(356d) Protein-Polypeptide Complex As Oxygen Carrier

Protein delivery strategies must preserve bioactivity, structural integrity, and sufficient circulation time due to the inherent complexity of therapeutic proteins. These proteins are often highly susceptible to enzymatic degradation and exhibit short circulation half-lives in vivo. Although PEGylation has improved circulation stability, the emergence of anti-PEG antibodies can significantly reduce its effectiveness by accelerating blood clearance. Proteins with complex quaternary structures, such as hemoglobin, face additional delivery challenges due to their susceptibility to subunit dissociation and oxidative degradation.

Objective

This study introduces a delivery approach tailored for complex quaternary proteins, using hemoglobin as a model system. The objective is to form stable protein–polypeptide nanocomplexes by electrostatically interacting negatively modified hemoglobin with polycationic carriers such as poly(L-arginine) and poly(L-lysine). To enhance biocompatibility and colloidal stability, these intermediate complexes will be further layered with anionic polypeptides such as poly(L-aspartic acid) or poly(L-glutamic acid).

Method

By varying the ratio of hemoglobin to polycation, the formulation will transition from a protein–polypeptide coacervate to nanoscale complexes. Turbidity measurements will be used to distinguish between coacervate and nanocomplex regimes. In the nanoscale range, dynamic light scattering (DLS) and zeta potential analyses will be employed to determine particle size and surface charge, and stability will be evaluated after 48 hours. UV–VIS spectroscopy will characterize the hemoglobin oxidation state (oxyhemoglobin vs. methemoglobin), while circular dichroism (CD) spectroscopy will verify the preservation of secondary structure post-complexation. Nanoparticle serum stability will be assessed in fetal bovine serum (FBS). To evaluate oxygen-carrying capacity, oxygen dissociation curves will be generated. Cytotoxicity will be investigated in human umbilical vein endothelial cells (HUVECs) using PrestoBlue and LDH assays. Lastly, nitric oxide (NO) binding behavior, a critical factor due to its potential for inducing vasoconstriction, will also be examined post-modification.

This study aims to develop a biocompatible, stable hemoglobin-based oxygen carrier with therapeutic potential, providing an alternative in protein drug delivery.