Prioritization of Natural Extracts by LC–MS-PCA for the Identification of New Photosensitizers for Photodynamic Therapy

Photodynamic therapy (PDT) is an alternative treatment for cancer that involves administration of a photosensitive drug or photosensitizer that localizes at the tumor tissue followed by in situ excitation at an appropriate wavelength of light. Tumour tissues are then killed by cytotoxic reactive oxygen species generated by the photosensitizer. Targeted excitation and photokilling of affected tissues is achieved through focal light irradiation, thereby minimizing systemic side effects to the normal healthy tissues. Currently, there are only a small number of photosensitizers that are in the clinic and many of these share the same structural core based on cyclic tetrapyrroles. This paper describes how metabolic tools are utilized to prioritize natural extracts to search for structurally new photosensitizers from Malaysian biodiversity. As proof of concept, we analyzed 278 photocytotoxic extracts using a hyphenated technique of liquid chromatography–mass spectrometry coupled with principal component analysis (LC–MS-PCA) and prioritized 27 extracts that potentially contained new photosensitizers for chemical dereplication using an in-house UPLC-PDA-MS-Photocytotoxic assay platform. This led to the identification of 2 new photosensitizers with cyclic tetrapyrrolic structures, thereby demonstrating the feasibility of the metabolic approach

Cytotoxicity of GMG-ITC on differentiated neuronal cells at different concentrations (0.313 to 10) μg/ml.

<p>(A) display 24 h, (B) 48 h and (C) 72 h of treatment. Whereas (D) is a cytotoxic analysis result of H₂O₂ used in this study with IC₅₀ = 300 μM. Values are presented in means ± SD of triplicate experiments and means with different letters varies significantly (p<0.05).</p

Ultrastructural analysis of differentiated neuronal cells by transmission electron microscopy.

<p>(a) 4 h H₂O₂ treated cells, (b) 72 h myrosinase pre-treated plus 4 h H₂O₂ exposure cells, (c) 72 h GMG-ITC pre-treated plus 4 h H₂O₂ exposure cells, (d) untreated (normal control) cells. CM = chromatin margination, IN = intact nucleus, LD = lipid droplet, NC = nuclei convolution. Magnification (x 3000).</p

Acridine orange (AO, green) and propidium iodide (PI, red) double staining fluorescent micrographs of differentiated neuronal cells.

<p>(a) 4 h H₂O₂ treated cells, (b) 72 h myrosinase pre-treated plus 4 h H₂O₂ exposed cells, (c) 72 h 1.25 μg/ml GMG-ITC pre-treated plus 4 h H₂O₂ exposed cells, (d) untreated cells (normal control). The images were captured in multiple times and x20 magnification was used.</p

Annexin V-FITC assay of differentiated neuronal cells analysed by flow cytometry.

<p>Where (a) 4 h H₂O₂ treated cells, (b) 72 h myrosinase pre-treated plus 4 h H₂O₂ exposure cells, (c) untreated (normal control) cells, (d) GMG-ITC pre-treated for 24 h plus 4 h H₂O₂ exposure, (e) GMG-ITC pre-treated for 48 h plus 4 h H₂O₂ exposure and (f) GMG-ITC pre-treated for 72 h plus 4 h H₂O₂ exposure. Whereas (g) represent distribution of cells at death. Values are presented in means ± SD of triplicate experiments and means of viable cells with different letters varies significantly (p<0.05).</p

Surface morphological analysis of differentiated neuronal cells by scanning electron microscopy.

<p>(a) 4 h H₂O₂ treated cells, (b) 72 h myrosinase pre-treated plus 4 h H₂O₂ exposure cells, (c) 72 h GMG-ITC pre-treated plus 4 h H₂O₂ exposure cells, (d) untreated (normal control) cells. AB = apoptotic body, IDVC = intact differentiated viable cells, FN = folded neurites, MB = membrane blabbing, NDAC = neurite disrupted apoptotic cells. Magnification (x 5000).</p

Micrographs of neuronal cells differentiation by 10 μM all trans retinoic acid (ATRA).

<p>(a) Undifferentiated cells cultured in 10% complete growth media for seven (7) days and viewed under phase contrast, (b) Differentiated cells cultured in 3% heat-inactivated FBS complete growth media containing 10 μM ATRA for seven (7) days and viewed under phase contrast, and (d) expressed tuj-1 in both cytoplasm and neurites. SN = short neurites, EN = extended neurites, CYP = cytoplasm,. Magnification (x 20).</p