Fantao Meng

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.

PEG-albumin supraplasma expansion is due to increased vessel wall shear stress induced by blood viscosity shear thinning

We studied the extreme hemodilution to a hematocrit of 11% induced by three plasma expanders: polyethylene glycol (PEG)-conjugated albumin (PEG-Alb), 6% 70-kDa dextran, and 6% 500-kDa dextran. The experimental component of our study relied on microelectrodes and cardiac output to measure both the rheological properties of plasma-expander blood mixtures and nitric oxide (NO) bioavailability in vessel walls. The modeling component consisted of an analysis of the distribution of wall shear stress (WSS) in the microvessels. Our experiments demonstrated that plasma expansion with PEG-Alb caused a state of supraperfusion with cardiac output 40% above baseline, significantly increased NO vessel wall bioavailability, and lowered peripheral vascular resistance. We attributed this behavior to the shear thinning nature of blood and PEG-Alb mixtures. To substantiate this hypothesis, we developed a mathematical model of non-Newtonian blood flow in a vessel. Our model used the Quemada rheological constitutive relationship to express blood viscosity in terms of both hematocrit and shear rate. The model revealed that the net effect of the hemodilution induced by relatively low-viscosity shear thinning PEG-Alb plasma expanders is to reduce overall blood viscosity and to increase the WSS, thus intensifying endothelial NO production. These changes act synergistically, significantly increasing cardiac output and perfusion due to lowered overall peripheral vascular resistance.

Increased Inter Dimeric Interaction of Oxy Hemoglobin is Necessary for Attenuation of Redutive Pegylation Promoted Dissociation of Tetramer

Abstract The propensity of site-specific carboxymethylation and Propylation of Val-1(α) of Hb to attenuate the reductive hexaPEGylation-induced dissociation of tetramers has been investigated. Only reductive Propylation of Val-1(α), which increases the stability of oxy Hb, attenuates the reductive hexaPEGylation-induced dissociation. Increasing the stability of the oxy conformation of Hb by chemical or genetic approaches is a strategy to generate PEGylated Hbs with native-like tetramer stability using direct PEGylation platforms. This new approach and EAF-PEGylation are the only two alternate PEGylation strategies available to design stable second-generation vasoinactive uncrosslinked PEGylated Hbs with native-like tetramer stability.

Packing Density of the PEG-Shell in PEG-Albumins: PEGylation Induced Viscosity and COP are Inverse Correlate of Packing Density

Abstract: PEG-Alb represents a new class of low viscogenic plasma expanders that achieve super perfusion in vivo by mimicking the vasodilatory influence of high viscogenic plasma expanders. PEGylation-engineered structure of PEG albumin can be envisaged as a deformable molecular domain around the rigid central protein core. The correlation between the structure of PEG-shell in terms of packing of the PEG inside the PEG shell and PEGylation induced plasma expander (PE)-like properties of albumin has been investigated as a function of the number and length of the PEG-chain. The increase in molecular radius of albumin on PEGylation is non-linear as a function of the number of PEG chains conjugated. The packing density of PEG within the PEG-shell is an inverse correlate of PEG-chain size; i.e. the shorter chains pack more compactly than the longer ones. The PEGylation induced increase in the viscosity and COP of albumin is an exponential correlation of the number of ethylene oxide units (-CH2-CH2-O-) conjugated and is also a function of the PEG-chain length. At equivalence of PEG mass conjugated, the viscosity and COP of PEG-albumin adducts correlate inversely with packing density of PEG. All PEGylated albumins are not equivalent on the basis of total PEG mass conjugated. Accordingly, the structure of PEG albumin and its solution properties can be engineered to optimize a given total PEG mass for the application of PEG albumin as a resuscitation fluid. The extension arms minimize the influence of PEG shell on the structure of the protein core. We speculate that EAF-PEGylation is a preferable platform for PEGylation of protein therapeutics and is expected to generate products with better therapeutic efficacy.

Tissue oxidative metabolism after extreme hemodilution with PEG-conjugated hemoglobin

NADH-localized fluorometry was used as a noninvasive technique to monitor changes in the energy state of intact tissue (muscle and connective tissue), without anesthesia, as a function of blood plasma O(2)-carrying capacity in the hamster window chamber model. Acute moderate isovolemic hemodilution was induced by two isovolemic hemodilution steps: in the first step, 6% 70-kDa dextran (Dex70) was used to induce an acute anemic state (18% Hct); in the second step, exchange transfusion of polyethylene glycol (PEG) maleimide-conjugated Hb (4 g/dl, PEG-Hb) or Dex70 (6 g/dl) was used to reduce erythrocytes to 75% of baseline (11% Hct). PEG-Hb had six copies of PEG (5 kDa) conjugated to each human Hb (0.48 g PEG/g Hb) through extension arm-facilitated chemistry. Systemic parameters, microvascular perfusion, functional capillary density, intravascular and interstitial Po(2), and intracellular NADH fluorescence were monitored. Mean arterial blood pressure after extreme hemodilution was statistically significantly reduced for Dex70 compared with PEG-Hb. The presence of PEG-Hb in the circulation maintained positive acid-base balance. While microvascular blood flows were not different, functional capillary density was significantly higher for PEG-Hb than Dex70. Arteriolar Po(2) was higher in the presence of PEG-Hb than Dex70, but tissue and venular Po(2) were not different. Cellular energy metabolism (intracellular O(2)) in the tissues was improved with PEG-Hb. Moderate hemodilution to 18% Hct (6.4 g Hb/dl) brings tissue O(2) delivery to the verge of inadequacy. Extreme hemodilution to 11% Hct (3.7 g Hb/dl) produces tissue anoxia, and high-O(2)-affinity PEG-Hb (Po(2) at which blood is 50% saturated with O(2) = 4 Torr, 1.1 g Hb/dl) only partially decreases anaerobic metabolism without increasing tissue Po(2).

PEGylation of αα-Hb using succinimidyl propionic acid PEG 5K: Conjugation chemistry and PEG shell structure dictate respectively the oxygen affinity and resuscitation fluid like properties of PEG αα-Hbs

Abstract PEGylation of intramolecularly crosslinked Hb has been studied here to overcome the limitation of dissociation of Hb tetramers. New hexa and deca PEGylated low oxygen affinity PEG-ααHbs have been generated. Influence of PEG conjugation chemistry and the PEG shell structure on the functional properties as well as PEGylation induced plasma expander like properties of the protein has been delineated. The results have established that in the design of PEG-Hbs as oxygen therapeutics, the influence of conjugation chemistry and the PEG shell structure on the oxygen affinity of Hb needs to be optimized independently besides optimizing the PEG shell structure for inducing resuscitation fluid like properties.

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

Enhancing the molecular size/volume Hb was introduced, as a simple molecular approach to overcome or attenuate the extravasation mediated NO scavenging activity of Hb in vivo. Conjugation of Hb, as a chemical tool to achieve this objective, has now advanced beyond this initial concept. It requires us now to recognize that engineering of resuscitation fluids like properties to Hb is a novel concept and simple approach to neutralize/attenuate the in vivo hypertensive activity of acellular Hb. Development of conjugated hemoglobins is reviewed here as it lead to the development of this concept along with future perspectives. Extension Arm chemistry, that integrates the concepts of click chemistry into the various conjugation approaches of relevance to this aspect of blood substitute research is discussed. Bridging these approaches to increase the efficacy of engineering resuscitation fluid like properties to Hb is suggested as the future direction of designing conjugated Hbs as oxygen therapeutics. The integration of site directed mutagenesis to tame the molecular properties of Hb with the extension arm facilitated PEGylation to attenuate the impact of conjugation chemistry and/or conjugated polymer on the tertiary/quaternary structure of the Hb molecule is discussed in detail as applied to PEGylation of Hb as the conjugation approach of choice to generate nonhypertensive Hb as oxygen therapeutics. We conclude that such a synergistic approach will result in the attenuation of heme degradation product dependent and oxidative stress mediated in vivo toxicity as well as facilitating an increase in the oxygen delivering capacity of the conjugated Hb.