Oxygen therapeutics has a range of applications in transfusion medicine and disease treatment. Synthetic molecules and all‐natural or semisynthetic hemoglobin‐based oxygen carriers (HBOCs) have seen success as potential circulating oxygen carriers. However, many early HBOC products stalled in development due to side effects from excess hemoglobin in the blood stream and hemoglobin entering the tissue. To overcome these issues, research has focused on increasing the molecular diameter of hemoglobin by polymerizing hemoglobin molecules or encapsulating hemoglobin in liposomal carriers. This work leverages the properties of silk fibroin, a cytocompatible and nonthrombogenic biopolymer, known to entrap protein‐based cargo, to engineer a fully protein‐based oxygen carrier. Herein, an all‐aqueous solvent evaporation technique is used to form silk particles via phase separation from a bulk polyvinyl alcohol phase. Particle size is tuned, and particles are formed with and without hemoglobin. The encapsulation efficiency and ferrous state of hemoglobin are analyzed, resulting in 60% encapsulation efficiency and a maximum of 20% ferric hemoglobin, yielding 100 μg mL−1 active hemoglobin in certain silk fibroin‐HBOCs formulations. The system does not elicit a strong inflammation response in vitro, demonstrating the potential for this particle system to serve as an injectable HBOC.

Oxygen therapeutics have a range of applications in transfusion medicine and disease treatment. Synthetic molecules and all-natural or semi-synthetic hemoglobin-based oxygen carriers (HBOCs) have seen success as potential circulating oxygen carriers. However, many early HBOC products were removed from the market due to side effects from excess hemoglobin in the blood stream and hemoglobin entering the tissue. To overcome these issues, research has focused on increasing the molecular diameter of hemoglobin by polymerizing hemoglobin molecules or encapsulating hemoglobin in liposomal carriers, where immune responses and circulation times remain a challenge. This work looks to leverage the properties of silk fibroin, a cytocompatible and non-thrombogenic biopolymer, known to entrap protein-based cargo, to engineer a silk fibroin-hemoglobin-based oxygen carrier (sfHBOC). Herein, an all-aqueous solvent evaporation technique was used to form silk fibroin particles with and without hemoglobin to tailor the formulation for specific particle sizes. The encapsulation efficiency and ferrous state of hemoglobin were analyzed, resulting in 60% encapsulation efficiency and a maximum of 20% ferric hemoglobin, yielding 100 µg/mL active hemoglobin in certain sfHBOC formulations. The system did not elicit a strong inflammation responsein vitro, demonstrating the potential for this particle system to serve as an injectable HBOC.Table of Contents FigureTable of Contents Figure:In this manuscript, we generate silk fibroin particles using an all-aqueous processing technique starting from silk fibroin polymer systems of differing molecular weights. We analyze the extent to which silk concentration and extraction time affect particle size. Further, we analyze the encapsulation of hemoglobin in the particle system and assess immune activation in macrophage-like cultures.