Surface-Templated Nanobubbles Protect Proteins from Surface-Mediated Denaturation

Abstract

In this Letter, we report that surface-bound nanobubbles reduce protein denaturation on methylated glass by irreversible protein shell formation. Single-molecule total internal reflection fluorescence (SM-TIRF) microscopy was combined with intramolecular Forster resonance energy transfer (FRET) to study the conformational dynamics of nitroreductase (NfsB) on nanobubble-laden methylated glass surfaces, using reflection brightfield microscopy to register nanobubble locations with NfsB adsorption. First, NfsB adsorbed irreversibly to nanobubbles with no apparent desorption after 5 h. Moreover, virtually all (96%) of the NfsB molecules that interacted with nanobubbles remained folded, whereas less than 50% of NfsB molecules remained folded in the absence of nanobubbles on unmodified silica or methylated glass surfaces. This trend was confirmed by ensemble-average fluorometer TIRF experiments. We hypothesize that nanobubbles reduce protein damage by passivating strongly denaturing topographical surface defects. Thus, nanobubble stabilization on surfaces may have important implications for antifouling surfaces and improving therapeutic protein storage. P roteins are ubiquitous in biomedical and biotechnological applications, including protein therapeutics, biosensing, biocatalysis, food processing, and detergents. 1−3 Unfortunately, proteins are notoriously unstable over long-term storage as a result of air oxidation, thermal unfolding, mechanical shearing by ice crystals, and many other potential processes. 4 Beyond simple loss of activity, in some cases therapeutic proteins form large aggregates that can provoke an immune response, diminishing long-term efficacy and potentially even endangering the patient's life. 5−9 Thus, a considerable amount of research and effort has been devoted to maintaining protein stability during storage. 6,10,11 Even under optimal conditions, proteins still adsorb and desorb from surfaces that contact liquid formulations, in some cases desorbing from the surface in an unfolded state. Recent advances using single-molecule (SM) fluorescence microscopy methods have led to the identification of anomalous surface sites, or "hot spots," that are responsible for the majority of unfolding events. 12 These anomalous denaturing sites are thought to be caused by random heterogeneities in the surface (e.g., glass) structure such as step edges or grain boundaries. Even nominally smooth glass exhibits topographical nonuniformities with protruding or depressed surface features, which can, in turn, alter the interaction of the protein with the surface relative to homogeneous surfaces. 12−14 Because these features may arise from many sources, such as demixing, crystallization, contamination, and/or the processing history of the glass, 15 and are thus generally unavoidable, an understanding of these sites and strategies to mediate their impact on protein stability are critical.

Nanobubble technologies: Applications in therapy from molecular to cellular level

“…5E) (Argenziano et al, 2022a;Houthaeve et al, 2022), delivery to the intracellular environment (Tian et al, 2018), changing protein behavior via nanobubbleprotein interactions (Fig. 5A and 5B) (Babu and Amamcharla, 2022;Bull et al, 2019;Snell et al, 2016;Zhang and Seddon, 2016), altering protein self-assembly (Yan et al, 2022) or lipid self-assembly (Jin et al, 2020), and shielding lipids from their surroundings (Zhang and Lemay, 2019). The addition of surfactants and shell materials aid in stability, targeting, tracking, and release, but may initiate unwanted side effects.…”

Hydrogel Coatings on Container Surfaces Reduce Protein Aggregation Caused by Mechanical Stress and Cavitation

“…Such nuclei may persist within the micro-/ nanoscale structures found on glass surfaces due to contact line pinning and may expand to form cavitation bubbles when mechanical shocks are imposed on the container. 26,27 Based on these observations, we hypothesize that a cavitationresistant surface should have low energy at the water interface, low roughness, and potentially a fluid-like structure to prevent stable contact line pinning. Hydrogels, three-dimensional hydrophilic polymeric networks, represent a material that possesses all of these attributes.…”

“…Even when surfaces are treated to increase hydrophilicity, the surface roughness inherent to glass containers allows the heterogeneous nucleation of gas bubbles, which in turn facilitate cavitation and subsequently protein damage and aggregation. Such nuclei may persist within the micro-/nanoscale structures found on glass surfaces due to contact line pinning and may expand to form cavitation bubbles when mechanical shocks are imposed on the container. , …”

The Influence of Air Nanobubbles on Controlling the Synthesis of Calcium Carbonate Crystals

“…Therefore, numerous reports have been published on the application of nanobubbles, including medicine [ 10 , 11 ], environment [ 9 , 12 ], flotation [ 13 , 14 ], and materials [ 15 ]. In medicine, nanobubbles have been used to protect proteins from surface-mediated denaturation [ 16 ] as well as for ultrasound imaging and intracellular drug delivery [ 17 ]. H 2 nanobubbles in water were discovered to inhibit tumor cell growth [ 18 ].…”