Insulin Aggregation at a Dynamic Solid–Liquid–Air Triple Interface

Abstract

Therapeutic proteins are privileged in drug development because of their exquisite specificity, which is due to their three-dimensional conformation in solution. During their manufacture, storage, and delivery, interactions with material surfaces and air interfaces are known to affect their stability. The growing use of automated devices for handling and injection of therapeutics increases their exposure to protocols involving intermittent wetting, during which the solid-liquid and liquid-air interfaces meet at a triple contact line, which is often dynamic. Using a microfluidic setup, we analyze the effect of a moving triple interface on insulin aggregation in real time over a hydrophobic surface. We combine thioflavin T fluorescence and reflection interference microscopy to concomitantly monitor insulin aggregation and the morphology of the liquid as it dewets the surface. We demonstrate that insulin aggregates in the region of a moving triple interface and not in regions submitted to hydrodynamic shear stress alone, induced by the moving liquid. During dewetting, liquid droplets form on the surface anchored by adsorbed proteins, and the accumulation of amyloid aggregates is observed exclusively as fluorescent rings growing eccentrically around these droplets. The fluorescent rings expand until the entire channel surface sweeped by the triple interface is covered by amyloid fibers. On the basis of our experimental results, we propose a model describing the growth mechanism of insulin amyloid fibers at a moving triple contact line, where proteins adsorbed at a hydrophobic surface are exposed to the liquid-air interface.

Structure and Aggregation Mechanisms in Amyloids

“…Gas-liquid interface Hydrophobic-hydrophilic interfaces may induce aggregation reactions. [175][176][177][178][179][180][181] Mechanical stress (agitation, stirring, pumping, or shaking) has been associated with cavitation which generates air bubbles and, consequently, the formation of an air-water interface which facilitates protein denaturation and aggregation. [176,[182][183][184][185][186][187][188][189] The use of beads during agitation accelerates the aggregation process by enhancing cavitation.…”

“…might lead to protein partial unfolding and aggregation. [181,[191][192][193][194] In vitro and in vivo, flow through tubes and vessels produce shear forces that may lead to protein partial unfolding and aggregation.…”

Insulin aggregation starts at dynamic triple interfaces, originating from solution agitation

“…Indeed, upon shaking or stirring, the solution meniscus moves up and down the container wall, thus creating a triple interface zone that is transiently wet and dry. Surface-adsorbed proteins in this zone are therefore exposed to partial dehydration which can induce their aggregation [21].…”

Role of Characteristics of External Mechanical Stress and Substrate on the Characteristics of Insulin Aggregates

“…It is difficult to relate the two for our system due to aggregation of the protein. Researchers have found the effect of shaking of aggregation of the protein in many cases, see for example references [25,[49][50][51][52][53]. Researchers reported the effect additives on the mechanism of aggregation of insulin, which follows a sigmoidal curve.…”

“…Effect of interfaces has been explored in various previous studies [25,50,51]. Reports suggest that the presence interfaces triggers the aggregation at a fixed agitation speed.…”