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DOI 10.24412/cl-37136-2023-1-118-128
ALUMINIUM PHTHALOCYANINE-GOLD NANOPARTICLE ENHANCE THE THERAPEUTIC EFFECT OF PDT IN OESOPHAGEAL CANCER.
ONYISI CHRISTIANA DIDAMSON1, RAHUL CHANDRAN1 AND HEIDI ABRAHAMSE 1
1 Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, South Africa
christydidamson@gmail.com
ABSTRACT
BACKGROUND
Gold nanoparticles mediated photodynamic therapy (PDT) has been reported to boost the efficiency and specificity of cancer treatment; however, their impact on oesophageal cancer is limited. In this study, we performed an in vitro assessment of aluminium phthalocyanine (AlPcS4Cl)-gold nanoparticle (AuNPs) mediated PDT targeting oesophageal cancer cells. The AlPcS4Cl-AuNPs conjugate was synthesized through a non-covalent method. The synthesized AlPcS4Cl-AuNPs were confirmed by ultraviolet-visible (UV-vis) absorption spectral analysis and high-resolution transmission electron microscopy. In vitro, effects of AlPcS4Cl-AuNPs based PDT in an oesophageal cancer cell line (HKESC-1) was examined. Cellular parameters which include cell viability, cellular proliferation, and cytotoxicity, were assessed by MTT assay, ATP cell proliferation assay, and lactate dehydrogenase (LDH) assay respectively. Fluorescent microscopy was used to determine the internalisation of the conjugates in cellular organelles. Furthermore, Mitochondrial membrane potential (MMP) integrity and reactive oxygen species (ROS) generation indications of cell death were also examined. The findings showed that PDT with aluminium (III) phthalocyanine chloride tetra sulfonic acid (AlPcS4Cl) conjugated gold nanoparticle (AuNPs)(AlPcS4Cl-AuNPs) significantly inhibit cell viability/cellular proliferation, increase cytotoxicity, and ROS generation. Fluorescent microscopy revealed that AlPcS4Cl-AuNPs was localised in the mitochondria and Endoplasmic reticulum (ER), suggesting that the induction of biochemical cell death pathways could be mitochondria and ER-dependent. More importantly, AlPcS4Cl-AuNPs significantly altered the mitochondrial membrane integrity in HKESC-1 cells. This further means the mitochondria-dependent cell death pathway may be involved. In conclusion, our findings demonstrated that AlPcS4Cl-AuNPs conjugates improved the anti-cancer effects of PDT in oesophageal cancer cells, proposing a better measure to boost the therapeutic efficiency of PDT in oesophageal cancer.
INTRODUCTION
Oesophageal cancer is a lethal digestive tract tumour, representing the eighth cancer-associated illness and the sixth cancer-associated death worldwide [1]. Risk factors associated with the emergence of oesophageal cancer include tobacco intake, alcohol ingestion, poor oral health, ingestion of carcinogenic agents, gastroesophageal reflux disease (GORD), obesity, and genetic and epigenetic changes [2]. The conventional therapy for oesophageal cancer consists of surgery, chemotherapy, and radiotherapy or combinations of these therapies. These treatments have tremendously improved the treatment of oesophageal. However, conventional treatments are faced with unfavourable side effects, ineffective treatment outcomes, treatment resistance, tumour relapse, low survival rate [3]. Hence, the search for an effective treatment measure, efficient at eliminating tumour cells without relapse, improving prognosis and without adverse effects, is critical. Photodynamic therapy (PDT), an efficient, non-invasive modality, has emerged for the treatment of cancer and other neoplastic disorders. Concerning conventional treatments, PDT has minimal side effects, providing safe and efficient measures that preferentially eradicate cancer cells from non-cancerous cells. Photodynamic therapy consists of three vital parameters, a photoactive agent called a photosensitiser (PS), molecular oxygen and light of the correct wavelength. These three parameters work together to eliminate tumour cells [3, 4]. Various PSs have been employed for PDT of different tumours, including Aluminium (III) Phthalocyanine Chloride Tetra Sulfonic Acid (AlPcS4Cl). The PS AlPcS4Cl has attracted much attention due to its high tissue penetration and increased reactive oxygen species (ROS) production. Studies have investigated the effects of AlPcS4Cl-
mediated PDT in different cancers [5-7], including oesophageal cancer [8]. Nevertheless, cellular internalisation is slow due to the repulsion that is created between the negative charges of the sulfonated group on the PS and the cell membrane. Hence an efficient delivery of AlPcS4Cl into tumour cells is needed.
With the emergence of nanotechnology, the application of nanoparticles as carriers to deliver drugs has been investigated. Several nanoparticles have been used as carriers for drug delivery in PDT. Gold nanoparticle (AuNPs) has gained much interest among several nanoparticles as a nanocarrier due to their chemical uncreativeness, efficient optical features and biocompatibility [4]. The effects of AuNPs mediated PDT have been reported on different cancer types [9-12]; however, its impact on oesophageal cancer is limited. Therefore, in this study, we evaluated the effects of AlPcS4Cl-AuNPs conjugates in enhancing the therapeutic efficiency of PDT in oesophageal cancer using in vitro assays.
MATERIALS AND METHODS
Reagent Preparation and Characterisation of AlPcS4Cl-AuNPs
The AlPcS4Cl (Frontier Scientific, AlPcS-834) with a molecular weight of 895.21 g/mol was reconstituted to a concentration of 1.0 mM, covered with aluminium foil and kept in the dark at room temperature. AuNPs (Sigma Aldrich, 765457), with a diameter size of 10 nm, functionalised with PEGylated carboxylic acid end (AuNP-PEG3000-COOH) with a stock concentration of 3 mg/ml were used in this study. The AlPcS4Cl was adsorbed onto AuNPs as previously described [10]. Briefly, the AlPcS4Cl and AuNPs were first filtered using a filter of 0.22-mm. The conjugation was achieved by adding AlPcS4Cl and AuNPs in a 1:2 ratio and vortexed for five minutes without a wash step. This method was adopted to retain the concentration of the conjugates. The ultraviolet-visible (UV-vis) absorption spectral of AlPcS4Cl, AuNPs, and AlPcS4Cl-AuNPs conjugates were evaluated using a UV-vis spectrophotometer (Jenway, GENOVA NANO Spectrophotometer, 67912). A high-resolution transmission electron microscope (JEOL Ltd., Tokyo, Japan, HR-TEM; JEM-2100) was used to determine the size and shape of AuNPs, and AlPcS4Cl-AuNPs using varying magnification using a method previously demonstrated [12].
Culture of oesophageal cancer cells, HKESC-1 and WS1 cells (control cells)
The human oesophageal cancer cell line (HKESC-1) (Cellonex, Johannesburg, South Africa) was propagated in T75 culture flasks in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, D5796) supplement with 10% Fetal Bovine Serum (FBS) (Biochrom, S0615), 1mM sodium pyruvate 1% antibiotic (Amphotericin B and Penicillin-streptomycin). The human skin fibroblast cell line (WS1) (ATCC®, CRL-1502™) was used as normal control cells. The cells were cultured in a complete growth medium composed of Minimum Essential Medium (MEM) from SAFC (51412C). The medium was supplemented with 0.1 mM MEM Non-essential Amino Acid Solution (NEAA) from Sigma-Aldrich (M7145), 2 mM L-Glutamine solution (L-Glut) from Sigma-Aldrich (G7513), 1 mM Sodium pyruvate solution, 10% fetal bovine serum (FBS), and 1% antibiotics. All cells were cultured under 5% CO2, 37°C and 80% humidity. The culture medium was changed every two days intervals. Experimental cells were seeded at a density of 5 x 105 cells in 35mm diameter culture plates containing 2mL of pre-warmed DMEM.
Intracellular Localisation of AlPcS4Cl-AuNPs conjugate
Fluorescent microscopy was used to determine the cellular localisation of AlPcS4Cl-AuNPs in oesophageal cancer with the aid of organelle trackers labelled with fluorescence dyes. Oesophageal cancer cells (HKESC-1) were cultured at a seeding concentration of 5 X 105 cells in 35mm culture plates in pre-warmed growth media containing glass coverslips. Following the attachment, 20 ^M of AlPcS4Cl-AuNPs conjugate was added and incubated for four hours. After incubation, cells were washed with Hank's balanced salt solution (HBSS). Cellular organelles in the cytoplasm were immediately stained with Mito-Tracker (100 nM) (M7514, Invitrogen™),) Lyso-Tracker (65 nM) (L7526, Invitrogen™), and ER-Tracker (65 nM) (E12353, Invitrogen™). After staining, the cytoplasmic organelle cells were rinsed with HBSS, and the nuclei were stained using 40-6-Diamidino-2 phenylindole (DAPI) (ID1306, Invitrogen™) and then rinsed with HBSS. Coverslips were removed and mounted onto sterile glass microscope slides. The slides were observed, and images were taken with a live imaging microscope (Carl Zeiss Axio Z1, Gottingen, Germany).
In vitro Photodynamic treatment
HKESC-1 cells were cultured at a concentration of 5 x105 concentration in 35mm cell culture plates containing 2 mL of complete culture medium and were grouped into control and test groups. Cells were incubated with DMEM, AlPcS4Cl, (1,25, 2.5, 5, 10, and 20 ^M) and AlPcS4Cl-AuNPs (1,25, 2.5, 5, 10, and 20 ^M) for four hours in the dark. After incubation, the cells were irradiated at 673.2 nm with 5 J/cm2 fluence using a continuous wave semiconductor diode laser (Oriel Corporation). The cells were further incubated for 24 hours post-irradiation, after which biochemical assays were performed.
MTT assay
Cellular proliferation was measured using the CyQUANT™ MTT Cell Proliferation Assay (V13154, Invitrogen™)). This assay relies on the ability of the mitochondria from live cells to hydrolyse MTT (dimethyl thiazolyl diphenyltetrazolium bromide) to an insoluble formazan resulting in yellow-to-purple colour formation. Briefly, twenty-four hours following treatment, 100 ^L of cell suspension was added in a 96 microwell plate, and 10 ^L of 12nM MTT solution was pipetted to each well, and the cells were then incubated at 37oC for three hours. After incubation, 150 ^L of DMSO (dimethyl sulfoxide) was added, mixed properly, and incubated for 10 minutes at 37oC. Absorbance at 570 nm was read using a plate reader PerkinElmer, VICTOR NivoTM.
ATP proliferation assay
Adenosine Triphosphate (ATP) level, which is directly proportional to cellular proliferation and cell viability, was evaluated using the CellTiter-Glo® 3D luminescence (G968A, Promega) according to the manufactural instruction as previously described [8].
Cytotoxicity assay
A cytotoxicity assay was conducted to measure the lactate dehydrogenase (LDH) enzyme released by the damaged cell membrane. Following the manufacturer's instructions, the test was achieved using the Cyto Tox 96® Non-Radioactive Cytotoxicity assay (Promega, G179A).
Reactive oxygen species measurement
To determine the levels of reactive oxygen species (ROS) within the cells, the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFDA/H2DCFD) was utilised. The cellular ROS assay (ab113851, Abcam) was employed for this test. HKESC-1 cells were grown in 96-well tissue culture plates and treated with 5 ^M AlPcS4Cl for 4 hours, followed by photodynamic therapy (PDT). After a 24-hour post-PDT incubation, the cells were incubated with DCFH-DA (5 ^M) at 37 °C for 10 minutes. Subsequently, the cells were transferred to a 96-well black plate. The fluorescence intensity was measured using the PerkinElmer VICTOR NivoTM plate reader with an excitation/emission filter set at 485 nm/538 nm. The amount of ROS produced in the treated cells was estimated as a ratio in relation to the control cells.
Mitochondrial membrane potential assay
The rhodamine-123 efflux fluorimetry assay (ab275545, Abcam) was employed to assess the mitochondrial membrane potential (MMP) on oesophageal cancer cells. Rhodamine-123, a fluorescent dye that localises in mitochondria, was used for this purpose. When the mitochondrial membrane potential is compromised, rhodamine leaks out of the mitochondria, resulting in reduced cell fluorescence intensity. After 24 hours of PDT treatment, the cells were detached, harvested, and washed twice with 1x PBS. The cells were then resuspended in 100 ^L of 1x PBS and stained with a 25 ^M solution of rhodamine-123 for 15 minutes at room temperature, protected from light. Following staining, the cells were washed twice with 1x PBS, and 400 ^l of PBS was added, mixed, and transferred into a 96-well black plate. The cells were then incubated for 30 minutes at room temperature, protected from light. The fluorescence signal of rhodamine-123 was quantified using the PerkinElmer VICTOR NivoTM plate reader with a filter of 485 nm excitation and 538 nm emission.
Results
Characterisation of AlPcS4Cl-AuNP
AlPcS4Cl-AuNPs were synthesised as previously described [10] and characterised using a UV-vis absorption spectroscopy and TEM. Following the successful adsorption of AlPcS4Cl to AuNPs, the absorption band of AuNPs was 520nm Figure 1(b) before adsorption was shifted to 540nm after adsorption, as demonstrated in Figure 1(c). The TEM analysis revealed that the AuNPs exhibited a uniform spherical shape with a smooth surface, and there was no evidence of agglomeration observed Figure 19(e). Both the AlPcS4Cl-AuNPs and the AuNPs displayed similar surface characteristics. However, the AlPcS4Cl-AuNP had a larger
diameter (33.20 ±1.93 nm) compared to the AuNPs (14.74±3.37 nm) Figure 1(f), indicating that the combination of AlPcS4Cl with the AuNPs was successful and stable.
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Figure 1. UV-Vis spectral characterisation of (a) AlPcS4Cl (Xmax 675nm), (b) AuNPs (Xmax 520nm), (c) AlPcS4Cl-AuNPs (Xmax 672nm and 540nm) and (d) combinations of (a)-(c). Transmission electron microscopy images of (e) AuNP and (f)AlPcS4Cl-AuNPs. Scale bars, 20 nm.
Subcellular Localisation of AlPcS4Cl-AuNPs in oesophageal cancer
Localisation of the AlPcS4Cl-AuNPs in HKESC-1 oesophageal cancer cells was examined using the Zen Pro (3.1) Carl Zeiss software on a Carl Zeiss fluorescent microscope. A remarkable accumulation of AlPcS4Cl-AuNPs was observed in the nucleus, mitochondria, and the ER of HKESC-1 oesophageal cancer cells, as depicted in Figure 2. This was clearly demonstrated by the merging of the blue fluorescence from the nuclei, green fluorescence from the mito tracker and ER tracker and red fluorescence from the AlPcS4Cl-AuNPs,
resulting in a yellow colour. The yellow colour showed that the AlPcS4Cl-AuNPs were specifically accumulating in the nucleus, mitochondria, and ER. This
Figure 2. AlPcS4Cl-AuNPs localisation in HKESC-1 oesophageal cancer cells displaying intracellular organelle localisation. The nuclei are counterstained with DAPI (blue), mitochondria, ER stained with FITC (green), and AlPcS4Cl-AuNPs auto fluoresces (red). The merged yellow/white colour showed an accumulation of' AlPcS4Cl-AuNPs in the mitochondria, ER and the nucleus. Scale bar:50^m
In Vitro Effects of AlPcS4Cl-AuNPs Conjugates Mediated PDT on Oesophageal Cancer Dose-Response Evaluation of AlPcS4Cl-AuNPs Conjugates
A dose-response was performed on HKESC-1 cells to obtain the appropriate concentration of AlPcS4Cl, and the conjugates required for the downstream application of PDT on HKESC-1 cells. The IC50 values were evaluated by using MTT cell viability assay post 24 h treatment with increasing concentrations of AlPcS4Cl and of AlPcS4Cl-AuNPs (1.25, 2.5, 5, 10, and 20^M). The results showed that AlPcS4Cl-AuNPs have a low IC50 of 2.88 ^M (p<0.001) when compared to AlPcS4Cl with an IC50 of 5.21 ^M as depicted in figure 3.
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Figure 3. Determination of 50% inhibitory concentration (IC50) using MTT Cell Viability assay of AlPcS4Cl-AuNPs and the free AlPcS4Cl in Oesophageal cancer. The results are depicted as ± SEM (n = 3);
(***p <0.001).
Cytotoxicity Evaluation
Cytotoxicity assay was performed by evaluating the quantity of lactate dehydrogenase (LDH) enzyme leakage in the cell culture media of HKESC-1 oesophageal cancer cells and normal cells (WS1, fibroblast cell). Cells that have damaged cell membrane integrity leak out LDH indicative of cytotoxicity. LDH release was measured following PDT at a light dose of 5 J/cm2 at concentrations of 5 ^M each for AlPcS4Cl-AuNPs and AlPcS4Cl. At 24 hours post-PDT, statistically significant results were observed for all PDT concentrations tested in the HKESC-1 oesophageal cancer cells (AlPcS4Cl 5 ^M at 5 J/cm2, and AlPcS4Cl-AuNPs 5 ^M at 5 J/cm2), as depicted in Figure 4(a). The significance levels were reported as ***p < 0.001, compared to the control group untreated. No statistical significance was found when comparing the AlPcS4Cl-AuNPs dark cells and AlPcS4Cl dark cells to the control dark cells. Conversely, no statistical significance was demonstrated in the normal control cells both between the treated and untreated group and within the treated and untreated cells (Figure 4(b)).
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Figure 4. The cytotoxic effects of AlPcS4Cl-AuNPs and AlPcS4Cl were determined by LDH release cytotoxicity assay. (a) The cytotoxic effects of AlPcS4Cl-AuNPs and AlPcS4Cl on HKESC-1 oesophageal cancer cells. The amount of LDH release after AlPcS4Cl-AuNPs and AlPcS4Cl with 5 J/cm2 irradiation, with AlPcS4Cl-
AuNPs conjugate showing increased LDH release compared to AlPcS4Cl and the control. (b) The cytotoxic effects of AlPcS4Cl-AuNPs and AlPcS4Cl on normal cells (WS1 fibroblast cells). No statistical significance was observed in the. The results are depicted as ± SEM (n = 3); (**p < 0.01, ***p <0.001).
ATP Cellular Proliferation Assessment
Oesophageal cancer cells were evaluated for cellular proliferation ability with and without photoactivation using ATP proliferation assay. Proliferation results showed that the non-irradiated cancer cells displayed high proliferation activity with increased ATP levels. While the irradiated cells at a concentration of 5 ^M at 5 J/cm2 showed a significant decrease of ATP activities in the cells administered with the AlPcS4Cl-AuNPs conjugate (**p<0.01) and AlPcS4Cl (*p<0.05), with the conjugate showing a more ATP inactivity (Figure 5).
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Figure 5. The cellular proliferative effects of AlPcS4Cl-AuNPs and AlPcS4Cl on oesophageal cancer cells. Blue: the non-irradiated cells showed increased proliferative ATP activities with no significant difference between the control cells and cells administered with AlPcS4Cl and the conjugate. Red: the irradiated cells at 5 J/cm2 demonstrated reduced proliferative actions with reduced ATP levels between the conjugate and the free PS (*p < 0.05 ***p < 0.001). The values shown are ± SEM (standard error of the mean) (n=3).
Mitochondria membrane potential Assessment
Oesophageal cancer cells were evaluated for MMP function using the rhodamine-123 efflux fluorimetry assay. The MMP results revealed that the untreated cancer cells displayed high MMP integrity. While the treated irradiated cells at a concentration of 5 ^M at 5 J/cm2 showed a significant decrease of MMP integrity in the cells administered with the AlPcS4Cl-AuNPs conjugate (***p<0.001) and AlPcS4Cl (***p<0.001), with the conjugate showing a more MMP impairment (Figure 6).
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Figure 6. AlPcS4Cl-AuNPs conjugate and AlPcS4Cl mediated PDT effects on Mitochondrial Membrane potential (MMP) Integrity of oesophageal cancer cells. Blue: the non-irradiated control cells showed decrease MMP impairment. Red: the irradiated cells treated with AlPcS4Cl-AuNPs conjugate and AlPcS4Cl at 5 J/cm2
demonstrated significantMMP alterations (**p < 0.01 ***p < 0.001). The values shown are ± SEM (standard
error of the mean) (n=3).
Measurement of Reactive Oxygen Species (ROS)
Fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFDA/H2DCFD), cellular reactive oxygen species (ROS) probe was used to quantify intracellular ROS generation of AlPcS4Cl-AuNPs conjugate and AlPcS4Cl based PDT in HKESC-1 cells. The ROS generated was measured post-PDT at a light dose of 5 J/cm2 at concentrations of 5 ^M for both AlPcS4Cl-AuNPs and AlPcS4Cl. The results showed a statistically significant increase in ROS production PDT treated group AlPcS4Cl (**p<0.01) and AlPcS4Cl-AuNPs (***p<0.001) when compared with the untreated controls as seen in Figure 7.
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Figure 7. Intracellular ROS production of AlPcS4Cl-AuNPs conjugate and AlPcS4Cl mediated PDT in HKESC-loesophageal cancer cells. Blue: the non-irradiated control cells showed decreased ROS generation. Red: the irradiated cells treated with AlPcS4Cl-AuNPs conjugate and AlPcS4Cl at 5 J/cm2 increase ROS generation (**p < 0.01 ***p < 0.001). The values shown are ± SEM (standard error of the mean) (n=3).
DISCUSSION
In this study, we investigated the effects of AlPcS4Cl-AuNPs conjugates in enhancing the therapeutic efficiency of photodynamic therapy (PDT) in oesophageal cancer. Nanoparticles, specifically gold nanoparticles (AuNPs), have garnered significant interest as potential nanocarriers for drug delivery due to their chemical inertness, efficient optical properties, and biocompatibility. While the use of AuNPs as carriers for drug delivery in PDT has been extensively studied in various cancer types, its impact on oesophageal cancer remains limited. Therefore, we evaluated the therapeutic efficiency of PDT using AlPcS4Cl-AuNPs conjugates by assessing various parameters such as intracellular localisation, cell viability, LDH enzyme release, MMP integrity and ROS production.
The synthesised AlPcS4Cl-AuNPs were confirmed by UV-Vis spectral absorption spectroscopy and TEM analysis. The results showed a red shift in the maximum absorption band of the AuNPs from 520nm before synthesis to 540nm after synthesis (Figure 1 (b) and (c). While the AlPcS4Cl showed a left shift from 675nm before synthesis to 672nm after synthesis (figure 1(a) and (c). The TEM analysis was performed to determine the morphological variation of the AuNPs before and after synthesis. The results showed a change in the diameter of AuNPs from 14.74 nm ± 3.37 before synthesis to 33.20 nm ±1.93 after synthesis. This result confirms the successful adsorption of AlPcS4Cl-AuNPs (Figure 1(e)(f).
Intracellular localisation evaluation was performed to determine the preferential accumulation sites of the AlPcS4Cl-AuNPs in oesophageal cancer cells. Understanding the impact of AlPcS4Cl-AuNPs on organelle-specific responses to cell death machinery can be modulated to boost the therapeutic efficacy of PDT. For instance, targeting photosensitisers to specific organelles through nanoparticle conjugation or other delivery strategies can potentially enhance the selectivity and effectiveness of PDT. In this investigation, we discovered that AlPcS4Cl-AuNPs selectively internalised in the mitochondria, ER, and nucleus (Figure 2). This suggests that these organelles may be necessary for the AlPcS4Cl-AuNPs enhanced PDT action of cell damage. This is consistent with a study by Mkhobongo and colleagues that showed that AlPcS4Cl-AuNPs are localised in the mitochondria and lysosomes of melanoma cells [11].
Findings from cell viability analysis using MTT assay revealed that AlPcS4Cl-AuNPs and the free AlPcS4Cl mediated PDT significantly inhibited cell viability (Figure 3). However, the AlPcS4Cl-AuNPs mediated PDT group had enhanced reduction in the cell viability when compared to the AlPcS4Cl group. The results also showed that AlPcS4Cl-AuNPs mediated PDT has a statistically significant lower IC50 concentration of 2.88 ^M when compared to the IC50 of AlPcS4Cl-mediated PDT of 5.02 ^M on HKESC-1 oesophageal cancer cells (Figure 3).
The cytotoxicity evaluation of AlPcS4Cl-AuNPs and AlPcS4Cl mediated PDT on HKESC-1 oesophageal cancer cells and on normal control cells was conducted using the LDH release cytotoxicity assay. This assay evaluates the extent to which LDH enzymes leak out after cellular insult. We observed statistically significant high efflux of LDH enzyme in the AlPcS4Cl-AuNPs and AlPcS4Cl mediated PDT group when compared with the control cells as shown in Figure 4(a). However, the AlPcS4Cl-AuNPs PDT group has more enhanced cytotoxicity effects. This aligned with the finding by Mkhobongo and co-worker which showed that AlPcS4Cl-AuNPs mediated PDT enhanced the cytotoxicity effects in Melanoma cells [11] and lung cancer cells and cancer stem cells [9, 13]. We further determine the effects of the conjugates and the free PS on normal noncancerous cells using the concentration of 5uM, to ascertain if it is toxic to normal cells. Our finding showed that the AlPcS4Cl-AuNPs and the free AlPcS4Cl do not induce any cytotoxic effects on normal cells. This implies that the conjugates and the free PS preferentially accumulation in tumour cells.
The effects of AlPcS4Cl-AuNPs on ATP cellular production levels in oesophageal cancer cell which serves as an indicator of cellular metabolic activity was evaluated. ATP is an essential molecule that serves as a primary energy source for cellular processes. Tumour cells rely on aerobic glycolysis, a metabolic pathway known as the Warburg effect, to generate ATP. Mitochondria play a crucial role in producing ATP during cellular respiration [14]. The decrease in ATP production observed in oesophageal cancer cells following the photodynamic action of the AlPcS4Cl-AuNPs conjugate and AlPcS4Cl photosensitiser suggests that the photosensitiser localised in specific subcellular organelles involved in maintaining cellular homeostasis. By targeting these organelles, the photosensitiser disrupts their normal functioning and leads to the inhibition of oesophageal cancer cells. This observation is similar to the finding of AlPcS4Cl-AuNPs conjugates inhibiting the ATP cellular proliferation activity in lung cancer stem cells [13].
The mitochondrial is known to be the powerhouse of cellular ATP production and damage to this organelle can alter its function. In this study we investigated the effects of AlPcS4Cl-AuNPs mediated PDT on the MMP activity of oesophageal cancer cells. The results demonstrated that AlPcS4Cl-AuNPs and AlPcS4Cl mediated PDT significantly alter the MMP of the cell. The AlPcS4Cl-AuNPs mediated PDT group has more MMP dysfunction. This finding agrees with the reduced ATP cellular production observed in this study. Finally, the effects of AlPcS4Cl-AuNPs mediated PDT in promoting ROS generation was examined. Our results showed increased ROS production with the AlPcS4Cl-AuNPs mediated PDT group than the free AlPcS4Cl mediated PDT. Suggesting the conjugate enhances the PDT effects in oesophageal cancer cells. CONCLUSION
The findings showed that PDT with aluminium (III) phthalocyanine chloride tetra sulfonic acid (AlPcS4Cl) conjugated gold nanoparticle (AuNPs)(AlPcS4Cl-AuNPs) significantly inhibit cell viability/cellular proliferation, increase cytotoxicity, and ROS generation. Fluorescent microscopy revealed that (AlPcS4Cl-AuNPs) was localised in the nuclei, mitochondria and ER suggesting the biochemical cell death pathways induction could be mitochondria and ER dependent. More importantly, AlPcS4Cl-AuNPs significantly altered
the mitochondrial membrane integrity in HKESC-1 cells. Further confirming the mitochondria dependent cell death pathway. In conclusion, our findings demonstrated that AlPcS4Cl-AuNPs conjugates improved the anticancer effects of PDT in oesophageal cancer cells, proposing a better measure to boost the therapeutic efficiency of PDT in oesophageal cancer.
Author Contributions: Conceptualisation, OCD; methodology, OCD; validation, OCD, RC and HA; formal analysis, OCD; investigation, OCD; resources, RC and HA; writing—original draft preparation, OCD; writing—review and editing, OCD, RC and HA; supervision, RC and HA; project administration, OCD, RC and HA; funding acquisition, OCD, RC and HA. All authors have read and agreed to the published version of the manuscript.
Funding: The research was financially supported by various entities. These included the Department of Science and Innovation (DSI) funded African Laser Centre (grant number HLHA23X task ALC-R007), the University Research Council (grant number 2022URC00513), the Department of Science and Technology's South African Research Chairs Initiative (grant number 98337). Additionally, the Article Processing Charges (APC) were covered by the University of Johannesburg Faculty of Health Sciences. Furthermore, funding was contributed by the University of Johannesburg Global Excellence and Stature, Fourth Industrial Revolution (GES 4.0) Doctoral Scholarship.
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated during and/or analysed during the current study are available from the authors upon request.
Acknowledgments: The authors sincerely thank the University of Johannesburg and the Laser Research Centre for their facilities and equipment.
Conflicts of Interest: The authors declare no conflict of interest. REFERENCES
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[10] Chi, Y.-f., et al., Enhanced anti-tumor efficacy of 5-aminolevulinic acid-gold nanoparticles-mediated photodynamic therapy in cutaneous squamous cell carcinoma cells. Brazilian Journal of Medical and Biological Research, 2020. 53.
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