Degradable PEGylated protein conjugates utilizing RAFT polymerization

Abstract

Poly(ethylene glycol) (PEG)-protein therapeutics exhibit enhanced pharmacokinetics, but have drawbacks including decreased protein activities and polymer accumulation in the body. Therefore a major aim for second-generation polymer therapeutics is to introduce degradability into the backbone. Herein we describe the synthesis of poly(poly(ethylene glycol methyl ether methacrylate)) (pPEGMA) degradable polymers with protein-reactive end-groups via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the subsequent covalent attachment to lysozyme through a reducible disulfide linkage. RAFT copolymerization of cyclic ketene acetal (CKA) monomer 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) with PEGMA yielded two polymers with number-average molecular weight (Mn) (GPC) of 10.9 and 20.9 kDa and molecular weight dispersities (Ð) of 1.34 and 1.71, respectively. Hydrolytic degradation of the polymers was analyzed by 1H-NMR and GPC under basic and acidic conditions. The reversible covalent attachment of these polymers to lysozyme, as well as the hydrolytic and reductive cleavage of the polymer from the protein, was analyzed by gel electrophoresis and mass spectrometry. Following reductive cleavage of the polymer, an increase in activity was observed for both conjugates, with the released protein having full activity. This represents a method to prepare PEGylated proteins, where the polymer is readily cleaved from the protein and the main chain of the polymer is degradable.

A guide to maximizing the therapeutic potential of protein–polymer conjugates by rational design

“…Our group has conjugated a copolymer of 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), a commonly used CKA, and PEGMA to lysozyme whose amines have been thiolated via disulfide exchange. 20 The resulting conjugate was active and the polymer was degradable under a basic condition (5% KOH), which suggests that the polymer could be degraded by ester cleavage in vivo.…”

Synthesis and Biological Evaluation of a Degradable Trehalose Glycopolymer Prepared by RAFT Polymerization

“…The M n by 1 H NMR could not be determined accurately because a complete loss of the aldehyde end group was observed. However, we believe that the low molecular weight was not due to cleavage of the backbone during these mild deprotection conditions because treatment with KOH further reduces the molecular weight (Figure S6, Supporting Information), and we have previously shown that BMDO copolymers are stable for greater than 7 d in carbonate solution . Instead, the low M n is likely a result of interactions of the polymer with the GPC column, as we have previously observed with other trehalose polymers …”

“…Degradation of p(BMDO‐ co ‐BMA‐trehalose) was evaluated by exposure to a 5% KOH solution. KOH is commonly used to accelerate conditions for polymer degradation . The trehalose copolymer was found to be hydrolytically degradable, and degradation was observed within 24 h as demonstrated by a decrease in GPC molecular weight to 1900 Da (Figure S6, Supporting Information).…”

“…We were thus interested to investigate other degradable protein stabilizers, specifically, those that could be made by controlled radical polymerizations (CRP). We had previously prepared degradable comb PEG polymers by CRP and conjugated them to proteins . However, these polymers were not protein stabilizers.…”

Development of novel bioconjugation strategies for creating cancer-targeting nanomaterials

“…However, it is presumed that one side of DBCO reacted with 27 kDa scFv and another side of DBCO reacted with lower MW of degraded scFv which has around 19 kDa based on SDS PAGE gel result. in polymer MW [212,213] . Following protein-polymer bioconjugation, we then carried out flow…”