Sonication Effectively Reduces Nanoparticle Size in Hemoglobin-Based Oxygen Carriers (HBOCs) Produced Through Coprecipitation: Implications for Red Blood Cell Substitutes

Abstract

Engineered hemoglobin (Hb) particles are O2 delivery vehicles that may be used as red blood cell (RBC) substitutes when blood is unavailable. One promising method produces high-purity Hb particles through coprecipitation with a removable inorganic template that yields porous Hb particles; however, this method traditionally results in particle diameters too large for applications in transfusion medicine. The current study investigates the incorporation of a sonication operation during Hb particle synthesis to eliminate aggregates and decrease mean particle diameter. We report an improved synthesis method for Hb nanoparticles, having optimized reagent ratios for increased Hb entrapment. The highest entrapment was observed at an equimolar ratio of 0.65 M inorganic precursors and a Hb concentration of 7.5 mg/mL. Sonication drastically reduced Hb particle diameter from a range of several micrometers to a mean diameter of ∼250 nm. The benefits of increased sonication duration and intensity rapidly plateaued, being able to achieve an order of magnitude reduction in particle size after 5 min of moderate sonication. We evaluated the Hb nanoparticles for in vitro O2 transport to validate their potential in transfusion medicine applications, revealing an O2 offloading rate constant of 4.2 s–1, and O2 affinity (i.e., P 50) of 8.66 mmHg, which was comparable to previously reported Hb-based O2 carriers (HBOCs). Taken together, these data describe a synthesis method capable of reliably producing Hb nanoparticles of reduced size, without the presence of large particles or aggregates while maintaining O2 transport characteristics.

Synthesis of Nanoparticles Fully Made of Hemoglobin with Antioxidant Properties: A Step toward the Creation of Successful Oxygen Carriers

“…These results are in agreement with the observations of the Palmer group. 43 Specifically, McDonel et al reported slight decreases in the size of Hb-NPs for the higher sonication values in terms of time and amplitude. However, in their study, the Hb-NPs were prepared by the coprecipitation, cross-linking, and dissolution method.…”

“…However, in their study, the Hb-NPs were prepared by the coprecipitation, cross-linking, and dissolution method. 43 3.1.4. Characterization of Hb-NPs.…”

“…15 However, an important drawback of these Hb-NPs is the low throughput of the electrospraying technique, which creates challenges for their production at clinically relevant scales. 16 Using a different approach, the Palmer group was able to synthesize Hb-NPs by borrowing the techniques currently used for the fabrication of protein NPs. Specifically, using the desolvation precipitation method, the authors reported Hb-NPs with a diameter of ∼250 nm that were able to bind and release oxygen.…”

“…We have recently reported the fabrication of small, solid NPs fully made of Hb (Hb-NPs) using the electrospraying technique, resulting in Hb-NPs with a diameter ranging from 370 to 480 nm . However, an important drawback of these Hb-NPs is the low throughput of the electrospraying technique, which creates challenges for their production at clinically relevant scales . Using a different approach, the Palmer group was able to synthesize Hb-NPs by borrowing the techniques currently used for the fabrication of protein NPs.…”

Biomedical Applications of Metal–Organic Frameworks at the Subcellular Level

“…[ 86–88 ] As free Hb is unstable and has limited blood circulation times, efforts have been made to develop Hb‐based nanocarriers that could be used as an oxygen delivery system and a potential alternative for donor blood. [ 86,89 ] An MOF‐based system was first reported by Yu and co‐workers. They successfully encapsulated Hb within ZIF‐8 through biomimetic mineralization and prevented Hb from thermal‐, chemical‐, and biological degradation (Figure 4b).…”