Seetharama A. Acharya

Early difference in tissue pH and microvascular hemodynamics in hemorrhagic shock resuscitation using polyethylene glycol-albumin- and hydroxyethyl starch-based plasma expanders

The hamster chamber window model was subjected to hemorrhagic shock by the withdrawal of 50% of blood volume (BV). BV was restored 1 h after hemorrhage with a single volume infusion (resuscitation) of 25% BV with polyethylene glycol (PEG)-conjugated bovine serum albumin (Alb) and hydroxyethyl starch (HES). Hemorrhage, shock, and resuscitation were monitored continuously in terms of mean arterial pressure (MAP), microvascular blood flow, capillary perfusion, and tissue pH. Blood samples for laboratory parameters were taken at baseline, shock, and resuscitation. Intravascular and tissue pO2 were assessed after resuscitation, and microvascular oxygen supply and extraction were calculated and corrected for pH effect on hemoglobin saturation. Resuscitation with PEG-Alb restored systemic and microvascular parameters up to the end of the observation period (90 min). HES was identical to PEG-Alb resuscitation during the initial 10 to 15 min, but was not sustained subsequently. The trend of recovery in MAP for HES persisted beyond the time when both function capillary density and tissue pH decreased, thus MAP was not indicative of early microvascular dysfunction. Hemoglobin oxygen saturation estimation showed a significant pH dependence. However, oxygen-dependant parameters corrected for pH varied less than 10% from uncorrected data. Early differences found at the microvascular levels suggest that decisions to amend end-result of resuscitation may be short and on the order of minutes. Furthermore, PEG-Alb appears to provide early and long-term sustained systemic and microvascular recovery when used to restitute perfusion and metabolic conditions after resuscitation from hemorrhagic shock.

Surface decoration of red blood cells with maleimidophenyl‐polyethylene glycol facilitated by thiolation with iminothiolane: an approach to mask A, B, and D antigens to generate universal red blood cells

BACKGROUND: The surface decoration of red blood cells (RBCs) by polyethylene glycol (PEG) chains has been an approach developed to camouflage the blood group antigens from their antibodies. A PEGylation protocol, however, that can mask the antigens appropriately to inhibit the agglutination of RBCs with the respective antibodies is not available so far.

Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin

The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly(ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an alphaalpha-fumaryl cross-link at Lys-99(alpha). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)(6)-alphaalpha-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)(6)-Hb. Perturbation of the haem microenvironment and the alpha1beta2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)(6)-Hb and (propyl-PEG5K)(6)-alphaalpha-Hb sediment as stable dimeric and tetrameric molecules, respectively. The betabeta-succinimidophenyl PEG-2000 cross-link at Cys-93(beta) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the alphaalpha-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. alphaalpha-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.

PEGylation of Human Serum Albumin : Reaction of PEG-Phenyl-Isothiocyanate with Protein

Successful and cost-effective PEGylation protocols require pure functionalized PEG reagents, which can be synthesized by simple and efficient procedures, exhibit high stability against hydrolysis, and maintain a level of reactivity with protein functional groups under mild reaction conditions. PEG-phenyl-isothiocyanate (PIT-PEG) is a new functionalized PEG having these characteristics, and has been synthesized by condensation of the bifunctional reagent 4-isothiocyanato phenyl isocyanate with monomethoxy PEG (mPEG). The data of (1)H NMR and colormetric analysis of the new PEG reagent establish that the mPEG has been quantitatively functionalized. The t 1/4 values for the hydrolysis of PIT-PEG5K in 100 mM phosphate solution at pH 6.5 and 9.2 are about 95 and 40 h, respectively. Incubation of human serum albumin (HSA, 0.5 mM) with a 10-fold molar excess of PIT-PEG (3K or 5K) at pH 6.5 and 9.2 generated PEG-HSA conjugates with average of 3.5 and 6.0 PEG chains per HSA molecule, respectively. The circular dichroism spectra of the conjugates showed that PEGylation of HSA has little influence on the secondary structure of HSA. The hexaPEGylated HSA, (TCP-PEG5K) 6-HSA, exhibited very high hydrodynamic volume, and the molecular radius of HSA increased from 3.95 to 6.57 nm on hexaPEGylation. The hexaPEGylation also increased the viscosity of 4% HSA from 1.05 to 2.10 cP, and the colloid osmotic pressure from 15.2 to 48.0 mmHg. The large increase in the hydrodynamic volume and the solution properties of (TCP-PEG5K) 6-HSA suggest that it could be a potential candidate as a plasma volume expander. PIT-PEG is a useful addition to the spectrum of functionalized PEG reagents available for surface decoration of proteins with PEG.

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive.

Recent studies have suggested that the “pressor effect” of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry–based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the ε-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256 kDa, (ii) a molecular radius of ∼6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the “pressor effect” of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.

PEGylation of Val-1(α) Destabilizes the Tetrameric Structure of Hemoglobin†

A hexaPEGylated hemoglobin (Hb), (Propyl-PEG5K)(6)-Hb, is essentially in alphabeta dimers (Hu et al. (2007) Biochem. J. 402, 143-151). In order to provide a biochemical insight into the tetramer-dimer dissociation of this PEGylated Hb, we prepared and characterized two PEGylated Hbs site-specifically modified at Val-1(alpha) and at Val-1(beta), respectively. PEGylation at Val-1(alpha) and at Val-1(beta) increase the tetramer-dimer dissociation constant (K(d)) of Hb by 2 and 1 order of magnitude, respectively. Accordingly, the sites of PEGylation can determine the tetramer stability of the PEGylated Hb. In order to determine the role of the polyethylene glycol (PEG) chains on the tetramer stability of Hb, we prepared a propylated Hb site-specifically modified at Val-1(alpha). Interestingly, site-specific propylation of Hb at Val-l(alpha) stabilizes the Hb tetramer by 1 order of magnitude. Therefore, conjugation of the PEG chains at Val-1(alpha) can greatly destabilize the tetramer stability of Hb. On the structural aspects, the PEG chains conjugated at Va-1(alpha) unfavorably alter the heme environment and quaternary structure and destabilize the alpha1beta2 interface of Hb. On the functional aspects, the PEG chains conjugated at Val-1(alpha) decrease the Hill coefficient, the Bohr effect of Hb and the sensitization to the presence of the allosteric effectors. In contrast, PEGylation of Hb at Val-1(beta) gives rise to less pronounced structural alteration and different functional change.

Microvascular effects following treatment with polyethylene glycol-albumin in lipopolysaccharide-induced endotoxemia

Objective:To determine whether resuscitation with polyethylene glycol conjugated bovine serum albumin (2.5% weight/volume) infused at 16 mL/kg/hr (PEG-BSA-16) or at 24 mL/kg/hr (PEG-BSA-24) for 1 hr improves microcirculatory conditions in endotoxemia compared with dextran 70 (6% weight/volume) infused at 24 mL/kg/hr (Dex). Design:Prospective study. Setting:University research laboratory. Subjects:Male Golden Syrian hamsters. Interventions:Hamsters implemented with a skinfold window chamber were given an intravenous injection of lipopolysaccharide and resuscitated within 10 mins with Dex, PEG-BSA-16, or PEG-BSA-24. Measurements and Main Results:Hamsters were observed during 24 hrs after lipopolysaccharide injection. Systemic variables measured included mean arterial pressure, heart rate, and systemic arterial blood gas. Microvascular function was characterized by measuring vessel diameter; red blood cell velocity; functional capillary density (FCD); Po2 in arterioles, venules, and tissue; and perivascular nitric oxide concentration 6 hrs after lipopolysaccharide injection. At 6 hrs, animals with no treatment had the lowest FCD (6.7 ± 5.7% of baseline). PEG-BSA provided significantly improved microvascular conditions as shown by restoration of FCD. Recovery of FCD was related to improved microvascular flow and perivascular and tissue Po2, normalization of shear rate, and decreased perivascular nitric oxide concentration. These effects were related to improved fluid retention using PEG-BSA-24 as evidenced by the significantly lower hematocrit at 24 hrs after resuscitation. Nitric oxide at 6 hrs after induction of sepsis achieved perivascular millimolar concentrations, which were reduced to normal values by PEG-BSA-24 treatment. At 6 hrs there were significant differences in FCD, tissue Po2, and perivascular nitric oxide concentration following PEG-BSA treatment by comparison with Dex treatment, although there were no differences in systemic variables between Dex and PEG-BSA groups. Conclusions:PEG-BSA produces improved microcirculatory conditions in the treatment of endotoxemia when compared with dextran 70.

Autoxidation of the site-specifically PEGylated hemoglobins: role of the PEG chains and the sites of PEGylation in the autoxidation.

The PEGylated hemoglobin (Hb) has been evaluated as a potential blood substitute. In an attempt to understand the autoxidation of the PEGylated Hb, we have studied the autoxidation of the PEGylated Hb site-specifically modified at Cys-93(beta) or at Val-1(beta). PEGylation of Hb at Cys-93(beta) perturbed the heme environment and increased the autoxidation rate of Hb, which is at a higher level than that caused by PEGylation at Val-1(beta). The perturbation of the heme environment of Hb is attributed to the maleimide modification at Cys-93(beta) and not due to conjugation of the PEG chains. However, the PEG chains enhance the autoxidation and the H 2O 2 mediated oxidation of Hb. Accordingly, the PEG chains are assumed to increase the water molecules in the hydration layer of Hb and enhance the autoxidation by promoting the nucleophilic attack of heme. The autoxidation rate of the PEGylated Hb does not show an inverse correlation with the oxygen affinity. The H 2O 2 mediated structural loss and the heme loss of Hb are increased by maleimide modification at Cys-93(beta) and further decreased by conjugation of the PEG chains. The autoxidation of the PEGylated Hbs is attenuated significantly in the plasma, possibly due to the presence of the antioxidant species in the plasma. This result is consistent with the recent suggestion that there is no direct correlation between the in vitro and in vivo autoxidation of the PEGylated Hb. Therefore, the pattern of PEGylation can be manipulated for the design of the PEGylated Hb with minimal autoxidation.

Labeling of monoclonal antibodies with radionuclides

Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as ethylenediaminepentaacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides.

Extension arm facilitated pegylation of αα-hemoglobin with modifications targeted exclusively to amino groups: Functional and structural advantages of free cys-93(β) in the PEG-Hb adduct

Cys-93(beta) of hemoglobin (Hb) was reversibly protected as a mixed disulfide with thiopyridine during extension arm facilitated (EAF) PEGylation and its influence on the structural and functional properties of the EAF-PEG-Hb has been investigated. Avoiding PEGylation of Cys-93(beta) in the EAF-PEG-Hb lowers the level of perturbation of heme pocket, alpha1beta2 interface, autoxidation, heme loss, and the O(2) affinity, as compared to the EAF-PEG-Hb with PEGylation of Cys-93(beta).The structural and functional advantages of reversible protection of Cys-93(beta) during EAF PEGylation of oxy-Hb has been compared with Euro PEG-Hb generated by EAF PEGylation of deoxy Hb where Cys-93(beta) is free in the final product. The alphaalpha-fumaryl cross-linking and EAF PEGylation targeted exclusively to Lys residues has been combined together for generation of second-generation EAF-PEG-Hb with lower oxygen affinity. The PEG chains engineered on Lys as well as PEGylation of Cys-93(beta) independently contribute to the stabilization of oxy conformation of Hb and hence increase the oxygen affinity of Hb. However, oxygen affinity of the EAF-PEG-alphaalpha-Hb is more sensitive to the presence of PEGylation on Cys-93(beta) than that of the EAF-PEG-Hb. The present modified EAF PEGylation platform is expected to facilitate the design of novel versions of the EAF-PEG-Hbs that can now integrate the advantages of avoiding PEGylation of Cys-93(beta).