Characterization of insulin interaction with positively charged liposomes

James Anthony Wesley, Purdue University

Abstract

The interaction between porcine zinc insulin and small unilamellar vesicle populations composed of dipalmitoylphosphatidylcholine (DPPC) and 0 mol% to 10 mol% stearylamine (SA) was studied at neutral pH conditions. The stearylamine was incorporated into the vesicle membrane to provide a positive charge at the liposomal surface. Insulin mediated liposomal aggregation was monitored using turbidimetric measurements. The results show there is an increase in both the rate and extent of liposomal aggregation as the membrane stearylamine concentrations increase from 0 mol% to 10 mol%. The extent of insulin adsorption to the membrane surface also increased as the concentration of stearylamine in the membrane increased with the adsorptive capacity increasing from 16 $\mu$g/m$\sp2$ for liposomes composed of 0 mol% SA to 625 $\mu$g/m$\sp2$ for liposomes containing 10 mol% SA. Particle size measurements after insulin induced aggregation showed the presence of both SUV (30 nm) and aggregates (5 $\mu$m) indicating an apparent equilibrium exists between aggregated and non-aggregated liposomes. The change in permeability upon insulin binding to liposomes was assessed by measuring the loss of entrapped carboxyfluorescein (CF). A definite relationship exists between the loss of entrapped CF and the quantity of insulin bound to the membrane surface. Liposomes composed of 0 mol% SA released approximately 25% of the entrapped CF after insulin adsorption. The release of CF increased steadily as the mol% SA incorporated into the membrane increased until, at 10 mol% SA, the liposomes released approximately 78% of the entrapped CF. The release of the entrapped CF appears due to disorder created between the fatty acyl chains of the bilayer and stearylamine molecules when insulin electrostatically binds to stearylamine containing liposomes. Hydrophobic interactions between insulin and the bilayer may also contribute to the CF release. Neither fusion nor complete destruction of the liposomes, as measured by a fluorescent liposomal lipid mixing assays, was deemed responsible for the CF release. Insulin adsorbed on the membrane surface appears to be less susceptible to $\alpha$-chymotrypsin degradation than insulin free in solution. The half-life of insulin degradation in the presence of liposomes increases from 16 minutes to 107 minutes as the membrane stearylamine increases from 0 mol% to 10 mol%.

Degree

Ph.D.

Advisors

Kildsig, Purdue University.

Subject Area

Pharmaceuticals

COinS