Protein Modification with Amphiphilic Block Copoly(2-oxazoline)s as a New Platform for Enhanced Cellular Delivery

Abstract

Several homo-, random and block copolymers based on poly(2-oxazoline)s (POx) were synthesized and conjugated to horseradish peroxidase (HRP) using biodegradable and non-biodegradable linkers. These conjugates were characterized by amino group titration, polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, enzymatic activity assay and conformation analysis. The conjugates contained on average from about one to two polymer chains per enzyme. From 70% to 90% of enzymatic activity was retained in most cases. Circular dichroism (CD) analysis revealed that HRP modification affected the secondary structure of the apoprotein but did not affect the tertiary structure and heme environment. Enhanced cellular uptake was found in the conjugates of two block copolymers using both MDCK cells and Caco-2 cells, but not in the conjugates of random copolymer and homopolymer. Conjugation with a block copolymer of 2-methyl-2-oxazoline and 2-butyl-2-oxazoline led to the highest cellular uptake as compared to other conjugates. Our data indicates that modification with amphiphilic POx has the potential to modulate and enhance cellular delivery of proteins.

Design Strategies for Functionalized Poly(2-oxazoline)s and Derived Materials

“…For both systems, the poly(ethylene glycol)-as well as the pEtOx-based one, similar results were obtained in terms of preserving the enzymatic activity, increasing the drug hydrodynamic volume and being protein-repellant. [58]. The synthetic routine comprised end-capping of the poly(2-oxazoline)s with piperazine, reaction of its remaining secondary amine with either di-succinimidyl proprionate or dithiobis(succinimidyl proprionate), and the reaction of amine groups of horseradish peroxidase with the succinimide-activated carboxylic acid at the poly(2-oxazoline)s' end-groups.…”

Session IV – Rapid Fire Presentations (V22–V31)

“…In contrast, via attachment of amphiphilic block copolymers, 4 we have observed a marked increase of intracellular protein concentration and activity. 5,6 This effect is dependent on the polymer structure. Thus, the excellent control over the polymerization possible with the living cation ring-opening polymerization of poly(2-oxazoline)s will allow fine-tuning of the interaction of therapeutically relevant proteins with biological barriers, such as the blood brain barrier.…”

Polymeric lipid vesicles with pH-responsive turning on–off membrane for programed delivery of insulin in GI tract

“…Samples were prepared by mixing sample powder with KBr and scanned at 2 cm À1 resolution. The degree of substitution (DS) of the amino groups in e-PLL was determined by the TNBS method (Tong et al, 2010), in which quantitative determination of the number of amines contained within a sample can be accomplished through comparison to a standard curve generated by the use of an amine-containing compound dissolved in a series of known concentrations. The critical aggregation concentration (CAC) of grafted polymers was determined by fluorescence probe technique that involves the use of pyrene based hydrophobic fluorescence dye which exhibits different fluorescence characteristics depending upon the polarity of the local environment (Du et al, 2012).…”