Optimization of aqueous remote loading of leuprolide in poly(lactic-co-glycolic acid) microspheres

Cationic peptides, such as leuprolide and octreotide, have been shown to strongly interact with free acid-terminated poly(lactic-co-glycolic acid) (PLGA-COOH). This interaction involves peptide absorption rather than adsorption at the surface. Here, we optimized the aqueous remote loading paradigm to load preformed 50/50 PLGA-COOH microspheres utilizing a model peptide, leuprolide acetate, in a 0.1 M HEPES buffer (pH 7.4) at elevated encapsulation efficiency and loading. Given the quasi-equilibrium absorption of leuprolide, a prediction for encapsulation efficiency (EE) and loading (l) was derived. This theory implies that EE is dependent on the binding strength and capacity, the polymer water content and initial polymer concentration, and l depends on all the factors controlling the EE as well as peptide/polymer mass ratio. Initial studies with microspheres without optimization were able to achieve loading of ∼9.8 % but had a low EE (∼38 %). These microspheres continuously released in vitro over 1 month with a low initial burst. In order to increase EE, loading conditions such as microsphere concentration in leuprolide solution, duration of loading, inner water phase volume and porosity were studied and optimized. We found that loading from high microsphere concentrations (e.g., 180-240 mg/mL) strongly improved EE with encapsulation rapidly occurring in the first 8 h to achieve a quasi-equilibrium. As the inner water phase volume was increased from 0 to 350 µL the porosity increased from 38 to 60 %, and the initial burst was minimal at low porosity values. The drug loading and EE were not strongly affected by porosity once above 50 %. Hence, by applying the theoretical analysis as described here, drug loading and EE can be manipulated to optimal levels by first monitoring quasi-equilibrium binding isotherms, indicating the potential for both general translational remote loading of therapeutic peptides and future applications for peptides with limited aqueous solubility and for encapsulation on the small scale.