Info
Background:
Tumor hypoxia is a key microenvironmental factor that impairs the efficacy of oxygen-dependent photodynamic therapy (PDT), a major limitation for the clinical application of this minimally invasive cancer treatment strategy. Conventional photosensitizers are additionally constrained by shallow tissue imaging depth, inadequate photostability, and the absence of adaptive therapeutic functions to accommodate the hypoxic tumor microenvironment (TME). Second near-infrared window fluorescence imaging (NIR-II FLI), fluorine-19 magnetic resonance imaging (¹⁹F MRI), and photoacoustic imaging (PAI) represent advanced multimodal imaging modalities with advantages of deep-tissue penetration, high spatial resolution, and background-free visualization, yet their integration with hypoxia-responsive phototherapeutic capabilities within a single nanoplatform remains insufficiently explored. Aza-BODIPY dyes are attractive candidates for theranostic applications owing to their strong near-infrared absorption and tunable photophysical properties, but their practical use is limited by inherent aggregation tendencies and narrow emission windows. To date, fluorinated aza-BODIPY-based systems that combine switchable PDT/photothermal therapy (PTT) and integrated NIR-II FLI/¹⁹F MRI/PAI have not been reported.
Hypothesis/Objective:
The present study hypothesized that a fluorinated N-oxide aza-BODIPY (OFBD) nanoemulsion (OFBD-NP), co-formulated with fluorinated oil (Foil), could mediate hypoxia-triggered switching between PDT and PTT, while integrating NIR-II FLI/¹⁹F MRI/PAI for multimodal imaging-guided phototherapy of lung cancer. The primary objective was to construct this multifunctional nanoplatform and investigate its potential to address the core limitations of conventional photosensitizers, including hypoxia-induced PDT inefficiency and insufficient deep-tissue imaging capacity.
Methods:
OFBD was synthesized via the selective oxidation of a fluorinated aza-BODIPY precursor (FBD), and OFBD-NP as well as FBD-NP nanoemulsions were formulated using Foil, egg yolk lecithin, DSPE-PEG₂₀₀₀, and cholesterol; surface modification with cRGD was performed to enable αᵥβ₃ integrin-mediated tumor targeting. The physicochemical properties of the photosensitizers and nanoemulsions were characterized via nuclear magnetic resonance (NMR) spectroscopy, dynamic light scattering (DLS), cryo-transmission electron microscopy (cryo-TEM), and zeta potential analysis. Optical and phototherapeutic properties were evaluated using UV-vis absorption, fluorescence emission spectroscopy, singlet oxygen generation assays, and photothermal conversion efficiency measurements. In vitro studies were conducted on A549 lung cancer cells to assess cellular uptake (confocal laser scanning microscopy, ¹⁹F NMR/MRI), hypoxia-dependent CYP450-mediated bioreduction (HPLC, PAI), and phototoxicity (CCK-8, flow cytometry, live/dead staining). In vivo theranostic performance was investigated in BALB/c nude mice bearing subcutaneous A549 tumors, with multimodal imaging (NIR-II FLI, ¹⁹F MRI, PAI, photothermal imaging) used to track nanoemulsion distribution and tumor localization. Tumor growth was monitored for 20 days post-treatment, and systemic biocompatibility was evaluated via body weight tracking, hemolysis assays, and hematoxylin-eosin (H&E) staining of major organs.
Results:
OFBD-NP exhibited a hydrodynamic diameter of approximately 117 nm with a low polydispersity index and moderate negative zeta potential, maintaining colloidal stability in aqueous and biological media for at least 15 days. Nanoemulsion formulation induced J-aggregation of OFBD and FBD, resulting in red-shifted absorption and fluorescence emission spectra that extended into the NIR-II window (up to 1007 nm). Under normoxic conditions, OFBD efficiently generated singlet oxygen for PDT, while hypoxic conditions triggered CYP450-mediated reduction of OFBD to FBD, which switched the therapeutic modality to PTT with a photothermal conversion efficiency of 72% and concurrently activated PAI signals. Foil incorporation produced a unified ¹⁹F NMR signal to enhance ¹⁹F MRI sensitivity, while also improving tumor oxygen delivery to alleviate local hypoxia. cRGD modification promoted selective accumulation of OFBD-NP in tumor tissues, with peak multimodal imaging signals detected at 12–24 h post-intravenous injection. In vitro, OFBD-NP showed low dark cytotoxicity and significant phototoxicity under both normoxic (IC₅₀ = 6.92 μM) and hypoxic (IC₅₀ = 8.65 μM) conditions, inducing cancer cell apoptosis via PDT/PTT effects. In vivo, laser irradiation of OFBD-NP-treated tumor-bearing mice resulted in complete regression of subcutaneous A549 tumors with 100% tumor growth inhibition. No significant changes in body weight, hemolysis, or histopathological damage to major organs were observed in treated mice throughout the study period.
Conclusion:
This study developed a fluorinated aza-BODIPY nanoemulsion (OFBD-NP) that integrates NIR-II FLI/¹⁹F MRI/PAI multimodal imaging with hypoxia-responsive PDT/PTT switching for lung cancer theranostics. Hypoxia-triggered CYP450-mediated bioreduction of OFBD to FBD enables adaptive phototherapeutic switching, while Foil incorporation enhances ¹⁹F MRI sensitivity and tumor oxygen delivery, and cRGD modification facilitates active tumor targeting. The nanoplatform achieves precise multimodal imaging-guided phototherapy and complete tumor ablation in a subcutaneous A549 lung cancer model, with favorable in vitro and in vivo biocompatibility. This design provides a rational strategy for constructing multifunctional nanotheranostics that address the limitations of tumor hypoxia and shallow imaging depth in conventional phototherapy. Limitations of the study include the use of a subcutaneous tumor model, and future research will focus on enhancing ¹⁹F MRI sensitivity and evaluating the platform in orthotopic lung cancer models to support further preclinical and clinical translation.
Author
Zhongxing Jiang
Chinese Academy of Sciences