Browsing by Author "Koch, Marcus"
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- ItemAgeing-associated small RNA cargo of extracellular vesicles(Philadelphia, Pa. : Taylor & Francis, 2023) Kern, Fabian; Kuhn, Thomas; Ludwig, Nicole; Simon, Martin; Gröger, Laura; Fabis, Natalie; Aparicio-Puerta, Ernesto; Salhab, Abdulrahman; Fehlmann, Tobias; Hahn, Oliver; Engel, Annika; Wagner, Viktoria; Koch, Marcus; Winek, Katarzyna; Soreq, Hermona; Nazarenko, Irina; Fuhrmann, Gregor; Wyss-Coray, Tony; Meese, Eckart; Keller, Verena; Laschke, Matthias W.; Keller, AndreasPrevious work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of RNAs. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory RNAs (sncRNAs) in two mouse plasma fractions at five time points across the lifespan from 2–18 months: (1) sncRNAs that are free-circulating (fc-RNA) and (2) sncRNAs bound outside or inside EVs (EV-RNA). Different sncRNA classes exhibit unique ageing patterns that vary between the fcRNA and EV-RNA fractions. While tRNAs showed the highest correlation with ageing in both fractions, rRNAs exhibited inverse correlation trajectories between the EV- and fc-fractions. For miRNAs, the EV-RNA fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncRNAs and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR.
- ItemBacteriomimetic Liposomes Improve Antibiotic Activity of a Novel Energy-Coupling Factor Transporter Inhibitor(Basel : MDPI, 2021) Drost, Menka; Diamanti, Eleonora; Fuhrmann, Kathrin; Goes, Adriely; Shams, Atanaz; Haupenthal, Jörg; Koch, Marcus; Hirsch, Anna K. H.; Fuhrmann, GregorLiposomes have been studied for decades as nanoparticulate drug delivery systems for cytostatics, and more recently, for antibiotics. Such nanoantibiotics show improved antibacterial efficacy compared to the free drug and can be effective despite bacterial recalcitrance. In this work, we present a loading method of bacteriomimetic liposomes for a novel, hydrophobic compound (HIPS5031) inhibiting energy-coupling factor transporters (ECF transporters), an underexplored antimicrobial target. The liposomes were composed of DOPG (18:1 (Δ9-cis) phosphatidylglycerol) and CL (cardiolipin), resembling the cell membrane of Gram-positive Staphylococcus aureus and Streptococcus pneumoniae, and enriched with cholesterol (Chol). The size and polydispersity of the DOPG/CL/± Chol liposomes remained stable over 8 weeks when stored at 4 °C. Loading of the ECF transporter inhibitor was achieved by thin film hydration and led to a high encapsulation efficiency of 33.19% ± 9.5% into the DOPG/CL/Chol liposomes compared to the phosphatidylcholine liposomes (DMPC/DPPC). Bacterial growth inhibition assays on the model organism Bacillus subtilis revealed liposomal HIPS5031 as superior to the free drug, showing a 3.5-fold reduction in CFU/mL at a concentration of 9.64 µM. Liposomal HIPS5031 was also shown to reduce B. subtilis biofilm. Our findings present an explorative basis for bacteriomimetic liposomes as a strategy against drug-resistant pathogens by surpassing the drug-formulation barriers of innovative, yet unfavorably hydrophobic, antibiotics.
- ItemCell-Derived Vesicles for Antibiotic Delivery—Understanding the Challenges of a Biogenic Carrier System(Weinheim : Wiley-VCH, 2023) Heinrich, Eilien; Hartwig, Olga; Walt, Christine; Kardani, Arefeh; Koch, Marcus; Jahromi, Leila Pourtalebi; Hoppstädter, Jessica; Kiemer, Alexandra K.; Loretz, Brigitta; Lehr, Claus‐Michael; Fuhrmann, GregorRecently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs’ technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro–in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics.
- ItemChemical and Structural Comparison of Different Commercial Food Supplements for Silicon Uptake(Basel : MDPI, 2023) Curto, Yannic; Koch, Marcus; Kickelbick, GuidoVarious food supplements for silicon uptake were compared in terms of their structures and chemical compositions. In particular, we analyzed the silanol group content, which can be an indicator of the uptake of the siliceous species in the human body. We analyzed the commercial products Original Silicea Balsam®, Flügge Siliceous Earth Powder, Pure Colloidal Silicon, and BioSil® by applying various methods such as FTIR, 29Si NMR, and TGA. The Si-OH group content of the samples containing pure silica was the highest for the Original Silicea Balsam followed by the Pure Colloidal Silicon. The siliceous earth powder revealed the lowest content of such groups and the densest structure. BioSil® contained a considerable concentration of organic molecules that stabilized orthosilicic acid. The study may help to understand the silicon uptake behavior of different food supplements depending on their chemical structure.
- ItemA correlative analysis of gold nanoparticles internalized by A549 cells(Hoboken, NJ : Wiley, 2014) Böse, Katharina; Koch, Marcus; Cavelius, Christian; Kiemer, Alexandra K.; Kraegeloh, AnnetteFluorescently labeled nanoparticles are widely used to investigate nanoparticle cell interactions by fluorescence microscopy. Owing to limited lateral and axial resolution, nanostructures (<100 nm) cannot be resolved by conventional light microscopy techniques. Especially after uptake into cells, a common fate of the fluorescence label and the particle core cannot be taken for granted. In this study, a correlative approach is presented to image fluorescently labeled gold nanoparticles inside whole cells by correlative light and electron microscopy (CLEM). This approach allows for detection of the fluorescently labeled particle shell as well as for the gold core in one sample. In this setup, A549 cells are exposed to 8 nm Atto 647N-labeled gold nanoparticles (3.3 × 109 particles mL−1, 0.02 μg Au mL−1) for 5 h and are subsequently imaged by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Eight fluorescence signals located at different intracellular positions are further analyzed by TEM. Five of the eight fluorescence spots are correlated with isolated or agglomerated gold nanoparticles. Three fluorescence signals could not be related to the presence of gold, indicating a loss of the particle shell.
- ItemCrystalline Carbosilane-Based Block Copolymers: Synthesis by Anionic Polymerization and Morphology Evaluation in the Bulk State(Weinheim : Wiley-VCH, 2022) Hübner, Hanna; Niebuur, Bart‐Jan; Janka, Oliver; Gemmer, Lea; Koch, Marcus; Kraus, Tobias; Kickelbick, Guido; Stühn, Bernd; Gallei, MarkusBlock copolymers (BCPs) in the bulk state are known to self-assemble into different morphologies depending on their polymer segment ratio. For polymers with amorphous and crystalline BCP segments, the crystallization process can be influenced significantly by the corresponding bulk morphology. Herein, the synthesis of the amorphous-crystalline BCP poly(dimethyl silacyclobutane)-block-poly(2vinyl pyridine), (PDMSB-b-P2VP), by living anionic polymerization is reported. Polymers with overall molar masses ranging from 17 400 g to 592 200 g mol−1 and PDMSB contents of 4.8–83.9 vol% are synthesized and characterized by size-exclusion chromatography and NMR spectroscopy. The bulk morphology of the obtained polymers is investigated by means of transmission electron microscopy and small angle X-ray scattering, revealing a plethora of self-assembled structures, providing confined and nonconfined conditions. Subsequently, the influence of the previously determined morphologies and their resulting confinement on the crystallinity and crystallization behavior of PDMSB is analyzed via differential scanning calorimetry and powder X-ray diffraction. Here, fractionated crystallization and supercooling effects are observable as well as different diffraction patterns of the PDMSB crystallites for confined and nonconfined domains.
- ItemDifferential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages(London : BioMed Central, 2010) Koch, Marcus; Hoppstädter, Jessica; Diesel, Britta; Zarbock, Robert; Breinig, Tanja; Monz, Dominik; Meyerhans, Andreas; Gortner, Ludwig; Lehr, Claus-Michael; Huwer, Hanno; Kiemer, Alexandra K.Background Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue. Methods Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay. Results IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6. Conclusion AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.
- ItemDimethylaminoethyl methacrylate copolymer-siRNA nanoparticles for silencing a therapeutically relevant gene in macrophages(Cambridge : Royal Society of Chemistry, 2015) Jain, Ratnesh; Dandekar, Prajakta; Loretz, Brigitta; Koch, Marcus; Lehr, Claus-MichaelTherapeutic gene silencing using small-interfering RNA (siRNA) for treatment of bacterial infections has been neglected in comparison with cancer and viral infections. The aim of our investigation was to formulate siRNA-loaded nanoparticles, using an established cationic polymethacrylate polymer, to enhance the delivery of siRNA into the cytoplasm of macrophages that host many pathogenic bacterial species, including tuberculosis. Nanoparticles of cationic dimethylaminoethyl methacrylate copolymer (Eudragit[registered sign] E 100) were successfully formulated and were found to efficiently bind the siRNA molecules (Cy3-siRNA, Bfl1/A1 siRNA). The efficiency of nanoparticles in overcoming cellular barriers to intracellular siRNA delivery and the precise pathway of endocytosis of nanoparticles were both confirmed using confocal microscopy. Through efficient siRNA release into the cytoplasm, the siRNA-loaded nanoparticles enabled a five-fold enhancement in the knockdown efficiency of the endogenous host gene Bfl1/A1, when the formulation was compared with free siRNA. Persistence of Bfl1/A1 is useful for phagolysosomal survival of tuberculosis bacteria in macrophages, and the nanoparticles offer a promising concept for exploitation as an anti-tuberculosis therapy.
- ItemDistribution of SiO2 nanoparticles in 3D liver microtissues(Macclesfield : Dove Medical Press, 2019) Fleddermann, Jana; Susewind, Julia; Peuschel, Henrike; Koch, Marcus; Tavernaro, Isabella; Kraegeloh, AnnetteIntroduction: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. Materials and methods: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. Results: Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size. Conclusion: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation.
- ItemEnhanced uptake and siRNA-mediated knockdown of a biologically relevant gene using cyclodextrin polyrotaxane(Cambridge : Royal Society of Chemistry, 2015) Dandekar, P.; Jain, R.; Keil, M.; Loretz, B.; Koch, Marcus; Wenz, G.; Lehr, Claus-MichaelIdeal cationic polymers for siRNA delivery could result in its enhanced cellular internalization, escape from endosomal degradation, and rapid release in cell cytoplasm, to facilitate knockdown of the target gene. In this study, we have investigated the ability of an in-house synthesized cationic polyrotaxane to bind siRNA into nanometric complexes. This polymer, which had earlier shown improved transfection of model siRNA (luciferase), was used to improve the cellular internalization of the siRNA molecule with therapeutic implications. In cellular assays, the polymer enhanced the knockdown of a gene involved in the pathogenesis of tuberculosis, when the nanocomplexes were compared with free siRNA. The efficacy and cellular non-toxicity of this polymer encourage its further exploitation in animal models of tuberculosis and other intracellular bacterial infections.
- ItemEnhancing the Stabilization Potential of Lyophilization for Extracellular Vesicles(Weinheim : Wiley-VCH, 2021) Trenkenschuh, Eduard; Richter, Maximilian; Heinrich, Eilien; Koch, Marcus; Fuhrmann, Gregor; Friess, WolfgangExtracellular vesicles (EV) are an emerging technology as immune therapeutics and drug delivery vehicles. However, EVs are usually stored at −80 °C which limits potential clinical applicability. Freeze-drying of EVs striving for long-term stable formulations is therefore studied. The most appropriate formulation parameters are identified in freeze-thawing studies with two different EV types. After a freeze-drying feasibility study, four lyophilized EV formulations are tested for storage stability for up to 6 months. Freeze-thawing studies revealed improved colloidal EV stability in presence of sucrose or potassium phosphate buffer instead of sodium phosphate buffer or phosphate-buffered saline. Less aggregation and/or vesicle fusion occurred at neutral pH compared to slightly acidic or alkaline pH. EVs colloidal stability can be most effectively preserved by addition of low amounts of poloxamer 188. Polyvinyl pyrrolidone failed to preserve EVs upon freeze-drying. Particle size and concentration of EVs are retained over 6 months at 40 °C in lyophilizates containing 10 mm K- or Na-phosphate buffer, 0.02% poloxamer 188, and 5% sucrose. The biological activity of associated beta-glucuronidase is maintained for 1 month, but decreased after 6 months. Here optimized parameters for lyophilization of EVs that contribute to generate long-term stable EV formulations are presented. © 2021 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH
- ItemFailure mechanism analysis based on laser-based surface treatments for aluminum-polyamide laser joining(Amsterdam [u.a.] : Elsevier, 2021) Elahi, Amne; Koch, Marcus; Bardon, Julien; Addiego, Frédéric; Plapper, PeterThe development of strong metal to polymer assemblies is currently an important research subject thanks to its prominence to develop lightweight structures. Furthermore, laser welding is known to be a fast, reliable, and versatile joining process, and it was demonstrated recently that it can be applied to such metal to polymer systems. To enhance the mechanical properties of the laser-joined aluminum-polyamide (Al-PA) specimens, laser polishing and laser ablation processes have been implemented on the aluminum surface before joining. The polyamide surface was also treated with the laser beam, separately. The surfaces were tested by several characterization techniques before and after each surface treatment. Then aluminum and polyamide samples with different surface treatments have been joined with an identical laser joining process. The mechanical properties of the joints in single lap shear configuration are reported and the failure mechanisms are discussed based on micro-computed x-ray tomography imaging of joined specimens and microscopic analysis before failure. Results show that both surface treatments of aluminum significantly improve the shear load of the joint; however, with different failure mechanisms. Polyamide surface treatment and increasing degree of crystallinity are effective when combined with the laser polishing of the Al surface. This combination is responsible for further enhancement of the shear load of the joint to the limit of base metal strength which is approximately 60 % improvement compared to the untreated samples. Finally, energy dispersive X-ray mapping shows the physicochemical bonding between aluminum oxide and polyamide at the interface.
- ItemFriction force microscopy of tribochemistry and interfacial ageing for the SiOx/Si/Au system(Frankfurt am Main : Beilstein-Institut, 2018) Petzhold, Chritinane; Koch, Marcus; Bennewitz, RolandFriction force microscopy was performed with oxidized or gold-coated silicon tips sliding on Au(111) or oxidized Si(100) surfaces in ultrahigh vacuum. We measured very low friction forces compared to adhesion forces and found a modulation of lateral forces reflecting the atomic structure of the surfaces. Holding the force-microscopy tip stationary for some time did not lead to an increase in static friction, i.e., no contact ageing was observed for these pairs of tip and surface. Passivating layers from tip or surface were removed in order to allow for contact ageing through the development of chemical bonds in the static contact. After removal of the passivating layers, tribochemical reactions resulted in strong friction forces and tip wear. Friction, wear, and the re-passivation by oxides are discussed based on results for the temporal development of friction forces, on images of the scanned area after friction force microscopy experiments, and on electron microscopy of the tips.
- ItemGivinostat-Liposomes: Anti-Tumor Effect on 2D and 3D Glioblastoma Models and Pharmacokinetics(Basel : MDPI, 2022) Taiarol, Lorenzo; Bigogno, Chiara; Sesana, Silvia; Kravicz, Marcelo; Viale, Francesca; Pozzi, Eleonora; Monza, Laura; Carozzi, Valentina Alda; Meregalli, Cristina; Valtorta, Silvia; Moresco, Rosa Maria; Koch, Marcus; Barbugian, Federica; Russo, Laura; Dondio, Giulio; Steinkühler, Christian; Re, FrancescaGlioblastoma is the most common and aggressive brain tumor, associated with poor prognosis and survival, representing a challenging medical issue for neurooncologists. Dysregulation of histone-modifying enzymes (HDACs) is commonly identified in many tumors and has been linked to cancer proliferation, changes in metabolism, and drug resistance. These findings led to the development of HDAC inhibitors, which are limited by their narrow therapeutic index. In this work, we provide the proof of concept for a delivery system that can improve the in vivo half-life and increase the brain delivery of Givinostat, a pan-HDAC inhibitor. Here, 150-nm-sized liposomes composed of cholesterol and sphingomyelin with or without surface decoration with mApoE peptide, inhibited human glioblastoma cell growth in 2D and 3D models by inducing a time-and dose-dependent reduction in cell viability, reduction in the receptors involved in cholesterol metabolism (from −25% to −75% of protein levels), and reduction in HDAC activity (−25% within 30 min). In addition, liposome-Givinostat formulations showed a 2.5-fold increase in the drug half-life in the bloodstream and a 6-fold increase in the amount of drug entering the brain in healthy mice, without any signs of overt toxicity. These features make liposomes loaded with Givinostat valuable as potential candidates for glioblastoma therapy.
- ItemImpact of mucus modulation by N-acetylcysteine on nanoparticle toxicity(Amsterdam : Elsevier, 2023) Meziu, Enkeleda; Shehu, Kristela; Koch, Marcus; Schneider, Marc; Kraegeloh, AnnetteHuman respiratory mucus is a biological hydrogel that forms a protective barrier for the underlying epithelium. Modulation of the mucus layer has been employed as a strategy to enhance transmucosal drug carrier transport. However, a drawback of this strategy is a potential reduction of the mucus barrier properties, in particular in situations with an increased exposure to particles. In this study, we investigated the impact of mucus modulation on its protective role. In vitro mucus was produced by Calu-3 cells, cultivated at the air-liquid interface for 21 days and used for further testing as formed on top of the cells. Analysis of confocal 3D imaging data revealed that after 21 days Calu-3 cells secrete a mucus layer with a thickness of 24 ± 6 μm. Mucus appeared to restrict penetration of 500 nm carboxyl-modified polystyrene particles to the upper 5–10 μm of the layer. Furthermore, a mucus modulation protocol using aerosolized N-acetylcysteine (NAC) was developed. This treatment enhanced the penetration of particles through the mucus down to deeper layers by means of the mucolytic action of NAC. These findings were supported by cytotoxicity data, indicating that intact mucus protects the underlying epithelium from particle-induced effects on membrane integrity. The impact of NAC treatment on the protective properties of mucus was probed by using 50 and 100 nm amine-modified and 50 nm carboxyl-modified polystyrene nanoparticles, respectively. Cytotoxicity was only induced by the amine-modified particles in combination with NAC treatment, implying a reduced protective function of modulated mucus. Overall, our data emphasize the importance of integrating an assessment of the protective function of mucus into the development of therapy approaches involving mucus modulation.
- ItemImproving the electrical and structural stability of highly piezoresistive nickel–carbon sensor thin films(Göttingen : Copernicus Publ., 2022) Schultes, Günter; Cerino, Mario; Lellig, Angela; Koch, MarcusThe family of sputter deposited granular metal-based carbon-containing sensor films is known for their high sensitivity transforming force-dependent strain into electrical resistance change. Among them nickel–carbon thin films possess a gauge factor of up to 30, compared to only 2 for traditional sensor films of metal alloys. This high sensitivity is based on disordered interparticle tunneling through barriers of graphite-like carbon walls between metal–carbon particles of columnar shape. Force and pressure sensors would benefit a lot from the elevated piezoresistivity. A disadvantage, however, is a disturbing temporal creep and drift of the resistance under load and temperature. This contribution shows how to stabilize such sensor films. A significant stabilization is achieved by partially replacing nickel with chromium, albeit at the expense of sensitivity. The more chromium used in these NixCr1−x-C layers, the higher the optimum annealing temperature can be selected and the better the electrical stabilization. A good compromise while maintaining sensitivities well above the standard of 2 is identified for films with x=0.5 to 0.9, stabilized by optimized temperature treatments. The stabilizing effect of chromium is revealed by transmission electron microscopy with elemental analysis. The post-annealing drives segregation processes in the layer material. While the interior of the layer is depleted of chromium and carbon, boundary layers are formed. Chromium is enriched near the surface boundary, oxidized in air and forms chromium-rich oxide sub-layers, which are chemically very stable and protect against further reactions and corrosion. As a result, creep and drift errors are greatly reduced, so that the optimized sensor coatings are now suitable for widespread use.
- ItemKinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles(London : Biomed Central, 2018) Yasar, Hanzey; Biehl, Alexander; De Rossi, Chiara; Koch, Marcus; Murgia, Xabi; Loretz, Brigitta; Lehr, Claus-MichaelBackground: Messenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. A variety of biodegradable nanoparticles (NPs) has been developed including lipid-based and polymer-based systems for mRNA delivery. However, both systems still lack in achieving an efficient transfection rate and a detailed understanding of the mRNA transgene expression kinetics. Therefore, quantitative analysis of the time-dependent translation behavior would provide a better understanding of mRNA's transient nature and further aid the enhancement of appropriate carriers with the perspective to generate future precision nanomedicines with quick response to treat various diseases. Results: A lipid-polymer hybrid system complexed with mRNA was evaluated regarding its efficiency to transfect dendritic cells (DCs) by simultaneous live cell video imaging of both particle uptake and reporter gene expression. We prepared and optimized NPs consisting of poly (lactid-co-glycolid) (PLGA) coated with the cationic lipid 1, 2-di-O-octadecenyl-3-trimethylammonium propane abbreviated as LPNs. An earlier developed polymer-based delivery system (chitosan-PLGA NPs) served for comparison. Both NPs types were complexed with mRNA-mCherry at various ratios. While cellular uptake and toxicity of either NPs was comparable, LPNs showed a significantly higher transfection efficiency of ~ 80% while chitosan-PLGA NPs revealed only ~ 5%. Further kinetic analysis elicited a start of protein translation after 1 h, with a maximum after 4 h and drop of transgene expression after 48 h post-transfection, in agreement with the transient nature of mRNA. Conclusions: Charge-mediated complexation of mRNA to NPs enables efficient and fast cellular delivery and subsequent protein translation. While cellular uptake of both NP types was comparable, mRNA transgene expression was superior to polymer-based NPs when delivered by lipid-polymer NPs.
- ItemLight emission intensities of luminescent Y2O3:Eu and Gd2O3:Eu particles of various sizes(Basel : MDPI, 2017) Adam, Jens; Metzger, Wilhelm; Koch, Marcus; Rogin, Peter; Coenen, Toon; Atchison, Jennifer S.; König, PeterThere is great technological interest in elucidating the effect of particle size on the luminescence efficiency of doped rare earth oxides. This study demonstrates unambiguously that there is a size effect and that it is not dependent on the calcination temperature. The Y2O3:Eu and Gd2O3:Eu particles used in this study were synthesized using wet chemistry to produce particles ranging in size between 7 nm and 326 nm and a commercially available phosphor. These particles were characterized using three excitation methods: UV light at 250 nm wavelength, electron beam at 10 kV, and X-rays generated at 100 kV. Regardless of the excitation source, it was found that with increasing particle diameter there is an increase in emitted light. Furthermore, dense particles emit more light than porous particles. These results can be explained by considering the larger surface area to volume ratio of the smallest particles and increased internal surface area of the pores found in the large particles. For the small particles, the additional surface area hosts adsorbates that lead to non-radiative recombination, and in the porous particles, the pore walls can quench fluorescence. This trend is valid across calcination temperatures and is evident when comparing particles from the same calcination temperature.
- ItemLipid droplets as a novel cargo of tunnelling nanotubes in endothelial cells(London : Nature Publishing Group, 2015) Astanina, Ksenia; Koch, Marcus; Jüngst, Christian; Zumbusch, Andreas; Kiemer, Alexandra K.Intercellular communication is a fundamental process in the development and functioning of multicellular organisms. Recently, an essentially new type of intercellular communication, based on thin membrane channels between cells, has been reported. These structures, termed intercellular or tunnelling nanotubes (TNTs), permit the direct exchange of various components or signals (e.g., ions, proteins, or organelles) between non-adjacent cells at distances over 100 μm. Our studies revealed the presence of tunnelling nanotubes in microvascular endothelial cells (HMEC-1). The TNTs were studied with live cell imaging, environmental scanning electron microscopy (ESEM), and coherent anti-Stokes Raman scattering spectroscopy (CARS). Tunneling nanotubes showed marked persistence: the TNTs could connect cells over long distances (up to 150 μm) for several hours. Several cellular organelles were present in TNTs, such as lysosomes and mitochondria. Moreover, we could identify lipid droplets as a novel type of cargo in the TNTs. Under angiogenic conditions (VEGF treatment) the number of lipid droplets increased significantly. Arachidonic acid application not only increased the number of lipid droplets but also tripled the extent of TNT formation. Taken together, our results provide the first demonstration of lipid droplets as a cargo of TNTs and thereby open a new field in intercellular communication research.
- ItemLipid–Polymer Hybrid Nanoparticles for mRNA Delivery to Dendritic Cells: Impact of Lipid Composition on Performance in Different Media(Basel : MDPI, 2022) Kliesch, Lena; Delandre, Simon; Gabelmann, Aljoscha; Koch, Marcus; Schulze, Kai; Guzmán, Carlos A.; Loretz, Brigitta; Lehr, Claus-MichaelTo combine the excellent transfection properties of lipids with the high stability of polymeric nanoparticles, we designed a hybrid system with a polymeric core surrounded by a shell of different lipids. The aim is to use this technology for skin vaccination purposes where the transfection of dendritic cells is crucial. Based on a carrier made of PLGA and the positively charged lipid DOTMA, we prepared a panel of nanocarriers with increasing amounts of the zwitterionic phospholipid DOPE in the lipid layer to improve their cell tolerability. We selected a nomenclature accordingly with numbers in brackets to represent the used mol% of DOPE and DOTMA in the lipid layer, respectively. We loaded mRNA onto the surface and assessed the mRNA binding efficacy and the degree of protection against RNases. We investigated the influence of the lipid composition on the toxicity, uptake and transfection in the dendritic cell line DC 2.4 challenging the formulations with different medium supplements like fetal calf serum (FCS) and salts. After selecting the most promising candidate, we performed an immune stimulation assay with primary mouse derived dendritic cells. The experiments showed that all tested lipid–polymer nanoparticles (LPNs) have comparable hydrodynamic parameters with sizes between 200 and 250 nm and are able to bind mRNA electrostatically due to their positive zetapotential (20–40 mV for most formulations). The more of DOPE we add, the more free mRNA we find and the better the cellular uptake reaching approx. 100% for LPN(60/40)–LPN(90/10). This applies for all tested formulations leading to LPN(70/30) with the best performance, in terms of 67% of live cells with protein expression. In that case, the supplements of the medium did not influence the transfection efficacy (56% vs. 67% (suppl. medium) for live cells and 63% vs. 71% in total population). We finally confirmed this finding using mouse derived primary immune cells. We can conclude that a certain amount of DOTMA in the lipid coating of the polymer core is essential for complexation of the mRNA, but the zwitterionic phospholipid DOPE is also important for the particles’ performance in supplemented media.
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