Bibliografische Beschreibung Name: Elena Wolfram Titel: Differenzierung humaner neuraler Vorläuferzellen auf Beschichtungen mit Polydopamin und Collagen Institut: Medizinische Fakultät der Universität Leipzig, Dissertation zur Erlangung des akademischen Grades doctor medicinae (Dr.med.) 85 Seiten, 9 Abbildungen, 7 Tabellen, 194 Quellen Der Morbus Parkinson gehört zu den häufigsten neurodegenerativen Erkrankungen. Forschungen auf dem Gebiet der Zelltransplantation können einen vielversprechenden kurativen Ansatz aufzeigen. Im Interesse dieser Promotion standen die Generierung von dopaminergen Neuronen aus humanen neuralen Vorläuferzellen (hNPC) und die Untersuchung des Einflusses der extrazellulären Matrixproteine (EZM) Polydopamin (PDA) und Collagen auf diese. Aufgrund begrenzter Ressourcen und ethischer Bedenken im Hinblick auf die Gewinnung humaner neuraler Vorläuferzellen aus Embryonen war eine Steigerung der Genese dopaminerger Neuronen in vitro für eine Etablierung der Zelltransplantation als Behandlungsmethode notwendig. In dieser Promotionsarbeit wurde das Standardprotokoll für die Kultivierung humaner neuraler Vorläuferzellen durch Zugabe von PDA, Collagen I (Cl) und Collagen II (CIl) in unterschiedlichen Kombinationen ergänzt. Die verwendeten EZM wurden aufgrund ihrer bekannten Anhaftungseigenschaften ausgewählt, um ein gleichmäßig adhärentes Wachstum der hNPCs am Kulturflaschenboden zu erreichen. Die Untersuchungen auf Transkript- und Proteinebene zeigen, dass eine Kombination der Standardbeschichtung aus Poly-L-Ornithin/Fibronectin (PLO/FN) mit PDA sowie PDA als Monobeschichtung die dopaminerge Genese der hNPCs nicht steigern konnte. Eine niedrige Konzentration an PDA in den Beschichtungen führte zu einem nichtadhärenten Wachstum der hNPCs und zur Ausbildung von Spheres. Höhere Konzentrationen an PDA hatten ein adhärentes Wachstum der Zellen in clusterförmigen Anordnungen zur Folge. Auf den Beschichtungen mit PLO/FN und Cl sowie Cll ohne den Zusatz von PDA zeigte sich eine signifikant höhere Expression des dopaminergen Markers Thyrosinhydroxylase (TH) in den Analysen durch ICC und Westernblot. Cl und Cll führten in Kombination mit der Standardbeschichtung PLO/FN zu einem gleichmäßig adhärenten Wachstum. Eine Kombination von PDA, Cl und Cll sowie der Standardbeschichtung PLO/FN führte nur unter Verwendung von höheren Konzentra- tionen PDA zu einem adhärenten Wachstum, eine Steigerung der dopaminergen Diffe- renzierung konnte nicht beobachtet werden.

The oxidation of dopamine in aqueous solutions deposits thin conformal films on a wide variety of material surfaces. These films consist of a material known as polydopamine (PDA), and they exhibit chemical and structural similarities to melanin pigments and adhesive proteins secreted by mussels. The facile synthesis and versatile adhesion of PDA enable the functional modification of numerous material surfaces for applications in biomedical devices, energy storage, and water purification. This thesis details fundamental investigations into the deposition, oxidation, and adhesive mechanisms of PDA films. Depositing PDA films on substrates with different controlled chemistries revealed the importance of solution pH and initial deposition rates on the morphology of the films. The deposition of PDA molecules with increasing pH depends on two competing factors: increased generation rate of PDA molecules versus increased solubility due to catechol ionization. The areal density and coverage of three-dimensional PDA islands is influenced by the surface charge and hydrophobicity of the substrate in aqueous solutions. Spectroscopic and electrochemical characterization of PDA films revealed that redox-inactive metal cations can accelerate the oxidation of PDA. The generation of radicals of 5,6-dihydroxyindole were monitored in situ via ultraviolet-visible spectroscopy as a function of cation concentration and pH. The extent of oxidation was quantified by cyclic voltammetry. The resulting oxidation modifies the metal sorption properties of PDA by generating more carboxylic acid groups and enhancing the iron chelation of the films. The adhesive stability of PDA films was characterized by delamination kinetics of films on SiO2 and indium tin oxide (ITO). PDA film adhesion is a substrate, salt, and oxidation-dependent phenomenon. Long-term adhesive stability of PDA films can be promoted by use of higher dopamine concentrations during synthesis, incorporation of multivalent cations, and avoiding alkaline conditions and strongly oxidizing electrical bias. Elastic moduli of PDA films were quantified by compressive thin film wrinkling, and the measured value of 2.0 ± 0.9 GPA agrees with simulations of PDA based on an oligomeric aggregate model. This thesis helps develop a framework for understanding the synthesis, composition, microstructure, and stability of PDA films.

Dopamine is an interesting biomolecule that functions as a neurotransmitter in the brain. It has been found able to stick to almost all surfaces due to its unique catecholamine structure. Under alkaline conditions, the catechol functional group oxidizes to quinone allowing dopamine to self-polymerize and form thin films on support surfaces. The facts that dopamine can be coated to virtually any materials and the amine and catechol functional groups support a variety of reactions with organic species make polydopamine an attractive multifunctional bioadhesive/coating. To date, most of research on polydopamine has been focusing on its applications as thin films and little attention has been paid to the adhesion aspect of the material. In the study, we evaluated the properties of self-polymerized dopamine thin films as a bioadhesive. The thesis consists of three consecutive studies: (i) characterization of the adhesion properties of polydopamine thin films; (ii) investigation of the mechanical properties of polydopamine thin films; and (iii) exploration of the potential of polydopamine thin films as a wet adhesive. Fundamental insights on the wettability, adhesion behaviours, and mechanical properties of polydopamine thin films for both wet and dry conditions were derived through sets of well-designed contact angle, contact adhesion, and contact deformation experiments. It was found that dopamine is able to coat plastic, ceramic and metal surfaces, and join or bond rigid substrates but might not be suitable for joining soft or flexible parts as polydopamine is fairly rigid and the bonding might be too slow and too rigid for practical applications if polydopamine is directly used as an adhesive. Based on the understanding, a new strategy for fabricating underwater adhesive was proposed and tested. In the strategy, polydopamine with ferric ion as the oxidant was utilized as a cross-linker to alginate solution, effectively turning the solution into a wet adhesive, which demonstrated better practical performances than other studies; the adhesive was able to produce a permanent tensile adhesive strength of 80 kPa joining aluminum and glass with macroscopic roughness at the interface within 2 hours of curing time. Inspired by the results from the contact deformation experiments, we were able to extend the JKR theory with the well-known plate theory to accommodate the deformation of nanometer thin films, obtaining their elasticity. We termed this extended theory the “thin film contact mechanics” and validated the theory against gold thin films and found it predicted the mechanical behaviours of the thin films fairly well.

Dopamine is an interesting biomolecule that functions as a neurotransmitter in the brain. It has been found able to stick to almost all surfaces due to its unique catecholamine structure. Under alkaline conditions, the catechol functional group oxidizes to quinone allowing dopamine to self-polymerize and form thin films on support surfaces. The facts that dopamine can be coated to virtually any materials and the amine and catechol functional groups support a variety of reactions with organic species make polydopamine an attractive multifunctional bioadhesive/coating. To date, most of research on polydopamine has been focusing on its applications as thin films and little attention has been paid to the adhesion aspect of the material. In the study, we evaluated the properties of self-polymerized dopamine thin films as a bioadhesive. The thesis consists of three consecutive studies: (i) characterization of the adhesion properties of polydopamine thin films; (ii) investigation of the mechanical properties of polydopamine thin films; and (iii) exploration of the potential of polydopamine thin films as a wet adhesive. Fundamental insights on the wettability, adhesion behaviours, and mechanical properties of polydopamine thin films for both wet and dry conditions were derived through sets of well-designed contact angle, contact adhesion, and contact deformation experiments. It was found that dopamine is able to coat plastic, ceramic and metal surfaces, and join or bond rigid substrates but might not be suitable for joining soft or flexible parts as polydopamine is fairly rigid and the bonding might be too slow and too rigid for practical applications if polydopamine is directly used as an adhesive. Based on the understanding, a new strategy for fabricating underwater adhesive was proposed and tested. In the strategy, polydopamine with ferric ion as the oxidant was utilized as a cross-linker to alginate solution, effectively turning the solution into a wet adhesive, which demonstrated better practical performances than other studies; the adhesive was able to produce a permanent tensile adhesive strength of 80 kPa joining aluminum and glass with macroscopic roughness at the interface within 2 hours of curing time. Inspired by the results from the contact deformation experiments, we were able to extend the JKR theory with the well-known plate theory to accommodate the deformation of nanometer thin films, obtaining their elasticity. We termed this extended theory the “thin film contact mechanics” and validated the theory against gold thin films and found it predicted the mechanical behaviours of the thin films fairly well.

Polydopamine (PDA), a unique bioinspired polymer, has been a subject of interest in fields including orthopedic biomaterials and antibacterial surfaces. Its osteogenic effects and ability to control surface traits through precise variables (e.g., pH, temperature) have led to its use as a coating in the enhancement of a wide range of materials, including metals and ceramics. In this Thesis, two studies were carried out to better understand the capability and mechanism of PDA-mediated bioactivity. In the first study, we investigated whether PDA coatings can further enhance the bioactivity of nanoporous Titanium (NPTi). While physicochemical traits were in line with literature, PDA was effective in enhancing cell proliferation, beyond NPTi, as early as 8 hours with enhancement in cell spreading and focal adhesion prevalence as early as 1 hour. No changes in adsorptive capacity were found, suggesting a serum-independent component (SIC) of the surface. The second study was focused on (1) determining how treatment parameters influence the physiochemical makeup of PDA surfaces, (2) assessing how PDA surfaces influence stem cell behavior and (3) confirming and investigating the SIC of PDA effect. Results confirm that there is indeed a SIC of PDA coatings with enhancement in cell spreading that improves with the increased size and density of PDA particles. Our findings show that the SIC works in concert with circulating sera to elicit the bioactive effects of PDA. The novel rPDA surface, obtained by adding rotation during the coating deposition, is also shown to elevate bioactivity during normal culturing, beyond classical coatings, with ongoing work suggesting enhancement in the osteogenic differentiation of hMSCs. Taken together, this work has demonstrated novel aspects underlying the potential and mechanism of action for the bioactivity of PDA, ultimately providing new evidence supporting the use of PDA as a biomedical material.

Fouling is a major obstacle to the implementation of membranes in water purification applications. Hydrophilization of the membrane surface tends to mitigate fouling because hydrophobic interactions between foulants and the membrane are reduced. Polydopamine was deposited onto membranes to render their surfaces hydrophilic. The chemical structure of polydopamine, which was previously ambiguous, was investigated by many spectroscopic techniques. While previously thought to consist of covalently-linked monomers, polydopamine was found to be an aggregate of partly-oxidized dopamine units linked by strong, non-covalent secondary interactions. Polydopamine was also used as a platform for the molecular conjugation of other anti-fouling materials, such as poly(ethylene glycol), to the membrane surface. Membrane fouling was assessed by constant permeate flux crossflow filtration with an oil/water emulsion feed. The threshold flux--the flux at which the rate of fouling significantly increases--was determined by a well-established flux stepping technique. Membrane resistance evolution during fouling was compared for constant flux and constant transmembrane pressure operation using unmodified membranes. Below the threshold flux (slow fouling), good agreement in resistance evolution was found between the two operational modes; above the threshold flux, significant deviation was observed. The effect of polydopamine and polydopamine-g-poly(ethylene glycol) surface modifications was studied under constant flux crossflow fouling conditions. The surface modifications were found to increase the membrane resistance, resulting in higher transmembrane pressures in the modified membranes than in the unmodified membranes at fluxes below the threshold flux. Modified membranes were also compared to unmodified membranes with the same pure water permeance (same initial resistance). In this case, the modified membranes had lower transmembrane pressures during fouling than the unmodified membranes, suggesting that a preferred method of membrane surface modification is to begin with a membrane of higher permeance than required, and then surface-modify it to achieve the desired permeance. The efficacy of polydopamine and polydopamine-g-poly(ethylene glycol) surface modifications in reducing biofouling was also evaluated. Modified membranes showed reduced protein and bacterial adhesion in short-term tests, which are commonly used to assess biofouling propensity. However, long-term operation under hydrodynamic conditions mimicking those of an industrial module showed no benefit of the hydrophilic coatings in limiting biofouling. / text

No description available.

Cancer is a leading case of mortality worldwide. Governments spent multibillion expenses on treatment and palliative care of diseased people. Despite these generous funding and intensive research with aim to find a cure or efficient treatment for cancer, until now there is a lack in selective cancer management strategies. Conventional treatment strategies for cancer, such as surgery, cytotoxic chemotherapy, radiation therapy, hormone therapy don’t have selectivity toward cancer – the property of discrimination of healthy organs and tissues from the diseased site. Chemotherapy is very challenging as the difference between effective and lethal doses is very minuscule in most cases. Moreover, devastating side effects dramatically changes the quality of life for cancer patients. To address these issues two main strategies are intensively utilized in chemistry: (I) the design and synthesis of novel anticancer organic compounds with higher selectivity and low toxicity profiles and the second, design and preparation of biocompatible nanocarriers for imaging and anticancer compound selective delivery nanomedicine. The following dissertation combines the above two strategies as bellows: First project is related to the design and synthetic route development toward novel nature-inspired group of heterocyclic compounds – iso-Phidianidines. The second project focused on design, preparation and evaluation of hybrid theranostics (therapeutic and diagnostic in a single entity). Chapter 1 is a general background review of the major topics that will be discussed in this dissertation. The first efficient and high-yielding synthetic route toward iso-phidianidines, containing regioisomeric form of 1,2,4-oxadiazole linked to the indole via methylene bridge is reported in Chapter 2. In vitro test of the synthesized library of iso-phidianidines revealed micromolar range of cytotoxicity toward human cervical cancer cell line. Structure activity relationship revealed the importance of presence of monosubsituted amine in 3 position of oxadiazole to maintain activity. Moreover, gradual increase of activity was detected in increasing of the length of the diamine. Polyamine (spermidine) side chain demonstrated strongest anticancer activity, identified as lead compound and may be studied further as a good candidate for cervical cancer treatment. Finally, the remaining high activity of amino-terminated iso-phidianidines demonstrated that presence of guanidine group in termini is not necessary for high cytotoxicity. The second part of this dissertation (Chapter 3) discusses the rational design, wet protocol synthesis and complete characterization of the novel hybrid material – polydopamine coated iron-cobalt nanocubes (PDFCs). This material was loaded with anticancer model drug doxorubicin in one step procedure (PDFC-DOX) and the resulting drug-delivery vehicle was found to be successfully internalized by cervical cancer cells. The cytotoxicity test demonstrated inhibition of 50% of the cells at the concentration of 30μg/ml for PDFC-DOX. Moreover, the release was highly attenuated and pH-sensitive in acidic range. PDFC was also modified with fluorescein leading to green fluorescent nanoparticles PDFC-FITC, which demonstrated excellent intracellular molecular imaging property. PDFCs with one of the highest magnetic saturation among the materials used in biomedicine (226 emu/g based on core) showed the absence of any cytotoxicity in vitro and excellent MRI contrasting property (r2=186.44 mMs-1, higher than commercial contrast agents Ferridex® and Clio®), both in vitro and in vivo on mice. They were cleared out from the mice bodies in month without affecting their health. Due to the high density of core (8.3 g/cm3) they demonstrated ability to be contrast materials also for X-Ray CT diagnostic modality, increasing the tumor detection and visualization probability in combination with MRI. In addition to it’s diagnostic and drug-delivery modalities, PDFC was evaluated also for microwave-induced cytotoxicity as a novel concept in cancer treatment. As low as 10 μg/ml concentration of PDFCs in human cervical cancer cells caused extensive death above 73% upon exposure to 2,45 GHz of microwaves for one minute. Laser irradiation (808 nm, 15 minutes) of cancer cells with internalized PDFCs caused cell death above 60%. The specific absorption rate of PDFCs at 470 MHz frequency and 20 mT of the alternating magnetic field power was 180 W/g, which is nearly 100 W higher than for commercial nanoparticles (Ferridex®).

Bibliografische Beschreibung Name: Elena Wolfram Titel: Differenzierung humaner neuraler Vorläuferzellen auf Beschichtungen mit Polydopamin und Collagen Institut: Medizinische Fakultät der Universität Leipzig, Dissertation zur Erlangung des akademischen Grades doctor medicinae (Dr.med.) 85 Seiten, 9 Abbildungen, 7 Tabellen, 194 Quellen Der Morbus Parkinson gehört zu den häufigsten neurodegenerativen Erkrankungen. Forschungen auf dem Gebiet der Zelltransplantation können einen vielversprechenden kurativen Ansatz aufzeigen. Im Interesse dieser Promotion standen die Generierung von dopaminergen Neuronen aus humanen neuralen Vorläuferzellen (hNPC) und die Untersuchung des Einflusses der extrazellulären Matrixproteine (EZM) Polydopamin (PDA) und Collagen auf diese. Aufgrund begrenzter Ressourcen und ethischer Bedenken im Hinblick auf die Gewinnung humaner neuraler Vorläuferzellen aus Embryonen war eine Steigerung der Genese dopaminerger Neuronen in vitro für eine Etablierung der Zelltransplantation als Behandlungsmethode notwendig. In dieser Promotionsarbeit wurde das Standardprotokoll für die Kultivierung humaner neuraler Vorläuferzellen durch Zugabe von PDA, Collagen I (Cl) und Collagen II (CIl) in unterschiedlichen Kombinationen ergänzt. Die verwendeten EZM wurden aufgrund ihrer bekannten Anhaftungseigenschaften ausgewählt, um ein gleichmäßig adhärentes Wachstum der hNPCs am Kulturflaschenboden zu erreichen. Die Untersuchungen auf Transkript- und Proteinebene zeigen, dass eine Kombination der Standardbeschichtung aus Poly-L-Ornithin/Fibronectin (PLO/FN) mit PDA sowie PDA als Monobeschichtung die dopaminerge Genese der hNPCs nicht steigern konnte. Eine niedrige Konzentration an PDA in den Beschichtungen führte zu einem nichtadhärenten Wachstum der hNPCs und zur Ausbildung von Spheres. Höhere Konzentrationen an PDA hatten ein adhärentes Wachstum der Zellen in clusterförmigen Anordnungen zur Folge. Auf den Beschichtungen mit PLO/FN und Cl sowie Cll ohne den Zusatz von PDA zeigte sich eine signifikant höhere Expression des dopaminergen Markers Thyrosinhydroxylase (TH) in den Analysen durch ICC und Westernblot. Cl und Cll führten in Kombination mit der Standardbeschichtung PLO/FN zu einem gleichmäßig adhärenten Wachstum. Eine Kombination von PDA, Cl und Cll sowie der Standardbeschichtung PLO/FN führte nur unter Verwendung von höheren Konzentra- tionen PDA zu einem adhärenten Wachstum, eine Steigerung der dopaminergen Diffe- renzierung konnte nicht beobachtet werden.

Biofouling is an ongoing problem in the development and usage of biomaterials for biomedical implants, microfluidic devices, and water-based sensors. Antifouling coatings involving surface modification of biomaterials is widely utilized to reduce unwanted protein adsorption and cell adhesion. Surface modification strategies, however, are reliant on the working material’s chemical properties. Thus, published procedures are often not applicable to a wide range of material classes. This constitutes a serious limitation in using surface modification on assembled multi-material devices, i.e on whole device modification. The objective of this research is to develop an antifouling coating with non-aggressive reaction conditions that can universally modify polymers and other material classes. Two strategies using polydopamine (PDA) as an anchor for polyethylene glycol (PEG) surface attachment were investigated: (1) PDA-PEG backfilled with bovine serum albumin (BSA), and (2) PDA-PEG with light activated perfluorophenyl azide (PFPA) conjugated to the PEG. Three materials varying in surface wettability were studied to evaluate the coatings for multi-material applications: porous polycarbonate membrane (PC), polydimethyl siloxane (PDMS), and soda lime glass cover slips. Atomic force microscopy (AFM) and ellipsometry studies revealed substantial structural differences of PDA. Differences in PDA surface roughness affected PEG grafting in solution (the first method), with higher PEG coverage achieved on PC with intermediate surface roughness to PDMS and glass. Radiolabeled Fg adsorption and E. coli adhesion experiments showed reduced fouling on all PDA-PEG modified materials when backfilled with BSA. The ability for BSA to penetrate the PEG layer indicated that low PEG grafting densities were achieved using this grafting-to approach. The use of a photoactive labeling agent, PFPA, to tether PEG was proposed to improve PEG grafting on PDA. The PFPA-PEG modification protocol was optimized by quantifying Fg adsorption. Two treatments of PFPA-PEG were required to fully block PDA active sites. Fg adsorption was not significantly improved on PFPA-PEG modified PC and glass when backfilled with BSA, indicating sufficient PEG coverage of PDA. High Fg adsorption on PFPA-PEG surfaces indicate that high density PEG brushes were still not achieved with this method. PDMS surfaces were damaged with this procedure due to increased surface handling in the protocol. This is the first, to our knowledge, successful demonstration of PFPA modification on PDA surfaces. Photopatterning of polymer-based materials can be achieved, providing opportunities for utilising new materials in cell patterned platforms. Due to low PEG coverage on PDA surfaces from solution and using PFPA, ultra-low protein adsorption cannot be achieved using these aqueous-based methods. Antifouling modifications using PDA and PEG should be applied for short-term cell studies. / Thesis / Master of Applied Science (MASc)