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5-ALA/PpIX fluorescence signal of glioblastoma cells in correlation with early and late photodynamic-induced apoptosis

Ekaterina Borisova, Institute of Electronics, Bulgarian Academy of Sciences, Bulgaria
Dobroslav Kyurkchiev, Laboratory of Clinical immunology, University Hospital " St. Ivan Rilski", Department of clinical laboratory and clinical immunology, Medical University - Sofia
Kalina Tumangelova-Yuseir, Laboratory of Clinical immunology, University Hospital " St. Ivan Rilski", Department of clinical laboratory and clinical immunology, Medical University - Sofia
Ekaterina Ivanova-Todorova, Laboratory of Clinical immunology, University Hospital " St. Ivan Rilski", Department of clinical laboratory and clinical immunology, Medical University - Sofia
Ivan Angelov, Institute of Electronics, Bulgarian Academy of Sciences, Bulgaria
Lidia Zaharieva, Institute of Electronics, Bulgarian Academy of Sciences, Bulgaria
Peter Karazapryanov, Neurosurgery Department, University Hospital " St. Ivan Rilski", Bulgaria
Krassimir MInkin, Neurosurgery Department, University Hospital " St. Ivan Rilski", Bulgaria

Abstract

The most significant clinical application for fluorescence diagnosis and photodynamic therapy of glioblastoma multiforme (GBM) to date is with the use of delta-aminolevulinic acid / protoporphyrin IX. 5-ALA is a natural precursor to heme, which can also be found endogenously. The exogenous introduction of 5-ALA (i.e., its additional administration in cells) results in excessive accumulation of fluorescent protoporphyrin IX in malignant brain tissues. Its concentration for grade III and IV glioma is almost 6 times higher than that in normal brain tissue. The metabolite protoporphyrin IX can be excited by irradiation with light in the range 400 - 410 nm (Soret line), and its emission is in the red spectral range. This fluorescence could be observed at in vivo irradiation of tumor tissues, and could serve as a tumor marker for real-time intraoperative resection.
The aim of this work was to investigate the photodynamic properties of 5-ALA/PpIX and AlPc on stem cell cultures isolated from glioblastoma. Sixteen GBM tissue samples were obtained after surgical excisions from patients in the period October 2019 to June 2020 and used for in vitro investigations using extracted GBM cells from these samples, grown as adherent cellular layers in 6-wall plates. However, in the case of treatment of adherent layers of cell samples from resected glioblastoma tumours, the fluorescent signals that were observed were significantly weaker than obtained at in vivo detection. As a result the supernatant of the GBM cells was used for fluorescence studies, to determine the trace concentrations of protoporphyrin IX there, and to correlate its amount and the efficiency of induction of apoptosis and / or necrosis in the studied cell samples. Several variables, such as histochemical and genetic variants of glioblastoma, various photosensitisers’ concentrations and light-emitting parameters were studied. The results of this in vitro work will be used as a basis for a further in vivo GBM-PDT application.
Acknowledgments: This work is supported by the NSF-Bulgaria under grant # KP06-N23/8/18.12.18 “Innovative photodynamic methods for treatment of stem cells cultivated from glioblastoma tumours”.

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Ekaterina Borisova
Institute of Electronics, Bulgarian Academy of Sciences
Bulgaria

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