CC BY
13DOI 10.24412/cl-37136-2023-1-30-34
ALUMINIUM PHTHALOCYANINE-GOLD NANOPARTICLE CONJUGATES 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
ABSTRACT BACKGROUND
Gold nanoparticles mediated photodynamic therapy (PDT) have been reported to boost the efficiency and specificity of cancer treatment on various tumour (1-5), 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.
METHOD
The AlPcS4Cl-AuNPs conjugate was synthesis through a non-covalent method. The synthesized AlPcS4Cl-AuNPs was confirmed by ultraviolet-visible (UV-vis) absorption spectral analysis and highresolution transmission electron microscopy. In vitro effects of AlPcS4Cl-AuNPs based PDT in an oesophageal cancer cell line (HKESC-1) such as 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.
RESULTS
AlPcS4Cl-AuNP Synthesis and Characterization
Figure 1: a) The AlPcS4Cl-AuNPs conjugates was synthesized using a non-covalent conjugation method. b) Transmission electron microscopy images of AuNP andAlPcS4Cl-AuNPs. Scale bars, 100 nm. c) UV-Vis spectral characterization of
AuNP, AlPcS4Cl andAlPcS4Cl-AuNPs Conjugate.
Subcellular Localisation of A1PcS4C1-AuNPs in oesophageal cancer
AIPcS4CI auto fluoresces (AF660)
Nucleus (Dapi)
Mitochondrla/Endoplasmic reticulum (ER) FITC (green)
Merge
Figure 2: The nuclei are counterstained with DAPI (blue), mitochondria, ER stained with FITC (green), and AlPcS4Cl-AuNPs auto fluoresces (red). The merged yellow/white and pink colour showed accumulation of AlPcS4Cl-AuNPs the
mitochondria, ER and the nucleus.
In Vitro Effects of AlPcS4Cl-AuNPs Conjugates Mediated PDT on Oesophageal Cancer Dose-Response Evaluation of AlPcS4Cl-AuNPs Conjugates
Dose response was performed on HKESC-1 cells to obtain the appropriate concentration of AlPcS4Cl, and the conjugates required for downstream application of PDT on HKESC-1 cells. The IC50 values was 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).
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 < 0.01, ***p <0.001).
Cytotoxicity Evaluation
Cytotoxicity assay was performed by evaluating the quantity of lactate dehydrogenase (LDH) enzyme leakage in the cell culture media. Cells that have damaged cell membrane integrity leaks out LDH indicative of cytotoxicity.
4.5
E 3.5
C
O .
CTi 3
©J 2.5 CU
u 2 c
-O 1.5
o
-Q 1 <
0.5
Non-Irradiated Irradiated
**
III!
tv^
^ a1» f ,«> of
C o/ C J?
Figure 4: The cytotoxic effects of AlPcS4Cl-AuNPs and AlPcS4Cl were determined by LDH release cytotoxicity assay. 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. 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 5 J/cm2 showed significant decrease of ATP activities in the cells administered with the AlPcS4Cl-AuNPs conjugate and AlPcS4Cl, with the conjugate showing a more ATP inactivity.
7000000
6000000
Non-Irradiated Irradiated (a) 5 J/cm2
^ 5000000 fc
g 4000000
S 3000000 c
s
s
2000000
1000000
Control
AlPcS4Cl AlPcS4Cl-AuNPs
Control
AlPcS4Cl AlPcS4Cl-AuNPs
Figure 5: The cellular proliferative effects of AlPcS4Cl-AuNPs and AlPcS4Cl on oesophageal cancer cells. Blue: the non-irradiated cells showed increase 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 conjugate and the free PS (*p < 0.05 ***p < 0.001). The values
shown are ± SEM (standard error of the mean) (n=3).
Mitochondrial Membrane potential (MMP) Integrity
Mitochondrial membrane potential (MMP) was estimated using rhodamine-123. The HKESC-1 oesophageal cancer cells treated with AlPcS4Cl-AuNPs conjugate and AlPcS4Cl showed different effects on the
MMP integrity. AlPcS4Cl-AuNPs conjugate significantly alter the MMP of the HKESC-1 cells compared to the free AlPcS4Cl.
1.6E+10
QJ
1 1.4E+10
QJ
1 1.2E+10
lu
= 1E+10 E
•S 8E+09 fi a
rsj aj
2 "S 6E+09
I " 4E+09 S
« 2E+09
I »
Non-Irradiated Irradiated (a), 5 J/cm2
I
Control
AlPcS4CL AlPcS4Cl-AuNPs 5uM 2.91V1
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 significant MMP 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.
Figure 7: Intracellular ROS production of AlPcS4Cl-AuNPs conjugate and AlPcS4Cl mediated PDT in HKESC-1oesophageal cancer cells. Blue: the non-irradiated control cells showed decrease 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 AND 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 localized in the mitochondria and Endoplasmic reticulum (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 anti-cancer effects of PDT in oesophageal cancer cells, proposing a better measure to boost the therapeutic efficiency of PDT in oesophageal cancer.
REFERENCES
[1] Chi Y-f, Qin J-j, Li Z, Ge Q, Zeng W-h. 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.
[2] Crous A, Abrahamse H. Photodynamic Therapy with an AlPcS4Cl Gold Nanoparticle Conjugate Decreases Lung Cancer's Metastatic Potential. Coatings. 2022;12(2):199.
[3] Didamson OC, Chandran R, Abrahamse H. A Gold Nanoparticle Bioconjugate Delivery System for Active Targeted Photodynamic Therapy of Cancer and Cancer Stem Cells. Cancers. 2022;14(19):4558.
[4] Mkhobongo B, Chandran R, Abrahamse H. In Vitro Photodynamic Treatment Modality for A375 Melanoma Cell Line Using a Sulphonated Aluminum Phthalocyanine Chloride-Photosensitizer-Gold Nanoparticle Conjugate. Pharmaceutics. 2022;14(11).
[5] Mokoena D, George BP, Abrahamse H. Conjugation of Hypericin to Gold Nanoparticles for Enhancement of Photodynamic Therapy in MCF-7 Breast Cancer Cells. Pharmaceutics. 2022;14(10):2212.
