Endowing textiles with self-repairing ability through the fabrication of composites with a bacterial biofilm.

To address the increasing environmental footprint of the fast-growing textile industry, self-repairing textile composites have been developed to allow torn or damaged textiles to restore their morphological, mechanical, and functional features. A sustainable way to create these textile composites is to introduce a coating material that is biologically derived, biodegradable, and can be produced through scalable processes. Here, we fabricated self-repairing textile composites by integrating the biofilms of Escherichia coli (E. coli) bacteria into conventional knitted textiles. The major structural protein component in E. coli biofilm is a matrix of curli fibers, which has demonstrated extraordinary abilities to self-assemble into mechanically strong macroscopic structures and self-heal upon contact with water. We demonstrated the integration of biofilm through three simple, fast, and scalable methods: adsorption, doctor blading, and vacuum filtration. We confirmed that the composites were breathable and mechanically strong after the integration, with improved Young's moduli or elongation at break depending on the fabrication method used. Through patching and welding, we showed that after rehydration, the composites made with all three methods effectively healed centimeter-scale defects. Upon observing that the biofilm strongly attached to the textiles by covering the extruding textile fibers from the self-repair failures, we proposed that the strength of the self-repairs relied on both the biofilm's cohesion and the biofilm-textile adhesion. Considering that curli fibers are genetically-tunable, the fabrication of self-repairing curli-expressing biofilm-textile composites opens new venues for industrially manufacturing affordable, durable, and sustainable functional textiles.

Fabrication of Curli Fiber-PEDOT:PSS Biomaterials with Tunable Self-Healing, Mechanical, and Electrical Properties.

Poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS) is a highly conductive, easily processable, self-healing polymer. It has been shown to be useful in bioelectronic applications, for instance, as a biointerfacing layer for studying brain activity, in biosensitive transistors, and in wearable biosensors. A green and biofriendly method for improving the mechanical properties, biocompatibility, and stability of PEDOT:PSS involves mixing the polymer with a biopolymer. Via structural changes and interactions with PEDOT:PSS, biopolymers have the potential to improve the self-healing ability, flexibility, and electrical conductivity of the composite. In this work, we fabricated novel protein-polymer multifunctional composites by mixing PEDOT:PSS with genetically programmable amyloid curli fibers produced byEscherichia coli bacteria. Curli fibers are among the stiffest protein polymers and, once isolated from bacterial biofilms, can form plastic-like thin films that heal with the addition of water. Curli-PEDOT:PSS composites containing 60% curli fibers exhibited a conductivity 4.5-fold higher than that of pristine PEDOT:PSS. The curli fibers imbued the biocomposites with an immediate water-induced self-healing ability. Further, the addition of curli fibers lowered the Young's and shear moduli of the composites, improving their compatibility for tissue-interfacing applications. Lastly, we showed that genetically engineered fluorescent curli fibers retained their ability to fluoresce within curli-PEDOT:PSS composites. Curli fibers thus allow to modulate a range of properties in conductive PEDOT:PSS composites, broadening the applications of this polymer in biointerfaces and bioelectronics.

Bacterial collagen-templated synthesis and assembly of inorganic particles.

Collagen has been used as a common template for mineralization and assembly of inorganic particles, because of the special arrangement of its fibrils and the presence of charged residues. Streptococcal bacterial collagen, which is inherently secreted on the surface ofStreptococcus pyogenes, has been progressively used as an alternative for type I animal collagen. Bacterial collagen is rich in charged amino acids, which can act as a substrate for the nucleation and growth of inorganic particles. Here, we show that bacterial collagen can be used to nucleate three different inorganic materials: hydroxyapatite crystals, silver nanoparticles, and silica nanoparticles. Collagen/mineral composites show an even distribution of inorganic particles along the collagen fibers, and the particles have a more homogenous size compared with minerals that are formed in the absence of the collagen scaffold. Furthermore, the gelation of silica occurring during mineralization represents a means to produce processable self-standing collagen composites, which is challenging to achieve with bacterial collagen alone. Overall, we highlight the advantage of simply combining bacterial collagen with minerals to expand their applications in the fields of biomaterials and tissue engineering, especially for bone regenerative scaffolds.

Extracellular Secretion and Simple Purification of Bacterial Collagen from Escherichia coli.

Because of structural similarities with type-I animal collagen, recombinant bacterial collagen-like proteins have been progressively used as a source of collagen for biomaterial applications. However, the intracellular expression combined with current costly and time-consuming chromatography methods for purification makes the large-scale production of recombinant bacterial collagen challenging. Here, we report the use of an adapted secretion pathway, used natively byEscherichia colito secrete curli fibers, for extracellular secretion of the bacterial collagen. We confirmed that a considerable fraction of expressed collagen (∼70%) is being secreted freely into the extracellular medium, with an initial purity of ∼50% in the crude culture supernatant. To simplify the purification of extracellular collagen, we avoided cell lysis and used cross-flow filtration or acid precipitation to concentrate the voluminous supernatant and separate the collagen from impurities. We confirmed that the secreted collagen forms triple helical structures, using Sirius Red staining and circular dichroism. We also detected collagen biomarkers via Raman spectroscopy, further supporting that the recombinant collagen forms a stable triple helical conformation. We further studied the effect of the isolation methods on the morphology and secondary structure, concluding that the final collagen structure is process-dependent. Overall, we show that the curli secretion system can be adapted for extracellular secretion of the bacterial collagen, eliminating the need for cell lysis, which simplifies the collagen isolation process and enables a simple cost-effective method with potential for scale-up.

Curli-Mediated Self-Assembly of a Fibrous Protein Scaffold for Hydroxyapatite Mineralization.

Nanostructures formed by self-assembled peptides have been increasingly exploited as functional materials for a wide variety of applications, from biotechnology to energy. However, it is sometimes challenging to assemble free short peptides into functional supramolecular structures, since not all peptides have the ability to self-assemble. Here, we report a self-assembly mechanism for short functional peptides that we derived from a class of fiber-forming amyloid proteins called curli. CsgA, the major subunit of curli fibers, is a self-assembling ß-helical subunit composed of five pseudorepeats (R1-R5). We first deleted the internal repeats (R2, R3, R4), known to be less essential for the aggregation of CsgA monomers into fibers, forming a truncated CsgA variant (R1/R5). As a proof-of-concept to introduce functionality in the fibers, we then genetically substituted the internal repeats by a hydroxyapatite (HAP)-binding peptide, resulting in a R1/HAP/R5 construct. Our method thus utilizes the R1/R5-driven self-assembly mechanism to assemble the HAP-binding peptide and form hydrogel-like materials in macroscopic quantities suitable for biomineralization. We confirmed the expression and fibrillar morphology of the truncated and HAP-containing curli-like amyloid fibers. X-ray diffraction and TEM showed the functionality of the HAP-binding peptide for mineralization and formation of nanocrystalline HAP. Overall, we show that fusion to the R1 and R5 repeats of CsgA enables the self-assembly of functional peptides into micron long fibers. Further, the mineral-templating ability that the R1/HAP/R5 fibers possesses opens up broader applications for curli proteins in the tissue engineering and biomaterials fields.

Fabrication of fluorescent pH-responsive protein-textile composites.

Wearable pH sensors are useful tools in the healthcare and fitness industries, allowing consumers to access information related to their health in a convenient manner via the monitoring of body fluids. In this work, we tailored novel protein-textile composites to fluorescently respond to changing pH. To do so, we used amyloid curli fibers, a key component in the extracellular matrix of Escherichia coli, as genetic scaffold to fuse a pH-responsive fluorescent protein, pHuji. Engineered amyloids form macroscopic and environmentally resistant aggregates that we isolated to use as stand-alone hydrogel-based sensors, and that we trapped within textile matrices to create responsive bio-composites. We showed that these composites were mechanically robust and vapor-permeable, thus exhibiting favorable characteristics for wearable platforms. CsgA-pHuji fibers integrated in the textile allowed the final device to respond to pH changes and distinguish between alkaline and acidic solutions. We demonstrated that the resulting composites could sustain their fluorescence response over days, and that their sensing ability was reversible for at least 10 high/low pH cycles, highlighting their potential for continuous monitoring. Overall, we introduced a biosynthesized amyloid-based textile composite that could be used as biosensing patch for a variety of applications in the smart textile industry.

Bacteria-Responsive Single and Core-Shell Nanofibrous Membranes Based on Polycaprolactone/Poly(ethylene succinate) for On-Demand Release of Biocides.

Traditional antibacterial dressings continuously elute biocides, even if there are no bacteria. This unneeded release can cause cytotoxicity, increase costs, and delay healing. We designed a bacteria-responsive nanofibrous wound dressing, which can be degraded in the presence of bacteria to release antimicrobial agents. A model biocide, benzyl dimethyl tetradecyl ammonium chloride (BTAC), was incorporated into bacteria-degradable polymers [polycaprolactone and poly(ethylene succinate)] in two ways: evenly distributed inside the polymers as single nanofibers and encapsulated in a core surrounded by the same polymers as core-shell nanofibers. Because of bacterial activity (both lipase secretion and acidic pH), degradation of the fibers was facilitated and caused the release of incorporated BTAC. BTAC-loaded single and core-shell nanofibers presented >1 log reduction of both Staphylococcus aureus and Escherichia coli within 2 h. Additionally, the core-shell structure provided a more controlled release of BTAC with prolonged antibacterial properties than single nanofibers. The core-shell nanofibers also exhibited minimal cytotoxicity against human fibroblast cells (>80% viable cells after 24 h contact). These nanofibrous mats have the potential to selectively release antibacterial agents to prevent wound infections without delaying wound healing.

Salinomycin-loaded Nanofibers for Glioblastoma Therapy.

Salinomycin is an antibiotic that has recently been introduced as a novel and effective anti-cancer drug. In this study, PLGA nanofibers (NFs) containing salinomycin (Sali) were fabricated by electrospinning for the first time. The biodegradable PLGA NFs had stability for approximately 30 days and exhibited a sustained release of the drug for at least a 2-week period. Cytotoxicity of the NFs + Sali was evaluated on human glioblastoma U-251 cells and more than 50% of the treated cells showed apoptosis in 48 h. Moreover, NFs + Sali was effective to induce intracellular reactive oxygen species (ROS) leading to cell apoptosis. Gene expression studies also revealed the capability of the NFs + Sali to upregulate tumor suppressor Rbl1 and Rbl2 as well as Caspase 3 while decreasing Wnt signaling pathway. In general, the results indicated anti-tumor activity of the Sali-loaded NFs suggesting their potential applications as implantable drug delivery systems in the brain upon surgical resection of the tumor.