(555d) Silk Fibroin Nano- and Micro- Particles As a Potential System for a Novel Hemoglobin-Based Oxygen Carrier (HBOC)

Traumatic injury, diabetes, anemia, stroke, and tumor growth can lead to local or systemic lack of oxygen^1,2. Blood transfusions are the gold-standard for delivery of O₂ when enriched respiratory delivery is not sufficient; however, blood donations and human-derived blood products are in limited supply, require extensive pathogen screening, and require cold-chain storage³. Because of these limitations, many options for therapeutic O₂ carriers (OCs) are under investigation. These include hemoglobin-based oxygen carriers (HBOCs) that make use of recombinantly produced hemoglobin or hemoglobin from other species, as well as synthetic perfluorocarbons^4-6. Many of these current systemically delivered OCs struggle with issues such as long-term organ toxicity, dose inefficiency, and rapid clearance rates (<12 hours)⁷. Thus, despite years of efforts, alternative O₂ carrying therapeutics are needed that prolong O₂ delivery without leading to systemic toxicity.

For the development of an all-natural HBOC, we investigate a novel source of hemoglobin. The hemoglobin from teleost fish (salmon, tuna, etc.) is unique due to the Root effect. The Root effect is a phenomenon in which an acidic pH decreases the affinity of hemoglobin for O₂, leading to efficient delivery of hemoglobin-bound O₂ to the hypoxic microenvironments⁸. Teleost fish leverage the Root effect to regulate their buoyancy, improve their vision, and maintain proper oxygenation of their tissues⁹. We aim to leverage this evolved advantage of salmon hemoglobin to locally and efficiently deliver O₂ to hypoxic tissues within humans. To improve shelf-life and dose efficiency, as well as avoid toxicity, the hemoglobin proteins are encapsulated in a matrix of an all-natural protein carrier. Silk fibroin is a protein isolated from Bombyx mori cocoons that is well established as a cytocompatible, inert natural polymer^10,11. Silk fibroin can be processed into multiple biomaterial formats including nano- and micro- particles, and subsequently degraded into simple amino acids¹².

In this work, we analyze the separation and purification of salmon hemoglobin from whole blood, establish a design space to control silk particle size, analyze hemoglobin encapsulation, assess the stability of the silk-hemoglobin particles as a function of their size and storage condition, and measure oxygen loading and release in vitro using a custom plug-flow reactor system equipped with dissolved oxygen sensors.

Methods: Hemoglobin is isolated and purified from whole salmon blood using immobilized metal affinity chromatography (IMAC). Purity is assessed using SDS-PAGE, while UV-vis is used to assess levels of methemoglobin. Silk particles are formed via phase separation with polyvinyl alcohol (PVA) as described previously^13,14. The phase separation occurs as a function of solvent evaporation. To test for shifts in particle size, we varied the sonication amplitude (0, 8, 12, 16, 25, 40%), silk concentration (2, 20, 50, 70 mg/mL), silk degumming time (30, 60, 90 min, inversely proportional to silk molecular weight) and hemoglobin concentration. Samples were assessed for particle size, stability, and morphology using dynamic light scattering (DLS) and scanning electron microscopy (SEM). To visualize hemoglobin encapsulation, hemoglobin was combined into the soluble silk solution prior to particle formation. Immunofluorescent imaging was then performed to confirm colocalization of the hemoglobin into the silk particles. To test the stability of the silk particles containing hemoglobin, we incubate both liquid and lyophilized conditions at temperatures ranging from -80°C to 40°C. Weekly, samples are assessed for changes in hemoglobin conformation using UV-vis and changes in particle size, stability and morphology using DLS and SEM.

Results: We found that hemoglobin was isolated with less than 25% of total isolated protein being methemoglobin. When attempting to control particle size, we found that we can generate particles ranging from 200 nm–10 um. We also observed the following trends: (i) increases in sonication amplitude and/or time lead to smaller particle sizes (ii) silk concentration is negatively correlated to average particle size, and (iii) silk degumming time is positively correlated to average size. Moderate levels of polydispersity (range of 0.2-0.4) were observed across all samples, prompting investigation into strategies to narrow polydispersity. While altering variables involved in particle formation like the sonication probe diameter and probe to sample volume ratios can impact polydispersity, we are also investigating post-processing strategies, like filtration. Hemoglobin was observed to be incorporated in particles by fluorescent microscopy and no significant changes in particle size were observed as a function of the hemoglobin inclusion.

These results show the control of particle size and level of hemoglobin incorporation, indicating the potential of this system to serve as a novel HBOC. These results are also being used in parallel efforts that seek to simulate O₂ release from the hemoglobin rich regions of the particles using a two tier COMSOL® reaction-diffusion model. Data from this model can drive experimental design for in vitro characterization assays and release experiments. Release experiments aim to evaluate O₂ delivery profiles in unique microenvironments using an in vitro perfusion flow system equipped with Presens® O₂ sensors. Preliminary results suggest that acidic microenvironments characteristic of injury or tumor sites prompt burst release of oxygen, loss of hemoglobin cooperativity, and particle degradation in both nano- and micro- particles, while in healthy physiological pH ranges the particles persist in solution and are capable of sustained release. Current work in the field still aims to understand how to optimize particle size for different targets and circulation times¹⁵, so we continue to model and test both silk micro- and nano- particles in preliminary modeling and in vitro work. Future work aims to assess safety of the key candidate particle formulations in vivo via intravenous (IV) injections and subsequent clearance by the kidneys, liver, and spleen along with the systemic immune response to injection.

References

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