Universität Stuttgart
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Item Open Access Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics(2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, MichaelCellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.Item Open Access Nano-in-micro-particles consisting of PLGA nanoparticles embedded in chitosan microparticles via spray-drying enhances their uptake in the olfactory mucosa(2021) Spindler, Lena Marie; Feuerhake, Andreas; Ladel, Simone; Günday, Cemre; Flamm, Johannes; Günday-Türeli, Nazende; Türeli, Emre; Tovar, Günter E. M.; Schindowski, Katharina; Gruber-Traub, CarmenIntranasal delivery has gained prominence since 1990, when the olfactory mucosa was recognized as the window to the brain and the central nervous system (CNS); this has enabled the direct site specific targeting of neurological diseases for the first time. Intranasal delivery is a promising route because general limitations, such as the blood-brain barrier (BBB) are circumvented. In the treatment of multiple sclerosis (MS) or Alzheimer’s disease, for example, future treatment prospects include specialized particles as delivery vehicles. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are well known as promising delivery systems, especially in the area of nose-to-brain (N2B) delivery. Chitosan is also broadly known as a functional additive due to its ability to open tight junctions. In this study, we produced PLGA nanoparticles of different sizes and revealed for the first time their size-time-dependent uptake mechanism into the lamina propria of porcine olfactory mucosa. The intracellular uptake was observed for 80 and 175 nm within only 5 min after application to the epithelium. After 15 min, even 520 nm particles were detected, associated with nuclei. Especially the presence of only 520 nm particles in neuronal fibers is remarkable, implying transcellular and intracellular transport via the olfactory or the trigeminal nerve to the brain and the CNS. Additionally, we developed successfully specialized Nano-in-Micro particles (NiMPs) for the first time via spray drying, consisting of PLGA nanoparticles embedded into chitosan microparticles, characterized by high encapsulation efficiencies up to 51%, reproducible and uniform size distribution, as well as smooth surface. Application of NiMPs accelerated the uptake compared to purely applied PLGA nanoparticles. NiMPs were spread over the whole transverse section of the olfactory mucosa within 15 min. Faster uptake is attributed to additional paracellular transport, which was examined via tight-junction-opening. Furthermore, a separate chitosan penetration gradient of ∼150 µm caused by dissociation from PLGA nanoparticles was observed within 15 min in the lamina propria, which was demonstrated to be proportional to an immunoreactivity gradient of CD14. Due to the beneficial properties of the utilized chitosan-derivative, regarding molecular weight (150-300 kDa), degree of deacetylation (80%), and particle size (0.1-10 µm) we concluded that M2-macrophages herein initiated an anti-inflammatory reaction, which seems to already take place within 15 min following chitosan particle application. In conclusion, we demonstrated the possibility for PLGA nanoparticles, as well as for chitosan NiMPs, to take all three prominent intranasal delivery pathways to the brain and the CNS; namely transcellular, intracellular via neuronal cells, and paracellular transport.Item Open Access Patterns of autologous and nonautologous interactions between core nuclear egress complex (NEC) proteins of α-, β- and γ-herpesviruses(2020) Häge, Sigrun; Sonntag, Eric; Borst, Eva Maria; Tannig, Pierre; Seyler, Lisa; Bäuerle, Tobias; Bailer, Susanne M.; Lee, Chung-Pei; Müller, Regina; Wangen, Christina; Milbradt, Jens; Marschall, ManfredNuclear egress is a regulated process shared by α-, β- and γ-herpesviruses. The core nuclear egress complex (NEC) is composed of the membrane-anchored protein homologs of human cytomegalovirus (HCMV) pUL50, murine cytomegalovirus (MCMV) pM50, Epstein-Barr virus (EBV) BFRF1 or varicella zoster virus (VZV) Orf24, which interact with the autologous NEC partners pUL53, pM53, BFLF2 or Orf27, respectively. Their recruitment of additional proteins leads to the assembly of a multicomponent NEC, coordinately regulating viral nucleocytoplasmic capsid egress. Here, the functionality of VZV, HCMV, MCMV and EBV core NECs was investigated by coimmunoprecipitation and confocal imaging analyses. Furthermore, a recombinant MCMV, harboring a replacement of ORF M50 by UL50, was analyzed both in vitro and in vivo. In essence, core NEC interactions were strictly limited to autologous NEC pairs and only included one measurable nonautologous interaction between the homologs of HCMV and MCMV. A comparative analysis of MCMV-WT versus MCMV-UL50-infected murine fibroblasts revealed almost identical phenotypes on the levels of protein and genomic replication kinetics. In infected BALB/c mice, virus spread to lung and other organs was found comparable between these viruses, thus stating functional complementarity. In conclusion, our study underlines that herpesviral core NEC proteins are functionally conserved regarding complementarity of core NEC interactions, which were found either virus-specific or restricted within subfamilies.Item Open Access The microalgae phaeodactylum tricornutum Is well suited as a food with positive effects on the intestinal microbiota and the generation of SCFA : results from a pre-clinical study(2022) Stiefvatter, Lena; Neumann, Ulrike; Rings, Andreas; Frick, Konstantin; Schmid-Staiger, Ulrike; Bischoff, Stephan C.Microalgae such as Phaeodactylum tricornutum (PT) are a sustainable source of nutrients, especially eicosapentaenoic acid (EPA), fucoxanthin (Fx), and chrysolaminarin (Chrl), the concentrations of which can vary depending on the culture conditions. We generated three types of diets containing either an EPA- and Fx-rich (EPA/Fx) or Chrl-rich microalgae (with 5, 15, or 25% added to the diet) or an isocaloric control diet (CD). These diets were evaluated over 14 days in young C57BL/6J mice for safety and bioavailability, short-chain fatty acid (SCFA) production, and microbiome analysis. Both microalgae diets increased body weight gain dose-dependently compared to the CD. Microalgae-derived EPA was well absorbed, resulting in increased liver and fat tissue levels and a decrease in the n-6:n-3 ratio in liver tissue. Both microalgae diets increased the production of selected SCFA and decreased the Firmicutes/Bacteriodota ratio, whereas the Chrl-rich diet led to an increase in Akkermansia. Doses of up to 4621 mg Chrl, 920 mg EPA, and 231 mg Fx per kg body weight daily were tolerated without adverse effects. This pre-clinical study shows that PT is suitable for mouse feed, with positive effects on microbiota composition and SCFA production, suggesting beneficial effects on gut health.Item Open Access Coumarin-4-ylmethyl- and p-hydroxyphenacyl-based photoacid generators with high solubility in aqueous media: synthesis, stability and photolysis(2020) Adatia, Karishma K.; Halbritter, Thomas; Reinfelds, Matiss; Michele, Andre; Tran, Michael; Laschat, Sabine; Heckel, Alexander; Tovar, Günter E. M.; Southan, Alexander(Coumarin‐4‐yl)methyl (c4m) and p‐hydroxyphenacyl (pHP)‐based compounds are well known for their highly efficient photoreactions, but often show limited solubility in aqueous media. To circumvent this, we synthesized and characterized the two new c4m and pHP‐based photoacid generators (PAGs), 7‐[bis(carboxymethyl)amino]‐4‐(acetoxymethyl)coumarin (c4m‐ac) and p‐hydroxyphenacyl‐2,5,8,11‐tetraoxatridecan‐13‐oate (pHP‐t), and determined their solubilities, stabilities and photolysis in aqueous media. The two compounds showed high solubilities in water of 2.77 mmol L−1±0.07 mmol L−1 (c4m‐ac) and 124.66 mmol L−1±2.1 mmol L−1 (pHP‐t). In basic conditions at pH 9, solubility increased for c4m‐ac to 646.46 mmol L−1±0.63 mmol L−1, for pHP‐t it decreased to 34.68 mmol L−1±0.62 mmol L−1. Photochemical properties of the two PAGs, such as the absorption maxima, the maximum molar absorption coefficients and the quantum yields, were found to be strongly pH‐dependent. Both PAGs showed high stabilities s24h ≥95 % in water for 24 h, but decreasing stability with increasing pH value due to hydrolysis. The present study contributes to a clearer insight into the synthesis, solubilities, stabilities, and photolysis of c4m and pHP‐based PAGs for further photochemical applications when high PAG concentrations are required, such as in polymeric foaming.Item Open Access Monodopsis subterranea is a source of α‐tocomonoenol, and its concentration, in contrast to α‐tocopherol, is not affected by nitrogen depletion(2023) Montoya‐Arroyo, Alexander; Muñoz‐González, Alejandra; Lehnert, Katja; Frick, Konstantin; Schmid‐Staiger, Ulrike; Vetter, Walter; Frank, Janα‐Tomonoenols (αT1) are tocochromanols structurally related to tocopherols (T) and tocotrienols (T3), the bioactive members of the vitamin E family. However, limited evidence exists regarding the sources and biosynthesis of tocomonoenols. Nitrogen depletion increases the content of α‐tocopherol (αT), the main vitamin E congener, in microalgae, but little is known regarding its effect on other tocochromanols, such as tocomonoenols and tocotrienols. We therefore quantified the concentrations of T, T1, and T3, in freeze‐dried biomass from nitrogen‐sufficient, and nitrogen‐depleted Monodopsis subterranea (Eustigmatophyceae). The identities of isomers of αT1 were confirmed by LC-MS and GC-MS. αT was the predominant tocochromanol (82% of total tocochromanols). αT1 was present in higher quantities than the sum of all T3 (6% vs. 1% of total tocochromanols). 11′‐αT1 was the main αT1 isomer. Nitrogen depletion increased αT, but not αT1 or T3 in M. subterranea. In conclusion, nitrogen depletion increased the content of αT, the biologically most active form of vitamin E, in M. subterranea without affecting αT1 and T3 and could potentially be used as a strategy to enhance its nutritional value but not to increase αT1 content, indicating that αT1 accumulation is independent of that of αT in microalgae.Item Open Access Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods(2020) Keller, Silke; Liedek, Anke; Shendi, Dalia; Bach, Monika; Tovar, Günter E. M.; Kluger, Petra J.; Southan, AlexanderAzide-bearing cell-derived extracellular matrices (“clickECMs”) have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.Item Open Access Proton-conducting membranes for the artificial leaf(2023) Bosson, Karell; Tovar, Günter E. M. (Prof.)With the aim of producing proton conducting membranes with improved proton conductivity and mechanical properties, the poly(pentafluorostyrene)-b-(butyl acrylate) (PPFS-b-PBuA) system was investigated. The study mainly focuses on the influence of the forming polymer nanostructures on the conductivity properties of the membranes. A series of well-defined PPFS-b-PBuA block copolymers (BCPs) were synthesized via nitroxide-mediated controlled radical polymerization (NMP). Spontaneous self-assembly of the BCP element was induced via a targeted change in polymer composition. Moreover, by adjusting the molar composition via enrichment of one of the blocks after synthesis, controlled self-assembly of the BCPs was realized. This was done by combining the corresponding homopolymer with the block copolymer to form a polymer blend - one of the blocks mixed to the BCP. Forming such polymer blends expanded the range of available techniques for tailoring the morphology for desired applications. Sulfonation of BCPs for the preparation of proton-conducting membranes was carried out by a para-fluoro thiol "click" reaction using sodium 3-mercapto-1-propanesulfonate (SMPS). The accessibility of fluorine in the para position of the phenylene group of PPFS provides countless opportunities for polymer functionalization by nucleophilic substitution. After modification of BCP, the self-assembly ability was retained, and higher conductivities were obtained compared to random copolymers. In addition, complementary studies were conducted on the use of printing techniques for membrane upscaling and evaluation of their life cycle.Item Open Access Oral bioavailability of omega-3 fatty acids and carotenoids from the microalgae Phaeodactylum tricornutum in healthy young adults(2021) Stiefvatter, Lena; Lehnert, Katja; Frick, Konstantin; Montoya-Arroyo, Alexander; Frank, Jan; Vetter, Walter; Schmid-Staiger, Ulrike; Bischoff, Stephan C.The microalgae Phaeodactylum tricornutum (PT) contains valuable nutrients such as proteins, polyunsaturated omega-3 fatty acids (n-3 PUFA), particularly eicosapentaenoic acid (EPA) and some docosahexaenoic acid (DHA), carotenoids such as fucoxanthin (FX), and beta-glucans, which may confer health benefits. In a randomized intervention trial involving 22 healthy individuals, we administered for two weeks in a crossover manner the whole biomass of PT (5.3 g/day), or fish oil (FO) containing equal amounts of EPA and DHA (together 300 mg/day). In an additional experiment, sea fish at 185 g/week resulting in a similar EPA and DHA intake was administered in nine individuals. We determined the bioavailability of fatty acids and carotenoids and assessed safety parameters. The intake of PT resulted in a similar increase in the n-3 PUFA and EPA content and a decrease in the PUFA n-6:n-3 ratio in plasma. PT intake caused an uptake of FX that is metabolized to fucoxanthinol (FXOH) and amarouciaxanthin A (AxA). No relevant adverse effects occurred following PT consumption. The study shows that PT is a safe and effective source of EPA and FX—and likely other nutrients—and therefore should be considered as a future sustainable food item.Item Open Access Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels(2021) Rebers, Lisa; Reichsöllner, Raffael; Regett, Sophia; Tovar, Günter E. M.; Borchers, Kirsten; Baudis, Stefan; Southan, AlexanderGelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.