Enhanced Photodynamic Cancer Treatment by Mitochondria-Targeting and Brominated Near-Infrared Fluorophores

Abstract

A noninvasive and selective therapy, photodynamic therapy (PDT) is widely researched in clinical fields; however, the lower efficiency of PDT can induce unexpected side effects. Mitochondria are extensively researched as target sites to maximize PDT effects because they play crucial roles in metabolism and can be used as cancer markers due to their high transmembrane potential. Here, a mitochondria targeting photodynamic therapeutic agent (MitDt) is developed. This photosensitizer is synthesized from heptamethine cyanine dyes, which are conjugated or modified as follows. The heptamethine meso-position is conjugated with a triphenylphosphonium derivative for mitochondrial targeting, the N-alkyl side chain is modified for regulation of charge balance and solubility, and the indolenine groups are brominated to enhance reactive oxygen species generation (ROS) after laser irradiation. The synthesized MitDt increases the cancer uptake efficiency due to the lipo-cationic properties of the triphenylphosphonium, and the PDT effects of MitDt are amplified after laser irradiation because mitochondria are susceptible to ROS, the response to which triggers an apoptotic anticancer effect. Consequently, these hypotheses are demonstrated by in vitro and in vivo studies, and the results indicate strong potential for use of MitDts as efficient single-molecule-based PDT agents for cancer treatment.

Keywords: cancer therapy; heptamethine cyanine dye; mitochondria targeting; near‐infrared (NIR) dye; photodynamic therapy.

Scheme 1

Structure and synthetic routes for MitDt groups. A) Precursor MitDt group (Pre‐MitDt group) was synthesized by substitution of R₁ and R₂ indolenine groups reacted with Vilsmeier–Haack reagent. B) TPP‐NH₂ (3‐aminopropyl)triphenylphosphonium was synthesized by substitution of bromine in 3‐bromopropylamine with triphenylphosphine. C) The MitDt group was synthesized through the S_RN1 mechanism of the pre‐MitDt group and the TPP‐NH₂ group.

Figure 1

The dependence of methanol content (in water) on the absorption and emission spectra of MitDt compounds.

Figure 2

Photodynamic efficacy of MitDt compounds. Time‐dependent singlet oxygen generation rate for 5 min A) and singlet oxygen generation rate after 5 min irradiation B) of MitDt compounds (150 × 10⁻⁶ m) were detected using singlet oxygen sensor green upon NIR laser irradiation in DPBS. C) ROS production of MitDt compounds (10 × 10⁻⁶ m) in NCI‐H460 tumor cells compared to nontreated group using 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA) without laser or after 5 min NIR laser irradiation. (D) and (E) are visualizations of in vitro ROS production level of brominated MitDt group using DCFH‐DA without laser, or after 5 min NIR laser irradiation in NCI‐H460 tumor cells measured by confocal laser scanning microscopy. A single irradiation from the NIR laser at 662 nm was given (100 mW cm⁻² power density) in all the in vitro tests.

Figure 3

Subcellular localization of MitDt compounds. Mitochondrial localization of MitDt‐1, MitDt‐2, and MitDt‐3 was evaluated using confocal laser scanning microscopy. NCI‐H460 A) and MCF‐7 B) cancer cells were stained with mitotracker (MitoTracker Orange CMTMRos) and incubated. After that, both cell lines were incubated with MitDt‐1 and MitDt‐3, followed by fixation using 4% paraformaldehyde. Nuclei were counterstained with DAPI. The distances in the fluorescence profiles were indicated by dash lines (Scale bar = 10 µm). P (Pearson's correlation coefficient) and M (Manders overlap coefficient) between the Mito‐tracker and NIR signal were obtained using the ZEN2 program (Carl Zeiss, Germany). Quantification of MitDt compounds on mitochondria was confirmed using mitochondria isolation kit (W TPP: MitDt compounds, W/O TPP: Pre‐MitDt compounds).

Figure 4

In vitro photoinduced therapeutic efficacy of MitDt‐1. Cell viability for NCI‐H460 A) and MCF‐7 D) were investigated using MTT assay. The relative mitochondrial membrane potential of NCI‐H460 B) and MCF‐7 E) cell lines were investigated using MitDt‐1 (35 × 10⁻⁶ m) and JC‐1 dye without or after NIR laser irradiation. The photodynamic therapeutic mechanisms in NCI‐H460 C) and MCF‐7 F) at different concentrations of MitDt‐1 were determined using flow cytometry. Cells were costained with propidium iodide (PI) and Annexin V‐FITC for flow cytometry analysis. A single 662 nm NIR laser irradiation was given (100 mW cm⁻² power density) for 5 min in all the in vitro tests.

Figure 5

In vivo photoinduced therapeutic efficacy of MitDt‐1. NIR fluorescence images of NCI‐H460 tumor bearing mice over time A), and ex vivo NIR fluorescence images B, C) after sacrifice of mice 6 h after injection were measured using IVIS. The contrast index D) was obtained by dividing the tumor NIR signal by the muscle signal obtained from Figure 5C. To investigate the antiproliferative activity of MitDt‐1 with laser irradiation, four groups were created: control, control + laser, MitDt‐1, and MitDt‐1 + laser. 100 × 10⁻⁶ m of MitDt‐1 (200 µL) was injected in a tail vein into NCI‐H460 tumor bearing mice. One hour after injection, the laser was used to irradiate the tumor in the “+ laser” groups for 5 min. A) Change in relative tumor size (mm³) and B) body weights of HuCCT1 xenograft‐bearing mice were monitored at predetermined times for 26 d (n = 5, error bars represent the standard deviation). Tumor tissue sections were excised from different treatment groups of mice after the 26th day. H) Excised tumor weights from different treatment groups and E) excised NCI‐H460 tumor images. I) Histology analysis after hematoxylin and eosin staining and TUNEL assay was performed for the different treatment groups using a virtual microscope (Scale bar = 60 µm).