Accepted Manuscript

Cervical cancer cells (HeLa) response to photodynamic therapyusing a zinc phthalocyanine photosensitizer

Natasha Hodgkinson, Cherie Ann Kruger, Mpho Mokwena, HeidiAbrahamse

PII: S1011-1344(17)30103-3DOI: doi:10.1016/j.jphotobiol.2017.10.004Reference: JPB 11007

To appear in: Journal of Photochemistry & Photobiology, B: Biology

Received date: 23 January 2017Revised date: 25 August 2017Accepted date: 2 October 2017

Please cite this article as: Natasha Hodgkinson, Cherie Ann Kruger, Mpho Mokwena,Heidi Abrahamse , Cervical cancer cells (HeLa) response to photodynamic therapy usinga zinc phthalocyanine photosensitizer. The address for the corresponding author wascaptured as affiliation for all authors. Please check if appropriate. Jpb(2017), doi:10.1016/j.jphotobiol.2017.10.004

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Cervical Cancer Cells (HeLa) Response to Photodynamic Therapy using a Zinc

Phthalocyanine Photosensitizer

Natasha Hodgkinson1*, Cherie Ann Kruger1, Mpho Mokwena1, Heidi Abrahamse1

1Laser Research Centre, Faculty of Health Sciences, University of Johannesburg,

Doornfontein, 2028, South Africa

All Correspondence and reprints should be addressed to:

*Dr. Natasha Hodgkinson PhD Biomedical Technology Laser Research Centre Faculty of Health Sciences University of Johannesburg P.O. Box 17011 Doornfontein 2028 South Africa Tel: +27 11 559-6926 Fax: +27 11 559-6558 Email: tashar@uj.ac.za Running title:

Cervical Cancer Cells Response to Photodynamic therapy

Declaration

This manuscript has not been published, nor has it been submitted elsewhere for

publication.

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Abstract

Cervical cancer is the most common gynecological malignancy worldwide, and the

leading cause of cancer related deaths among females. Conventional treatment for

early cervical cancer is radical hysterectomy. In locally advanced cancer the

treatment of choice is concurrent chemo radiation. Although such treatment methods

show promise, they do have adverse side effects. To minimize these effects, as well

as prevent cancer re-occurrence, new treatment methods are being investigated.

Photodynamic therapy (PDT) involves the selective uptake of a photosensitizer (PS)

by cancer cells, illumination with light of an appropriate wavelength that triggers a

photochemical reaction leading to the generation of reactive oxygen and subsequent

tumor regression. The effect of PDT on a cervical cancer cell line (HeLa) was

assessed by exposing cultured cells to a sulphonated zinc phthalocyanine PS

(ZnPcSmix) and irradiating the cells using a 673 nm diode laser. The effects were

measured using the Trypan blue viability assay, adenosine triphosphate assay (ATP)

luminescence assay for proliferation, Lactate Dehydrogenase (LDH) membrane

integrity cytotoxicity assay, and fluorescent microscopy to assess PS cellular

localization and nuclear damage. Fluorescent microscopy revealed localization of the

PS in the cytoplasm and perinuclear region of HeLa cells. PDT treated cellular

responses showed dose dependent structural changes, with decreased cell viability

and proliferation, as well as considerable membrane damage. Hoechst stained cells

also revealed DNA damage in PDT treated cells. The final findings from this study

suggest that ZnPcSmix is a promising PS for the PDT treatment of cervical cancer in

vitro, where a significant 85% cellular cytotoxicity with only 25% cellular viability was

noted in cells which received 1µM ZnPcSmix when an 8 J/cm2 fluence was applied.

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Keywords: Cervical cancer, photodynamic therapy, zinc phthalocyanine

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1 Introduction

Cervical cancer is one of most common causes of cancer related deaths in women

worldwide (Chen et al., 2015). It is the third most common cancer that develops in

women, and nearly half a million new cases are reported each year (Ordikhani et al.,

2016). Despite advances in current therapies for the treatment of cervical cancer,

roughly 35% of women diagnosed with cervical cancer have recurrent disease, in

both advanced and early stage patients, with 90% of these found within 3 years after

the initial treatment (Hou et al., 2015; Lopez et al., 2012). There are also adverse

side effects of current treatment modalities which could decrease patient quality of

life (Ordikhani et al., 2016).

Radical hysterectomy is the treatment is currently the treatment of choice in early

cervical cancer. Concurrent chemo radiation is the favored modality for the cure of

locally advanced cancer (Lee et al., 2016). Although these modalities have shown

promise, the side effects are vast. Radiation therapy has been shown to induce DNA

damage in cells, leading to the loss of cell recovery, arrest of cell cycle, and

consequently cell destruction (Lomax et al., 2013). Chemotherapy, a primary

treatment of metastatic cancer, and alternative treatment for recurrent cervical

cancer, also comes with toxicity and adverse side effects.

In search of alternative, methods of treatment to effectively treat cancer, reduce side

effects and prevent cancer recurrence and metastasis, alternative methods of

treatment are being investigated (Portilho et al., 2013). One such treatment modality

is photodynamic therapy (PDT). PDT is an emerging therapy that is non-invasive and

involves a photosensitizer (PS), a drug which is taken up readily by cancer cells, and

an external light source which activates the drug and can treat diseases (Huang et

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al., 2015). The three components that are involved in PDT are: light, a

photosensitizer (PS), and oxygen (Wan and Lin, 2014). After a PS drug has been

administered either topically or systematically, and absorbed by the tumour cells, the

PS is irradiated by a light of a particular wavelength (Portilho et al., 2013; de Paula et

al., 2015). The excited PS’s then generate singlet oxygen and other reactive oxygen

species (ROS), which can damage different biomolecules, including proteins, DNA

and lipids and so leads to tumor cell death (Fang et al., 2015; Calixto et al., 2016).

Currently PDT is being used to treat patients who want to preserve their fertility and

those who would rather avoid having surgery. Previous studies have utilized PS’s

Photofrin and 5-ALA in the treatment/prevention of cervical cancer. Although the use

of systemic Photofrin was effective, Photofrin caused skin photosensitivity.

Conversely, 5-ALA was used topically to treat cervical lesions which could lead to

cancer, as well as to eradicate Human Papilloma Virus (HPV) infection (Shishkova et

al., 2012). Phthalocyanines are common PS’s used in PDT due to their high tumor

uptake efficiencies, their high ROS production and strong absorption in the

wavelength range between 650 and 850 nm (Pereira et al., 2014). A second

generation PS, Zinc (II) phthalocyanine has absorption Q bands at longer

wavelengths (670 - 770 nm) that allows maximum penetration of the light into the

tissues (Ocakoglu et al., 2016). There is limited literature on the use of

phthalocyanines in PDT and the treatment of cervical cancer, therefore, this study

aimed to investigate the PDT effects of a sulphonated zinc phthalocyanine PS

(ZnPcSmix) on the survival of HeLa cells, in vitro, to determine its potential as a

cancer treatment option.

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2 Materials and Methods

2.1 Cell culture

Cervical cancer cells (HeLa, (ATCC® CCL2™) were grown in Eagles Minimum

Essential Medium (MEM) (Sigma Aldrich: M2279) medium supplemented with Foetal

Bovine Serum (FBS) 10% (Sigma Aldrich: F0804), penicillin and streptomycin (100

mg) (Sigma Aldrich: P4333-100ML), and Amphotericin B (100 mg) (Sigma Aldrich:

A2942-100ML). All cells were incubated at 37°C with 5% CO2 and 85% humidity.

2.2 Treatment with Photosensitizer

The ZnPcSmix PS used in this study is a mixed isomer of sulfonated phthalocyanines,

and was synthesized from (OH2) ZnPc and fuming sulfuric acid (30% SO3) at

Rhodes University in South Africa and donated by Prof Tebello Nyokong (Ogunsipe

and Nyokong, 2005). The photochemical and photophysical properties were

determined and the PS had a fluorescence quantum yield of 0.16 (Φ??); triplet

quantum yield of 0.53 (Φ??); singlet oxygen quantum yield of 0.45 (ΦΔ), and triplet

lifetime (????) of 2.95 μs (Ogunsipe and Nyokong, 2005). Stock solutions of 0.0005 M

ZnPcSmix re-suspended in Phosphate Buffered Saline (PBS: Sigma Aldrich P5493-

1L) has a peak absorbance of 680 nm. Three different concentrations of ZnPcSmix

(0.25; 0.5; and 1 µM) diluted in supplemented cell culture media was used to

determine which would be the most toxic and induce cell death when PDT was

applied.

2.3 Cell preparation and Photosensitizer doses

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Cells from culture were seeded into 3.3 cm2 culture plates at a density of 5 x 105

cells/cm2 and incubated for 4 hours in supplemented growth medium to allow for

attachment. After 4 hours the growth medium was removed from all culture plates

and replaced, however within PS experimental plates, supplemented culture media

with ZnPcSmix was added at varying concentrations (0.25; 0.5; and 1 µM) and both

control groups not treated with ZnPcSmix and experimental groups were incubated, in

the dark, for 24 hours at 37˚C with 5% CO2 and 85% humidity.

2.4 Subcellular Localization

Fluorescent staining was performed to determine subcellular localization and uptake

of the PS (ZnPcSmix). HeLa cells were grown on glass coverslips in 35 mm culture

dishes in the conditions previously described. An hour prior to observation, cells

were fixed with 200 µl of 3.5% (v/v) Paraformaldehyde (Sigma Aldrich P6148) in

DMEM, and permeabilized with 200 µl of 0.5% (v/v) TrixtonX-100 (Sigma–Aldrich

T9284) in distilled water, then washed three times with HBSS. Fifty microliters of 1

µg/ml 40-6-Diamidino-2-phenylindole (DAPI: Invitrogen, D1306) was used to

counter-stain the nuclei. After 5 min incubation, the samples were rinsed with HBSS

and the coverslips were inverted onto glass microscope slides onto which a 30 µl of

20% Fluoromount™ Aqueous Mounting Medium (Sigma Aldrich, F4680) in distilled

water had been added. Coverslip borders were sealed with nail polish and slides

were examined using the fluorescent settings of a Carl Zeiss Axio Z1 Observer. The

358Ex/461Em filter was used to detect blue DAPI counter stained nuclei, and the

589Ex/610Em filter was used to detect any Texas red auto fluorescent signal

produced from cells that had absorbed ZnPcSmix.

2.5 Laser Irradiation

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Semi-confluent monolayers of HeLa cells in supplemented medium were irradiated

from above with the lid off in the dark at room temperature with a 673 nm diode laser

(Oriel, USA). On average a power output of 96 mW was measured using the

Coherent Fieldmate detector and sensor, this value was used to calculate the

duration (time) of each exposure for the different irradiation fluences (2, 4 and 8

J/cm2) which were used. All the laser parameters are described in Table i. Non-

irradiated cells (0 J/cm2) were used as controls and were kept under the same

conditions. Both irradiated and non-irradiated samples were re-incubated at 37°C in

a humidified atmosphere of 5% CO2 for 24, 48 or 72 hrs.

Table i: Laser Parameters used for Irradiation using the 673 nm Diode Laser

Parameter Description/Value

Laser Type Semiconductor diode

Wavelength (nm) 673 nm

Wave Emission Continuous

Power Output (mW) 96 mW

Power Density (mW/cm2) 10.5 mW/cm2

Spot Size (cm2) 9.1 cm2

Fluence (J/cm2) 2; 4; 8

Irradiation times 3 mins; 6 mins; 13 mins

2.6 Cell Morphology

Changes in cellular morphology in control and experimental groups of cervical cells

were observed using an inverted light microscope (Wirsam, Olympus CKX41) 24

hours post-irradiation. Pictures were taken with the SC30 Olympus camera. Images

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of the treated cells were compared to those of the control cells and any

morphological changes were recorded.

2.7 Trypan Blue assay

The Trypan blue staining method was used to determine the percentage of viable

cells. The assay is used to identify dead cells. Cells that are viable have intact

membranes and can effectively exclude the dye, whereas dead cells, with damaged

membranes take up the stain. Equal volumes of 0.4% Trypan blue (Sigma Aldrich:

T8154-20ML) and cell suspension were mixed and loaded into a counting chamber,

where the number of viable and dead cells were counted using the CountessTM

Automated Cell Counter (Invitrogen, C10227). Percent viability was determined by

calculating the number of viable cells from the total number of cells counted.

2.8 Adenosine Triphosphate (ATP) assay

The CellTiter-Glo luminescent cell viability assay (AnaTech: Promega, PRG7571)

was used to determine the number of metabolically active cells post irradiation. The

assay employs the properties of a proprietary thermostable luciferase, which

generates a luminescent signal proportional to the amount of ATP released upon cell

lysis. According to the manufacturer’s protocol, 50 µl of reconstituted reagent was

added to an equal volume of cell suspension. The contents was added into a white

walled 96 well plate and mixed on a shaker for 2 min to induce cell lysis. The plate

was then incubated at room temperature for 10 min to stabilize the luminescent

signal. The amount of ATP was quantified, and luminescence was recorded using

the Perkin Elmer, VICTOR3™ Multilabel Counter (Model 1420).

2.9 Lactose Dehydrogenase (LDH) Assay

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Membrane damage post irradiation was evaluated using the Cyto-Tox96 X assay

(Anatech: Promega, PRG1780). The assay quantitatively measures lactate

dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis.

LDH catalyses the conversion of lactate to pyruvate via NAD+ reduction to NADH.

Diaphorase then uses NADH to reduce a tetrazolium salt (INT) to a red formazan

product which is measured at 490 nm. The level of formazan formation is directly

proportional to the amount of LDH released into the medium, which indicates

cytotoxicity. Fifty µl of reconstituted reagent and culture medium post irradiation was

transferred into a clear 96 well-plate, incubated for 30 min in dark and the

colorimetric complex was measured at 490 nm using Perkin Elmer, VICTOR3™

Multilabel Counter (Model 1420).

2.10 DNA Damage Induced by PDT

HeLa cells were grown on glass coverslips in 35 mm culture dishes and categorized

into two groups: [1] control of cells only and [2] cells + 1 µg/ml ZnPcSmix + 4 J/cm2.

Cells were incubated at 37°C with 5% CO2 and 80% humidity for 4 hours to allow for

cellular attachment. Both groups were incubated in the dark for an additional 20

hours. Cells were then washed (3x with HBSS) and fixed with 200 µl of 3.5% (v/v)

Paraformaldehyde (Sigma Aldrich P6148) in DMEM for 10 min and permeabilized for

8 min with 200 µl of 0.5% (v/v) TrixtonX-100 (Sigma Aldrich T9284) in distilled water.

The samples were rinsed with HBSS and 1 ml of culture media that contained 1 µl of

1ug/ml (w/v) Hoechst dye was added to both groups for 15 min. The coverslips were

then inverted onto glass microscope slides onto which a 30 µl of 20% Fluoromount™

Aqueous Mounting Medium (Sigma Aldrich, F4680) in distilled water had been

added. Coverslip borders were sealed with nail polish and slides were examined at

40X magnification using the Carl Zeiss Axio Z1 Observer. Hoechst 33258

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352Ex/461Em blue filter was used to track this fluorescent dye uptake in nucleic

acids and so quantitatively analyse DNA damage in cells which received PDT when

compared to control cells only.

2.11 Statistical Analysis

Experiments were repeated three times (n = 3). All assays were performed in

triplicate, and the average was used for statistical calculations. Results were

analysed using Sigma Plot Version 12, and the mean, standard deviation, and

standard error were calculated. The student t test and one way ANOVA were

performed to detect differences between control groups and experimental groups.

Statistical significances are indicated in the figures as p < 0.05 (*), p < 0.01, (**) and

p < 0.001 (***).

3 Results

Subcellular Localization

Fluorescent microscopy revealed significant uptake of the photosensitizer by the

HeLa cells, as seen in Figure i below. The PS (stained in red) appeared to

accumulate within the cytoplasm and perinuclear region of the cells (nuclei stained in

blue). A similar study by Avaștar et al., 2016 showed ZnPcSmix PS localized in

the cytoplasm of MCF-7 cells. Previous studies have also confirmed perinuclear

localization of PS’s in HeLa cells (Soriano et al., 2014). Additionally, studies by Ge et

al. (2013) have noted that the cytoplasmic localization of PSs in HeLa cells is usually

restricted to mitochondrial cells only.

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Figure i: Texas red auto fluorescent signal produced from HeLa cells which

absorbed ZnPcSmix is shown in red. HeLa cells nuclei were counter

stained with DAPI as shown in blue. The merged image showing

localisation of the PS in relation to the nucleus. Magnification 40X and

Scale bar: 20 µm.

Cell Morphology

HeLa cells were examined for morphological changes after treatment, as per the

relevant experimental groups shown in Figure ii. Untreated HeLa cells remained

intact and maintained normal cell morphology. Cells treated with either laser

irradiation or non-activated ZnPcSmix presented morphological changes of cellular

damage as the dosage of PS and fluence increased. The most significant

morphological changes were observed within cells which received either 1 µM alone

or 8 J/cm2 alone. The experimental groups that received PDT yielded the most

significant dose dependent morphological changes indicative of cellular damage, as

the cells began to lose their characteristic shape, became rounded up, detached and

appeared to be free-floating structures in the culture medium. Studies by Manoto et

al. (2015 and 2012), reported similar morphological changes in human lung (A549)

and colon cancer cells (DLD-1) when exposed to 680 nm PDT induced zinc

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sulfophthalocyanine photosensitization at a concentration of 10 µM, and a fluence of

5 J/cm2.

Figure ii: Human cervical cancer cells (HeLa), showing a reduction in cell

sustainability with a dose dependent trend. PDT groups at a higher

fluence and increased PS concentration showed a visible difference in

cellular morphology when compared to control or experimental cells

which only received PS, whereby cell rounding, shrinkage and

detachment from the flask were observed, indicative of cell death.

Magnification 40X.

Trypan Blue assay

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The viability assay (Trypan Blue) showed a significant dose dependent decrease in

cellular viability within experimental groups, which were exposed to increasing

concentrations of either ZnPcSmix only or laser light alone, when compared to control

cells (Figure iii). In the experimental groups which were exposed to PDT, the change

in percentage viability when compared to control cells already noted 50 % cell

damage when HeLa cells received 1 µM of ZnPcSmix at a fluence of 4 J/cm2. These

results became increasingly significant (P < 0.001) in cells that received 1 µM of

ZnPcSmix when the fluence was increased to 8 J/cm2, as only 25% of cells were

reported to be viable. A similar study by Chen et al. (2015) applied 1.5 µM of ZnPcS

to cervical cancer cells and noted a 40% decrease in cell viability, when using a

480nm light emitting diode.

Figure iii: Trypan blue cellular viability assay. The cell viability decreased in all

experimental groups, and showed a dose dependent trend. Both the

ZnPcSmix and laser irradiation groups alone had an effect on the cellular

viability. However, when combined in the PDT application of 2 J/cm2 slight

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proliferation of cells was reported, and as the fluence of laser light and PS

increased so the viability of cells decreased, when compared to control

cells.

Adenosine Triphosphate assay

Cellular proliferation, as shown in Figure iv, was measured using the ATP assay,

significantly decreased in a dose dependent manner in HeLa experimental groups

which were exposed to ZnPcSmix alone as its concentration increased. However, in

experimental groups which were exposed to laser irradiation alone no significant

decrease in cell proliferation was noted as the fluence increased. The experimental

PDT groups which were exposed to a concentration of 1 µM ZnPcSmix in combination

with either 4 J/cm2 or 8 J/cm2, the most significant decrease (P < 0.001) in cellular

proliferation was noted when compared to any of the other experimental or control

groups. Similar findings were noted in studies performed by Tynga et al. (2014 and

2015), within MCF-7 human breast cancer cells when PDT was applied at 680nm,

with a 0.5 µM ZnPcSmix concentration using a fluence of 10 J/cm2. The study

concluded that ZnPcSmix mediated PDT led to an apoptotic cell death pathway,

which initiate programmed cell death in cells and so hindered further cellular

proliferation.

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Figure iv: Adenosine triphosphate assay (ATP) for proliferation. There was a very

significant decrease in cellular proliferation in experimental groups which

received 1 µM ZnPcSmix alone, however laser irradiation alone reported

no effects. The PDT combination of PS and laser irradiation indicated a

dose dependent decrease in proliferation, with the most significant

decrease being noted at a PDT combination concentration of 1 µM

ZnPcSmix at 8 J/cm2.

Lactose Dehydrogenase assay

Cellular damage induced by PDT was measured by evaluating the level of LDH

released into the culture media post irradiation. After 24 hours incubation,

experimental groups that received PDT treatment showed an increase in cell

membrane damage, as noted by the significant increase in the level of LDH

detected, when compared to control cells. In the experimental groups which received

either ZnPcSmix or laser dose treatment alone, no significant cellular cytotoxicity was

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noted. Overall, the cytotoxic effects of ZnPcSmix mediated PDT were dependent on

the concentration of PS used, as well as the fluence that was applied, with the most

significant cytotoxicity of 90% (P < 0.0002) being noted within cells, which received a

concentration of 1 µM ZnPcSmix and were irradiated at a fluence of 8 J/cm2.

Similarly, within studies performed by Manoto et al. (2012), significant in vitro

phototoxicity was noted in human lung (A549) and colon cancer cells (DLD-1) when

exposed to 680nm PDT 10µM induced ZnPcSmix photosensitization at fluence of 5

J/cm2, when compared to inactivated ZnPcSmix control cells.

Figure v: Lactose dehydrogenase assay for cytotoxicity. The ZnPcSmix and laser

irradiation alone showed little cytotoxicity when compared to control cells.

The PDT combination experimental groups reported a dose dependent

increase in cytotoxicity, with the most significant cytotoxicity observed in

cells which received 1 µM PS at a fluence of 8 J/cm2.

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Fluorescent Imaging for DNA damage

The blue Hoechst stained nuclear damage of HeLa cells post incubation which

received 1 µM ZnPcSmix at a PDT fluence of 4 J/cm2, when compared to control cells

is shown in Figure vi. Control cells showed no morphological changes. In contrast,

HeLa cells which received ZnPcSmix PDT treatment presented a small nucleus with

highly condensed chromatin, suggesting DNA damage was present. Studies

performed by Soriano et al (2014) and Acedo et al. (2014) in HeLa cells described

cell shrinkage, chromatin condensation, and nuclear fragmentation, which are typical

apoptotic features of PS laser activated induced cell damage. Similar studies

performed by El-Hussein et al. (2012), noted marked PDT induced DNA damage at

wavelength of 636nm when 10 µM ZnPcSmix was applied to human lung (A549),

breast (MCF-7), and esophageal (SNO) cancer cells at a fluence of 10 J/cm2. This

study proved that cytotoxic singlet oxygen, which is produced by the activation of the

ZnPcSmix during PDT, directly damaged the DNA in cells by causing single and

double-strand breaks which lead to cell death.

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Figure vi: Hoechst DNA detected damage after PDT (blue stain). Untreated cells

showed no morphological changes in the nuclei, however cells which

received ZnPcSmix and laser irradiation, showed highly condensed

chromatin granules, indicative of cellular DNA damage.

Conclusion

In conclusion, the results presented in this paper exhibit the effective

phototherapeutic activities of ZnPcSmix to induce cell damage in cervical cancer cells,

in vitro. This is supported by findings such as obvious changes in cell morphology,

with significantly decreased cellular viability and proliferation, increased cytotoxicity,

and DNA fragmentation noted in experimental groups which received PDT treatment

in comparison to those that didn’t. The significant effects on cellular morphology,

viability, cytotoxicity and DNA damage were seen to increase and proliferation

decrease in dose dependent manner with the most significant effect being observed

at a concentration of 1 µM PS when a fluence of 8 J/cm2 was applied. Hence,

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ZnPcSmix is an effective photodynamic agent, in vitro, for the treatment of cervical

cancer cells. Studies reported by Tynga and Abrahamse (2015), have noted that

metallated phthalocyanine-mediated PDT has been shown to induce autophagy

mode of cell death in various types of cancer cells and this study is suggestive of

this. However, in terms of the future applications of this sulphonated metal

phthalocyanine, further investigation in terms of its in vitro and in vivo drug delivery

(Abrahamse and Hamblin, 2016) through structural modifications (Dabroski et al.

2016), such as the addition of biocompatible antibodies (Iqbal et al. 2016) and

biodegradable nanoparticles (Sundar et al. 2016) should be evaluated. Additionally,

further investigation will include the incorporation of a control cervical cell line for

comparative PS cellular uptake studies to determine the retention rate in normal

versus cancerous cells as well as elucidation of the cell death induction mechanism

which will require extensive comparative research with normal cervical cells. Only

once this is complete could this form of specifically targeted PS PDT induced cell

damage be entirely considered as a prospective means of managing cancer.

Acknowledgements

The authors would like to acknowledge the following institutions for their

contributions: The Laser Research Centre, the National Research foundation,

National Laser Centre, and the University of Johannesburg.

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Highlights

Photodynamic therapy a new less invasive treatment for cervical cancer.

Photosensitizers of choice inlude phthalocyanines due to high tumour uptake.

Zinc phthalocyanine is a promising new photosensitizer for cervical cancer

treatment.

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