Biocompatibility of the oxygen carrier polymerized human hemoglobin towards HepG2/C3A cells.

Hemoglobin (Hb) based oxygen carriers (HBOCs) are designed to minimize the toxicity of extracellular Hb, while preserving its high oxygen-carrying capacity for oxygen delivery to cells. Polymerized human Hb (PolyHb) is a novel type of nanosized HBOC synthesized via glutaraldehyde-mediated crosslinking of free Hb, and which preserves the predominant quaternary state during the crosslinking reaction (low oxygen affinity tense (T) quaternary state PolyHb is synthesized at 0% Hb oxygen saturation, and high oxygen affinity relaxed (R) quaternary state PolyHb is synthesized at 100% Hb oxygen saturation). Major potential applications for PolyHbs, and HBOCs in general, include oxygenation of bioreactor systems containing large liver cell masses, and ex-vivo perfusion preservation of explanted liver grafts. The toxicity of these compounds toward liver cells must be evaluated before testing their use in these complex systems for oxygen delivery. Herein, we characterized the effect of PolyHbs on the hepatoma cell line HepG2/C3A, used as a model hepatocyte and as a cell line used in some investigational bioartificial liver support devices. HepG2/C3A cells were incubated in cell culture media containing PolyHbs or unmodified Hb at concentrations up to 50 mg/mL and for up to 6 days. PolyHbs were well tolerated at a dose of 10 mg/mL, with no significant decrease in cell viability; however, proliferation was inhibited as much as 10-fold after 6 days of exposure at 50 mg/mL. Secretion of albumin, and urea, as well as glucose and ammonia removal were measured in presence of 10 mg/mL of PolyHbs or unmodified Hb. In addition, methoxy- and ethoxy-resorufin deacetylase (MROD and EROD) activities, which reflect cytochrome P450 metabolism, were measured. R-state PolyHb displayed improved or intact activity in 3 out of 7 functions compared to unmodified Hb. T-state PolyHb displayed improved or intact activity in 4 out of 7 functions compared to unmodified Hb. Thus, PolyHbs, both in the R-state and T-state, are safer to use at a concentration of 10 mg/mL as compared to unmodified Hb in static culture liver-related applications.

Photocatalytic Synthesis of a Polydopamine-Coated Acellular Mega-Hemoglobin as a Potential Oxygen Therapeutic with Antioxidant Properties.

Hemoglobin-based oxygen carriers (HBOCs) are being developed to overcome limitations associated with transfusion of donated red blood cells (RBCs) such as potential transmission of blood-borne pathogens and limited ex vivo storage shelf-life. Annelid erythrocruorin (Ec) derived from the worm Lumbricus terrestris (Lt) is an acellular mega-hemoglobin that has shown promise as a potential HBOC due to the large size of its oligomeric structure, thus overcoming limitations of unmodified circulating cell-free hemoglobin (Hb). With a large molecular weight of 3.6 MDa compared to 64.5 kDa for human Hb (hHb) and 144 oxygen-binding globin subunits compared to the 4 globin subunits of hHb, LtEc does not extravasate from the circulation to the same extent as hHb. LtEc is stable in the circulation without RBC membrane encapsulation and has a lower rate of auto-oxidation compared to acellular hHb, which allows the protein to remain functional for longer periods of time in the circulation compared to HBOCs derived from mammalian Hbs. Surface coatings, such as poly(ethylene glycol) (PEG) and oxidized dextran (Odex), have been investigated to potentially reduce the immune response and improve the circulation time of LtEc in vivo. Polydopamine (PDA) is a hydrophilic, biocompatible, bioinspired polymer coating used for biomedical nanoparticle assemblies and coatings and has previously been investigated for the surface coating of hHb. PDA is typically synthesized via the self-polymerization of dopamine (DA) under alkaline (pH > 8.0) conditions. However, at pH > 8.0, the oligomeric structure of LtEc begins to dissociate. Therefore, in this study, we investigated a photocatalytic method of PDA polymerization on the surface of LtEc using 9-mesityl-10-methylacridinium tetrafluoroborate (Acr-Mes) to drive PDA polymerization under physiological conditions (pH 7.4, 25 °C) over 2, 5, and 16 h in order to preserve the size and structure of LtEc. The resulting structural, biophysical, and antioxidant properties of PDA surface-coated LtEc (PDA-LtEc) was characterized using various techniques. PDA-LtEc showed an increase in measured particle size, molecular weight, and surface ζ-potential with increasing reaction time from t = 2 to 16 h compared to unmodified LtEc. PDA-LtEc reacted for 16 h was found to have reduced oxygen-binding cooperativity and slower deoxygenation kinetics compared to PDA-LtEc with lower levels of polymerization (t = 2 h), but there was no statistically significant difference in oxygen affinity. The thickness of the PDA coating can be controlled and in turn the biophysical properties can be tuned by changing various reaction conditions. PDA-LtEc was shown to demonstrate an increased level of antioxidant capacity (ferric iron reduction and free-radical scavenging) when synthesized at a reaction time of t = 16 h compared to LtEc. These antioxidant properties may prove beneficial for oxidative protection of PDA-LtEc during its time in the circulation. Hence, we believe that PDA-LtEc is a promising oxygen therapeutic for potential use in transfusion medicine applications.

Scalable manufacturing platform for the production of PEGylated heme albumin.

Cell-free heme, which was previously shown to have adverse effects on the innate immune system, does not induce inflammation when bound to a protein carrier via overexpression of the enzyme heme-oxygenase 1 (HO-1). Studies in mouse macrophage cell culture and human endothelial cells have confirmed HO-1 catalyzed breakdown of protein bound heme into biliverdin, iron, and carbon monoxide (CO), which elicits anti-inflammatory effects. However, to fully realize the anti-inflammatory therapeutic effects of heme, a colloidally stable heme protein carrier must be developed. To accomplish this goal, we incorporated multiple heme molecules into human serum albumin (HSA) via partial unfolding of HSA at basic pH followed by refolding at neutral pH, and subsequently conjugated the surface of the heme-HSA complex with polyethylene glycol (PEG) to stabilize heme-HSA. Quantification studies confirmed that a maximum of 5-6 hemes could be bound to HSA without precipitation or degradation of heme-HSA. Dynamic light scattering, size exclusion-high performance liquid chromatography (SEC-HPLC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the increase in hydrodynamic diameter and molecular weight (MW), respectively, upon PEGylation of heme-HSA. Furthermore, PEG-heme-HSA was stable upon exposure to different pH environments, freeze-thaw cycles, and storage at 4°C. Taken together, we devised a synthesis and purification platform for the production of PEGylated heme-incorporated HSA that can be used to test the potential anti-inflammatory effects of heme in vivo.

Lyophilized annelid mega-hemoglobin retains its' quaternary structure and oxygen equilibrium properties after room temperature storage for over 6 months.

The long-term storage stability and portability of hemoglobin (Hb)-based oxygen carriers are important design criteria in the development of these therapeutics. Lyophilization or storing proteins in a freeze-dried form is known to increase storage lifetime and reduce overall weight. In this study, we lyophilized the extracellular mega-hemoglobin of the annelid Lumbricus terrestris and tested the storage stability at different temperatures and oxygenation conditions. Storage in refrigerated conditions for over 6 months in the presence of N2 reduced oxidation by 50% while storage at room temperature in the presence of N2 reduced oxidation by 60%, all while maintaining the structural stability of the mega-hemoglobin. We also demonstrated a reliable strategy to freeze dry Hbs in the presence of a minimally non-reducing disaccharide sugar that could be easily re-solubilized in deionized water. Overall, this study made significant advances towards long term storage stability of oxygen therapeutics for direct applications in transfusion medicine.

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.

Structural Stability and Biophysical Properties of the Mega-Protein Erythrocruorin Are Regulated by Polyethylene Glycol Surface Coverage.

A wide variety of hemoglobin-based oxygen carriers (HBOCs) have been designed for use as red blood cell (RBC) substitutes in transfusion medicine, ex vivo organ perfusion, oxygen delivery to hypoxic tissues, and a myriad of other applications. However, hemoglobin (Hb) derived from annelids (erythrocruorins [Ecs]) comprise a natural class of HBOC, since they are larger in size (30 nm in diameter) and contain more heme groups per molecule (144 heme groups) compared to human Hb (hHb; 5 nm in diameter and 4 heme groups). The larger size of Ec compared to hHb reduces tissue extravasation from the vascular space, thus, reducing vasoconstriction, systemic hypertension, and tissue oxidative injury when used as an RBC substitute. In addition, prior research has shown that Ecs possess slower auto-oxidation rates than hHb at physiological temperature, thus, making them attractive candidates for use as RBC substitutes. Unfortunately, it was also observed that Ecs have a much lower circulatory half-life in vivo compared to other HBOCs. Hence, conjugating polyethylene glycol (PEG) to the surface of Ec was proposed as a simple strategy to increase Ec circulatory half-life. Therefore, in order to inform future in vivo studies with PEGylated Ec, we decided to investigate the structural stability and biophysical properties of variable PEG surface coverage on Ec compared to native Ec. We observed an increase in PEG-Ec diameter and molecular weight (MW) and changes to the quaternary structure, secondary structure, and surface hydrophobicity after PEGylation. There was also an increase in oxygen binding affinity, reduction in oxygen offloading rate, and increase in auto-oxidation rate for increasing PEGylation ratios. Weak dissociation of Ec was also observed after dense PEGylation caused by steric repulsion of the conjugated PEG chains. Hence, we determined an optimum Ec PEGylation ratio that resulted in a substantial size and MW increase along with preservation of oxygen binding properties. In future studies, these materials will be tested in animal models to evaluate pharmacodynamics, pharmacokinetics, tissue oxygenation, microcirculatory responses, and overall safety.

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.

Purification of Lumbricus terrestris Mega-Hemoglobin for Diverse Oxygen Therapeutic Applications.

Oxygen therapeutics are being developed for a variety of applications in transfusion medicine. In order to reduce the side-effects (vasoconstriction, systemic hypertension, and oxidative tissue injury) associated with previous generations of oxygen therapeutics, new strategies are focused on increasing the molecular diameter of hemoglobin obtained from mammalian sources via polymerization and encapsulation. Another approach towards oxygen therapeutic design has centered on using naturally occurring large molecular diameter hemoglobins (i.e. erythrocruorins) derived from annelid sources. Therefore, the goal of this study was to purify erythrocruorin from the terrestrial worm Lumbricus terrestris for diverse oxygen therapeutic applications. Tangential flow filtration (TFF) was used as a scalable protein purification platform to obtain a >99% pure LtEc product, which was confirmed by size exclusion high performance liquid chromatography and SDS-PAGE analysis. In vitro characterization concluded that the ultra-pure LtEc product had oxygen equilibrium properties similar to human red blood cells, and a lower rate of auto-oxidation compared to human hemoglobin, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultra-pure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (~86% purity). In summary, we purified an LtEc product with favorable biophysical properties that performed well in an animal model using a reliable and scalable purification platform to eliminate undesirable proteins.

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.

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.