Towards the clinical translation of extracellular vesicles – surface engineering and optimized vesicle-storage

Extracellular vesicles (EVs) are membrane-bound nanoparticles produced by cells. Their main function is the intercellular transport of information. Their natural biological activity and ability to interact with target cells drives the effort for their evaluation as therapeutic agents and their clinical translation. Thus, the present work aimed at evaluating two important topics for the development of EV-therapeutics, engineering the surface of bacterial EVs to introduce targeting moieties and optimizing the lyophilisation of mammalian EVs. Different methods for the surface engineering of bacterial EVs were tested. While cholesterol insertion and activated esters were not successful in our setup, diazotransfer to install azide groups on the EV surface followed by click-chemistry demonstrated high efficiency with low impact on vesicle-integrity. Based on these results modular linker-molecules to tether targeting moieties to the vesicle surface were synthesized and evaluated in proof-of-concept experiments that afforded important insights for future optimization of their design. Initial studies on the lyophilisation and storage of mammalian EVs revealed the high stress of freezing and dehydration. Thus, optimized protocols for the lyophilisation of EVs were developed and successfully produced formulations with long-term stability. Taken together, effective surface engineering by diazotransfer and optimized lyophilisation of EVs were achieved.Extrazelluläre Vesikel (EVs) sind von Zellen produzierte Nanopartikel, deren Hauptaufgabe der interzelluläre Transport von Informationen ist. Ihre natürliche biologische Aktivität und ihre Fähigkeit mit Zielzellen zu interagieren, waren Auslöser für die Untersuchung von EVs als Therapeutika mit dem Ziel ihrer klinischen Translation. Ziel der vorliegenden Arbeit war die Evaluierung zweier wichtiger Faktoren für die Entwicklung von EV-Therapeutika, die Oberflächenmodifikation bakterieller EVs mit Molekülen für aktives Targeting und die Lyophilisation von aus Säuger-EVs. Verschiedene Methoden zur Oberflächenmodifikation von bakteriellen EVs wurden getestet. Während Cholesterol-Insertion und aktivierte Ester wenig erfolgreich waren, zeigte Diazotransfer zur Einführung von Azid-Gruppen gefolgt von Click-Chemie-Reaktionen eine hohe Effektivität mit geringem Einfluss auf die EV-Stabilität. Zur Einführung von Targeting- Molekülen wurden verschiedene modulare Linker-Moleküle synthetisiert und in Machbarkeitsstudien evaluiert, diese lieferten wichtige Erkenntnisse für ihre zukünftige Optimierung. Erste Untersuchungen der Lyophilisation und Lagerung von Säuger-EVs zeigten eine hohe Beanspruchung der Vesikel durch Einfrieren und Trocknung. Daher wurden die Protokolle für die EV-Lyophilisation optimiert und EV-Formulierungen mit Langzeitstabilität erfolgreich hergestellt. Insgesamt wurden sowohl bei der Oberflächenmodifikation als auch Lyophilisation von EVs wichtige Fortschritte erzielt.Promotionsstipendium Studienstiftung des Deutschen Volke

Controlled self-organisation using learning classifier systems

The complexity of technical systems increases, breakdowns occur quite often. The mission of organic computing is to tame these challenges by providing degrees of freedom for self-organised behaviour. To achieve these goals, new methods have to be developed. The proposed observer/controller architecture constitutes one way to achieve controlled self-organisation. To improve its design, multi-agent scenarios are investigated. Especially, learning using learning classifier systems is addressed

Dirac Landau levels for surfaces with constant negative curvature

Studies of the formation of Landau levels based on the Schr\"odinger equation for electrons constrained to curved surfaces have a long history. These include as prime examples surfaces with constant positive and negative curvature, the sphere [Phys. Rev. Lett. 51, 605 (1983)] and the pseudosphere [Annals of Physics 173, 185 (1987)]. Now, topological insulators, hosting Dirac-type surface states, provide a unique platform to experimentally examine such quantum Hall physics in curved space. Hence, extending previous work we consider solutions of the Dirac equation for the pseudosphere for both, the case of an overall perpendicular magnetic field and a homogeneous coaxial, thereby locally varying, magnetic field. For both magnetic-field configurations, we provide analytical solutions for spectra and eigenstates. For the experimentally relevant case of a coaxial magnetic field we find that the Landau levels split and show a peculiar scaling $\propto B^{1/4}$, thereby characteristically differing from the usual linear $B$ and $B^{1/2}$ dependence of the planar Schr\"odinger and Dirac case, respectively. We compare our analytical findings to numerical results that we also extend to the case of the Minding surface

Controlled self-organisation using learning classifier systems

The complexity of technical systems increases, breakdowns occur quite often. The mission of organic computing is to tame these challenges by providing degrees of freedom for self-organised behaviour. To achieve these goals, new methods have to be developed. The proposed observer/controller architecture constitutes one way to achieve controlled self-organisation. To improve its design, multi-agent scenarios are investigated. Especially, learning using learning classifier systems is addressed

Classical and Quantum Signatures of Quantum Phase Transitions in a (Pseudo) Relativistic Many-Body System

We identify a (pseudo) relativistic spin-dependent analogue of the celebrated quantum phase transition driven by the formation of a bright soliton in attractive one-dimensional bosonic gases. In this new scenario, due to the simultaneous existence of the linear dispersion and the bosonic nature of the system, special care must be taken with the choice of energy region where the transition takes place. Still, due to a crucial adiabatic separation of scales, and identified through extensive numerical diagonalization, a suitable effective model describing the transition is found. The corresponding mean-field analysis based on this effective model provides accurate predictions for the location of the quantum phase transition when compared against extensive numerical simulations. Furthermore, we numerically investigate the dynamical exponents characterizing the approach from its finite-size precursors to the sharp quantum phase transition in the thermodynamic limit

Late Life-Threatening Hemorrhage after Percutaneous Tracheostomy

Purpose. Formation of a tracheoinnominate artery fistula (TIF) and consecutive hemorrhage is a rare and life-threatening complication with high mortality. Warning symptoms can be absent. The current literature contains only few considerations for misleading signs, especially in cases where the contact between the tissue and the cannula is tight. Method and Results. We report two cases of life-threatening hemorrhages that appeared six days and two months after percutaneous dilatational tracheostomy (PDT) in two patients, respectively. In these cases, diagnosis of tracheoinnominate artery fistula (TIF) was difficult. Tracheal ring fracture after PDT and pressure ulceration caused by cannula were implicated in TIF formation. The cannula was overblocked to buy time before surgical closure. Both patients survived without any additional neurological deficiency. Conclusion. Massive hemorrhage in patients after tracheostomy is likely due to TIF. Ultrasound scanning before PDT and careful periodical followup of the trachea are required

Enhancing the Stabilization Potential of Lyophilization for Extracellular Vesicles

Extracellular 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 Gmb