The effects of droplet stabilization by surfactants and nanoparticles on leakage, cross-talk, droplet stability, and cell adhesion.

Partially fluorinated nanoparticles (FNPs) have been proposed as a promising alternative for stabilising aqueous droplets in fluorinated oils. The exceptional energetic stability of FNPs at the droplet interface holds the potential for minimising leakage, enhancing stability, and promoting improved cell adhesion. However, their lower diffusion coefficient compared to surfactants presents challenges in achieving rapid droplet stabilisation, which is important in microfluidics applications. While several studies have focused on some of these aspects, a comprehensive study and direct comparison with conventional fluorosurfactants is still missing. In this manuscript, we undertake an examination and comparison of four crucial facets of both FNP- and surfactant-stabilised droplets: leakage of compounds, emulsion stability, droplet formation dynamics and cell adhesion. Contrary to what has previously been claimed, our findings demonstrate that FNPs only reduce leakage and cross-talk in very specific cases (e.g., resorufin), failing to provide enhanced compartmentalisation for highly hydrophobic dyes (e.g., rhodamine dyes). On the other hand, FNP-stabilised droplets indeed exhibit greater long-term stability compared to their surfactant-stabilised counterparts. Regarding the size of droplets generated via a diversity of microfluidic methods, no significant differences were observed between FNP-stabilised and surfactant-stabilised droplets. Finally, the previously reported improvements in cell adhesion and spreading on FNP-stabilised interfaces is limited to flat oil/water (o/w) interfaces and could not be observed within droplets. These comprehensive analyses shed light on the nuanced performance of FNPs and commercial fluorosurfactants as stabilising agents for aqueous droplets in fluorinated oils, contributing valuable insights for choosing the correct formulation for specific droplet-based microfluidics applications.

Artificial cells for in vivo biomedical applications through red blood cell biomimicry.

Recent research in artificial cell production holds promise for the development of delivery agents with therapeutic effects akin to real cells. To succeed in these applications, these systems need to survive the circulatory conditions. In this review we present strategies that, inspired by the endurance of red blood cells, have enhanced the viability of large, cell-like vehicles for in vivo therapeutic use, particularly focusing on giant unilamellar vesicles. Insights from red blood cells can guide modifications that could transform these platforms into advanced drug delivery vehicles, showcasing biomimicry's potential in shaping the future of therapeutic applications.

Lipid vesicle formation by encapsulation of SMALPs in surfactant-stabilised droplets.

Understanding the intricate functions of membrane proteins is pivotal in cell biology and drug discovery. The composition of the cell membrane is highly complex, with different types of membrane proteins and lipid species. Hence, studying cellular membranes in a complexity-reduced context is important to enhance our understanding of the roles of these different elements. However, reconstitution of membrane proteins in an environment that closely mimics the cell, like giant unilamellar vesicles (GUVs), remains challenging, often requiring detergents that compromise protein function. To address this challenge, we present a novel strategy to manufacture GUVs from styrene maleic acid lipid particles (SMALPs) that utilises surfactant-stabilised droplets as a template. As a first step towards the incorporation of membrane proteins, this work focusses on the conversion of pure lipid SMALPs in GUVs. To evaluate the method, we produced a new form of SMA linked to fluorescein, referred to as FSMA. We demonstrate the assembly of SMALPs at the surfactant-stabilised droplet interface, resulting in the formation of GUVs when released upon addition of a demulsifying agent. The released vesicles appear similar to electroformed vesicles imaged with confocal light microscopy, but a fluorescein leakage assay and cryo-TEM imaging reveal their porous nature, potentially as a result of residual interactions of SMA with the lipid bilayer. Our study represents a significant step towards opening new avenues for comprehensive protein research in a complexity-reduced, yet biologically relevant, setting.

Building Synthetic Cells─From the Technology Infrastructure to Cellular Entities.

The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.

Formation of Giant Unilamellar Vesicles Assisted by Fluorinated Nanoparticles.

In the quest to produce artificial cells, one key challenge that remains to be solved is the recreation of a complex cellular membrane. Among the existing models, giant unilamellar vesicles (GUVs) are particularly interesting due to their intrinsic compartmentalisation ability and their resemblance in size and shape to eukaryotic cells. Many techniques have been developed to produce GUVs all having inherent advantages and disadvantages. Here, the authors show that fluorinated silica nanoparticles (FNPs) used to form Pickering emulsions in a fluorinated oil can destabilise lipid nanosystems to template the formation of GUVs. This technique enables GUV production across a broad spectrum of buffer conditions, while preventing the leakage of the encapsulated components into the oil phase. Furthermore, a simple centrifugation process is sufficient for the release of the emulsion-trapped GUVs, bypassing the need to use emulsion-destabilising chemicals. With fluorescent FNPs and transmission electron microscopy, the authors confirm that FNPs are efficiently removed, producing contaminant-free GUVs. Further experiments assessing the lateral diffusion of lipids and unilamellarity of the GUVs demonstrate that they are comparable to GUVs produced via electroformation. Finally, the ability of incorporating transmembrane proteins is demonstrated, highlighting the potential of this method for the production of GUVs for artificial cell applications.