Whereas nanobubble stability on solid surfaces is thought to be based on local surface structure, in this work, we show that nanobubble stability on polymer brushes does not appear to require contact-line pinning. Glass surfaces were functionalized with copolymer brushes containing mixtures of hydrophobic and hydrophilic segments, exhibiting water contact angles ranging from 10 to 75°. On unmodified glass, dissolution and redeposition of nanobubbles resulted in reformation in mostly the same locations, consistent with the contact line pinning hypothesis. However, on polymer brushes, the nucleation sites were random, and nanobubbles formed in new locations upon redeposition. Moreover, the presence of stable nanobubbles was correlated with global surface wettability, as opposed to local structure, when the surface exceeded a critical water contact angle of 50 or 60° for polymers containing carboxyl or sulfobetaine groups, respectively, as hydrophilic side chains. The critical contact angles were insensitive to the identity of the hydrophobic segments.

Aggregation of therapeutic proteins can result from a number of stress conditions encountered during their manufacture, transportation, and storage. This work shows the effects of two interrelated sources of protein aggregation: the chemistry and structure of the surface of the container in which the protein is stored, and mechanical shocks that may result from handling of the formulation. How different mechanical stress conditions (dropping, tumbling, and agitation) and container surface passivation affect the stability of solutions of intravenous immunoglobulin are investigated. Application of mechanical shock causes cavitation to occur in the protein solution, followed by bubble collapse and the formation of high‐velocity fluid microjets that impinged on container surfaces, leading to particle formation. Cavitation was observed after dropping of vials from heights as low as 5 cm, but polyethylene glycol (PEG) grafting provided temporary protection against drop‐induced cavitation. PEG treatment of the vial surface reduced the formation of protein aggregates after repeated dropping events, most likely by reducing protein adsorption to container surfaces. These studies enable the development of new coatings and surface chemistries that can reduce the particulate formation induced by surface adsorption and/or mechanical shock.

This work demonstrates that hydrogen peroxide (HO) is generated in weak polyacrylamide hydrogels due to mechanochemical reactions to osmotic swelling. Hydrogels are important tools and materials for many biomedical applications, particularly for growth of stem cells. However, swollen gels are under constant tension, which makes their individual chains susceptible to mechanochemical bond breakage. In this work, an assay was developed to measure the generation of HO as a result of hydrogel swelling. Polyacrylamide hydrogels with both weak disulfide and strong PEG-diacrylate crosslinkers were synthesized and swelled. HO generation increased in the presence of weaker crosslinkers, up to 30 μM HO, whereas stronger crosslinkers reduced this to 5 μM HO. HO levels decreased when swelled in the presence of dextran to reduce osmotic stress or increased if the gels were conjugated to an acrylated surface. Finally, HO continued to form for days after the gels had reached their equilibrium sizes, independently of dissolved oxygen. The results of this work impact those working in the 3D cell culture community and demonstrate that even well-characterized systems undergo mechanochemical processes in mild environments.

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.

No abstract