Analysis of chitosan/tripolyphosphate micro- and nanogel yields is key to understanding their protein uptake performance.

Chitosan/tripolyphosphate (TPP) micro- and nanogels are widely explored as vehicles for protein drug and vaccine delivery. Yet, aside from the consensus that protein uptake into these particles is enhanced by stronger protein/particle binding, factors that control their uptake performance, such as differences in the chitosan, TPP and protein concentrations, remain poorly understood. Here, we show that many of the differences in the reported association efficiencies (AE-values) for protein uptake likely reflect the largely-ignored variability in the particle yield (XAgg), which is the fraction of the added chitosan that self-assembles into particles and (like the AE) varies with the chitosan, TPP and protein concentrations. Factors affecting XAgg are first systematically explored. The AE is then shown to scale almost linearly with the XAgg (which increases with the TPP and protein-to-chitosan ratios) until all chitosan aggregates into particles. Remarkably, the data collected at variable TPP and protein concentrations collapses onto a single AE∝XAgg curve for each protein type. Further analysis of protein/particle binding reveals this rise in AE with XAgg to reflect: (1) an increase in binding sites within the particles; and (2) a decrease in soluble (non-particulate) chitosan molecules, which form soluble protein/chitosan complexes and compete with the chitosan/TPP particles for the unassociated protein. These findings highlight the need to carefully analyze the effects of formulation parameters on chitosan/TPP particle yields and can likely be extended to other ionically crosslinked colloidal drug carriers.