Die Herstellung von Scaffolds zur Gewebezucht auf Basis von mesoporösen Silicananopartikeln sowie die detaillierte Charakterisierung PEGylierter Partikel

In der vorliegenden Dissertation wird die Entwicklung von Substraten, in denen mesoporöse Silicananopartikel (MSN) verarbeitet sind, für das Tissue Engineering in Hinblick auf biomedizinische Anwendungen beschrieben. Da der Einsatz von mesenchymalen Stammzellen bei der Gewebezucht vielseitige Einsatzmöglichkeiten verspricht, wurde in dieser Arbeit mit murinen Myoblasten (C2C12-Zellen) eine Zelllinie gewählt, die in der Lage ist, zu differenzieren und mehrkernige Myotuben auszubilden. Der Schwerpunkt bei der Untersuchung der hergestellten Plattformen liegt auf der Eignung zur Steuerung der Proliferation und Differenzierung von murinen Myoblasten. Die verwendeten mesoporösen Silicananopartikel sind ein erprobtes, durch die einfach realisierbare Modifizierbarkeit der Partikeloberfläche vielfältig einsetzbares Trägermaterial für Wirkstoffe und können durch adsorptive oder kovalente Anbindung problemlos für die verschiedenen Anwendungsbereiche des Tissue Engineerings angepasst und eingesetzt werden. Im ersten Schritt wird die Herstellung von Beschichtungen aus MSN auf planaren Oberflächen realisiert. Anschließend erfolgt deren Modifikation mit einem Hydrogel sowie durch die kovalente Anbindung der MSN auf einer funktionalisierten Oberfläche. Als Weiterentwicklung werden dreidimensionale Substrate auf Basis elektrogesponnener Polymere mit adsorbierten MSN hergestellt und untersucht. Abschließend wird die Oberflächenmodifikation der MSN mit Polyethylenglykol (PEG) vorgestellt, wobei die beobachtete Veränderung der Proteinadsorption in Abhängigkeit von der PEG-Menge untersucht wird. Bei der Herstellung partikulärer Filme werden die mesoporösen Silicananopartikel als Wirkstoffreservoir und als Transporter für wasserunlösliche Wirkstoffe eingesetzt. Die Eignung der MSN wird durch die Untersuchung der zellulären Aufnahme durch murine Myoblasten und der Bestimmung der Vitalität der Zellen bestätigt. Zunächst wird die Herstellung von Filmen aus calcinierten MSN sowie die Abhängigkeit der Zytokompatibilität der Substrate für murine Myoblasten von Partikelgröße und Filmdicke gezeigt. Zudem wird die Auswirkung des Einsatzes von MSN, deren   Zetapotential durch die Veränderung des Verhältnisses der Amino- und Carboxyfunktionen auf der Partikeloberfläche gesteuert wird, auf die Biokompatibilität beschrieben. Der Nachweis des Wirkstofftransports und der Aufnahme der MSN wird durch die Verwendung von mit einem Fluoreszenzfarbstoff markierten MSN in Kombination mit einem hydrophoben, fluoreszenten Modellmedikament erbracht. Zudem belegt die Beschleunigung der Differenzierung von murinen Myoblasten zu Myofasern auf einem mit dem γ-Sekretasehemmer N-[N-(3,5-Difluorphenacetyl)- ʟ -alanyl]-S-phenylglycin-tert-butylester (DAPT) beladenen Film, dass mit Hilfe des partikulären Trägersystems eine Steuerung der Entwicklung von Stamm- bzw. deren Vorläuferzellen realisierbar ist. Um die Adhärenz der Zellen, die gerade bei den mit Amino- und Carboxyfunktionen versehenen MSN als ein Problem bei der Handhabung der Filme identifiziert wurde, zu verbessern, wird eine Beschichtung der Filme mit der extrazellulären Matrix Matrigel® durchgeführt. Durch die Verzögerung der zellulären Aufnahme der MSN durch murine Myoblasten kann mit der Matrigel®-Beschichtung zudem die Kinetik des Wirkstofftransports beeinflusst werden. Insbesondere für die Arbeit mit Stammzellen ist die zeitliche Steuerung der Wirkstofffreisetzung von entscheidender Bedeutung, da die Differenzierung der Zellen erst nach einer ausreichenden Proliferationsphase eingeleitet werden darf. Um eine bessere Kontrolle über die Kinetik des Wirkstofftransports zu gewinnen, werden die MSN auf eine mit Hyaluronsäure funktionalisierte Oberfläche eines Glassubstrates aufgebracht. Kovalent angebundene MSN zeigen im Gegensatz zu adsorptiv gebundenen MSN eine deutlich verzögerte Aufnahme durch die murinen Myoblasten. Das Ziel des Tissue Engineerings ist allerdings die Heilung von großvolumigen Schäden oder die Nachzucht ganzer Organe. Dies setzt freistehende dreidimensionale Substrate voraus. In dieser Arbeit wird daher die Kombination der MSN mit bereits etablierten Gerüstmaterialien realisiert. Hierfür wird die Adsorption der MSN auf kommerziellen Polyurethansubstraten und auf elektrogesponnenen Fasern durchgeführt. Anschließend wird die Fähigkeit der Hybridmaterialien, in Interaktion mit murinen Myoblasten sowohl MSN als auch Wirkstoff freizusetzen, belegt. Die Einsatzfähigkeit des letztgenannten Systems wurde zudem in uovo belegt. Die Herstellung von funktionalisierten MSN erfolgt im Rahmen dieser Arbeit vor dem Hintergrund der Anwendung zur Herstellung von Substraten. Da die Zelladhärenz durch die auf einer Oberfläche adsorbierten Proteine gesteuert wird, werden sowohl MSN mit variablem Zetapotential als auch PEGylierte MSN hergestellt. Für die Herstellung von MSN, deren Proteinadsorption durch eine PEG-Hülle modifiziert ist, wird ein Monomethoxy-Trialkoxysilyl-PEG aus jeweils einem PEG mit 750 und 5 kDa herstellt und auf den MSN kovalent angebunden. Zusätzlich zur Bestimmung der PEG-Menge auf der Oberfläche wird die Hydratisierung des PEGs unter Verwendung des Alexander - de Gennes Modells berechnet und die Proteinadsorption untersucht. Hierbei kommen die Einzelproteine bovines Serumalbumin (BSA), Trypsin und Lysozym sowie das Proteingemisch fötales Kälberserum (fetal calf serum, FCS) zum Einsatz. Zusammengefasst können mesoporöse Silicananopartikel als Trägermaterial für ein fluoreszentes Modellmedikament sowie für DAPT eingesetzt werden. Der Aufbau von planaren Substraten sowie die Herstellung von freistehenden dreidimensionalen Scaffolds ermöglicht eine Anpassung an die Anforderungen der jeweiligen biomedizinischen Applikation. Da neben der adsorptiven Anbindung der MSN die Möglichkeit der Verzögerung der Wirkstofffreisetzung durch die Beschichtung mit Hydrogelen oder durch die kovalente Anbindung an Hyaluronsäure besteht, zeigt diese Arbeit an ausgewählten Beispielen das Potential der MSN-Hybridsubstrate für das Tissue Engineering auf.In the present thesis the development of scaffolds containing mesoporous silica nanoparticles for tissue engineering applications is reported. Due to the great potential of mesenchymal stem cells for the different aspects of tissue engineering, murine myoblasts (C2C12) were used within this work as this cell line shows differentiation to myotubes if the cells are treated with appropriate drugs. Within the examined platform the mesoporous silica nanoparticles show the ability to deliver (model) drugs to the cells. Furthermore, the surface of the biodegradable particles can be modified and therefore rationally designed through covalent linking of organic molecules, typically silanes carrying one or more functional groups, to the silica surface. This allows tuning of the particle-substrate interactions and further covalent linking of the particles to the substrate. Thus, the kinetics by which the particles are taken up by cells can be tuned. The development of the scaffold was realized in several steps. First, flat glass substrates were coated with MSN of different sizes and different surface chemistries. To improve the system, on the one hand a hydrogel coating was added and on the other hand the particles were linked to a modified glass substrate. Furthermore, the flat glass was exchanged by 3D-scaffolds made from electrospun fibres. Finally, the influence of PEGylation on serum protein adsorption was examined. The films of MSN were used to transport a hydrophobic model drug into C2C12-cells. For that purpose, MSN with different functionalisation were synthesised and the biocompatibility to C2C12 cells as well as the cellular uptake was demonstrated. First, calcined particles with different diameters which showed the best biocompatibility at high particle concentrations were employed to make films with various numbers of layers and therefore different thicknesses. The adhesion of C2C12-cells was found to depend on both particle size and film thickness, but nevertheless cells on all films comprising calcined MSN showed the expected morphology after 72 h of incubation. The successful drug delivery and cellular uptake of MSN was confirmed by using particles labelled with a covalently bound fluorescent dye and by loading a hydrophobic drug model (DiI, DiO) into the mesopores through physisorption. In addition, the differentiation of C2C12 cells was accelerated by using the drug delivery ability of a film of MSN loaded with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)- ʟ -alanyl]-S-phenylglycine tert-butyl ester (DAPT). Moreover, the adherence of C2C12 cells on films made of MSN with a functionalised surface was investigated. The results show that the cell survival and proliferation was poor due to problems in cell attachment and particle-induced cytotoxicity. To enhance the cell adherence a coating made from commercially available extracellular matrix (Martrigel®) was established on top of the particular film’s surface. This coating influenced the drug delivery kinetics by causing a delayed MSN uptake. Especially regarding the work with stem cells this property is very interesting, because the differentiation should be triggered not before a long enough proliferation phase which provides enough cells. A second approach to control the kinetics of the drug delivery is linking the MSN to the scaffold’s surface. Therefore, amino functionalised MSN were covalently linked to a surface coated with hyaluronic acid by EDC/NHS-coupling. A significant delay of the cellular uptake of the MSN by C2C12 cells was observed for covalently linked particles as compared to physisorbed ones. Especially for engineering of muscles there is a need of freestanding 3D scaffolds. The best way to create such a scaffold is to combine established scaffolds with the MSN. Here, commercial polyurethane scaffolds and electrospun fibres of polycaprolactone were used. The interaction of the hybrid material and a successful drug delivery was demonstrated. The evaluation of the second system was conducted in uovo. The synthesis of MSN with a variety of surface functionalisations was done with regard to the possibility to use them to create scaffolds. An important aspect to control the cell adhesion is the adsorption of proteins on the particles’ surface. To influence this property MSN with different zeta potentials were produced as well as particles with a PEG coating. The PEGylation was performed using either 750 Da and 5 kDa PEG-silanes or a mixture of both. The MSN were evaluated with respect to the amount of PEG on their surface, and the hydration state of the PEG layer was calculated by using the Alexander-de Gennes model for the thickness of the PEG layer. Finally this was correlated with the adsorption of single proteins BSA, lysozyme and trypsin and the protein mixture of fetal calf serum (FCS). In conclusion the results point out that MSN show the ability to deliver hydrophobic drugs like DAPT if they are used to create scaffolds for tissue engineering applications. The adjustment of this system for different biomedical applications is possible as there is a simple way to prepare coatings on plane substrates with a high particle concentration as well as 3D-scaffolds. Furthermore, controlling the kinetics of drug delivery can be realised by hydrogel coatings or covalent binding of the MSN to the scaffold

A miniaturized method for fast, simple, and sensitive pesticide analysis in soils

Purpose: Organochlorine pesticides (OCPs) like lindane and DDT have been used extensively after World War II until the 1990s. Still, residues of these pesticides can be found in agricultural soils all over the world, especially in developing countries. Often, they occur in extensive areas and elevated concentrations so that food safety is jeopardized. Hence, simple, cheap, and fast analytical methods are needed for a straight-forward assessment of risks. A miniaturized solid–liquid extraction combined with solid-phase microextraction (SPME) based on a proven ISO method is presented. Methods: The performance of the method is evaluated by extracting three different soils which were spiked with HCH and DDT congeners, and trifluralin, and aged for 35 days. The results are compared with those of a modified quick, easy, cheap, efficient, rugged, and safe (QuEChERS) method. For further validation, both methods are applied to three environmental soil samples. Results: Validation results show limits of detection and quantification as well as recovery rates in good agreement with standard requirements. The new method was found to be quicker than QuEChERS, which requires time-consuming preparation of reagents. Conclusion: Merits include low time and sample volume requirements (0.5 g) and the possibility to extract many samples simultaneously, which allows the screening of large sample sizes to determine the pollution status of whole landscape regions. However, access to an automated SPME apparatus is assumed. The authors can recommend this method as a cheap and fast alternative where SPME is available.bundesministerium für bildung und forschung http://dx.doi.org/10.13039/501100002347Justus-Liebig-Universität Gießen (3114

A miniaturized method for fast, simple, and sensitive pesticide analysis in soils

PURPOSE: Organochlorine pesticides (OCPs) like lindane and DDT have been used extensively after World War II until the 1990s. Still, residues of these pesticides can be found in agricultural soils all over the world, especially in developing countries. Often, they occur in extensive areas and elevated concentrations so that food safety is jeopardized. Hence, simple, cheap, and fast analytical methods are needed for a straight-forward assessment of risks. A miniaturized solid–liquid extraction combined with solid-phase microextraction (SPME) based on a proven ISO method is presented. METHODS: The performance of the method is evaluated by extracting three different soils which were spiked with HCH and DDT congeners, and trifluralin, and aged for 35 days. The results are compared with those of a modified quick, easy, cheap, efficient, rugged, and safe (QuEChERS) method. For further validation, both methods are applied to three environmental soil samples. RESULTS: Validation results show limits of detection and quantification as well as recovery rates in good agreement with standard requirements. The new method was found to be quicker than QuEChERS, which requires time-consuming preparation of reagents. CONCLUSION: Merits include low time and sample volume requirements (0.5 g) and the possibility to extract many samples simultaneously, which allows the screening of large sample sizes to determine the pollution status of whole landscape regions. However, access to an automated SPME apparatus is assumed. The authors can recommend this method as a cheap and fast alternative where SPME is available

Mesoporous silica nanoparticle-based substrates for cell directed delivery of Notch signalling modulators to control myoblast differentiation

Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with ¿-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery

Mesoporous silica nanoparticle-based substrates for cell directed delivery of Notch signalling modulators to control myoblast differentiation

Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with ¿-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery

Mesoporous silica particle-PLA-PANI hybrid scaffolds for cell-directed intracellular drug delivery and tissue vascularization

\u3cp\u3eInstructive materials are expected to revolutionize stem cell based tissue engineering. As many stem cell cues have adverse effects on normal tissue homeostasis, there is a need to develop bioactive scaffolds which offer locally retained and cell-targeted drug delivery for intracellular release in targeted cell populations. Further, the scaffolds need to support vascularization to promote tissue growth and function. We have developed an electrospun PLA-PANI fiber scaffold, and incorporated mesoporous silica nanoparticles within the scaffold matrix to obtain cell-targeted and localized drug delivery. The isotropy of the scaffold can be tuned to find the optimal morphology for a given application and the scaffold is electroactive to support differentiation of contractile tissues. We demonstrate that there is no premature drug release from particles under physiological conditions over a period of one week and that the drug is released upon internalization of particles by cells within the scaffold. The scaffold is biocompatible, supports muscle stem cell differentiation and cell-seeded scaffolds are vascularized in vivo upon transplantation on the chorioallantoic membrane of chicken embryos. The scaffold is a step towards instructive biomaterials for local control of stem cell differentiation, and tissue formation supported by vascularization and without adverse effects on the homeostasis of adjacent tissues due to diffusion of biological cues.\u3c/p\u3

Controlled drug release performance of plasma modified slab and mat matrices: A model study with “Ampicillin”

Two types of ampicillin carrier platforms were prepared with polycaprolactone (PCL) and the release behavior of a hydrophilic model drug (ampicillin sodium salt) from those matrices was investigated. Spin coating and electrospinning techniques were used to prepare slab and mat platforms, respectively. Ampicillin sodium salt (ASS) at 5% (w:w) concentration was loaded into the slab or mat structures of PCL. The thickness of the slab was measured 3.349 ± 0.345 ?m and surface morphology of the slabs showed uniform PCL spherulites. On the other hand, fiber diameter of PCL and ASS loaded PCL (ASSLPCL) was measured 604 ± 176 nm and 549 ± 119 nm, respectively. The dynamic behavior of the controlled release was improved by a very thin film (<100 nm) formation of sulfur hexafluoride (SF6) over the surface via plasma polymerization. Plasma coating was facilitated and speed up the drug diffusion, then led to 45.60 ± 6.46% and 63.67 ± 4.33% enhancement of drug from slab and mat, respectively. Transport mechanism from all matrices showed a Fickian diffusion behavior and plasma modification of the surface did not affected the mechanism. The in vitro antibacterial property of ASS loaded matrices against S. aureus and E. coli was studied through the comparison of bacterial inhibition zones and ASS showed antibacterial effect after all processes