Synthetic Cells Revisited: Artificial Cells Construction Using Polymeric Building Blocks.

The exponential growth of research on artificial cells and organelles underscores their potential as tools to advance the understanding of fundamental biological processes. The bottom-up construction from a variety of building blocks at the micro- and nanoscale, in combination with biomolecules is key to developing artificial cells. In this review, artificial cells are focused upon based on compartments where polymers are the main constituent of the assembly. Polymers are of particular interest due to their incredible chemical variety and the advantage of tuning the properties and functionality of their assemblies. First, the architectures of micro- and nanoscale polymer assemblies are introduced and then their usage as building blocks is elaborated upon. Different membrane-bound and membrane-less compartments and supramolecular structures and how they combine into advanced synthetic cells are presented. Then, the functional aspects are explored, addressing how artificial organelles in giant compartments mimic cellular processes. Finally, how artificial cells communicate with their surrounding and each other such as to adapt to an ever-changing environment and achieve collective behavior as a steppingstone toward artificial tissues, is taken a look at. Engineering artificial cells with highly controllable and programmable features open new avenues for the development of sophisticated multifunctional systems.

Microfluidic Giant Polymer Vesicles Equipped with Biopores for High-Throughput Screening of Bacteria.

Understanding the mechanisms of antibiotic resistance is critical for the development of new therapeutics. Traditional methods for testing bacteria are often limited in their efficiency and reusability. Single bacterial cells can be studied at high throughput using double emulsions, although the lack of control over the oil shell permeability and limited access to the droplet interior present serious drawbacks. Here, a straightforward strategy for studying bacteria-encapsulating double emulsion-templated giant unilamellar vesicles (GUVs) is introduced. This microfluidic approach serves to simultaneously load bacteria inside synthetic GUVs and to permeabilize their membrane with the pore-forming peptide melittin. This enables antibiotic delivery or the influx of fresh medium into the GUV lumen for highly parallel cultivation and antimicrobial efficacy testing. Polymer-based GUVs proved to be efficient culture and analysis microvessels, as microfluidics allow easy selection and encapsulation of bacteria and rapid modification of culture conditions for antibiotic development. Further, a method for in situ profiling of biofilms within GUVs for high-throughput screening is demonstrated. Conceivably, synthetic GUVs equipped with biopores can serve as a foundation for the high-throughput screening of bacterial colony interactions during biofilm formation and for investigating the effect of antibiotics on biofilms.

Current Perspectives on Synthetic Compartments for Biomedical Applications.

Nano- and micrometer-sized compartments composed of synthetic polymers are designed to mimic spatial and temporal divisions found in nature. Self-assembly of polymers into compartments such as polymersomes, giant unilamellar vesicles (GUVs), layer-by-layer (LbL) capsules, capsosomes, or polyion complex vesicles (PICsomes) allows for the separation of defined environments from the exterior. These compartments can be further engineered through the incorporation of (bio)molecules within the lumen or into the membrane, while the membrane can be decorated with functional moieties to produce catalytic compartments with defined structures and functions. Nanometer-sized compartments are used for imaging, theranostic, and therapeutic applications as a more mechanically stable alternative to liposomes, and through the encapsulation of catalytic molecules, i.e., enzymes, catalytic compartments can localize and act in vivo. On the micrometer scale, such biohybrid systems are used to encapsulate model proteins and form multicompartmentalized structures through the combination of multiple compartments, reaching closer to the creation of artificial organelles and cells. Significant progress in therapeutic applications and modeling strategies has been achieved through both the creation of polymers with tailored properties and functionalizations and novel techniques for their assembly.

Block Copolymer Giant Unilamellar Vesicles for High-Throughput Screening.

Bottom-up synthetic cells offer the potential to study cellular processes with reduced complexity. Giant unilamellar vesicles (GUVs) can mimic cells in their morphological characteristics because their architecture is precisely controllable. We propose a block copolymer-based GUV system that can be used for high-throughput screening. Through droplet microfluidic methods, we produce double emulsions that then serve as templates for GUVs with adjustable inner, polymer membrane, and outer composition. Using flow cytometry, we are able to analyze tens of thousands of GUVs in a short amount of time, enabling their use for screening assays.

Antibacterial, Cytocompatible, Sustainably Sourced: Cellulose Membranes with Bifunctional Peptides for Advanced Wound Dressings.

Progressive antibiotic resistance is a serious condition adding to the challenges associated with skin wound treatment, and antibacterial wound dressings with alternatives to antibiotics are urgently needed. Cellulose-based membranes are increasingly considered as wound dressings, necessitating further functionalization steps. A bifunctional peptide, combining an antimicrobial peptide (AMP) and a cellulose binding peptide (CBP), is designed. AMPs affect bacteria via multiple modes of action, thereby reducing the evolutionary pressure selecting for antibiotic resistance. The bifunctional peptide is successfully immobilized on cellulose membranes of bacterial origin or electrospun fibers of plant-derived cellulose, with tight control over peptide concentrations (0.2 ± 0.1 to 4.6 ± 1.6 µg mm-2 ). With this approach, new materials with antibacterial activity against Staphylococcus aureus (log4 reduction) and Pseudomonas aeruginosa (log1 reduction) are developed. Furthermore, membranes are cytocompatible in cultures of human fibroblasts. Additionally, a cell adhesive CBP-RGD peptide is designed and immobilized on membranes, inducing a 2.2-fold increased cell spreading compared to pristine cellulose. The versatile concept provides a toolbox for the functionalization of cellulose membranes of different origins and architectures with a broad choice in peptides. Functionalization in tris-buffered saline avoids further purification steps, allowing for translational research and multiple applications outside the field of wound dressings.

Enhanced Antimicrobial Activity and Structural Transitions of a Nanofibrillated Cellulose-Nisin Biocomposite Suspension.

Resistance to antibiotics has posed a high demand for novel strategies to fight bacterial infections. Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. However, their poor solubility in water and sensitivity to degradation has limited their application. Here, we report the design of a smart, pH-responsive antimicrobial nanobiocomposite material based on the AMP nisin and 2,2,6,6-tetramethyl-1-piperidinyloxyl-oxidized nanofibrillated cellulose (TONFC). Morphological transformations of the nanoscale structure of nisin functionalized-TONFC fibrils were discovered at pH values between 5.8 and 8.0 using small-angle X-ray scattering. Complementary ζ potential measurements indicate that electrostatic attractions between the negatively charged TONFC surface and the positively charged nisin molecules are responsible for the integration of nisin. Modification of the pH level or increasing the ionic strength reduces the nisin binding capacity of TONFC. Biological evaluation studies using a bioluminescence-based reporter strain of Bacillus subtilis and a clinically relevant strain of Staphylococcus aureus indicated a significantly higher antimicrobial activity of the TONFC-nisin biocomposite compared to the pure nisin against both strains under physiological pH and ionic strength conditions. The in-depth characterization of this new class of antimicrobial biocomposite material based on nanocellulose and nisin may guide the rational design of sustainable antimicrobial materials.