ZIF-8 metal organic framework nanoparticle loaded with tense quaternary state polymerized bovine hemoglobin: potential red blood cell substitute with antioxidant properties.

Due to several limitations associated with blood transfusion, such as the relatively short shelf life of stored blood, low risk of developing acute immune hemolytic reactions and graft-versus-host disease, many strategies have been developed to synthesize hemoglobin-based oxygen carriers (HBOCs) as universal red blood cell (RBC) substitutes. Recently, zeolite imidazole framework-8 (ZIF-8), a metal-organic framework, has attracted considerable attention as a protective scaffold for encapsulation of hemoglobin (Hb). Despite the exceptional thermal and chemical stability of ZIF-8, the major impediments to implementing ZIF-8 for Hb encapsulation are the structural distortions associated with loading large quantities of Hb in the scaffold as the Hb molecule has a larger hydrodynamic diameter than the pore size of ZIF-8. Therefore to reduce the structural distortion caused by Hb encapsulation, we established and optimized a continuous-injection method to synthesize nanoparticle (NP) encapsulated polymerized bovine Hb (PolybHb) using ZIF-8 precursors (ZIF-8P-PolybHb NPs). The synthesis method was further modified by adding EDTA as a chelating agent, which reduced the ZIF-8P-PolybHb NP size to <300 nm. ZIF-8P-PolybHb NPs exhibited lower oxygen affinity (36.4 ± 3.2 mm Hg) compared to unmodified bovine Hb, but was similar in magnitude to unencapsulated PolybHb. The use of the chemical cross-linker glutaraldehyde to polymerize bovine Hb resulted in the low Hill coefficient of PolybHb, indicating loss of Hb's oxygen binding cooperativity, which could be a limitation when using PolybHb as an oxygen carrier for encapsulation inside the ZIF-8 matrix. ZIF-8P-PolybHb NPs exhibited slower oxygen offloading kinetics compared to unencapsulated PolybHb, demonstrating successful encapsulation of PolybHb. ZIF-8P-PolybHb NPs also exhibited favorable antioxidant properties when exposed to H2O2. Incorporation of PolybHb into the ZIF-8 scaffold resulted in reduced cytotoxicity towards human umbilical vein endothelial cells compared to unloaded ZIF-8 NPs and ZIF-8 NPs loaded with bovine Hb. We envisage that such a monodisperse and biocompatible HBOC with low oxygen affinity and antioxidant properties may broaden its use as an RBC substitute.

Biophysical Analysis and Preclinical Pharmacokinetics-Pharmacodynamics of Tangential Flow Filtration Fractionated Polymerized Human Hemoglobin as a Red Blood Cell Substitute.

Red blood cell (RBC) substitutes tested in late-phase clinical trials contained low-molecular-weight hemoglobin species (<500 kDa), resulting in vasoconstriction, hypertension, and oxidative tissue injury; therefore, contributing to poor clinical outcomes. This work aims to improve the safety profile of the RBC substitute, polymerized human hemoglobin (PolyhHb), via in vitro and in vivo screening of PolyhHb fractionated into four molecular weight brackets (50-300 kDa [PolyhHb-B1]; 100-500 kDa [PolyhHb-B2]; 500-750 kDa [PolyhHb-B3]; and 750 kDa to 0.2 µm [PolyhHb-B4]) using a two-stage tangential flow filtration purification process. Analysis showed that PolyhHb's oxygen affinity, and haptoglobin binding kinetics decreased with increasing bracket size. A 25% blood-for-PolyhHb exchange transfusion guinea pig model suggests that hypertension and tissue extravasation decreased with increasing bracket size. PolyhHb-B3 demonstrated extended circulatory pharmacokinetics, no renal tissue distribution, no aberrant blood pressure, or cardiac conduction effects, and may therefore be appropriate material for further evaluation.

Pilot scale production and characterization of next generation high molecular weight and tense quaternary state polymerized human hemoglobin.

Polymerized human hemoglobin (PolyhHb) is being studied as a possible red blood cell (RBC) substitute for use in scenarios where blood is not available. While the oxygen (O2 ) carrying capacity of PolyhHb makes it appealing as an O2 therapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc.) was never approved for clinical use due to the presence of large quantities of low molecular weight (LMW) polymeric hemoglobin (Hb) species (<500 kDa), which have been shown to elicit vasoconstriction, systemic hypertension, and oxidative tissue injury in vivo. Previous bench-top scale studies in our lab demonstrated the ability to synthesize and purify PolyhHb using a two-stage tangential flow filtration purification process to remove almost all undesirable Hb species (>0.2 µm and <500 kDa) in the material, to create a product that should be safer for transfusion. Therefore, to enable future large animal studies and eventual human clinical trials, PolyhHb synthesis and purification processes need to be scaled up to the pilot scale. Hence in this study, we describe the pilot scale synthesis and purification of PolyhHb. Characterization of pilot scale PolyhHb showed that PolyhHb could be successfully produced to yield biophysical properties conducive for its use as an RBC substitute. Size exclusion high performance liquid chromatography showed that pilot scale PolyhHb yielded a high molecular weight Hb polymer containing a small percentage of LMW Hb species (<500 kDa). Additionally, the auto-oxidation rate of pilot scale PolyhHb was even lower than that of previous generations of PolyhHb. Taken together, these results demonstrate that PolyhHb has the ability to be seamlessly manufactured at the pilot scale to enable future large animal studies and clinical trials.

Boosting Monocular 3D Human Pose Estimation With Part Aware Attention.

Monocular 3D human pose estimation is challenging due to depth ambiguity. Convolution-based and Graph-Convolution-based methods have been developed to extract 3D information from temporal cues in motion videos. Typically, in the lifting-based methods, most recent works adopt the transformer to model the temporal relationship of 2D keypoint sequences. These previous works usually consider all the joints of a skeleton as a whole and then calculate the temporal attention based on the overall characteristics of the skeleton. Nevertheless, the human skeleton exhibits obvious part-wise inconsistency of motion patterns. It is therefore more appropriate to consider each part's temporal behaviors separately. To deal with such part-wise motion inconsistency, we propose the Part Aware Temporal Attention module to extract the temporal dependency of each part separately. Moreover, the conventional attention mechanism in 3D pose estimation usually calculates attention within a short time interval. This indicates that only the correlation within the temporal context is considered. Whereas, we find that the part-wise structure of the human skeleton is repeating across different periods, actions, and even subjects. Therefore, the part-wise correlation at a distance can be utilized to further boost 3D pose estimation. We thus propose the Part Aware Dictionary Attention module to calculate the attention for the part-wise features of input in a dictionary, which contains multiple 3D skeletons sampled from the training set. Extensive experimental results show that our proposed part aware attention mechanism helps a transformer-based model to achieve state-of-the-art 3D pose estimation performance on two widely used public datasets. The codes and the trained models are released at https://github.com/thuxyz19/3D-HPE-PAA.

Scalable manufacturing platform for the production of methemoglobin as a non-oxygen carrying control material in studies of cell-free hemoglobin solutions.

Methemoglobin (metHb) arises from the oxidation of ferrous hemoglobin (HbFe2+, Hb) to ferric hemoglobin (HbFe3+, metHb), which is unable to bind gaseous ligands such as oxygen (O2) and carbon monoxide (CO), and binds to nitric oxide (NO) significantly slower compared to Hb. Therefore, metHb does not elicit vasoconstriction and systemic hypertension in vivo due to its extremely slow NO scavenging rate in comparison to cell-free Hb, but will induce oxidative tissue injury, demonstrating the potential of using metHb as a control material when studying the toxicity of cell-free Hb. Hence, the goal of this work was to develop a novel manufacturing strategy for production of metHb that is amenable to scale-up. In this study, small scale (e.g. 1 mL reaction volume) screening experiments were initially conducted to determine the optimal molar ratio of Hb to the oxidization agents hydrogen peroxide (H2O2) or sodium nitrite (NaNO2) to achieve the highest conversion of Hb into metHb. A spectral deconvolution program was employed to determine the molar fraction of various species (hemichrome, metHb, oxyHb, metHb-[Formula: see text], and NaNO2) in solution during the oxidation reaction. From this analysis, either a 1:1 or 1:5 molar ratio was identified as optimal molar ratios of Hb:NaNO2 (heme basis) that yielded the highest conversion of Hb into metHb with negligible amounts of side products. Hence in order to reduce the reaction time, a 1:5 molar ratio was chosen for large scale (i.e. 1.5 L reaction volume) synthesis of bovine metHb (metbHb) and human metHb (methHb). The biophysical properties of metHb were then characterized to elucidate the potential of using the synthesized metHb as a non-O2 carrying control material. The haptoglobin binding kinetics of metHb were found to be similar to Hb. Additionally, the synthesized metHb was stable in phosphate buffered saline (PBS, 50 mM, pH 7.4) at 4°C for approximately one week, indicating the high stability of the material.

Tangential flow filtration facilitated fractionation and PEGylation of low and high-molecular weight polymerized hemoglobins and their biophysical properties.

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P50 than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.

Early Intervention in Ischemic Tissue with Oxygen Nanocarriers Enables Successful Implementation of Restorative Cell Therapies.

BACKGROUND: Tissue ischemia contributes to necrosis and infection. While angiogenic cell therapies have emerged as a promising strategy against ischemia, current approaches to cell therapies face multiple hurdles. Recent advances in nuclear reprogramming could potentially overcome some of these limitations. However, under severely ischemic conditions necrosis could outpace reprogramming-based repair. As such, adjunctive measures are required to maintain a minimum level of tissue viability/activity for optimal response to restorative interventions. METHODS: Here we explored the combined use of polymerized hemoglobin (PolyHb)-based oxygen nanocarriers with Tissue Nano-Transfection (TNT)-driven restoration to develop tissue preservation/repair strategies that could potentially be used as a first line of care. Random-pattern cutaneous flaps were created in a mouse model of ischemic injury. PolyHbs with high and low oxygen affinity were synthesized and injected into the tissue flap at various timepoints of ischemic injury. The degree of tissue preservation was evaluated in terms of perfusion, oxygenation, and resulting necrosis. TNT was then used to deploy reprogramming-based vasculogenic cell therapies to the flaps via nanochannels. Reprogramming/repair outcomes were evaluated in terms of vascularity and necrosis. RESULTS: Flaps treated with PolyHbs exhibited a gradual decrease in necrosis as a function of time-to-intervention, with low oxygen affinity PolyHb showing the best outcomes. TNT-based intervention of the flap in combination with PolyHb successfully curtailed advanced necrosis compared to flaps treated with only PolyHb or TNT alone. CONCLUSIONS: These results indicate that PolyHb and TNT technologies could potentially be synergistically deployed and used as early intervention measures to combat severe tissue ischemia.

Comprehensive characterization of tense and relaxed quaternary state glutaraldehyde polymerized bovine hemoglobin as a function of cross-link density.

Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this study, we prepared PolybHb in the T and relaxed (R) quaternary state with ultrahigh MW (>500 kDa) with varying cross-link densities, and investigated the effect of MW on key biophysical properties (i.e., O2 affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, auto-oxidation, and haptoglobin [Hp]-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e., cross-linker:bovine hemoglobin (bHb) molar ratio, gas-liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T- and R-state PolybHbs. Our results showed that, the duration of the fast-step auto-oxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHbs exhibited significantly higher bimolecular rate constants for binding to Hp and unimolecular O2 offloading rate constants compared to R-state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.