(357am) Hemoglobin Encapsulated Metal Organic Framework Nanoparticles As an Oxygen Therapeutic with Tunable Size Distribution

Research Interests: My current research focused on developing universal hemoglobin based oxygen carriers (HBOCs) that can replace the oxygen storage and transport functions of red blood cells to improve clinical outcomes both for trauma victims and patients undergoing high-blood-loss surgical procedures. We combined polymerization, pegylation, and encapsulation approaches to design and engineer HBOCs. Our design strategy is based on the observation that transfusion of cell free hemoglobin results in vasoconstriction and the development of systemic hypertension. Thus, increasing the size of HBOCs and surface shielding of the nanocomposite would be the key to address those safety concerns induced by cell free hemoglobin.

Motivation: Hemoglobin (Hb)-based oxygen carriers (HBOCs) constitute one major class of artificial red blood cell (RBC) substitute, which has gained considerable attention in treating hemorrhagic shock, ameliorating traumatic brain injury and suppressing tumor growth via oxygenation of hypoxic tissues. Unfortunately, prior generations of HBOCs yielded disappointing clinical outcomes due to significant safety concerns. Various strategies have been developed to reduce/prevent extravasation of cell-free Hb including Hb polymerization, surface conjugation of Hb, and liposome encapsulation of H. All of these strategies increase the molecular radius of Hb, so that it is unable to extravasate through the blood vessel wall. It was observed that chemical modification of Hb via polymerization and surface conjugation approaches drastically decrease the flexibility of Hb, which affects cooperative oxygen (O₂) binding and release. Although liposome encapsulation had a negligible impact on cooperative O₂ binding/release, low Hb encapsulation efficiency remains a significant issue that impeded scale-up and potential commercialization. Those underlying flaws associated with liposome encapsulated Hb nanoparticles underscore the need to develop next generation Hb nanoparticles with enhanced uniformity, high Hb content, and high encapsulation efficiency without jeopardizing cooperative O₂ binding and release. Metal organic frameworks (MOFs) are crystalline materials synthesized by the coordination of metal ions and organic linkers. Zeolitic imidazolate frameworks (ZIFs) are a subclass of MOF, which exhibits outstanding solvothermal stability due to their unique structure compared to other MOFs. ZIF-8, the prototypical ZIF, preserves its crystallinity and zeolite-like porosity when exposed to boiling water, organic solvents, and several biological buffers. Very recently, ZIF-8 has emerged as a promising material in biological sensing, drug delivery, and HBOC fabrication. In this study, bovine Hb (bHb) was successfully encapsulated using ZIF-8 precursors into ZIF-8P-Hb nanoparticles as a potential O₂ therapeutics.

Methods: Bovine Hb (bHb) was purified via tangential flow filtration (TFF). Briefly, Hb was purified via a two-stage TFF system with hollow fiber cartridges with MWCOs of 500 and 50 kDa (Repligen Corporation, Rancho Dominguez, CA). The purified bHb was concentrated to > 200 mg/mL and stored at −80 °C for future use. To synthesize bHb encapsulated ZIF-8 precursors-based (ZIF-8P-Hb) nanoparticle. Initially, 50 mL of deionized (DI) water was used to fully dissolve 100 mg of Zn(NO₃)₂·6H₂O, chased by a 1 mL bolus addition of bHb solution (250 mg/mL) with continuous stirring (500 rpm) for 5 min. 827.9 mg of Hmim powder was then added to the above mixture. The reaction proceeded for 1 h at 25 °C followed by overnight stabilization at 4 °C. Then, the formed ZIF-8P-Hb nanoparticles were purified by washing with TFF for 6~10 diacycles using a hollow fiber cartridge with 500 kDa pore size first with DI water and buffered exchanged into phosphate buffered saline (PBS, 0.1 M) for further use. Bare ZIF-8 nanoparticles were synthesized following a similar protocol without the addition of bHb and were suspended in DI water.

Results: The size of ZIF-8P-Hb nanoparticles was primarily controlled by Hmim:Zn molar ratio, flow rate during TFF processing, concentration of EDTA, and concentration of zinc nitrate. It was found that adding Hmim directly into the reaction vessel regulated particle size, which also affected the crystalline structure of the particle. Furthermore, we demonstrated that the high molar ratio of Hmim:Zn could be used to better control nucleation of ZIF-8P-Hb nanoparticles. The monodisperse size distribution was a result of the rapid nucleation rate facilitated by the relatively high molar ratio of Hmim:zinc, and TFF operated at relatively low flow rate did not exert a strong impact on the size distribution.

Takeaways: In this study, we established a scalable purification platform to manufacture ZIF-8 and ZIF-8P-Hb nanoparticles via TFF. The optimized synthesis protocol yielded relatively low batch-to-batch variance with respect to most biophysical properties including hydrodynamic diameter, zeta potential, oxygen equilibria, and oxygen offloading rate constant. The higher bHb loading and bHb encapsulation efficiency of ZIF-8P-Hb nanoparticles compared to prior attempts in the literature is critical in order to potentially use these materials to treat hemorrhagic shock.