Preclinical Evaluation of a Peptide-amphiphile Derived Toroidal Oxygen Carrier

Abstract There is need for an artificial oxygen (O2) carrier to substitute for banked blood in settings where stored blood is unavailable or undesirable. Our goal is to design and optimize a blood substitute prototype (Nano-RBC/nRBC), that is based on a deformable nanoparticle, is morphologically similar to red blood cells (RBCs) and incorporates high per particle payloads of hemoglobin (Hb). This project will use machine learning tools (ML), computational studies, synthetic and biological experiments to design a 'smart' optimized O2-delivery biomaterial with physiological binding and dissociation properties, biodegradability, and no complement activation problems. Prior hemoglobin-based oxygen carriers (HBOCs) have failed because they do not preserve physiologic interactions of hemoglobin (Hb) with O2 and nitric oxide (NO). Nano-RBC design avoids these weaknesses by: 1) encapsulating Hb, 2) controlling O2 binding/release with a novel RSR-13/2,3-DPG shuttle, 3) attenuating NO uptake, and 4) retarding metHb formation. Moreover, Nano-RBC is designed for sterile lyophilization. This design constitutes a new class of Hb-encapsulated, toroidal-shaped nanoparticle formulated by self-assembly of amphiphilic lipopeptides. Toroidal shape affords increased stability, enhanced surface area for gas exchange, and improved rheology and vascular interactions. The toroidal particles are developed from amine rich β-turn peptide amphiphiles. The inherent cationic nature of the peptides enables RSR13 retention via electrostatic interaction. Moreover, these functionalities exhibit significant buffering centered upon physiologic pH; as pH falls below 7.4 (e.g., as in tissue), protonation of the amines increases, displacing RSR13 from the inner shell, thereby linking free [RSR] in the Nano-RBC cavity to ambient pH. Therefore, (as Hb O2 affinity falls in proportion to RSR availability) pH shifts encountered during circulatory transit facilitate O2 release at physiologically appropriate gas tensions. This physiologically responsive RSR reservoir-shuttle differentiates Nano-RBC from all prior HBOC designs. This project will focus upon refining the O2 affinity control system designed to enhance O2 transport under conditions of physiologic stress. Thus, our Aims are: Sp Aim 1: Design and syntheses of β-turn peptide amphiphiles and characterize self-assembly. Sp Aim 2: Develop computational tools to optimize self-assembly and functional properties of Nano-RBC. Sp Aim 3: Analyze and optimize Nano-RBC composition for efficient O2 delivery in vitro. Sp. Aim 4: Exploratory pharmacokinetic (PK) profiling and bio-distribution (Bio-D) studies. Upon project completion, we expect to have successfully optimized and evaluated key Nano-RBC functions: 1) O2 binding/release; 2) NO sequestration; and 3) optimized shell character. As such, we will have prepared Nano- RBC for the next stage of funding, preclinical evaluation, and development, readying the particle for: 1) formulation scale-up and 2) formal evaluation in large animal models of hemorrhage and hypoxemia (completion of both will be necessary prior to Phase I Trials in humans).