Engineered human tissue and disease models can decrease the cost and time of developing new drugs and treatments, facilitate personalized medicine, and eliminate the need for animal models that poorly represent the human body and are ethically problematic. However, the current conventional cell expansion methods using 2D culture flasks cannot enable the development of such complex multi-cellular 3D models. In general, hydrogels are considered promising materials that can make the biofabrication of tissue models possible. Hydrogels are highly hydrated materials comprised of either synthetic or naturally derived polymers, or a combination of both, and can form an environment mimicking the biomacromolecular network surrounding cells in the body. This network of biopolymers, known as extracellular matrix (ECM), is comprised of proteins such as collagen, laminin, fibronectin, and polysaccharides such as hyaluronan (HA), heparan, keratan, and chondroitin sulfate. The design of hydrogels representing the physical and biochemical properties of the ECM and which can be used for biofabrication is challenging but of increasing interest due to the rapid progress in the development of 3D and 4D bioprinting techniques. As the ECM properties differ between various tissues and disease conditions and change over time, a dynamic modular hydrogel system is needed to that can be optimized for each cell and tissue type. This thesis aims to develop modular enzyme-responsive polysaccharide-based hydrogels for 3D cell culture and biofabrication. The natural polysaccharides, hyaluronic acid (HA) and alginate (Alg) were used as the main backbone in the hydrogels developed in this thesis. HA was modified by conjugating bicyclo[6.1.0]non-4-yne (BCN) to the backbone to form HA-BCN-based hydrogels by a bioorthogonal strain-promoted alkyne-azide cycloaddition. The click reaction between BCN and azide groups allowed for modulating the biochemical and mechanical properties of the HA-BCN hydrogels. HA-BCN was further decorated with peptides to explore peptide folding and dimerization-mediated dynamic cross-linking and biofunctionalization. This system was further used to explore possibilities to dynamically alter the properties of 3D bioprinted structures, mimicking the biomineralization process in bone tissue. In a different study, a tumor model comprising fibroblast and breast cancer cells (MCF7) was bioprinted using HA-BCN cross-linked by matrix metalloporotease (MMP) cleavable and PEG-diazide MMP-resistant cross-linkers, demonstrating the synergistic relationship between hydrogel degradability and cancer cell growth, intensified by the presence of fibroblasts. The possibility of incorporating a conductive module into this hydrogel system was explored using the enzyme-assisted polymerization of ETE-S to form an interpenetrating conductive network inside HA-BCN hydrogel. The in situ and user-triggered polymerization of conductive ETE-S was demonstrated after 3D printing HA-BCN bioink containing ETE-S monomers into a lattice shape structure. We also demonstrated that cellulose nanofibrils (CNF) improved the printability of HA-BCN bioinks, and this hybrid bioink was used for printing self-standing cell-laden 3D structures. Besides these studies, a novel enzymatically triggered thiol-based chemistry was developed to address the unwanted oxidation of thiol-containing hydrogels and decrease the off-target thiol reactions during hydrogel synthesis and formation. Alginate containing sulfhydryl moieties, protected by an enzyme-labile Phacm group (AlgCP), was treated with penicillin G acylase and subsequently formed a disulfide cross-linked hydrogel. We studied the gelation kinetics and rheological properties of AlgCP and different modes of cross-linking by reversible disulfide bonds, a thiol-maleimide Micheale-type addition reaction, and ionic interactions between alginate and Ca²⁺ ions. MCF7 breast cancer cells cultured in the AlgCP hydrogels formed spheroids that could be harvested by GSH dissolution of the hydrogels. Finally, this novel chemistry enabled bioprinting of multi-material 3D structures with control over the printed structure's physiochemical properties, including the type and density of cross-linkers. Bioprinted fibroblasts formed extended morphology, and MCF7 cells formed spheroids in the bioprinted lattice structures.   

The hydrogel systems developed and explored in this thesis are modular and exhibit dynamic and tunable properties, and are applicable for a wide range of 3D cell culture and bioprinting applications. The hydrogels were either formed in response to the activity of an enzyme or remodeled by enzymes. Both enzyme-responsive HA-BCN and AlgCP hydrogel systems are promising bioinks for generating more elaborate and spatially defined cell-laden 3D structures whose features can be altered post-printing by cell-secreted and extrinsic reagents. These hydrogel-based toolkits can play a vital role in developing tissue and disease models that can make the drug discovery process faster, cheaper, and animal-free. 

Bioprinting of hydrogel-based bioinks can allow for thefabricationof elaborate, cell-laden 3D structures. In addition to providing anadequate extracellular matrix mimetic environment and high cell viability,the hydrogels must offer facile extrusion through the printing nozzleand retain the shape of the printed structure. We demonstrate a strategyto incorporate cellulose oxalate nanofibrils in hyaluronan-based hydrogelsto generate shear thinning bioinks that allowed for printing of free-standingmultilayer structures, covalently cross-linked after bioprinting,yielding long-term stability. The storage modulus of the hydrogelswas tunable between 0.5 and 1.5 kPa. The nanocellulose containinghydrogels showed good biocompatibility, with viability of primaryhuman dermal fibroblasts above 80% at day 7 after seeding. The cellswere also shown to tolerate the printing process well, with viabilityabove 80% 24 h after printing. We anticipate that this hydrogel systemcan find broad use as a bioink to produce complex geometries thatcan support cell growth.

Thiolated polymers are widely used in hydrogels for drug delivery, tissue engineering, and biofabrication. The oxidation of thiols is spontaneous, resulting in the formation of disulfide bridges and cross linking of polymers. The cross-linking process is, however, difficult to control and is initiated directly when the thiolated components are exposed to ambient conditions, which significantly complicates handling of the materials. Here, we show a fully bioorthogonal enzyme-mediated thiol-based chemistry for dynamic covalent cross-linking of carbohydrate-based hydrogels that circumvents the problems with uncontrolled thiol oxidation. Alginate was modified with cysteine residues, protected by an enzyme-labile thiol-protecting group (Phacm). Releasing the Phacm group by penicillin G acylase generates free thiols that oxidize under physiological conditions, resulting in a reversible cross-linking and formation of hydrogels with tunable stiffness. Prior to deprotection, the components can be exposed to ambient conditions. The enzyme-triggered deprotection and subsequent gelation allows for encapsulation of cells and 3D bioprinting of cell-laden hydrogel structures. Remaining deprotected thiols enabled postprinting modifications and hydrogel self-healing. The proposed hydrogel synthesis strategy significantly increases the versatility of thiol-based cross-linking chemistries and provides new possibilities to generate dynamic covalent hydrogels for a broad range of biomedical applications.

Coiled coils are characterized by an arrangement of two or more alpha-helices into a superhelix and one of few protein motifs where the sequence-to-structure relationship to a large extent have been decoded and understood. The abundance of both natural and de novo designed coil coils provides a rich molecular toolbox for self assembly of elaborate bespoke molecular architectures, nanostructures, and materials. Leveraging on the numerous possibilities to tune both affinities and preferences for polypeptide oligomerization, coiled coils offer unique possibilities to design modular and dynamic assemblies that can respond in a predictable manner to biomolecular interactions and subtle physicochemical cues. In this review, strategies to use coiled coils in design of novel therapeutics and advanced drug delivery systems are discussed. The applications of coiled coils for generating drug carriers and vaccines, and various aspects of using coiled coils for controlling and triggering drug release, and for improving drug targeting and drug uptake are described. The plethora of innovative coiled coil-based molecular systems provide new knowledge and techniques for improving efficacy of existing drugs and can facilitate development of novel therapeutic strategies. (c) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). 1. Introduction 27 2. The coiled coil motif 27 3. Coiled coils and coiled coil-hybrids for drug delivery and therapeutics 30 3.1. Coiled coils in liposome drug delivery systems 30 3.2. Lipidated coiled coils for assembly of virus-like particles 31 3.3. Coiled coil nanoparticles 31 3.4. Coiled coil nanocarriers 33 3.5. Coiled coil polymer-hybrids 33 3.6. Coiled coil-based hydrogels 36 3.7. Coiled coil inorganic nanoparticle hybrids 37 3.8. Coiled coils combined with cell penetrating peptides 37 3.9. Coiled coils for improved targeting 38

Hydrogels are used in a wide range of biomedical applications, including three-dimensional (3D) cell culture, cell therapy and bioprinting. To enable processing using advanced additive fabrication techniques and to mimic the dynamic nature of the extracellular matrix (ECM), the properties of the hydrogels must be possible to tailor and change over time with high precision. The design of hydrogels that are both structurally and functionally dynamic, while providing necessary mechanical support is challenging using conventional synthesis techniques. Here, we show a modular and 3D printable hydrogel system that combines a robust but tunable covalent bioorthogonal cross-linking strategy with specific peptide-folding mediated interactions for dynamic modulation of cross-linking and functionalization. The hyaluronan-based hydrogels were covalently cross-linked by strain-promoted alkyne-azide cycloaddition using multi-arm poly(ethylene glycol). In addition, ade novodesigned helix-loop-helix peptide was conjugated to the hyaluronan backbone to enable specific peptide-folding modulation of cross-linking density and kinetics, and hydrogel functionality. An array of complementary peptides with different functionalities was developed and used as a toolbox for supramolecular tuning of cell-hydrogel interactions and for controlling enzyme-mediated biomineralization processes. The modular peptide system enabled dynamic modifications of the properties of 3D printed structures, demonstrating a novel route for design of more sophisticated bioinks for four-dimensional bioprinting.