Multi-layer nanoencapsulation of human pancreatic islets

A major problem in human islet (HI) allotransplantation is the need for chronic immunosuppression, that could be overcome if HI are protected against immunological attacks by biocompatible, semipermeable membranes. Whereas macro/microencapsulation techniques have given conflicting results, an attractive strategy is the use of conformal nanocoating, with reduced volumes and possible implantation into any suitable site. We coated HI by a multilayer nanoencapsulation procedure and assessed several features of the encapsulated cells. HI were isolated from 12 donors (age: 68±13 yrs, BMI: 27.9±3.0 kg/m2, mean blood glucose during ICU stay: 163±69 mg/dl). Chitosan [(Poly(D-glucosamine) deacetylated chitin)] and PSS [(Poly(styrenesulfonic acid sodium salt)] were dissolved (1 mg/ml) in culture medium at 37oC for 36 h. HI were then incubated in each polymer for 15 min and nanoencapsulation was achieved through layer-by-layer electrostatic binding. Morphology, ultrastructure, viability and insulin secretion (IS) studies were performed and initial in-vivo work was also accomplished by transplanting the islets under the kidney capsule of non-diabetic Sprague-Dawley rats. The procedure provided full coating of all the islets, as assessed by confocal and fluorescence microscopy examination, with membrane thickness (measured by electron microscopy, EM) of 104.2±4.2 nm. Vital staining showed ≥90% cell survival; this was confirmed by EM, that also demonstrated well maintained beta cell ultrastructure with unchanged morphometry of intracellular organelles. IS (uU/ml) from coated islets was 44±10 at 3.3 mM glucose and increased to 147±29 at 16.7 mM glucose (p<0.01), with stimulation index of 4.7±1.1 (all values similar to those of uncoated HI). Insulin, glucagon and chromogranin stainings revealed better preservation of nanocoated HI up to 7 days from implantation. HI were efficiently encapsulated by layer-by-layer nanocoating, providing a tool for experimental transplantation studies