Category: Formulation and Quality
Purpose: Effective tissue preservation strategies are key to improving outcomes of extremity salvage and limb transplantation. Static cold storage (SCS) at 4 °C is the current “gold standard” for preserving tissues. However, cold preservation contributes to damage associated with hypothermic storage and fails to eliminate ischemia-reperfusion injury (IRI) (Giwa et al. 2017). Hypoxia during SCS leads to anaerobic metabolism with loss of cell membrane integrity, reduced interstitial osmotic pressure, and mitochondrial disruption with cell death. One crucial element to maintaining tissue integrity is providing adequate oxygen (O2) to support aerobic metabolism and avoid IRI and its associated repercussions. Normothermic conditions are ideal for avoiding IRI during tissue preservation, but strategies must guarantee availability of an adequate oxygen supply. Whole blood (WB) supplementation can be used to treat patients, but risk of infection, religious beliefs, and WB shortages can limit practicality (Castro and Briceno 2010). Artificial oxygen carriers (AOC) derived from perfluorocarbon (PFC) materials can replenish oxygen in lieu of WB without risk of infection, can be commercially manufactured, are chemically and biologically inert, and are resistant to temperature or prolonged storage (Riess 2001). Furthermore, during the reperfusion stage of IRI, reactive oxygen species (ROS) generated by resident tissue cells react with other species such as nitric oxide (NO) to produce more potent or reactive molecules that may initiate denaturation of proteins. Demand for a platform capable of serving simultaneous oxygen supply and ROS scavenger delivery functions delivery exists in tissue preservation. No clinically available PFC AOC exists even though several have entered clinical trials. Design of PFC AOC has historically used one-factor-at-a-time (OFAT) approaches, and development currently is stagnant. We present a novel, more systematic methodology of formulating PFC AOC. The proposed approach comprises methodical experimental design to prepare PFC-in-hydrocarbon-in-water nanoemulsions, thorough colloidal stability characterization, and multivariate modeling which we hypothesize will accelerate progress in discovering a clinically viable AOC.
Methods: We developed complex nanoemulsions consisting of three distinct liquid phases: PFC oil, hydrocarbon oil, and aqueous component. Nanoemulsions were prepared using an ultrasonication pre-emulsification step (Model 500 Ultrasonic Dismembrator, FisherScientific, Pittsburgh, PA) followed by processing on a microfluidizer (M110S, Microfluidics, MA, USA). A rational formulation approach utilizing design of experiments (DoE) and statistical multivariate modeling was used to evaluate the effects of nanoemulsion composition on critical quality attributes. Statistical treatment of data was performed using JMP Pro 13.0.0 (SAS, Cary, NC). Colloidal properties (droplet diameter, polydispersity, zeta potential, stability) of nanoemulsion formulations were thoroughly characterized by dynamic light scattering (Zetasizer Nano, Malvern Instruments, Worcestershire, UK). In vitro biological assessment included cell toxicity and cellular uptake in RAW 264.7 macrophage cell line. Oxygen carrying capacity was examined with a polarographic dissolved oxygen probe (HI5421 Dissolved Oxygen Bench Meter, Hanna Instruments, Woonsocket, RI) after bubbling oxygen to degas the nanoemulsion.
Results: Colloidal stability data identified a robust PFC nanoemulsion formulation that exhibits adequate shelf-life. The droplet diameter was < 200 nm and the size distribution narrow (polydispersity index < 0.2), ensuring that the droplet dimensions are suitable for distributing their payload into the microvasculature. A formulation design space was identified in which droplet size could be manipulated by adjusting nanoemulsion composition (Figure 1). Scale-up of the nanoemulsion from 25mL to 100mL was demonstrated which displayed properties comparable to those at small scale (Figure 2). Oxygen capacity and oxygen release kinetics were documented.
Conclusion: The proposed approach to design of a PFC nanoemulsion oxygen-carrier seeks to make advancements in bringing a clinically viable oxygen therapeutic onto the market by branching beyond conventional OFAT investigations. This rational approach is directed by DoE and process modeling to understand the critical quality attributes and how they are affected by critical process parameters.
Castro, C. I., and J. C. Briceno. 2010. 'Perfluorocarbon-based oxygen carriers: review of products and trials', Artif Organs, 34: 622-34.
Giwa, Sebastian, Jedediah K. Lewis, Luis Alvarez, Robert Langer, Alvin E. Roth, George M. Church, et al. 2017. 'The promise of organ and tissue preservation to transform medicine', Nature Biotechnology, 35: 530-42.
Lambert, E., and J. M. Janjic. 2019. 'Multiple linear regression applied to predicting droplet size of complex perfluorocarbon nanoemulsions for biomedical applications', Pharm Dev Technol: 1-11.
Riess, J. G. 2001. 'Oxygen carriers ("blood substitutes")--raison d'etre, chemistry, and some physiology', Chem Rev, 101: 2797-920.