Hb Polymer Research Articles

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) polymerichemoglobin (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 filtrationpurification 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.

Effects of BCL11A Shmir-Induced Post-Transcriptional Silencing on Hemoglobin Polymer Inhibition in Single Red Blood Cells at Physiologic Oxygen Tension

IntroductionFetal hemoglobin (HbF) induction is a successful strategy for treating sickle cell disease. Higher levels of total HbF% are associated with better clinical outcomes, and a more uniform distribution of HbF across individual red blood cells (RBCs) is predicted to lead to better outcomes by inhibiting hemoglobin (Hb) polymerization in a higher fraction of individual RBCs. 1 Post-transcriptional silencing of BCL11A using lentivirus expression of a shRNA embedded in a microRNA architecture (shmiR) to re-activate γ-globin expression has been safe and demonstrated high levels of total HbF expression broadly distributed in red cells in a pilot clinical study (NCT 03282656). 2 We assessed the effects of BCL11A down-regulation on Hb polymer formation at the single-RBC level in 7 subjects in this trial (BCL) using novel assays of single-RBC Hb polymer content levels and single-RBC HbF.MethodsHb polymer was detected in individual RBCs using a previously published microfluidic system. 3 The system detects Hb polymer based on RBC morphology and on the principle that Hb polymer lowers RBC oxygen saturation in proportion to polymer concentration. 4,5 Single-RBC HbF mass was measured by flow cytometry using a previously published protocol. 6 These two measurements of distributions of single-RBC HbF mass and single-RBC Hb polymer were then integrated at the cell-population level to estimate the threshold of single-RBC HbF needed to inhibit formation of a detectable level of polymer as function of oxygen tension. Blood samples from BCL subjects (HbF% range 23 - 44, after stabilizing post-therapy) were compared to 14 hydroxyurea (HU) responsive patients (mean HbF% = 17, range 6 - 32), including 3 highly-responsive patients (HbF% = 25, 27, 31) as well as 4 individuals with sickle cell trait (AS).ResultsAt 3.7% oxygen tension typical of the human microcirculation 7 and some regions of the central nervous system, 7 the typical BCL subject had detectable levels of Hb polymer in 52% (median) of RBCs vs. 66% for HU (p = 0.02) and 6% for AS (Figure 1A). At lower oxygen tensions of 1.7% typical of the bone marrow 7 or kidney medulla 7 BCL subjects had a polymerized RBC fraction of 68%, compared to 79% for HU (p<0.001) and 30% for AS. Fractions of RBC with polymer in BCL subjects stabilized about 3-6 months after treatment. By combining measurements of single-RBC HbF mass and single-RBC polymer at the population level, we estimated the threshold HbF mass or fraction required to inhibit detectable levels of polymer as a function of oxygen tension. We compared single-RBC HbF thresholds for the BCL cohort to the three HU high responders. The mean HbF threshold for polymerization inhibition estimated for the BCL cohort was lower (18%) than that for the HU high responders (25%) but was not statistically significant (p = 0.07) in this initial analysis.ConclusionsBCL11A down-regulation in seven clinical trial subjects is associated with favorable distributions of single-RBC polymer at multiple physiologic oxygen tensions compared to HU. Future study is needed to determine whether these favorable Hb polymer distributions are simply a result of higher total HbF or also due to lower single-RBC HbF thresholds for polymer inhibition.

Entropy-Driven Supramolecular Ring-Opening Polymerization of a Cyclic Hemoglobin Monomer for Constructing a Hemoglobin-PEG Alternating Polymer with Structural Regularity.

Our earlier report described that a cyclic hemoglobin (Hb) monomer with two β subunits of a Hb molecule (α2β2) bound through a flexible polyethylene glycol (PEG) chain undergoes reversible supramolecular ring-opening polymerization (S-ROP) to produce a supramolecular Hb polymer with a Hb-PEG alternating structure. In this work, we polymerized cyclic Hb monomers with different ring sizes (2, 5, 10, or 20 kDa PEG) to evaluate the thermodynamics of S-ROP equilibrium. Quantification of the produced supramolecular Hb polymers and the remaining cyclic Hb monomers in the equilibrium state revealed a negligibly small enthalpy change in S-ROP (ΔHp ≤ 1 kJ·mol-1) and a markedly positive entropy change increasing with the ring size (ΔSp = 26.8-33.2 J·mol-1·K-1). The results suggest an entropy-driven mechanism in S-ROP: a cyclic Hb monomer with the larger ring size prefers to form a supramolecular Hb polymer. The S-ROP used for this study has the potential to construct submicrometer-sized Hb-PEG alternating polymers having structural regularity.

Validation of BCL11A As Therapeutic Target in Sickle Cell Disease: Results from the Adult Cohort of a Pilot/Feasibility Gene Therapy Trial Inducing Sustained Expression of Fetal Hemoglobin Using Post-Transcriptional Gene Silencing

Validation of BCL11A As Therapeutic Target in Sickle Cell Disease: Results from the Adult Cohort of a Pilot/Feasibility Gene Therapy Trial Inducing Sustained Expression of Fetal Hemoglobin Using Post-Transcriptional Gene Silencing

Ring-Opening Polymerization of Hemoglobin.

Hemoglobin (Hb), an oxygen-carrying protein, has an α2β2 tetrameric structure that dissociates reversibly into two αβ dimers (α2β2 ⇄ 2αβ). We synthesized a cyclic Hb-ring monomer with two β subunits bound through a 10 kDa polyethylene glycol (PEG) chain. The monomer induced ring-opening polymerization to produce a supramolecular polymer via intersubunit interaction of αβ dimers of an Hb molecule at the PEG terminals. Both the ring-closed monomer and the ring-opened supramolecular polymer were then fixed covalently by intramolecular cross-linking of two β subunits. Quantification of fixed products at various monomer concentrations revealed the equilibrium constant ( K), a ratio of propagation and depropagation rate constants, as 5.68 mM-1. The average degree of polymerization ([Formula: see text]) increased proportionally, concomitantly with the initial monomer concentration. Hb polymer with [Formula: see text] = 13.2 ( Mn = ca. 1 MDa) was obtained by cross-linking at 2.33 mM. Our novel strategy of ring-opening polymerization of Hb will eventually realize a highly aligned and efficiently polymerized Hb for creating artificial oxygen carriers for a clinical use.

Synthesis and properties of hyperbranched polymers by polymerization of an AB3-type incompletely condensed cage silsesquioxane (IC-POSS) monomer

An incompletely condensed cage silsesquioxane (IC-POSS)-based AB3-type monomer, tris(dimethylsilyl)-p-vinylhexaisobutyl-IC-POSS (3), was prepared by the corner-cleaved reaction of vinylheptaisobutyl-POSS (1) and subsequent capping reaction with chlorodimethylsilane. The structure of 3 was confirmed by FT-IR; 1H-, 13C-, and 29Si-NMR; and HR-FAB-MS analyses. These analyses also suggested 12.5 mol% contamination of tris(dimethylsilyl)-heptaisobutyl-IC-POSS (4). The hydrosilylation polymerization of 3 as an AB3-type IC-POSS monomer in the presence of Karstedt’s catalyst in toluene at 80 °C overnight provided a soluble polymer with a number average molecular weight (Mn) and molecular weight distribution (Mw/Mn) of the polymer that are 3.9 × 103 and 1.39, respectively. The resulting hyperbranched polymer (HB) reacted with two molar equivalents of isoprenol or allylheptaisobutyl-POSS against the Si–H groups in the presence of Karstedt’s catalyst in toluene at 80 °C for 7 h. The HB polymers before and after post functionalization with isoprenol or allylheptaisobutyl-POSS showed 1% weight loss at 298, 285, and 358 °C, respectively, under N2. Introducing POSS units at the terminus significantly increased the thermal stability. The XRD pattern of the HB polymers showed a denser structure formed in the allylheptaisobutyl-POSS-terminated HB polymer. The refractive index of the HB polymer before post functionalization was 1.4376. Post functionalization with isoprenol and allylheptaisobutyl-POSS increased the refractive index to 1.4464 and 1.4573, respectively. An incompletely condensed cage silsesquioxane (IC-POSS)-based AB3-type monomer, tris(dimethylsilyl)-p-vinylhexaisobutyl-IC-POSS (3), was prepared by the corner-cleaved reaction of vinylheptaisobutyl-POSS (1) and subsequent capping reaction with chlorodimethylsilane. The hydrosilylation polymerization of 3 in the presence of Karstedt’s catalyst in toluene provided a soluble polymer. The refractive index of the unmodified polymer film was 1.4376. Post functionalization with isoprenol increased the dispersibility in polar organic solvents and significantly increased the glass-transition temperature. Post functionalization with allylheptaisobutyl-POSS significantly increased the thermal stability. Their refractive index varied from 1.4464 to 1.4573 upon post functionalization.

Density and dynamic viscosity measurement and correlation of binary solutions containing hyperbranched polymer Boltorn H2004 and propanone, ethanol and dimethylformamide

In this study the density and dynamic viscosity of solutions containing Boltorn H2004 as a hyperbranched polymer (HB) and three various solvents (propanone, ethanol and dimethylformamide) has been measured at a temperature range of 298.15K to 313.15K and different mass fractions of HB polymer. Using the measured densities, the excess volumes of solutions were obtained which were negative values indicating interaction between solvent and the HB polymer. In addition, excess volumes were modeled applying the Redlich-Kister equation with an AARD% of 2.4224. The dynamic viscosities of binary solutions were also calculated using Vogel-Fulcher-Tammann (VFT), Redlich-Kister (RK) and the modified two suffix Eyring-Margules (E-MTSM) models successfully with the AARD% values of 4.0759, 0.1793 and 8.3676 respectively.

Hyperbranched polyhedral oligomeric silsesquioxane (HB‐POSS) nanomaterials for high transmission and radiation‐resistant space and solar applications

ABSTRACTHyperbranched polycarbosiloxanes and polysiloxanes with octafunctional polyhedral oligomeric silsesquioxane (POSS) branchpoints and curable alkoxysilane or silanol end‐groups were formulated with linear polysiloxanes to fabricate transparent and robust nanostructured POSS‐containing materials for use in a range of high performance space and solar applications. The effect of methyl vs. phenyl content, architecture and linear polysiloxane mass on transmission, thermal, physical, and proton, electron and UV radiation resistance properties was determined, and the physical properties of the nanomaterials were tailored to produce adhesives, or rigid or flexible coatings as desired. The methyl formulations showed superior electron resistance relative to a commercial space control material and to a POSS‐free HB polymer control material, even when directly exposed to radiation in coating form, whereas the phenyl formulations were shown to have inferior electron and UV resistance. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 3849–3861, 2013

Enhanced catalytic activity in organic solvents using molecularly dispersed haemoglobin–polymer surfactant constructs

The surface of haemoglobin (Hb) is chemically modified to produce molecular dispersions of discrete core-shell Hb-polymer surfactant bionanoconjugates in water and organic solvents. The hybrid nanoconstructs exhibit peroxidase-like catalytic activity with enhanced turnover rates compared with native Hb in water.

Endothelin rather than 20-HETE contributes to loss of pial arteriolar dilation during focal cerebral ischemia with and without polymeric hemoglobin transfusion

Partial exchange transfusion with a cell-free hemoglobin (Hb) polymer during transient middle cerebral artery occlusion (MCAO) reduces infarct volume but fails to increase blood flow, as might be expected with the induced decrease in hematocrit. In ischemic brain, endothelin antagonists are known to produce vasodilation. In nonischemic brain, pial arterioles constrict after Hb exchange transfusion, and the constriction is blocked by an inhibitor of 20-HETE synthesis. We tested the hypothesis that a 20-HETE synthesis inhibitor and an endothelin A receptor antagonist increase pial arteriolar dilation after Hb exchange transfusion during MCAO. Pial arteriolar diameter was measured in the ischemic border region of the distal MCA border region through closed cranial windows in anesthetized rats subjected to the filament model of MCAO. During 2 h of MCAO, pial arteriolar dilation gradually subsided from 37 +/- 3 to 7 +/- 5% (+/-SE). Compared with residual dilation at 2 h of MCAO with vehicle superfusion (14 +/- 3%), loss of dilation was not prevented by superfusion of a 20-HETE synthesis inhibitor (21 +/- 5%), partial Hb exchange transfusion (7 +/- 5%) that decreased hematocrit to 23%, or a combination of the two (5 +/- 5%). However, loss of dilation was prevented by superfusion of an endothelin A receptor antagonist with (35 +/- 4%) or without (32 +/- 5%) Hb transfusion. Pial artery constriction during reperfusion was attenuated by HET0016 alone and by BQ610 with or without Hb transfusion. Systemic administration of the endothelin antagonist during prolonged MCAO increased blood flow in the border region. Thus loss of pial arteriolar dilation in the ischemic border region during prolonged MCAO depends on endothelin A receptor activation, and this effect was independent of the presence of cell-free Hb polymers in the plasma. In contrast to previous work in nonischemic brain, inhibition of oxygen-dependent 20-HETE synthesis does not significantly influence the pial arteriolar response to polymeric Hb exchange transfusion during focal ischemia.

Synthesis of a Hemoglobin Polymer Containing Antioxidant Enzymes Using Complementary Chemistry of Maleimides and Sulfhydryls

To increase the overall size of hemoglobin (Hb), we developed a novel system of polymerization based on the complementary chemistry between sulfhydryls and maleimides. The maleimides were introduced onto the protein through N-(-maleimidobutyryloxy) succinimide, while the sulfhydryls were added using 2-iminothiolane hydrochloride (Trauts reagent). Resulting polymers showed SDS-PAGE bands with molecular weights as high as 96 kDa. Size exclusion chromatography has demonstrated species with molecular weight > 700 kDa. The flexibility of the sulfhydryl-maleimide chemistry has also allowed insertion of two antioxidant enzymes, catalase (Cat) and superoxide dismutase (SOD), into the Hb polymer. Cat was incorporated into the heavier fractions of the polymer, while SOD was found throughout the molecular weight range.

The Pharmacokinetics of Hemoglobin-Based Oxygen Carrier Hemoglobin Glutamer-200 Bovine in the Horse

Hemoglobin-glutamer-200 (HBOC-200) is a hemoglobin (Hb)-based oxygen carrier (HBOC) comprising glutaraldehyde-polymerized bovine Hb. In this study, we sought to determine the pharmacokinetics of this first generation HBOC after IV infusion of 32.5 g of HBOC-200 solution in horses. Quantification of HBOC-200 in equine plasma and urine was performed using a method recently developed by our laboratory. The elimination from plasma was based on size distribution of the bovine Hb polymer. The decline of plasma concentration-time curve of HBOC-200 was described by a noninterchanging 2-compartmental model. The median elimination half-lives of the small and large aggregates were 1.3 and 12.0 h, respectively. Of the HBOC-200 infused, 47.0% was eliminated as the smaller molecular weight and 53% as the larger molecular weight polymers. The area under the plasma concentration-time curve was 5143.1 microg.h(-1).mL(-1). The volumes of distribution of the small and large aggregates were 86.9 and 63.9 mL/kg and the clearances were 42.1 and 3.8 mL.kg(-1).h(-1), respectively. In conclusion, elimination of first generation HBOCs was shown to be more complex than previously assumed because of the heterogeneous nature of these solutions. Mammalian species dispose of Hb using similar mechanisms, and there is no unique metabolic process in the horse that would not allow a logical extension of the general interpretation of this study.

O2 Binding and Dissociation and Ligand Exchange Reaction of O2 with CO in Polymer Composite Films of Hemoglobin

A polymer composite film of hemoglobin (Hb–polymer film) was prepared by the casting of an Hb–polymer mixed solution (weight ratio of Hb to polymer is 1 to 1). The percentages of O2 and CO saturation of the Hb–dextran film were 46% and 70%, respectively. In the Hb solution, 100% saturation was observed for both ligands, and a humidified Hb–dextran film also showed 100% saturation. Water molecules would provide flexibility to the matrix polymer and promote a structural change in the Hb from a tense state (T) to a relaxed state (R). Thus the ligand binding to the Hb in the polymer films was strongly affected by the degree of interaction of Hb with the matrix polymers and the physical properties of the polymers. Inositol hexaphosphate (IHP) worked as an allosteric effector even in the solid polymer film and lowered the oxygen affinity of Hb. The O2 transport through an Hb–polyethyleneimine (PEI) film with IHP showed the facilitated O2 transport in comparison with the film without IHP because of the high dissociation rate of O2 from Hb with IHP. © 1997 John Wiley & Sons, Ltd.

O2 Binding and Dissociation and Ligand Exchange Reaction of O2 with CO in Polymer Composite Films of Hemoglobin

A polymer composite film of hemoglobin (Hb–polymer film) was prepared by the casting of an Hb–polymer mixed solution (weight ratio of Hb to polymer is 1 to 1). The percentages of O2 and CO saturation of the Hb–dextran film were 46% and 70%, respectively. In the Hb solution, 100% saturation was observed for both ligands, and a humidified Hb–dextran film also showed 100% saturation. Water molecules would provide flexibility to the matrix polymer and promote a structural change in the Hb from a tense state (T) to a relaxed state (R). Thus the ligand binding to the Hb in the polymer films was strongly affected by the degree of interaction of Hb with the matrix polymers and the physical properties of the polymers. Inositol hexaphosphate (IHP) worked as an allosteric effector even in the solid polymer film and lowered the oxygen affinity of Hb. The O2 transport through an Hb–polyethyleneimine (PEI) film with IHP showed the facilitated O2 transport in comparison with the film without IHP because of the high dissociation rate of O2 from Hb with IHP. © 1997 John Wiley & Sons, Ltd.

The effect of speed of deoxygenation on the percentage of aligned hemoglobin in sickle cells. Application of differential polarization microscopy.

We have used differential polarization microscopy, which provides images of linear dichroism, to measure the percentage of aligned hemoglobin (Hb) in 1086 deoxygenated red blood cells from subjects with sickle cell anemia. The percentage was found to be only slightly dependent on the speed of deoxygenation, thus showing that the percentage of aligned Hb was thermodynamically controlled (as has been found previously for the percentage of polymerized Hb in vitro). A slight decrease in the percentage of aligned Hb due to increasing speed of deoxygenation is primarily due to the increase in the number of cells containing no detectable aligned Hb. This class of cells was also the most variable between the different subjects studied. We were able to identify two other groups of cells that contain different numbers of domains of aligned Hb and show that these groups contain statistically different percentages of aligned Hb. The differences between these classes of cells was shown to be primarily due to different numbers of initial nucleation sites within each cell. It appears that the presence of preformed nucleation sites within cells at ambient oxygen tensions results in the thermodynamic control of aligned Hb polymer.

Ion transport in rabbit ileal mucosa. I. Na and Cl fluxes and short-circuit current.

Ion transport in rabbit ileal mucosa. I. Na and Cl fluxes and short-circuit current.

Characterization of HB polymer with gel permeation chromatography

AbstractA low molecular weight liquid polymer, polybutadiene‐acrylic acid‐acrylonitrile (HB), is used as a binder‐fuel fraction in solid composite propellants. The molecular weight distribution of this polymer, previously undetermined and uncontrolled, is being determined routinely with gel permeation chromatography (GPC). Analytical conditions are described and the precision and accuracy of the determination are discussed. Correlation of GPC determinations of low molecular weight materials in HB polymer (100 through 500 molecular weight range) with final propellant physical properties is statistically significant. These low molecular weight materials are being characterized, and studies of their role in determining propellant mechanical properties are being conducted. Use of a Waters Model 100 instrument for fractionating and isolating low molecular weight species in the molecular weight range of 100–500 yielded milligram quantities. Analysis of these materials with semimicrotechniques has permitted tentative indentification of the molecular species present.