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.

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.

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.

[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.

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.

Octamers and nanoparticles as hemoglobin based blood substitutes.

Progress in developing a blood substitute is aided by new biotechnologies and a better understanding of the circulatory system. For Hb based solutions, there is still a debate over the best set of fundamental parameters concerning the oxygen affinity which is correlated with the oxidation rate, the cooperativity, the transporter size, and of course the final source of material. Genetic engineering methods have helped discover novel globins, but not yet the quantity necessary for the high demand of blood transfusions. The expanding database of globin properties has indicated that certain individual parameters are coupled, such as the oxygen affinity and the oxidation rate, indicating that one must accept a compromise of the best parameters. After a general introduction of these basic criteria, we will focus on two strategies concerning the size of the oxygen transporter: Hb octamers, and Hb integrated within a nanoparticle.

HemoCD as an artificial oxygen carrier: oxygen binding and autoxidation.

Despite many attempts to construct completely artificial systems for carrying oxygen (O(2)) in aqueous solution, no successful example had been reported until quite recently except for picket fence porphinatoiron(II) embedded in liposomal membrane. We newly prepared a 1:1 complex (hemoCD) of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(II) (Fe[II]TPPS) and a per-O-methylated beta-cyclodextrin dimer having a pyridine linker (Py3CD). HemoCD binds O(2) reversibly in aqueous solution. The oxygen affinity corresponding to the partial O(2) pressure, at which half of the hemoCD molecules are oxygenated, was 16.9 torr in phosphate buffer at pH 7.0 and 25 degrees C. Oxy-hemoCD was gradually autoxidized (t(1/2) = 30.1 h) due to nucleophilic attack of a water molecule to the O(2)-Fe bond. Encapsulation of the iron center of Fe(II)TPPS by two cyclodextrin truncated cones is essential for binding of O(2) to the ferrous center of the porphyrin. This manuscript reports the basic characteristics of hemoCD and the possible future utility of a totally artificial O(2) carrier.

[Research on prerequisite for using bovine hemoglobin modified by polyethylene glycol as a red blood cell substitute].

This paper aims to raise concerns about the modification of bovine hemoglobin with the use of SC-mPEG as modifying reagent. The yield rates of SC-mPEG-BHB under dissimilar conditions are studied, and more important, a series of methods used to characterize the SC-mPEG-BHB mixtures are built. The best reactive system has been confirmed, i.e., at 4 degrees C, pH9; SC-mPEG and BHB are mixed according to the proportion of 60:1 and be submitted to reaction for 2 h.

[Effects of surfactant and solvent on the encapsulation efficiency and size in using double emulsion method for preparing bovine hemoglobin loaded nanoparticles as blood substitutes].

On the basis of previous researches, we have prepared Bovine hemoglobin-loaded nanoparticles (HbP), using the double emulsion method. More mild dispersing treatment was employed during the primary and secondary emulsion; over 97% encapsulation efficiency (EE%) and an average size about 286 nm were achieved by using surfactants, screening solvents, as well as avoiding the traditional strong dispersing process. The value of Hydrophile-lipophile balance in oil phase exerted a significant effect on EE% and led to higher EE% when matched with the surfactants in outer aqueous phase. When compared with the sole solvent Span80, the mixed surfactants such as Poloxemer188/Span80 stabilized the emulsion more efficiently and increased the EE%. The higher concentration of surfactants resulted in higher EE% and narrower size distribution. But over some amount, the surfactants had no significant effect on EE%, resulting in larger size and polydispersity index (PDI). The appropriate removal rate of solvents contributes to higher EE%, smaller size and PDI.

Role of extension arm in PEG-Hb conjugates on the stability of the tetramer: non-conservative EAF maleimide thio-PEG mediated PEGylation.

PEGylation induced increase in colloidal osmotic pressure (COP) of Hb, one of the unique molecular properties of PEG-Hb conjugates, facilitates the neutralization of the vasoconstrictive activity of acellular Hb is a function of chemistry of conjugation of PEG-chains. The dependence of COP with the chemistry is a consequence of PEGylation induced weakening of interdimeric interactions. The conservative Extension Arm Facilitated (EAF) PEGylation exerts least influence on lowering the tetramer stability as compared to direct PEGylation. We have now designed a new, non-conservative EAF PEGylation that uses acylation chemistry to introduce the extension arm onto proteins to delineate the role of the extension arm and of the charge at the site of attachment in PEG-Hb conjugate on tetramer stability. The non-conservative EAF PEGylation does not lower the tetramer stability just as the non-conservative direct PEGylation of Hb. The impact of the extension arm in PEGylated Hb in terms of their structure and potential significance of higher tetramer stability of PEG-Hb conjugates generated by EAF-PEGylation in the in vivo toxicity and in the design of oxygen carrying plasma expander has been discussed.

[Study of polymerizing hemoglobin on cation exchange chromatography].

Poly-hemoglobin is the active component of hemoglobin-based blood substitutes. The excess reactivity of glutaraldehyde with hemoglobin in solution leads to poly-hemoglobin of a wide molecular weight distribution and a high average molecular weight. A new polymerization method has been tested to decrease the molecular weight distribution and the average molecular weight. The poly-hemoglobin with lower degree of modification (polymerization) was found enriched on the cation exchange columns and further polymerized with glutaraldehyde. The poly-hemoglobin of narrower molecular weight distribution has been prepared in this way.

[Current status of researches in the development of blood tissue engineering product].

The term "blood substitutes" includes plasma substitutes and blood cell substitutes in the broad sense, but in its narrow sense, it means red blood cell (RBC) substitutes, platelet substitutes and white blood cell (WBC) substitutes. The RBC substitutes includes perfluorocarbon, hemoglobin-based and encapsuled substitutes. The hemoglobin-based substitutes which was widely researched in the world includes human hemoglobin-based, animal hemoglobin-based and gene recombined hemoglobin based substitutes. The function and immunology of WBC is very complicated, so it is rarely used in clinic. Nowadays the platelet substitutes pursued by the researches and developments includes mainly the liposome and collagenic fiber species substitutes.

[Study on mass transfer behavior of hemoglobin-based nanocapsule surface].

Three dimensional structure of the surface is an important factor that influences the mass transfer behavior of hemoglobin-based nanocapsule surface. In this paper, the modified double emulsion method was used to fabricate the blood substitute of hemoglobin-based nanocapsules, and with the use of different molecular weight of PEG as probes, the effects of different technical conditions (such as primary emulsion, double emulsion, polymer, solvent, et al) in the processing on the three dimensional structure of the nanocapsule surface were investigated in details. Researches indicated that the water-soluable solvent, such as ethyl acetate and acetone could effectively modulate the pore size of the nanocapsule surface. With the increasing of the ratio of water-soluble solvent, the pore size of the nanocapsules firstly increased and then decreased. The increasing of the extra-water volume, the prolongation of the solvent evaporation time, and the improvement of the stirring speed resulted in a bigger pore size, but the increasing of the solvent volume and PEG polymer could reduce the pore size of nanocapsule surface.

[Preparation of hemoglobin-loaded nanoparticles and safety evaluation in vitro and in vivo].

Hemoglobin-loaded nano-sized particles with oxygen carrying capacity were prepared. All experiments were performed using biodegradable polymer poly (polyepsilon-caprolactone) (PCL) as matrix polymer. Optimized preparation parameters led to nanoparticles with well-defined characteristics such as size <200 nm, P50 27 mmHg and high encapsulation efficiency up to 99.4%. The results of in vitro and vivo studies suggested that Hb-loaded particles did not activate complements. After the nanoparticles suspension was injected into the mice via tail vein, the particles did not cause significant changes in total platelet counts. Apparently, the hemoglobin-loaded nanoparticles can serve as a potential candidate in substitution for red cells.

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.

Polypeptide multilayer nanofilm artificial red blood cells.

Reliable encapsulation of hemoglobin (Hb) within polypeptide multilayer nanofilms has been achieved by a template-based approach, and protein functionality has been demonstrated postencapsulation. The method is general in scope and could be useful for many other encapsulants. Met-Hb was adsorbed onto 5 microm-diameter CaCO3 microparticles, and the Hb-coated particles were encapsulated within a multilayer nanofilm of poly(L-glutamic acid) (PLGA) and poly(L-lysine) (PLL) by layer-by-layer assembly. The CaCO3 templates were then dissolved within the PLGA/PLL nanofilms by addition of ethylenediaminetetraacetic acid. Encapsulation of Hb was proved by fluorescence microscopy, the pH-dependence of retention of Hb was determined by visible wavelength absorbance, and conversion of the encapsulated met-Hb to deoxy-Hb and oxy-Hb was demonstrated by spectroscopic analysis of the Soret absorption peak under various conditions. It thus has been shown that control of Hb oxygenation within polypeptide multilayer nanofilm artificial cells is possible, and that Hb thus encapsulated can bind, release, and subsequently rebind molecular oxygen. This work therefore represents an advance in the development of polypeptide multilayer film artificial red blood cells.

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.