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A versatile system for studying the enzymatic degradation of multi-block poly(ester amide)s.

Journal of chromatography. A (2013-03-21)
A Ghaffar, G J J Draaisma, G Mihov, P J Schoenmakers, Sj van der Wal
ABSTRACT

The suitability of biomaterials for specific biomedical applications can be investigated through their in vitro biodegradability with selected enzymes and through their degradation kinetics. A system was developed for studying the enzymatic degradation of poly(ester amide)s (PEAs) coatings under sink conditions, with on-stream analysis of degradation products by liquid chromatography coupled to time-of-flight mass spectrometry (LC-ToF-MS). A coated capillary was treated by an enzyme solution in pulses (pulse-feed mode) or continuously (continuous-feed mode) with different flow rates. The water-soluble products resulting from the interaction of enzyme with the PEA coating were deposited on-line on a reversed-phase LC column, separated by gradient-elution LC, ionized by electrospray ionization (ESI), and identified based on ToF-MS data. The experiments underline the benefits of the experimental set-up, which requires only small amounts of coating and enzyme and produces detailed results rapidly. The system was investigated using different injection volumes (pulses) of an α-chymotrypsin solution in varying concentrations, different flow rates, and different lengths of coated capillary. The versatility of the system makes it easy to follow the course of degradation and to differentiate between primary and secondary degradation products. The system was applied to study the degradation of a di-block and a tri-block PEA. Specific degradation products showed different time profiles than the (more gradual) overall weight loss. Continuous-feed-mode analysis allowed the convenient determination of highly stable amide-bond-containing fragments, while pulse-feed-mode analysis revealed benzyl ester-containing products as primary degradation products.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
α-Chymotrypsin from human pancreas, lyophilized powder
Sigma-Aldrich
α-Chymotrypsin from bovine pancreas, ≥40 units/mg protein, vial of 5 mg
Sigma-Aldrich
α-Chymotrypsin from bovine pancreas, suitable for protein sequencing, salt-free, lyophilized powder
Sigma-Aldrich
α-Chymotrypsin from bovine pancreas, Type I-S, essentially salt-free, lyophilized powder
Sigma-Aldrich
α-Chymotrypsin from bovine pancreas, Type II, lyophilized powder, ≥40 units/mg protein
Sigma-Aldrich
α-Chymotrypsin from bovine pancreas, (TLCK treated to inactivate residual tryspin activity), Type VII, essentially salt-free, lyophilized powder, ≥40 units/mg protein