Perfluorocarbon Nanoemulsions for Simultaneous Delivery of Oxygen and Antioxidants During Machine Perfusion Supported...

Background: Solid organ transplantation (SOT) is a life-saving treatment for patients with end-stage diseases and/or organ failure. However, access to healthy organs is often limited by challenges in organ preservation. Furthermore, upon transplantation, ischemia-reperfusion injury (IRI) can lead to increased organ rejection or graft failures. The work presented aims to address both challenges using an innovative nanomedicine platform for simultaneous drug and oxygen delivery. In recent studies, resveratrol (RSV), a natural antioxidant, anti-inflammatory, and reactive oxygen species (ROS) scavenging agent, has been reported to protect against IRI by inhibiting ferroptosis. Here, we report the design, development, and scalable manufacturing of the first-in-class dual-function perfluorocarbon-nanoemulsion (PFC-NE) perfusate for simultaneous oxygen and antioxidant delivery, equipped with a near-infrared fluorescence (NIRF) reporter, longitudinal, non-invasive NIRF imaging of perfusate flow through organs/tissues during machine perfusion. Methods: A Quality-by-Design (QbD)-guided optimization was used to formulate a triphasic PFC-NE with 30% w/v perfluorooctyl bromide (PFOB). Drug-free perfluorocarbon nanoemulsions (DF-NEs) and RSV-loaded nanoemulsions (RSV-NEs) were produced at 250-1000 mL scales using M110S, LM20, and M110P microfluidizers. Colloidal attributes, fluorescence stability, drug loading, and RSV release were evaluated using DLS, NIRF imaging, and HPLC, respectively. PFC-NE oxygen loading and release kinetics were evaluated during perfusion through the BMI OrganBank^® machine with the MEDOS HILITE^® oxygenator and by controlled flow of oxygen. The in vitro antioxidant activity of RSV-NE was measured using the oxygen radical scavenging antioxidant capacity (ORAC) assay. The cytotoxicity and ferroptosis inhibition of RSV-NE were evaluated in RAW 264.7 macrophages. Results: PFC-NE batches maintained a consistent droplet size (90-110 nm) and low polydispersity index (<0.3) across all scales, with high reproducibility and >80% PFOB loading. Both DF-NE and RSV-NE maintained colloidal and fluorescence stability under centrifugation, serum exposure at body temperature, filtration, 3-month storage, and oxygenation. Furthermore, RSV-NE showed high drug loading and sustained release (63.37 ± 2.48% at day 5) compared with the rapid release observed in free RSV solution. In perfusion studies, the oxygenation capacity of PFC-NE consistently exceeded that of University of Wisconsin (UW) solution and demonstrated stable, linear gas responsiveness across flow rates and FiO₂ (fraction of inspired oxygen) inputs. RSV-NE displayed strong antioxidant activity and concentration-dependent inhibition of free radicals. RSV-NE maintained higher cell viability and prevented RAS-selective lethal compound 3 (RSL3)-induced ferroptosis in murine macrophages (macrophage cell line RAW 264.7), compared to the free RSV solution. Morphological and functional protection against RSL3-induced ferroptosis was confirmed microscopically. Conclusions: This study establishes a robust and scalable PFC-NE platform integrating antioxidant and oxygen delivery, along with NIRF-based non-invasive live monitoring of organ perfusion during machine-supported preservation. These combined features position PFC-NE as a promising next-generation acellular perfusate for preventing IRI and improving graft viability during ex vivo machine perfusion.