Core-Shell Nanoparticles as a Platform Technology for the Treatment of Proliferative Ocular Diseases

Core-shell Nanoparticles offer a highly flexible platform technology for the design of biocompatible, nanoparticulate therapeutics. These core-shell Nanoparticles were decorated with losartan carboxylic acid (EXP3174), that is an antagonist for angiotensin II type 1 receptors (AT1R). It was the superior goal to develop a therapeutical system to inhibit ocular neovascularization during progressive age-related macular degeneration (AMD) or diabetic retinopathy (DR). Viruses use spike proteins on their surface to target host cells. This architecture was used as a blueprint for the nanoparticle design. EXP3174 molecules were linked to longer polymer chains to raise the ligands out of the nanoparticle shell. Polyethylene glycol (PEG)- polylactide (PLA) block-co-polymers were synthesized with tailored properties. Various functional end groups offered the opportunities to introduce charges and different reactivities. EXP3174 was covalently linked to amino-PEG-PLA block-co-polymers. The synthetized PEG-PLA block-co-polymers as well as a purchased poly(lactic-co-glycolic acid) (PLGA) were utilized to manufacture core-shell nanoparticles via nanoprecipitation. PLGA was linked to fluorescent dyes or ultra-small gold particles, so that the manufactured nanoparticles were traceable via various analytical methods (e.g. flow cytometry, confocal laser scanning microscopy and inductively coupled plasma mass spectrometry). Moreover, the core-shell nanoparticles were successfully tagged with radioactive indium ions. This means that core-shell nanoparticles are traceable via a multitude of analysis techniques and can be applied therapeutically as an untagged preparation. The designed nanoparticles were extensively characterized. It is worth to mention the established formula for the calculation of the particle number concentration. The formula was based on the total polymer content and the hydrodynamic diameter. This calculation strategy was an essential milestone, since it facilitated the comparison of results obtained from differently sized nanoparticles. Furthermore, it was the basis for numerous binding studies at an AT1R-carrying cell line. Different methods evidenced a sub nanomolar avidity. Consequently, EXP3174-decorated core-shell nanoparticles outperformed their naïve ligand EXP3174 itself. The explanation could be found in the multivalent binding behavior. It was determined that only a single-digit number of ligands bound to the target cells. This is in accordance with the number of spikes per virus. Eventually the EXP3174-decorated core-shell nanoparticles were tested at healthy laboratory animals. With the help of computed tomography assisted single-photon emission computed tomography (SPECT-CT) the biodistribution of radioactively tagged core-shell nanoparticles was followed in real time. The results unveiled a renal elimination pathway and a very short half-life. An accumulation in the ocular vasculature could not be substantiated. Hence further development is needed for this field of application. Beyond that it was focused on the long-term stability of core-shell nanoparticles. Since they were composed of biodegradable polymers, core-shell nanoparticles degrade in an aqueous dispersion. Consequently, the dispersions must be freshly prepared prior every experiment. This aspect indicated the need for the development of an anhydrous formulation. It was possible to remove the dispersing agent water quantitatively via freeze drying into a stable lyophilizate. It is worth to mention that the core-shell nanoparticles retained their full avidity after redispersion. It was necessary to just add sucrose as the only excipient (cryo- and lyoprotector). Finally, the block-co-polymers allowed to tailor the properties of the needed core-shell nanoparticles precisely. It is possible to decorate the nanoparticles with different ligand densities or to create hetero multivalent nanoparticles as well. Especially the different labelling techniques must be highlighted. They gave access to a multitude of analysis techniques without changing the nanoparticle properties substantially. All in all, a highly promising nanoparticulate platform technology with the potential to become a therapeutical application was developed

Atomic scale light-matter interaction: From ultrafast physics to photoexcitation phenomena

In this thesis various methods to study light-matter interaction at the atomic scale are explored. By employing scanning tunneling microscopy (STM) and atomic force microscopy (AFM) the interaction of single molecules with electromagnetic radiation can be investigated with sub-molecular spatial resolution. The first part of this study analyzes the interaction of a single magnesium phthalocyanine (MgPc) molecule with the electric field of an ultrafast terahertz laser pulse. Due to its adsorption geometry on a thin sodium chloride (NaCl) film this molecule acts as a bistable switch, which can be coherently controlled by exerting ultrafast forces with atomic scale precision. The second topic investigated in this thesis is the direct photoexcitation and subsequent toggling motion of a single MgPc molecule. Here the synchronization of optical laser pulses with the oscillating motion of an AFM cantilever allows probing the effects of the direct photoexcitation on the molecule. It was found that the molecule can be switched between its two stable adsorption geometries via photoexcitation, which is attributed to charge exchange between the excited molecule and the STM electrodes. In the third section the effects of photoexcitation on single copper phthalocyanine (CuPc) molecules deposited onto bulk-like NaCl are explored. By employing laser pulses synchronized with the AFM cantilever oscillation single electron photocurrents from individual molecules could be generated and spatially resolved. The final part of this thesis outlines the development and construction of a novel STM design, which is specifically tailored to facilitate experiments with unprecedented spatio-temporal resolution

Brain stars take the lead during critical periods of early postnatal brain development: relevance of astrocytes in health and mental disorders

In the brain, astrocytes regulate shape and functions of the synaptic and vascular compartments through a variety of released factors and membrane-bound proteins. An imbalanced astrocyte activity can therefore have drastic negative impacts on brain development, leading to the onset of severe pathologies. Clinical and pre-clinical studies show alterations in astrocyte cell number, morphology, molecular makeup and astrocyte-dependent processes in different affected brain regions in neurodevelopmental (ND) and neuropsychiatric (NP) disorders. Astrocytes proliferate, differentiate and mature during the critical period of early postnatal brain development, a time window of elevated glia-dependent regulation of a proper balance between synapse formation/elimination, which is pivotal in refining synaptic connectivity. Therefore, any intrinsic and/or extrinsic factors altering these processes during the critical period may result in an aberrant synaptic remodeling and onset of mental disorders. The peculiar bridging position of astrocytes between synaptic and vascular compartments further allows them to “compute” the brain state and consequently secrete factors in the bloodstream, which may serve as diagnostic biomarkers of distinct healthy or disease conditions. Here, we collect recent advancements regarding astrogenesis and astrocyte-mediated regulation of neuronal network remodeling during early postnatal critical periods of brain development, focusing on synapse elimination. We then propose alternative hypotheses for an involvement of aberrancies in these processes in the onset of ND and NP disorders. In light of the well-known differential prevalence of certain brain disorders between males and females, we also discuss putative sex-dependent influences on these neurodevelopmental events. From a translational perspective, understanding age- and sex-dependent astrocyte-specific molecular and functional changes may help to identify biomarkers of distinct cellular (dys)functions in health and disease, favouring the development of diagnostic tools or the selection of tailored treatment options for male/female patients

Probabilistic Closed-Loop Active Grasping

Picking a specific object is an essential task of assistive robotics. While the majority of grasp detection approaches focus on grasp synthesis from a single depth image or point cloud, this approach is often not viable in an unstructured, uncontrolled environment. Due to occlusion, heavy influence of noise or simply because no collision-free grasp is visible from some perspectives, it is beneficial to collect additional information from other views before opting for grasp execution. We present a closed-loop approach that selects and navigates towards the next-best-view by minimizing the entropy of the volume under consideration. We use a local measure of estimation uncertainty of the surface reconstruction, to sample grasps and estimate their success probabilities in an online fashion. Our experiments show that our algorithm achieves better grasp success rates than comparable approaches, when presented with challenging household objects

Therapist versus Machine—Immediate Effects of Manual versus Mechanical Lymphatic Drainage in Patients with Secondary Lymphedema

Background: Complex decongestive therapy (CDT) is the standard and basic therapy for lymphedema. The central component of CDT is manual lymphatic drainage (MLD). In addition to CDT, other measures such as intermittent pneumatic compression therapy (IPCT) (active compression machine therapy) are available. In this prospective research study, the objective and subjective effects of MLD and IPCT on lymphedema of the lower extremity were investigated and both therapies were directly compared. Furthermore, the patients’ body mass index (BMI) and stage of lymphedema were tested for their effect on the respective therapy. Methods: Patients participating in the study received both therapies (MLD and IPCT) on the same lymphedema-affected limb at an interval of two days. The objective volumetric therapy effect was measured by the digital volume measurement of the affected limb. The subjective effects of the therapies were measured using two specially designed questionnaires. Results: A total of 40 patients were included in the study. There was no significant difference in the volume differences between the interventions, BMI categories, lymphedema, or treatment order regarding the immediate and two-day effect. Conclusions: No significant difference was found in the subjective or objective therapy efficacy of the two methods. Intermittent pneumatic compression therapy is considered a comparable therapeutic procedure when properly indicated

Unravelling White Phosphorus: Experimental and Computational Studies Reveal the Mechanisms of P4 Hydrostannylation

The hydrostannylation of white phosphorus (P4) allows this crucial industrial precursor to be easily transformed into useful P1 products via direct, ‘one pot’ (or even catalytic) procedures. However, a thorough mechanistic understanding of this transformation has remained elusive, hindering attempts to use this rare example of successful, direct P4 functionalization as a model for further reaction development. Here, we provide a deep and generalizable mechanistic picture for P4 hydrostannylation by combining DFT calculations with in situ31P NMR reaction monitoring and kinetic trapping of previously unobservable reaction intermediates using bulky tin hydrides. The results offer important insights into both how this reaction proceeds and why it is successful and provide implicit guidelines for future research in the field of P4 activation

The German Cliometrics Database

This short article introduces the German Cliometrics Database as the foundation for an article by Jopp and Spoerer (2024) who trace cliometric research on German history. This newly constructed database of every publication that (1) contributes to the historiography of Germany and (2) employs, as a baseline, inferential statistics enables researchers to find cliometric studies related to their own work much more efficiently. Even though no full texts are provided alongside the data file, the collected abstracts or, respectively, summaries for every publication in the database allow for some baseline text mining approaches. Along with the remaining information provided, they may also form the basis for broader bibliometric or historiographical studies

Membrane-Free Lateral Flow Assay with the Active Control of Fluid Transport for Ultrasensitive Cardiac Biomarker Detection

Membrane-based lateral flow immunoassays (LFAs) have been employed as early point-of-care (POC) testing tools in clinical settings. However, the varying membrane properties, uncontrollable sample transport in LFAs, visual readout, and required large sample volumes have been major limiting factors in realizing needed sensitivity and desirable precise quantification. Addressing these challenges, we designed a membrane-free system in which the desirable three-dimensional (3D) structure of the detection zone is imitated and used a small pump for fluid flow and fluorescence as readout, all the while maintaining a one-step assay protocol. A hydrogel-like protein–polyelectrolyte complex (PPC) within a polyelectrolyte multilayer (PEM) was developed as the test line by complexing polystreptavidin (pSA) with poly(diallyldimethylammonium chloride) (PDDA), which in turn was layered with poly(acrylic acid) (PAA) resulting in a superior 3D streptavidin-rich test line. Since the remainder of the microchannel remains material-free, good flow control is achieved, and with the total volume of 20 μL, 7.5-fold smaller sample volumes can be used in comparison to conventional LFAs. High sensitivity with desirable reproducibility and a 20 min total assay time were achieved for the detection of NT-proBNP in plasma with a dynamic range of 60–9000 pg·mL–1 and a limit of detection of 56 pg·mL–1 using probe antibody-modified fluorescence nanoparticles. While instrument-free visual detection is no longer possible, the developed lateral flow channel platform has the potential to dramatically expand the LFA applicability, as it overcomes the limitations of membrane-based immunoassays, ultimately improving the accuracy and reducing the sample volume so that finger-prick analyses can easily be done in a one-step assay for analytes present at very low concentrations

Optimierung der Chlorid-Kanalfunktion von mutantem Bestrophin-1

Das retinale Pigmentepithel (RPE) stellt eine wichtige einschichtige Zellschicht zwischen der äußeren Netzhaut (Retina) und der Aderhaut (Choroidea) im menschlichen Auge dar. Transportprozesse und Stoffwechselvorgänge dieser Zellen tragen dazu bei, die Funktionalität der Sinnesrezeptoren der Netzhaut aufrechtzuerhalten. Ein wichtiger Akteur in diesen Transportvorgängen ist das im RPE basolateral lokalisierte Kanalprotein BEST1. Dieses setzt sich aus fünf gleichen BEST1 Untereinheiten zusammen und bildet einen Ionenkanal, der Chloridionen aus den RPE Zellen abtransportiert. Mutationen im zugrunde liegenden hBEST1-Gen sind ursächlich für Funktionseinschränkungen als Folge einer fehlerhaften Cl--Leitfähigkeit, die je nach Art und Lokalisation der Mutation zu unterschiedlichen monogenen Netzhauterkrankungen führen können. Dazu zählen die autosomal-dominante Best´sche vitelliforme Makuladegeneration (Morbus Best), die autosomal-dominante Vitreoretinochoroidopathie (ADVIRC) und die autosomal-rezessive Bestrophinopathie (ARB). Bislang besteht für keine dieser Erkrankungen eine effektive Therapie. Studien der letzten Jahre zeigen bereits erste vielversprechende positive Effekte für einige Wirkstoffe. Auch am Institut für Humangenetik der Universität Regensburg wurde eine dieser Wirkstoffgruppen entdeckt, deren Effektivität zur Verbesserung der BEST1-Funktionalität mit Hilfe der Patch-Clamp Technik in dieser Arbeit direkt bestätigt werden sollte. Dafür standen aus Vorarbeiten vier Zelllinien aus humanen induzierten pluripotenten Stammzellen zur Verfügung, die zu RPE-Zellen ausdifferenziert worden waren. Die beiden Linien hiPSC-RPE BEST +/+ und die hiPSC-RPE LRRC8A -/- stellten dabei Positivkontrollen mit endogen exprimierten nativen BEST1 Untereinheiten dar, während hiPSC-RPE BEST -/- eine Negativkontrolle darstellte, die kein BEST1 synthetisieren konnte. In der Patienten-abgeleiteten Linie hiPSC-RPE BEST +/R218C sollte dann die gefundene Wirkstoffgruppe überprüft werden. Zudem wurden auch Messungen an zwei weiteren Zelllinien (HEK293T und Y79) vorgenommen, um die Kapazität, den Anstieg der Stromantwort im Zeitverlauf und die Stromdichte bei Verwendung einer 0,38 µM Ca2+ enthaltenden Intrazellulärlösung zur Induktion einer Cl--Leitfähigkeit charakterisieren und einordnen zu können. Signifikante Unterschiede zwischen den hiPSC-RPE Linien zeigten sich hierbei in der durchschnittlichen Größe der Zellen, welche über die Kapazität bestimmt werden konnte. Die beiden Knock-out Linien fielen durch ein im Vergleich zur Kontrolllinie BEST +/+ oder der Patienten-abgeleiteten Linie BEST +/R218C grundsätzlich erhöhtes Zellvolumen auf. In drei verschiedenen Versuchsansätzen mit unterschiedlichen Temperaturen und Ca2+-Konzentrationen konnten allgemeine Aktivierungseffekte, jedoch keine BEST1-assoziierten Unterschiede des Stromanstiegs oder der Stromdichte festgestellt werden. Bereits an diesem Punkt konnte somit keine Unterscheidung der unterschiedlichen hiPSC-RPE Linien festgestellt werden. Die Erhöhung der Ca2+-Konzentration führte zudem zu Messfehlern, die einen starken Einfluss auf die Verwertbarkeit der hier beobachteten Stromkurven hatten. Dadurch konnten Effekte von Wirkstoffderivaten auf die mutierten Kanaluntereinheiten nicht überprüft werden. Die Stromentwicklung in den hiPSC-RPE Linien unterschied sich dabei aber deutlich von den Aktivierungsverläufen, die in HEK293T und Y79 Zellen festgestellt werden konnten, und in diesen anderen Zellsystemen merklich geringer ausfielen. Eine intensive Fehlersuche zeigte, dass vermutlich eine unzureichende Aktivierung von BEST1 dem Messproblem zugrunde lag. Alternativ könnte ein Überlagern der Effekte durch unabhängige endogen exprimierte Kanäle in den hiPSC-RPE Zellen aufgetreten sein. In weiterführenden Untersuchungen sollte zunächst im Überexpressionssystem ein stabiles BEST1-Signal mithilfe des Patch Clamp Messverfahrens abgeleitet werden können, bevor die Fortsetzung von weiteren Messreihen mit den verfügbaren Wirkstoffderivaten sinnvoll erscheint

Cytokine Response of Natural Killer Cells to Hepatitis B Virus Infection Depends on Monocyte Co-Stimulation

Hepatitis B virus (HBV) is a major driver of chronic hepatic inflammation, which regularly leads to liver cirrhosis or hepatocellular carcinoma. Immediate innate immune cell response is crucial for the rapid clearance of the infection. Here, natural killer (NK) cells play a pivotal role in direct cytotoxicity and the secretion of antiviral cytokines as well as regulatory function. The aim of this study was to further elucidate NK cell responses triggered by an HBV infection. Therefore, we optimized HBV in vitro models that reliably stimulate NK cells using hepatocyte-like HepG2 cells expressing the Na+-taurocholate co-transporting polypeptide (NTCP) and HepaRG cells. Immune cells were acquired from healthy platelet donors. Initially, HepG2-NTCP cells demonstrated higher viral replication compared to HepaRG cells. Co-cultures with immune cells revealed increased production of interferon-γ and tumor necrosis factor-α by NK cells, which was no longer evident in isolated NK cells. Likewise, the depletion of monocytes and spatial separation from target cells led to the absence of the antiviral cytokine production of NK cells. Eventually, the combined co-culture of isolated NK cells and monocytes led to a sufficient cytokine response of NK cells, which was also apparent when communication between the two immune cell subpopulations was restricted to soluble factors. In summary, our study demonstrates antiviral cytokine production by NK cells in response to HBV+ HepG2-NTCP cells, which is dependent on monocyte bystander activation