Directed Evolution of Material Binding Peptide for Polylactic Acid-specific Degradation in Mixed Plastic Wastes.

In order to preserve our livelihood for future generations, responsible use of plastics in a climate-neutral and circular economy has to be developed so that plastics can be used in an environmentally friendly way by future generations. The prerequisite is that bioplastic polymers such as polylactic acid (PLA) can be efficiently recycled from petrochemical based plastic. Here, a concept in which accelerated PLA degradation in the mixed suspension of PLA and polystyrene (PS) nanoparticles has been achieved through an engineered material binding peptide. After comparison of twenty material binding peptides, Cg-Def is selected due to its PLA binding specificity. Finally, a suitable high-throughput screening system is developed for enhancing material-specific binding toward PLA in presence of PS. Through KnowVolution campaign, a variant Cg-Def YH (L9Y/S19H) with 2.0-fold improved PLA binding specificity compared to PS is generated. Contact angle and surface plasmon resonance measurements validated higher surface coverage of Cg-Def YH on PLA surface and the fusion of Cg-Def YH with PLA degrading enzyme confirmed the accelerated PLA depolymerization (two times higher than only enzyme) in mixed PLA/PS plastics.

A Competitive High-Throughput Screening Platform for Designing Polylactic Acid-Specific Binding Peptides.

Among biobased polymers, polylactic acid (PLA) is recognized as one of the most promising bioplastics to replace petrochemical-based polymers. PLA is typically blended with other polymers such as polypropylene (PP) for improved melt processability, thermal stability, and stiffness. A technical challenge in recycling of PLA/PP blends is the sorting/separation of PLA from PP. Material binding peptides (MBPs) can bind to various materials. Engineered MBPs that can bind in a material-specific manner have a high potential for material-specific detection or enhanced degradation of PLA in mixed PLA/PP plastics. To obtain a material-specific MBP for PLA binding (termed PLAbodies ), protein engineering of MBP Cg-Def for improved PLA binding specificity is reported in this work. In detail, a 96-well microtiter plate based high-throughput screening system for PLA specific binding (PLABS) was developed and validated in a protein engineering (KnowVolution) campaign. Finally, the Cg-Def variant V2 (Cg-Def S19K/K10L/N13H) with a 2.3-fold improved PLA binding specificity compared to PP was obtained. Contact angle and surface plasmon resonance measurements confirmed improved material-specific binding of V2 to PLA (1.30-fold improved PLA surface coverage). The established PLABS screening platform represents a general methodology for designing PLAbodies for applications in detection, sorting, and material-specific degradation of PLA in mixed plastics.

Dual Labeling of Primary Cells with Fluorescent Gadolinium Oxide Nanoparticles.

The interest in mesenchymal stromal cells as a therapy option is increasing rapidly. To improve their implementation, location, and distribution, the properties of these must be investigated. Therefore, cells can be labeled with nanoparticles as a dual contrast agent for fluorescence and magnetic resonance imaging (MRI). In this study, a more efficient protocol for an easy synthesis of rose bengal-dextran-coated gadolinium oxide (Gd2O3-dex-RB) nanoparticles within only 4 h was established. Nanoparticles were characterized by zeta potential measurements, photometric measurements, fluorescence and transmission electron microscopy, and MRI. In vitro cell experiments with SK-MEL-28 and primary adipose-derived mesenchymal stromal cells (ASC), nanoparticle internalization, fluorescence and MRI properties, and cell proliferation were performed. The synthesis of Gd2O3-dex-RB nanoparticles was successful, and they were proven to show adequate signaling in fluorescence microscopy and MRI. Nanoparticles were internalized into SK-MEL-28 and ASC via endocytosis. Labeled cells showed sufficient fluorescence and MRI signal. Labeling concentrations of up to 4 mM and 8 mM for ASC and SK-MEL-28, respectively, did not interfere with cell viability and proliferation. Gd2O3-dex-RB nanoparticles are a feasible contrast agent to track cells via fluorescence microscopy and MRI. Fluorescence microscopy is a suitable method to track cells in in vitro experiments with smaller samples.

BioAdhere: tailor-made bioadhesives for epiretinal visual prostheses.

Introduction: Visual prostheses, i.e. epiretinal stimulating arrays, are a promising therapy in treating retinal dystrophies and degenerations. In the wake of a new generation of devices, an innovative method for epiretinal fixation of stimulator arrays is required. We present the development of tailor-made bioadhesive peptides (peptesives) for fixating epiretinal stimulating arrays omitting the use of traumatic retinal tacks. Materials and methods: Binding motifs on the stimulating array (poly[chloro-p-xylylene] (Parylene C)) and in the extracellular matrix of the retinal surface (collagens I and IV, laminin, fibronectin) were identified. The anchor peptides cecropin A (CecA), KH1, KH2 (author's initials) and osteopontin (OPN) were genetically fused to reporter proteins to assess their binding behavior to coated microtiter plates via fluorescence-based assays. Domain Z (DZ) of staphylococcal protein A was used as a separator to generate a bioadhesive peptide. Following ISO 10993 "biological evaluation of medical materials", direct and non-direct cytotoxicity testing (L-929 and R28 retinal progenitor cells) was performed. Lastly, the fixating capabilities of the peptesives were tested in proof-of-principle experiments. Results: The generation of the bioadhesive peptide required evaluation of the N- and C-anchoring of investigated APs. The YmPh-CecA construct showed the highest activity on Parylene C in comparison with the wildtype phytase without the anchor peptide. eGFP-OPN was binding to all four investigated ECM proteins (collagen I, laminin > collagen IV, fibronectin). The strongest binding to collagen I was observed for eGFP-KH1, while the strongest binding to fibronectin was observed for eGFP-KH2. The selectivity of binding was checked by incubating eGFP-CecA and eGFP-OPN on ECM proteins and on Parylene C, respectively. Direct and non-direct cytotoxicity testing of the peptide cecropin-A-DZ-OPN using L-929 and R28 cells showed good biocompatibility properties. Proof-of-concept experiments in post-mortem rabbit eyes suggested an increased adhesion of CecA-DZ-OPN-coated stimulating arrays. Conclusion: This is the first study to prove the applicability and biocompatibility of peptesives for the fixation of macroscopic objects.