- Email: [email protected]
Nuclear imaging potential and in vitro photodynamic activity of Boron subphthalocyanine on colon carcinoma cells
Journal Pre-proof Nuclear imaging potential and in vitro photodynamic activity of Boron subphthalocyanine on colon carcinoma cells Fatma Yurt, Tayfun Arslan, Zekeriya Biyiklioglu, Ayça Tunçel, Derya Ozel, Kasim Ocakoglu PII:
S1773-2247(19)31809-X
DOI:
https://doi.org/10.1016/j.jddst.2020.101567
Reference:
JDDST 101567
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 25 November 2019 Revised Date:
30 January 2020
Accepted Date: 2 February 2020
Please cite this article as: F. Yurt, T. Arslan, Z. Biyiklioglu, Ayç. Tunçel, D. Ozel, K. Ocakoglu, Nuclear imaging potential and in vitro photodynamic activity of Boron subphthalocyanine on colon carcinoma cells, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/ j.jddst.2020.101567. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Authors Statement Tayfun Arslan, Ayça Tunçel, Derya Ozel: Performed the experiments; Analyzed and interpreted the data. Tayfun Arslan, Ayça Tunçel, Derya Ozel: Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Fatma Yurt, Zekeriya Biyiklioglu, Kasim Ocakoglu: Contributed reagents, materials, analysis tools or data; Wrote the paper. *
Nuclear imaging potential and in vitro photodynamic activity of Boron substituted-phthalocyanine on colon carcinoma cells
Fatma Yurta*, Tayfun Arslanb, Zekeriya Biyikliogluc, Ayça Tunçela, Derya Ozela, Kasim Ocakoglud
Author address aInstitute of Nuclear Science, Department of Nuclear Applications, Ege University, Izmir, 35100, Turkey. b
Department of Chemistry, Faculty of Science, Giresun University, 28200, Giresun, Turkey.
c
Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, 61080, Turkey.
d
Department of Energy Systems Engineering, Faculty of Technology, Tarsus University, Mersin, 33400, Turkey.
Nuclear imaging potential and in vitro photodynamic activity of Boron subphthalocyanine on colon carcinoma cells
Fatma Yurta*, Tayfun Arslanb, Zekeriya Biyikliogluc, Ayça Tunçela, Derya Ozela, Kasim Ocakoglud
Author address aInstitute of Nuclear Science, Department of Nuclear Applications, Ege University, Izmir, 35100, Turkey. b
c
Department of Chemistry, Faculty of Science, Giresun University, 28200, Giresun, Turkey.
Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, 61080, Turkey.
d
Department of Energy Systems Engineering, Faculty of Technology, Tarsus University, Mersin, 33400, Turkey.
ABSTRACT: Photodynamic therapy (PDT) has been a promising clinical agent in various types of cancer in recent years. In this study, in vitro nuclear imaging and PDT potential of Es-SubPc (Boron subphthalocyanine) were evaluated in colon adenocarcinoma cell line (HT-29) and human healthy lung fibroblast cell line (WI-38). For this purpose, the Es-SubPc was labeled with
131
I using the iodogen method
under the optimum conditions resulting in labeled with high yield (98.9 ± 1.2 %). In addition, the uptake rate of
131
I-Es-SubPc was determined in HT-29 and WI-38 cell lines. In comparison to the healthy cell line, the
uptake of 131I-Es-SubPc was found to be 2-fold higher in the HT-29 cell line. For PDT studies, both cells were exposed to white light at 30-90 J/cm2 in the presence of Es-SubPc. The results showed that Es-SubPc was a good PDT agent likely to be used in HT-29 cell line. As a result, Es-SubPc can be promising nuclear imaging and PDT agent for colon carcinoma.
KEYWORDS: Boron subphthalocyanines, Photodynamic therapy, colon adenocarcinoma, 131I
*Correspondence to: Fatma YURT, E-mail addresses: [email protected]
1.
Introduction
Colorectal cancer (CRC) is the third most common cancer in men and the second for women around the world. In addition, this is the fourth most common cause of cancer death in the world, covering 8.5% of all cancer deaths envisaged each year [1,2]. Apart from early diagnosis, some patients carrying colorectal cancer have already metastasized at the time of diagnosis, while the rest have the potential to develop metastasis [3]. Because of this reason, to develop the new treatment options and prognosis for CRC patients have great importance with the development of new drugs [3–6]. However, due to the increased resistance of tumor cells to chemotherapeutics used in CRC treatment and these drugs leading to nonspecific toxicity in healthy tissues, it is necessary to find new methods for CRC therapy [5,7–11]. PDT is the formation of tissue and cellular damage by combined effects of the three components containing a photosensitizer (PS), light and oxygen. In a clinical setting, PDT usually involves a two-step procedure. First, PS is applied, then exposed to a certain wavelength which allows the PS to absorb a photon and cause an electronic transition from the ground state.[12] PSs act as catalysts by absorbing visible light and then converting molecular oxygen into a series of reactive oxygen species (ROS). ROS produced during PDT destroys tumors with multi-factor mechanisms.[13][14] PDT causes direct cell death by necrosis and/or apoptosis. PDT also has an effect on the vascular structure of the tumor, thereby causing illumination and ROS production to close the vessels and then depriving the tumor of oxygen and nutrients[15][16]. In addition, PDT has a significant effect on the immune system.[17] PDT has many advantages over conventional approaches due to its non-invasive nature, natural site selectivity, PS being only activated by light irradiation, activated systemic toxicity, ability to be used in repeated processes without resistance, and activated at the site of rapid recovery [18– 23]. In addition, repeated administration of PDT to the same individuals is possible in the treatment of tumors. In particular, the non-invasiveness of the method and the negligible side effects are of great importance in the treatment of a disease with high recurrence potential such as cancer [24,25].
A key component of PDT is a photosensitizer (PS) and the choice of the appropriate one changes the treatment efficacy depending on the type of cancer. Therefore, in studies comparing with different photodynamic effects for a particular cancer type, the importance of the selection of PSs has been frequently emphasized. Among these PSs such as porphyrins, chlorines, bacteriochlorines, and phthalocyanines photodynamic effects on the HT-29 cell line have been reported in the literature [21,26–32]. In photodynamic therapy, a promising cancer treatment method, phthalocyanines and their derivatives are widely used. Thus, the phthalocyanines provide a more effective PDT agent. Boron subphthalocyanines (SubPcs) are regarded as the lower homologs of phthalocyanines with coordination of the three constituent diimino-isoindoles to central tetrahedral boron. Unlike the related planar phthalocyanines, they have a cone-shaped structure. This feature, together with their delocalized 14-π-electron cores, leads to strong absorption and fluorescence emission in the orange-yellow visible region at around 550 to 600 nm and a broad peak typically below 400 nm [33,34]. Due to their versatile modifications at the axial and peripheral positions, SubPcs, which exclusive photophysical and electrochemical features have been shown to play important roles in various optoelectronics including organic field-effect transistors (OFETs), organic thin-film transistors, organic photovoltaics (OPVs), nonlinear optics [35,36] and also in PDT [37]. PDT treatment of fungal and bacterial infections has been widely utilized for years [38][39]. Over the last decades, PDT has increased care in clinical
applications for the treatment of tumors, such as skin, lung, head and neck, bone, esophagus, bladder, prostate, brain, cervix, and ovarian carcinomas [40][41][42].
We recently reported the synthesis of Es-SubPc and examine the potential effects of newly synthesized Es-SubPc against S. aureus and E. coli in the field of antimicrobial photodynamic therapy (aPDT) [43]. In this study, the potential of EsSubPc for nuclear imaging as in vitro, and ıts cytotoxic/phototoxic anticancer activities were investigated. Nuclear imaging or treatment potential of 131I labeled Es-SubPc molecule was examined at the human colon adenocarcinoma cell line (HT29) and healthy human lung cell line (WI38).
2.
Material and Methods
Es-SubPc was synthesized according to the reported literature [43]. The chemicals used for the in vitro studies were supplied from Biological Industries and all other chemicals were purchased from Merck. Na131I was supplied from the Nuclear medicine department of Ege University Hospital. Iodogen was obtained from Sigma-Aldrich. Thin-layer chromatography-cellulose gel (ITLC-F plastic sheets 20x20) was purchased from Merck to determine the radiolabeling yield. Radiochromatographs were analyzed using a Bioscan AR2000 TLC Scanner. A Thermo MSC Advantage 1.2 laminar airflow cabinet was used to performed cell culture studies. An Olympus Japan inverted light microscope was used for counting cells. IC50 values of cell culture were determined via the Thermo Multimode microplate reader. 2.1. Optimization Conditions of 131I-Es-SubPc The optimal conditions for pH, iodogen amount and reaction time parameters were tested for Es-SubPc. The pH values were adjusted to 3, 5, 7 and 9 to analyze the effect of reaction pH on the radiolabeling yield of Es-SubPc. Es-SubPc with different pH values were labeled with
131
I (5.5-7.4 MBq) and the quality control of the radiolabeling was determined with
the TLRC method. After the pH parameters optimization, the effects of the iodogen amount on radiolabeling yield were examined. Es-SubPc solution was adjusted at optimal pH and was added to 0.25, 0.5, and 1 mg iodogen containing tubes and labeled with 131I. The radiolabeling efficiency was determined according to the TLRC method. The effect of incubation time on radiolabeling yield was investigated at an optimal condition of pH and iodogen amount. Radiolabeled Es-SubPc was collected the 15th, 30th, 45th and 60th min at different incubation time and the quality control of the radiolabeling was defined according to the TLRC method. (n=3)
2.2. Radiolabeling Study of Es-SubPc Water-insoluble Es-SubPc was dissolved in dimethyl sulfoxide (DMSO) (1ml) at room temperature. Es-SubPc was radiolabeled with 5.5-7.4 MBq Iodine-131 (131I) using the iodogen method [44],[45],[46]. Es-SubPc (50 nmol) solution at optimum pH condition was added to tubes that contain 1 mg iodogen. After the mixture solution was incubated for 45 minutes at room temperature and incubation time, quality control of 131I labeled Es-SubPc (131I-Es-SubPc) was determined via Thin-Layer Radio Chromatography (TLRC). Na131I and radiolabeled Es-SubPc were spotted on TLRC sheets [cellulosecoated plastic (ITLC-cellulose 1×10 cm; Merck)] and the TLRC sheets were soaked in two solvent systems. The first solvent mixture was n-butanol-water-acetic acid (4:2:1) and the second solvent mixture was isopropanol-n-butanolammonium hydroxide (2:1:1)]. Then the TLRC sheets were removed from the mobile phases and left dried, later the TLRC
sheets were analyzed by TLC Scanner (BioScan AR-2000) to determine the radiolabeling yield and the Rf values of the radiolabeled Es-SubPc. All radiolabeling experiments were carried out at room temperature (22 ± 1 ° C). 2.3. In vitro Stability Studies of 131I-Es-SubPc The stability assays of
131
I -Es-SubPc were performed in phosphate buffer saline (pH=7.2) to decide shelf life. The
radiolabeled compound samples were taken and were dropped separately to the TLRC sheets for 30 min, 1h, 2h, 4h, and 24 h for determining radiolabeled yields of the compound in optimum conditions via TLRC method.
2.3.1.
Serum Stability
Human serum albumin (HSA) which is commercial availability and suitable for injection, was used in the serum stability study. 50 µL sample from the radiolabeled compounds in optimum conditions were added into the HSA solution diluted in 25% PBS (phosphate-buffered saline) at 37 °C. Serum stability assays were performed at the same conditions with in vitro stability assays. After the radiolabeling efficiency was calculated for different incubation time periods (30 min, 1h, 2h, 4h, and 24h at 37°C), (n=3). 2.4. Lipophilicity Test of 131I-Es-SubPc To decide the lipophilicity of 131I-Es-SubPc was calculated the n-octanol to water ratio. Radiolabeled 131I -Es-SubPc (200 µl) in optimum conditions were added in 3 ml n-octanol and 3 ml water in a tube. The mixture solution was stirred for 1 h and then centrifuged 5 min. at 2500 rpm. 500µl samples were taken from each phase in the tube to three eppendorf. The radioactivities of these samples were counted by Cd(Te)-RAD-501 single-channel analyzer and the ratios of n-octanol/water (log P value) were determined.
2.5. In vitro Studies In vitro studies were carried out by using HT29 (human colon adenocarcinoma cell line) and WI-38 (healthy human lung cell line) cell lines. HT29 and WI-38 cell lines were cultured in Eagle’s Minimum Essential Medium (EMEM) and Dulbecco’s Modified Eagle Medium (DMEM) which comprised 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, essential amino acids and 1% penicillin-streptomycin respectively. The cells were produced in culture flasks (75 cm2) and incubated at temperature 37 °C, 95% humidity and 5% CO2 until adequate proliferation was obtained in a CO2 cell culture incubator. The cytotoxicity of Es-SubPc on HT29 and WI-38 cell lines was determined with a colorimetric method using the 3(4,5-dimethylthiazol2-yl) -2,5-diphenyltetrazolium-bromide (MTT) kit. Moreover, for MTT assay, HT29 and WI-38 cells were seeded (10,000 cells/well) in 96 well cell culture plates. After cytotoxicity studies, photodynamic therapy potentials of Es-SubPc were evaluated. In addition to this, the intracellular uptake study of
131
I-Es-SubPc was carried out in HT29 and
WI-38 cell lines. Cells were seeded (50.000 cells/well) in 24 well cell culture plates. The uptake of the radiolabeled 131I-EsSubPc in these cells was calculated as a percentage.
2.5.1.
Cytotoxicity and in vitro Photodynamic Therapy Study of Es-SubPc
In our study, cell proliferation assay was performed to determine the number of cells/mL of HT29, and WI-38 cell lines for 24 h. The IC50 value, known as the concentration range causing 50% mortality, was obtained by using the MTT kit. The
serial concentrations of Es-SubPc of 3.13, 6.25, 12.50, 25.00 and 50.00 were treated to the cells to determine the IC50 value. This value was determined by calculating the percentage of cell viability according to the untreated control group. HT29 and WI-38 cells (10.000 cells in each well) were seeded to 96-well culture plates in EMEM and DMEM medium, respectively and they were incubated for 24 hours at 37 °C incubator. After the incubation period, the medium was discarded, and the wells were washed with PBS twice. Then, serum-free medium was added to the cells and the serial concentrations of Es-SubPc of 3.13, 6.25, 12.50, 25.00, 50.00 µM and incubated at 37 °C for 24 hours. At the end of the incubation period, the medium was discarded again and 100 µl of MTT solution was added to the medium then cells were incubated for 3 h in an incubator (37°C, 5% CO2). After 3 hours the MTT solution was aspirated, and 100 µl of DMSO was added to each well. Absorbance values of each well were measured at a microplate reader at 550 nm (EL800, Bio-Tek Instruments, Inc.). The cell viability was calculated as the ratio of the mean of absorbance values measured for each group to the control group viability without any compound. Each parameter of the assay was performed in triplicate.
2.5.2.
Intracellular Uptake of 131I-Es-SubPc
In vitro Photodynamic therapy (PDT) experiments were carried out after determining the IC50 value of Es-SubPc. PDT experiments were performed via a white LED light source (17.41 mW/cm2). The cells were seeded in a 96-well E-plate for different light doses at the indicated cell/ml density. The cells were incubated 24 h. After 24 h incubation, the medium is replaced with new mediums DMEM /MEM which containing at varying concentrations (3.13, 6.25,12.5, 25.00) Es-SubPc for HT29 and WI-38, respectively. The cells were exposed to white light at doses of 30, 60, and 90 J/cm2 and the untreated group were kept in the dark as the control. After photodynamic therapy, the cells were incubated at 37 °C in an incubator containing 5% CO2 and 95% humidity for 24 h. Lastly, cell viability was measured after the 24 h of PDT treatment. The intracellular uptake studies were performed to examine for the binding capacity of the 131I-Es-SubPc in HT29 tumor cell line and WI-38 healthy cell line. Uptake study was performed below the IC50 values that 25 µM concentration for 131IEs-SubPc. Furthermore, the intracellular uptake of Na131I was determined as the control in both cell lines. HT29, WI-38 cells were seeded in 24-well culture plates (50.000 cells in each well) and they were incubated throughout 48 h at 37 °C temperature in the incubator. After the incubation periods, the medium on the cells was removed and washed three times with PBS. The 131I-Es-SubPc (25 µM concentration) which was diluted with their medium without fetal bovine serum, were added to each well on the cells and radiolabeled with 131I which the activity of 0.37 MBq. The intracellular uptake of Na131I as the control group was performed with the same conditions to checked whether the uptake was caused by free iodine or radioiodinated Es-SubPc. The radioactivity of each well was counted via the Cd(Te)RAD 501 signal channel analyzer thus the first activity value A0 (cps) was obtained. At each count, the wells were counted 3 times for 10 sec via the Cd(Te) detector. After the incubation times for 1, 2, 4, and 24 h at 37 oC, the medium on the wells was removed and washed with PBS (n= 2). The wells were counted again with the detector, and the second count values A1 (cps) were determined. The first and second count values were analyzed, and the percentage of intracellular uptake was calculated using the following equation (n=3).
% intracellular uptake =
3.
Results and Discussion
A1 x100 A
3.1. Synthesis of Es-SubPc Es-SubPc was synthesized according to the reported literature [43] and used in this study (Fig. 1).
3.2. Optimum Conditions of Radiolabeling Efficiency During the radiolabeling of Es-SubPc with
131
I, experiments were performed with parameters such as ph, amount of
iodogen reagent and reaction time to determine optimum conditions. The radiolabeling efficiency of Es-SubPc was determined by the TLRC method and three replicate experiments were performed for each parameter. In order to determine the appropriate ph value for maximum radiolabeling efficiency with
131
I, Es-SubPc standard solution was prepared at
different ph values (3, 5, 7 and 9). When the results were evaluated, the maximum labeling efficiency of 131I-Es-SubPc was 98.9 ± 1.2 % at ph 9. Experiments were also conducted with iodogen tubes in prepared using different amounts of iodogen (0.25, 0.50 and 1.00 mg) to test the effect of oxidation of Na131I on the labeling yield. The results showed that radiolabeling efficacy was highest for Es-SubPc in a tube containing 1.00 mg iodogen. Finally, the effect of reaction time on radiolabeling efficiency was tested at various times (15, 30, 45 and 60 minutes) and the highest radiolabeling efficiency of Es-SubPc was determined to be 45 minutes.
3.3. Quality Control of Labeled Compounds by Thin Layer Radio Chromatography (TLRC) Radiochemical purity and radiolabeling efficiency of 131I-Es-SubPc were determined by the TLRC method. According to the method, Na131I,
131
I-Es-SubPc, and oxidized
131
I were added in dropwise to the TLRC sheets separately. Then, these
compounds on the sheets were moved in different mobile phases [mobile phase 1: n-butanol-water-acetic acid (4-2-1), mobile phase 2: isopropanol-n-butanol-ammonium hydroxide (2-1-1))]. Following this, the sheets were removed from the baths and were left drying later their radioactivity counted by TLRC Scanner. Rf (retention front) values and radiolabeling efficiencies were calculated from these chromatograms. According to the results, Na131I, oxidized 131I, and 131I-Es-SubPc Rf values in mobile phase-1 were determined as 0.36, 0.03, 0.90 respectively. In mobile phase-2, Na131I, oxidized 131I, and 131IEs-SubPc thus Rf values were found as 0.017, 0.39 and 0.88, respectively. When TLRC results were evaluated,
131
I-Es-
SubPc radiolabeling efficiency was calculated as 97.2 ± 1.7%. 3.4. In vitro and Serum Stability of 131I-Es-SubPc 131
I-Es-SubPc shelf life was determined via in vitro stability tests considering the optimum conditions. The results show
that the radiolabeling efficiency in 24 hours was determined quite high and was found as 92.5 ± 1.2 %. In addition, it is observed to be nearly constant for 24 hours. Also, serum stability experiments were performed under the same conditions applied in vitro stability assays and each test was repeated three times. In this experiment, 50 µL of the radiolabeled compound was left to incubate in serum solution prepared by dilution with 25% PBS at 37 °C. According to the results, the radiolabeling efficacy of 131I-Es-SubPc in the serum solution was 75.0 ± 2.5% within 24 hours. In addition, the radiolabeling yields of the compound were defined as stable for 24 hours. 3.5. Lipophilicity of 131I-Es-SubPc An amount of sample from labeled 131I-Es-SubPc was left to incubate in n-octanol / water solution. At the end of 1-hour incubation, the samples separated as lower and upper phases and the activities were counted with Cd (Te) detector as a
result of lipophilicity was determined. When results were evaluated, the lipophilicity of 131I-Es-SubPc was found to be 5.5 ± 1.4 and it was confirmed to be lipophilic.
3.6. In vitro Studies Cytotoxicity of Es-SubPc was determined by the MTT method and HT29 tumor cells and WI-38 healthy cell lines were used in all in vitro studies. The photodynamic therapy potential of Es-SubPc was further evaluated by selecting concentrations below IC50 values obtained by the completion of cytotoxicity studies. In addition, the results were supported by intracellular uptake studies of 131I-Es-SubPc performed in these cell lines.
3.6.1.
Cytotoxicity and Photodynamic Therapy Studies of Es-SubPc
In our previous study, the potential of antimicrobial photodynamic therapy of Es-SubPc was evaluated and found to be effective on Gram-positive and Gram-negative bacteria (Biyiklioğlu et al., 2019). In this study, the PDT activity of EsSubPc was evaluated on HT29 (human colon adenocarcinoma) and WI-38 (healthy) cell lines. First, both cell lines were plated in 96-well plates and kept at 37 °C for 24 hours. After a 24-hour incubation period, the water-insoluble Es-SubPc was dissolved in 1 mL of dimethyl sulfoxide (DMSO). Dilution was carried out considering that the DMSO content of the compound to be added to the cell medium and it was specified as 1 ‰. After dilution, various concentrations (3.13, 6.25, 12.5, 25 µM) of the compound were added to the medium via suspending cell culture media. In the HT29 and WI-38 cell lines, the IC50 values of Es-SubPc obtained by MTT experiments. These values were determined as 50 µM for both cell lines. After obtained IC50 values, cells were left in the dark for 24 hours incubation with these various concentrations. According to the results, the dark toxicity of DMSO was negligible and PBS was not exhibited dark toxicity in both cell lines. Besides, no significant changes were observed in both cell lines where the only light was applied. By performing PDT, plates with both cell lines were measured after 24 hours to determine cell viability. In the PDT experiment group, the cells had exposed the white LED light to various intensities (30, 60, 90 J/cm2) and various compound concentrations. As shown in Fig. 2, when PDT was applied, at the maximum concentration of Es-SubPc on HT29 cell, the cell viability was decreased under to 60% which means its slightly dose-dependent manner. The following illumination, this compound exhibited varying degrees of photocytotoxicity due to a slight dose increase. In fact, it has been found that all concentrations of this compound exhibit phototoxic effects on the HT29 cell line. The results of PDT studies show that while the compound causes significant phototoxicity in the HT29 cell line, it is found to be less effective in WI-38 cell line. (Fig. 2). In addition, when applied to the highest light dose and the highest concentration of Es-SubPc on HT29 cells, this caused reduced cell viability to less than 40%. If we evaluate both cell lines under the same conditions, the HT29 cell line is more sensitive to PDT than WI-38 cells. In other words, the highest phototoxic effect of Es-SubPc was found as 30% of cell viability in HT29 at maximum light power, while the cell viability was determined as 60% in the WI-38 cell line under the same conditions. These results have high similarities between our previous studies [44,47]. Those studies also showed similar PDT results of SubPc in the same cell lines and it was found highly phototoxic. At the same time, the best phototoxic effect of SubPc was found at the highest concentration (6.25 µM) and the highest light power (90 J/cm2) when the emergence of PDT, this data also supports our findings. In addition, it was emphasized that SubPc-TiO2 caused significant phototoxicity at quite high concentrations; 1.1 mg/ml, 2.2 mg/ml and 30 J/cm2, 60 J/cm2 light power was concluded on the WI-38 cell
line, whereas it was found to be less toxic compared to HT29 and HepG2 cell lines [44]. In concluding, in vitro, photodynamic therapy studies suggest that Es-SubPc is an appropriate agent for PDT in tumor treatment. Intracellular Uptake Efficiencies of 131I-Es-SubPc
3.6.2.
At this stage, the Cd (Te) detector system was used to determine the intracellular uptake potential of 131I-Es-SubPc. It was noted that all uptake experiments were performed at concentrations below the IC50 values
of Es-SubPc. The
131
intracellular uptake potential of Na I also was evaluated as a control in these cell lines. According to the results, the maximum uptake of 131I-Es-SubPc in the HT-29 cell line was determined as 12.1 ± 2.85% at 1 hour and this percentage was found to decrease to 4.88 ± 0.69% after 24 hours was completed (p<0.05) (Fig. 3). In addition, the maximum uptake of 131IEs-SubPc in the WI-38 cell line was determined as 5.99 ± 0.53% at 1 hour and it was found that this decrease of the cell viability continued over increasing time periods. This study has shown that the uptake value of
131
I-Es-SubPc in the HT29
131
cell line is about 20-fold higher than the uptake of Na I. Considering literature, it must be at least 2-fold higher target/nontarget ratio than a healthy cell line and our results support this finding [48]. Taking into account the 1 hour and 24 hours of uptake of
131
I-Es-SubPc in the WI-38 cell line, no difference was observed as the time increased. In the previous study, it
was observed that the highest uptake of 131I-SubPc in the HT29 cell line at 1 hour (4.0±0.7 %) and it was found to be the as stable end of the 24 hours [44]. These findings quite similar to our previous study also. This is seeming to indicate that this decreased uptake was due to differences in the cellular penetration and the aggregation of the Es-SubPc as in vitro. In the literature, it is suggested that intracellular uptake of the phthalocyanines depends on leading factors such as shape, charge, size, cell membrane differentiation between cancer and healthy cells [49]. Furthermore, new substituents can influence cell uptake or subcellular localization so that it interacts with cell membranes to facilitate the cellular uptake. Therefore, a balance of hydrophilic/ lipophilic characteristics is usually required for optimum photosensitizer uptake by cells. Several studies have shown that uptake of certain phthalocyanines demonstrated correlation with their lipophilicity parameter as in our studies [50–52][53]. In this respect, in our study, the uptake results of
131
I-Es-SubPc on the WI-38 cell line were
supported by the conclusion that the uptake detected in healthy cell lines is lower than HT29 cells and it could be a candidate as a nuclear imaging agent for HT29 tumor.
Conclusion Es-SubPc, previously synthesized by our group, was radiolabeled with studies have shown that
131
I in high efficiency. Radiolabeled in vitro
131
I-Es-SubPc accumulates 2 times more in the HT-29 cell line than in the WI-38 cell line. Results
It was concluded that Es-SubPc can be a nuclear imaging agent when labeled with an appropriate imaging radionuclide. In addition, HT-29 has been shown to be promising as a suitable agent for PDT. As a result, Es-SubPc is promising to become an effective agent for photodynamic therapy and nuclear imaging.
Statistical analyses Statistical analyses were performed by the GraphPad program using a t-test, and p˂0.5 values were determined as significant. Acknowledgment This study was not supported by any organization.
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FİGURES
Figure 1. The synthesis of Es-SubPc.
Figure 2. Photodynamic therapy efficiency of Es-SubPc in HT29 & WI-38 cell lines. PDT assays were performed with EsSubPc prepared at concentrations of 3.13 µM, 6.25 µM, 12.5 µM and 25 µM using 30, 60 and 90 J/cm2 light doses on both cell line (p<0.05).
Figure 3. Intracellular uptake of 131I-Es-SubPc in HT29 and WI-38 cell lines
•
The efficacy of Es-SubPc (Boron-substituted subphthalocyanines) in vitro nuclear imaging and Photodynamic therapy (PDT) was evaluated in HT-29 and WI-38 cell lines.
•
The uptake of 131I-Es-SubPc was found to be 2-fold higher in the HT-29 cell line compared to the WI-38 cell line.
•
The results showed that Es-SubPc can be promising agent for nuclear imaging and PDT for in colon cancer. .
No Conflict of Interest
S1773-2247(19)31809-X
DOI:
https://doi.org/10.1016/j.jddst.2020.101567
Reference:
JDDST 101567
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 25 November 2019 Revised Date:
30 January 2020
Accepted Date: 2 February 2020
Please cite this article as: F. Yurt, T. Arslan, Z. Biyiklioglu, Ayç. Tunçel, D. Ozel, K. Ocakoglu, Nuclear imaging potential and in vitro photodynamic activity of Boron subphthalocyanine on colon carcinoma cells, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/ j.jddst.2020.101567. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Authors Statement Tayfun Arslan, Ayça Tunçel, Derya Ozel: Performed the experiments; Analyzed and interpreted the data. Tayfun Arslan, Ayça Tunçel, Derya Ozel: Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Fatma Yurt, Zekeriya Biyiklioglu, Kasim Ocakoglu: Contributed reagents, materials, analysis tools or data; Wrote the paper. *
Nuclear imaging potential and in vitro photodynamic activity of Boron substituted-phthalocyanine on colon carcinoma cells
Fatma Yurta*, Tayfun Arslanb, Zekeriya Biyikliogluc, Ayça Tunçela, Derya Ozela, Kasim Ocakoglud
Author address aInstitute of Nuclear Science, Department of Nuclear Applications, Ege University, Izmir, 35100, Turkey. b
Department of Chemistry, Faculty of Science, Giresun University, 28200, Giresun, Turkey.
c
Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, 61080, Turkey.
d
Department of Energy Systems Engineering, Faculty of Technology, Tarsus University, Mersin, 33400, Turkey.
Nuclear imaging potential and in vitro photodynamic activity of Boron subphthalocyanine on colon carcinoma cells
Fatma Yurta*, Tayfun Arslanb, Zekeriya Biyikliogluc, Ayça Tunçela, Derya Ozela, Kasim Ocakoglud
Author address aInstitute of Nuclear Science, Department of Nuclear Applications, Ege University, Izmir, 35100, Turkey. b
c
Department of Chemistry, Faculty of Science, Giresun University, 28200, Giresun, Turkey.
Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, 61080, Turkey.
d
Department of Energy Systems Engineering, Faculty of Technology, Tarsus University, Mersin, 33400, Turkey.
ABSTRACT: Photodynamic therapy (PDT) has been a promising clinical agent in various types of cancer in recent years. In this study, in vitro nuclear imaging and PDT potential of Es-SubPc (Boron subphthalocyanine) were evaluated in colon adenocarcinoma cell line (HT-29) and human healthy lung fibroblast cell line (WI-38). For this purpose, the Es-SubPc was labeled with
131
I using the iodogen method
under the optimum conditions resulting in labeled with high yield (98.9 ± 1.2 %). In addition, the uptake rate of
131
I-Es-SubPc was determined in HT-29 and WI-38 cell lines. In comparison to the healthy cell line, the
uptake of 131I-Es-SubPc was found to be 2-fold higher in the HT-29 cell line. For PDT studies, both cells were exposed to white light at 30-90 J/cm2 in the presence of Es-SubPc. The results showed that Es-SubPc was a good PDT agent likely to be used in HT-29 cell line. As a result, Es-SubPc can be promising nuclear imaging and PDT agent for colon carcinoma.
KEYWORDS: Boron subphthalocyanines, Photodynamic therapy, colon adenocarcinoma, 131I
*Correspondence to: Fatma YURT, E-mail addresses: [email protected]
1.
Introduction
Colorectal cancer (CRC) is the third most common cancer in men and the second for women around the world. In addition, this is the fourth most common cause of cancer death in the world, covering 8.5% of all cancer deaths envisaged each year [1,2]. Apart from early diagnosis, some patients carrying colorectal cancer have already metastasized at the time of diagnosis, while the rest have the potential to develop metastasis [3]. Because of this reason, to develop the new treatment options and prognosis for CRC patients have great importance with the development of new drugs [3–6]. However, due to the increased resistance of tumor cells to chemotherapeutics used in CRC treatment and these drugs leading to nonspecific toxicity in healthy tissues, it is necessary to find new methods for CRC therapy [5,7–11]. PDT is the formation of tissue and cellular damage by combined effects of the three components containing a photosensitizer (PS), light and oxygen. In a clinical setting, PDT usually involves a two-step procedure. First, PS is applied, then exposed to a certain wavelength which allows the PS to absorb a photon and cause an electronic transition from the ground state.[12] PSs act as catalysts by absorbing visible light and then converting molecular oxygen into a series of reactive oxygen species (ROS). ROS produced during PDT destroys tumors with multi-factor mechanisms.[13][14] PDT causes direct cell death by necrosis and/or apoptosis. PDT also has an effect on the vascular structure of the tumor, thereby causing illumination and ROS production to close the vessels and then depriving the tumor of oxygen and nutrients[15][16]. In addition, PDT has a significant effect on the immune system.[17] PDT has many advantages over conventional approaches due to its non-invasive nature, natural site selectivity, PS being only activated by light irradiation, activated systemic toxicity, ability to be used in repeated processes without resistance, and activated at the site of rapid recovery [18– 23]. In addition, repeated administration of PDT to the same individuals is possible in the treatment of tumors. In particular, the non-invasiveness of the method and the negligible side effects are of great importance in the treatment of a disease with high recurrence potential such as cancer [24,25].
A key component of PDT is a photosensitizer (PS) and the choice of the appropriate one changes the treatment efficacy depending on the type of cancer. Therefore, in studies comparing with different photodynamic effects for a particular cancer type, the importance of the selection of PSs has been frequently emphasized. Among these PSs such as porphyrins, chlorines, bacteriochlorines, and phthalocyanines photodynamic effects on the HT-29 cell line have been reported in the literature [21,26–32]. In photodynamic therapy, a promising cancer treatment method, phthalocyanines and their derivatives are widely used. Thus, the phthalocyanines provide a more effective PDT agent. Boron subphthalocyanines (SubPcs) are regarded as the lower homologs of phthalocyanines with coordination of the three constituent diimino-isoindoles to central tetrahedral boron. Unlike the related planar phthalocyanines, they have a cone-shaped structure. This feature, together with their delocalized 14-π-electron cores, leads to strong absorption and fluorescence emission in the orange-yellow visible region at around 550 to 600 nm and a broad peak typically below 400 nm [33,34]. Due to their versatile modifications at the axial and peripheral positions, SubPcs, which exclusive photophysical and electrochemical features have been shown to play important roles in various optoelectronics including organic field-effect transistors (OFETs), organic thin-film transistors, organic photovoltaics (OPVs), nonlinear optics [35,36] and also in PDT [37]. PDT treatment of fungal and bacterial infections has been widely utilized for years [38][39]. Over the last decades, PDT has increased care in clinical
applications for the treatment of tumors, such as skin, lung, head and neck, bone, esophagus, bladder, prostate, brain, cervix, and ovarian carcinomas [40][41][42].
We recently reported the synthesis of Es-SubPc and examine the potential effects of newly synthesized Es-SubPc against S. aureus and E. coli in the field of antimicrobial photodynamic therapy (aPDT) [43]. In this study, the potential of EsSubPc for nuclear imaging as in vitro, and ıts cytotoxic/phototoxic anticancer activities were investigated. Nuclear imaging or treatment potential of 131I labeled Es-SubPc molecule was examined at the human colon adenocarcinoma cell line (HT29) and healthy human lung cell line (WI38).
2.
Material and Methods
Es-SubPc was synthesized according to the reported literature [43]. The chemicals used for the in vitro studies were supplied from Biological Industries and all other chemicals were purchased from Merck. Na131I was supplied from the Nuclear medicine department of Ege University Hospital. Iodogen was obtained from Sigma-Aldrich. Thin-layer chromatography-cellulose gel (ITLC-F plastic sheets 20x20) was purchased from Merck to determine the radiolabeling yield. Radiochromatographs were analyzed using a Bioscan AR2000 TLC Scanner. A Thermo MSC Advantage 1.2 laminar airflow cabinet was used to performed cell culture studies. An Olympus Japan inverted light microscope was used for counting cells. IC50 values of cell culture were determined via the Thermo Multimode microplate reader. 2.1. Optimization Conditions of 131I-Es-SubPc The optimal conditions for pH, iodogen amount and reaction time parameters were tested for Es-SubPc. The pH values were adjusted to 3, 5, 7 and 9 to analyze the effect of reaction pH on the radiolabeling yield of Es-SubPc. Es-SubPc with different pH values were labeled with
131
I (5.5-7.4 MBq) and the quality control of the radiolabeling was determined with
the TLRC method. After the pH parameters optimization, the effects of the iodogen amount on radiolabeling yield were examined. Es-SubPc solution was adjusted at optimal pH and was added to 0.25, 0.5, and 1 mg iodogen containing tubes and labeled with 131I. The radiolabeling efficiency was determined according to the TLRC method. The effect of incubation time on radiolabeling yield was investigated at an optimal condition of pH and iodogen amount. Radiolabeled Es-SubPc was collected the 15th, 30th, 45th and 60th min at different incubation time and the quality control of the radiolabeling was defined according to the TLRC method. (n=3)
2.2. Radiolabeling Study of Es-SubPc Water-insoluble Es-SubPc was dissolved in dimethyl sulfoxide (DMSO) (1ml) at room temperature. Es-SubPc was radiolabeled with 5.5-7.4 MBq Iodine-131 (131I) using the iodogen method [44],[45],[46]. Es-SubPc (50 nmol) solution at optimum pH condition was added to tubes that contain 1 mg iodogen. After the mixture solution was incubated for 45 minutes at room temperature and incubation time, quality control of 131I labeled Es-SubPc (131I-Es-SubPc) was determined via Thin-Layer Radio Chromatography (TLRC). Na131I and radiolabeled Es-SubPc were spotted on TLRC sheets [cellulosecoated plastic (ITLC-cellulose 1×10 cm; Merck)] and the TLRC sheets were soaked in two solvent systems. The first solvent mixture was n-butanol-water-acetic acid (4:2:1) and the second solvent mixture was isopropanol-n-butanolammonium hydroxide (2:1:1)]. Then the TLRC sheets were removed from the mobile phases and left dried, later the TLRC
sheets were analyzed by TLC Scanner (BioScan AR-2000) to determine the radiolabeling yield and the Rf values of the radiolabeled Es-SubPc. All radiolabeling experiments were carried out at room temperature (22 ± 1 ° C). 2.3. In vitro Stability Studies of 131I-Es-SubPc The stability assays of
131
I -Es-SubPc were performed in phosphate buffer saline (pH=7.2) to decide shelf life. The
radiolabeled compound samples were taken and were dropped separately to the TLRC sheets for 30 min, 1h, 2h, 4h, and 24 h for determining radiolabeled yields of the compound in optimum conditions via TLRC method.
2.3.1.
Serum Stability
Human serum albumin (HSA) which is commercial availability and suitable for injection, was used in the serum stability study. 50 µL sample from the radiolabeled compounds in optimum conditions were added into the HSA solution diluted in 25% PBS (phosphate-buffered saline) at 37 °C. Serum stability assays were performed at the same conditions with in vitro stability assays. After the radiolabeling efficiency was calculated for different incubation time periods (30 min, 1h, 2h, 4h, and 24h at 37°C), (n=3). 2.4. Lipophilicity Test of 131I-Es-SubPc To decide the lipophilicity of 131I-Es-SubPc was calculated the n-octanol to water ratio. Radiolabeled 131I -Es-SubPc (200 µl) in optimum conditions were added in 3 ml n-octanol and 3 ml water in a tube. The mixture solution was stirred for 1 h and then centrifuged 5 min. at 2500 rpm. 500µl samples were taken from each phase in the tube to three eppendorf. The radioactivities of these samples were counted by Cd(Te)-RAD-501 single-channel analyzer and the ratios of n-octanol/water (log P value) were determined.
2.5. In vitro Studies In vitro studies were carried out by using HT29 (human colon adenocarcinoma cell line) and WI-38 (healthy human lung cell line) cell lines. HT29 and WI-38 cell lines were cultured in Eagle’s Minimum Essential Medium (EMEM) and Dulbecco’s Modified Eagle Medium (DMEM) which comprised 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, essential amino acids and 1% penicillin-streptomycin respectively. The cells were produced in culture flasks (75 cm2) and incubated at temperature 37 °C, 95% humidity and 5% CO2 until adequate proliferation was obtained in a CO2 cell culture incubator. The cytotoxicity of Es-SubPc on HT29 and WI-38 cell lines was determined with a colorimetric method using the 3(4,5-dimethylthiazol2-yl) -2,5-diphenyltetrazolium-bromide (MTT) kit. Moreover, for MTT assay, HT29 and WI-38 cells were seeded (10,000 cells/well) in 96 well cell culture plates. After cytotoxicity studies, photodynamic therapy potentials of Es-SubPc were evaluated. In addition to this, the intracellular uptake study of
131
I-Es-SubPc was carried out in HT29 and
WI-38 cell lines. Cells were seeded (50.000 cells/well) in 24 well cell culture plates. The uptake of the radiolabeled 131I-EsSubPc in these cells was calculated as a percentage.
2.5.1.
Cytotoxicity and in vitro Photodynamic Therapy Study of Es-SubPc
In our study, cell proliferation assay was performed to determine the number of cells/mL of HT29, and WI-38 cell lines for 24 h. The IC50 value, known as the concentration range causing 50% mortality, was obtained by using the MTT kit. The
serial concentrations of Es-SubPc of 3.13, 6.25, 12.50, 25.00 and 50.00 were treated to the cells to determine the IC50 value. This value was determined by calculating the percentage of cell viability according to the untreated control group. HT29 and WI-38 cells (10.000 cells in each well) were seeded to 96-well culture plates in EMEM and DMEM medium, respectively and they were incubated for 24 hours at 37 °C incubator. After the incubation period, the medium was discarded, and the wells were washed with PBS twice. Then, serum-free medium was added to the cells and the serial concentrations of Es-SubPc of 3.13, 6.25, 12.50, 25.00, 50.00 µM and incubated at 37 °C for 24 hours. At the end of the incubation period, the medium was discarded again and 100 µl of MTT solution was added to the medium then cells were incubated for 3 h in an incubator (37°C, 5% CO2). After 3 hours the MTT solution was aspirated, and 100 µl of DMSO was added to each well. Absorbance values of each well were measured at a microplate reader at 550 nm (EL800, Bio-Tek Instruments, Inc.). The cell viability was calculated as the ratio of the mean of absorbance values measured for each group to the control group viability without any compound. Each parameter of the assay was performed in triplicate.
2.5.2.
Intracellular Uptake of 131I-Es-SubPc
In vitro Photodynamic therapy (PDT) experiments were carried out after determining the IC50 value of Es-SubPc. PDT experiments were performed via a white LED light source (17.41 mW/cm2). The cells were seeded in a 96-well E-plate for different light doses at the indicated cell/ml density. The cells were incubated 24 h. After 24 h incubation, the medium is replaced with new mediums DMEM /MEM which containing at varying concentrations (3.13, 6.25,12.5, 25.00) Es-SubPc for HT29 and WI-38, respectively. The cells were exposed to white light at doses of 30, 60, and 90 J/cm2 and the untreated group were kept in the dark as the control. After photodynamic therapy, the cells were incubated at 37 °C in an incubator containing 5% CO2 and 95% humidity for 24 h. Lastly, cell viability was measured after the 24 h of PDT treatment. The intracellular uptake studies were performed to examine for the binding capacity of the 131I-Es-SubPc in HT29 tumor cell line and WI-38 healthy cell line. Uptake study was performed below the IC50 values that 25 µM concentration for 131IEs-SubPc. Furthermore, the intracellular uptake of Na131I was determined as the control in both cell lines. HT29, WI-38 cells were seeded in 24-well culture plates (50.000 cells in each well) and they were incubated throughout 48 h at 37 °C temperature in the incubator. After the incubation periods, the medium on the cells was removed and washed three times with PBS. The 131I-Es-SubPc (25 µM concentration) which was diluted with their medium without fetal bovine serum, were added to each well on the cells and radiolabeled with 131I which the activity of 0.37 MBq. The intracellular uptake of Na131I as the control group was performed with the same conditions to checked whether the uptake was caused by free iodine or radioiodinated Es-SubPc. The radioactivity of each well was counted via the Cd(Te)RAD 501 signal channel analyzer thus the first activity value A0 (cps) was obtained. At each count, the wells were counted 3 times for 10 sec via the Cd(Te) detector. After the incubation times for 1, 2, 4, and 24 h at 37 oC, the medium on the wells was removed and washed with PBS (n= 2). The wells were counted again with the detector, and the second count values A1 (cps) were determined. The first and second count values were analyzed, and the percentage of intracellular uptake was calculated using the following equation (n=3).
% intracellular uptake =
3.
Results and Discussion
A1 x100 A
3.1. Synthesis of Es-SubPc Es-SubPc was synthesized according to the reported literature [43] and used in this study (Fig. 1).
3.2. Optimum Conditions of Radiolabeling Efficiency During the radiolabeling of Es-SubPc with
131
I, experiments were performed with parameters such as ph, amount of
iodogen reagent and reaction time to determine optimum conditions. The radiolabeling efficiency of Es-SubPc was determined by the TLRC method and three replicate experiments were performed for each parameter. In order to determine the appropriate ph value for maximum radiolabeling efficiency with
131
I, Es-SubPc standard solution was prepared at
different ph values (3, 5, 7 and 9). When the results were evaluated, the maximum labeling efficiency of 131I-Es-SubPc was 98.9 ± 1.2 % at ph 9. Experiments were also conducted with iodogen tubes in prepared using different amounts of iodogen (0.25, 0.50 and 1.00 mg) to test the effect of oxidation of Na131I on the labeling yield. The results showed that radiolabeling efficacy was highest for Es-SubPc in a tube containing 1.00 mg iodogen. Finally, the effect of reaction time on radiolabeling efficiency was tested at various times (15, 30, 45 and 60 minutes) and the highest radiolabeling efficiency of Es-SubPc was determined to be 45 minutes.
3.3. Quality Control of Labeled Compounds by Thin Layer Radio Chromatography (TLRC) Radiochemical purity and radiolabeling efficiency of 131I-Es-SubPc were determined by the TLRC method. According to the method, Na131I,
131
I-Es-SubPc, and oxidized
131
I were added in dropwise to the TLRC sheets separately. Then, these
compounds on the sheets were moved in different mobile phases [mobile phase 1: n-butanol-water-acetic acid (4-2-1), mobile phase 2: isopropanol-n-butanol-ammonium hydroxide (2-1-1))]. Following this, the sheets were removed from the baths and were left drying later their radioactivity counted by TLRC Scanner. Rf (retention front) values and radiolabeling efficiencies were calculated from these chromatograms. According to the results, Na131I, oxidized 131I, and 131I-Es-SubPc Rf values in mobile phase-1 were determined as 0.36, 0.03, 0.90 respectively. In mobile phase-2, Na131I, oxidized 131I, and 131IEs-SubPc thus Rf values were found as 0.017, 0.39 and 0.88, respectively. When TLRC results were evaluated,
131
I-Es-
SubPc radiolabeling efficiency was calculated as 97.2 ± 1.7%. 3.4. In vitro and Serum Stability of 131I-Es-SubPc 131
I-Es-SubPc shelf life was determined via in vitro stability tests considering the optimum conditions. The results show
that the radiolabeling efficiency in 24 hours was determined quite high and was found as 92.5 ± 1.2 %. In addition, it is observed to be nearly constant for 24 hours. Also, serum stability experiments were performed under the same conditions applied in vitro stability assays and each test was repeated three times. In this experiment, 50 µL of the radiolabeled compound was left to incubate in serum solution prepared by dilution with 25% PBS at 37 °C. According to the results, the radiolabeling efficacy of 131I-Es-SubPc in the serum solution was 75.0 ± 2.5% within 24 hours. In addition, the radiolabeling yields of the compound were defined as stable for 24 hours. 3.5. Lipophilicity of 131I-Es-SubPc An amount of sample from labeled 131I-Es-SubPc was left to incubate in n-octanol / water solution. At the end of 1-hour incubation, the samples separated as lower and upper phases and the activities were counted with Cd (Te) detector as a
result of lipophilicity was determined. When results were evaluated, the lipophilicity of 131I-Es-SubPc was found to be 5.5 ± 1.4 and it was confirmed to be lipophilic.
3.6. In vitro Studies Cytotoxicity of Es-SubPc was determined by the MTT method and HT29 tumor cells and WI-38 healthy cell lines were used in all in vitro studies. The photodynamic therapy potential of Es-SubPc was further evaluated by selecting concentrations below IC50 values obtained by the completion of cytotoxicity studies. In addition, the results were supported by intracellular uptake studies of 131I-Es-SubPc performed in these cell lines.
3.6.1.
Cytotoxicity and Photodynamic Therapy Studies of Es-SubPc
In our previous study, the potential of antimicrobial photodynamic therapy of Es-SubPc was evaluated and found to be effective on Gram-positive and Gram-negative bacteria (Biyiklioğlu et al., 2019). In this study, the PDT activity of EsSubPc was evaluated on HT29 (human colon adenocarcinoma) and WI-38 (healthy) cell lines. First, both cell lines were plated in 96-well plates and kept at 37 °C for 24 hours. After a 24-hour incubation period, the water-insoluble Es-SubPc was dissolved in 1 mL of dimethyl sulfoxide (DMSO). Dilution was carried out considering that the DMSO content of the compound to be added to the cell medium and it was specified as 1 ‰. After dilution, various concentrations (3.13, 6.25, 12.5, 25 µM) of the compound were added to the medium via suspending cell culture media. In the HT29 and WI-38 cell lines, the IC50 values of Es-SubPc obtained by MTT experiments. These values were determined as 50 µM for both cell lines. After obtained IC50 values, cells were left in the dark for 24 hours incubation with these various concentrations. According to the results, the dark toxicity of DMSO was negligible and PBS was not exhibited dark toxicity in both cell lines. Besides, no significant changes were observed in both cell lines where the only light was applied. By performing PDT, plates with both cell lines were measured after 24 hours to determine cell viability. In the PDT experiment group, the cells had exposed the white LED light to various intensities (30, 60, 90 J/cm2) and various compound concentrations. As shown in Fig. 2, when PDT was applied, at the maximum concentration of Es-SubPc on HT29 cell, the cell viability was decreased under to 60% which means its slightly dose-dependent manner. The following illumination, this compound exhibited varying degrees of photocytotoxicity due to a slight dose increase. In fact, it has been found that all concentrations of this compound exhibit phototoxic effects on the HT29 cell line. The results of PDT studies show that while the compound causes significant phototoxicity in the HT29 cell line, it is found to be less effective in WI-38 cell line. (Fig. 2). In addition, when applied to the highest light dose and the highest concentration of Es-SubPc on HT29 cells, this caused reduced cell viability to less than 40%. If we evaluate both cell lines under the same conditions, the HT29 cell line is more sensitive to PDT than WI-38 cells. In other words, the highest phototoxic effect of Es-SubPc was found as 30% of cell viability in HT29 at maximum light power, while the cell viability was determined as 60% in the WI-38 cell line under the same conditions. These results have high similarities between our previous studies [44,47]. Those studies also showed similar PDT results of SubPc in the same cell lines and it was found highly phototoxic. At the same time, the best phototoxic effect of SubPc was found at the highest concentration (6.25 µM) and the highest light power (90 J/cm2) when the emergence of PDT, this data also supports our findings. In addition, it was emphasized that SubPc-TiO2 caused significant phototoxicity at quite high concentrations; 1.1 mg/ml, 2.2 mg/ml and 30 J/cm2, 60 J/cm2 light power was concluded on the WI-38 cell
line, whereas it was found to be less toxic compared to HT29 and HepG2 cell lines [44]. In concluding, in vitro, photodynamic therapy studies suggest that Es-SubPc is an appropriate agent for PDT in tumor treatment. Intracellular Uptake Efficiencies of 131I-Es-SubPc
3.6.2.
At this stage, the Cd (Te) detector system was used to determine the intracellular uptake potential of 131I-Es-SubPc. It was noted that all uptake experiments were performed at concentrations below the IC50 values
of Es-SubPc. The
131
intracellular uptake potential of Na I also was evaluated as a control in these cell lines. According to the results, the maximum uptake of 131I-Es-SubPc in the HT-29 cell line was determined as 12.1 ± 2.85% at 1 hour and this percentage was found to decrease to 4.88 ± 0.69% after 24 hours was completed (p<0.05) (Fig. 3). In addition, the maximum uptake of 131IEs-SubPc in the WI-38 cell line was determined as 5.99 ± 0.53% at 1 hour and it was found that this decrease of the cell viability continued over increasing time periods. This study has shown that the uptake value of
131
I-Es-SubPc in the HT29
131
cell line is about 20-fold higher than the uptake of Na I. Considering literature, it must be at least 2-fold higher target/nontarget ratio than a healthy cell line and our results support this finding [48]. Taking into account the 1 hour and 24 hours of uptake of
131
I-Es-SubPc in the WI-38 cell line, no difference was observed as the time increased. In the previous study, it
was observed that the highest uptake of 131I-SubPc in the HT29 cell line at 1 hour (4.0±0.7 %) and it was found to be the as stable end of the 24 hours [44]. These findings quite similar to our previous study also. This is seeming to indicate that this decreased uptake was due to differences in the cellular penetration and the aggregation of the Es-SubPc as in vitro. In the literature, it is suggested that intracellular uptake of the phthalocyanines depends on leading factors such as shape, charge, size, cell membrane differentiation between cancer and healthy cells [49]. Furthermore, new substituents can influence cell uptake or subcellular localization so that it interacts with cell membranes to facilitate the cellular uptake. Therefore, a balance of hydrophilic/ lipophilic characteristics is usually required for optimum photosensitizer uptake by cells. Several studies have shown that uptake of certain phthalocyanines demonstrated correlation with their lipophilicity parameter as in our studies [50–52][53]. In this respect, in our study, the uptake results of
131
I-Es-SubPc on the WI-38 cell line were
supported by the conclusion that the uptake detected in healthy cell lines is lower than HT29 cells and it could be a candidate as a nuclear imaging agent for HT29 tumor.
Conclusion Es-SubPc, previously synthesized by our group, was radiolabeled with studies have shown that
131
I in high efficiency. Radiolabeled in vitro
131
I-Es-SubPc accumulates 2 times more in the HT-29 cell line than in the WI-38 cell line. Results
It was concluded that Es-SubPc can be a nuclear imaging agent when labeled with an appropriate imaging radionuclide. In addition, HT-29 has been shown to be promising as a suitable agent for PDT. As a result, Es-SubPc is promising to become an effective agent for photodynamic therapy and nuclear imaging.
Statistical analyses Statistical analyses were performed by the GraphPad program using a t-test, and p˂0.5 values were determined as significant. Acknowledgment This study was not supported by any organization.
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FİGURES
Figure 1. The synthesis of Es-SubPc.
Figure 2. Photodynamic therapy efficiency of Es-SubPc in HT29 & WI-38 cell lines. PDT assays were performed with EsSubPc prepared at concentrations of 3.13 µM, 6.25 µM, 12.5 µM and 25 µM using 30, 60 and 90 J/cm2 light doses on both cell line (p<0.05).
Figure 3. Intracellular uptake of 131I-Es-SubPc in HT29 and WI-38 cell lines
•
The efficacy of Es-SubPc (Boron-substituted subphthalocyanines) in vitro nuclear imaging and Photodynamic therapy (PDT) was evaluated in HT-29 and WI-38 cell lines.
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The uptake of 131I-Es-SubPc was found to be 2-fold higher in the HT-29 cell line compared to the WI-38 cell line.
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The results showed that Es-SubPc can be promising agent for nuclear imaging and PDT for in colon cancer. .
No Conflict of Interest