Improving Photodynamic Therapy Anticancer Activity of a Mitochondria-Targeted Coumarin Photosensitizer Using a Polyurethane-Polyurea Hybrid Nanocarrier

Abstract

Integration of photosensitizers (PSs) within nanoscale delivery systems offers great potential for overcoming some of the "Achiles' heels" of photodynamic therapy (PDT). Herein, we have encapsulated a mitochondria-targeted coumarin PS into amphoteric polyurethane-polyurea hybrid nanocapsules (NCs) with the aim of developing novel nanoPDT agents. The synthesis of coumarin-loaded NCs involved the nanoemulsification of a suitable prepolymer in the presence of a PS without needing external surfactants, and the resulting small nanoparticles showed improved photostability compared with the free compound. Nanoencapsulation reduced dark cytotoxicity of the coumarin PS and significantly improved in vitro photoactivity with red light toward cancer cells, which resulted in higher phototherapeutic indexes compared to free PS. Importantly, this nanoformulation impaired tumoral growth of clinically relevant three-dimensional multicellular tumor spheroids. Mitochondrial photodamage along with reactive oxygen species (ROS) photogeneration was found to trigger autophagy and apoptotic cell death of cancer cells.

Figure 1

Structure of COUPY-based PSs investigated in this work.

Scheme 1. Schematic Representation of the Synthesis of the Amphiphilic Polyurethane–Polyurea Prepolymer (Steps 1–4) Followed the Nanoemulsion and Nanoencapsulation Processes (Steps 5–8)

Puzzle pieces codes: black for isophorone diisocyanate; blue for YMER N-120; green for N-(3-dimethylaminopropyl)-N,N′-diisopropanolamine; yellow for 2,2′-dihydroxyethyl disulfide; red for 1,3-diamino-N-octadecylpropane; pink for l-lysine, and orange for diethylenetriamine.

Figure 2

Schematic representation of the different moieties incorporated in the polyurethane-polyurea backbone of the NCs’ shell structure.

Figure 3

Characterization of NC-COUPY-2. (a) TEM micrograph (left). (b) UV–vis and emission spectra in water solution. (c) Photographic images of free and encapsulated COUPY 2 in daylight and in the dark upon irradiation with a green LED source.

Figure 4

Emission spectra of COUPY 2 (a) and NC-COUPY-2 (b) after green LED irradiation at different times.

Figure 5

Cellular uptake of COUPY 2 and NC-COUPY 2 at 37 and 4 °C. Single confocal planes of HeLa cells incubated with the compounds at 1 μM for 30 min at 37 °C or 4 °C. Scale bar: 20 μm.

Figure 6

Dose–response curves of COUPY 2 (a) and NC-COUPY 2 (b) in HeLa cells. (c) Comparison of half-maximal inhibitory concentration (IC₅₀) and phototoxic index (PI) values for light-activated COUPY compounds (0.5 h in dark +1 h red light irradiation followed by 48 h drug-free recovery period) in HeLa cells.

Figure 7

Comparison of half-maximal inhibitory concentration (IC₅₀) and PI values for light-activated COUPY compounds (0.5 h in dark + 1 h visible light irradiation followed by 48 h drug-free recovery period) under normoxia (21% O₂) and hypoxia (2% O₂) in HeLa cells.

Figure 8

Fluorescence microscopy images of HeLa spheroids treated with COUPY 2 and NC-COUPY 2 at 2 μM for 2 h. Scale bar: 100 μm.

Figure 9

Normalized volume of HeLa MCTS over a span of 9 days. MCTS were treated on day 3 with COUPY 2 or NC-COUPY 2 (2 μM) for 6 h in the dark and then exposed to red light irradiation (630 nm, 0.5 h, 89 mW/cm²). Data expressed as mean ± SD from three replicates. An independent unpaired t-test was used to define statistical differences between the values obtained on day 9 (***p < 0.001).

Figure 10

ROS generation in HeLa cells after light irradiation treatments with COUPY 2 and NC-COUPY 2 at 2 μM (1 h incubation + 1 h visible light irradiation). (a) ROS levels of HeLa cells on 2D monolayer cells or 3D MCTS stained with DCFH-DA for 0.5 h at 310 K after phototreatments and imaged on a Zeiss Axiovert inverted microscope; menadione (50 μM) being used as positive control. Scale bar: 200 μm. (b) Quantitation of oxidative stress based on DCF fluorescence after irradiation treatments. Three independent experiments were performed, and the error bars were calculated as the SD from the mean. Statistical significance control vs treatments determined via one-way ANOVA test (*p < 0.05; **p < 0.01 and ***p < 0.001).

Figure 11

Phototoxic mechanism of action in HeLa cancer cells after treatments with COUPY 2 or NC-COUPY 2 at IC₅₀^LIGHT concentrations (0.5 h incubation + 1 h visible light irradiation and 24 h recovery). (a) Flow cytometry analysis of the MMP using JC-1 dye. The mitochondrial phosphorylation inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP 50 μM, 24 h) was used as a positive control for mitochondrial dysfunction. (b) Apoptosis induction upon exposure to COUPY 2 or NC-COUPY 2 in the dark or after visible light irradiation treatments detected by flow cytometry as Annexin V-FITC fluorescence on the FL1-H channel; cisplatin (20 μM) was used as the positive control. (c) Number of autophagic processes detected in HeLa cells as quantified by confocal microscopy imaging through monodansylcadaverine (MDC) staining from >10 cells; resveratrol (50 μM, 2 h) was used as the positive control. (d) Mitochondrial oxidative phosphorylation on the basis of the OCR after 2 h treatment with tested complexes (10 μM) in the dark using the Seahorse XFe analyzer. All data represented as mean ± SD from three independent experiments. Statistical significance was determined via two-way ANOVA tests (*p < 0.05; ***p < 0.001).