Oxygen-ReleasingNanodroplets Relieve IntratumoralHypoxia and Potentiate Photodynamic Therapy in 3D Head and Neck CancerSpheroids

Hypoxia in solid tumors, including head and neck cancer (HNC), contributes to treatment resistance, aggressive tumor phenotypes, and poorer clinical outcomes. Perfluorocarbon nanodroplets have emerged as promising drugs to alleviate tumor hypoxia. These versatile nanocarriers can also encapsulate and deliver various therapeutic agents, offering a multifunctional approach to cancer treatment. However, a detailed characterization of hypoxia alleviation, particularly the duration of hypoxia treatment drug residence, has not been thoroughly investigated. In this study, we developed and characterized perfluoropentane nanodroplets (PFP NDs) for the codelivery of oxygen and the photoactivatable drug benzoporphyrin derivative (BPD) to hypoxic HNC spheroids. The PFP NDs exhibited excellent stability, efficient oxygen loading/release, and biocompatibility. Using 3D multicellular tumor spheroids of FaDu and SCC9 HNC cells, we investigated the spatiotemporal dynamics of hypoxia within these spheroids and the ability of oxygenated PFP NDs to alleviate hypoxia. Our results showed that oxygen-loaded PFP NDs effectively penetrated the core of tumor spheroids, significantly reducing hypoxia, as evidenced by the downregulation of hypoxia-inducible factors HIF-1α and HIF-2α. Importantly, we demonstrated sustained hypoxia alleviation for up to 3 h post-treatment with PFP NDs. BPD-loaded PFP NDs successfully delivered the photosensitizer into the spheroid core in a time-dependent manner. Furthermore, we evaluated the efficacy of oxygen-dependent treatment modality, namely, photodynamic therapy (PDT) with BPD and oxygen-loaded PFP NDs compared to free BPD. The NDs formulation exhibited superior PDT outcomes, which were attributed to improved oxygen availability during the treatment. This study provides comprehensive evidence for the potential of PFP NDs as a codelivery platform to overcome hypoxia-mediated treatment resistance and enhance PDT efficacy in HNC. Our findings pave the way for further investigation of this promising approach in more complex <i>in vivo</i> models, potentially leading to improved therapeutic strategies for hypoxic solid tumors.