Fuat Topuz

Embedding of Active Proteins and Living Cells in Redox‐Sensitive Hydrogels and Nanogels through Enzymatic Cross‐Linking

Redox-sensitive materials have received dramatically increasing interest over the past years.1 Disulfide cross-linked colloidal networks are particularly appealing, as they are rapidly reduced to thiols under the reductive environment inside cells, allowing the quantitative release of the payload incorporated within the particles.2 Moreover, disulfide cross-linked hydrogels as three-dimensional cell-culture scaffolds can be degraded under cytocompatible mild reductive conditions without affecting the vitality of the embedded cells.3

Magnesium ions and alginate do form hydrogels: a rheological study

Our study shows that magnesium ions which have so far been considered as non-gelling ions for alginate do induce alginate gelation. Rheology is used to examine effects of alginate chemical composition as well as alginate and magnesium ion concentration. Gelation in this system occurs at ca. 5–10 times higher concentration of ions than reported for calcium-based gels. Alginate network formation with magnesium ions is very slow and is typically accomplished within 2–3 hours. Gelation with magnesium ions is also strongly dependent on alginate chemical composition as the presence of long guluronic units privileges faster gel formation.

Molecular response of liver sinusoidal endothelial cells on hydrogels

There is a high demand for the isolation of primary endothelial cells for biomaterial endotheliazation studies, tissue engineering, and artificial organ development. Further, biomarkers for monitoring the response of endothelial cells in biomaterials science are required. We systematically compared two strategies for isolating liver sinusoidal endothelial cells (LSEC) from mouse liver. We demonstrate that fluorescence-activated cell sorting results in a considerably higher purity (~97%) compared to magnetic-assisted cell sorting (~80%), but is associated with a lower yield and recovery rate. Cell repellent polyethylene glycol (PEG) substrates affected the morphology of primary LSEC in culture and significantly downregulated the intracellular adhesion molecule (ICAM) and upregulated the vascular cell adhesion molecule (VCAM). This molecular response could partially be reverted by further modification with arginylglycylaspartic acid (RGD). Thus, usage of PEGylated materials may reduce, while applying RGD may support endotheliazation of materials, and we could relate LSEC attachment to their expression of ICAM and VCAM mRNA, suggesting their usage as biomarkers for endothelialization.

3.329 – Hydrogels in Biosensing Applications

This chapter gives an overview of the use of hydrogels in biosensing. Many intrinsic properties of hydrogels predetermine their use in this field of application. The most important points are their high water content that renders them biocompatible and allows diffusion of water-soluble compound through the polymer network as well as their chemical diversity and the ability to tune their properties both regarding the chemical nature of the polymer backbone and the introduction of (functional) side groups. This allows the generation and design of stimuli-sensitive hydrogels that may act as sensors themselves, for example, by introduction of hydrophobic groups for temperature sensitivity or protic groups for pH sensitivity. It also enables functionalization of the hydrogel with moieties that act as sensors. The range of molecules that can be and has been used for this purpose is very broad and comprises (fluorescence) dyes, luminescent compounds, enzymes, biochemical recognition sites such as oligonucleotides, antibodies, lectins, and very specific recognition mechanisms.

DNA Nanogels To Snare Carcinogens: A Bioinspired Generic Approach with High Efficiency.

Polycyclic aromatic hydrocarbons (PAHs) are combustion-related pollutants and are ubiquitous in the environment, including in sources of drinking water. Upon contact with DNA, stable PAH-DNA adducts form rapidly as the first step towards their toxic effects. In this work, we prepared hydrophilic DNA nanogels to exploit this generic complexation process as a biomimetic scavenging method. This approach relies on interaction between PAHs and the complete network that constitutes the water-swollen nanogels, and is not restricted to interfacial adsorption. Up to 720 μg of PAH per gram of DNA nanogel are taken up, meaning that 1 mg of DNA nanogel is sufficient to purify a liter of water containing the critical PAH concentration for cancer risk (600 ng L(-1) ). As a result of short diffusion pathways, PAH uptake is rapid, reaching 50 % loading after 15 minutes. Beyond PAHs, DNA nanogels may be useful for the generic detoxification of water containing genotoxins, since most known molecules that strongly associate with DNA are mutagenic.

Covalently layer-by-layer assembled homogeneous nanolayers with switchable wettability

Layer-by-layer (LbL) assembly is a practical and versatile approach to build up ultrathin hydrogel networks using mostly polyelectrolytes via alternate adsorption of oppositely charged molecules. It has recently been applied covalently by means of many different types of molecules, particularly those having low molecular weights or linear polymer structures. Using isocyanate (NCO) end-functional star-type polyethers (NCO-sP(EO-stat-PO)) in such covalent assemblies is a great challenge, since they are prone to form a smooth but non-reactive layer that precludes chemisorption of a subsequent layer. To overcome this problem, we developed a protocol where oligomers (e.g., dimers, trimers) act as building blocks for a monolayer instead of single star shaped molecules. Since these are larger and multifunctional but still flexible, smooth layers thicker than a monomolecular film (ca. 10 nm) result with sufficient mobility of the building blocks to bear enough reactive groups for covalent binding of a subsequent layer. As a second component for the chemical LbL layer buildup, a high molecular weight copolymer of vinylformamide/vinylamine (PVFA-co-PVAm) was used. The first layer was obtained by treating aminosilylated surfaces with NCO-s(EO-stat-PO) followed by incubation with PVFA-co-PVAm and chemical cross-linking with the first layer via urea links. The cycle was repeated to achieve the desired layer growth, and the resulting layers were characterized by ellipsometry, contact angle analysis, X-ray photoelectron spectroscopy (XPS), and scanning force microscopy (SFM). The amorphous structures of the polymers were revealed by WAXS analysis, suggesting the lack of the long-range order, which led to structural degree of freedom available to the polymer (i.e., molecular flexibility) on the surface. Thus, multilayers were obtained with homogeneous structure together with low roughness values, and the water contact angles of the layers switched between 37 and 45° depending on the terminal layer. The layers were stable over three months under humid conditions during which no significant changes could be observed in thickness and hydrophilicity.

Stimuli-Sensitive Microgels from Native Elastin: An Easy Approach for a Drug Release System

Thermo- and pH-responsive microgels were prepared from solubilized native elastin by crosslinking of the elastin lysine residues with poly(ethylene glycol) diglycidyl ether (PEG-DGE) and with bis(sulfosuccinimidyl) suberate (BS3). In the first case, a peptide-PEG conetwork was obtained whereas, in the second case, the elastin peptides were interlinked with hydrophobic bridges. The structure of the microgels was controlled by the ratio of crosslinker to elastin and by performing the crosslinking reaction in an inverse minielemulsion, yielding particles with a diameter in the submicron range. Depending on the degree of crosslinking, the hybrid microgels exhibited a volume change transition at 37 and 35.5°C and pH responsivity in the range of 5–7 for microgels crosslinked with PEG-DE and BS3, respectively. This temperature- and pH-responsive behavior can be assigned to the well-investigated coacervation of elastin peptides, demonstrating that the elastin functionality is abolished only by rather dense crosslinking. In spite of the broad distribution in the molecular weight of the elastin molecules, the microgels remained soluble. Light scattering and sedimentation experiments demonstrated that the coacervation occurred predominantly intramolecularly, i.e., by collapse in the core while the corona stabilized the colloidal dispersion against precipitation. Preliminary experiments were conducted to evaluate the suitability of these microgels for use as a drug-release system and demonstrated cytocompatibility, enzymatic degradability by elastase, and entrapping and slow release of a water-soluble biopolymer (Texas Red-labeled dextran with M w = 70,000). In summary, we present an easy entry to functional biohybrid microgels, where the responsiveness to temperature and pH can be exploited further for application of the microgel as a drug carrier.