Investigating the Molecular Mechanism of Protein–Polymer Binding with Direct Saturation Compensated Nuclear Magnetic Resonance

Abstract

Herein, we describe a new technique, direct saturation compensated transfer (DISCO) NMR, to characterize protein−macromolecule interactions. DISCO enables the direct observation of intermolecular interactions and is used to investigate mucoadhesion, a type of polymer−protein interaction that is widely implemented in drug delivery but remains poorly understood. In a model system of bovine submaxillary mucin and poly(acrylic acid), DISCO identifies selective backbone interactions that facilitate mucoadhesion through chain interpenetration. DISCO demonstrated distinct patterns of molecular selectivity between mucoadhesive polymers when applied to hydroxypropyl cellulose and carboxymethyl cellulose and that functionalizing adhesive polymers with strongly interacting moieties may be detrimental to the overall adhesive interaction. Additionally, DISCO was used to estimate polymer−protein dissociation constants using individual proton signals as reporters. Overall, DISCO can be used as a label-free screening tool to generate polymer-specific binding fingerprints to map and quantify interactions between macromolecules.

Mechanistic Influence of Polymer Species, Molecular Weight, and Functionalization on Mucin–Polymer Binding Interactions

“…The value DISCO AF0 ( t = 0) is a quantitative measure of a proton’s interaction intimacy with mucin . To obtain the DISCO AF0 ( t = 0), the saturation time-dependent buildup curve of each proton is collected and fit to an exponential binding model before taking its derivative at t = 0 as we reported previously .…”

“…The value DISCO AF0 ( t = 0) is a quantitative measure of a proton’s interaction intimacy with mucin . To obtain the DISCO AF0 ( t = 0), the saturation time-dependent buildup curve of each proton is collected and fit to an exponential binding model before taking its derivative at t = 0 as we reported previously . The parameters of the exponential binding model are obtained through nonlinear curve fitting (using the Levenburg–Marquardt method SciPy.Optimize library), and the model’s derivative then yields DISCO AF0 ( t = 0) (eq S1).…”

“…While these results provide clues to the underlying mechanisms at play, the direct observation and quantification of these interactions with atomic precision remains limited. In our previous work, we developed direct saturation compensated (DISCO) NMR and demonstrated that we could map interactions in a model macromolecular system with atomic precision . In the context of mucoadhesion, many research groups have identified a need for standardized experiments using a broad range of mucoadhesive polymers to deepen the general understanding of the mechanism of mucoadhesion, , we sought to use DISCO to do so.…”

Untangling Mucosal Drug Delivery: Engineering, Designing, and Testing Nanoparticles to Overcome the Mucus Barrier

“…Similarly, NMR uses magnetic excitation to monitor interactions between different species in solution and generally encounters the same challenges as FTIR . Nevertheless NMR-based methods continue to evolve, and some progress has been made in understanding polymer–mucin interactions using relaxometry, evolution of line width, and macromolecular saturation transfer …”

“…52 Nevertheless NMR-based methods continue to evolve, and some progress has been made in understanding polymer−mucin interactions using relaxometry, 292 evolution of line width, 293 and macromolecular saturation transfer. 294 While previously mentioned techniques are used preferentially for mucoadhesive systems, multiple particle tracking (MPT) can be implemented for both mucoadhesive and mucopenetrating formulations. Usually accomplished by tagging nanoparticles with a fluorescent label, time-resolved translocation of nanoparticles through a gel (usually mucus or a mucus mimic) can be tracked using a fluorescence video microscope.…”