Improving the biocompatibilty of alginate/poly-l-ornithine capsules through the masking of amines and pegylation

Abstract

Living Cell Technologies (LCT) Ltd uses encapsulation to isolate porcine pancreatic islets from the recepient’s immune system to prevent rejection in xenotransplantation. This capsule is composed of an alginate bead coated with poly-L-orithine (PLO) and alginate (APA). One of the main problems with this capsule is the exposure of PLO which has a net positive charge and exposed amine groups. Here we present the approach LCT is using to modify the surface of PLO by masking exposed amine groups and the positive charge, using Sulfo-NHS-acetate and PEGylated compounds. Sulfo-NHS-acetate is commonly used to block amine groups on proteins by capping amino groups with a more favourable acyl group. PEGylated compounds covalently attach through amine esters, shielding the surface of the microcapsule with a non-ionic water-soluble polymer. Both approaches aim to reduce protein adsorption and and the onset of the immune response. In vitro experiments have investigated modifications to the encapsulation procedure using the following coating scheme: AlginatePLO/PLO/x, where x is sulfo-NHS-acetate, BS (PEG) 9 or MS (PEG) 12 at concentrations over the range of 1-10 mM. Micro-beads are formed by pumping the alginate, porcine islet mix through a needle supplied with a coaxial airflow into a CaCl2 bath, PLO is used to coat the external surface of the bead, followed by the amine masking reagent. Capsules are then treated with sodium citrate to remove the alginate core. Capsules were characterised using light and fluorescent microscopy, flourescent imaging and Transmission electron microscopy (TEM). Capsule size, uniformity and defects were also assessed. Islet viability and insulin release were monitored over the course of a month. All conditions tested showed excellent islet viabilty and no reduction in insulin release when compared to the control.