Synthesis and Characterization of Ceramide-Containing Liposomes as Membrane Models for Different T Cell Subpopulations

A fine balance of regulatory (Treg) and conventional CD4+ T cells (Tconv) is required to prevent harmful immune responses, while at the same time ensuring the development of protective immunity against pathogens. As for many cellular processes, sphingolipid metabolism also crucially modulates the Treg/Tconv balance. However, our understanding of how sphingolipid metabolism is involved in T cell biology is still evolving and a better characterization of the tools at hand is required to advance the field. Therefore, we established a reductionist liposomal membrane model system to imitate the plasma membrane of mouse Treg and Tconv with regards to their ceramide content. We found that the capacity of membranes to incorporate externally added azide-functionalized ceramide positively correlated with the ceramide content of the liposomes. Moreover, we studied the impact of the different liposomal preparations on primary mouse splenocytes in vitro. The addition of liposomes to resting, but not activated, splenocytes maintained viability with liposomes containing high amounts of C16-ceramide being most efficient. Our data thus suggest that differences in ceramide post-incorporation into Treg and Tconv reflect differences in the ceramide content of cellular membranes

Synthesis and Characterization of II-VI-Semiconductor Nanoparticles in Selected Environment

Gegenstand dieser Arbeit ist die Synthese und Charakterisierung von II-VI-Halbleiter-Nanopartikeln (NP) in unterschiedlicher Umgebung. Aufgrund des großen Oberfläche-zu-Volumen-Verhältnisses werden Partikeleigenschaften stark durch ihre Oberfläche und die Wechselwirkung mit der Umgebung beeinflusst. Zuerst wurden strukturierte CdSe und CdSe/ZnS-Kern-Schale Nanopartikel durch eine organometallische Synthese in koordinierenden Lösungsmitteln hergestellt. Die optischen und elektronischen Eigenschaften wurden mittels Absorptions-(UV/VIS)-, Fluoreszenz- und konfokaler Fluoreszenz-Korrelations-Spektroskopie (FCS) untersucht. Die Ermittlung der Kristallstruktur erfolgte durch hochauflösende Transmissionselektronenmikroskopie (HRTEM) und Röntgenpulverdiffraktometrie (XRD). Die experimentellen XRD Resultate wurden durch Simulationen mittels der Debye-Formel sowie Berechnung einer Paarverteilungsfunktion (PDF) für die verschiedenen Nanopartikel-Modelle ausgewertet. Somit konnten die Partikelgröße, -form und die Kristallstruktur ermittelt werden. Ramanspektroskopische Untersuchungen ergaben Informationen über die Zusammensetzung des anorganischen Partikelkerns sowie seiner stabilisierenden Ligandenhülle. Aufbauend auf diesen Ergebnissen aus unterschiedlichen spektroskopischen und mikroskopischen Methoden konnte ein Struktur-Modell für die Kern-Schale Nanopartikel entwickelt werden. Dabei ist ein prolater wurtzitischer CdSe-Kern mit einer segmentartigen, lückenhaften ZnS-Schale beschichtet, die eine Zinkblende-Struktur aufweist. Zur Untersuchung der Umgebungseffekte wurden die CdSe- und CdSe/ZnS-Halbleiter-NP mit hydrophilen Liganden funktionalisiert, reversibel mit einer Polymerhülle beschichtet sowie kontrolliert in Silica-Kolloide eingebettet (Multikernpartikel). Somit konnten die Nanopartikel in unterschiedlich polaren und apolaren Lösungsmitteln stabilisiert und charakterisiert werden. Im Hinblick auf die Anwendungen von Halbleiter-NP als Marker in den Lebenswissenschaften wurde die Biokompatibilität und die lichtinduzierte Fluoreszenzverstärkung von Polymer-beschichteten II-VI-Halbleiter-NP und CdSe/ZnS-dotierten Silica-Kolloiden in unterschiedlichen Umgebungen untersucht. Mit Hilfe der erhaltenen Resultate ist ein neues qualitatives Modell für die lichtinduzierten Aktivierungs- und Desaktivierungsprozesse in Multikernpartikeln entwickelt worden. Ein weiterer Aspekt dieser Arbeit war die Untersuchung der lokalen elektronischen Struktur von II-VI-Halbleiter-NP in unterschiedlichen Umgebungen durch elementspezifische Anregung mit weicher Röntgenstrahlung. Dazu wurde ein Verfahren weiterentwickelt, das es erlaubt, einzelne gespeicherte feste und flüssige Nanopartikel substratfrei mit Hilfe von Synchrotronstrahlung zu analysieren. Darüber hinaus wurde die Röntgenabsorptionsfeinstruktur von deponierten CdSe/ZnS-dotierten Silica-Kolloiden durch die Messung der röntgenangeregten optischen Fluoreszenz (XEOL) bzw. durch die Bestimmung der totalen Elektronenausbeute (TEY) untersucht.Subject of this thesis is the synthesis and characterization of II-VI-semiconductor nanoparticles (quantum dots, QD) in selected environments. Structured CdSe and CdSe/ZnS core-shell nanoparticles have been prepared by syntheses that are based on the high temperature thermolysis of organometallic precursors in the presence of stabilizing agents. The influence of the local environment on the optical properties of the QD is studied by optical absorption (UV/VIS), photoluminescence, and confocal fluorescence correlation spectroscopy (FCS). The crystal structure of the nanoparticles is characterized by high-resolution electron microscopy (HRTEM) and X-ray diffraction (XRD). The diffraction patterns are fitted directly by modeling the nanocrystals using the Debye equation and pair distribution function (PDF), which models the interatomic distances within the sample. These results allowed us to determine fundamental parameters, such as size, shape, and crystal structure. Raman spectroscopy probes the lattice vibrations of the nanocrystals. This approach is applied to investigate the composition of the core-shell particles and especially to study the bonding between the stabilizing ligands and the nanoparticle surface by analyzing the internal vibrational modes. The results of the several characterization methods allowed us to develop a new model for the core-shell particle structure: ZnS with a zincblende structure forms an irregularly shaped shell around the elongated CdSe core which has a wurtzite structure. Further, the particles are subsequently functionalized either by an exchange of their ligands, or by the reversible adsorption of an amphiphilic polymer. Alternatively, they are embedded in silica colloids in a controlled way (multicore particles). As a result, the nanoparticle properties can be studied in various solvents ranging from apolar to polar liquids as well as in solid environments. Moreover, multicore particles have the potential to be used as selective labels for biological studies. Therefore, the cytotoxicity and the light induced enhancement of the luminescence of the multicore particles in various environments are investigated. Based on the present results, a new qualitative model for the mechanism of the photoactivation and -deactivation of CdSe/ZnS dotted silica colloids in various solvents is developed. The local electronic structure of the QD is studied by element-specific excitation using soft X-rays. For this purpose, a recently developed approach has been improved, allowing the investigation of single solid and liquid particles trapped in an electrodynamic particle trap, which are probed by monochromatic synchrotron radiation. In addition, the near edge X-ray absorption fine structure of deposited CdSe/ZnS doped silica colloids is determined by the measurement of the X-ray excited optical luminescence (XEOL) and the total electron yield (TEY)

Synthesis and characterization of NIR dye-doped nanoparticles for in vivo tumor diagnostics

Nanoparticles (NPs) are promising tools for a wide spectrum of biological and medical applications. They can be used as carrier and delivery systems for active agents such as biomolecules, dyes and a wide range of sensitive substances and also contribute to the stabilization of these compounds in vivo. Real time non-destructive imaging screening in vivo can be performed by means of fluorescent based methods. Near infrared (NIR) dyes are perfectly suited for this purpose. They are very promising for tissue labeling because of the fact that in the IR range there is significantly lower background fluorescence than in the visible range. Another feature of tissue is the so called transparent “NIR-window” at wavelengths from 650 nm to 1350 nm. One major disadvantage of most organic NIR dyes is their very fast degradation in vivo, so long-term investigations are not feasible. To stabilize these dyes, one option is to encapsulate the dye molecules into a NP matrix. Here, we present our recent research activities in the field of medical diagnostics concerning the encapsulation of NIR dyes into NPs for in vivo tumor diagnostics. Our work is focused on the synthesis and characterization of NP carrier systems on the basis of amorphous silica with mean particle sizes in the range of 60 to 150 nm. These NPs are synthesized via wet-chemical synthesis and doped with different NIR dyes. The choice of silica as a basis of the NPs is motivated by their high biocompatibility, biodegradability and the possibility of surface modifications. The characterization of the NPs is done by conventional methods such as transmission electron microscopy (TEM) and dynamic light scattering (DLS). Dye-doped NPs were characterized by fluorescence spectroscopy, measuring of absorption and emission with a plate reader and elemental analysis. The focus here was on the stability of the encapsulated NIR dyes under different storage conditions. In summary, the synthesis of different NP systems on the basis of amorphous silica and the encapsulation of different NIR dyes was successfully demonstrated. With the confirmation of the stability of the encapsulated dyes in the NP matrix they have shown their potential in the field of medical imaging

Multifunctional Nanoparticles for Medical Applications

Extended Abstract Well-tailored multifunctional nanoparticles (NPs), which are in the focus of our R&D work, play a major role in the development of future-oriented, advanced functional materials for life science applications such as in contrast agents for medical imaging, for in vitro and in vivo diagnostics, in drug delivery as well as in tissue engineering. Inorganic-organic biohybrid NPs in particular are considered to be important for the development of smart materials and novel technologies for medical applications. We prepare our NPs by wet-chemical methods: sol-gel, precipitation or hydrothermal synthesis. Our preferred particle materials are inorganic and hybrid materials, e.g. materials based on silicate, calcium fluoride and phosphate, TiO2 and iron oxide The great potential of novel NPs as assay labels for immunodetection, contrast agents for medical imaging or tools for tumor therapy has been demonstrated in different research projects In close collaboration with the Fraunhofer translational center "Regenerative therapies in oncology and musculoskeletal diseases", a special application of NPs for in vitro and in vivo monitoring of wound healing process is investigated. To this end, artificial wound models, which help to increase the effectiveness of a medication by testing it in an earlier stage to significantly reduce the number of medications selected for clinical tests, have been developed. In general, the 3D tissue models can be used as a preliminary stage to animal experiments for investigations of functional parameters like the distribution and metabolization of substances in different tissue layers. The demographic change in the industrialized countries manifests itself in a constantly growing number of humans in need of care and, at the same time, a declining number of available care forces. This effect is strengthened by the fact that cost-intensive illnesses such as cardiovascular diseases, diabetes, dementia, cancer diseases and chronic wounds are growing in numbers with increasing age of the population. This predictable dramatic development can only be countered with improved pre-diagnostics, a marked cost reduction in therapeutic treatment through better efficiency and extended self-care by patients. Here, we present the development and evaluation of economical and innovative therapy procedures for chronic skin diseases, which enable patients to check whether their chronically open wounds are healing. Central elements in the development phase are modifiable in-vitro skin models, biodegradable CE-certified wound dressings, implementation of immunotherapeutics in the wound dressing, targeted nanoparticles for smart diagnostics of biomarker

Screening applications to test cellular fitness in transwell® models after nanoparticle treatment

Nanoparticles (NPs) in biotechnology hold great promise for revolutionizing medical treatments and therapies. In order to bring NPs into clinical application there is a number of preclinical in vitro and in vivo tests, which have to be applied before. The initial in vitro evaluation includes a detailed physicochemical characterization as well as biocompatibility tests, among others. For determination of biocompatibility at the cellular level, the correct choice of the in vitro assay as well as NP pretreatment is absolutely essential. There are a variety of assay technologies available that use standard plate readers to measure metabolic markers to estimate the number of viable cells in culture. Each cell viability assay has its own set of advantages and disadvantages. Regardless of the assay method chosen, the major factors critical for reproducibility and success include: (1) choosing the right assay after comparing optical NP properties with the read-out method of the assay, (2) verifying colloidal stability of NPs in cell culture media, (3) preparing a sterile and stable NP dispersion in cell culture media used in the assay, (4) using a tightly controlled and consistent cell model allowing appropriate characterization of NPs. This chapter will briefly summarize these different critical points, which can occur during biocompatibility screening applications of NPs

Effect of ph on the synthesis and properties of luminescent sio2/calcium phosphate:eu3+ core-shell nanoparticles

A novel method for the synthesis of luminescent SiO(2)/calcium phosphate (CaP):Eu(3+) core-shell nanoparticles (NPs) was developed via a sol-gel route followed by annealing at a temperature of 800 degrees C. The object of this study was the investigation of the effect of pH on the formation of a CaP shell around the silica core. The resulting annealed NPs exhibited an amorphous SiO(2) core and a crystalline luminescent shell. The formation of a CaP layer was possible at pH below 4.5 and above 6.5 during the coating step. The crystal structure of the shell was studied by X-ray diffraction analysis. Hydroxyapatite (HAp) and alpha-tricalcium phosphate were detected as crystal phases of the surrounding layer. However, NPs produced under basic conditions exhibited a higher crystallinity of the CaP layer than did samples coated at pH below 4.5. In the pH interval between 4.5 and 6.5, no shell growth but the formation of secondary NPs containing CaO and Ca(OH)(2) was observed. Furthermore, SiO(2)/CP:Eu(3+) core-shell NPs were investigated by transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy, inductively coupled plasma optical emission spectrometry, and photoluminescence spectroscopy. The resulting HAp-coated NPs were successfully tested by a cell-culture-based viability assay with respect to a later application as a luminescent marker for biomedical applications

Assessment of the Impact of Nanowarming on Microstructure of Cryopreserved Fibroblast-Containing 3D Tissue Models Using Mueller Polarimetry

International audienceWe studied the impact of two different thawing mechanisms on the microstructure of defrosted cryopreserved 3D tissue models using transmission Mueller microscopy and a statistical analysis of polarimetric images of thin histological sections of defrosted tissue models. The cryopreserved 3D tissue models were thawed by using either a 37 °C water bath or radio-frequency inductive heating with the magnetic nanoparticles embedded into the 3D tissue model during the preparation process. Polarimetric measurements were conducted at 700 nm and the acquired Mueller matrices of the samples were post-processed using the differential decomposition and the statistical analysis of the maps of the azimuth of the optic axis. Our results indicate the sensitivity of polarimetry to the changes in thawed tissue morphology compared to that of reference non-frozen tissue. Thus, Mueller microscopy can be used as a fast complementary technique to the currently accepted gold standard methods for the assessment of the cryopreserved tissue microstructure after thawing

Digital histology of tissue with Mueller microscopy and FastDBSCAN

International audienceWe present the results of the automated post-processing of Mueller microscopy images of skin tissue models with a new fast version of the algorithm of density-based spatial clustering of applications with noise (FastDBSCAN) and discuss the advantages of its implementation for digital histology of tissue. We demonstrate that using the FastDBSCAN algorithm, one can produce the diagnostic segmentation of high resolution images of tissue by several orders of magnitude faster and with high accuracy ( > 97 % ) compared to the original version of the algorithm

Sol-Gel-Derived Fibers Based on Amorphous α-Hydroxy-Carboxylate-Modified Titanium(IV) Oxide as a 3-Dimensional Scaffold

The development of novel fibrous biomaterials and further processing of medical devices is still challenging. For instance, titanium(IV) oxide is a well-established biocompatible material, and the synthesis of TiOx particles and coatings via the sol-gel process has frequently been published. However, synthesis protocols of sol-gel-derived TiOx fibers are hardly known. In this publication, the authors present a synthesis and fabrication of purely sol-gel-derived TiOx fiber fleeces starting from the liquid sol-gel precursor titanium ethylate (TEOT). Here, the α-hydroxy-carboxylic acid lactic acid (LA) was used as a chelating ligand to reduce the reactivity towards hydrolysis of TEOT enabling a spinnable sol. The resulting fibers were processed into a non-woven fleece, characterized with FTIR, 13C-MAS-NMR, XRD, and screened with regard to their stability in physiological solution. They revealed an unexpected dependency between the LA content and the dissolution behavior. Finally, in vitro cell culture experiments proved their potential suitability as an open-mesh structured scaffold material, even for challenging applications such as therapeutic medicinal products (ATMPs)