Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

The assessment of drug-dose responses is vital for the prediction of unwanted toxicological effects in modern medicine. Three-dimensional (3D) cell cultures techniques can provide in vivo-like spheroids and microtissues that resemble natural tumor function. However, formation of necrotic core and diffusion limitation of chemical compounds within these models can reduce the reproducibility and precision of standard bioassay protocols used to test two-dimensional (2D) cell cultures. Nonetheless, the accurate prediction of detrimental effects of test compounds based on functional bioassays is essential for the development of new efficient therapeutic strategies. For instance, alamarBlue® is a widely-used commercially available redox indicator dye that can evaluate metabolic activity and cellular health status in a single-step procedure however, suitability and optimization of this bioassay must be determined for each individual application scenario. Here, we optimized the standard alamarBlue® proliferation/viability protocol for tumor spheroid cultures to enhance assay precision during toxicological drug screening. We optimized the original protocol of alamarBlue® assay that usually suggests an incubation time of 2-4 hours. The key modifications of the protocol for spheroid cultures are as follows: •Aspiration of cell culture medium before drug exposure.•Replacement of drug-supplemented medium with 10% (v/v) alamarBlue® reagent mixed with culture medium.•Increase of incubation period to 24 h at 37 °C protected from light.

A unique combination of surface chemistry and self-assembly of amphiphilic block copolymers was employed to obtain—for the first time—solid-supported biomimetic polymer bilayers. An organized monolayer from sulfur-functionalized poly(butadiene)-b-poly(ethylene oxide) was covalently attached to ultrasmooth gold upon Langmuir-Blodgett transfer. Hydrophobic interactions, on the other hand, were exploited to attach the second monolayer. As a result, we obtained a homogeneous hydrophilic-hydrophobic-hydrophilic structure, similar to supported lipid bilayers by architecture, stability and fluidity. Our polymer bilayers, however, outperform such lipid membranes with regard to tunability of thickness and stability in gaseous environments. As characterized by surface analysis tools (AFM, SPR), solid-supported polymer membranes are smooth with a thickness of ca. 11 nm, resistant to rinsing with aqueous solutions and stable upon drying and rehydration. These properties could be attractive for nanotechnological applications, such as immobilization of functional molecules or nanoparticles, sensor development or preparation of chemically responsive functional surfaces.