Next-Generation Photosensitizers: Cyanine-Carborane Salts for Superior Photodynamic Therapy of Metastatic Cancer

Abstract

Photodynamic therapy (PDT) has emerged as a promising targeted treatment for cancer. However, current PDT is limited by low tissue penetration, insufficient phototoxicity (toxicity with light irradiation), and undesirable cytotoxicity (toxicity without light irradiation). Here, we report the discovery of cyanine-carborane salts as potent photosensitizers (PSs) that harness the near-infrared (NIR) absorbing [cyanine⁺] with the inertness of [carborane^-]. The implementation of [cyanine⁺] [carborane^-] salts dramatically enhance cancer targeting of the PSs and decrease cytotoxicity. We characterize the cellular uptake of the cyanine-carborane PSs, organelle localization, generation of reactive oxygen species (ROS) with the ability to cogenerate multiple ROS species, suppression of pro-metastatic pathways, and activation of apoptotic pathways. We further demonstrate the ability of optimized PSs to eliminate tumors in vivo using an orthotopic mouse model of breast cancer. These newly developed [cyanine⁺] [carborane^-] salt PSs introduce a potent therapeutic approach against aggressive breast cancer while decreasing side effects.

Keywords: Carborane anion; Cyanine; Metastatic breast cancer; Near-infrared; Photodynamic therapy.

Figure 1

Cyanine‐carborane photosensitizers are potent inhibitors of breast cancer cellular viability. (A) Photoactive heptamethine cyanine cation (Cy⁺) was paired with carborane anions CB₉H₁₀ ⁻, CB₁₁H₆Cl₆ ⁻, CB₉HCl₉ ⁻ _, C₁₀B₁₁H₃₂Si⁻, and C₅B₂₂N₂H₂₅ ⁻ to tune the energy level of organic salt PSs. (B) Frontier energy levels of cyanine‐carboranes determined by ultraviolet photoelectron spectroscopy (UPS). Cellular viability of 4T1 mouse mammary cancer cells treated with indicated concentrations of (C) CyCB₁₁H₆Cl₆ or (D) CyCB₉HCl₉ with or without 30 min of daily 850 nm light irradiation over a 5‐day duration. Crystal violet staining was used to visualize the cellular confluency on day 5. Scale bars: 300 μm. The number of viable cells was quantified using trypan blue and presented as log₂ fold changes. Data are presented as the mean ± S. D. (n=3, *p <0.05, **p<0.01, ****p <0.001).

Figure 2

Cyanine‐carboranes preferentially localize to the mitochondria and their uptake relies on organic‐anion transporting polypeptides. (A) Representative microscopic images showing PSs (red) and respective target tracker (green) distribution in 4T1 cells. The r‐value corresponds to the Pearson correlation coefficient, reflecting the level of colocalization between cyanine‐carboranes and their respective target tracker. Scale bars: 100 μm. (B) Fluorescent images of targeted endocytosis inhibitors showing the nucleus (Hoechst; blue) and PS (red) distribution in 4T1 cells. Scale bars: 100 μm. Control cells (vehicle; Veh) were treated with 0.1 % DMSO.

Figure 3

Photoactivation of cyanine‐carboranes generates simultaneous mitochondrial and cytoplasmic reactive oxygen species in breast cancer cells. After 24 h incubation of 4T1 cells with or without PSs (CyCB₁₁H₆Cl₆ and CyCB₉HCl₉), cells were briefly washed with PBS and irradiated with 850 nm or remained in dark conditions (Veh). Following light irradiation, cells were immediately incubated with three ROS‐sensing dyes: (A) a general cytoplasmic ROS‐sensing dye, (B) a singlet oxygen sensing dye, and (C) a mitochondrial ROS‐sensing dye. Scale bars: 100 μm. The white arrows in the phase contrast indicate early cell death (apoptosis). The ROS levels were measured in relative fluorescence units and normalized to the mean ROS level of control cells (Veh) at the 30‐min mark. Data are presented as the mean ± S. D. (n=3, *p <0.05, **p <0.01, ***p <0.001). ns; not significant.

Figure 4

Photoactivation of cyanine‐carboranes suppresses metastatic capacity of breast cancer cells. (A) Representative microscopic images after 24 h of wound closure in cells treated with or without PSs under 850 nm light exposure or dark conditions. (B) Real‐time wound closure of the cells for 24 h immediately following irradiation with 850 nm light. (C) western blot analysis of metastatic markers E‐cadherin, N‐cadherin, Vimentin, and Snail expression levels in 4T1 cells treated with or without PSs under 850 nm light exposure (30 min) or dark conditions harvested at 6 h time point. (D) Representative microscopic images of F‐actin filaments (rhodamine–phalloidin) and nucleus (Hoechst) in 4T1 cells treated with or without PSs under 850 nm light exposure (30 min) or dark conditions at 6 h time point. Scale bars: 400 μm. (E) The total GTP/ATP ratio of cells was quantified using liquid chromatography‐mass spectrometry at 6 h time point. All data are shown as the mean ± S. D. (n=3, *p<0.05). ns; not significant.

Figure 5

Photoactivation of cyanine‐carboranes induces early apoptosis and disrupts mitochondrial function in breast cancer cells. (A) Cells were stained with annexin V conjugated to fluorescein isothiocyanate (FITC) and propidium iodide (PI) and analyzed by flow cytometry with or without PSs under 850 nm light exposure (30 min) or in dark conditions at 6 h time point. (B) Quantitative RT‐PCR was employed to assess the messenger ribonucleic acid (mRNA) levels of Bax, Bak, and Bid. Data were normalized to the β‐actin reference gene expression. (C) western blot analysis of p‐ERK 1/2, T‐ERK 1/2, p‐MLKL, PARP, Cytochrome c, and Bax expression levels in 4T1 cells at 6 h time point. (D) Representative fluorescence images of mitochondrial membrane potentials (ΔΨm) in 4T1 cells. N.I: Normalized intensity; intensities of mitochondrial membrane potential were normalized against the no‐treatment cell conditions in dark or under 850 nm exposure. Scale bar: 100 μm. Data are presented as the mean ± S. D. (n=3, *p <0.05, **p <0.01, ***p <0.001).

Figure 6

PDT using cyanine‐carboranes arrests tumor growth in a mouse model of breast cancer. (A) Representative microscopic images showing the precise localization of PSs within breast tumor cells. (B) The fluorescence intensity of cyanine‐carboranes uptake within the tumor site was monitored over a period of 5 days following the administration of the PSs. (C) The tumor volume was measured every other day following the initiation of PDT in mice. Data are presented as the mean ± S. D. (n=5, ****p <0.001). (D) Schematic illustration of the mechanism of action of cyanine‐carborane PSs in breast cancer cells.