The impact of calcium phosphate on FITC-BSA loading of sonochemically prepared PLGA nanoparticles for inner ear drug delivery elucidated by two different fluorimetric quantification methods

Highlights • Fluorimetric quantification of entrapped FITC-BSA is possible. • Two separate methods allow its quantification in the nanogram range. • Dequenching of FITC-BSA by enzymatic degradation or alkaline unfolding. • Calcium phosphate increases FITC-BSA loading of PLGA nanoparticles by 2.7-fold. • Nanoparticle characteristics were optimized for inner ear drug delivery.
Although therapeutically active proteins are highly efficacious, their content in protective nanoparticles is often too low to elicit adequate plasma levels. A strategy to increase protein loading is the in-situ generation of calcium phosphate as a protein adsorbent. To verify this approach, a highly sensitive and reliable fluorimetric method for quantification of incorporated fluorescein-labelled bovine serum albumin (FITC-BSA) as a model protein drug was developed. Dequenching the fluorescein label by pronase E, which digests the protein backbone, and dissolving the nanoparticle matrix in acetonitrile enabled FITC-BSA quantification in the nanogram per milliliter range. This test was confirmed by a second assay involving alkaline hydrolysis of FITC-BSA and the matrix. Nanoparticles prepared with calcium phosphate contained 40 µg FITC-BSA/mg and nanoparticles without calcium phosphate only 15 µg FITC-BSA/mg, representing a 2.7-fold increase in model protein loading. In this work the nanoparticle preparation procedure was optimized in terms of size for administration in the inner ear, but the range of applications is not limited.