Aza-BODIPY based carbonic anhydrase IX: Strategy to overcome hypoxia limitation in photodynamic therapy

Abstract

Hypoxia caused by photodynamic therapy (PDT) is a major hurdle to cancer treatment since it can promote recurrence and progression by activating angiogenic factors, lowering therapeutic efficacy dramatically. In this work, AZB-I-CAIX₂ was developed as a carbonic anhydrase IX (CAIX)-targeting NIR photosensitizer that can overcome the challenge by utilizing a combination of CAIX knockdown and PDT. AZB-I-CAIX₂ showed a specific affinity to CAIX-expressed cancer cells and enhanced photocytotoxicity compared to AZB-I-control (the molecule without acetazolamide). Moreover, selective detection and effective cell cytotoxicity of AZB-I-CAIX₂ by PDT in hypoxic CAIX-expressed murine cancer cells were achieved. Essentially, AZB-I-CAIX₂ could minimize tumor size in the tumor-bearing mice compared to that in the control groups. The results suggested that AZB-I-CAIX₂ can improve therapeutic efficiency by preventing PDT-induced hypoxia through CAIX inhibition.

Keywords: PDT; acetazolamide; aza-BODIPY; carbonic anhydrase IX (CA9); hypoxia.

FIGURE 1

Structures of targeting probe (AZB-I-CAIX₂) and control (AZB-I-control) in this study.

SCHEME 1

Synthetic scheme of AZB-I-CAIX₂ and AZB-I-control.

FIGURE 2

Normalized Vis-NIR absorption and emission spectra of AZB-I-CAIX₂ in various solvents.

FIGURE 3

Selective CAIX-dependent uptake of AZB-I-CAIX₂ on various cells. (A) Time-dependent cell internalization of AZB-I-CAIX₂ tested on CAIX+ (MDA-MB-231) and CAIX- (MCF-7, HeLa, A549, HEK293) cells. (B) Corrected Total Cell Fluorescence (CTCF) of signals from experiment (A) (C) Confocal images of AZB-I-CAIX₂ internalized CAIX+ (MDA-MB-231) and CAIX- (MCF-7) in the co-culture system. Statistical analysis: One-way ANOVA followed by Tukey’s analysis was used for comparison between multiple groups using GraphPad Prism9 software. p values of less than 0.05 (95% confidence interval) are considered significant (ns p < 0.12, * p < 0.033, ** p < 0.002, *** p < 0.001). Scale bar = 20 μm.

FIGURE 4

Inhibitory effect of CAIX inhibitor (acetazolamide) on the cellular uptake of AZB-I-CAIX₂. (A) Confocal images of MDA-MB-231 cells incubated with AZB-I-CAIX₂ (5 μM) in the absence and presence of acetazolamide (0.5 mM and 1.0 mM) for 6 h. (B) Corrected Total Cell Fluorescence (CTCF) of signals from experiment (A) (C) Colocalization of AZB-I-CAIX₂ with organelle trackers (MitoTracker, LysoTracker, Golgi and ER markers) in MDA-MB-231 cells with Pearson’s coefficients of 0.47, 0.22, 0.28 and 0.74, respectively. Scale bar = 20 μm.

FIGURE 5

Half maximal inhibitory concentration (IC₅₀) curves of AZB-I-CAIX₂ tested on CAIX+ (MDA-MB-231) and CAIX- (MCF-7, HeLa, A549, HEK293) cells under various light exposure times (0 min, 5 min, 10 min, 15 min). The cells were incubated with various concentrations of AZB-I-CAIX₂ (0–10 μM) for 6 h and irradiated with a lamp (660 nm, power density of 8.7 mW cm⁻²). IC₅₀ was evaluated using GraphPad Prism9 software.

FIGURE 6

Viability/Cytotoxicity assay and intracellular ROS detection. (A) Live/dead cell imaging of CAIX+ (MDA-MB-231) and CAIX- (MCF-7, HeLa, A549, HEK-293) cells incubated with AZB-I-CAIX₂ (0.5 μM) under 10 min light irradiation. (B) Confocal images of MDA-MB-231 cells incubated with AZB-I-CAIX₂ (0.5 μM) and light irradiation for 10 min in the presence of ROS detection probe, DCFH-DA, and ROS scavengers, NAC or NaN₃. The green emission signal indicated the existence of ROS inside the cells. (C) Flow cytometry Annexin V fluorescein isothiocynate (FITC)/propidium iodide (PI) apoptosis analysis.

FIGURE 7

(A) Time-dependent cell internalization of AZB-I-CAIX₂ under normoxia and hypoxia conditions of 4T1 and 67NR cells. (B) IC₅₀ curves of 4T1 cells incubated with AZB-I-CAIX₂ for 6 h before light illumination for 0 min, 5 min, 10 min, and 15 min.

FIGURE 8

In vivo acute toxicity and antitumor efficacy of AZB-I-CAIX₂, AZB-I-Control, and acetazolamide without irradiation. (A) Percentage of mice body weight (n = 2) for in vivo acute toxicity study throughout 14 days of observation. (B) Gross observation of tumor at days 0, 3, and 7 post-PDT. The figure in each group is representative of n = 6 mice. (C) 4T1 tumor volume post-AZB-I-CAIX₂, AZB-I-Control and acetazolamide treatment, and PDT. ** p < 0.0001 for AZB-I-CAIX₂ vs control saline, AZB-I-Control, and acetazolamide at all time points. (D) The area under the tumor volume in AZB-I-CAIX₂, AZB-I-Control, and acetazolamide throughout 21 days of analysis. The graph represents mean ± SEM (n = 6), * p < 0.001 and ** p < 0.0001 compared to control saline, using One Way ANOVA (Dunnett’s). (E) H&E histological analysis of 4T1 tumor tissue post-three days of PDT. (Left) Control saline and PDT. (Right) 30 mg/kg AZB-I-CAIX2 and PDT with necrotic tissue. The picture shown is a representative of each group with similar results. Magnification: ×40.