Efficacy of photochemical internalisation using disulfonated chlorin and porphyrin photosensitisers: An in vitro study in 2D and 3D prostate cancer models

Abstract

This study shows the therapeutic outcome of Photochemical Internalisation (PCI) in prostate cancer in vitro surpasses that of Photodynamic Therapy (PDT) and could improve prostate PDT in the clinic, whilst avoiding chemotherapeutics side effects. In addition, the study assesses the potential of PCI with two different photosensitisers (TPCS_2a and TPPS_2a) in prostate cancer cells (human PC3 and rat MatLyLu) using standard 2D monolayer culture and 3D biomimetic model. Photosensitisers were used alone for photodynamic therapy (PDT) or with the cytotoxin saporin (PCI). TPPS_2a and TPCS_2a were shown to be located in discrete cytoplasmic vesicles before light treatment and redistribute into the cytosol upon light excitation. PC3 cells exhibit a higher uptake than MatLyLu cells for both photosensitisers. In the 2D model, PCI resulted in greater cell death than PDT alone in both cell lines. In 3D model, morphological changes were also observed. Saporin-based toxicity was negligible in PC3 cells, but pronounced in MatLyLu cells (IC50 = 18 nM). In conclusion, the study showed that tumour features such as tumour cell growth rate or interaction with drugs determine therapeutic conditions for optimal photochemical treatment in metastatic prostate cancer.

Keywords: Photochemical internalisation (PCI); Photodynamic therapy (PDT); Prostate cancer; TPCS(2a); TPPS(2a).

Fig. 1

Uptake of TPPS_2a& TPCS_2ain PC3 and MatLyLu cells. PC3 and MatLyLu cells were exposed to increasing doses of photosensitiser (0.2 μg/ml – 0.8 μg/ml) during 24 h and uptake was related to fluorescence emission.

Fig. 2

Reduction of Cell viability of PC3 and MatLyLu cells upon exposure to PDT & PCI. (A, B) Relative cell viability of PC3 cells following incubation to 0.2 μg/ml TPPS_2a or TPCS_2a alone (PDT) or combined with Saporin 2 nM (PCI) during 24 h and posterior illumination during 3 min. Cell viability was measured through the MTT assay either 24 h (A) or 96 h (B) after photosensitiser light-excitation. (C, D) Relative cell viability of MatLyLu cells following incubation to 0.4 μg/ml TPCS_2a alone (PDT) or combined with Saporin 20 pM (PCI) during 24 h and posterior illumination during 3 min (1.3 J/cm²). Cell viability was measured through the MTT assay either 24 h (C) or 48 h (D) after photosensitiser light-excitation.

Fig. 3

PDT & PCI in PC3 cells seeded in 3D collagen hydrogels. Fluorescence microscopy of PDT and PCI treated PC3 cells in 3D cultures. (A) PC3 control cells. (B) TPPS_2a (0.2 μg/ml), 96 h after no light conditions. (C) TPPS_2a (0.2 μg/ml) 96 h after 5 min light (2.1 J/cm²) (PDT). (D) TPPS_2a (0.2 μg/ml), Saporin (2 nM), 96 h after no light conditions. (E) TPPS_2a (0.2 μg/ml), Saporin (2 nM), 96 h after 5 min light (2.1 J/cm²) (PCI). (F) Saporin (2 nM), (K) higher magnification of (F). (G) TPCS_2a (0.2 μg/ml), 96 h after no light conditions. (H) TPCS_2a (0.2 μg/ml) 96 h after 5 min light (2.1 J/cm²) (PDT), (L) higher magnification of (H). (I) TPCS_2a (0.2 μg/ml), Saporin (2 nM), 96 h after no light conditions. (J) TPCS_2a (0.2 μg/ml), Saporin (2 nM), 96 h after 5 min light (2.1 J/cm²) (PCI). Scale bars shown as 200 μm.

Fig. 4

Subcellular localisation of TPPS_2a& TPCS_2ain MatLyLu and PC3 cells. Optical microscopy of TPPS_2a & TPCS_2a in PC3 and MatLyLu cells. (A) TPCS_2a (0.35 μg/ml) in MatLyLu cells prior to light-excitation. (B) TPCS_2a (0.35 μg/ml) in MatLyLu cells 3 min post light-excitation. (C) TPPS_2a (0.35 μg/ml) in PC3 cells prior to light-excitation. (D) TPPS_2a (0.35 μg/ml) in PC3 3 min post light-excitation. Scale bars shown as 50 μm.

Fig. 5

Saporin-Alexa-Fluor488^®subcellular localisation & redistribution upon excitation of TPPS_2a. Fluorescence microscopy of Saporin-Alexa Fluor488^® in PC3 cells. (A) Saporin-Alexa Fluor488^® (400 nM) prior to illumination with 405 nm laser. (B) Saporin-Alexa Fluor488^® (400 nM) post exposure to 405 nm laser. (C) Saporin-Alexa Fluor488^® (400 nM) and TPPS_2a (0.4 μg/ml) prior to illumination with 405 nm laser. (D) Saporin-Alexa Fluor488^® (400 nM) and TPPS_2a (0.4 μg/ml) post exposure to 405 nm laser. (E) Saporin-Alexa Fluor488^® (400 Nm). (F) LysoTracker^® Red DND-99 (75 nM). (G) Merged Saporin-Alexa Fluor488^® (E) & LysoTracker^® Red DND-99 (F). Scale bars shown as 50 μm.