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.

Improved oxygen storage capacity of haemoglobin submicron particles by one-pot formulation.

The coprecipitation-cross-linking-dissolution (CCD) technique for protein submicron particle fabrication was improved by omitting one preparation step using the macromolecular cross-linker, periodate-oxidized dextran (Odex, M.W. of 40 and 70 kDa). The coprecipitation and cross-linking of haemoglobin (Hb) were combined in one single step since the cross-linker is incorporated into the inorganic template, MnCO3, together with the protein. After removal of the MnCO3 templates by EDTA, the amount of entrapped Hb was 60 to 70% of the initial amount. This technique provides deformable Hb submicron particles (HbMP) with narrow size distribution between 800 and 1000 nm, uniform morphology and negative zeta-potential. More than 40% of Hb in the particles was able to carry oxygen over a storage period of 90 days. The results suggest that our new protein submicron particle fabrication technique minimizes the fabrication time and is very efficient and cost-effective.

Manipulating hemoglobin oxygenation using silica nanoparticles: a novel prospect for artificial oxygen carriers.

Recently, nanoparticles have attracted much attention as new scaffolds for hemoglobin-based oxygen carriers (HBOCs). Indeed, the development of bionanotechnology paves the way for the rational design of blood substitutes, providing that the interaction between the nanoparticles and hemoglobin at a molecular scale and its effect on the oxygenation properties of hemoglobin are finely controlled. Here, we show that human hemoglobin has a high affinity for silica nanoparticles, leading to the adsorption of hemoglobin tetramers on the surface. The adsorption process results in a remarkable retaining of the oxygenation properties of human adult hemoglobin and sickle cell hemoglobin, associated with an increase of the oxygen affinity. The cooperative oxygen binding exhibited by adsorbed hemoglobin and the comparison with the oxygenation properties of diaspirin cross-linked hemoglobin confirmed the preservation of the tetrameric structure of hemoglobin loaded on silica nanoparticles. Our results show that silica nanoparticles can act as an effector for human native and mutant hemoglobin. Manipulating hemoglobin oxygenation using nanoparticles opens the way to the design of novel HBOCs.

Negligible interaction of [Ru(Phen)3]2+ with human serum albumin makes it promising for a reliable invivo assessment of the tissue oxygenation.

The interaction between a ruthenium - based water soluble oxygen probe ([Ru(Phen)3]2+, phen - phenanthroline) and human serum albumin (HSA) was investigated with the aim of describing the influence of HSA on the [Ru(Phen)3]2+ luminescence properties. Nowadays, several oxygen sensitive luminescent probes are used to determine the oxygen level in different compartments of living organisms. However, they can interact, depending on their hydrophilic/hydrophobic characters, with various serum proteins, and/or lipids, during their utilization for invivo oxygen measurement. Since HSA is the most abundant serum protein in most biological organisms, its presence may affect the spectral properties of the employed probes and, consequently, the determination of the oxygen concentration. Having this in mind, we have applied several spectroscopic and calorimetric techniques to study [Ru(Phen)3]2+ - HSA mixtures. Only a negligible effect of HSA on the absorption and luminescence spectra of [Ru(Phen)3]2+ was observed. In addition, differential scanning calorimetric studies showed that [Ru(Phen)3]2+ does not significantly influence HSA thermal stability. Importantly, [Ru(Phen)3]2+ retained a reliable luminescence lifetime sensitivity to the oxygen concentration in solutions supplemented with HSA and in U87 MG cancer cells. Finally, the biodistribution of [Ru(Phen)3]2+ in the presence of serum proteins in the blood stream of chick embryo's chorioallantoic membrane (CAM) was investigated. Fast [Ru(Phen)3]2+ and similar extravasations were observed in the presence or absence of CAM-serum. We can conclude that HSA-[Ru(Phen)3]2+ complex interaction does not significantly influence the potential of [Ru(Phen)3]2+ to be a suitable candidate for a reliable oxygen probe in living organisms.

HemoCD as a Totally Synthetic Artificial Oxygen Carrier: Improvements in the Synthesis and O2 /CO Discrimination.

HemoCD, which is composed of an iron(II)porphyrin such as 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(II) (Fe(II)TPPS) and a cyclodextrin (CD) dimer having a pyridine linker, represents a synthetic hemoglobin (Hb) model compound that exhibits reversible oxygen (O2 ) binding ability in aqueous solution at an ambient temperature. Therefore, hemoCD has the potential to be used as a totally synthetic artificial oxygen carrier. In this article, we describe the improvements of hemoCD related to its synthesis and O2 /CO selectivity. The synthesis procedure of the CD dimer of hemoCD was re-examined, and the CD dimer was successively synthesized from inexpensive ß-CD with a 38% yield (three-steps), which enabled us to obtain the CD dimer in gram-quantities. The O2 /CO selectivity of hemoCD was also markedly improved using an iron(II)porphyrin having a carboxylate group at the distal site of hemoCD.

"Inside-Out" PEGylation of Bovine ß-Cross-Linked Hemoglobin.

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm maleimide-PEG (Mal-PEG) to ß-cross-linked (ßXL-Hb) hemoglobin (Hb) tetramers through the Cys ß93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three ßXL-Hb to one Mal-PEG. Thermal denaturation studies showed that the cross-linked and PEGylated species were more stable than native Hb. Cross-linking under oxy-conditions produced a high oxygen affinity Hb species (P50  = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P50  = 9.67 Torr). Inside-out PEGylation can be used to produce a hemoglobin-based oxygen carrier and potentially for other multiprotein complexes.

Copolymerization of recombinant Phascolopsis gouldii hemerythrin with human serum albumin for use in blood substitutes.

Hemerythrin is an oxygen-carrying protein found in marine invertebrates and may be a promising alternative to hemoglobin for use in blood substitutes, primarily due to its negligible peroxidative toxicity. Previous studies have shown that glutaraldehyde-induced copolymerization of hemoglobin with bovine serum albumin increases the half-life of the active oxy form of hemoglobin (i.e. decreases the auto-oxidation rate). Here, we describe a protocol for glutaraldehyde copolymerization of Hr with human serum albumin and the dioxygen-binding properties of the co-polymerized products. The copolymerization with HSA results in alteration of hemerythrin's dioxygen-binding properties in directions that may be favorable for use in blood substitutes.

Biomacromolecules based core/shell architecture toward biomedical applications.

Polyelectrolyte multilayer capsules have become a novel and promising class of hybrid materials with great potential since they can be applied in various areas, such as pharmaceutical sciences, biotechnology, and biomedicine. The concept of using such carriers for biology application is diagnosis and treatment of diseases for convenience, safety and specific targeting. Therefore, the development of biocompatible, biodegradable and specific characteristic nanostructure material is highly desirable. Much effort has been devoted to exploring innovative and effective techniques to fabricate such materials. Among the available techniques, layer-by-layer (LbL) assembly capsules have attracted considerable attention attributing to the flexibly controlled size, shape, composition, wall thickness and functions. Protein, as the large class of biomacromolecules, was incorporated into capsules for improving the biocompatibility and specific function. In this review we provide an overview of the recent progress in biomacromolecular capsules or core/shell architecture with different diameters for the variety of purposes. The size ranging from micro-, sub-micro to nano scale based on the choice of the template. Their advantages are discussed here. The applications of these biomacromolecular capsules in biotechnological fields have also been summarized, for instance blood substitute, ATP carriers, photodynamic therapy and nanomedicines.

Polymerization of modified diaspirin cross-linked hemoglobin (DCLHb) with 1,6-bismaleimic-hexane.

Increasing the size of hemoglobin (Hb) by polymerization offers the benefits of reduced renal clearance and increased duration in the vascular circulation. With this goal, diaspirin cross-linked hemoglobin (DCLHb) was modified in order to keep one thiol group on the surface and then polymerized with 1,6-bismaleimic-hexane (1,6-BMH) to increase the molecular weight. The HPLC results indicated that approximate 20% dimers to tetramers of DCLHb desired were achieved after the polymerization. It was also demonstrated that the oxygen-carrying capacity of the products was similar to natural heme. The present study is expected to improve the efficacy of the DCLHb as an oxygen therapeutic agent.

Processing of ferulic acid modified hemoglobin.

The application of hemoglobin-based oxygen carriers (HBOCs) remains stagnant because a large number of reactive oxygen species (ROS) will be produced in organisms when HBOCs are used. Ferulic acid (FA) has a known effect on quenching oxygen free radicals and relieving vasospasm. Thus a kind of FA modified hemoglobin (BAEGF-Hb) is expected to produce the anti-oxidation effect on HBOCs spontaneously, and reduce toxicity. Isoelectric focusing electrophoresis, UV-vis wavelength scanning, and oxygen affinity curve showed that hemoglobin (Hb) was successfully modified and this modification had no effect on the structure of Hb and P50. The modification degree of Hb is 4.

Influence of Molecular Structure on O2-Binding Properties and Blood Circulation of Hemoglobin‒Albumin Clusters.

A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, is an artificial O2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater than that of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity compared to the others because of the chemical linkage between the Cys-93(ß) residues in Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4. Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior.

Nanoscale Metal-Organic Framework-Hemoglobin Conjugates.

A metal-organic framework (MOF)-protein conjugate, NH2 -MIL-125(Ti)-hemoglobin [MIL-125(Ti)-Hb], was synthesized by a covalent postmodification strategy. The crystalline structure was maintained after chemical and protein modification. The content of grafted Hb was tuned by the stoichiometric ratio and reached 50 wt % if the mass ratio of MIL-125(Ti)/Hb was 1:1.25 in the feed. The oxygen-transporting capacity of grafted Hb was kept, and the P50 (the half O2 pressure saturated with O2 ) and Hill coefficients of the MIL-125(Ti)-Hb conjugate were found to be 22.9 mm Hg and 2.35, respectively, which are close to the respective values of free Hb. All the results indicate that the MIL-125(Ti)-Hb conjugate could be potentially used as an oxygen carrier.

Optimization and characterization of stable lipid-based, oxygen-filled microbubbles by mixture design.

Tissue hypoxia is a final common pathway that leads to cellular injury and death in a number of critical illnesses. Intravenous injections of self-assembling, lipid-based oxygen microbubbles (LOMs) can be used to deliver oxygen gas, preventing organ injury and death from systemic hypoxemia. However, current formulations exhibit high polydispersity indices (which may lead to microvascular obstruction) and poor shelf-lives, limiting the translational capacity of LOMs. In this study, we report our efforts to optimize LOM formulations using a mixture response surface methodology (mRSM). We study the effect of changing excipient proportions (the independent variables) on microbubble diameter and product loss (the dependent variables). By using mRSM analysis, the experimental data were fit using a reduced Scheffé linear mixture model. We demonstrate that formulations manufactured from 1,2-distearoyl-sn-glycero-3-phosphocholine, corn syrup, and water produce micron-sized microbubbles with low polydispersity indices, and decreased product loss (relative to previously described formulations) when stored at room temperature over a 30-day period. Optimized LOMs were subsequently tested for their oxygen-releasing ability and found to have similar release kinetics as prior formulations.

Modification of a dioxygen carrier, hemoCD, with PEGylated dendrons for extension of circulation time in the bloodstream.

A supramolecular diatomic receptor, hemoCD, was modified with PEGylated dendrons to extend its circulation time in the bloodstream. The core component was 4-oxo-4-[[4-(10,15,20-tris(4-sulfonatophenyl)-21H,23H-porphin-5-yl)phenyl]amino]butanoic acid (Por-COOH). The building block of the dendrons was Fmoc-4-amino-4-(2-carboxyethyl)heptanedioic acid (FmocTA), which was condensed with α-amino-ω-methoxy-poly(ethylene glycol) (PEG(5000)-NH(2)) to yield an FmocG1-dendron. After deprotection, the G1-dendron was condensed with Por-COOH to yield G1-Por. A precursor (FmocNA) of an FmocG2-dendron was prepared via a condensation reaction of 4-amino-4-(2-t-butoxycarbonylethyl)heptanedioic acid di-t-butyl ester (TA-E) with FmocTA followed by hydrolysis of the resultant nona-carboxylic acid nona-t-butyl ester. Condensation of FmocNA with PEG(5000)-NH(2) yielded an FmocG2-dendron. After deprotection, the G2-dendron was condensed with Por-COOH to yield G2-Por. The ferrous complexes of G1- and G2-Pors formed stable 1:1 inclusion complexes with Py3CD, a per-O-methylated ß-cyclodextrin dimer with a pyridine linker, in aqueous solution yielding supramolecular complexes designated as G1-hemoCD and G2-hemoCD, respectively. Both G1- and G2-hemoCDs bound molecular oxygen, with the O(2) affinities (P(1/2)) of hemoCD, G1-, and G2-hemoCDs at pH 7.4 and 37 °C being 22, 20, and 20 Torr, respectively. The modification of hemoCD with the dendrons did not cause destabilization of the O(2) adducts via autoxidation, as indicated by their half-lives (t(1/2)) of 6.8, 6.1, and 5.5 h for hemoCD, G1-, and G2-hemoCDs, respectively. The blood concentration-time curves of G1- and G2-hemoCDs injected into the bloodstream of rats exhibited two phases, with the half-lives of the fast and slow decays being 0.45 and 5.3 h, respectively, for G1-hemoCD, and 0.20 and 12.8 h, respectively, for G2-hemoCD. The half-lives of hemoCD were 0.02 and 0.50 h, respectively. The circulation time of hemoCD was markedly extended by its modification with the PEGylated dendrons, which was very effective in protecting hemoCD against opsonization for uptake by the reticuloendothelial system.

Low modulus biomimetic microgel particles with high loading of hemoglobin.

We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT (particle replication in nonwetting templates) technique. Low cross-linking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained without a significant effect on particle stability and shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen, as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with a Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.

Highly loaded hemoglobin spheres as promising artificial oxygen carriers.

Seeking safe and effective artificial blood substitutes based on hemoglobin (Hb) as oxygen carriers is an important topic. A significant challenge is to enhance the loading content of Hb in a well-defined structure. Here we report a facile and controllable avenue to fabricate Hb spheres with a high loading content by templating decomposable porous CaCO(3) particles in collaboration with covalent layer-by-layer assembly technique. The surface of the Hb spheres was further chemically modified by biocompatible polyethylene glycol to protect and stabilize the system. Multiple characterization techniques were employed to reveal the loading and density of Hb in an individual CaCO(3) particle. The results demonstrate that the strategy developed in this work is effective and flexible for construction of the highly loaded Hb spheres. More importantly, such Hb spheres retain their carrying-releasing oxygen function. It may thus have great potential to develop Hb spheres with highly loaded content as realistic artificial blood substitutes in the future.

[Influence of Perftoran nanoemulsion on blood plasma concentrations of lipophilic drugs].

The influence of perfluorocarbon blood substitute Perfloran on the plasma concentrations of bendazole, drotaverine, ketorolac and verapamil upon intravenous introduction after Perfloran infusion (5 ml/kg) has been investigated on rabbits. It has been found that the plasma concentrations of verapamil, drotaverine and bendazole (highly lipophilic drugs with log(P) = 4.5, 4.9 and 3.5, respectively) increased in the presence of Perfloran. The influence of Perfloran on the concentration of weakly lipophilic ketorolac was less significant. Perfloran effectively bound drotaverine, ketorolac and verapamil in vitro, whereas the binding of ketorolac by the emulsion particles was weak. Evidently, the infusion of hydrophobic nanoemulsion Perftoran elevates the sorption capacity of plasma and creates prerequisites for the redistribution drugs and favors increase in their concentrations.

Preparation and function of poly(acrylic acid)s modified by supramolecular complex composed of porphinatoiron and a cyclodextrin dimer that bind diatomic molecules (O2 and CO) in aqueous solution.

Poly(acrylic acid) (PAA) is modified by 5-(4-ß-alanylaminophenyl)-10,15,20-tris(4-sulfonatophenyl) porphinatoiron(III) to yield iron porphyrin-bearing PAAs (FeP(n)s) through a condensation reaction. FeP(n)s were further functionalized by Py3CD, which is a per-O-methylated ß-cyclodextrin (CD) dimer with a pyridine linker and includes the porphyrin pendants to form ferric hemoCD-P(n)s. Ferrous hemoCD-P(3), having three porphyrin chromophores in a polymer chain, is shown to bind molecular oxygen (P(1/2)=7.9±1.4 Torr) in aqueous solution at pH 7.0 and 25 °C, affording oxy-hemoCD-P(3). Oxy-hemoCD-P(3) is biphasically autoxidized to ferric hemoCD-P(3), with 27% of the dioxygen adducts being rapidly oxidized. The rate of autoxidation of oxy-hemoCD-P(15), having 15 porphyrin chromophores in a polymer chain, was much faster than that of oxy-hemoCD-P(3), thus suggesting self-catalyzed autoxidation of oxy-hemoCD-P(n)s. Oxy-hemoCD-P(n)s are markedly stabilized by catalase, thereby indicating that hydrogen peroxide generated from oxy-hemoCD-P(n) accelerates the autoxidation. Most of the hemoCD-P(3) molecules injected into the femoral vein of a rat remained in the body, though about 16% of the hemoCD-P(3) molecules were excreted in the urine as a carbon monoxide adduct.

Improvement of gas transfer by hemosome.

Intravascular oxidation is a respiratory assist method used to treat acute respiratory distress syndrome (ARDS). However intravascular oxidation through higher gas exchange is needed for successful clinical applications. In this study, an attempt was made to improve the gas exchange of an intravascular lung assist device by decreasing the level of damage to the blood through the microencapsulation of hemoglobin. The results showed that a hemosome 0.8 microm in diameter could be produced by microencapsulating the hemoglobin extracted from fresh bovine blood with the phospholipids extracted from egg yolk. The oxygen saturation curve of hemosome was S-shaped, which is similar to that found in normal blood, and the P50 was 24 mmHg. The oxygen saturation in the mixed solution of hemosome and blood at a 1:4 (v/v%) ratio was similar to that of normal blood. The gas exchange of the blood-hemosome mixed solution was more effective than whole blood. Therefore, the hemosome solution is expected to improve oxygen transfer.