@phdthesis{Tunn2020,
  author    = {Tunn, Isabell},
  title     = {From single molecules to bulk materials: tuning the viscoelastic properties of coiled coil cross-linked hydrogels},
  doi       = {10.25932/publishup-47595},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-475955},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XVI, 140},
  year      = {2020},
  abstract  = {The development of bioinspired self-assembling materials, such as hydrogels, with promising applications in cell culture, tissue engineering and drug delivery is a current focus in material science. Biogenic or bioinspired proteins and peptides are frequently used as versatile building blocks for extracellular matrix (ECM) mimicking hydrogels. However, precisely controlling and reversibly tuning the properties of these building blocks and the resulting hydrogels remains challenging. Precise control over the viscoelastic properties and self-healing abilities of hydrogels are key factors for developing intelligent materials to investigate cell matrix interactions. Thus, there is a need to develop building blocks that are self-healing, tunable and self-reporting. This thesis aims at the development of α-helical peptide building blocks, called coiled coils (CCs), which integrate these desired properties. Self-healing is a direct result of the fast self-assembly of these building blocks when used as material cross-links. Tunability is realized by means of reversible histidine (His)-metal coordination bonds. Lastly, implementing a fluorescent readout, which indicates the CC assembly state, self-reporting hydrogels are obtained. Coiled coils are abundant protein folding motifs in Nature, which often have mechanical function, such as in myosin or fibrin. Coiled coils are superhelices made up of two or more α-helices wound around each other. The assembly of CCs is based on their repetitive sequence of seven amino acids, so-called heptads (abcdefg). Hydrophobic amino acids in the a and d position of each heptad form the core of the CC, while charged amino acids in the e and g position form ionic interactions. The solvent-exposed positions b, c and f are excellent targets for modifications since they are more variable. His-metal coordination bonds are strong, yet reversible interactions formed between the amino acid histidine and transition metal ions (e.g. Ni2+, Cu2+ or Zn2+). His-metal coordination bonds essentially contribute to the mechanical stability of various high-performance proteinaceous materials, such as spider fangs, Nereis worm jaws and mussel byssal threads. Therefore, I bioengineered reversible His-metal coordination sites into a well-characterized heterodimeric CC that served as tunable material cross-link. Specifically, I took two distinct approaches facilitating either intramolecular (Chapter 4.2) and/or intermolecular (Chapter 4.3) His-metal coordination. Previous research suggested that force-induced CC unfolding in shear geometry starts from the points of force application. In order to tune the stability of a heterodimeric CC in shear geometry, I inserted His in the b and f position at the termini of force application (Chapter 4.2). The spacing of His is such that intra-CC His-metal coordination bonds can form to bridge one helical turn within the same helix, but also inter-CC coordination bonds are not generally excluded. Starting with Ni2+ ions, Raman spectroscopy showed that the CC maintained its helical structure and the His residues were able to coordinate Ni2+. Circular dichroism (CD) spectroscopy revealed that the melting temperature of the CC increased by 4 °C in the presence of Ni2+. Using atomic force microscope (AFM)-based single molecule force spectroscopy, the energy landscape parameters of the CC were characterized in the absence and the presence of Ni2+. His-Ni2+ coordination increased the rupture force by ~10 pN, accompanied by a decrease of the dissociation rate constant. To test if this stabilizing effect can be transferred from the single molecule level to the bulk viscoelastic material properties, the CC building block was used as a non-covalent cross-link for star-shaped poly(ethylene glycol) (star-PEG) hydrogels. Shear rheology revealed a 3-fold higher relaxation time in His-Ni2+ coordinating hydrogels compared to the hydrogel without metal ions. This stabilizing effect was fully reversible when using an excess of the metal chelator ethylenediaminetetraacetate (EDTA). The hydrogel properties were further investigated using different metal ions, i.e. Cu2+, Co2+ and Zn2+. Overall, these results suggest that Ni2+, Cu2+ and Co2+ primarily form intra-CC coordination bonds while Zn2+ also participates in inter-CC coordination bonds. This may be a direct result of its different coordination geometry. Intermolecular His-metal coordination bonds in the terminal regions of the protein building blocks of mussel byssal threads are primarily formed by Zn2+ and were found to be intimately linked to higher-order assembly and self-healing of the thread. In the above example, the contribution of intra-CC and inter-CC His-Zn2+ cannot be disentangled. In Chapter 4.3, I redesigned the CC to prohibit the formation of intra-CC His-Zn2+ coordination bonds, focusing only on inter-CC interactions. Specifically, I inserted His in the solvent-exposed f positions of the CC to focus on the effect of metal-induced higher-order assembly of CC cross-links. Raman and CD spectroscopy revealed that this CC building block forms α-helical Zn2+ cross-linked aggregates. Using this CC as a cross-link for star-PEG hydrogels, I showed that the material properties can be switched from viscoelastic in the absence of Zn2+ to elastic-like in the presence of Zn2+. Moreover, the relaxation time of the hydrogel was tunable over three orders of magnitude when using different Zn2+:His ratios. This tunability is attributed to a progressive transformation of single CC cross-links into His-Zn2+ cross-linked aggregates, with inter-CC His-Zn2+ coordination bonds serving as an additional, cross-linking mode. Rheological characterization of the hydrogels with inter-CC His-Zn2+ coordination raised the question whether the His-Zn2+ coordination bonds between CCs or also the CCs themselves rupture when shear strain is applied. In general, the amount of CC cross-links initially formed in the hydrogel as well as the amount of CC cross-links breaking under force remains to be elucidated. In order to more deeply probe these questions and monitor the state of the CC cross-links when force is applied, a fluorescent reporter system based on F{\"o}rster resonance energy transfer (FRET) was introduced into the CC (Chapter 4.4). For this purpose, the donor-acceptor pair carboxyfluorescein and tetramethylrhodamine was used. The resulting self-reporting CC showed a FRET efficiency of 77 \% in solution. Using this fluorescently labeled CC as a self-reporting, reversible cross-link in an otherwise covalently cross-linked star-PEG hydrogel enabled the detection of the FRET efficiency change under compression force. This proof-of-principle result sets the stage for implementing the fluorescently labeled CCs as molecular force sensors in non-covalently cross-linked hydrogels. In summary, this thesis highlights that rationally designed CCs are excellent reversibly tunable, self-healing and self-reporting hydrogel cross-links with high application potential in bioengineering and biomedicine. For the first time, I demonstrated that His-metal coordination-based stabilization can be transferred from the single CC level to the bulk material with clear viscoelastic consequences. Insertion of His in specific sequence positions was used to implement a second non-covalent cross-linking mode via intermolecular His-metal coordination. This His-metal binding induced aggregation of the CCs enabled for reversibly tuning the hydrogel properties from viscoelastic to elastic-like. As a proof-of-principle to establish self-reporting CCs as material cross-links, I labeled a CC with a FRET pair. The fluorescently labelled CC acts as a molecular force sensor and first preliminary results suggest that the CC enables the detection of hydrogel cross-link failure under compression force. In the future, fluorescently labeled CC force sensors will likely not only be used as intelligent cross-links to study the failure of hydrogels but also to investigate cell-matrix interactions in 3D down to the single molecule level.},
  language  = {en}
}
@phdthesis{Niedl2015,
  author    = {Niedl, Robert Raimund},
  title     = {Nichtlineare Kinetik und responsive Hydrogele f{\"u}r papierbasierte Schnelltestanwendungen},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-77735},
  school      = {Universit{\"a}t Potsdam},
  pages     = {iv, 128},
  year      = {2015},
  abstract  = {Viele klinische Schnelltestsysteme ben{\"o}tigen vorpr{\"a}parierte oder aufgereinigte Analyte mit frisch hergestellten L{\"o}sungen. Fernab standardisierter Laborbedingungen wie z.B. in Entwicklungsl{\"a}ndern oder Krisengebieten sind solche Voraussetzungen oft nur unter einem hohen Aufwand herstellbar. Zus{\"a}tzlich stellt die erforderliche Sensitivit{\"a}t die Entwicklung einfach zu handhabender Testsysteme vor große Herausforderungen. Autokatalytische Reaktionen, die sich mit Hilfe sehr geringer Initiatorkonzentrationen ausl{\"o}sen lassen, k{\"o}nnen hier eine Perspektive f{\"u}r Signalverst{\"a}rkungsprozesse bieten. Aus diesem Grund wird im ersten Teil der vorliegenden Arbeit das Verhalten der autokatalytischen Arsenit-Jodat-Reaktion in einem mikrofluidischen Kanal untersucht. Dabei werden insbesondere die diffusiven und konvektiven Einfl{\"u}sse auf die Reaktionskinetik im Vergleich zu makroskopischen Volumenmengen betrachtet. Im zweiten Teil werden thermoresponsive Hydrogele mit einem kanalstrukturierten Papiernetzwerk zu einem neuartigen, kapillargetriebenen, extern steuerbaren Mikrofluidik-System kombiniert. Das hier vorgestellte Konzept durch Hydrogele ein papierbasiertes LOC-System zu steuern, erm{\"o}glicht zuk{\"u}nftig die Herstellung von komplexeren, steuerbaren Point-Of-Care Testsystemen (POCT). Durch z.B. einen thermischen Stimulus, wird das L{\"o}sungsverhalten eines Hydrogels so ver{\"a}ndert, dass die gespeicherte Fl{\"u}ssigkeit freigesetzt und durch die Kapillarkraft des Papierkanals ins System transportiert wird. Die Eigenschaften dieses Gelnetzwerks k{\"o}nnen dabei so eingestellt werden, dass eine Freisetzung von Fl{\"u}ssigkeiten sogar bei K{\"o}rpertemperatur m{\"o}glich w{\"a}re und damit eine Anwendung g{\"a}nzlich ohne weitere Hilfsmittel denkbar ist. F{\"u}r die Anwendung notwendige Chemikalien oder Enzyme lassen sich hierbei bequem in getrocknetem Zustand im Papiersubstrat vorlagern und bei Bedarf in L{\"o}sung bringen. Im abschließenden dritten Teil der Arbeit wird ein durch Hydrogele betriebener, Antik{\"o}rper-basierter Mikroorganismenschnelltest f{\"u}r Escherichia coli pr{\"a}sentiert. Dar{\"u}ber hinaus wird weiterf{\"u}hrend eine einfache Methode zur Funktionalisierung eines Hydrogels mit Biomolek{\"u}len {\"u}ber EDC/NHS-Kopplung vorgestellt.},
  language  = {de}
}
@article{TartivelBehlSchroeteretal.2012,
  author    = {Tartivel, Lucile and Behl, Marc and Schr{\"o}ter, Michael and Lendlein, Andreas},
  title     = {Hydrogel networks based on ABA triblock copolymers},
  series = {Journal of applied biomaterials \& functional materials},
  volume    = {10},
  journal   = {Journal of applied biomaterials \& functional materials},
  number    = {3},
  publisher = {Wichtig},
  address   = {Milano},
  issn      = {2280-8000},
  doi       = {10.5301/JABFM.2012.10295},
  pages     = {243 -- 248},
  year      = {2012},
  abstract  = {Background: Triblock copolymers from hydrophilic oligo(ethylene glycol) segment A and oligo(propylene glycol) segment B, providing an ABA structure (OEG-OPG-OEG triblock), are known to be biocompatible and are used as self-solidifying gels in drug depots. A complete removal of these depots would be helpful in cases of undesired side effects of a drug, but this remains a challenge as they liquefy below their transition temperature. Therefore we describe the synthesis of covalently cross-linked hydrogel networks. Method: Triblock copolymer-based hydrogels were created by irradiating aqueous solutions of the corresponding macro-dimethacrylates with UV light. The degree of swelling, swelling kinetics, mechanical properties and morphology of the networks were investigated. Results: Depending on precursor concentration, equilibrium degree of swelling of the films ranged between 500\% and 880\% and was reached in 1 hour. In addition, values for storage and loss moduli of the hydrogel networks were in the 100 Pa to 10 kPa range. Conclusion: Although OEG-OPG-OEG triblocks are known for their micellization, which could hamper polymer network formation, reactive OEG-OPG-OEG triblock oligomers could be successfully polymerized into hydrogel networks. The degree of swelling of these hydrogels depends on their molecular weight and on the oligomer concentration used for hydrogel preparation. In combination with the temperature sensitivity of the ABA triblock copolymers, it is assumed that such hydrogels might be beneficial for future medical applications -e.g., removable drug release systems.},
  language  = {en}
}
@article{NeffeLoebusZaupaetal.2011,
  author    = {Neffe, Axel T. and Loebus, Axel and Zaupa, Alessandro and St{\"o}tzel, Christian and M{\"u}ller, Frank A. and Lendlein, Andreas},
  title     = {Gelatin functionalization with tyrosine derived moieties to increase the interaction with hydroxyapatite fillers},
  series = {Acta biomaterialia},
  volume    = {7},
  journal   = {Acta biomaterialia},
  number    = {4},
  publisher = {Elsevier},
  address   = {Oxford},
  issn      = {1742-7061},
  doi       = {10.1016/j.actbio.2010.11.025},
  pages     = {1693 -- 1701},
  year      = {2011},
  abstract  = {Combining gelatins functionalized with the tyrosine-derived groups desaminotyrosine or desaminotyrosyl tyrosine with hydroxyapatite (HAp) led to the formation of composite materials with much lower swelling ratios than those of the pure matrices. Shifts of the infra-red (IR) bands related to the free carboxyl groups could be observed in the presence of HAp, which suggested a direct interaction of matrix and filler that formed additional physical cross-links in the material. In tensile tests and rheological measurements the composites equilibrated in water had increased Young's moduli (from 200 kPa up to 2 MPa) and tensile strengths (from 57 kPa up to 1.1 MPa) compared with the matrix polymers without affecting the elongation at break. Furthermore, an increased thermal stability of the networks from 40 to 85 degrees C could be demonstrated. The differences in the behaviour of the functionalized gelatins compared with pure gelatin as a matrix suggested an additional stabilizing bond between the incorporated aromatic groups and the HAp as supported by the IR results. The composites can potentially be applied as bone fillers.},
  language  = {en}
}