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In dieser Dissertation konnten erfolgreich mechanisch stabile Hydrogele über eine freie radikalische Polymerisation (FRP) in Wasser synthetisiert werden. Dabei diente vor allem das Sulfobetain SPE als Monomer. Dieses wurde mit dem über eine nukleophile Substitution erster bzw. zweiter Ordnung hergestellten Vernetzer TMBEMPA/Br umgesetzt.
Die entstandenen Netzwerke wurden im Gleichgewichtsquellzustand im Wesentlichen mittels Niederfeld-Kernresonanzspektroskopie, Röntgenkleinwinkelstreuung (SAXS), Rasterelektronenmikroskopie mit Tieftemperaturtechnik (Kryo-REM), dynamisch-mechanische Analyse (DMA), Rheologie, thermogravimetrische Analyse (TGA) und dynamische Differenzkalorimetrie (DSC) analysiert.
Das hierarchisch aufgebaute Netzwerk wurde anschließend für die matrixgesteuerten Mineralisation von Calciumphosphat und –carbonat genutzt. Über das alternierende Eintauchverfahren (engl. „alternate soaking method“) und der Variation von Mineralisationsparametern, wie pH-Wert, Konzentration c und Temperatur T konnten dann verschiedene Modifikationen des Calciumphosphats generiert werden. Das entstandene Hybridmaterial wurde qualitativ mittels Röntgenpulverdiffraktometrie (XRD), abgeschwächte Totalreflexion–fouriertransformierte Infrarot Spektroskopie (ATR-FTIR), Raman-Spektroskopie, Rasterelektronenmikroskopie (REM) mit energiedispersiver Röntgenspektroskopie (EDXS) und optischer Mikroskopie (OM) als auch quantitative mittels Gravimetrie und TGA analysiert.
Für die potentielle Verwendung in der Medizintechnik, z.B. als Implantatmaterial, ist die grundlegende Einschätzung der Wechselwirkung zwischen Hydrogel bzw. Hybridmaterial und verschiedener Zelltypen unerlässlich. Dazu wurden verschiedene Zelltypen, wie Einzeller, Bakterien und adulte Stammzellen verwendet. Die Wechselwirkung mit Peptidsequenzen von Phagen komplettiert das biologische Unterkapitel.
Hydrogele sind mannigfaltig einsetzbar. Diese Arbeit fasst daher weitere Projektperspektiven, auch außerhalb des biomedizinischem Anwendungsspektrums, auf. So konnten erste Ansätze zur serienmäßige bzw. maßgeschneiderte Produktion über das „Inkjet“ Verfahren erreicht werden. Um dies ermöglichen zu können wurden erfolgreich weitere Synthesestrategien, wie die Photopolymerisation und die redoxinitiierte Polymerisation, ausgenutzt. Auch die Eignung als Filtermaterial oder Superabsorber wurde analysiert.
While click chemistry reactions for biopolymer network formation are attractive as the defined reactions may allow good control of the network formation and enable subsequent functionalization, tailoring of gelatin network properties over a wide range of mechanical properties has yet to be shown. Here, it is demonstrated that copper-catalyzed alkyne-azide cycloaddition of alkyne functionalized gelatin with diazides gave hydrogel networks with properties tailorable by the ratio of diazide to gelatin and diazide rigidity. 4,4′-diazido-2,2′-stilbenedisulfonic acid, which has been used as rigid crosslinker, yielded hydrogels with Young’s moduli E of 50–390 kPa and swelling degrees Q of 150–250 vol.%, while the more flexible 1,8-diazidooctane resulted in hydrogels with E = 125–280 kPa and Q = 225–470 vol.%. Storage moduli could be varied by two orders of magnitude (G′ = 100–20,000 Pa). An indirect cytotoxicity test did not show cytotoxic properties. Even when employing 1:1 ratios of alkyne and azide moieties, the hydrogels were shown to contain both, unreacted alkyne groups on the gelatin backbone as well as dangling chains carrying azide groups as shown by reaction with functionalized fluorescein. The free groups, which can be tailored by the employed ratio of the reactants, are accessible for covalent attachment of drugs, as was demonstrated by functionalization with dexamethasone. The sequential network formation and functionalization with click chemistry allows access to multifunctional materials relevant for medical applications.
Polymer degradation occurs under physiological conditions in vitro and in vivo, especially when bonds susceptible to hydrolysis are present in the polymer. Understanding of the degradation mechanism, changes of material properties over time, and overall rate of degradation is a necessary prerequisite for the knowledge-based design of polymers with applications in biomedicine. Here, hydrolytic degradation studies of gelatin-based networks synthesized by copper-catalyzed azide-alkyne cycloaddition reaction are reported, which were performed with or without addition of an enzyme. In all cases, networks with a stilbene as crosslinker proofed to be more resistant to degradation than when an octyl diazide was used. Without addition of an enzyme, the rate of degradation was ruled by the crosslinking density of the network and proceeded via a bulk degradation mechanism. Addition of Clostridium histolyticum collagenase resulted in a much enhanced rate of degradation, which furthermore occurred via surface erosion. The mesh size of the hydrogels (>7nm) was in all cases larger than the hydrodynamic radius of the enzyme (4.5nm) so that even in very hydrophilic networks with large mesh size enzymes may be used to induce a fast surface degradation mechanism. This observation is of general interest when designing hydrogels to be applied in the presence of enzymes, as the degradation mechanism and material performance are closely interlinked. Copyright (c) 2016 John Wiley & Sons, Ltd.
Proteins are natural polypeptides produced by cells; they can be found in both animals and plants, and possess a variety of functions. One of these functions is to provide structural support to the surrounding cells and tissues. For example, collagen (which is found in skin, cartilage, tendons and bones) and keratin (which is found in hair and nails) are structural proteins. When a tissue is damaged, however, the supporting matrix formed by structural proteins cannot always spontaneously regenerate. Tailor-made synthetic polypeptides can be used to help heal and restore tissue formation.
Synthetic polypeptides are typically synthesized by the so-called ring opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCA). Such synthetic polypeptides are generally non-sequence-controlled and thus less complex than proteins. As such, synthetic polypeptides are rarely as efficient as proteins in their ability to self-assemble and form hierarchical or structural supramolecular assemblies in water, and thus, often require rational designing. In this doctoral work, two types of amino acids, γ-benzyl-L/D-glutamate (BLG / BDG) and allylglycine (AG), were selected to synthesize a series of (co)polypeptides of different compositions and molar masses.
A new and versatile synthetic route to prepare polypeptides was developed, and its mechanism and kinetics were investigated. The polypeptide properties were thoroughly studied and new materials were developed from them. In particular, these polypeptides were able to aggregate (or self-assemble) in solution into microscopic fibres, very similar to those formed by collagen. By doing so, they formed robust physical networks and organogels which could be processed into high water-content, pH-responsive hydrogels. Particles with highly regular and chiral spiral morphologies were also obtained by emulsifying these polypeptides. Such polypeptides and the materials derived from them are, therefore, promising candidates for biomedical applications.
A concentrated solution of a symmetric triblock copolymer with a thermoresponsive poly(methoxy diethylene glycol acrylate) (PMDEGA) middle block and short hydrophobic, fully deuterated polystyrene end blocks is investigated in D2O where it undergoes a lower critical solution temperature-type phase transition at ca. 36 A degrees C. Small-angle neutron scattering (SANS) in a wide temperature range (15-50 A degrees C) is used to characterize the size and inner structure of the micelles as well as the correlation between the micelles and the formation of aggregates by the micelles above the cloud point (CP). A model featuring spherical core-shell micelles, which are correlated by a hard-sphere potential or a sticky hard-sphere potential together with a Guinier form factor describing aggregates formed by the micelles above the CP, fits the SANS curves well in the entire temperature range. The thickness of the thermoresponsive micellar PMDEGA shell as well as the hard-sphere radius increase slightly already below the cloud point. Whereas the thickness of the thermoresponsive micellar shell hardly shrinks when heating through the CP and up to 50 A degrees C, the hard-sphere radius decreases within 3.5 K at the CP. The volume fraction decreases already significantly below the CP, which may be at the origin of the previously observed gel-sol transition far below the CP (Miasnikova et al., Langmuir 28: 4479-4490, 2012). Above the CP, small, and at higher temperatures, large aggregates are formed by the micelles.
Biopolymers of the extracellular matrix are attractive starting materials for providing degradable and biocompatible biomaterials. In this study, hyaluronic acid-based hydrogels with tunable mechanical properties were prepared by the use of copper-catalyzed azide-alkyne cycloaddition (known as "click chemistry"). Alkyne-functionalized hyaluronic acid was crosslinked with linkers having two terminal azide functionalities, varying crosslinker density as well as the lengths and rigidity of the linker molecules. By variation of the crosslinker density and crosslinker type, hydrogels with elastic moduli in the range of 0.5-4 kPa were prepared. The washed materials contained a maximum of 6.8 mg copper per kg dry weight and the eluate of the gel crosslinked with diazidostilbene did not show toxic effects on L929 cells. The hyaluronic acid-based hydrogels have potential as biomaterials for cell culture or soft tissue regeneration applications.
Novel hydrogels based on hydroxyethyl starch modified with polyethylene glycol methacrylate (HES-P(EG)(6)MA) were developed as delivery system for the controlled release of proteins. Since the drug release behavior is supposed to be related to the pore structure of the hydrogel network the pore sizes were determined by cryo-SEM, which is a mild technique for imaging on a nanometer scale. The results showed a decreasing pore size and an increase in pore homogeneity with increasing polymer concentration. Furthermore, the mesh sizes of the hydrogels were calculated based on swelling data. Pore and mesh size were significantly different which indicates that both structures are present in the hydrogel. The resulting structural model was correlated with release data for bulk hydrogel cylinders loaded with FITC-dextran and hydrogel microspheres loaded with FITC-IgG and FITC-dextran of different molecular size. The initial release depended much on the relation between hydrodynamic diameter and pore size while the long term release of the incorporated substances was predominantly controlled by degradation of the network of the much smaller meshes.
Thick poly(styrene-b-monomethoxydiethylenglycol-acrylate-b-styrene) [P(S-b-MDEGA-b-S)] films (thickness 5 mu m) are prepared from different solvents on flexible substrates by solution casting and investigated with small-angle X-ray scattering. As the solvents are either PS- or PMDEGA-selective, micelles with different core-shell micellar structures are formed. In PMDEGA-selective solvents, the PS block is the core and PMDEGA is the shell, whereas in PS-selective solvents, the order is reversed. After exposing the films to liquid D2O, the micellar structure inside the films prepared from PMDEGA-selective solvents remains unchanged and only the PMDEGA (shell part) swells. On the contrary, in the films prepared from PS-selective solvents, the micelles revert the core and the shell. This reversal causes more entanglements of the PMDEGA chains between the micelles. Moreover, the thermal collapse transition of the PMDEGA block in liquid D2O is significantly broadened. Irrespective of the solvent used for film preparation, the swollen PMDEGA shell does not show a prominent shrinkage when passing the phase transition, and the transition process occurs via compaction. The collapsed micelles have a tendency to densely pack above the transition temperature.
In this thesis, a route to temperature-, pH-, solvent-, 1,2-diol-, and protein-responsive sensors made of biocompatible and low-fouling materials is established. These sensor devices are based on the sensitivemodulation of the visual band gap of a photonic crystal (PhC), which is induced by the selective binding of analytes, triggering a volume phase transition.
The PhCs introduced by this work show a high sensitivity not only for small biomolecules, but also for large analytes, such as glycopolymers or proteins. This enables the PhC to act as a sensor that detects analytes without the need of complex equipment.
Due to their periodical dielectric structure, PhCs prevent the propagation of specific wavelengths. A change of the periodicity parameters is thus indicated by a change in the reflected wavelengths. In the case explored, the PhC sensors are implemented as periodically structured responsive hydrogels in formof an inverse opal.
The stimuli-sensitive inverse opal hydrogels (IOHs) were prepared using a sacrificial opal template of monodispersed silica particles. First, monodisperse silica particles were assembled with a hexagonally packed structure via vertical deposition onto glass slides. The obtained silica crystals, also named colloidal crystals (CCs), exhibit structural color. Subsequently, the CCs templates were embedded in polymer matrix with low-fouling properties. The polymer matrices were composed of oligo(ethylene glycol) methacrylate derivatives (OEGMAs) that render the hydrogels thermoresponsive. Finally, the silica particles were etched, to produce highly porous hydrogel replicas of the CC. Importantly, the inner structure and thus the ability for light diffraction of the IOHs formed was maintained.
The IOH membrane was shown to have interconnected pores with a diameter as well as interconnections between the pores of several hundred nanometers. This enables not only the detection of small analytes, but also, the detection of even large analytes that can diffuse into the nanostructured IOH membrane. Various recognition unit – analyte model systems, such as benzoboroxole – 1,2-diols, biotin – avidin and mannose – concanavalin A, were studied by incorporating functional
comonomers of benzoboroxole, biotin and mannose into the copolymers. The incorporated recognition units specifically bind to certain low and highmolar mass biomolecules, namely to certain saccharides, catechols, glycopolymers or proteins.
Their specific binding strongly changes the overall hydrophilicity, thus modulating the swelling of the IOH matrices, and in consequence, drastically changes their internal periodicity. This swelling is amplified by the thermoresponsive properties of the polymer matrix. The shift of the interference band gap due to the specific molecular recognition is easily visible by the naked eye (up to 150 nm shifts). Moreover, preliminary trial were attempted to detect even larger entities. Therefore anti-bodies were immobilized on hydrogel platforms via polymer-analogous esterification. These platforms incorporate comonomers made of tri(ethylene glycol) methacrylate end-functionalized with a carboxylic acid. In these model systems, the bacteria analytes are too big to penetrate into the IOH membranes, but can only interact with their surfaces. The selected model bacteria, as Escherichia coli, show a specific affinity to anti-body-functionalized hydrogels. Surprisingly in the case functionalized IOHs, this study produced weak color shifts, possibly opening a path to detect directly living organism, which will need further investigations.
Diese Arbeit befasst sich mit der Synthese und der Charakterisierung von thermoresponsiven Polymeren und ihrer Immobilisierung auf festen Oberflächen als nanoskalige dünne Schichten. Dabei wurden thermoresponsive Polymere vom Typ der unteren kritischen Entmischungstemperatur (engl.: lower critical solution temperature, LCST) verwendet. Sie sind bei niedrigeren Temperaturen im Lösungsmittel gut und nach Erwärmen oberhalb einer bestimmten kritischen Temperatur nicht mehr löslich; d. h. sie weisen bei einer bestimmten Temperatur einen Phasenübergang auf. Als Basismaterial wurden verschiedene thermoresponsive und biokompatible Polymere basierend auf Diethylenglykolmethylethermethacrylat (MEO2MA) und Oligo(ethylenglykol)methylethermethacrylat (OEGMA475, Mn = 475 g/ mol) über frei radikalische Copolymerisation synthetisiert. Der thermoresponsive Phasenübergang der Copolymere wurde in wässriger Lösung und in gequollenen vernetzten dünnen Schichten beobachtet. Außerdem wurde untersucht, inwiefern eine selektive Proteinbindung an geeignete funktionalisierte Copolymere die Phasenübergangstemperatur beeinflusst. Die thermoresponsiven Copolymere wurden über photovernetzbare Gruppen auf festen Oberflächen immobilisiert. Die nötigen lichtempfindlichen Vernetzereinheiten wurden mittels des polymerisierbaren Benzophenonderivates 2 (4 Benzoylphenoxy)ethylmethacrylat (BPEM) in das Copolymer integriert. Dünne Filme der Copolymere mit ca. 100 nm Schichtdicke wurden über Rotationsbeschichtung auf Siliziumwafer aufgeschleudert und anschließend durch Bestrahlung mit UV Licht vernetzt und auf der Oberfläche immobilisiert. Die Filme sind stabiler je größer der Vernetzeranteil und je größer die Molmasse der Copolymere ist. Bei einem Waschprozess nach der Vernetzung wird beispielsweise aus einem Film mit moderater Molmasse und geringem Vernetzeranteil mehr unvernetztes Copolymer ausgewaschen als bei einem höhermolekularen Copolymer mit hohem Vernetzeranteil. Die Quellbarkeit der Polymerschichten wurde mit Ellipsometrie untersucht. Sie ist größer je geringer der Vernetzeranteil in den Copolymeren ist. Schichten aus thermoresponsiven OEG Copolymeren zeigen einen Volumenphasenübergang vom Typ der LCST. Der thermoresponsive Kollaps der Schichten ist komplett reversibel, die Kollapstemperatur kann über die Zusammensetzung der Copolymere eingestellt werden. Für einen Vergleich dieser Eigenschaften mit dem gut charakterisierten und derzeit wohl am häufigsten untersuchten thermoresponsiven Polymer Poly(N-isopropylacrylamid) (PNIPAM) wurden zusätzlich photovernetzte Schichten aus PNIPAM hergestellt und ebenfalls ellipsometrisch vermessen. Im Vergleich zu PNIPAM verläuft der Phasenübergang der Schichten aus den Copolymeren mit Oligo(ethylenglykol)-seitenketten (OEG Copolymere) über einen größeren Temperaturbereich. Mit Licht einer Wellenlänge > 300 nm wurden die photosensitiven Benzophenongruppen selektiv angeregt. Bei der Verwendung kleinerer Wellenlängen vernetzten die Copolymerschichten auch ohne die Anwesenheit der lichtempfindlichen Benzophenongruppen. Dieser Effekt ließ sich zur kontrollierten Immobilisierung und Vernetzung der OEG Copolymere einsetzen. Als weitere Methode zur Immobilisierung der Copolymere wurde die Anbindung über Amidbindungen untersucht. Dazu wurden OEG Copolymere mit dem carboxylgruppenhaltigen 2 Succinyloxyethylmethacrylat (MES) auf mit 3 Aminopropyldimethylethoxysilan (APDMSi) silanisierte Siliziumwafer rotationsbeschichtet, und mit dem oligomeren α, ω Diamin Jeffamin® ED 900 vernetzt. Die Vernetzungsreaktion erfolgte ohne weitere Zusätze durch Erhitzen der Proben. Die Hydrogelschichten waren anschließend stabil und zeigten neben thermoresponsivem auch pH responsives Verhalten. Um zu untersuchen, ob die Phasenübergangstemperatur durch eine Proteinbindung beeinflusst werden kann, wurde ein polymerisierbares Biotinderivat 2 Biotinyl-aminoethylmethacrylat (BAEMA) in das thermoresponsive Copolymer eingebaut. Der Einfluss des biotinbindenen Proteins Avidin auf das thermoresponsive Verhalten des Copolymers in Lösung wurde untersucht. Die spezifische Bindung von Avidin an das biotinylierte Copolymer verschob die Übergangstemperatur deutlich zu höheren Temperaturen. Kontrollversuche zeigten, dass dieses Verhalten auf eine selektive Proteinbindung zurückzuführen ist. Thermoresponsive OEG Copolymere mit photovernetzbaren Gruppen aus BPEM und Biotingruppen aus BAEMA wurden über Rotationsbeschichtung auf Gold- und auf Siliziumoberflächen aufgetragen und durch UV Strahlung vernetzt. Die spezifische Bindung von Avidin an die Copolymerschicht wurde mit Oberflächenplasmonenresonanz und Ellipsometrie untersucht. Die Bindungskapazität der Schichten war umso größer, je kleiner der Vernetzeranteil, d. h. je größer die Maschenweite des Netzwerkes war. Die Quellbarkeit der Schichten wurde durch die Avidinbindung erhöht. Bei hochgequollenen Systemen verursachte eine Mehrfachbindung des tetravalenten Avidins allerdings eine zusätzliche Quervernetzung des Polymernetzwerkes. Dieser Effekt wirkt der erhöhten Quellbarkeit durch die Avidinbindung entgegen und lässt die Polymernetzwerke schrumpfen.