Entwicklung und Charakterisierung biokompatibler Kern-Schale-Hybridmaterialien anhand der gesteuerten Erzeugung einer Biomolekülcorona auf magnetischen Nanopartikeln
Im Rahmen dieser Arbeit wurden Protokolle zur wiederholbaren Herstellung von Dispersionen aus biokompatiblen, gezielt mit einer Biomolekülcorona umgebenen magnetischen Hybrid-Partikeln entwickelt, die sich sowohl in dest. Wasser als auch biologischem Medium wie fetalem Kälberserum (FCS) stabil verhalten und praktisch nutzbare Eigenschaften wie eine Konzentration von β ≥ 1,0 mg/ml und eine enge Partikelgrößenverteilung (Polydispersitätsindex PDI ≤ 0,100) aufweisen. Für die Experimente wurde die wiederholbare Herstellung von vergleichbaren Partikelsystemen aus superferrimagnetischen Eisenoxid-Multikern-Partikeln mit einer Ankerhüllschicht aus Dextranen mit unterschiedlichen Funktionalisierungen, aber gleichem Rückgrat etabliert (Carboxymethyldextran (CMD) – negativ und Diethylaminoethyldextran (DEAE) – positiv), um Abhängigkeiten von der Oberflächenladung nachvollziehen zu können. Zusätzlich wurden Stärke-Kern-Hülle-Partikelchargen als Vertreter einer überwiegend sterisch stabilisierenden Hülle genutzt.
Protokolle zur wiederholbaren Überführung der Kern-Hülle-Partikelchargen in Hybrid-Partikelchargen mit „harter“ Biomolekülcorona durch kontrollierte Inkubation in FCS, eine angepasste Waschprozedur sowie eine sich anschließende Fraktionierung mittels Zentrifugation, Überstandsnahme und Probenselektion wurden entwickelt und angewandt. Einschätzungen zu Verfahrensschritten sowie Ausbeuten wurden gegeben. Die hergestellten Kern-Hülle- und Hybrid-Partikelchargen wurden hinsichtlich ihrer Eigenschaften wie Konzentration, Partikelgrößenverteilung und Zeta-Potential charakterisiert und untereinander verglichen. Neben Standardmethoden wie dynamischer Lichtstreuung (DLS), elektrophoretischer Lichtstreuung (ELS) und Thermogravimetrieanalyse (TGA) kamen magnetische Messverfahren wie AC-Suszeptibilität (ACS) und Vibrationsmagnetometrie (VSM) zum Einsatz. Für mindestens eine Hybrid-Partikelcharge jeder Ankerhüllschicht konnten die Zieleigenschaften (β ≥ 1,0 mg/ml, PDI ≤ 0,100) eingehalten werden. Mithilfe der Kombination von TGA, VSM, DLS und ACS war es mit spezifischen Modellen (z. B. Multikern-Eindomänenmodell (MultiCore-SingleDomain-Model – MCSDM)) möglich für die meisten Partikelsysteme quantitative Abschätzungen zu den Schichtdicken der Ankerhüllschicht und der Biomolekülcorona, teilweise mit Unterscheidung zwischen „hartem“ und „weichem“ Anteil, vorzunehmen. Die entstandenen Biomolekülcoronen wurden mittels Gelelektrophorese (SDS-PAGE) mit anschließender Silberfärbung weiterhin auf ihre Proteinzusammensetzung untersucht. Ein Zusammenhang zwischen dem Betrag des Zeta-Potentials der Ankerhüllschicht und der entstehenden Schichtdicke der Biomolekülcorona, insbesondere durch die Anlagerung von Proteinen der kleinsten Molekulargrößenklasse (Mw < 30 kDa), wurde gefunden. Über UV-VIS-Spektrometrie und visuelle Beobachtung sowie ACS und DLS konnte die kolloidale Stabilität aller untersuchten Partikelchargen für mind. 1 d bei Verdünnung 1 zu 10 (v/v) in destilliertem Wasser nachgewiesen werden. Für die (Re-)Inkubation 1 zu 10 (v/v) in FCS zeigten sich teilweise starke Instabilitäten. Mittels ACS konnte eine quantitative Abschätzung der Menge sich kolloidal instabil verhaltender Partikel vorgenommen werden. Die DEAE-Hybrid-Partikel blieben als einziges Partikelsystem für mind. 7 d kolloidal stabil in destilliertem Wasser und FCS.
Erste Experimente zu biologischen Wechselwirkungen zeigten in vitro mittels Celltiter Glo®-Test an BeWo-Zellen mit Testkonzentrationen bis zu 100 μg/cm² (entspricht β = 0,378 mg/ml) für die meisten Partikelchargen mäßige, schwache oder gar keine Toxizität. Die Unterschiede in der Toxizität konnten in allen Fällen mit einem Größeneffekt durch Agglomeration und Clusterbildung aufgrund der Interaktion mit Biomolekülen interpretiert werden. In vivo-Experimente mittels schalenlosem ex ovo Hühnerei-Modells (HET-CAV) mit Konzentrationen bis zu β = 1 mg/ml zeigten für alle hergestellten Partikelsysteme eine gute Biokompatibilität.
Für die DEAE-Hybrid-Partikel wurden exemplarisch Experimente zur Haltbarmachung durch Gefrieren, Tiefgefrieren und Gefriertrocknung mit unterschiedlichen Additiven sowie zur Sterilisation durch Autoklavieren, Sterilfiltrieren und UV-Sterilisation durchgeführt. Die besten Resultate zeigten für Lagerzeiten bis zu 6 Wochen Gefriertrocknung mit Polyethylenglycol (PEG) als Additiv und zur Haltbarmachung Sterilisation mit UV-C-Licht.
In this work, protocols were developed for the reproducible preparation of dispersions of biocompatible magnetic hybrid particles intentionally coated with a biomolecular corona, which are stable in both distilled water as well as biological medium such as fetal calf serum (FCS) and have practically useful properties such as a concentration of β ≥ 1.0 mg/ml and a narrow particle size distribution (polydispersity index PDI ≤ 0.100). For the experiments, the reproducible preparation of comparable particle systems of superferrimagnetic iron oxide multicore particles with an anchor shell layer of dextrans with different functionalisations but the same backbone was established (carboxymethyldextran (CMD) - negative and diethylamino-ethyldextran (DEAE) - positive) in order to determine dependencies on surface charge. In addition, starch core-shell particle batches were used as representatives of a predominantly sterically stabilising shell. Protocols for the reproducible conversion of core-shell particle batches into hybrid particle batches featuring a "hard" biomolecule corona by controlled incubation in FCS, an adapted washing procedure and subsequent fractionation by centrifugation, collection of supernatant and sample selection were developed and applied. Estimations on process steps as well as yields were given. The produced core-shell and hybrid particle batches were characterised with respect to their properties such as concentration, particle size distribution and zeta potential and compared with each other. In addition to standard methods such as dynamic light scattering (DLS), electrophoretic light scattering (ELS) and thermogravimetric analysis (TGA), magnetic measurement methods such as AC susceptibility (ACS) and vibrating-sample magnetometry (VSM) were used. For at least one hybrid particle batch of each anchor coating layer, the target properties (β ≥ 1.0 mg/ml, PDI ≤ 0.100) could be met. Using the combination of TGA, VSM, DLS and ACS, it was possible with specific models (e.g. MultiCore Single Domain Model (MCSDM)) to quantitatively estimate the thicknesses of the anchor shell layer and the biomolecule corona for most of the particle systems, in some cases with differentiation between "hard" and "soft" corona. The resulting biomolecule coronae were further analysed with respect to their protein composition by gel electrophoresis (SDS-PAGE) followed by silver staining. A correlation between the height of the zeta potential of the anchor shell layer and the resulting layer thickness of the biomolecule corona was found, in particular due to the accumulation of proteins of the smallest molecular size class (Mw < 30 kDa). Using UV-VIS spectrometry and visual observation as well as ACS and DLS, the colloidal stability of all particle batches investigated could be demonstrated for at least 1 d at dilution 1 to 10 (v/v) in distilled water. For the (re)incubation 1 to 10 (v/v) in FCS, partly strong instabilities were found. By means of ACS, a quantitative estimation of the amount of colloidally unstable particles could be made. The DEAE hybrid particles were the only particle system that remained colloidally stable for at least 7 d in distilled water and FCS. Initial experiments on biological interactions showed moderate, weak or no toxicity for most particle batches in vitro using the Celltiter Glo® assay on BeWo cells with test concentrations up to 100 μg/cm² (corresponding to β = 0.378 mg/ml). The differences in toxicity could in all cases be interpreted with a size effect due to agglomeration and clustering due to interaction of the particles with biomolecules. In vivo experiments using shell-less ex ovo hen’s egg model (HET-CAV) with concentrations up to β = 1.0 mg/ml showed good biocompatibility for all particle systems produced. For the DEAE hybrid particles, exemplary experiments were carried out on preservation by freezing, deep-freezing and freeze-drying with different additives as well as sterilisation by autoclaving, sterile filtration and UV sterilisation. The best results were achieved for storage times of up to 6 weeks by freeze-drying with polyethylene glycol (PEG) as an additive and for preservation by sterilisation with UV C light. In this work, protocols were developed for the reproducible preparation of dispersions of biocompatible magnetic hybrid particles intentionally coated with a biomolecular corona, which are stable in both distilled water as well as biological medium such as fetal calf serum (FCS) and have practically useful properties such as a concentration of β ≥ 1.0 mg/ml and a narrow particle size distribution (polydispersity index PDI ≤ 0.100). For the experiments, the reproducible preparation of comparable particle systems of superferrimagnetic iron oxide multicore particles with an anchor shell layer of dextrans with different functionalisations but the same backbone was established (carboxymethyldextran (CMD) - negative and diethylamino-ethyldextran (DEAE) - positive) in order to determine dependencies on surface charge. In addition, starch core-shell particle batches were used as representatives of a predominantly sterically stabilising shell. Protocols for the reproducible conversion of core-shell particle batches into hybrid particle batches featuring a "hard" biomolecule corona by controlled incubation in FCS, an adapted washing procedure and subsequent fractionation by centrifugation, collection of supernatant and sample selection were developed and applied. Estimations on process steps as well as yields were given. The produced core-shell and hybrid particle batches were characterised with respect to their properties such as concentration, particle size distribution and zeta potential and compared with each other. In addition to standard methods such as dynamic light scattering (DLS), electrophoretic light scattering (ELS) and thermogravimetric analysis (TGA), magnetic measurement methods such as AC susceptibility (ACS) and vibrating-sample magnetometry (VSM) were used. For at least one hybrid particle batch of each anchor coating layer, the target properties (β ≥ 1.0 mg/ml, PDI ≤ 0.100) could be met. Using the combination of TGA, VSM, DLS and ACS, it was possible with specific models (e.g. MultiCore Single Domain Model (MCSDM)) to quantitatively estimate the thicknesses of the anchor shell layer and the biomolecule corona for most of the particle systems, in some cases with differentiation between "hard" and "soft" corona. The resulting biomolecule coronae were further analysed with respect to their protein composition by gel electrophoresis (SDS-PAGE) followed by silver staining. A correlation between the height of the zeta potential of the anchor shell layer and the resulting layer thickness of the biomolecule corona was found, in particular due to the accumulation of proteins of the smallest molecular size class (Mw < 30 kDa). Using UV-VIS spectrometry and visual observation as well as ACS and DLS, the colloidal stability of all particle batches investigated could be demonstrated for at least 1 d at dilution 1 to 10 (v/v) in distilled water. For the (re)incubation 1 to 10 (v/v) in FCS, partly strong instabilities were found. By means of ACS, a quantitative estimation of the amount of colloidally unstable particles could be made. The DEAE hybrid particles were the only particle system that remained colloidally stable for at least 7 d in distilled water and FCS. Initial experiments on biological interactions showed moderate, weak or no toxicity for most particle batches in vitro using the Celltiter Glo® assay on BeWo cells with test concentrations up to 100 μg/cm² (corresponding to β = 0.378 mg/ml). The differences in toxicity could in all cases be interpreted with a size effect due to agglomeration and clustering due to interaction of the particles with biomolecules. In vivo experiments using shell-less ex ovo hen’s egg model (HET-CAV) with concentrations up to β = 1.0 mg/ml showed good biocompatibility for all particle systems produced. For the DEAE hybrid particles, exemplary experiments were carried out on preservation by freezing, deep-freezing and freeze-drying with different additives as well as sterilisation by autoclaving, sterile filtration and UV sterilisation. The best results were achieved for storage times of up to 6 weeks by freeze-drying with polyethylene glycol (PEG) as an additive and for preservation by sterilisation with UV C light.
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