Polyelectrolytes have gained significant attention for their unique, potentially specific association with living cells. Particularly, amino-acid-derived polyzwitterions feature specificity to cancer cells, highlighting their potential for biomedical applications. Here, the synthesis of lysine- and phenylalanine-derived polyelectrolytes via post-polymerisation modification is presented. The amidation of activated ester prepolymers with protected basic amino acids and their subsequent deprotection yield tailored amino acid-functionalised polyzwitterions comprising side chain chirality. Similarly, the use of achiral neutral amino acids results in polyanionic analogues of these polyzwitterions. Via this strategy, a small library of five polymers was created, which enables the careful evaluation of structure-property relationships of polyelectrolytes and their interactions with cells. In particular, we study the influence of side chain motifs: (i) aliphatic vs. aromatic side chains, (ii) permanent vs. non-permanent charge, and (iii) zwitterionic vs. anionic patterns. High-performance liquid chromatography measurements are performed to investigate the influence of both the charge character and aromaticity on the hydrophilicity of the polyelectrolytes. Response to changes in the pH value is studied by dynamic light scattering (DLS) and electrophoretic light scattering at different pH values. Furthermore, polyelectrolyte stability under biologically relevant conditions, i.e. in the presence of model proteins and more complex protein mixtures from foetal bovine serum, is assessed by DLS. Association with cells from different cancer and non-cancer cell lines provides information regarding structural driving forces for association efficiency and specificity. Furthermore, fluorescence imaging shows fast uptake of polyanions into targeted cells, making them interesting candidates for drug-delivery applications.

The earthworm Eisenia fetida is a widely used model organism for environmental toxicity assessment, including microplastic particles (MPP). Impacts of MPP at the organismic level have been extensively documented. Effects on the cellular level and in particular the immune system of E. fetida are less well understood. Here we exposed earthworms outside soil for six days to food pellets containing 10% (w/w) pure polystyrene (PS) MPP (25 – 75 µm) or PS MPP supplemented with 0.5% (w/w) of the antioxidant Irgafos (PSIrgafos). Immune cells (coelomocytes) were isolated from E. fetida using a non-invasive extrusion method, and their number, viability, and subpopulation distribution (amoebocytes and eleocytes) were assessed by flow cytometry. Husbandry outside soil was found to generally increase propensity for apoptosis in particular of amoebocytes with little difference between experimental setups. The total number of extruded cells was not affected by MPP, while coelomocyte viability was significantly reduced. PSIrgafos ingestion further decreased the number of living cells per mg fresh weight. Ex vivo analysis of coelomocyte viability confirmed an enhanced toxicity of PSIrgafos compared to PS. Our findings show that MPP and their additives cause physiological changes and alter immune cell distribution, posing potential risks to soil invertebrates.

Microplastic particles (MPP) arise from mismanaged plastic waste, plastic degradation (i.e., agricultural films, textiles, or packaging), and direct use in products such as cosmetics. Their environmental persistence raises concerns for ecosystems, wildlife, and human health. However, most in vitro studies focus on pristine spherical particles and short-term exposure, limiting environmental relevance. Here, we conducted a comprehensive multi-parametric analysis of short- (24 h) and long-term (21 days) exposure of murine macrophages to pristine (PSpristine) and artificially weathered (PSaged) polystyrene (PS) MPP. PSpristine were largely non-toxic during short-term exposure but induced adaptive pro- and anti-inflammatory responses, with minimal cytotoxicity, after repeated long-term exposure. PSaged triggered acute oxidative stress, reduced metabolic activity, induced DNA damage, and elicited early pro-inflammatory activation, while long-term exposure resulted in pronounced cellular stress, functional impairment, and minimal macrophage polarization. These results demonstrate that particle ageing and exposure period critically shape macrophage responses and highlight the need for long-term in vitro studies using environmentally relevant, well-characterized MPP.

The magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1 synthesizes membrane-enclosed magnetite (Fe3O4) nanocrystals, known as magnetosomes. Owing to their uniform size, purity and superior magnetic properties, magnetosomes represent highly attractive nanomaterials for biotechnological and biomedical applications. However, their bioproduction is limited by demanding cultivation requirements, largely because magnetite biomineralization is highly sensitive to environmental parameters, particularly oxygen. While elevated oxygen concentrations are known to inhibit magnetosome formation, quantitative analyses under defined low-oxygen conditions are scarce. Here, we cultivated MSR-1 in bioreactors under precisely controlled dissolved oxygen (DO) levels and quantified growth behaviour, substrate uptake and magnetosome characteristics. Cells harvested during late exponential growth revealed that magnetite crystal numbers per cell were similar across a wide DO range (0%-5%), whereas crystal sizes decreased with increasing oxygen levels. The data further indicate that oxygen inhibits biomineralization primarily through direct oxidative interference rather than indirect metabolic effects. These findings provide a mechanistic basis for optimizing oxygen control strategies in MTB cultivation and demonstrate that fine-tuning DO levels enables targeted modulation of magnetosome size and properties. This advances both the bioprocess development of high-yield magnetosome production and the application of tailored magnetic nanoparticles in biotechnology and medicine.

Nonviral gene delivery offers promise for treating age-related macular degeneration (AMD), a major cause of blindness. Genetic modification of retinal pigment epithelium (RPE) cells is a potential therapeutic strategy for AMD. This study presents a multiparametric approach to enhance nonviral transfection of human ARPE-19 cells using linear poly-(ethylenimine) (l-PEI, 25 kDa) as a delivery agent for plasmid DNA (pDNA). The transfection protocol was optimized by adjusting the N/P ratio through nucleic acid concentration, varying polymer density, reducing transfection volume, and minimizing contact time between cells and polyplexes. Under optimized conditions, transfection efficiency (TE) reached 88% with ∼85% viability. A semi-automated method for polyplex formation was developed using a 3D-printed microfluidic system, thereby enabling standardized production. This optimized protocol was successfully adapted to the microfluidic system without compromising TE or viability. This semi-automated approach represents a step toward the scalable and reproducible application of l-PEI-based transfection technologies for future therapeutic use.

Bioprinting of microtissues has become a standard technique in medical and biotechnological research, offering a more accurate replication of thein vivosetting than conventional 2D cell culture. However, widespread adoption is limited by the absence of a universally accepted printing benchmark-common in standard fused deposition modeling (FDM) printing, as well as the high cost and restricted customizability of commercial bioprinters. This study introduces a method to convert a standard FDM printer into a bioprinter. All cell-contacting components are biocompatible and autoclavable, while the printer body can be UV-sanitized. Using a heated FDM printhead, we used the thermal properties of alginate-gelatin bioinks to achieve high-resolution 3D printing. A key achievement was the developed print quality index (PQI) method, which correlates nozzle temperature with bioink flow behavior, streamlining optimization of slicer settings. Guided by PQI, we reproducibly bioprinted complex alginate-gelatin structures with high quality and dimensional/geometric accuracy. A case study using recombinant HuH7EGFPcell-laden hydrogels demonstrated long-term cell proliferation, confirming high viability. Given its efficiency, the PQI method has the potential to become the missing printing benchmark for slicer optimization in bioprinting. The presented approach significantly advances the accessibility of sophisticated bioprinting technology to interested research groups worldwide.

Amino-acid-derived polyzwitterions and polybetaines (PBs) are two promising alternatives to non-ionic polymers, for example, to increase tumor permeability. In this study, amino-acid-derived polyzwitterions are synthesized and a strategy to quarternize the amine in the side chain functional group is developed to combine the advantages of both types. The functional monomer is polymerized via reversible addition-fragmentation chain-transfer polymerization for which a kinetic study is performed. Further, the impact of the permanent positive charge on amino-acid-derived polyzwitterions is studied based on two zwitterionic polymers obtained via post-polymerization modification (PPM) of Poly(N-acryloxysuccinimide) to allow good comparison between methylated and non-methylated polymers. Circular dichroism shows that the stereocenter remains intact during PPM. pH titration and zeta-potential measurements show that the methylated polymer has a negative zeta-potential over the measured pH range and, therefore, the polymer remains zwitterionic over a broader pH range than its non-methylated equivalent. Both polymers are well tolerated by mammalian cells up to concentrations of 1 mg mL-1. The study introduces a path to a new polymer class that combines the advantages of both PBs and amino-acid-derived polyzwitterions and highlights the impact a permanent charge has on the physiochemical properties. Polyzwitterions are promising materials for applications at the bio-interface as they can combine both - low fouling properties and target specificity. Prominent examples of polyzwitterions include non-chiral polybetaines and chiral amino-acid-derived polyzwitterions. The synthesis and characterization of chiral amino-acid-derived polyzwitterions are reported. Chiral polybetaines are highlighted as novel materials with potential for future applications in biological environments. image

Microplastics (MP) have become a well-known and widely investigated environmental pollutant. Despite the huge amount of new studies investigating the potential threat posed by MP, the possible uptake and trophic transfer in lower trophic levels of freshwater ecosystems remains understudied. This study aims to investigate the internalization and potential trophic transfer of fluorescent polystyrene (PS) beads (0.5 mu m, 3.6 x 10(8 )particles/ mL; 6 mu m, 2.1 x 10(5) particles/mL) and fragments (<30 m, 5 x 10(3) particles/mL) in three unicellular eukaryotes. This study focuses on the size-dependent uptake of MP by two freshwater Ciliophora, Tetrahymena pyriformis, Paramecium caudatum and one Amoebozoa, Amoeba proteus, serving also as predator for experiments on potential trophic transfer. Size-dependent uptake of MP in all three unicellular eukaryotes was shown. P. caudatum is able to take up MP fragments up to 27.7 mu m, while T. pyriformis ingests particles up to 10 mu m. In A. proteus, small MP (PS0.5 mu m and PS6 mu m) were taken up via pinocytosis and were detected in the cytoplasm for up to 14 days after exposure. Large PS-MP (PS<30m) were detected in A. proteus only after predation on MP-fed Ciliophora. These results indicate that A. proteus ingests larger MP via predation on Ciliophora (PS<30m), which would not be taken up otherwise. This study shows trophic transfer of MP at the base of the aquatic food web and serves as basis to study the impact of MP in freshwater ecosystems.

Due to global pollution derived from plastic waste, the research on microplastics is of increasing public interest. Until now, most studies addressing the effect of microplastic particles on vertebrate cells have primarily utilized polystyrene particles (PS). Other studies on polymer microparticles made, e.g., of polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), or poly (ethylene terephthalate) (PET), cannot easily be directly compared to these PS studies, since the used microparticles differ widely in size and surface features. Here, effects caused by pristine microparticles of a narrow size range between 1 - 4 µm from selected conventional polymers including PS, PE, and PVC, were compared to those of particles made of polymers derived from biological sources like polylactic acid (PLA), and cellulose acetate (CA). The microparticles were used to investigate cellular uptake and assess cytotoxic effects on murine macrophages and epithelial cells. Despite differences in the particles' properties (e.g. ζ-potential and surface morphology), macrophages were able to ingest all tested particles, whereas epithelial cells ingested only the PS-based particles, which had a strong negative ζ-potential. Most importantly, none of the used model polymer particles exhibited significant short-time cytotoxicity, although the general effect of environmentally relevant microplastic particles on organisms requires further investigation.

The impact of microplastic particles on organisms is currently intensely researched. Although it is well established that macrophages ingest polystyrene (PS) microparticles, little is known about the subsequent fate of the particles, such as entrapment in organelles, distribution during cell division, as well as possible mechanisms of excretion. Here, submicrometer (0.2 and 0.5 µm) and micron-sized (3 µm) particles were used to analyze particle fate upon ingestion of murine macrophages (J774A.1 and ImKC). Distribution and excretion of PS particles was investigated over cycles of cellular division. The distribution during cell division seems cell-specific upon comparing two different macrophage cell lines, and no apparent active excretion of microplastic particles could be observed. Using polarized cells, M1 polarized macrophages show higher phagocytic activity and particle uptake than M2 polarized ones or M0 cells. While particles with all tested diameters were found in the cytoplasm, submicron particles were additionally co-localized with the endoplasmic reticulum. Further, 0.5 µm particles were occasionally found in endosomes. Our results indicate that a possible reason for the previously described low cytotoxicity upon uptake of pristine PS microparticles by macrophages may be due to the preferential localization in the cytoplasm.

Polystyrene (PS) is an important model polymer for the investigation of effects of microplastic (MP) and nanoplastic (NP) particles on living systems. Aqueous dispersions of PS MP or NP contain residual monomers of styrene. In consequence, it is not clear if the effects observed in standard (cyto)toxicity studies are evoked by the polymer (MP/NP) particle or by residual monomers. We addressed that question by comparing standard PS model particle dispersions with in-house synthesized PS particle dispersions. We proposed a rapid purification method of PS particle dispersions by dialysis against mixed solvents and developed a simple method of UV-vis spectrometry to detect residual styrene in the dispersions. We found that standard PS model particle dispersions, which contain residual monomers, exerted a low but significant cytotoxicity on mammalian cells, while the in-house synthesized PS, after rigorous purification to reduce the styrene content, did not. However, the PS particles per se but not the residual styrene in both PS particle dispersions resulted in immobilization of Daphnia. Only by using freshly monomer-depleted particles, will it be possible in the future to assess the (cyto)toxicities of PS particles, avoiding an otherwise not controllable bias effect of the monomer.

Biodegradable electrospun sponges are of interest for various applications including tissue engineering, drug release, dental therapy, plant protection, and plant fertilization. Biodegradable electrospun poly(l-lactide)/poly(ε-caprolactone) (PLLA/PCL) blend fiber-based sponge with hierarchical pore structure is inherently hydrophobic, which is disadvantageous for application in tissue engineering, fertilization, and drug delivery. Contact angles and model studies for staining with a hydrophilic dye for untreated, plasma-treated, and surfactant-treated PLLA/PCL sponges are reported. Thorough hydrophilization of PLLA/PCL sponges is found only with surfactant-treated sponges. The MTT assay on the leachates from the sponges does not indicate any cell incompatibility. Furthermore, the cell proliferation and penetration of the hydrophilized sponges are verified by in vitro cell culture studies using MG63 and human fibroblast cells.

Abstract Background One of the most complex prokaryotic organelles are magnetosomes, which are formed by magnetotactic bacteria as sensors for navigation in the Earth’s magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense magnetosomes consist of chains of magnetite crystals (Fe3O4) that under microoxic to anoxic conditions are biomineralized within membrane vesicles. To form such an intricate structure, the transcription of > 30 specific structural genes clustered within the genomic magnetosome island (MAI) has to be coordinated with the expression of an as-yet unknown number of auxiliary genes encoding several generic metabolic functions. However, their global regulation and transcriptional organization in response to anoxic conditions most favorable for magnetite biomineralization are still unclear. Results Here, we compared transcriptional profiles of anaerobically grown magnetosome forming cells with those in which magnetosome biosynthesis has been suppressed by aerobic condition. Using whole transcriptome shotgun sequencing, we found that transcription of about 300 of the > 4300 genes was significantly enhanced during magnetosome formation. About 40 of the top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. The mam and mms gene clusters, specifically controlling magnetosome biosynthesis, were highly transcribed, but constitutively expressed irrespective of the growth condition. By Cappable-sequencing, we show that the transcriptional complexity of both the MAI and the entire genome decreased under anaerobic conditions optimal for magnetosome formation. In addition, predominant promoter structures were highly similar to sigma factor σ70 dependent promoters in other Alphaproteobacteria. Conclusions Our transcriptome-wide analysis revealed that magnetite biomineralization relies on a complex interplay between generic metabolic processes such as aerobic and anaerobic respiration, cellular redox control, and the biosynthesis of specific magnetosome structures. In addition, we provide insights into global regulatory features that have remained uncharacterized in the widely studied model organism M. gryphiswaldense, including a comprehensive dataset of newly annotated transcription start sites and genome-wide operon detection as a community resource (GEO Series accession number GSE197098).

Human bone morphogenetic protein 2 (hBMP2) is routinely used in medical applications as an inducer of osteoformation. The recombinant production of BMP2 is typically performed using stable Chinese hamster ovary (CHO) cell lines. However, this process is inefficient, resulting in low product titers. In contrast, transient gene expression (TGE), which also enables the production of recombinant proteins, suffers from short production times and hence limited total product amounts. Here, we show that TGE-based BMP2 production is more efficient in HEKsus than in CHOsus cells. Independently of the cell lines, a bicistronic plasmid co-expressing EGFP and BMP2 facilitated the determination of the transfection efficiency but led to inferior BMP2 titers. Finally, we used a high cell density transient transfection (HCD-TGE) protocol to improve and extend the BMP2 expression by performing four rounds of serial transfections on one pool of HEKsus cells. This repeated transient transfection (RTT) process in HEKsus cells was implemented using EGFP as a reporter gene and further adapted for BMP2 production. The proposed method significantly improves BMP2 production (up to 509 ng/106 cells) by extending the production phase (96–360 h). RTT can be integrated into the seed train and is shown to be compatible with scale-up to the liter range.

Electrospun nanofibers can be effectively used as a surrogate for extracellular matrices (ECMs). However, in the context of cellular mechanobiology, their mechanical performances can be enhanced by using nanofibrous materials with a high level of structural organization. Herein, this work develops multifibrillar yarns with superior mechanical performance based on biocompatible polyacrylonitrile (PAN) as surrogate ECM. Nearly perfect aligned nanofibers along with the axis of the multifibrillar yarn are prepared. These highly aligned yarns exhibit high strength, high toughness, good stress relaxation behavior, and are robust enough for technical or medical applications. Further, this work analyzes the influence of the highly aligned-hierarchical topological structure of the material on cell proliferation and cell orientation using cells derived from epithelial and connective tissues. Compared to nonoriented electrospun multifibrillar yarns and flat films, the well-ordered topology in the electrospun PAN multifibrillar yarns triggers an improved proliferation of fibroblasts and epithelial cells. Fibroblasts acquire an elongated morphology analogous to their behavior in the natural ECM. Hence, this heterogeneous multifibrillar material can be used to restore or reproduce the ECM for tissue engineering applications, notably in the skeletal muscle and tendon.

Microplastic particles (MP), arising from the gradual decomposition of plastics in the environment, have been identified as a global problem. Most investigations of MP cytotoxicity use pristine spherical particles available from commercial sources when evaluating their impact on mammalian cells, while only limited data is available for the more relevant "weathered microplastic". In this study, we exposed murine macrophages to polystyrene MP either after up to 130 days of accelerated ageing or in pristine condition. Weathered and pristine MP were physicochemically characterized, and their cytotoxicity was investigated using biological assays, transcriptome analysis, and metabolic pathways prediction. Whereas the response to pristine MP is mainly dominated by a TNF-α release, sharp-edged weathered MP induce broader adverse cellular reactions. This study stresses the importance of including more realistic test particles (e.g., weathered particles) in combination with a broad range of biological assays when evaluating the potential risk of microplastic exposure.

Microplastics and its putative adverse effects on environmental and human health increasingly gain scientific and public attention. Systematic studies on the effects of microplastics are currently hampered by using rather poorly characterised particles, leading to contradictory results for the same particle type. Here, surface properties and chemical composition of two commercially available nominally identical polystyrene microparticles, frequently used in effect studies, were characterised. We show distinct differences in monomer content, ζ-potentials and surface charge densities. Cells exposed to particles showing a lower ζ-potential and a higher monomer content displayed a higher number of particle-cell-interactions and consequently a decrease in cell metabolism and proliferation, especially at higher particle concentrations. Our study emphasises that no general statements can be made about the effects of microplastics, not even for the same polymer type in the same size class, unless the physicochemical properties are well characterised.

Microplastic particles are pollutants in the environment with a potential impact on ecology and human health. As soon as microplastic particles get in contact with complex (biological) environments, they will be covered by an eco- and/or protein corona. In this contribution, protein corona formation was conducted under defined laboratory conditions on polystyrene (PS) microparticles to investigate the influence on surface properties, protein corona evolution, particle-cell interactions, and uptake in two murine epithelial cells. To direct protein corona formation, PS particles were preincubated with five model proteins, namely, bovine serum albumin (BSA), myoglobin, β-lactoglobulin, lysozyme, and fibrinogen. Subsequently, the single-protein-coated particles were incubated in a cell culture medium containing a cocktail of serum proteins to analyze changes in the protein corona profile as well as in the binding kinetics of the model proteins. Therein, we could show that the precoating step has a critical impact on the final composition of the protein corona. Yet, since proteins building the primary corona were still detectable after additional incubations in a protein-containing medium, backtracking of the particle's history is possible. Interestingly, whereas the precoating history significantly disturbs particle-cell interactions (PCIs), the cellular response (i.e., metabolic activity, MTT assay) stays unaffected. Of note, lysozyme precoating revealed one of the highest rates in PCI for both epithelial cell lines. Taken together, we could show that particle history has a significant impact on protein corona formation and subsequently on the interaction of particles with murine intestinal epithelial-like cells. However, as this study was limited to one cell type, further work is needed to assess if these observations can be generalized to other cell types.