34 research outputs found
Effective zero-thickness model for a conductive membrane driven by an electric field
The behavior of a conductive membrane in a static (DC) electric field is
investigated theoretically. An effective zero-thickness model is constructed
based on a Robin-type boundary condition for the electric potential at the
membrane, originally developed for electrochemical systems. Within such a
framework, corrections to the elastic moduli of the membrane are obtained,
which arise from charge accumulation in the Debye layers due to capacitive
effects and electric currents through the membrane and can lead to an
undulation instability of the membrane. The fluid flow surrounding the membrane
is also calculated, which clarifies issues regarding these flows sharing many
similarities with flows produced by induced charge electro-osmosis (ICEO).
Non-equilibrium steady states of the membrane and of the fluid can be
effectively described by this method. It is both simpler, due to the zero
thickness approximation which is widely used in the literature on fluid
membranes, and more general than previous approaches. The predictions of this
model are compared to recent experiments on supported membranes in an electric
field.Comment: 14 pages, 5 figure
Dendritic Core-Multishell Nanocarriers in Murine Models of Healthy and Atopic Skin
Dendritic hPG-amid-C18-mPEG core-multishell nanocarriers (CMS) represent a
novel class of unimolecular micelles that hold great potential as drug
transporters, e.g., to facilitate topical therapy in skin diseases. Atopic
dermatitis is among the most common inflammatory skin disorders with complex
barrier alterations which may affect the efficacy of topical treatment. Here,
we tested the penetration behavior and identified target structures of
unloaded CMS after topical administration in healthy mice and in mice with
oxazolone-induced atopic dermatitis. We further examined whole body
distribution and possible systemic side effects after simulating high dosage
dermal penetration by subcutaneous injection. Following topical
administration, CMS accumulated in the stratum corneum without penetration
into deeper viable epidermal layers. The same was observed in atopic
dermatitis mice, indicating that barrier alterations in atopic dermatitis had
no influence on the penetration of CMS. Following subcutaneous injection, CMS
were deposited in the regional lymph nodes as well as in liver, spleen, lung,
and kidney. However, in vitro toxicity tests, clinical data, and morphometry-
assisted histopathological analyses yielded no evidence of any toxic or
otherwise adverse local or systemic effects of CMS, nor did they affect the
severity or course of atopic dermatitis. Taken together, CMS accumulate in the
stratum corneum in both healthy and inflammatory skin and appear to be highly
biocompatible in the mouse even under conditions of atopic dermatitis and thus
could potentially serve to create a depot for anti-inflammatory drugs in the
skin
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DYAMOND: the DYnamics of the Atmospheric general circulation Modeled On Non-hydrostatic Domains
A review of the experimental protocol and motivation for DYAMOND, the first intercomparison project of global storm-resolving models, is presented. Nine models submitted simulation output for a 40-day (1 Augustâ10 September 2016) intercomparison period. Eight of these employed a tiling of the sphere that was uniformly less than 5 km. By resolving the transient dynamics of convective storms in the tropics, global storm-resolving models remove the need to parameterize tropical deep convection, providing a fundamentally more sound representation of the climate system and a more natural link to commensurately high-resolution data from satellite-borne sensors. The models and some basic characteristics of their output are described in more detail, as is the availability and planned use of this output for future scientific study. Tropically and zonally averaged energy budgets, precipitable water distributions, and precipitation from the model ensemble are evaluated, as is their representation of tropical cyclones and the predictability of column water vapor, the latter being important for tropical weather
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Tropical cyclones in global storm-resolving models
Recent progress in computing and model development has initiated the era of global storm-resolving modeling and with it the potential to transform weather and climate prediction. Within the general theme of vetting this new class of models, the present study evaluates nine global-storm resolving models in their ability to simulate tropical cyclones (TCs). Results show that, broadly speaking, the models produce realistic TCs and remove longstanding issues known from global models such as the deficiency to accurately simulate TC intensity. However, TCs are strongly affected by model formulation, and all models suffer from unique biases regarding the number of TCs, intensity, size, and structure. Some models simulated TCs better than others, but no single model was superior in every way. The overall results indicate that global storm-resolving models are able to open a new chapter in TC prediction, but they need to be improved to unleash their full potential
Anwendung und ZellulÀre Interaktionen von Kationischen Nanopartikeln
1 Introduction 5 1.1 Nanoparticles 5 1.2 Advantages and Applications of
Cationic Nanoparticles 8 1.2.1 Enhanced Cellular Uptake and Endosomal Escape 8
1.2.2 Complexation of Nucleic Acids 11 1.3 Adverse Effects of Nanoparticles 13
1.3.1 Systemic Effects 13 1.3.2 Cellular and Subcellular Mechanisms 15 1.4
Strategies to overcome adverse effects 22 1.4.1 Nanoparticle Materials 22
1.4.2 Biodegradability 24 2 Scientific Goal 26 3 Publications and Manuscripts
28 3.1 Systematic Adjustment of Charge Densities and Size of Polyglycerol
Amines Reduces Cytotoxic Effects and Enhances Cellular Uptake 28 3.2
Crosslinked Redox-Responsive Micelles Based on Lipoic Acid-Derived Amphiphiles
for Enhanced siRNA Delivery 42 3.3 Synthesis of pH-Cleavable dPG-Amines for
Gene Delivery Application 71 3.4 Defined pH-Sensitive Nanogels as Gene
Delivery Platform for siRNA Mediated in vitro Gene Silencing 106 3.5 FLIM-ROX
as a Highly Sensitive Fluorescence Lifetime Based Approach for Reliable
Reactive Oxygen Species Detection in vitro and in vivo 122 4 Conclusions and
Outlook 163 5 Kurzzusammenfassung 165 6 References 168 7 Appendices 179 7.1
List of other Publications 179 7.2 Abbreviations 180The goals of this work were the development of new cationic carrier systems
that are able to transport sensitive nucleic acid cargo into cells while at
the same time de-creasing their adverse effects. This was realized with the
use of PG as the polymeric backbone of the carrier systems and a cleavable
linker that enables the particles bio-degradability in the cytosol and also a
release of the cargo material. The first project was a systematic approach to
find a best candidate in the frame of hyperbranched polyglycerol nanocarriers
by tweaking both the size and the hydroxyl-to-amino ratio on the particle
surface. The particles were tested for their performance to transfect plasmid
DNA in vitro. Among several candidates, PG with a size of 14 kDa and 90 % of
amine surface groups and PG 200 kDa with 30 % amine groups were the most
promising candidates. These findings are in accord with those of Zeng et al.,
where 14 kDa particles worked best with 50 % amines and 55 kDa particles
worked best with 35 % amines for siRNA delivery. The best candidate with
similar transfection efficacies as PEI, 200 kDa HPG and 30 % amines, were
further studied to elaborate their exact mechanism of toxicity. Compared to 25
kDa branched PEI (bPEI), the cell viability was less affected. Additional
tests revealed no evidence for apoptosis but a slight membrane disruption
which was more prominent in PEI than in the HPG. In the second project,
micelles based on lipoic acid and PG were combined with a redox sensitive
disulfide crosslinker for the delivery of siRNA. All biocompatible build-ing
blocks were selected so that there will be no toxicity upon particle
degradation and four different structure were generated. Acceptable cell
viabilities (> 70 %) were ob-served for all but one construct up to an N/P
ratio of 120. One of the three candidates showed comparable transfection
efficacy to the commercial control LipofectamineÂź but was still lower. Two
other approaches featured the efficacy of different PG poly-mer structures for
transfection: hyperbranched PG with an amine shell and a PG nano-gel with
small 600 Da PEI units. Both these systems incorporated a pH sensitive cleav-
able linker. The HPG carriers contained a cleavable linker (50 % cleaved after
12 hours) and a fast-cleavable variety of that (50 % cleaved after 4 hours).
The cell viabil-ity of the non-degradable carrier and the two degradable ones
was comparable and the cleavable one also showed similar transfection
efficacy. However, the fast-cleavable carriers showed a decreased performance,
indicating that the degradation process needs to be delayed for efficient
transfection. The other benzacetal-linker sys-tem was a crosslinked nanogel
comprising linear PG and small linear PEI molecules. This system was able to
physically encapsulate siRNA during the synthesis process and showed a
transfection efficacy similar to that of 25 kDa bPEI. Also, the cell viability
was higher, especially at higher concentrations and a subsequent test using
red blood cells showed high biocompatibility with biological membranes,
whereas PEI showed a visi-ble disruptive effect. The nanogel approach also
eliminated the need for complexation of nucleic acids and the carrier system
prior to application, making them more feasi-ble in practical use. This
feature and the increased biocompatibility makes these sys-tem promising
candidates for further in vivo tests. Another project focused on the sensitive
detection of reactive oxygen species upon nanoparticle exposure. For this,
fluorescence lifetime imaging microscopy (FLIM) was utilized because it
increased the sensitivity of the commercial CellROX GreenÂź dye. Calibration
curves proved the high functionality of the method in fixed and live cells.
Further studies on ex vivo skin tissue demonstrated the methods applicability
for more complex systems. Also, to consolidate the picture that the ROS
generation by model particles made of bioinert gold with an amine shell, other
biological studies were per-formed. These studies revealed induction of
cellular senescence and genotoxic effect at subtoxic concentrations as
determined by cell viability studies. The results confirm the overall picture
of cationic nanoparticle induced adverse effects on cells and legiti-mate this
FLIM method for further routine application for toxicological tests. For the
future, these polycationic nanoparticle delivery system will be further ex-
plored for their in vitro and in vivo application of nucleic acid delivery, or
gene deliv-ery, and the delivery of other drugs. The challenge of
outperforming viral carrier sys-tems or decreasing the adverse effects
completely have not yet been entirely accom-plished. Further work will be done
on the nanogels due to their flexible size range and the ability to
encapsulate a great variety of different cargoes. Also, the interactions of
nanoparticles and cells regarding adverse effects needs to be further
elucidated. As a sensitive method for the detection of ROS was established in
the last project using FLIM, further effort can be taken to eliminate subtoxic
oxidative stress and possibly outperform current drug delivery systems.Das Ziel dieser Arbeit bestand aus der Entwicklung kationischer Nanopartikel-
TrĂ€gersysteme, die in der Lage sind, sensible Gast-MolekĂŒle wie NukleinsĂ€uren
in Zel-len zu transportieren und gleichzeitig ihre adversen Effekte zu
reduzieren. Realisiert wurde das durch die Verwendung von Polyglycerin als
Polymergrundlage des TrÀger-systems und spaltbarer, Stimuli-responsiver
Querverbindungen. Diese ermöglichten die Bioabbaubarkeit der Partikel im
Zytosol und den Lysosomen und die Freisetzung der GastmolekĂŒle. Das erste
Projekt bestand aus einer systematischen Herangehensweise, um das beste
Konstrukt eines hochverzweigten, Polyglycerin-basierten NanotrÀgers zu finden.
Hierzu wurden sowohl die GröĂe als auch das VerhĂ€ltnis von Hydroxyl- zu
Aminogrup-pen auf der PartikeloberflĂ€che moduliert. AnschlieĂen wurden die
Partikel auf ihr FĂ€-higkeit getestet, in vitro Plasmid DNA in Zellen zu
transfizieren. Unter mehreren Kan-didaten stellten sich PG mit einer GröĂe von
14 kDa mit 90 % Aminogruppen und 200 kDa mit 30 % Aminogruppen als die besten
heraus. Damit konnten die Ergebnisse von Zeng et al bestÀtigt werden, wonach
14 kDa Partikel mit 50 % Aminogruppen und 55 kDa partikel mit 35 %
Aminogruppen am besten fĂŒr die Transfektion von siRNA geeig-net waren.[152]
Der beste Kandidat mit einer vergleichbaren Transfektionseffizienz wie PEI,
200 kDa HPG mit 30 % Aminogruppen, wurde des Weiteren auf die Mechanismen der
ToxizitĂ€t geprĂŒft. Im Vergleich zu 25 kDa bPEI (bPEI) war die ZellviabilitĂ€t
allge-mein weniger beeintrÀchtigt. ZusÀtzliche Tests zeigten keine Anzeichen
von Apoptose-Induktion, aber eine leichte Störung der MembranintegritÀt, wobei
der Effekt durch PEI deutlicher ausgeprÀgt war. Im zweiten Projekt wurden
LiponsĂ€ure und PG mit einer redox-sensitiven VerknĂŒp-fung kombiniert, um siRNA
zu transfizieren. Alle Grundbausteine wurden so gewÀhlt, dass bei einer
Spaltung keine toxischen Abbauprodukte freigesetzt werden und vier
verschiedene Konstrukte untersucht. FĂŒr drei von vier Konstrukte wurde eine
akzeptab-le ZellviabilitÀt festgestellt, bis zu einem N/P-VerhÀltnis von 120.
Einer der drei Kandi-daten zeigte eine Transfektioneffizienz, die mit der
kommerziell erhÀltlichen Kontrolle Lipofectamine Ÿ vergleichbar war, wenn auch
etwas niedriger. Zwei andere Herange-hensweisen befassten sich mit der
Effizienz anderer PG Polymerkonstrukte fĂŒr Trans-fektionen: hochverzweigtes PG
mit einer Amin-Schale und ein PG-Nanogel mit kleinen 600 Da PEI Einheiten.
Beide Systeme beinhalten eine pH-sensitiven Querverbindung. Die HPG TrÀger
wurden mit einer spaltbaren Queverbindung und einer schnell-spaltbaren
Variante davon ausgestattet, wobei 50 % der TrÀger nach 12 Stunden bzw. 4
Stunden gespalten wurden. Die ZellviabilitÀt des TrÀgers ohne spaltbare
Querverbin-dung und dieser beiden Konstrukte waren sehr Àhnlich, aber nur das
spaltbare zeigte eine vergleichbare Transfektionseffizienz zu dem nicht-
spaltbaren. Die schnell-spaltbare Variante allerdings zeigte eine schlechtere
Performance, was darauf hindeu-tet, dass der Spaltungsvorgang fĂŒr eine
effiziente Transfektion langsamer von statten gehen muss. Das andere pH-
sensitive, Benzazetal-verknĂŒpfte System bestand aus Na-nogelen bestehend aus
PG und kleinen, linearen PEI Einheiten. In diesem System konn-te siRNA wÀhrend
der Synthese physikalisch eingekapselt werden. Die Transfektioneffi-zienz
dieses Konstrukts war vergleichbar, wenn auch niedriger, mit der von 25 kDa
bPEI. Andererseits war die ZellviabilitÀt höher, vor allem bei hohen
Konzentrationen. Ein zusÀtzlicher Test an roten Blutzellen demonstrierte eine
hohe BiokompatibilitĂ€t gegenĂŒber biologischen Membranen, wĂ€hrend PEI einen
deutlichen schÀdlichen Effekt zeigte. Der Nanogel-Ansatz als TrÀger-System
eliminiert auĂerdem die Notwendigkeit fĂŒr eine Komplexierung von NukleinsĂ€uren
vor der Anwendung, da diese bereits ver-kapselt sind. Das macht sie in der
Anwendung vergleichsweise praktikabel. Insgesamt machen die Vorteile der
erhöhten BiokompatibilitÀt und der Einfachheit der Anwen-dung dieses System zu
einem vielversprechenden Kandidaten fĂŒr spĂ€tere in vivo Versu-che. Ein anderes
Projekt war auf die sensitive Detektion von reaktiven Sauerstoffspe-
ziesfokussiert, welche bei Kontakt mit Nanopartikeln freigesetzt werden
können. DafĂŒr wurde der Farbstoff CellROX Green Âź in Kombination mit
Fluorescence Lifetime Ima-ging Microscopy (FLIM) verwendet, um dessen
SensitivitÀt zu erhöhen. Durch Kalibra-tionskurven konnte die FunktionalitÀt
der Methode sowohl in fixierten als auch in le-benden Zellen gezeigt werden.
Des Weiteren konnte die Anwendbarkeit der Methode fĂŒr komplexere biologische
Systeme anhand von ex vivo Versuchen demonstriert wer-den. Um das Gesamtbild
um den gemessen oxidativen Stress zu vervollstÀndigen, wur-den Modell-Partikel
aus Gold mit einer Amin-Schale verwendet und weitere biologi-sche
Untersuchungen durchgefĂŒhrt. Dazu wurden ZellviabilitĂ€tsstudien durchgefĂŒhrt
und subtoxische Konzentrationen verwendet. Diese demonstrierten neben den der
Ge-nerierung von ROS auch die Induktion von zellulÀrer Seneszenz und einen
genotoxi-schen Effekt. Die Ergebnisse bestÀtigten das allgemeine Gesamtbild
der adversen Ef-fekte von kationischen Nanopartikeln auf Zellen und
legitimierten damit diese neue FLIM-Methode und deren möglichen Einsatz in
Routine-Kontrollen im Zusammenhang mit medizinischen Anwendungen. FĂŒr die
Zukunft werden diese vielversprechenden kationischen Nanopartikel-
TrĂ€gersysteme weiter erforscht, sowohl fĂŒr die Anwendung mit NukleinsĂ€uren als
auch anderen Arzneistoffen und das sowohl in vitro als auch in vivo. Die
Herausforderung, virale Vektorsysteme zu ĂŒbertreffen oder die adversen Effekte
komplett zu eliminie-ren, ist noch nicht gemeistert. Weitere Arbeit sollte in
die Nanogel-Systeme gesteckt werden, da diese durch ihre flexible GröĂe und
der Möglichkeit zur Einkapselung ver-schiedenster Arzneimittel ein besonders
groĂen Potential haben. ZusĂ€tzlich sollten die Interaktionen zwischen
kationischen Nanopartikeln und Zellen sowie die adversen Ef-fekte weiter
aufgeklĂ€rt werden. Da mit dieser Arbeit eine sensitive Methode fĂŒr die Messung
von ROS mittels FLIM-Technologie etabliert wurde, sollte diese genutzt wer-
den, um die adversen Effekte bei toxischen und subtoxischen Konzentrationen
weiter zu eliminieren. Auf lange Sicht könnte das eine aussagekrÀftige Methode
sein, aktuelle TrÀgersysteme weiter zu untersuchen und zu verbessern
Tissue specificity of the initiation of immunoglobulin k gene transcription
Falkner FG, Neumann E, Zachau HG. Tissue specificity of the initiation of immunoglobulin k gene transcription. Hoppe-Seyler's Zeitschrift fĂŒr physiologische Chemie. 1984;365:1331-1343.The transient transcription of a rearranged mouse immunoglobulin k gene was studied in a monkey fibroblast cell line. The gene was inserted into an SV40 expression vector and the calcium phosphate coprecipitation method was used for transfection. The transcripts were correctly spliced; transcription, however, was initiated within the vector and not at the correct site 23-26 bp upstream of the gene, irrespective of the length of the upstream sequences (90, 160, 370, and 870 bp) in the plasmid constructs. In contrast, accurately initiated transcripts were observed when a plasmid containing the k gene with 870 bp of its upstream sequence was introduced into a lymphoid cell line; the plasmid was constructed from the pSV2-gpt vector and the electric impulse method was used fro gene transfer in most experiments. Tissue-specific expression of k light chain genes in lymphoid cells is known to depend on the presence of an enhancer element in the J-C intron. The results reported in this paper suggest that the sequence elements pd and dc which are located upstream of the leader gene segment also act in a tissue-specific manner ant that it is the initiation of transcription which is a tissue-specific event
Potential Analysis Of Flexible Small Series Production Of Spare Parts By Direct Polymer Additive Tooling
In recent decades, there has been an increasing creation of vehicle model variants and product
individualization for customer needs. There is a need for a greater variety of spare parts which is leading to
high requirements on their logistics. As a result of this trend, automotive production companies have to
ensure the provision of many different spare parts as part of their post-fulfillment obligations. However, the
surplus spare parts stock will increase the operation cost of the company. This implies the need for a more
flexible approach to spare parts production and provision to improve profitability compared to existing
process chains. In this research paper, the potential of flexible spare parts production and provision is
discussed. For this purpose, the state of spare part production technologies and existing approaches for spare
part service are analyzed regarding their technological characteristics, flexibility as well as cost structure.
Following this, an approach for flexible production for spare parts with regard to its potential is analyzed
and demonstrated based on three exemplary automotive use cases using additively manufactured production
resources. In the casting use case, a Fused Filament Fabricated (FFF) additively manufactured speedometer
screw model will be used as a sand casting form. The production of deep-drawn car body parts using
polymer-based FFF forming tools is investigated in the second use case. Lastly, the production of ignition
distributor caps by PolyJet Modelling (PJM) manufactured Injection Molding (PUR-RIM) molds is
presented