TBA-MDA test for lipid peroxidation detection induced by UV-A and UV-B irradiation of Protoporphyrin IX in SUV liposomes

Protoporphyrin IX (PPIX) is a well-known photosensitiser with great potential for use in photodynamic therapy (PDT). However, PPIX is a highly hydrophobic and cannot be dissolved in water which represents an environment suitable for PDT applications. So liposomes (as multilamellar or unilamellar phospholipidic submicroscopic vesicles) allow the incorporation of lipophilic and hydrophilic drugs such as PPIX in their matrix with potential for the delivery of various bioactive molecules used for therapeutic applications. Because of that it can be very important to investigate how PPIX incorporated inside the liposomes lipid bilayers influences lipid peroxidation (LP) - process applied in various fields in medicine - PDT and pharmacy. The most prominent and currently used assay for detection and recording of LP is the spectrophotometric thiobarbituric acid-malondialdehyde test (TBA-MDA). PPIX-loaded liposomes were prepared using dry PPIX-lipid film method and extrusion through filters with 100 nm pores to obtain SUV (small unilamellar vesicles) liposomes. Lipid peroxidation was initiated by photosensitization reaction of PPIX-SUV system with continual UV-A and UV-B irradiation. The formation of the LP product malon-aldehyde (MDA), was measured spectrophotometrically (TBA-MDA test). UV-A and UV-B irradiation treatments of PPIX-SUV mixtures (with or without the photosensitizer PPIX) was performed in cylindrical photochemical reactor with emission maximum at 350 and 300 nm, with 10 symmetrically placed Hg-lamps and the corresponding flux at 12.9 Wm^-2 and 15 Wm^-2, respectively. The continual UV-A and UV-B irradiation of PPIX-SUV complex resulted in lipid peroxidation process - continuous increase in absorbance band responsible for LP product. Lipid peroxidation process obeys first-order kinetics. The calculated rate constants of LP products growth are 0.0105 min^-1 and 0.0439 min^-1 for UV-A and UV-B irradiation, respectively. Lipid peroxidation process was not observed in the samples without photosensitizer, or samples with photosensitizer kept in dark. The results are in accordance with the known fact that UV-B irradiation is more energetic in comparison with UV-A and due to that photosensitiser PPIX in SUV liposomes is more excited causing faster lipid peroxidation process. Further, it is shown that PPIX-SUV complex is a stable with PDT potential, and photosensitiser can express oxidative potential. (author)