Alexander Southan

Desmosine-Inspired Cross-Linkers for Hyaluronan Hydrogels

We designed bioinspired cross-linkers based on desmosine, the cross-linker in natural elastin, to prepare hydrogels with thiolated hyaluronic acid. These short, rigid cross-linkers are based on pyridinium salts (as in desmosine) and can connect two polymer backbones. Generally, the obtained semi-synthetic hydrogels are form-stable, can withstand repeated stress, have a large linear-elastic range, and show strain stiffening behavior typical for biopolymer networks. In addition, it is possible to introduce a positive charge to the core of the cross-linker without affecting the gelation efficiency, or consequently the network connectivity. However, the mechanical properties strongly depend on the charge of the cross-linker. The properties of the presented hydrogels can thus be tuned in a range important for engineering of soft tissues by controlling the cross-linking density and the charge of the cross-linker.

Side chain thiol-functionalized poly(ethylene glycol) by post-polymerization modification of hydroxyl groups: synthesis, crosslinking and inkjet printing

Polymers with a poly(ethylene glycol) backbone and mercaptomethyl side chains were synthesized by post-polymerization modification of hydroxymethyl side chains in three steps. As the starting point of the synthetic route, linear copolymers of ethylene oxide and glycidol with molar contents of glycidol repeating units of approximately 20, 40, 60, 80 and 100% were used. The polymer-bound hydroxyl groups were converted to thiol groups in three steps, comprising tosylation, introduction of a triphenylmethyl protected thiol and thiol deprotection by acid treatment. The degree of thiol-functionalization was controlled by the degree of functionalization of the starting material. The degree of conversion of hydroxyl groups to thiol groups determined by 1H NMR spectroscopy was quantitative for copolymers with approximately 20 and 40% glycidol repeating units and 92, 81 and 87% for copolymers with approximately 60, 80 and 100% glycidol repeating units, respectively. Exemplarily, poly(glycidylthiol) obtained by conversion of poly(glycidol) was crosslinked with poly(ethylene glycol) diacrylate (PEG-DA) to yield hydrogels which supported adhesion and proliferation of human fibroblasts 48 h after cell seeding. Spatially defined and surface attached gel structures were fabricated by subsequent inkjet printing of poly(glycidylthiol) and PEG-DA solutions onto acrylated glass slides.

Quantification of Substitution of Gelatin Methacryloyl: Best Practice and Current Pitfalls

Cross-linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix (ECM) in tissue engineering. However, the quantification of modified groups in GM is still an unsolved issue, although this is the key factor for tailoring the physicochemical material properties. In this contribution, 1H-13C-HSQC NMR spectra are used to gain detailed structural information on GMs and of 2-fold modified gelatin containing methacryloyl and acetyl groups (GMAs). Distinctive identification of methacrylate, methacrylamide, and acetyl groups present in GMs and GMAs revealed an overlap of methacrylamide and modified hydroxyproline signals in the 1H NMR spectrum. Considering this, we suggest a method to quantify methacrylate and methacrylamide groups in GMs precisely based on simple 1H NMR spectroscopy with an internal standard. Quantification of acetylation in GMAs is also possible, yet, 2D NMR spectra are necessary. The described methods allow direct quantification of modified groups in gelatin derivatives, making them superior to other, indirect methods known so far.

Charged Triazole Cross-Linkers for Hyaluronan-Based Hybrid Hydrogels

Polyelectrolyte hydrogels play an important role in tissue engineering and can be produced from natural polymers, such as the glycosaminoglycan hyaluronan. In order to control charge density and mechanical properties of hyaluronan-based hydrogels, we developed cross-linkers with a neutral or positively charged triazole core with different lengths of spacer arms and two terminal maleimide groups. These cross-linkers react with thiolated hyaluronan in a fast, stoichiometric thio-Michael addition. Introducing a positive charge on the core of the cross-linker enabled us to compare hydrogels with the same interconnectivity, but a different charge density. Positively charged cross-linkers form stiffer hydrogels relatively independent of the size of the cross-linker, whereas neutral cross-linkers only form stable hydrogels at small spacer lengths. These novel cross-linkers provide a platform to tune the hydrogel network charge and thus the mechanical properties of the network. In addition, they might offer a wide range of applications especially in bioprinting for precise design of hydrogels.

Adenosine triphosphate diffusion through poly(ethylene glycol) diacrylate hydrogels can be tuned by cross-link density as measured by PFG-NMR

The diffusion of small molecules through hydrogels is of great importance for many applications. Especially in biological contexts, the diffusion of nutrients through hydrogel networks defines whether cells can survive inside the hydrogel or not. In this contribution, hydrogels based on poly(ethylene glycol) diacrylate with mesh sizes ranging from ξ = 1.1 to 12.9 nm are prepared using polymers with number-average molecular weights between Mn = 700 and 8000 g/mol. Precise measurements of diffusion coefficients D of adenosine triphosphate (ATP), an important energy carrier in biological systems, in these hydrogels are performed by pulsed field gradient nuclear magnetic resonance. Depending on the mesh size, ξ, and on the polymer volume fraction of the hydrogel after swelling, ϕ, it is possible to tune the relative ATP diffusion coefficient D/D0 in the hydrogels to values between 0.14 and 0.77 compared to the ATP diffusion coefficient D0 in water. The diffusion coefficients of ATP in these hydrogels are compared with predictions of various mathematical expressions developed under different model assumptions. The experimental data are found to be in good agreement with the predictions of a modified obstruction model or the free volume theory in combination with the sieving behavior of the polymer chains. No reasonable agreement was found with the pure hydrodynamic model.

Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels

Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (Mn = 700 g mol−1) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.

Gelatin methacrylamide as coating material in cell culture

Unmodified gelatin (uG) is widely used as a coating material in cell culture for improving surface properties. In this study, the authors investigated if gelatin methacrylamide (GM) with a medium degree of methacrylamide modification (GM1.5) and a high degree of methacrylamide modification (GM4) are equally suitable for this purpose. Therefore, gold surfaces were coated with uG, GM1.5, and GM4 by adsorption of the polymers on the surfaces. Coating success was confirmed by spectroscopic ellipsometry, contact angle measurements, surface plasmon resonance spectroscopy (SPRS), and atomic force microscopy (AFM). The authors found that upon adsorption of uG, GM1.5, a nd GM4 on gold, thin films with thicknesses of 2.95 nm, 2.50 nm, and 2.26 nm were formed. The coated surfaces showed advancing contact angles of 46° (uG and GM1.5) and 52° (GM4) without alteration of the surface roughness determined by AFM. Protein adsorption taking place on the coated surfaces was measured during contact of the surfaces with fetal calf serum by SPRS. Protein adsorption on the coated surfaces was reduced by the factor of 6.4 (uG), 5.4 (GM1.5), and 4.6 (GM4) compared to gold surfaces. Human fibroblasts cultured on the surfaces showed excellent viability shown by water soluble tetrazolium salt assay as well as live/dead staining with propidium iodide and fluorescein diacetate. No cytotoxic effects of the GM coated surfaces were observed, giving rise to the conclusion that GMs are suitable materials as coatings in cell culture.

Triazole-based cross-linkers in radical polymerization processes: tuning mechanical properties of poly(acrylamide) and poly(N,N-dimethylacrylamide) hydrogels

Triazole-based cross-linkers with different spacer lengths and different functional end groups (acrylamides, methacrylamides, maleimides and vinylsulfonamides) were synthesized, investigated for cytotoxic and antibacterial activity, and incorporated into poly(acrylamide) (PAAm) and poly(N,N-dimethylacrylamide) (PDMAAm) hydrogels by free-radical polymerization. Hydrogels prepared with different cross-linkers and cross-linker contents between 0.2% and 1.0% were compared by gel yields, equilibrium degrees of swelling (S) and storage moduli (G′). Generally with increasing cross-linker content, G′ values of the hydrogels increased, while S values decreased. The different polymerizable cross-linker end groups resulted in a decrease of G′ in the following order for cross-linkers with C4 spacers: acrylamide > maleimide > methacrylamide > vinylsulfonamide. Longer cross-linker alkyl spacer lengths caused an increase in G′ and a decrease in S. Independent of the cross-linker used, a universal correlation between G′ and equilibrium polymer volume fraction ϕ was found. For PAAm hydrogels, G′ ranged between 4 kPa and 23 kPa and ϕ between 0.07 and 0.14. For PDMAAm hydrogels, G′ ranged between 0.1 kPa and 4.9 kPa and ϕ between 0.02 and 0.06. The collected data were used to establish an empirical model to predict G′ depending on ϕ. G′ of PAAm and PDMAAm hydrogels is given by G′ = 4034 kPa ϕ2.66 and G′ = 4297 kPa ϕ2.46, respectively.

Active Ester Containing Surfmer for One-Stage Polymer Nanoparticle Surface Functionalization in Mini-Emulsion Polymerization

Functional surface active monomers (surfmers) are molecules that combine the functionalities of surface activity, polymerizability, and reactive groups. This study presents an improved pathway for the synthesis of the active ester containing surfmer p-(11-acrylamido)undecanoyloxyphenyl dimethylsulfonium methyl sulfate (AUPDS). Further, the preparation of poly(methyl methacrylate) and polystyrene nanoparticles (NPs) by mini-emulsion polymerization using AUPDS is investigated, leading to NPs with active ester groups on their surface. By systematically varying reaction parameters and reagent concentrations, it was found that AUPDS feed concentrations between 2–4 mol% yielded narrowly distributed and stable spherical particles with average sizes between 83 and 134 nm for non-cross-linked NPs, and up to 163 nm for cross-linked NPs. By basic hydrolysis of the active ester groups in aqueous dispersion, the positive ζ-potential (ZP) was converted into a negative ZP and charge quantities determined by polyelectrolyte titrations before and after hydrolysis were in the same range, indicating that the active ester groups were indeed accessible in aqueous suspension. Increasing cross-linker amounts over 10 mol% also led to a decrease of ZP of NPs, probably due to internalization of the AUPDS during polymerization. In conclusion, by using optimized reaction conditions, it is possible to prepare active ester functionalized NPs in one stage using AUPDS as a surfmer in mini-emulsion polymerization.

Photoinduced Cleavage and Hydrolysis of o‐Nitrobenzyl Linker and Covalent Linker Immobilization in Gelatin Methacryloyl Hydrogels

Light-induced release systems can be triggered remotely and are of interest for many controlled release applications due to the possibility for spatio-temporal release control. In this study a biotin-functionalized photocleavable macromer is incorporated with an o-nitrobenzyl moiety into gelatin methacryloyl(-acetyl) hydrogels via radical cross-linking. Stronger immobilization of streptavidin-coupled horseradish peroxidase occurs in linker-functionalized hydrogels compared to pure gelatin methacryloyl(-acetyl) hydrogels, and a controlled release of the streptavidin conjugate upon UV-irradiation is possible. Liquid chromatography coupled to mass spectrometry (LC-MS) analysis of aqueous linker solutions allows the identification of the main cleavage products and the cleavage kinetics. Thus, it is shown that a significant hydrolysis of the linker occurs at 37 °C. Nevertheless the system reported here is a promising controlled release scaffold for proteins and application in tissue engineering, if background releases of the immobilized drug are tolerable.