Because peptides/protein-based drugs have strong immunogenicity, when they are used in the body, they will trigger the production of antibodies, which will reduce the efficacy and even cause allergic reactions. Therefore, researchers try to use soluble inert polymers, such as polyethylene glycol (PEG), combined with peptides/proteins to solve the dilemma, which can not only reduce its immunogenicity, but also can extend its half-life and retain its biological activity. PEGylation is a popular technology gradually developed in the late 1970s. At present, there are more than ten kinds of PEGylated protein drugs on the market with excellent clinical medical effects.
PEG contains a large number of ethoxy groups, which can form hydrogen bonds with water, has a high degree of hydrophilicity, and has a large hydrodynamic volume in an aqueous solution. It can change the biological distribution behavior of drugs in the aqueous solution and create a space barrier around the modified drugs to protect drugs from enzymatic hydrolysis, avoiding rapid elimination in the metabolism of the kidney. PEG is immunologically inert, even if its molecular weight (Mw) is as high as 5.9×106 Da, its immunogenicity is also very low. When using PEGylated protein in clinical treatment, no anti-PEG antibody was found. PEG is one of the few synthetic polymers approved by the Food and Drug Administration (FDA) that can be used for injectable medicine in the body.
Fig. 1 Several explanations on why PEG increases the stealth of nanocarriers. (Journal of Biomedical Nanotechnology 2008, 4 (2), 133-148.
Fig. 2 Illustration of N-terminal PEGylation. (Journal of controlled release 2014, 192, 67-81)
Site-selective PEGylation of N-terminal amino groups can be performed by simple adjustment of reaction pH to acidic condition where the lysine will be protonated. For this purpose, PEG aldehydes with low reactivity were preferred to minimize amount of lysine conjugate.
Fig. 3 Illustration of thiol and bridging PEGylation. (Journal of controlled release 2014, 192, 67-81)
PEG maleimide is the most commonly used reactive PEGylating agent for thiol PEGylation. A thioether bond can be formed by Michael's addition between the double bond of the maleimide ring and the thiol group of cysteine. In addition, another method based on use of specific cross-functionalized mono-sulfone PEG derivative containing double bond was developed, which can react first with one of the two thiols present in protein disulfide bridge.
Fig. 4 Illustration of PEGylation of proteins with unnatural amino acids. (Journal of controlled release 2014, 192, 67-81)
A novel strategy has been developed involving site-selectively introduce unnatural amino acids through recombinant technique, which are later PEGylated with specific PEGylating agents. The unnatural amino acids such as p-acetyl-L-phenylalanine and p-azidophenylalanine have been used for protein PEGylation.
In the field of PEGylation, enzymes due to their highly specificity and selectivity have also been considered as tools for PEGylation of peptides/proteins. Transglutaminase (TGase)-mediated PEGylation is the most common used method, which catalyzes addition of acyl residues to primary amines. Moreover, glycoPEGylation, involving two enzymes to mimic O-glycosylation, aims to solve the problem that threonine and serine in the case of O-glycosylation and aspargine cannot be PEGylated directly without affecting other traditional PEGylation sites.
Releasable and non-covalent PEGylation
Releasable and non-covalent PEGylation strives to overcome the loss of bioactivity resulting from PEG coupling at receptor binding site. For the Releasable PEGylation, tailor made PEGylating agents, containing hydrolysable linkers, are exploited to allow a slow and controlled protein releasein vivo. As regards to the non-covalent PEGylation, the polymer coupling does not involve a covalent bond but the polymer interacts with the protein through hydrophobic interactions or coordination bonds.
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