Novel high molecular weight polymerized hemoglobin in a non-obese model of cardiovascular and metabolic dysfunction.

The widespread adoption of high-calorie, high-fat, high-sucrose diets (HFHSD) has become a global health concern, particularly due to their association with cardiovascular diseases and metabolic disorders. These comorbidities increase susceptibility to severe outcomes from viral infections and trauma, with trauma-related incidents significantly contributing to global mortality rates. This context underscores the critical need for a reliable blood supply. Recent research has focused on high molecular weight (MW) polymerized human hemoglobin (PolyhHb) as a promising alternative to red blood cells (RBCs), showing encouraging outcomes in previous studies. Given the overlap of metabolic disorders and trauma-related health issues, it is crucial to assess the potential toxicity of PolyhHb transfusions, particularly in models that represent these vulnerable populations. This study evaluated the effects of PolyhHb exchange transfusion in guinea pigs that had developed metabolic disorders due to a 12-week HFHSD regimen. The guinea pigs, underwent a 20 % blood volume exchange transfusion with either PolyhHb or the lower molecular weight polymerized bovine hemoglobin, Oxyglobin. Results revealed that both PolyhHb and Oxyglobin transfusions led to liver damage, with a more pronounced effect observed in HFHSD-fed animals. Additionally, markers of cardiac dysfunction indicated signs of cardiac injury in both the HFHSD and normal diet groups following the Oxyglobin transfusion. This study highlights how pre-existing metabolic disorders can exacerbate the potential side effects of hemoglobin-based oxygen carriers (HBOCs). Importantly, the newer generation of high MW PolyhHb showed lower cardiac toxicity compared to the earlier generation low MW PolyhHb, known as Oxyglobin, even in models with pre-existing endothelial and metabolic challenges.

Polymerized human hemoglobin with low and high oxygen affinity in trauma models.

The present study aimed to compare the ability of tense (T) and relaxed (R) quaternary state polymerized human hemoglobin (PolyhHb) to restore hemodynamics after severe trauma in a rat model, and to assess their relative toxicity in a guinea pigs (GPs). To assess the efficacy of these PolyhHbs in restoring hemodynamics, Wistar rats were subjected to traumatic brain injury (TBI) followed by hemorrhagic shock (HS). Animals were separated into 3 groups based on the resuscitation solution: Whole blood, T-state or R-state PolyhHb, and followed for 2 hours after resuscitation. For toxicity evaluation, GPs were subjected to HS and the hypovolemic state was maintained for 50 minutes. Then, the GPs were divided randomly into 2 groups, and reperfused with T- or R-state PolyhHb. Rats resuscitated with blood and T-state PolyhHb had a higher recovery of MAP at 30 min after resuscitation when compared to R-state PolyhHb, demonstrating the greater ability of T-state PolyhHb to restore hemodynamics compared to R-state PolyhHb. Resuscitation with R-state PolyhHb in GPs increased markers of liver damage and inflammation, kidney injury and systemic inflammation compared to the T-state PolyhHb group. Finally, increased levels of cardiac damage markers, such as troponin were observed, indicating greater cardiac injury in GPs resuscitated with R-state PolyhHb. Therefore, our results showed that T-state PolyhHb exhibited superior efficacy in a model of TBI followed by HS in rats, and presented reduced vital organ toxicity in GPs, when compared to R-state PolyhHb.

Safety and efficacy of human polymerized hemoglobin on guinea pig resuscitation from hemorrhagic shock.

For the past thirty years, hemoglobin-based oxygen carriers (HBOCs) have been under development as a red blood cell substitute. Side-effects such as vasoconstriction, oxidative injury, and cardiac toxicity have prevented clinical approval of HBOCs. Recently, high molecular weight (MW) polymerized human hemoglobin (PolyhHb) has shown positive results in rats. Studies have demonstrated that high MW PolyhHb increased O2 delivery, with minimal effects on blood pressure, without vasoconstriction, and devoid of toxicity. In this study, we used guinea pigs to evaluate the efficacy and safety of high MW PolyhHb, since like humans guinea pigs cannot produce endogenous ascorbic acid, which limits the capacity of both species to deal with oxidative stress. Hence, this study evaluated the efficacy and safety of resuscitation from severe hemorrhagic shock with high MW PolyhHb, fresh blood, and blood stored for 2 weeks. Animals were randomly assigned to each experimental group, and hemorrhage was induced by the withdrawal of 40% of the blood volume (BV, estimated as 7.5% of body weight) from the carotid artery catheter. Hypovolemic shock was maintained for 50 min. Resuscitation was implemented by infusing 25% of the animal's BV with the different treatments. Hemodynamics, blood gases, total hemoglobin, and lactate were not different before hemorrhage and during shock between groups. The hematocrit was lower for the PolyhHb group compared to the fresh and stored blood groups after resuscitation. Resuscitation with stored blood had lower blood pressure compared to fresh blood at 2 h. There was no difference in mean arterial pressure between groups at 24 h. Resuscitation with PolyhHb was not different from fresh blood for most parameters. Resuscitation with PolyhHb did not show any remarkable change in liver injury, inflammation, or cardiac damage. Resuscitation with stored blood showed changes in liver function and inflammation, but no kidney injury or systemic inflammation. Resuscitation with stored blood after 24 h displayed sympathetic hyper-activation and signs of cardiac injury. These results suggest that PolyhHb is an effective resuscitation alternative to blood. The decreased toxicities in terms of cardiac injury markers, vital organ function, and inflammation following PolyhHb resuscitation in guinea pigs indicate a favorable safety profile. These results are promising and support future studies with this new generation of PolyhHb as alternative to blood when blood is unavailable.

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.

Biophysical properties of tense quaternary state polymerized human hemoglobins bracketed between 500 kDa and 0.2 µm in size.

Polymerized hemoglobin (Hb)-based oxygen carriers (HBOCs) are a scalable and cost-effective red blood cell (RBC) substitute. However, previous generations of commercial polymerized HBOCs elicited oxidative tissue injury in vivo due to the presence of low molecular weight polymeric Hb species (<500 kDa) and cell-free Hb (64 kDa). Polymerized human Hb (PolyhHb) locked in the tense quaternary state (T-state) exhibits great promise to meet clinical needs where past polymerized HBOCs failed. This work shows that separation of T-state PolyhHb via a two-stage tangential flow filtration (TFF) purification train such that the Hb polymers are bracketed between 500 kDa and 0.2 µm creates a uniform polymer size and largely eliminates the Hb species which elicit deleterious side effects in vivo. Biophysical characterization of these materials demonstrates their potential effectiveness as an RBC substitute and verifies the low percentage of low molecular weight Hb polymers and cell-free Hb. Size exclusion chromatography confirms that T-state PolyhHb can be consistently produced in a size range between 500 kDa and 0.2 µm. Furthermore, the average molecular weight of all PolyhHb species produced is one or two orders of magnitude larger than that of the commercial polymerized HBOCs Hemolink and Oxyglobin, respectively. Haptoglobin binding kinetics confirms that two-stage TFF processing of PolyhHb reliably removes cell-free Hb and low molecular weight polymeric Hb species. T-state PolyhHbs demonstrate lower auto-oxidation rates compared to unmodified Hb and prior generations of commercial polymerized HBOCs. These results demonstrate T-state PolyhHb's feasibility as a next-generation polymerized HBOC for potential use in transfusion medicine.

Resuscitation from hemorrhagic shock after traumatic brain injury with polymerized hemoglobin.

Traumatic brain injury (TBI) is often accompanied by hemorrhage, and treatment of hemorrhagic shock (HS) after TBI is particularly challenging because the two therapeutic treatment strategies for TBI and HS often conflict. Ischemia/reperfusion injury from HS resuscitation can be exaggerated by TBI-induced loss of autoregulation. In HS resuscitation, the goal is to restore lost blood volume, while in the treatment of TBI the priority is focused on maintenance of adequate cerebral perfusion pressure and avoidance of secondary bleeding. In this study, we investigate the responses to resuscitation from severe HS after TBI in rats, using fresh blood, polymerized human hemoglobin (PolyhHb), and lactated Ringer's (LR). Rats were subjected to TBI by pneumatic controlled cortical impact. Shortly after TBI, HS was induced by blood withdrawal to reduce mean arterial pressure (MAP) to 35-40 mmHg for 90 min before resuscitation. Resuscitation fluids were delivered to restore MAP to ~ 65 mmHg and animals were monitored for 120 min. Increased systolic blood pressure variability (SBPV) confirmed TBI-induced loss of autoregulation. MAP after resuscitation was significantly higher in the blood and PolyhHb groups compared to the LR group. Furthermore, blood and PolyhHb restored diastolic pressure, while this remained depressed for the LR group, indicating a loss of vascular tone. Lactate increased in all groups during HS, and only returned to baseline level in the blood reperfused group. The PolyhHb group possessed lower SBPV compared to LR and blood groups. Finally, sympathetic nervous system (SNS) modulation was higher for the LR group and lower for the PolyhHb group compared to the blood group after reperfusion. In conclusion, our results suggest that PolyhHb could be an alternative to blood for resuscitation from HS after TBI when blood is not available, assuming additional testing demonstrate similar favorable results. PolyhHb restored hemodynamics and oxygen delivery, without the logistical constraints of refrigerated blood.

Targeted Myoglobin Delivery as a Strategy for Enhancing the Sensitivity of Hypoxic Cancer Cells to Radiation.

The effectiveness of cancer radiotherapy is frequently hindered by the hypoxia of the tumor microenvironment. Direct delivery of oxygen to hypoxic tumor tissues is an attractive strategy to overcome this hypoxia-associated radioresistance. Herein, we report the generation of submicron-sized particles comprising myoglobin fused to the crystal-forming domain of Cry3Aa protein for the targeted delivery of oxygen to cancer cells. We demonstrate that myoglobin-containing particles were successfully produced in Bacillus thuringiensis with the assistance of the Cry3Aa domain I. Furthermore, these particles could be genetically modified to incorporate the cell penetrating peptide TAT and cell targeting peptide A549.1, resulting in particles that exhibited improved cellular uptake and targeting toward A549 cells. Notably, these myoglobin-containing particles increased the intracellular oxygen levels of A549 cells and thereby sensitized them to radiation. These findings suggest that the targeted delivery of O2-bound myoglobin could be an effective approach to enhance the efficacy of radiotherapy.

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.

Injectable biodegradable bi-layered capsule for sustained delivery of bevacizumab in treating wet age-related macular degeneration.

Vascular endothelial growth factor (VEGF) is a key regulator of abnormal blood vessel growth. As such, bevacizumab-based inhibition of VEGF has been the clinically adopted strategy to treat colorectal and breast cancers as well as age-related macular degeneration (AMD). However, as the treatment of vascular diseases often requires a high drug concentration for a long period, the burst release of bevacizumab remains a critical limitation in anti-VEGF-based therapies. Maintaining bevacizumab at high concentrations over extended periods remains challenging due to insufficient drug loading capacity and drug-device interactions. We report the development of a polymeric based bi-layered capsule that could address these challenges by extending the release over one year, thereby providing an effective platform enabling treatment of chronic vascular diseases. Remarkably, the developed capsules have a bi-layered structure which ensures the structural integrity of the injectable capsules and appropriate diffusion of bevacizumab by providing optimal physical trapping and electrostatic interaction. Meanwhile, the central hollow design enables a higher drug loading to meet the need for long-term release of bevacizumab for several months to one year. Using an in vitro drug release assay, we demonstrated that the bi-layered capsule could produce longer-term local drug administration by intravitreal injection compared to previously reported devices. The capsules also present minimal toxicity and maintain anti-VEGF potency, suggesting that our approach may have the potential to treat vascular-related diseases using bevacizumab.

Controlled Polymerization and Ultrafiltration Increase the Consistency of Polymerized Hemoglobin for Use as an Oxygen Carrier.

Polymerized human hemoglobins (PolyhHbs) are a promising class of red blood cell substitute for use in transfusion medicine. Unfortunately, the application of the commonly used glutaraldehyde cross-linking chemistry to synthesize these materials results in a complex mixture of PolyhHb molecules with highly varied batch-to-batch consistency. We implemented a controlled method of gas exchange and reagent addition that results in a homogeneous PolyhHb product. A fully coupled tangential flow filtration system was used to purify and concentrate the synthesized PolyhHb molecules. This improved method of PolyhHb production could be used to more precisely control the size and reduce the polydispersity of PolyhHb molecules, with minimal effects on the resulting oxygen-carrying capability. In addition to these factors, we assessed how the hemoglobin scavenging protein haptoglobin (Hp) would interact with PolyhHb molecules of varying sizes and quarternary states. Our results indicated that T-state PolyhHbs may be more efficiently detoxified by Hp compared with R-state PolyhHb and unmodified Hb.