Materialorientierte Synthetische Biologie

We report a straightforward methodology to access structurally well-defined hybrid assemblies of plasmonic and excitonic nanoparticles (NPs). The developed strategy is based on the incorporation of quantum dots (QDs) coated with zinc-sulfide shells into poly(ethylene glycol) (PEG) brushes at gold NP surfaces, without the necessity of incorporating specialized functional groups to drive the supracolloidal assembly. Based on control experiments involving PEGs with distinct polymeric architecture and Fourier-transform infrared spectroscopy analysis, we attribute the structure formation to attractive interactions between the QD surface and the monomeric repeat unit of the PEG brushes. This combination leads to short interparticle spacings and plasmon/exciton interactions, resulting in photoluminescence (PL) quenching upon assembly. However, using block-copolymers comprising a NP-adjacent spacer block in addition to a NP-remote PEG block, the distance between gold NPs and QDs can be controlled, which in turn affects the PL properties. The versatility of the structure-formation approach is demonstrated by the possibility of applying it to two distinct core/shell QDs (InP/ZnSe/ZnS and CdSe/CdS/ZnS). This offers new perspectives in the quest for efficient nanomaterial fabrication procedures.

Recombinant adeno-associated viral (rAAV) vectors are a leading platform for in vivo gene therapy, valued for their excellent safety, broad serotype diversity, and scalable production. Targeted delivery through capsid display of ligands holds great promise, yet current retargeting strategies often rely on extensive capsid re-engineering and restrict the use of ligands incompatible with intracellular expression systems. Here, we present a modular AAV retargeting platform that, for the first time, employs the SpyTag/SpyCatcher system via genetic integration into the AAV2 capsid. SpyTag is a small peptide that forms a covalent, irreversible bond with its protein partner, SpyCatcher, allowing site-specific ligand coupling under physiological conditions. Inserting SpyTag into surface-exposed capsid sites enabled postassembly functionalization of AAVs with SpyCatcher-fused targeting proteins. As proof of concept, we used SpyCatcher fusions with designed ankyrin repeat proteins (DARPins) specific for EGFR, EpCAM, and HER2. This conferred highly specific transduction of corresponding cancer cell lines with minimal off-target activity. Therapeutic potential was demonstrated by delivering a suicide gene, inducing selective cancer cell killing upon prodrug administration. This “one-fits-all” platform allows rapid and flexible retargeting without significantly altering the underlying vectors genome or production process. It supports the incorporation of large or complex ligands not amenable to genetic fusion and facilitates high-throughput preclinical evaluation strategies. By uniting capsid engineering with modular ligand display, our approach provides a scalable and versatile framework for precision gene delivery, broadening the applicability of rAAV in both therapeutic and discovery settings.

Discrimination of self from non-self RNA is a critical requirement for any cell to respond to infections and to maintain cellular integrity. We report novel functions for two RNA-dependent RNA polymerases (RDRs) in Paramecium. In RNAinterference (RNAi), RDRs are normally involved in the production of large amounts of secondary small interfering RNAs (siRNAs). To characterize the function of RDRs in context of exogenous RNA recognition, we developed a novel double-stranded RNA (dsRNA) application system using dextran nanoparticles to deliver heteroduplex dsRNA to cells as food particles, mimicking the natural phagosomal entry. Small RNA sequencing allows to dissect siRNAs produced from exogenous RNA or RDR transcripts. Contrary to expectations, our data show that Dicer is unable to directly cleave exogenous dsRNA while two RDRs, RDR1 and RDR2, are required for the initial steps of dsRNA-induced RNAi. Paradoxically, these two RDRs must replicate dsRNA before Dicer cleavage. This system works efficiently also with exogenous single-stranded RNA (ssRNA), although RDR2 is dispensable for ssRNA conversion. The function of RDRs is in contrast to that in animals, plants and fungi and extends the functional diversity of these polymerases as RDR-associated complexes appear to control the entry of food RNA into the RNAi machinery.

Catechol-modified polymers, such as DOPA-functionalized systems, have recently gained significant interest for a variety of biomedical applications, particularly in their role as antibacterial adjuvants due to their oxidative activity and ability to generate reactive oxygen species (ROS). Current catechol-functionalized polymers, however, often suffer from a restricted number of catechol groups, limited biocompatibility and solubility, and low stability due to the rapid oxidation under physiological conditions. In this study, we developed a water-soluble, biocompatible DOPA-modified biodynamer (DOPA-BD), leveraging the principles of constitutional dynamic chemistry (CDC). DOPA-BD was synthesized via polycondensation of DOPA-hydrazide and the hexaethylene glycol-conjugated carbazole dialdehyde (CA-HG), forming dynamic imine and acylhydrazone linkages between the monomers. As a result of its dynamic covalent backbone, DOPA-BD exhibits biodegradability and undergoes pH-responsive degradation under mildly acidic conditions typically found at infection sites, leading to a more than 3-fold increase in DOPA-hydrazide release compared to physiological pH. Interestingly, driven by CDC, DOPA-BD folds into a nanorod structure with a hydrodynamic diameter of ∼7.8 nm, surrounded by HG chains that offer water solubility and biocompatibility. Moreover, the incorporation of the DOPA-derivative in each repeating unit yields a polymer with exceptionally high catechol content, which remains stable and resistant to oxidation for 72 h in physiological buffer conditions. Regarding its antibacterial applicability, DOPA-BD demonstrated synergistic antibacterial activity with Azithromycin (AZM) against AZM-resistant E. coli, enhancing the antibiotic’s efficacy by 4-fold. Our study indicates that DOPA-BD induces ROS production in the respective bacterial strain, suggesting ROS generation as one of the possible mechanisms contributing to the observed synergy. Overall, DOPA-BD represents a promising alternative strategy to potentiate antibacterial activity against resistant strains, holding strong potential for future antibacterial applications.

Chronic lung infections caused by Pseudomonas aeruginosa are a major contributor to morbidity and mortality in patients with cystic fibrosis. Biofilm formation and the emergence of antibiotic resistance limit the effectiveness of current inhaled therapies, highlighting the need for innovative formulation strategies that enhance local antibacterial efficacy while preserving epithelial integrity. This study explores a nano-in-micro inhalable formulation design that combines azithromycin (AZM) with the antibiotic adjuvant menadione (MEN) to enhance antibiofilm activity under biorelevant pulmonary exposure conditions. Nano-embedded microparticles (NEMs) were developed as an inhalable “Trojan particle” approach, combining AZM in a microparticle matrix with MEN-loaded polymeric nanoparticles. The formulations were produced by spray drying and characterized in terms of morphology, aerodynamic performance, solid-state properties and redispersibility. Antibiofilm activity against P. aeruginosa biofilms and epithelial safety in Calu-3 cells were evaluated using biorelevant aerosol exposure models, including nebulization and dry powder atomization. NEMs exhibited favorable aerodynamic properties suitable for bronchial delivery. Incorporation of MEN into the NEM system enhanced the antibiofilm efficacy of AZM compared with the microparticle matrix-only formulation, indicating that the adjuvant effect of MEN was retained upon translation into an inhalable formulation. Cytotoxicity studies demonstrated that neither nebulized nor dry powder–delivered formulations compromised the membrane integrity of Calu-3 cells. These results indicate that the combination of azithromycin and menadione within a NEM design preserves drug activity and adjuvant efficacy while enabling effective aerosol delivery. Future studies in more complex and disease-relevant models will further strengthen the understanding of the translational potential of this approach.

Engineering cells to fabricate and program bio-based materials presents a sustainable alternative to petroleum-derived composites while simultaneously enabling the integration of advanced functionality. This is particularly relevant for engineered wood composites, which are widely used in construction, yet rely on petrol-based, non-biodegradable binders. Here, we develop bio-programmed wood composites using engineered bacteria, addressing both functional enhancement and sustainability. To navigate the large design space linking genetic programs, material composition, and processing conditions to mechanical performance, we combine lab automation with a pretrained transformer model for in-context prediction. This approach enables rapid identification of formulations yielding desired mechanical properties. Beyond mechanical properties, we introduce programmable features such as optogenetically patterned in situ pigmentation, local porosity control, and autonomous damage reporting. We demonstrate the applicability of our approach through manufacturing macro-scale furniture prototypes. Our platform establishes a blueprint for the accelerated, AI-driven development of sustainable, living multifunctional materials with applications in construction and beyond.

The functional integration of biological switches with synthetic building blocks enables the design of modular, stimulus-responsive biohybrid materials. By connecting the individual modules via diffusible signals, information-processing circuits can be designed. Such systems are, however, mostly limited to respond to either small molecules, proteins, or optical input thus limiting the sensing and application scope of the material circuits. Here, a highly modular biohybrid material is design based on CRISPR/Cas13a to translate arbitrary single-stranded RNAs into a biomolecular material response. This system exemplified by the development of a cascade of communicating materials that can detect the tumor biomarker microRNA miR19b in patient samples or sequences specific for SARS-CoV. Specificity of the system is further demonstrated by discriminating between input miRNA sequences with single-nucleotide differences. To quantitatively understand information processing in the materials cascade, a mathematical model is developed. The model is used to guide systems design for enhancing signal amplification functionality of the overall materials system. The newly designed modular materials can be used to interface desired RNA input with stimulus-responsive and information-processing materials for building point-of-care suitable sensors as well as multi-input diagnostic systems with integrated data processing and interpretation.

Antibiotic resistance in chronic lung infections caused by Pseudomonas aeruginosa requires alternative approaches to improve antibiotic efficacy. One promising approach is the use of adjuvant compounds that complement antibiotic therapy. This study explores the potential of menadione as an adjuvant to azithromycin against planktonic cells and biofilms of P. aeruginosa, focusing on its mechanisms of action and cytotoxicity in pulmonary cell models. Methods: The effect of menadione in improving the antibacterial and antibiofilm potency of azithromycin was tested against P. aeruginosa. Mechanistic studies in P. aeruginosa and AZMr-E. coli DH5α were performed to probe reactive oxygen species (ROS) production and bacterial membrane disruption. Cytotoxicity of antibacterial concentrations of menadione was assessed by measuring ROS levels and membrane integrity in Calu-3 and A549 lung epithelial cells. Results: Adding 0.5 µg/mL menadione to azithromycin reduced the minimum inhibitory concentration (MIC) by four-fold and the minimum biofilm eradication concentration (MBEC) by two-fold against P. aeruginosa. Adjuvant mechanisms of menadione involved ROS production and disruption of bacterial membranes. Cytotoxicity tests revealed that antibacterial concentrations of menadione (≤64 µg/mL) did not affect ROS levels or membrane integrity in lung cell lines. Conclusions: Menadione enhanced the efficacy of azithromycin against P. aeruginosa while exhibiting a favorable safety profile in lung epithelial cells at antibacterial concentrations. These findings suggest that menadione is a promising antibiotic adjuvant. However, as relevant data on the toxicity of menadione is sparse, further toxicity studies are required to ensure its safe use in complementing antibiotic therapy.

Living therapeutic and diagnostic materials based on engineered microorganisms are emerging as a novel approach with the perspective of providing patient-tailored, sustainable, and cost-effective healthcare solutions. In this review, we focus on recent advances in using genetically or chemically engineered microorganisms as living diagnostics, therapeutics, and as a means of prevention for various diseases. We also highlight the applications of living therapeutics for acute and chronic diseases, and the role of micro/macro-encapsulation of the engineered microorganisms. We further showcase the current success of engineered living therapeutics in clinical trials and discuss challenges and future trends in the field.