(256a) Variations in Chitosan/Tripolyphosphate Micro- and Nanogel Yield and Their Two Key Effects on Protein Uptake

Chitosan/tripolyphosphate (TPP) micro- and nanogels form spontaneously through ionotropic gelation in dilute aqueous solutions. Due to their biocompatible and mucoadhesive properties and mild formation conditions, they are widely explored as vehicles for ocular and intranasal delivery of protein drugs and vaccines. Aside from the knowledge that their protein uptake is enhanced by stronger protein/particle binding, however, factors that control protein association efficiency (AE) such as variations in the chitosan, TPP and protein concentrations used during protein loading, remain poorly understood. To this end, we hypothesized that many of the differences in the reported AE-values might reflect the largely-ignored variability in the particle yield (X_Agg), which also varies with the chitosan, TPP and protein concentrations, and is defined here as the fraction of the added chitosan that self-assembles into particles. To test this, we used bovine serum albumin (BSA) and α-lactalbumin (α-LA) as model proteins to analyze the correlation between AE and X_Agg. Consistent with our hypothesis, AE scaled linearly with the X_Agg (which increased with the TPP and drug-to-chitosan ratios) until all chitosan was aggregated into particles, and data collected at variable TPP and protein concentrations collapsed onto a single AE vs. X_Agg line. These trends were also quantitatively independent of the protein/particle mixing procedure, suggesting that protein uptake was governed by its equilibrium partitioning. Further analysis of the protein/particle binding isotherms revealed the rise in AE with X_Agg to reflect two effects: (1) an increase in the number of protein binding sites within the particles (due to an increase in the particle mass); and (2) a decrease in the number of soluble (unaggregated) chitosan molecules, which formed soluble protein/chitosan complexes and (through this competitive binding) limited protein uptake into the particles. These findings highlight the need to carefully analyze the effects of formulation parameters on X_Agg when designing chitosan/TPP drug carriers, and can likely be extended to other biopolymeric nanocarriers that form through ionotropic gelation.