(592e) Optimizing Silk Fibroin Hemoglobin Based Oxygen Carriers: Investigation of Crystallinity, Hemoglobin Encapsulation, Inflammatory Response, and Oxygen Binding Kinetics

Oxygen therapeutics have many applications in transfusion medicine and across disease treatment. Researchers have achieved some early translational success with the creation of stable emulsions of gas dissolving synthetic molecules and the engineering of carriers containing hemoglobin (Hb), the protein component of native red blood cells responsible for oxygen binding and transport. Limitations with synthetic technologies and early hemoglobin-based oxygen carriers (HBOCs) include rapid clearance, limited stability and major side effects resulting from excess hemoglobin extravasating through the vessel wall.¹ New advances in the creation of larger HBOCs (>100 nm) by polymerization or encapsulation has led to a reduction in side effects caused by HBOCs. In this work we evaluate the use of silk fibroin as an encapsulating biopolymer for the creation of novel HBOCs. Silk fibroin is a known cytocompatible and nonthrombogenic polymer that has been shown to stably encapsulate protein-based cargo in crystalline regions.² We approach this development of a silk fibroin hemoglobin-based oxygen carrier wholistically, first characterizing the silk fibroin polymer solution itself, then analyzing particle structure, proceeding with the encapsulation of hemoglobin in the created nanoparticle system, and finally characterizing its interaction with macrophage like-cells in vitro.³

Silk particles were formed by phase separation with polyvinyl alcohol (PVA).^4,5 The phase separation is induced via aqueous solvent evaporation following probe sonication. Using dynamic light scattering (DLS) and scanning electron microscopy (SEM), four formulations were found to yield tunable particle size (200 nm-5 µm) and thus were used for further experimentation. Hb was incorporated into the particles at concentrations of (0, 0.125, and 0.25, mg/mL) by reconstituting lyophilized Hb and combining with silk solution prior to sonication. To assess the encapsulation efficiency and the maintenance of the active ferrous form of Hb, the cyanmethemoglobin method was used.⁶ To assess immune activation by the silk carrier system, RAW 264.7 cells were stimulated with 50 ng of particles twice over 72 hours.⁷ Inflammatory activation was measured by immunohistological analysis of iNOS expression and cell morphology. To assess safety and particle fate, sfHBOCs conjugated with a fluorescent FITC label by an amine reaction were injected in C57BL/6J mice at a concentration of 2 mg/kg bw. Endpoint assessments at 1,4, 24, 48 hours and 4 and 7 days include Perkin Elmer IVIS optical imaging and standard histology for particle distributions, systemic immune response via cytokine panels of blood serum, and flow cytometry of whole blood components to access cell distributions and particles.

Overall, our results show that control of the silk polymer molecular weight is a critical parameter for achieving submicron particles. This was achieved by lengthening the duration of extraction time in silk solutions to 90 minutes (Figure 1A). We show that silk particles can be made using an aqueous solvent evaporation technique in the size range of 200-500 nm. The smallest particle sizes were made with low concentrations of the silk fibroin extracted for 90 minutes (Figure 1B). We further show persistence of >40% crystalline regions throughout the volume of the particles, indicating potential for protein encapsulation and stabilization within the particles crystalline regions (Figure 1C). Finally, we show ~60% hemoglobin encapsulation efficiency with <20% oxidation of human hemoglobin to an inactive form (Figure 1D). All formulations did not elicit an inflammatory immune response, confirming that further investigation into therapeutic efficiency and safety is indicated (Figure 1E).

To evaluate therapeutic efficiency in vitro, RAW 264.7 cells were cultured in hypoxic conditions, prompting a phenotypic shift associated with the HIF-2 pathway. sfHBOCs were introduced to cultures to evaluate if the phenotypic shift could be corrected. These studies were coupled with evaluation of O₂ binding and release kinetics in a packed bed reactor system and subsequent COMSOL® modeling, enabling a dose-dependent response in the in vitro cultures. Efficacy of sfHBOC formulations is evaluated by measuring the oxygen concentration gradient across a sfHBOC packed flow through reactor. The system has PreSens flow through sensors at the inlet and outlet, and by switching from O₂ rich to inert media O₂ release from particles can be observed. Ongoing work seeks to reconstruct oxygen association and disassociation curves for the sfHBOC system.

Future work seeks to prolong the circulation time of sfHBOC in vivo by conjugating stealth polymers, such as polyethylene glycol, to the surface via amine chemistry. We are working to improve this sfHBOC system’s potential to serve as a novel oxygen therapeutic. We are also developing protocols to isolate and purify different types and sources of Hb to modulate oxygen binding and release kinetics, allowing for potential application across a wide range of common injuries and diseases.

References

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2 Lee, K. Y., Kong, S. J., Park, W. H., Ha, W. S. & Kwon, I. C. Effect of surface properties on the antithrombogenicity of silk fibroin/S-carboxymethyl kerateine blend films. J Biomater Sci Polym Ed 9, 905-914 (1998). https://doi.org:10.1163/156856298x00235

3 Pacheco, M. O. et al. Silk Fibroin Particles as Carriers in the Development of All-Natural Hemoglobin-Based Oxygen Carriers (HBOCs). bioRxiv (2023). https://doi.org:10.1101/2023.03.01.530637

4 Rockwood, D. N. et al. Materials fabrication from Bombyx mori silk fibroin. Nature Protocols 6, 1612-1631 (2011). https://doi.org:10.1038/nprot.2011.379

5 Wang, X., Yucel, T., Lu, Q., Hu, X. & Kaplan, D. L. Silk nanospheres and microspheres from silk/pva blend films for drug delivery. Biomaterials 31, 1025-1035 (2010). https://doi.org:https://doi.org/10.1016/j.biomaterials.2009.11.002

6 Whitehead, R. D., Jr., Mei, Z., Mapango, C. & Jefferds, M. E. D. Methods and analyzers for hemoglobin measurement in clinical laboratories and field settings. Ann N Y Acad Sci 1450, 147-171 (2019). https://doi.org:10.1111/nyas.14124

7 Totten, J. D., Wongpinyochit, T., Carrola, J., Duarte, I. F. & Seib, F. P. PEGylation-Dependent Metabolic Rewiring of Macrophages with Silk Fibroin Nanoparticles. ACS Applied Materials & Interfaces 11, 14515-14525 (2019). https://doi.org:10.1021/acsami.8b18716