Lipid droplet biogenesis comprises the emergence of cytosolic lipid droplets with a typical diameter 0.1–5 μm via synthesis of fat in the endoplasmatic reticulum, the formation of membrane-embedded lenses, and the eventual budding of lenses into solution as droplets. Lipid droplets in cells are increasingly being viewed as highly dynamic organelles with multiple functions in cell physiology. However, the mechanism of droplet formation in cells remains poorly understood, partly because their formation involves the rapid transformation of transient lipid structures that are difficult to capture. Thus, the development of controlled experimental systems that model lipid biogenesis is highly relevant for an enhanced mechanistic understanding. Here we prepare and characterize triolein (TO) lenses in a multilamellar spin-coated phosphatidyl­choline (POPC) film and determine the lens nucleation threshold to 0.25–0.5% TO. The TO lens shapes are characterized by atomic force microscopy (AFM) including their mean cap angle ⟨α⟩ = 27.3° and base radius ⟨a⟩ = 152.7 nm. A cross-correlation analysis of corresponding AFM and fluorescence images confirms that TO is localized to lenses. Hydration of the lipid/lens film induces the gel to fluid membrane phase transition and makes the lenses more mobile. The budding of free droplets into solution from membrane lenses is detected by observing a change in motion from confined wiggling to ballistic motion of droplets in solution. The results confirm that droplet budding can occur spontaneously without being facilitated by proteins. The developed model system provides a controlled platform for testing mechanisms of lipid droplet biogenesis in vitro and addressing questions related to lens formation and droplet budding by quantitative image analysis.

In this study, we systematically investigate the interactions between mobile ions generated from added salts and immobile charges within a sulfobetaine-based polyzwitterionic film in the presence of five salts (KCl, KBr, KSCN, LiCl, and CsCl). The sulfobetaine groups contain quaternary alkylammonium and sulfonate groups, giving the positive and negative charges. The swelling of the zwitterionic film in the presence of different salts is compared with the swelling behavior of a polycationic or polyanionic film containing the same charged groups. For such a comparative study, we design cross-linked terpolymer films with similar thicknesses, cross-link densities, and charge fractions, but with varying charged moieties. While the addition of salt in general leads to a collapse of both cationic and anionic films, the presence of specific types of mobile anions (Cl–, Br–, and SCN–) considerably influences the swelling behavior of polycationic films. We attribute this observation to a different degree of ion-pair formations between the different types of anionic counterions and the immobile cationic quaternary alkylammonium groups in the films where highly polarizable counterions such as SCN– lead to a high degree of ion pairing and less polarizable counterions, such as Cl–, cause a low degree of ion pairing. Conversely, we do not observe any substantial effect of varying the type of cationic counterions (K+, Li+, and Cs+), which we assign to the lack of ion pairing between the weakly polarizable cations and the immobile anionic sulfonate groups in the films. In addition, we observe that the zwitterionic films swell with increasing ionic strength and the degree of swelling is anion dependent, which is in agreement with previous reports on the “antipolyelectrolyte effect”. Herein, we explain this ion-specific swelling behavior with the different cation and anion abilities to form ion pairs with quaternary alkylammonium and sulfonate in the sulfobetaine groups.

Layer-by-layer assembled multilayer films have shown great potential for different applications owing to their responsive behavior. Herein, we systematically investigated the effects of composition, salt concentration, and ion specificity on the pH responsiveness of covalently crosslinked chitosan and alginate dialdehyde multilayer films. The changes in film swelling were measured using ellipsometry from low (0.01 mM) to high (3 M) salt (NaCl or NaSCN) concentrations at pH 3, 6, and 9. The swelling responses to increasing ionic strength matched the swelling responses observed for polyzwitterionic and weak monocomponent polyelectrolyte films and depended on the multilayer composition, pH, and ion specificity. Finally, we used the ellipsometric data to demonstrate that the pH responsiveness of such multilayer films, as measured using a quartz crystal microbalance with dissipation monitoring, strongly depends on the ionic condition under which the responses were measured. We thus show that erroneous conclusions about the pH responsiveness of polyelectrolyte multilayer films can be easily obtained if the ionic environment of the application does not closely resemble the ionic condition under which the pH responsiveness is tested.