Open Access

Herein an assay toward a rapid and reliable profiling of extracellular matrix of Escherichia coli (E. coli) utilizing a tandem of GC-MS as a tool for definition of the exact chemical nature of low molecular weight compounds and cyclic voltammetry for their high throughput detection is presented. Briefly, during a set of investigations the formation of glycerol in the extracellular matrix (ECM) of E. coli at physiological relevant conditions of cells was revealed. Based on the obtained knowledge, the electrochemical protocol allowing both qualitative and quantitative analyses of glycerol in E. coli ECMs at palladium ink-modified screen printed electrodes with precision values (RSD) <10 % and recovery rates ranged from 98 % to 102 % was proposed. The provided protocol for a rapid electrochemical profiling of the bacterial ECMs can readily be used as a guideline for the controlled electroanalysis of target electroactive signaling analytes in complex biological samples.

To combine the excellent transfection properties of lipids with the high stability of polymeric nanoparticles, we designed a hybrid system with a polymeric core surrounded by a shell of different lipids. The aim is to use this technology for skin vaccination purposes where the transfection of dendritic cells is crucial. Based on a carrier made of PLGA and the positively charged lipid DOTMA, we prepared a panel of nanocarriers with increasing amounts of the zwitterionic phospholipid DOPE in the lipid layer to improve their cell tolerability. We selected a nomenclature accordingly with numbers in brackets to represent the used mol% of DOPE and DOTMA in the lipid layer, respectively. We loaded mRNA onto the surface and assessed the mRNA binding efficacy and the degree of protection against RNases. We investigated the influence of the lipid composition on the toxicity, uptake and transfection in the dendritic cell line DC 2.4 challenging the formulations with different medium supplements like fetal calf serum (FCS) and salts. After selecting the most promising candidate, we performed an immune stimulation assay with primary mouse derived dendritic cells. The experiments showed that all tested lipid–polymer nanoparticles (LPNs) have comparable hydrodynamic parameters with sizes between 200 and 250 nm and are able to bind mRNA electrostatically due to their positive zetapotential (20–40 mV for most formulations). The more of DOPE we add, the more free mRNA we find and the better the cellular uptake reaching approx. 100% for LPN(60/40)–LPN(90/10). This applies for all tested formulations leading to LPN(70/30) with the best performance, in terms of 67% of live cells with protein expression. In that case, the supplements of the medium did not influence the transfection efficacy (56% vs. 67% (suppl. medium) for live cells and 63% vs. 71% in total population). We finally confirmed this finding using mouse derived primary immune cells. We can conclude that a certain amount of DOTMA in the lipid coating of the polymer core is essential for complexation of the mRNA, but the zwitterionic phospholipid DOPE is also important for the particles’ performance in supplemented media.

Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs’ technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro–in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics.

Messenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. A variety of biodegradable nanoparticles (NPs) has been developed including lipid-based and polymer-based systems for mRNA delivery. However, both systems still lack in achieving an efficient transfection rate and a detailed understanding of the mRNA transgene expression kinetics. Therefore, quantitative analysis of the time-dependent translation behavior would provide a better understanding of mRNA’s transient nature and further aid the enhancement of appropriate carriers with the perspective to generate future precision nanomedicines with quick response to treat various diseases.

The permeability of the human trabecular meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases such as glaucoma. Engineered HTMs could help to understand the structure–function relation in natural tissues and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125–500 μm and fiber diameters of 10 –12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa and a static compression modulus in the range of 6 –360 kPa are obtained by varying the scaffold design, that is, the density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8–14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW for reconstructing complex morphological features of natural tissues.