Toxic side-effects of diaspirin cross-linked human hemoglobin are attenuated by the apohemoglobin-haptoglobin complex.

Alpha-alpha diaspirin-crosslinked human hemoglobin (DCLHb or ααHb) was a promising early generation red blood cell (RBC) substitute. The DCLHb was developed through a collaborative effort between the United States Army and Baxter Healthcare. The core design feature underlying its development was chemical stabilization of the tetrameric structure of hemoglobin (Hb) to prevent Hb intravascular dimerization and extravasation. DCLHb was developed to resuscitate warfighters on the battlefield, who suffered from life-threatening blood loss. However, extensive research revealed toxic side effects associated with the use of DCLHb that contributed to high mortality rates in clinical trials. This study explores whether scavenging Hb and heme via the apohemoglobin-haptoglobin (apoHb-Hp) complex can reduce DCLHb associated toxicity. Awake Golden Syrian hamsters were equipped with a window chamber model to characterize the microcirculation. Each group was first infused with either Lactated Ringer's or apoHb-Hp followed by a hypovolemic infusion of 10% of the animal's blood volume of DCLHb. Our results indicated that animals pretreated with apoHb-Hb exhibited improved microhemodynamics vs the group pretreated with Lactated Ringer's. While systemic acute inflammation was observed regardless of the treatment group, apoHb-Hp pretreatment lessened those effects with a marked reduction in IL-6 levels in the heart and kidneys compared to the control group. Taken together, this study demonstrated that utilizing a Hb and heme scavenger protein complex significantly reduces the microvasculature effects of ααHb, paving the way for improved HBOC formulations. Future apoHb-Hp dose optimization studies may identify a dose that can completely neutralize DCLHb toxicity.

Human STING is a proton channel.

Proton leakage from organelles is a common signal for noncanonical light chain 3B (LC3B) lipidation and inflammasome activation, processes induced upon stimulator of interferon genes (STING) activation. On the basis of structural analysis, we hypothesized that human STING is a proton channel. Indeed, we found that STING activation induced a pH increase in the Golgi and that STING reconstituted in liposomes enabled transmembrane proton transport. Compound 53 (C53), a STING agonist that binds the putative channel interface, blocked STING-induced proton flux in the Golgi and in liposomes. STING-induced LC3B lipidation and inflammasome activation were also inhibited by C53, suggesting that STING's channel activity is critical for these two processes. Thus, STING's interferon-induction function can be decoupled from its roles in LC3B lipidation and inflammasome activation.

Engineering Therapeutics to Detoxify Hemoglobin, Heme, and Iron.

Hemolysis (i.e., red blood cell lysis) can increase circulatory levels of cell-free hemoglobin (Hb) and its degradation by-products, namely heme (h) and iron (Fe). Under homeostasis, minor increases in these three hemolytic by-products (Hb/h/Fe) are rapidly scavenged and cleared by natural plasma proteins. Under certain pathophysiological conditions, scavenging systems become overwhelmed, leading to the accumulation of Hb/h/Fe in the circulation. Unfortunately, these species cause various side effects such as vasoconstriction, hypertension, and oxidative organ damage. Therefore, various therapeutics strategies are in development, ranging from supplementation with depleted plasma scavenger proteins to engineered biomimetic protein constructs capable of scavenging multiple hemolytic species. In this review, we briefly describe hemolysis and the characteristics of the major plasma-derived protein scavengers of Hb/h/Fe. Finally, we present novel engineering approaches designed to address the toxicity of these hemolytic by-products.

STING Protein-Based In Situ Vaccine Synergizes CD4+ T, CD8+ T, and NK Cells for Tumor Eradication.

Stimulator of interferon genes (STING) signaling is a promising target in cancer immunotherapy, with many ongoing clinical studies in combination with immune checkpoint blockade (ICB). Existing STING-based therapies largely focus on activating CD8+ T cell or NK cell-mediated cytotoxicity, while the role of CD4+ T cells in STING signaling has yet to be extensively studied in vivo. Here, a distinct CD4-mediated, protein-based combination therapy of STING and ICB as an in situ vaccine, is reported. The treatment eliminates subcutaneous MC38 and YUMM1.7 tumors in 70-100% of mice and protected all cured mice against rechallenge. Mechanistic studies reveal a robust TH 1 polarization and suppression of Treg of CD4+ T cells, followed by an effective collaboration of CD4+ T, CD8+ T, and NK cells to eliminate tumors. Finally, the potential to overcome host STING deficiency by significantly decreasing MC38 tumor burden in STING KO mice is demonstrated, addressing the translational challenge for the 19% of human population with loss-of-function STING variants.

Synergistic combination therapy delivered via layer-by-layer nanoparticles induces solid tumor regression of ovarian cancer.

The majority of patients with high grade serous ovarian cancer (HGSOC) develop recurrent disease and chemotherapy resistance. To identify drug combinations that would be effective in treatment of chemotherapy resistant disease, we examined the efficacy of drug combinations that target the three antiapoptotic proteins most commonly expressed in HGSOC-BCL2, BCL-XL, and MCL1. Co-inhibition of BCL2 and BCL-XL (ABT-263) with inhibition of MCL1 (S63845) induces potent synergistic cytotoxicity in multiple HGSOC models. Since this drug combination is predicted to be toxic to patients due to the known clinical morbidities of each drug, we developed layer-by-layer nanoparticles (LbL NPs) that co-encapsulate these inhibitors in order to target HGSOC tumor cells and reduce systemic toxicities. We show that the LbL NPs can be designed to have high association with specific ovarian tumor cell types targeted in these studies, thus enabling a more selective uptake when delivered via intraperitoneal injection. Treatment with these LbL NPs displayed better potency than free drugs in vitro and resulted in near-complete elimination of solid tumor metastases of ovarian cancer xenografts. Thus, these results support the exploration of LbL NPs as a strategy to deliver potent drug combinations to recurrent HGSOC. While these findings are described for co-encapsulation of a BCL2/XL and a MCL1 inhibitor, the modular nature of LbL assembly provides flexibility in the range of therapies that can be incorporated, making LbL NPs an adaptable vehicle for delivery of additional combinations of pathway inhibitors and other oncology drugs.

Layer-by-layer interleukin-12 nanoparticles drive a safe and effective response in ovarian tumors.

Ovarian cancer is especially deadly, challenging to treat, and has proven refractory to known immunotherapies. Cytokine therapy is an attractive strategy to drive a proinflammatory immune response in immunologically cold tumors such as many high grade ovarian cancers; however, this strategy has been limited in the past due to severe toxicity. We previously demonstrated the use of a layer-by-layer (LbL) nanoparticle (NP) delivery vehicle in subcutaneous flank tumors to reduce the toxicity of interleukin-12 (IL-12) therapy upon intratumoral injection. However, ovarian cancer cannot be treated by local injection as it presents as dispersed metastases. Herein, we demonstrate the use of systemically delivered LbL NPs using a cancer cell membrane-binding outer layer to effectively target and engage the adaptive immune system as a treatment in multiple orthotopic ovarian tumor models, including immunologically cold tumors. IL-12 therapy from systemically delivered LbL NPs shows reduced severe toxicity and maintained anti-tumor efficacy compared to carrier-free IL-12 or layer-free liposomal NPs leading to a 30% complete survival rate.

Controlled Lipid Self-Assembly for Scalable Manufacturing of Next-Generation Immune Stimulating Complexes.

Immune stimulating complexes (ISCOMs) are safe and effective saponin-based adjuvants formed by the self-assembly of saponin, cholesterol, and phospholipids in water to form cage-like 30-40 nm diameter particles. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) in ISCOM particles yields a promising next-generation adjuvant termed Saponin-MPLA NanoParticles (SMNP). In this work, we detail protocols to produce ISCOMs or SMNP via a tangential flow filtration (TFF) process suitable for scalable synthesis and Good Manufacturing Practice (GMP) production of clinical-grade adjuvants. SMNP or ISCOM components were solubilized in micelles of the surfactant MEGA-10, then diluted below the critical micelle concentration (CMC) of the surfactant to drive ISCOM self-assembly. Assembly of ISCOM/SMNP particles using the purified saponin QS-21 used in clinical-grade saponin adjuvants was found to require controlled stepwise dilution of the initial micellar solution, to prevent formation of undesirable kinetically-trapped aggregate species. An optimized protocol gave yields of ~77% based on the initial feed of QS-21 and the final SMNP particle composition mirrored the feed ratios of the components. Further, samples were highly homogeneous with comparable quality to that of material prepared at lab scale by dialysis and purified via size-exclusion chromatography. This protocol may be useful for clinical preparation of ISCOM-based vaccine adjuvants and therapeutics.

Apohemoglobin-haptoglobin complex alleviates iron toxicity in mice with ß-thalassemia via scavenging of cell-free hemoglobin and heme.

ß-thalassemia is a genetic hemoglobin (Hb) disorder that affects millions of people world-wide. It is characterized by ineffective erythropoiesis and anemia. The resultant chronic anemia can require life-long blood transfusion regimens, leading to secondary hemochromatosis. Moreover, the abnormal red blood cells (RBCs) from ß-thalassemia patients are prone to hemolytic events that release cell-free Hb and heme causing a series of events that result in oxidative organ and tissue damage. In this study, ß-thalassemic mice were treated with a protein scavenger for six weeks, apohemoglobin-haptoglobin (apoHb-Hp), this protein scavenges cell free Hb and heme. We hypothesize that scavenging cell-free Hb and heme will lead to a positive therapeutic event. After the apoHb-hp treatment it was observed to reduce the weight of the liver and spleen and show an improvement in liver function by a drop in ALT, AST, and ALP markers. ApoHb-hp treatment also hints at an improved RBC half-life as the number of reticulocytes decreased, the mean corpuscular volume (MCV) increased, mean corpuscular hemoglobin increase and the RBC distribution width decreased. Furthermore, apoHb-Hp treatment reduced circulating serum iron concentration and transferrin saturation concentration. Based on these outcomes, introducing a scavenger protein can benefit ß-thalassemic mice. This study demonstrated that apoHb-Hp treatment may be a viable strategy to mitigate toxicities associated with cell free Hb and heme, a driver of ß-thalassemic issues.

Engineering Strategies for Immunomodulatory Cytokine Therapies - Challenges and Clinical Progress.

Cytokines are immunoregulatory proteins involved in many pathological states with promising potential as therapeutic agents. A diverse array of cytokines have been studied in preclinical disease models since the 1950s, some of which became successful biopharmaceutical products with the advancement of recombinant protein technology in the 1980s. However, following these early approvals, clinical translation of these natural immune signaling molecules has been limited due to their pleiotropic action in many cell types, and the fact that they have evolved to act primarily locally in tissues. These characteristics, combined with poor pharmacokinetics, have hindered the delivery of cytokines via systemic administration routes due to dose-limiting toxicities. However, given their clinical potential and recent clinical successes in cancer immunotherapy, cytokines continue to be extensively pursued in preclinical and clinical studies, and a range of molecular and formulation engineering strategies are being applied to reduce treatment toxicity while maintaining or enhancing therapeutic efficacy. This review provides a brief background on the characteristics of cytokines and their history as clinical therapeutics, followed by a deeper discussion on the engineering strategies developed for cytokine therapies with a focus on the translational relevance of these approaches.

Purification and analysis of a protein cocktail capable of scavenging cell-free hemoglobin, heme, and iron.

BACKGROUND: Hemolysis releases toxic cell-free hemoglobin (Hb), heme, and iron, which overwhelm their natural scavenging mechanisms during acute or chronic hemolytic conditions. This study describes a novel strategy to purify a protein cocktail containing a comprehensive set of scavenger proteins for potential treatment of hemolysis byproducts. STUDY DESIGN AND METHODS: Tangential flow filtration was used to purify a protein cocktail from Human Cohn Fraction IV (FIV). A series of in vitro assays were performed to characterize composition and biocompatibility. The in vivo potential for hemolysis byproduct mitigation was assessed in a hamster exchange transfusion model using mechanically hemolyzed blood plasma mixed with the protein cocktail or a control colloid (dextran 70 kDa). RESULTS: A basis of 500 g of FIV yielded 62 ± 9 g of a protein mixture at 170 g/L, which bound to approximately 0.6 mM Hb, 1.2 mM heme, and 1.2 mM iron. This protein cocktail was shown to be biocompatible in vitro with red blood cells and platelets and exhibits nonlinear concentration dependence with respect to viscosity and colloidal osmotic pressure. In vivo assessment of the protein cocktail demonstrated higher iron transport to the liver and spleen and less to the kidney and heart with significantly reduced renal and cardiac inflammation markers and lower kidney and hepatic damage compared to a control colloid. DISCUSSION: Taken together, this study provides an effective method for large-scale production of a protein cocktail suitable for comprehensive reduction of hemolysis-induced toxicity.

Apohemoglobin-haptoglobin complexes attenuate the hypertensive response to low-molecular-weight polymerized hemoglobin.

Polymerized hemoglobin (PolyHb) is a promising hemoglobin (Hb)-based oxygen carrier currently undergoing development as a red blood cell substitute. Unfortunately, commercially developed products are composed of low-molecular-weight (LMW) PolyHb molecules, which extravasate, scavenge nitric oxide, and result in vasoconstriction and hypertension. The naturally occurring Hb-scavenging species haptoglobin (Hp), combined with the purified heme-scavenging species apohemoglobin (apoHb), is a potential candidate to alleviate the pressor effect of PolyHb. This study evaluated the protective activity of administering the apoHb-Hp complex to mitigate the vasoactive response induced by the transfusion of LMW PolyHb. Hp binding to PolyHb was characterized in vitro. The effectiveness of apoHb-Hp administration on reducing the vasoconstriction and pressor effects of PolyHb was assessed by measuring systemic and microcirculatory hemodynamics. Transfusion of LMW PolyHb to vehicle control pretreated animals increased mean arterial pressure while decreasing arteriole diameter and functional capillary density. However, transfusion of LMW PolyHb to apoHb-Hp pretreated animals prevented changes in mean arterial pressure, heart rate, arteriole diameter, blood flow, and functional capillary density relative to before transfusion. These results indicate that the increased size of PolyHb after binding to the apoHb-Hp complex may help compartmentalize PolyHb in the vascular space and thus reduce extravasation, nitric oxide scavenging, and toxicity responsible for vasoconstriction and systemic hypertension.

Anti-inflammatory effects of haptoglobin on LPS-stimulated macrophages: Role of HMGB1 signaling and implications in chronic wound healing.

Nonhealing wounds possess elevated numbers of pro-inflammatory M1 macrophages, which fail to transition to anti-inflammatory M2 phenotypes that promote healing. Hemoglobin (Hb) and haptoglobin (Hp) proteins, when complexed (Hb-Hp), can elicit M2-like macrophages through the heme oxygenase-1 (HO-1) pathway. Despite the fact that nonhealing wounds are chronically inflamed, previous studies have focused on non-inflammatory systems, and do not thoroughly compare the effects of complexed vs individual proteins. We aimed to investigate the effect of Hb/Hp treatments on macrophage phenotype in an inflammatory, lipopolysaccharide (LPS)-stimulated environment, similar to chronic wounds. Human M1 macrophages were cultured in vitro and stimulated with LPS. Concurrently, Hp, Hb, or Hb-Hp complexes were delivered. The next day, 27 proteins related to inflammation were measured in the supernatants. Hp treatment decreased a majority of inflammatory factors, Hb increased many, and Hb-Hp had intermediate trends, indicating that Hp attenuated overall inflammation to the greatest extent. From this data, Ingenuity Pathway Analysis software identified high motility group box 1 (HMGB1) as a key canonical pathway-strongly down-regulated from Hp, strongly up-regulated from Hb, and slightly activated from Hb-Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. In vivo results in diabetic mice with biopsy punch wounds demonstrated accelerated wound closure with Hp treatment, and delayed wound closure with Hb treatment. This work specifically studied Hb/Hp effects on macrophages in a highly inflammatory environment relevant to chronic wound healing. Results show that Hp-and not Hb-Hp, which is known to be superior in noninflammatory conditions-reduces inflammation in LPS-stimulated macrophages, and HMGB1 signaling is also implicated. Overall, Hp treatment on M1 macrophages in vitro reduced the inflammatory secretion profile, and also exhibited benefits in in silico and in vivo wound-healing models.

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.

Apohemoglobin-haptoglobin complex attenuates the pathobiology of circulating acellular hemoglobin and heme.

Haptoglobin (Hp) is the plasma protein that binds and clears cell-free hemoglobin (Hb), whereas apohemoglobin (apoHb, i.e., Hb devoid of heme) can bind heme. Therefore, the apoHb-Hp protein complex should facilitate holoHb-apoHb αß-dimer exchange and apoHb-heme intercalation. Thus, we hypothesized that apoHb-Hp could facilitate both Hb and heme clearance, which, if not alleviated, could have severe microcirculatory consequences. In this study, we characterized apoHb-Hp and Hb/heme ligand interactions and assessed their in vivo consequences. Hb exchange and heme binding with the apoHb-Hp complex was studied with transfer assays using size-exclusion high-performance liquid chromatography coupled with UV-visible spectrophotometry. Exchange/transfer experiments were conducted in guinea pigs dosed with Hb or heme-albumin followed by a challenge with equimolar amounts of apoHb-Hp. Finally, systemic and microcirculatory parameters were studied in hamsters instrumented with a dorsal window chamber via intravital microscopy. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. Dosing with the apoHb-Hp complex reversed Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, reduced microvascular blood flow, and diminished functional capillary density. Therefore, this study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure.NEW & NOTEWORTHY This study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. The apoHb-Hp complex reverses Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, preserves microvascular blood flow, and functional capillary density. In summary, the unique properties of the apoHb-Hp complex prevent adverse systemic and microvascular responses to Hb and heme-albumin exposure and introduce a novel therapeutic approach to facilitate simultaneous removal of extracellular Hb and heme.

Tangential flow filtration of haptoglobin.

Haptoglobin (Hp) is a plasma glycoprotein that scavenges cell-free hemoglobin (Hb). Hp has various potential therapeutic applications, but it has been mainly studied for treatment of acute hemolytic conditions that can arise from situations such as massive blood transfusion, infusion of stored red blood cells, severe burns, trauma, sepsis, radiation injury, and others. Therefore, Hp may also be beneficial during chronic hemolytic disease states such as hereditary spherocytosis, nocturnal hemoglobinuria, sickle-cell anemia, and malaria. Various methods have been developed to purify Hp from plasma or plasma fractions. However, none of these methods have exploited the large molecular weight (MW) range distribution of Hp polymers to easily isolate Hp from other plasma proteins. The present study used tangential flow filtration (TFF) to isolate polymeric Hp from plasma proteins using human Fraction IV (FIV) as the starting material. After removal of insoluble material from a suspension of FIV paste, the protein mixture was clarified on a 0.2 µm hollow fiber (HF) TFF filter. The clarified protein solution was then bracketed based on protein MW using HF filters with MW cut-offs (MWCOs) of 750, 500, and 100 kDa. Using untreated FIV, the Hp purity of the main bracket was ~75% with a total Hb binding capacity (HbBC) yield of 1.2 g starting from 500 g of FIV paste. However, pretreatment of FIV with fumed silica to remove lipoproteins increased Hp purity to >95% with a HbBC yield of 1.7 g per 500 g of FIV. Taken together this study provides a novel and scalable method to purify Hp from plasma or plasma fractions.

Poly(ethylene glycol) Surface-Conjugated Apohemoglobin as a Synthetic Heme Scavenger.

Apohemoglobin (apoHb) contains vacant hydrophobic heme-binding pockets that can bind to a variety of hydrophobic molecules. Thus, apoHb is a promising protein for drug delivery, bioimaging, and heme scavenging. Unfortunately, apoHb has a short half-life and precipitates at physiological temperature. In this study, apoHb was surface-conjugated with poly(ethylene glycol) (PEG) to improve the therapeutic potential of apoHb. The scalable PEGylation process had >95% protein yield with ∼10 to 12 PEGs attached to each apoHb αß dimer. The resulting PEG-apoHb had an average molecular weight of ∼80 to 90 kDa and a hydrodynamic diameter of 11 nm. PEG-apoHb maintained high heme-binding affinity and 30-40% of the heme-binding activity. Moreover, heme-bound and heme-free PEG-apoHb bound to haptoglobin, enabling PEG-apoHb to potentially target CD163+ macrophages and monocytes. Finally, PEG-apoHb was stable at physiological temperature with minimal precipitation. In summary, the in vitro results shown demonstrate that PEG-apoHb could be an effective in vivo heme scavenger during states of hemolysis.

Enhanced Photodynamic Therapy Using the Apohemoglobin-Haptoglobin Complex as a Carrier of Aluminum Phthalocyanine.

Photodynamic therapy (PDT) has been shown to effectively treat cancer by producing cytotoxic reactive oxygen species via excitation of photosensitizer (PS). However, most PS lack tumor cell specificity, possess poor aqueous solubility, and cause systemic photosensitivity. Removing heme from hemoglobin (Hb) yields an apoprotein called apohemoglobin (apoHb) with a vacant heme-binding pocket that can efficiently bind to hydrophobic molecules such as PS. In this study, the PS aluminum phthalocyanine (Al-PC) was bound to the apoHb-haptoglobin (apoHb-Hp) protein complex, forming an apoHb-Al-PC-Hp (APH) complex. The reaction of Al-PC with apoHb prevented Al-PC aggregation in aqueous solution, retaining the characteristic spectral properties of Al-PC. The stability of apoHb-Al-PC was enhanced via binding with Hp to form the APH complex, which allowed for repeated Al-PC additions to maximize Al-PC encapsulation. The final APH product had 65% of the active heme-binding sites of apoHb bound to Al-PC and a hydrodynamic diameter of 18 nm that could potentially reduce extravasation of the molecule through the blood vessel wall and prevent kidney accumulation of Al-PC. Furthermore, more than 80% of APH's absorbance spectra were retained when incubated for over a day in plasma at 37 °C. Heme displacement assays confirmed that Al-PC was bound within the heme-binding pocket of apoHb and binding specificity was demonstrated by ineffective Al-PC binding to human serum albumin, Hp, or Hb. In vitro studies confirmed enhanced singlet oxygen generation of APH over Al-PC in aqueous solution and demonstrated effective PDT on human and murine cancer cells. Taken together, this study provides a method to produce APH for enhanced PDT via improved PS solubility and potential targeted therapy via uptake by CD163+ macrophages and monocytes in the tumor (i.e., tumor-associated macrophages). Moreover, this scalable method for site-specific encapsulation of Al-PC into apoHb and apoHb-Hp may be used for other hydrophobic therapeutic agents.

Novel manufacturing method for producing apohemoglobin and its biophysical properties.

Apohemoglobin (apoHb) is a dimeric globular protein with two vacant heme-binding pockets that can bind heme or other hydrophobic ligands. Purification of apoHb is based on partial hemoglobin (Hb) unfolding to facilitate heme extraction into an organic solvent. However, current production methods are time consuming, difficult to scale up, and use highly flammable and toxic solvents. In this study, a novel and scalable apoHb production method was developed using an acidified ethanol solution to extract the hydrophobic heme ligand into solution and tangential flow filtration to separate heme from the resultant apoprotein. Total protein and active protein yields were >95% and ~75%, respectively, with <1% residual heme in apoHb preparations and >99% purity from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Virtually no loss of apoHb activity was detected at 4°C, -80°C, and in lyophilized form during long term storage. Structurally, size exclusion chromatography (SEC) and circular dichroism indicated that apoHb was dimeric with a ~25% reduction of helical content compared to Hb. Furthermore, mass spectroscopy and reverse-phase chromatography indicated that the mass of the α and ß subunits were virtually identical to the theoretical mass of these subunits in Hb and had no detectable oxidative modifications upon heme removal from Hb. SEC confirmed that apoHb bound to haptoglobin at a similar ratio to that of native Hb. Finally, reconstituted Hb (rHb) was processed via a hemichrome removal method to isolate functional rHb for biophysical characterization in which the O2 equilibrium curve, O2 dissociation, and CO association kinetics of rHb were virtually identical to native Hb. Overall, this study describes a novel and improved method to produce apoHb, as well as presents a comprehensive biochemical analysis of apoHb and rHb.

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.

Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm.

Delayed ischemic neurological deficit (DIND) is a major driver of adverse outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH), defining an unmet need for therapeutic development. Cell-free hemoglobin that is released from erythrocytes into the cerebrospinal fluid (CSF) is suggested to cause vasoconstriction and neuronal toxicity, and correlates with the occurrence of DIND. Cell-free hemoglobin in the CSF of patients with aSAH disrupted dilatory NO signaling ex vivo in cerebral arteries, which shifted vascular tone balance from dilation to constriction. We found that selective removal of hemoglobin from patient CSF with a haptoglobin-affinity column or its sequestration in a soluble hemoglobin-haptoglobin complex was sufficient to restore physiological vascular responses. In a sheep model, administration of haptoglobin into the CSF inhibited hemoglobin-induced cerebral vasospasm and preserved vascular NO signaling. We identified 2 pathways of hemoglobin delocalization from CSF into the brain parenchyma and into the NO-sensitive compartment of small cerebral arteries. Both pathways were critical for hemoglobin toxicity and were interrupted by the large hemoglobin-haptoglobin complex that inhibited spatial requirements for hemoglobin reactions with NO in tissues. Collectively, our data show that compartmentalization of hemoglobin by haptoglobin provides a novel framework for innovation aimed at reducing hemoglobin-driven neurological damage after subarachnoid bleeding.