Design and Synthesis of Multifunctional Hybrid Hydrogels for Medicinal Applications

Abstract

Hydrogels have sparked great interest in life sciences as they are an ideal material class for many medical applications such as drug delivery and tissue engineering. In particular, the development of hybrid hydrogel materials is a major focus of current research efforts, since the combination of natural (macro-)molecules with synthetic compounds allows for engineering of application-oriented conjugates with complementing properties. Despite extensive research, many proposed hydrogels suffer from heterogeneity regarding their chemical composition, which complicates precise adjustment of their physical and pharmacological properties. Chemically defined hydrogels that are able to serve as biocompatible and -degradable cell matrices featuring controlled and rapid gelation, excellent self-healing, a well-defined and simple introduction of functionalities, as well as general stability during cell cultivation are currently still difficult to achieve. In this Doctoral thesis, the design and preparation of novel hybrid hydrogels mainly in the context of tissue engineering is explored, with an aim to improve material properties and incorporate the above-mentioned features in a single hydrogel system. The hydrogels are tested as artificial cell matrices for regeneration of diseased or traumatized tissue, with a potential modulation of endogenous metabolic processes via the delivery of therapeutically active compounds. By creating hybrid hydrogels, this work bridges existing gels made from either fully synthetic or natural systems. Taking the architecture of the natural ECM as an inspiration, the development of novel hybrid hydrogels is explored by employing supramolecular cross-linking of covalent backbones to overcome the current limitations of conventional hydrogels. Furthermore, this thesis provides a detailed investigation of material properties such as gelation, mechanical properties, release of active ingredients degradability and compatibility with different cell types. Here, biopolymers in combination with programmable structural elements such as DNA and peptides are investigated.

Nucleic acids are becoming increasingly important as versatile building blocks for next generation hydrogel materials. Within this work, a comprehensive and critical overview of the available examples of DNA-based hydrogels and their potential applications in life sciences is provided, describing how the structure of DNA materials influences their various properties. The most important synthetic concepts for the production of all-DNA hydrogels as well as various DNA-based hybrids are described and comprehensively reviewed in view of new concepts, properties, and applications. Excellent programmability and perfect sequence control are offered by DNA, making it an appealing candidate as a supramolecular cross-linker. However, to achieve a final material that is well-defined, all hydrogel components need to be produced with a high level of precision, including the covalent backbone. Denatured proteins, such as human serum albumin (HSA) represent a new class of precision polymers that have a pre-defined sequence and chain length and different functional groups for further chemical modifications. The combination of both materials for the production of highly defined hydrogels for biomedicine is investigated. In this context, the applicability of protein-DNA hydrogels for spatiotemporally controlled release of therapeutically active compounds is successfully demonstrated. To prepare the backbone of these hybrid hydrogels, HSA is transferred into a brush-like protein-derived precision copolymer. Subsequently, physical hydrogels are formed by sequence-specific DNA hybridization of backbone-grafted DNA tags with small, complementary, dendritic DNA linkers, which can simultaneously introduce therapeutically active compounds into the gel. In this way, a rapid, highly specific and stable loading as well as controlled release of DNA-tagged bioactive cargo is realized. In this context, deoxyribonuclease triggered C3 toxin release allows efficient and specific inhibition of osteoclast formation and bone resorption without affecting osteoblastic differentiation and mineralization in vitro, confirming the suitability for local treatment of bone diseases such as osteoporosis via local drug delivery on a controlled time scale. In a second example for hybrid hydrogels, self-assembling peptides (SAPs) capable of forming nanofibrous structures instead of DNA strands are implemented as supramolecular cross-linkers. While these SAP-based systems do not reach the same level of programmability as DNA, they are much easier and more cost-effective to synthesize on larger scale and still allow the preparation of highly defined 3D materials. Additionally, the formed peptide nanofibrils (PNFs) promise improved biological performance, since they resemble the fibrous morphology of natural ECM. Here, pH-responsive, amphiphilic peptide gelators, so-called depsi peptides, are created and investigated for their ability to self-assemble into various nanostructures and finally induce hydrogel cross-linking. These depsi peptides are unable to assemble into PNFs under acidic conditions, whereas upon increasing the pH to neutral, an intramolecular O–N–acyl migration reaction causes instantaneous PNF formation with β-sheet structures. Conjugating depsi peptides onto the HSA-derived backbone transfers the beneficial pH-responsive aggregation behavior onto the hybrid. At higher mass concentration, the hybrid forms a highly porous hydrogel with instantaneous gelation at neutral pH. The PNF cross-linked hybrids show thixotropic behavior with ultra-fast, near-quantitative recovery after multiple shear cycles. The gels are injectable and their mechanical properties can be adjusted by changing the number of peptide grafts or the solid content. The material is shown to support the survival and growth of various cell types. Even the active migration of endothelial cells into the material after topological seeding is demonstrated, being a distinguishing feature compared to many other types of hydrogels. In view of all these findings, the pH-responsive PNF cross-linked hydrogel offers excellent potential as a regenerative scaffold for biomedicine. To further evolve the depsi peptides as controllable nanomaterial building blocks, the responsiveness of assembly is extended to other physiologically relevant stimuli besides pH, while methionine-containing peptides provide an orthogonal “responsive unit” for controlled disassembly under oxidative conditions. To create a material that combines the scalability of peptides with the programmability of DNA, dynamic covalent interactions between boronic acids (BAs) and catechols (CAs) are developed into synthetic nucleobase analogs to encode molecular recognition in a stimuli-responsive way. Simulating DNA by creating and arranging BA or CA residues along a defined peptide backbone, their complementary binding is analyzed, and cytochrome c is functionalized with polyethylene glycol using trivalent tag-conjugated derivatives, indicating that such a synthetic code may also allow to program dynamic macromolecular architectures.