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- ItemWetting-Induced Polyelectrolyte Pore Bridging(Basel : MDPI, 2021) Kalde, Anna; Kamp, Johannes; Evdochenko, Elizaveta; Linkhorst, John; Wessling, MatthiasActive layers of ion separation membranes often consist of charged layers that retain ions based on electrostatic repulsion. Conventional fabrication of these layers, such as polyelectrolyte deposition, can in some cases lead to excess coating to prevent defects in the active layer. This excess deposition increases the overall membrane transport resistance. The study at hand presents a manufacturing procedure for controlled polyelectrolyte complexation in and on porous supports by support wetting control. Pre-wetting of the microfiltration membrane support, or even supports with larger pore sizes, leads to ternary phase boundaries of the support, the coating solution, and the pre-wetting agent. At these phase boundaries, polyelectrolytes can be complexated to form partially freestanding selective structures bridging the pores. This polyelectrolyte complex formation control allows the production of membranes with evenly distributed polyelectrolyte layers, providing (1) fewer coating steps needed for defect-free active layers, (2) larger support diameters that can be bridged, and (3) a precise position control of the formed polyelectrolyte multilayers. We further analyze the formed structures regarding their position, composition, and diffusion dialysis performance.
- ItemAll-Conjugated Polymer Core-Shell and Core-Shell-Shell Particles with Tunable Emission Profiles and White Light Emission(Weinheim : Wiley-VCH, 2021) Haehnle, Bastian; Schuster, Philipp A.; Chen, Lisa; Kuehne, Alexander J. C.Future applications of conjugated polymer particles (CPP) in medicine, organic photonics, and optoelectronics greatly depend on high performance and precisely adjustable optical properties of the particles. To meet these criteria, current particle systems often combine conjugated polymers with inorganic particles in core-shell geometries, extending the possible optical characteristics of CPP. However, current conjugated polymer particles are restricted to a single polymer phase composed of a distinct polymer or a polymer blend. Here, a synthetic toolbox is presented that enables the synthesis of monodisperse core-shell and core-shell-shell particles, which consist entirely of conjugated polymers but of different types in the core and the shells. Seeded and fed-batch dispersion polymerizations based on Suzuki-Miyaura-type cross-coupling are investigated. The different approaches allow accurate control over the created interface between the conjugated polymer phases and thus also over the energy transfer phenomena between them. This approach opens up completely new synthetic freedom for fine tuning of the optical properties of CPP, enabling, for example, the synthesis of individual white light-emitting particles.
- ItemOptimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations(London : BioMed Central, 2020) Keil, Timm; Dittrich, Barbara; Lattermann, Clemens; Büchs, JochenBackground: Small-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved. Results: Customization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24-0.90 mg/h per well. Conclusions: The optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates. © 2020 The Author(s).
- ItemRecycling and Separation of Homogeneous Catalyst from Aqueous Multicomponent Mixture by Organic Solvent Nanofiltration(Basel : MDPI, 2021) Schnoor, J.-Kilian; Bettmer, Jens; Kamp, Johannes; Wessling, Matthias; Liauw, Marcel A.Organic solvent nanofiltration (OSN) has evolved to an established recycling method for homogeneous catalysts. However, commercial availability has not circumvented the need for classification and the scoping of possible applications for specific solvent mixtures. Therefore, Evonik’s DuraMem® 300 was assessed for the recycling of magnesium triflate at two transmembrane pressures from a mixture of ethanol, ethyl acetate and water. Catalyst retention up to 98% and permeability of up to 4.44·10−1∙L∙bar−1∙m−2∙h−1 were possible when less than 25% ethyl acetate was in the mixture. The retention of some of the components in the ternary mixture was observed while others were enriched, making the membrane also suitable for fractioning thereof.
- ItemFrom In Vitro to Perioperative Vascular Tissue Engineering: Shortening Production Time by Traceable Textile-Reinforcement(Heidelberg : Springer, 2022) Mohapatra, Saurav Ranjan; Rama, Elena; Melcher, Christoph; Call, Tobias; Al Enezy-Ulbrich, Miriam Aischa; Pich, Andrij; Apel, Christian; Kiessling, Fabian; Jockenhoevel, StefanBackground: The production of tissue-engineered vascular graft (TEVG) usually involves a prolonged bioreactor cultivation period of up to several weeks to achieve maturation of extracellular matrix and sufficient mechanical strength. Therefore, we aimed to substantially shorten this conditioning time by combining a TEVG textile scaffold with a recently developed copolymer reinforced fibrin gel as a cell carrier. We further implemented our grafts with magnetic resonance imaging (MRI) contrast agents to allow the in-vitro monitoring of the TEVG’s remodeling process. Methods: Biodegradable polylactic-co-glycolic acid (PLGA) was electrospun onto a non-degradable polyvinylidene fluoride scaffold and molded along with copolymer-reinforced fibrin hydrogel and human arterial cells. Mechanical tests on the TEVGs were performed both instantly after molding and 4 days of bioreactor conditioning. The non-invasive in vitro monitoring of the PLGA degradation and the novel imaging of fluorinated thermoplastic polyurethane (19F-TPU) were performed using 7T MRI. Results: After 4 days of close loop bioreactor conditioning, 617 ± 85 mmHg of burst pressure was achieved, and advanced maturation of extracellular matrix (ECM) was observed by immunohistology, especially in regards to collagen and smooth muscle actin. The suture retention strength (2.24 ± 0.3 N) and axial tensile strength (2.45 ± 0.58 MPa) of the TEVGs achieved higher values than the native arteries used as control. The contrast agents labeling of the TEVGs allowed the monitorability of the PLGA degradation and enabled the visibility of the non-degradable textile component. Conclusion: Here, we present a concept for a novel textile-reinforced TEVG, which is successfully produced in 4 days of bioreactor conditioning, characterized by increased ECM maturation and sufficient mechanical strength. Additionally, the combination of our approach with non-invasive imaging provides further insights into TEVG’s clinical application.
- ItemEngineering robust cellulases for tailored lignocellulosic degradation cocktails(Basel : MDPI AG, 2020) Contreras, Francisca; Pramanik, Subrata; Rozhkova, Aleksandra M.; Zorov, Ivan N.; Korotkova, Olga; Sinitsyn, Arkady P.; Schwaneberg, Ulrich; Davari, Mehdi D.Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemMolecular Brightness Approach for FRET Analysis of Donor-Linker-Acceptor Constructs at the Single Molecule Level: A Concept(Lausanne : Frontiers, 2021) Kay, Taryn M.; Aplin, Cody P.; Simonet, Rowan; Beenken, Julie; Miller, Robert C.; Libal, Christin; Boersma, Arnold J.; Sheets, Erin D.; Heikal, Ahmed A.In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1-linker-mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery.
- ItemUnraveling the Mechanism and Kinetics of Binding of an LCI-eGFP-Polymer for Antifouling Coatings(Weinheim : Wiley-VCH, 2021) Söder, Dominik; Garay-Sarmiento, Manuela; Rahimi, Khosrow; Obstals, Fabian; Dedisch, Sarah; Haraszti, Tamás; Davari, Mehdi D.; Jakob, Felix; Heß, Christoph; Schwaneberg, Ulrich; Rodriguez-Emmenegger, CesarThe ability of proteins to adsorb irreversibly onto surfaces opens new possibilities to functionalize biological interfaces. Herein, the mechanism and kinetics of adsorption of protein-polymer macromolecules with the ability to equip surfaces with antifouling properties are investigated. These macromolecules consist of the liquid chromatography peak I peptide from which antifouling polymer brushes are grafted using single electron transfer-living radical polymerization. Surface plasmon resonance spectroscopy reveals an adsorption mechanism that follows a Langmuir-type of binding with a strong binding affinity to gold. X-ray reflectivity supports this by proving that the binding occurs exclusively by the peptide. However, the lateral organization at the surface is directed by the cylindrical eGFP. The antifouling functionality of the unimolecular coatings is confirmed by contact with blood plasma. All coatings reduce the fouling from blood plasma by 8894% with only minor effect of the degree of polymerization for the studied range (DP between 101 and 932). The excellent antifouling properties, combined with the ease of polymerization and the straightforward coating procedure make this a very promising antifouling concept for a multiplicity of applications.
- ItemNanovesicles displaying functional linear and branched oligomannose self-assembled from sequence-defined Janus glycodendrimers(Washington, DC : NAS, 2020) Xiao, Qi; Delbianco, Martina; Sherman, Samuel E.; Reveron Perez, Aracelee M.; Bharate, Priya; Pardo-Vargas, Alonso; Rodriguez-Emmenegger, Cesar; Kostina, Nina Yu; Rahimi, Khosrow; Söder, Dominik; Möller, Martin; Klein, Michael L.; Seeberger, Peter H.; Percec, VirgilCell surfaces are often decorated with glycoconjugates that contain linear and more complex symmetrically and asymmetrically branched carbohydrates essential for cellular recognition and communication processes. Mannose is one of the fundamental building blocks of glycans in many biological membranes. Moreover, oligomannoses are commonly found on the surface of pathogens such as bacteria and viruses as both glycolipids and glycoproteins. However, their mechanism of action is not well understood, even though this is of great potential interest for translational medicine. Sequence-defined amphiphilic Janus glycodendrimers containing simple mono- and disaccharides that mimic glycolipids are known to self-assemble into glycodendrimersomes, which in turn resemble the surface of a cell by encoding carbohydrate activity via supramolecular multivalency. The synthetic challenge of preparing Janus glycodendrimers containing more complex linear and branched glycans has so far prevented access to more realistic cell mimics. However, the present work reports the use of an isothiocyanate-amine “click”-like reaction between isothiocyanate-containing sequence-defined amphiphilic Janus dendrimers and either linear or branched oligosaccharides containing up to six monosaccharide units attached to a hydrophobic amino-pentyl linker, a construct not expected to assemble into glycodendrimersomes. Unexpectedly, these oligoMan-containing dendrimers, which have their hydrophobic linker connected via a thiourea group to the amphiphilic part of Janus glycodendrimers, self-organize into nanoscale glycodendrimersomes. Specifically, the mannose-binding lectins that best agglutinate glycodendrimersomes are those displaying hexamannose. Lamellar “raft-like” nanomorphologies on the surface of glycodendrimersomes, self-organized from these sequence-defined glycans, endow these membrane mimics with high biological activity. © 2020 National Academy of Sciences. All rights reserved.
- ItemMechanobiology of Epithelia From the Perspective of Extracellular Matrix Heterogeneity(Lausanne : Frontiers Media, 2020) Kozyrina, Aleksandra N.; Piskova, Teodora; Di Russo, JacopoUnderstanding the complexity of the extracellular matrix (ECM) and its variability is a necessary step on the way to engineering functional (bio)materials that serve their respective purposes while relying on cell adhesion. Upon adhesion, cells receive messages which contain both biochemical and mechanical information. The main focus of mechanobiology lies in investigating the role of this mechanical coordination in regulating cellular behavior. In recent years, this focus has been additionally shifted toward cell collectives and the understanding of their behavior as a whole mechanical continuum. Collective cell phenomena very much apply to epithelia which are either simple cell-sheets or more complex three-dimensional structures. Researchers have been mostly using the organization of monolayers to observe their collective behavior in well-defined experimental setups in vitro. Nevertheless, recent studies have also reported the impact of ECM remodeling on epithelial morphogenesis in vivo. These new concepts, combined with the knowledge of ECM biochemical complexity are of key importance for engineering new interactive materials to support both epithelial remodeling and homeostasis. In this review, we summarize the structure and heterogeneity of the ECM before discussing its impact on the epithelial mechanobiology. © Copyright © 2020 Kozyrina, Piskova and Di Russo.
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