(M1030-07-42) Optimization of Perfluorocarbon-Based Oxygen Carriers for Machine Perfusion with Controllable Oxygen Release

Purpose: As of 2024, approximately one hundred thousand people are on the national transplant waiting list, with one person being added to the list every 8 minutes. The need for life-saving organ transplantation drives the need for innovative and efficient preservation techniques to ensure the quality of donated organs. The preservation approach efficacy directly correlates to the graft survival after transplantation and maintaining organ viability before transplant. Static cold storage (SCS) remains the standard method for organ preservation, with outstanding drawbacks of SCS, such as ischemic reperfusion injury (IRI) and formation of reactive oxygen species (ROS), emphasizing the need for developing new, better techniques. Machine perfusion allows for organ preservation at various temperatures, such as hypothermic (0-10 °C), sub-normothermic (10-36 °C), and normothermic (36-37 °C), by pumping perfusates through the organ vasculature[1]. Perfusates are pivotal in supplying oxygen and therapeutic agents to protect organs from reperfusion injury. Artificial oxygen carriers (AOCs) such as hemoglobin and perfluorocarbon-based carriers have the capacity to load and deliver oxygen exactly where it is needed. Perfluorocarbon nanoemulsions (PFC-NE), being chemically and metabolically inert and with high oxygen-dissolving capacity, are a viable alternative to acellular hemoglobin, whole blood, and standard non-oxygenated perfusates[2, 3]. However, PFC-NEs proved to be challenging to manufacture on a large scale with controlled quality attributes. Further, it is challenging to balance PFC-NEs’ oxygen loading capacity with their colloidal stability and osmotic and metabolic requirements for successful preservation. To solve these problems, we adopted a quality by design (QbD) approach to both optimize their oxygen carrier and perfusate properties, while we can also achieving successful manufacturing with maintained quality control on a large scale needed for clinical translation[4]. The presented study focuses on optimizing near-infrared fluorescence (NIRF) dye-loaded PFC-NE formulation with hydrocarbon oil and binary mixtures of pluronic surfactants, adopting the QbD approach. The stable NIRF labeling of the PFC-NE droplets allows for in-line continuous imaging of the perfusate during machine perfusion. Two different hydrocarbon (HC) oils of distinct physicochemical properties (HC1 being solid and HC2 being liquid at room temperature), two different perfluorocarbons, and two different surfactant systems were used. Previously, we have studied the impact of perfluorocarbon core on oxygen loading and release[5]. Here, the primary purpose is to investigate the impact of nanoemulsion shell (hydrocarbon oil and surfactant system) on oxygen loading and release patterns.

Methods: All nanoemulsions in the present study were manufactured on a microfluidizer LM20 following previously published procedures[5-7]. A 5-factor D-optimal design of experiment (DoE) is implemented, and nanoemulsions are manufactured using a high-energy microfluidization method. Colloidal stability studies are performed under stress conditions like incubation in the presence of biological fluid and fluorescence stability after 72 hours of incubation at 37 °C, mimicking normothermic and sub-normothermic perfusion systems. Oxygen loading and release kinetics are assessed with fiber optic oxygen sensors using a machine perfusion system.

Results: Out of the 13 batches from the DoE, one batch met all the critical quality attributes criteria. From the multiple linear regression analysis, it is evident that the nanoemulsion shell significantly impacts the oxygen release pattern. With increasing hydrocarbon oil concentration, the slower release of oxygen is observed, which is prominent for HC1. Since the PFC type does not have a significant impact on oxygen release, changing the PFC type based on other attributes like formulation stability, organ retention, and specific use is feasible. One-way analysis of change in fluorescence intensity shows that formulation with the higher melting-point lipid (HC1) demonstrates significantly lower loss in fluorescence. The optimized formulation shows an insignificant change in particle size and intensity distribution after exposure to centrifugation and serum-containing medium. The smaller slope value of oxygen kinetics indicates a slower rate of oxygen release between the two nanoemulsions with two different hydrocarbon oils.

Conclusion: Here we present an optimized PFC-NE perfusate platform with colloidal and fluorescence stability necessary for machine perfusion. The increased concentration of hydrocarbon oil offers more room to load hydrophobic therapeutic agents, and the slower release of oxygen eliminates the need for continuous oxygen supply. The consistent fluorescence signal of clinical-grade NIRF dye potentiates real-time monitoring of perfused organ regions by ex vivo imaging techniques, even at normothermic perfusion. To the best of our knowledge, this is the first time perfluorocarbon-based perfusates have been optimized with a controllable oxygen release profile.

References: 1. Smith, T.B., M.L. Nicholson, and S.A. Hosgood, Advances in hypothermic and normothermic perfusion in kidney transplantation. Transplantology, 2021. 2(4): p. 460-477.
2. Tadros, T., et al., Formation and stability of nano-emulsions. Advances in colloid and interface science, 2004. 108: p. 303-318.
3. Riess, J.G., Oxygen carriers (“blood substitutes”) raison d'être, chemistry, and some physiology blut ist ein ganz besondrer saft. Chemical reviews, 2001. 101(9): p. 2797-2920.
4. Lambert, E., V.S. Gorantla, and J.M. Janjic, Pharmaceutical design and development of perfluorocarbon nanocolloids for oxygen delivery in regenerative medicine. Nanomedicine, 2019. 14(20): p. 2697-2712.
5. Lambert, E. and J.M. Janjic, Quality by design approach identifies critical parameters driving oxygen delivery performance in vitro for perfluorocarbon based artificial oxygen carriers. Scientific reports, 2021. 11(1): p. 5569.
6. Vichare, R., et al., Theranostic nanoemulsions suppress macrophage-mediated acute inflammation in rats. J Nanobiotechnology, 2025. 23(1): p. 80.
7. Vichare, R., et al., Folate-conjugated near-infrared fluorescent perfluorocarbon nanoemulsions as theranostics for activated macrophage COX-2 inhibition. Sci Rep, 2023. 13(1): p. 15229.

Acknowledgements: This work was supported by CDMRP award HT9425-24-1-0828 (PI: Janjic) and DHA contract W81XWH-21-C-0055 (PI: Srinivas).


Figure 1: Evaluation of the critical quality attributes (CQAs) of the DoE nanoemulsions. The row with green color meets all the CQA specifications.


Figure 2: Design of experiments (DoE) results. (A) Prediction variance surface of the design across HC% and surfactant%, (B) actual vs predicted % oxygen release plot with a significant p-value of 0.0032, (C) effect summary stating the significance of the each factor in the MLR model, (D) one-way analysis of % oxygen release by hydrocarbon (HC) oil type, (E) Bivariate fit of % oxygen release by hydrocarbon oil%, and (F) one-way analysis of change in fluorescence intensity by hydrocarbon (HC) oil type.


Figure 3: Fluorescence and colloidal stability, and in vitro oxygen loading and release of PFC NE: (A) Comparison of fluorescence intensity after 72 hours of incubation at 37 °C, (B) comparison of particle size at different centrifugation speed, (C) intensity distribution of PFC NE after incubating 72 hours in presence of 20% serum containing medium and (D) comparison of oxygen loading and release measured in a prototype perfusion machine.