Site-Specific Chemical Modification of Human Serum Albumin with Polyethylene Glycol Prolongs Half-life and Improves Intravascular Retention in Mice

Abstract

Human serum albumin (HSA) is used as an important plasma volume expander in clinical practice. However, the infused HSA may extravasate into the interstitial space and induce peripheral edema in treating the critical illness related to marked increase in capillary permeability. Such poor intravascular retention also demands a frequent administration of HSA. We hypothesize that increasing the molecular weight of HSA by PEGylation may be a potential approach to decrease capillary permeability of HSA. In the present study, HSA was PEGylated in a site-specific manner and the PEGylated HSA carrying one chain of polyethylene glycol (PEG) (20 kDa) per HSA molecule was obtained. The purity, PEGylated site and secondary structure of the modified protein were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), thiol group blockage method and circular dichroism (CD) measurement, respectively. In addition, the pharmacokinetics in normal mice was investigated, vascular permeability of the PEGylated HSA was evaluated in lipopolysaccharide (LPS)-induced lung injury mouse model and the pharmacodynamics was investigated in LPS-induced sepsis model with systemic capillary leakage. The results showed that the biological half-life of the modified HSA was approximately 2.3 times of that of the native HSA, PEG-HSA had a lower vascular permeability and better recovery in blood pressure and haemodilution was observed in rats treated with PEG-HSA. From the results it can be inferred that the chemically well-defined and molecularly homogeneous PEGylated HSA is superior to HSA in treating capillary permeability increase related illness because of its longer biological half-life and lower vascular permeability.

N-terminal PEGylation of human serum albumin and investigation of its pharmacokinetics and pulmonary microvascular retention

“…The results of the pharmacokinetic study and pulmonary microvascular permeability of N-terminal PEGylated HSA in this study are very similar to those of Cys34-specific PEGylated HSA as we described previously (29). The molecular weights of PEG reagents applied to these two conjugations targeting different modification sites were both 20 kDa.…”

“…It consists of a single non-glycosylated polypeptide chain of 585 amino acids forming a heartshaped molecule with molecular weight of 66.5 kDa (1). HSA is responsible for 80% of the colloid osmotic pressure of plasma (25)(26)(27)(28)(29)(30)(31)(32)(33). Because of this, its main clinical use is in maintaining colloid osmotic pressure and increasing circulating plasma volume.…”

Papaverine increases human serum albumin glycation

“…Virtually every polypeptide is prone to glycation because of the presence of amino groups at their N-terminus and their lysine residues. Because of the long half-life and high concentration (40 mg/ml) of human serum albumin in plasma, it is highly sensitive to glycation [15]. HSA is a lysine rich protein and as previously mentioned several specific lysine residues, which include lys-199, lys-281, lys-439 and lys-525, are more susceptible to glycation after 2 or 5 weeks of incubation (Fig.…”

“…Reduced eNOS activity and inactivation of NO by AGEs may play an important role in the defective vasodilatory responses that occur in diabetes [14]. Because of the long half-life and high concentration (40 mg/ml) of human serum albumin in plasma, it is highly sensitive to glycation [15]. Non-enzymatic glycation of serum albumin occurs at multiple sites, in vivo.…”

Cys34-PEGylated Human Serum Albumin for Drug Binding and Delivery

“…35,36 S -PEGylation of albumins can be performed with minimal perturbation to pre-existing disulfide bridges with subsequent retention of protein structure, as demonstrated in the case of PEG-(C34)BSA, 35 and can increase its circulation lifetime relative to unmodified albumins, as demonstrated in a rat model with PEG(C34)HSA. 36 These reports indicate that S -PEGylation at Cys-34 is an appealing alternative to amide-based ligations for developing albumin-based carriers with tailored pharmacological properties. It is worth mentioning that maleimide-based reagents have also been developed for PEGylation across disulfide bonds, 37,38 but are not immediately relevant for proteins bearing free thiols.…”