tumor cell death including tumor angiogenesis and modulation of the immune system [1,4,6,7]. Each photosensitizer is activated by light of a specific wavelength [8]. This wavelength determines how far the light can travel into the body [9,10]. An ideal photosensitizer should meet the following criteria that are clinically relevant: a commercially available pure chemical, possessing low dark toxicity but strong photocytotoxicity, good selectivity towards target cells, long-wavelength absorbing, rapid removal from the body and ease of administration through various routes. These criteria provide a general guideline for comparison. Although some photosensitizers satisfy all of or some of these
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criteria, there are currently only a few photosensitizers that have received official approval around the world. However, PDT with the currently FDA-approved photosensitizers is not without adverse effects. For example, PhotofrinÒ, the first systemic drug approved, is well known for causing an intense inflammatory and necrotic reaction at the treated site and prolonged widespread photosensitivity for up to several weeks post-PDT, thereby imposing severe limitations on the patient’s lifestyle [11,12]. Because of this and other drawbacks of PhotofrinÒ, many additional photosensitizers have been synthesized, and a few of them have developed into FDA-approved drugs or are under clinical trial. The aim of this study was to determine the photodynamic efficacy of one of the newly developed photosensitizer – Symmetrical diiodinated squaraine (SQDI) [13] – on Ehrlich’s Ascites Carcinoma (EAC) cells and also to assess the in vivo toxicity in the absence of light on Swiss Albino mice. The in vitro PDT efficacy was done on EAC cells developed in the peritoneum of mice. The in vivo toxicity evaluation was done after the administration of the dye through intraperitoneal cavity of mice and compared with control mice which were not administered with the dye. 2. Experimental 2.1. Chemicals All reagents used for the study were of analytical grade. All biochemicals used were obtained from M/s. Sigma, St. Louis, MO, USA. A 1000 W halogen lamp (Philips PF 811) was used as the light source for the PDT studies. 2.2. Synthesis of SQDI A mixture of N-sulfobutyl-6-iodo-2-methyl-1, 3 benzothiazole (200 mg, 0.48 mmol) and squaric acid (28 mg, 0.24 mmol) were refluxed for 6 h in 2:1 butanol:benzene mixture in presence of 1 ml of quinoline fitted with a Dean stark condenser for the azeotropic removal of water. The reaction mixture was cooled, concentrated to 2 ml, and precipitated by adding 20 times excess of hexane. It was then filtered and was purified by column chromatography using neutral alumina with 12:7:1 chloroform:methanol:triethylamine as the eluent to give SQDI as pure product. Yield: 80 mg (37%); m.p. = 208–210° C (decomp.); IR (in KBr): 2939.5, 2678.2, 1562.3, 1474.6, 1270.1, 1202.6, 1040.6, 963.4, 811.0, 518.8 cm1; 1H NMR (500 MHz, CD3OD): 8.06 (s, 2H); 7.73–7.75 (d, J = 8.5 Hz, 2H); 7.34–7.35 (d, J = 8.4 Hz, 2H); 6.61 (s, olefinic, 2H); 4.25 (t, 4H); 2.89–2.92 (t, 4H); 1.97 (m, 8H) ppm; 13 C NMR (125 MHz, CD3OD): 172.61, 160.60, 142.38, 140.96, 139.22, 137.54, 136.83, 133.34, 115.33, 95.87, 62.55, 53.77, 30.15, 23.73 ppm. MALDI-TOF: Calcd: 900.02; Found: 900.94. 2.3. Animal models The animal models for the in vivo study (Swiss Albino miceMale, weighing 20–25 g) were from the departmental animal house. The animals were housed in polypropylene cages in rooms maintained at 25 ± 1 °C. Drinking water was given ad libitum. Mice were fed with standard laboratory diet supplied by Lipton India Ltd. For maintaining the experimental animals, the Institutional Ethical guidelines were absolutely followed as per CPCSEA rules [Sanction No: IAEC- KU-4/2010-11-BC-AA (15)]. 2.4. Maintenance of Ehrlich’s Ascites Carcinoma (EAC) cells on Swiss Albino mice To maintain the cells, the Ehrlich’s Ascites Carcinoma (EAC) cells were injected into the intraperitoneal cavity of mice and
maintained up to 3 weeks in aseptic conditions in the departmental animal house. The matured cells were aspirated from the peritoneum to apply for in vitro studies. 2.5. In vitro cytotoxicity assessment on cancer cell lines The EAC cells were aspirated from peritoneal cavity of tumor bearing mice, washed three times with ice cold PBS and checked the viability using Trypan blue dye exclusion method [14] (Cell viability should be above 98%). Serial dilutions were made of 101, 102 and 103. Counted the cells in the 103 dilution on hemocytometer and adjusted the cell number to 1 106 cells/ml. The cells were incubated at 37 °C with different concentrations of SQDI dye with 1 106 cells for different time intervals. Another set of cells, after treatment with different concentrations of the dye were illuminated in ice for 10 min with a 1000 W halogen lamp kept at a distance of 33 cm. A control was set with the cells, which received the illumination alone (without treatment with the dye). 0.1 ml of Trypan blue was added and the number of dead cells was determined using a hemocytometer. The percentage of dead cells was calculated by the formula:
Percentage of dead cells ¼
Number of dead cells 100 Total number of cells
2.6. In vitro photodynamic therapy effects of SQDI on Ehrlich’s Ascites Carcinoma (EAC) cells From the in vitro cytotoxicity and PDT studies, it was found that the cytotoxicity was produced by the synergestic effect of the dye and light. Neither the dye alone nor light alone induced cytotoxicity on EAC cells nor therefore to study the mechanism of cell death, the following parameters were analyzed both in the presence and absence of light after treatment of the cells with the dye. A control was set with the EAC cells alone which neither treated with dye nor subjected to light treatment. The second group of cells received the illumination alone (without treatment with the dye). The third group of cells was incubated at 37 °C with SQDI (0.2 mg/ml) with 1 106 cells for 3 h. Another set of cells, after treatment with SQDI (0.2 mg/ml) were illuminated for 10 min with a 1000 W halogen lamp kept at a distance of 33 cm. The total light dose was not delivered at a single stretch. Instead the method of fractionated light delivery with short term intervals (dark periods) was used. This allows re-oxygenation of tumor tissue during the treatment and thus enhances efficacy of PDT by also preventing thermal injury. 2.6.1. Estimation of levels of ROS after PDT treatment The dichlorofluorescein (DCF) assay was used to measure the levels of ROS in cells according to the procedure as described previously [15]. Direct evidence of intracellular oxidation in cells using the oxidant-sensitive probe 20 ,70 - dichlorofluorescein diacetate (DCFDA) was used to measure of ROS, which is expressed as fluorescence intensity/mg protein. A 10% (w/v) cells was prepared in phosphate buffer and aliquots were taken from this for ROS determination and protein estimation. 150 ll DCFDA in ethanol was added and incubated at 37 °C for 30 min. Supernatant obtained after centrifugation at 10,000 rpm for 15 min was analyzed for fluorescence at an excitation wavelength of 502 nm and emission wavelength of 523 nm using a spectrofluorimeter (Luminescence Spectrometer-45, Perkin Elmer). 2.6.2. Measurement of caspase-3 activity Caspase-3 was estimated by Caspase-3 Assay Kit (Sigma Aldrich). The process of measurement was according to the instructions in the reagent kit.
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2.6.3. Estimation of levels of lipid peroxidation products-TBARS TBARS was estimated by thiobarbituric acid assay. Reagents (a) 0.9% NaCl (b) TCA (c) 0.1 N NaOH
S O I
S
O
N HO3S HO 3S
Procedure EAC cells were photodynamically treated with the photosensitizer. The cells were washed with phosphate-buffered saline solution (PBS), trypsinized and pelleted. The pellets (106 cells) were suspended in 2 ml of 0.9% NaCl and vortexed vigorously, and an aliquot was taken from each sample for a Lowry protein assay. The remaining samples were added with 230 ll of trichloroacetic acid-saturated solution, vortexed and centrifuged at 3000g for 10 min to precipitate the proteins. The supernatants (1.6 ml) were placed in glass test tubes, and 200 ll of 14.4 mg/ml 2-thiobarbituric acid and 0.1 N NaOH solution were added. The samples were incubated for 45 min at 75 °C. Standards were prepared from the hydrolysis of 1,1,2,2-ethoxypropane in a 40% trichloroacetic acid solution. After the samples were cooled, the absorbance of the samples was read at 535 nm by spectrophotometer. thiobarbituric acid reactive substances (TBARS) values were calculated as nmol/mg of total protein.
I
N
SQDI
Fig. 1. Structure of SQDI.
the light treatment, the percentage of cell death was found to be increasing in a concentration and dark incubation dependant manner.100% cytotoxicity was found for the sample incubated with SQDI at a concentration of 0.6 mg/ml for 1 h, whereas when the samples were dark incubated for 3 h, 100% cytotoxicity was found at very lower concentrations (0.2 mg/ml) (Fig. 2). Cytotoxicity was not observed in the samples which were subjected to either incubation with the photosensitizer alone or light treatment alone (data not shown). Cell death occurs only after combined action of light and photosensitizer. From this result, it can be stated that cytotoxicity is produced by the synergistic effect of both the photosensitizer and light. 3.3. In vitro photodynamic therapy effects of SQDI on Ehrlich’s Ascites Carcinoma EAC cells
2.8. In vivo studies of SQDI To determine whether there is any toxicity produced by SQDI in the body, the following studies were done. SQDI was administered through the peritoneum of mice at a concentration of 12.5 mg/kg body weight. The toxicity parameters SGPT, SGOT, LDH, CK and ALP were assayed at 4 h and 24 h after the administration of the dye (since from the biodistribution study it was found that the dye is retained at maximum concentration at 4 h in the tissue of mice and completely cleared out from the body by 24 h) [16]. The enzyme activity was assayed for both the groups and compared with control mice which were not subjected to dye treatment. Number of animals used per group is 7. 2.9. Statistical analysis The data were statistically analyzed using analysis of variance (ANOVA) and significant difference of means was determined using Duncan’s multiple range test [17] at the level of p < 0.05.
Cytotoxicity of different concentrations of SQDI with and without light treatment on EAC cells showed that maximum cell death was found at a concentration of 0.2 mg/ml. Hence, further PDT studies were carried out with this concentration (0.2 mg/ml). 3.3.1. Estimation of ROS levels ROS may act as signaling molecules for the initiation and execution of the apoptotic cell death program [18]. The mechanism of action of most photosensitizers revealed that, in the presence of molecular oxygen, the light irradiation of photosensitizer and energy transfer can lead to a series of photochemical reactions and thereby generation of various cytotoxic species (e.g. singlet oxygen and ROS) which consequently induce cellular damage by apoptosis/necrosis. In the present study the generation of ROS was evaluated both in the presence and absence of light after the treatment with SQDI and the result showed a significant increase in ROS when the EAC cells were treated with photosensitizers in 120
3. Results and discussion
The dye was synthesized as described in Section 2.2. The structure of the dye is shown in Fig. 1. 3.2. In vitro cytotoxicity assessment by Trypan blue dye exclusion method The in vitro cytotoxicity of SQDI at different concentrations was analyzed on Ehrlich’s Ascites Carcinoma (EAC) cells to evaluate the cytotoxicity in the absence and presence of light (phototoxicity). The cells were incubated with different concentration of the dye in phosphate buffered saline (PBS) for different time intervals. The in vitro photodynamic effect of SQDI was checked by exposing the EAC cells to visible light from a 1000 W halogen lamp, kept at a distance of 33 cm and provided illumination for 10 min corresponding to the light dose of 46 J/cm2. The percentage of cell death was then analyzed using trypan blue exclusion method respectively. After
Percentage of cytotoxicity
3.1. Synthesis of SQDI
100 80 60 40
1h 2h 3h
20 0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Concentration of the dye (mg/ml) Fig. 2. In vitro cytotoxicity assessment of SQDI on EAC cells. The in vitro cytotoxicity study was done by Trypan blue dye exclusion method. When the cells were subjected to light treatment with the different concentrations of the dye, there was a significant induction of cytotoxicity in a concentration dependent manner (after dark incubation of 1, 2 and 3 h). Neither the dye alone nor the light alone induced toxicity to EAC cells. Values are mean of seven estimations ± SD.
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the presence of light whereas no significant ROS level was observed in the dye alone treated group without light when compared to control (Fig. 3).
3.3.3. Estimation of levels of TBARS Free radicals cause lipid peroxidation which result in cellular damage. Thiobarbituric acid reactive substance assay is most commonly used for the quantification of the end products of lipid peroxidation, specifically malondialdehyde (MDA) [20]. Malondialdehyde is a reactive species that occurs naturally as a product of lipid peroxidation. Reactive Oxygen Species degrade polyunsaturated lipids, forming malondialdehyde. This compound is a reactive aldehyde and is one of the many reactive electrophile species that cause toxic stress in cells and form advanced glycation end products. The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism [21]. In the present study, the activity of malondialdehyde (TBARS) was assessed on EAC cells both in the absence and presence of light and it was found that, administration of SQDI induced significant change in the concentration of malondialdehyde (Fig. 5). 3.4. In vivo studies of SQDI
Fluorescence intensity/ mg protein
Since the results of the in vitro part of the study were encouraging, we decided to check whether the dye induces any toxicity to normal tissues in the absence of light. Before analyzing the 350 300 250 200 150 100 50 0 1
2
3
4
Groups Fig. 3. Estimation of ROS. Group I: control (EAC alone), Group II: EAC + light, Group III: EAC + SQDI, Group IV: EAC + SQDI + light. The result shows that when the cells were subjected to squaraine PDT, there was a significant increase in the level of ROS when compared to control. Samples in Group 2 and 3 did not show significant increase. Values are mean of 7 estimations per group ± SD.
Fig. 4. Activity of caspase. Group I: control (EAC alone), Group II: EAC + light, Group III: EAC + SQDI, Group IV: EAC + SQDI + light. The result shows that when the cells were subjected to squaraine PDT, there was a significant increase in the activity of caspase when compared to control. Samples in Group 2 and 3 did not show significant increase. Values are mean of 7 estimations per group ± SD.
0.03 0.025
nM/mg protein
3.3.2. Measurement of caspase-3 activity Caspases are a family of cysteine proteases that are activated during the execution phase of apoptotic process [19]. They are activated at an early stage of apoptosis and are responsible for triggering most of the changes observed during cell death. Caspases accomplish this feat by cleaving a selected group of essential proteins like protein kinases, cytoskeletal proteins etc. Caspase-3 is a key executioner of apoptosis whose activation is mediated by the initiator caspases such as caspase-9. It is responsible either partially or wholly for the proteolytic cleavage of many key proteins involved in the Apoptotic signaling pathways. Generation of ROS can trigger the intrinsic pathway of apoptosis. Induction of apoptosis in cancer cells is considered as an effective anticancer strategy, since it leads to marked damage to the highly proliferating cancer cells. In the present study, in presence of light, caspase-3 is found to be markedly increased in SQDI PDT treated groups whereas no significant change is observed in photosensitizer alone and light alone treated groups (Fig. 4). Thus it can be inferred that, in EAC cells, PDT mediated oxidative stress result in apoptosis, which was induced by combined action of SQDI and light.
0.02 0.015 0.01 0.005 0 1
2
3
4
Groups Fig. 5. Estimation of levels of TBARS. Group I: control (EAC alone), Group II: EAC + light, Group III: EAC + SQDI, Group IV: EAC + SQDI + light. The result shows that when the cells were subjected to squaraine PDT, there was a significant increase in the levels of TBARS when compared to control. Samples in Group 2 and 3 did not show significant increase. Values are mean of 7 estimations per group ± SD.
therapeutic efficacy of the compound as a photosensitizer, it should be confirmed that it does not elicit any toxic manifestations in the body, when not illuminated. Thus we have checked whether SQDI is an ideal photosensitizer in this aspect. Swiss Albino mice were taken as experimental models to study this. SQDI was administered through the intraperitoneal cavity of the mice and sacrificed at 4 and 24 h after the treatment with the dye (since from the biodistribution study it was found that SQDI is retained at maximum concentration at 4 h in the body of mice and completely cleared out from the body by 24 h). For the acute toxicity assessment, the activity of the enzymes SGOT, SGPT, CK, LDH and ALP were analyzed. Aspartate transaminase (AST) also called serum glutamic oxaloacetic transaminase (SGOT) or aspartate aminotransferase (ASAT/ AAT) is similar to alanine transaminase (ALT) in that it is another enzyme associated with liver parenchymal cells. It is raised in acute liver damage. It is also present in red blood cells and cardiac muscle, skeletal muscle, kidney and brain tissue, and elevated due to damage to those sources as well. AST (SGOT) is commonly measured clinically as a part of diagnostic liver function tests, to determine liver health. In the present study, the activity of SGPT was assessed in serum and it was found that the administration of SQDI induced no significant effect in the activity of SGOT in both 4 and 24 h when compared to the control (Fig. 6).
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Fig. 6. Activity of SGOT. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of GOT in the serum after the administration of SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
Serum glutamic pyruvic transaminase (SGPT) or alanine aminotransferase (ALAT) is found in serum and in various bodily tissues, but is most commonly associated with the liver. ALT is commonly used as a way of screening for liver problems. When elevated ALT levels are found in the blood, the possible underlying causes can be further narrowed down by measuring other enzymes. For example, elevated ALT levels due to liver-cell damage can be distinguished from biliary duct problems by measuring alkaline phosphatase. Several drugs elevate the ALT levels. In the present study, the activity of SGPT was assessed in serum and it was found that the administration of SQDI induced no significant effect in the activity of SGPT in both 4 and 24 h when compared to the control (Fig. 7). Lactate dehydrogenase is a cytoplasmic enzyme found in organs like heart, liver, kidney and skeletal muscle. Elevated LDH levels in serum are observed in hemolytic, cardiac, skeletal muscle and renal diseases. Other uses are assessment of tissue breakdown in general; this is possible when there are no other indicators of hemolysis. The activity of LDH was assessed in all the groups and it was found that the administration of SQDI induced no significant effect in the activity of the enzyme among the groups (Fig. 8). Creatine kinase (CK), also known as creatine phosphokinase (CPK) or phospho-creatine kinase, is an enzyme expressed by various tissues and cell types. Clinically, creatine kinase is assayed in blood tests as a marker of myocardial infarction (heart attack), rhabdomyolysis (severe muscle breakdown), muscular dystrophy, and in acute renal failure. Elevation of CK is an indication of damage to muscle. In the present study, the activity of CK was assessed in serum and it was found that the administration of SQDI induced no significant effect in the activity of the enzyme in SQDI treated groups when compared to the control (Fig. 9).
Fig. 8. Activity of LDH in serum. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of LDH in the serum after the administration of SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
350 Activity of CK (units/ mg protein)
48
345 340 335 330 325 320 315 310 305 300 1
2 Groups
3
Fig. 9. Activity of CK in serum. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of CK in the serum after the administration of the SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
Fig. 10. Activity of ALP in serum. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of ALP in the serum after the administration of the SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
Fig. 7. Activity of SGPT. Group 1: control, Group 2: mice sacrificed 4 h after dye administration, Group 3: mice sacrificed 24 h after dye administration. The results show that there is no significant change in the activity of GPT in the serum after the administration of the SQDI when compared to control. Values are mean of 7 estimations per group ± SD.
Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. Alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney and bone. Higher levels are seen in polycythemia vera (PV), essential thrombocytosis (ET), primary myelofibrosis (PM), and the leukemoid reaction. Lower levels are found in chronic myelogenous leukemia (CML) and paroxysmal nocturnal hemoglobinuria (PNH). In the present study, the activity of ALP was assessed in serum and it was found that the administration of SQDI induced no significant effect in the activity of the enzyme when compared to the control group (Fig. 10).
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4. Conclusions In the present study, we have assessed the in vitro and in vivo cytotoxicity effect of SQDI, a newly developed PDT sensitizer, on EAC Cells and Swiss Albino mice respectively. The in vitro cytotoxicity studies revealed that cytotoxicity is produced by the synergistic effect of SQDI and light on EAC cells. Neither the SQDI alone nor the light alone was found to be toxic. The in vivo part of the study dealt with the acute toxicity assessment of the SQDI on Swiss Albino mice and the results shown that the SQDI leaves no acute toxicity to the tissues in the absence of light. Thus, from the present study, it can be concluded that the dye selected for our study, SQDI, may be used as a promising agent for the application in photodynamic therapy, without any significant toxicity to the normal tissues as evident from the absence of clinical signs of toxicity in the mice after the administration of dye. Conflict of Interest None declared. Transparency Document The Transparency document associated with this article can be found in the online version.
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