04 Fakultät Energie-, Verfahrens- und Biotechnik
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5
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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 Physical interactions strengthen chemical gelatin methacryloyl gels(2019) Rebers, Lisa; Granse, Tobias; Tovar, Günter E. M.; Southan, Alexander; Borchers, KirstenChemically cross-linkable gelatin methacryloyl (GM) derivatives are getting increasing attention regarding biomedical applications. Thus, thorough investigations are needed to achieve full understanding and control of the physico-chemical behavior of these promising biomaterials. We previously introduced gelatin methacryloyl acetyl (GMA) derivatives, which can be used to control physical network formation (solution viscosity, sol-gel transition) independently from chemical cross-linking by variation of the methacryloyl-to-acetyl ratio. It is known that temperature dependent physical network formation significantly influences the mechanical properties of chemically cross-linked GM hydrogels. We investigated the temperature sensitivity of GM derivatives with different degrees of modification (GM2, GM10), or similar degrees of modification but different methacryloyl contents (GM10, GM2A8). Rheological analysis showed that the low modified GM2 forms strong physical gels upon cooling while GM10 and GM2A8 form soft or no gels. Yet, compression testing revealed that all photo cross-linked GM(A) hydrogels were stronger if cooling was applied during hydrogel preparation. We suggest that the hydrophobic methacryloyl and acetyl residues disturb triple helix formation with increasing degree of modification, but additionally form hydrophobic structures, which facilitate chemical cross-linking.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 Acid catalyzed cross‐linking of polyvinyl alcohol for humidifier membranes(2021) Michele, Andre; Paschkowski, Patrick; Hänel, Christopher; Tovar, Günter E. M.; Schiestel, Thomas; Southan, AlexanderPolyvinyl alcohol (PVA) is a hydrophilic polymer well known for good film forming properties, high water vapor permeance JW, and low nitrogen permeance. However, depending on molar mass and temperature, PVA swells strongly in water until complete dissolution. This behavior affects the usability of PVA in aqueous environments and makes cross‐linking necessary if higher structural integrity is envisaged. In this work, PVA networks are formed by thermal cross‐linking in the presence of p‐toluenesulfonic acid (TSA) and investigated in a design of experiments approach. Experimental parameters are the cross‐linking period tc, temperature ϑ and the TSA mass fraction wTSA. Cross‐linking is found to proceed via ether bond formation at all reaction conditions. Degradation is promoted especially by a combination of high wTSA, tc and ϑ. Thermal stability of the networks after preparation is strongly improved by neutralizing residual TSA. Humidification membranes with a JW of 6423 ± 63.0 gas permeation units (GPU) are fabricated by coating PVA on polyvinyliden fluoride hollow fibers and cross‐linking with TSA. Summarizing, the present study contributes to a clearer insight into the cross‐linking of PVA in presence of TSA, the thermal stability of the resulting networks and the applicability as selective membrane layers for water vapor transfer.Item Open Access Integrated process design and optimisation for the production of cellobiose lipids from Ustilago sp.(2024) Oraby, Amira; Rupp, Steffen (Prof. Dr.)Item Open Access Unravelling parameter interactions in calcium alginate/polyacrylamide double network hydrogels using a design of experiments approach for the optimization of mechanical properties(2024) Gorke, Oliver; Stuhlmüller, Marc; Tovar, Günter E. M.; Southan, AlexanderCalcium alginate/polyacrylamide double network hydrogels were reported to be exceptionally tough. However, literature reports so far varied the sample compositions mainly by one parameter at a time approaches, thus only drawing an incomplete picture of achievable material properties. In this contribution, sample compositions are varied according to a face-centered central composite experimental design taking into account the four parameters of alginate concentration cAlg, high/low molar mass alginate mixing ratio RP, acrylamide concentration cAAm, and N,N′-methylenebisacrylamide concentration cMBA. Each sample composition is investigated in triplicate. Thus, 75 samples were investigated by tensile testing, and a detailed analysis of the significant parameters and parameter interactions influencing the mechanical properties is conducted. The data shows that two parameter interactions, involving all four tested parameters, have a large effect on the Young's modulus, the strength, the toughness and the strain at material failure. As a consequence, it becomes evident that the experimental procedure from previous studies did not always result in optimum sample compositions. The results allow optimization of the mechanical properties within the studied parameter space, and a new maximum value of the strength of 710 kPa is reported. The data also give rise to the assumption that other parameters and parameter interactions ignored also in this study may allow further tailoring of mechanical properties.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 Factors affecting the synthesis of cellobiose lipids by Sporisorium scitamineum(2020) Oraby, Amira; Werner, Nicole; Sungur, Zehra; Zibek, SusanneCellobiose lipids (CL) are extracellular glycolipids that are produced by many microorganisms from the family Ustilaginaceae. The sugarcane smut fungus Sporisorium scitamineum has been long known as a producer of the glycolipids mannosylerythritol lipids (MEL) and was recently described to additionally secrete CL as a byproduct. In fact, we identified 11 homologous genes in S. scitamineum by in silico analysis sharing a high similarity to the CL biosynthesis gene cluster of Ustilago maydis. We here report the first systematic cultivation of S. scitamineum targeting the synthesis of CL with high product titers and its transfer to the bioreactor. In an initial screening we examined different fermentation media compositions, consisting of a mineral salts solution with vitamins and/or trace elements, three carbon sources (glucose, fructose, sucrose), three pH values (2.5, 4.0, 6.7) and three levels of C/N values (42.2, 83.8, 167.2 molC molN -1) with urea as nitrogen source. A pH of 2.5 proved to result in the highest product titers. An increase of urea concentration from 0.6 to 1.2 g L-1 had a positive effect on biomass formation, however the glycolipid formation was favored at a C/N ratio of 83.8 molC molN -1, using 0.6 g L-1 urea. Amongst the examined carbon sources, sucrose resulted in an increase in the secretion of cellobiose lipids, compared to glucose. Comparing different media compositions, vitamins were identified as not necessary for CL synthesis. We obtained a concentration of cellobiose lipids of 8.3 1.0 g L-1 in shaking flasks. This increased to 17.6 g L-1 in the 1 L bioreactor with additional feeding of carbon source, with a final purity of 85-93%. As a side product, erythritol and mannosylerythritol lipids (MEL) were also synthesized. Via HPTLC coupled MALDI-TOF MS we were able to analyze the secreted CL structures. S. scitamineum produces a mixture of acylated low molecular weight D-glucolipids, linked to a 2,15,16-trihydroxy-hexadecanoic acid via their &-hydroxyl group (CL-B). The produced cellobiose lipids precipitate as needle like crystals at an acidic pH value of 2.5.Item Open Access Influence of light conditions on the production of chrysolaminarin using Phaeodactylum tricornutum in artificially illuminated photobioreactors(2023) Frick, Konstantin; Ebbing, Tobias; Yeh, Yen‐Cheng; Schmid‐Staiger, Ulrike; Tovar, Günter E. M.The light conditions are of utmost importance in any microalgae production process especially involving artificial illumination. This also applies to a chrysolaminarin (soluble 1,3-β-glucan) production process using the diatom Phaeodactylum tricornutum. Here we examine the influence of the amount of light per gram biomass (specific light availability) and the influence of two different biomass densities (at the same amount of light per gram biomass) on the accumulation of the storage product chrysolaminarin during nitrogen depletion in artificially illuminated flat-panel airlift photobioreactors. Besides chrysolaminarin, other compounds (fucoxanthin, fatty acids used for energy storage [C16 fatty acids], and eicosapentaenoic acid) are regarded as well. Our results show that the time course of C-allocation between chrysolaminarin and fatty acids, serving as storage compounds, is influenced by specific light availability and cell concentration. Furthermore, our findings demonstrate that with increasing specific light availability, the maximal chrysolaminarin content increases. However, this effect is limited. Beyond a certain specific light availability (here: 5 µmolphotons gDW-1 s-1) the maximal chrysolaminarin content no longer increases, but the rate of increase becomes faster. Furthermore, the conversion of light to chrysolaminarin is best at the beginning of nitrogen depletion. Additionally, our results show that a high biomass concentration has a negative effect on the maximal chrysolaminarin content, most likely due to the occurring self-shading effects.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.