Understanding affinity-driven protein uptake into ionically crosslinked chitosan micro- and nanogels
July 21-24, 2019
Colloidal particles formed through the ionotropic gelation of chitosan with tripolyphosphate (TPP) have attracted widespread interest as protein drug and vaccine delivery vehicles. Yet, aside from the consensus that efficient protein uptake into these particles requires particle/protein affinity, factors affecting uptake performance of colloidal chitosan/TPP particles have, until recently, remained poorly understood, with many seemingly conflicting reports appearing in the literature. To this end, we have recently hypothesized that some of these differences in uptake might have stemmed from variations in the particle yield, which are frequently ignored and, for the purpose of this study, were quantified as fractions of the chitosan molecules assembled into particles (XAgg). Spectroscopic analysis revealed that XAgg increased sharply with both TPP:glucosamine molar ratio and pH until reaching 100%, whereupon further TPP addition coagulated the particles into macroscopic precipitates. Likewise, when particles were prepared from solutions at pH-values above the protein pI, XAgg increased with the protein concentration. Thus, the efficiency of particle formation depended strongly on chitosan/TPP mixture compositions. A corresponding analysis of the association efficiency (AE), defined as the fraction of the added protein taken up by the particles, revealed that the uptake of the model, bovine serum albumin (BSA; pI ≈ 4.7 – 4.9) and α-lactalbumin (α-LA; pI ≈ 4.2 – 4.5) proteins was also highly composition-dependent. When particle formation and protein uptake, for instance, were performed using parent chitosan and TPP solutions at pH 4.0 (i.e., below the protein pI), the chitosan/protein binding was weak and the AE was below 10%. Raising the parent solution pH to 5.5 (above the protein pI), however, strengthened the chitosan/protein binding and enabled efficient protein uptake (where at optimized TPP:glucosamine ratios the AE reached 90%). Hence, consistent with prior reports, significant chitosan/protein affinity was needed to achieve high AE. Conversely, TPP:glucosamine molar ratio effect was nonmonotonic. Regardless of the protein composition, the AE first increased with the TPP:glucosamine ratio (up to the ratio where XAgg first reached 100%), and then decreased with the TPP:glucosamine ratio. Additionally, AE often (albeit not always) increased with the protein concentration.
Despite this variable uptake, however, when AE-values obtained at pH > pI was plotted against XAgg, all the data collected for each model protein (at variable TPP:glucosamine ratios and protein concentrations) up to the TPP:glucosamine ratio where XAgg first reaches 100% collapsed onto a single line, indicating that, as long as there was substantial chitosan/protein affinity, XAgg was the primary determinant of protein uptake. Further, replotting the AE vs. XAgg data as binding isotherms (where the uptake was normalized by particulate chitosan mass) revealed that the apparent particle/protein association strength also increased sharply with XAgg, suggesting that soluble chitosan inhibited protein association with chitosan/TPP particles. This soluble chitosan effect was inferred to reflect the formation of soluble chitosan/protein complexes, which prevented protein molecules from binding to the chitosan/TPP particles (and whose presence was confirmed through dynamic light scattering). When the TPP content, however, exceeded that needed to maximize XAgg, its further addition eliminated the cationic chitosan/TPP particle binding sites, and thus weakened particle/protein association.
Moreover, the AE vs. XAgg and binding isotherm curves agreed well with a simple competitive binding model (derived based on linear chitosan/protein binding isotherms), where the binding constants for protein/particulate chitosan and protein/soluble chitosan association were the only adjustable parameters and had remarkably similar values. These experimental and model analyses indicate two reasons for the AE vs. XAgg scaling: (1) that, not surprisingly, higher XAgg-values increase the number of particulate binding sites; and (2) that higher XAgg-values reduce the formation of uptake-inhibiting soluble chitosan/protein complexes. Collectively, these findings provide essential guidelines for optimizing protein uptake into chitosan/TPP micro- and nanogels, and likely (at least in part) extend to other related drug carriers, such as micro- and nanogels prepared through the ionotropic gelation of alginate or complexation of oppositely charged polyelectrolytes.
Cai, Y.; Lapitsky, Y. Analysis of chitosan/tripolyphosphate micro- and nanogel yields is key to understanding their protein uptake performance. J. Colloid Interface Sci. 494, 242-254 (2017).
Yakov Lapitsky, "Understanding affinity-driven protein uptake into ionically crosslinked chitosan micro- and nanogels" in "Colloidal, Macromolecular and Biological Gels II", Samiul Amin, Manhattan College, USA Saad Khan, North Carolina State University, USA Srini Raghavan, University of Maryland College Park, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/cmb_gels_ii/5