Perfluorocarbon nanodroplets can reoxygenate hypoxic tumors in vivo without carbogen breathing

Abstract

Nanoscale perfluorocarbon (PFC) droplets have enormous potential as clinical theranostic agents. They are biocompatible and are currently used in vivo as contrast agents for a variety of medical imaging modalities, including ultrasound, computed tomography, photoacoustic and ¹⁹F-magnetic resonance imaging. PFC nanodroplets can also carry molecular and nanoparticulate drugs and be activated in situ by ultrasound or light for targeted therapy. Recently, there has been renewed interest in using PFC nanodroplets for hypoxic tumor reoxygenation towards radiosensitization based on the high oxygen solubility of PFCs. Previous studies showed that tumor oxygenation using PFC agents only occurs in combination with enhanced oxygen breathing. However, recent studies suggest that PFC agents that accumulate in solid tumors can contribute to radiosensitization, presumably due to tumor reoxygenation without enhanced oxygen breathing. In this study, we quantify the impact of oxygenation due to PFC nanodroplet accumulation in tumors alone in comparison with other reoxygenation methodologies, in particular, carbogen breathing. Methods: Lipid-stabilized, PFC (i.e., perfluorooctyl bromide, CF₃(CF₂)₇Br, PFOB) nanoscale droplets were synthesized and evaluated in xenograft prostate (DU145) tumors in male mice. Biodistribution assessment of the nanodroplets was achieved using a fluorescent lipophilic indocarbocyanine dye label (i.e., DiI dye) on the lipid shell in combination with fluorescence imaging in mice (n≥3 per group). Hypoxia reduction in tumors was measured using PET imaging and a known hypoxia radiotracer, [¹⁸F]FAZA (n≥ 3 per group). Results: Lipid-stabilized nanoscale PFOB emulsions (mean diameter of ~250 nm), accumulated in the xenograft prostate tumors in mice 24 hours post-injection. In vivo PET imaging with [¹⁸F]FAZA showed that the accumulation of the PFOB nanodroplets in the tumor tissues alone significantly reduced tumor hypoxia, without enhanced oxygen (i.e., carbogen) breathing. This reoxygenation effect was found to be comparable with carbogen breathing alone. Conclusion: Accumulation of nanoscale PFOB agents in solid tumors alone successfully reoxygenated hypoxic tumors to levels comparable with carbogen breathing alone, an established tumor oxygenation method. This study confirms that PFC agents can be used to reoxygenate hypoxic tumors in addition to their current applications as multifunctional theranostic agents.

Keywords: PET imaging; nanodroplets; perfluorocarbon; reoxygenation; tumor hypoxia.

Figure 1

(A) Schematic of DiI-loaded, PFOB nanodroplet with a PFOB core and a lipid shell comprised of lecithin and DPPE-PEG2000. The hydrophobic tails of lipids face inward towards the slightly lipophilic PFOB, while the hydrophilic heads of the lipids face outward along with the water-soluble PEG chains. (B) Hydrodynamic mean diameter (Z-average) distribution of both plain and DiI-labeled PFOB nanodroplet as measured by DLS immediately after preparation and purification. (C) Size stability of both plain and DiI-labeled PFOB nanodroplet at two different concentrations, 1 mg/mg and 200 mg/mL. All samples were tested at three temperatures (22 °C, 37 °C, and 4 °C) for 20 days. Error bars indicate the standard deviation of the replicated samples (n=3).

Figure 2

Biodistribution of DiI-labeled PFOB nanodroplets in NRG mice bearing DU145 tumor xenografts: (A) Representative ex vivo fluorescence images of organs and tumor tissues acquired at 3h, 6h, and 24h post-injection of PFOB nanodroplets.) (B) The quantitative fluorescence signal of DiI acquired from organs and tumors tissues acquired at 3h, 6h, and 24h post-injection (n≥3, p-value <0.005 from Tukey test was shown as ***).

Figure 3

(A) For each animal, the 3D anatomical (CT) and molecular (PET) modalities were co-registered and the tissue segmentations including muscle, heart, and tumor were performed based on the anatomical image for quantitative hypoxia analysis. (B) The signal of [¹⁸F]FAZA (expressed in %ID/g) from PET imaging detected in the muscle, heart, and tumor segmentation from each animal and the mean ± standard deviation. (C) The normalized tumor-to-muscle ratio for each animal (Black triangles) and the mean ratio ± standard deviation (Red circles) (n≥3, p values <0.05, <0.01, and <0.005 from Tukey test was shown as *, **, and ***).