Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation.

Intracellular compartments are functional units that support the metabolism within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer complex emulsion-based, multicompartment artificial cells, using microfluidics and acoustic levitation. Such levitated models provide free-standing, dynamic, definable droplet networks for the compartmentalisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and mechanosensitive channel of large-conductance. The assembly of droplet networks is three-dimensionally patterned with fluidic input configurations determining droplet contents and connectivity, whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. The mechanosensitive channel can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact, control of membrane protein function. Collectively, this expands our growing capability to program and operate increasingly sophisticated artificial cells as life-like materials.

Bilayer Networks within a Hydrogel Shell: A Robust Chassis for Artificial Cells and a Platform for Membrane Studies.

The ability to make artificial lipid bilayers compatible with a wide range of environments, and with sufficient structural rigidity for manual handling, would open up a wealth of opportunities for their more routine use in real-world applications. Although droplet interface bilayers (DIBs) have been demonstrated in a host of laboratory applications, from chemical logic to biosynthesis reaction vessels, their wider use is hampered by a lack of mechanical stability and the largely manual methods employed in their production. Multiphase microfluidics has enabled us to construct hierarchical triple emulsions with a semipermeable shell, in order to form robust, bilayer-bound, droplet networks capable of communication with their external surroundings. These constructs are stable in air, water, and oil environments and overcome a critical obstacle of achieving structural rigidity without compromising environmental interaction. This paves the way for practical application of artificial membranes or droplet networks in diverse areas such as medical applications, drug testing, biophysical studies and their use as synthetic cells.

Simple and Versatile 3D Printed Microfluidics Using Fused Filament Fabrication.

The uptake of microfluidics by the wider scientific community has been limited by the fabrication barrier created by the skills and equipment required for the production of traditional microfluidic devices. Here we present simple 3D printed microfluidic devices using an inexpensive and readily accessible printer with commercially available printer materials. We demonstrate that previously reported limitations of transparency and fidelity have been overcome, whilst devices capable of operating at pressures in excess of 2000 kPa illustrate that leakage issues have also been resolved. The utility of the 3D printed microfluidic devices is illustrated by encapsulating dental pulp stem cells within alginate droplets; cell viability assays show the vast majority of cells remain live, and device transparency is sufficient for single cell imaging. The accessibility of these devices is further enhanced through fabrication of integrated ports and by the introduction of a Lego®-like modular system facilitating rapid prototyping whilst offering the potential for novices to build microfluidic systems from a database of microfluidic components.

A new assay for rhamnolipid detection-important virulence factors of Pseudomonas aeruginosa.

Rhamnolipids (RLs) are heterogeneous glycolipid molecules that are composed of one or two L-rhamnose sugars and one or two ß-hydroxy fatty acids, which can vary in their length and branch size. They are biosurfactants, predominantly produced by Pseudomonas aeruginosa and are important virulence factors, playing a major role in P. aeruginosa pathogenesis. Therefore, a fast, accurate and high-throughput method of detecting such molecules is of real importance. Here, we illustrate the ability to detect RL-producing P. aeruginosa strains with high sensitivity, based on an assay involving phospholipid vesicles encapsulated with a fluorescent dye. This vesicle-lysis assay is confirmed to be solely sensitive to RLs. We illustrate a half maximum concentration for vesicle lysis (EC50) of 40 µM (23.2 µg/mL) using pure commercial RLs and highlight the ability to semi-quantify RLs directly from the culture supernatant, requiring no extra extraction or processing steps or technical expertise. We show that this method is consistent with results from thin-layer chromatography detection and dry weight analysis of RLs but find that the widely used orcinol colorimetric test significantly underestimated RL quantity. Finally, we apply this methodology to compare RL production among strains isolated from either chronic or acute infections. We confirm a positive association between RL production and acute infection isolates (p = 0.0008), highlighting the role of RLs in certain infections.

Advanced dextran based nanogels for fighting Staphylococcus aureus infections by sustained zinc release.

The growth in numbers and severity of hospital acquired infections has increased the need to target bacteria locally and specifically. Consequently, smart drug-delivery systems are being developed for local bactericidal action. The approach takes the concept of nanogels in drug delivery of small molecules to the next level by enclosing them in a shell. Versatile polysaccharide nanogels were loaded with zinc ions as antibacterial agents in a miniemulsion process, in order to target methicillin resistant strains of Staphylococcus aureus (MRSA). The encapsulation of drugs in nanogels is limited by the crosslinking density of the gel and the size of the drug. The characterization of the nanogels with inductively coupled plasma optical emission spectroscopy (ICP-OES) revealed that zinc ions cannot be retained within without an additional 'shell' layer. The nanogels were surrounded by a dextran-polyurethane shell, which can retain substances by reduction of water penetration. A delayed zinc release compared to the nanogels was confirmed by ICP-OES. Bacterial tests revealed an antibacterial effect of the shell enhanced nanogels against S. aureus. The studied nanogel system shows potential in locally addressing bacterial infections. The platform is extremely versatile and can be tailored to application as dextran and Zn(NO3)2 can be replaced by other polysaccharides (e.g. hyaluronic acid) and antibacterial agents, respectively.