UV-VIS Curable PEG Hydrogels for Biomedical Applications with Multifunctionality.

Multifunctional biomedical materials capable of integrating optical functions are highly desirable for many applications, such as advanced intra-ocular lens (IOL) implants. Therefore, poly(ethylene glycol)-diacrylate (PEG-DA) hydrogels are used with different photoinitiators (PI). In addition to standard UV PI Irgacure, Erythrosin B and Eosin Y are used as PI with high sensitivity in the optical range of the spectrum. The minimum PI concentrations for producing new hydrogels with PEG-DA and different PIs were determined. Hydrogel films were obtained, which were applicable for light-based patterning and, hence, the functionalization of surface and volume. Cytotoxicity tests confirm cytocompatibility of hydrogels and compositions. Exploiting the correlation of structure and function allows biomedical materials with multifunctionality.

Micropatterning of Au NPs on PEG Hydrogels Using Different Silanes To Control Cell Adhesion on the Nanocomposites.

Amino-silanization of silica-based substrates has proven to be effective in guiding the immobilization of citrate-stabilized Au NPs in a good, homogeneous fashion. This accomplishment has formed the basis of fabricating micropatterns of Au NPs on such substrates by patterning of oxidized silicon wafers with (3-aminopropyl)trimethoxysilane (amino-silane) using the microcontact printing (µCP) process. This micropattern of amino-silane is used to specifically adsorb Au NPs. To avoid unspecific adsorption to the nonsilanized areas on the silicon wafers, the nonstamped areas were backfilled with self-assembled monolayers of organosilanes, for example, with methyl- or perfluoro-end-groups. Finally, after having fabricated a micropattern of Au NPs on silicon wafers, the Au NP patterns were transferred onto poly(ethylene glycol) hydrogels by our newly developed procedures, and on these nanocomposite materials, controlled cell adhesion has been achieved. Furthermore, these materials are great candidates for plasmon-based biosensor applications and also for various medical applications, such as for drug delivery systems or photothermal therapies.

Self-assembly of porphyrin hexamers via bidentate metal-ligand coordination.

The supramolecular assembly of metal-porphyrin hexamers with bidentate ligands in chloroform solutions is demonstrated by UV/Vis and 1H NMR-titrations, and Small Angle Neutron Scattering (SANS) experiments. Titrations of zinc porphyrin hexamer Zn1 with 1,4-diazabicyclo[2,2,2]octane (DABCO) revealed that at a DABCO/Zn1 molar ratio of 3, intermolecular sandwich complexes are formed, which can be considered as "circular-shaped porphyrin ladders". These supramolecular complexes further aggregate into larger polymeric stacks, as a result of a combination of cooperativity effects, π-π stacking interactions, and chelate effects. The presence of rodlike assemblies in solution, formed by assembly of Zn1 and DABCO, is confirmed by SANS-experiments. Using a model for cylindrical assemblies, curve fitting calculations reveal that rods with an average length of 26 nm and a radius of 30-35 Å were formed, corresponding to columnar stacks of approximately 30 hexamer molecules. In contrast, the metal-free hexamer H21 did not form extended assemblies due to the absence of coordinative intermolecular interactions.

Influence of Network Structure on the Crystallization Behavior in Chemically Crosslinked Hydrogels.

The network structure of hydrogels is a vital factor to determine their physical properties. Two network structures within hydrogels based on eight-arm star-shaped poly(ethylene glycol)(8PEG) have been obtained; the distinction between the two depends on the way in which the macromonomers were crosslinked: either by (i) commonly-used photo-initiated chain-growth polymerization (8PEG⁻UV), or (ii) Michael addition step-growth polymerization (8PEG⁻NH3). The crystallization of hydrogels is facilitated by a solvent drying process to obtain a thin hydrogel film. Polarized optical microscopy (POM) results reveal that, while in the 8PEG⁻UV hydrogels only nano-scaled crystallites are apparent, the 8PEG⁻NH3 hydrogels exhibit an assembly of giant crystalline domains with spherulite sizes ranging from 100 to 400 µm. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) analyses further confirm these results. A model has been proposed to elucidate the correlations between the polymer network structures and the crystallization behavior of PEG-based hydrogels.

Micro-Patterning of PEG-Based Hydrogels With Gold Nanoparticles Using a Reactive Micro-Contact-Printing Approach.

In this work a novel, relatively simple, and fast method for patterning of gold nanoparticles (Au NPs) on poly(ethylene glycol) (PEG)-based hydrogels is presented. In the hereby exploited reactive micro-contact printing (reactive-µ-CP) process, the surface of a micro-relief patterned PDMS-stamp is first functionalized with an amino-silane self-assembled monolayer (SAM), which is then inked with Au NPs. The stamp is subsequently brought into conformal contact with thiol-functionalized PEG-based hydrogel films. Due to the strong gold-thiol interactions the Au NPs are adequately and easily transferred onto the surfaces of these soft, multifunctional PEG hydrogels. In this way, defined µ-patterns of Au NPs on PEG hydrogels are achieved. These Au NPs patterns allow specific biomolecular interactions on PEG surfaces, and cell adhesion has been studied. Cells were found to effectively adhere only on Au NPs micro-patterns and to avoid the anti-adhesive PEG background. Besides the cell adhesion studies, these Au NPs µ-patterns can be potentially applied as biosensors in plasmon-based spectroscopic devices or in medicine, e.g., for drug delivery systems or photothermal therapies.

Surface Patterning of Gold Nanoparticles on PEG-Based Hydrogels to Control Cell Adhesion.

We report on a versatile and easy approach to micro-pattern gold nanoparticles (Au NPs) on 8-arm poly(ethylene glycol)-vinyl sulfone thiol (8PEG-VS-SH) hydrogels, and the application of these patterned Au NPs stripes in controlling cell adhesion. Firstly, the Au NPs were patterned on silicon wafers, and then they were transferred onto reactive, multifunctional 8PEG-VS-SH hydrogels. The patterned, micrometer-sized Au NPs stripes with variable spacings ranging from 20 µm to 50 µm were created by our recently developed micro-contact deprinting method. For this micro-contact deprinting approach, four different PEG-based stamp materials have been tested and it was found that the triblock copolymer PEG-PPG-PEG-(3BC) stamp established the best transfer efficiency and has been used in the ongoing work. After the successful creation of micro-patterns of Au NPs stripes on silicon, the patterns can be transferred conveniently and accurately to 8PEG-VS-SH hydrogel films. Subsequently these Au NPs patterns on 8PEG-VS-SH hydrogels have been investigated in cell culture with murine fibroblasts (L-929). The cells have been observed to adhere to and spread on those nano-patterned micro-lines in a remarkably selective and ordered manner.

Novel Wet Micro-Contact Deprinting Method for Patterning Gold Nanoparticles on PEG-Hydrogels and Thereby Controlling Cell Adhesion.

In the present work we introduce a novel method to create linear and rectangular micro-patterns of gold nanoparticles (Au NPs) on poly(ethylene glycol) (PEG) hydrogels. The strategy consists of removing Au NPs from defined regions of the silicon wafer by virtue of the swelling effect of the hydrogel. Using this method, which we denote as "Wet Micro-Contact Deprinting", well-defined micro-patterns of Au NPs on silicon can be created. This resulting pattern is then transferred from the hard substrate to the soft surface of PEG-hydrogels. These unique micro- and nano-patterned hydrogels were cultured with mouse fibroblasts L929 cells. The cells selectively adhered on the Au NPs coated area and avoided the pure PEG material. These patterned, nanocomposite biointerfaces are not only useful for biological and biomedical applications, such as tissue engineering and diagnostics, but also, for biosensor applications taking advantage of surface plasmon resonance (SPR) or surface enhanced Raman scattering (SERS) effects, due to the optical properties of the Au NPs.

Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant.

The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report a simple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyl-tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate. We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology.

Geometric Control of Cell Alignment and Spreading within the Confinement of Antiadhesive Poly(Ethylene Glycol) Microstructures on Laser-Patterned Surfaces.

In this study, a mask-less laser-assisted patterning method is used to fabricate well-defined cell-adhesive microdomains delimited by protein-repellent poly(ethylene glycol) (PEG) microstructures prepared from multiarm (8-PEG) macromonomers. The response of murine fibroblasts (L-929) toward these microdomains is investigated, revealing effective cell confinement within the cell-adhesive areas surrounded by nonadhesive 8-PEG microstructures. Moreover, the spatial positioning of cells in microdomains of various sizes and geometries is analyzed, indicating control of cell density, size, and elongated cell shape induced by the size of the microdomains and the geometric confinement.

Enzymatically cross-linked hyperbranched polyglycerol hydrogels as scaffolds for living cells.

Although several strategies are now available to enzymatically cross-link linear polymers to hydrogels for biomedical use, little progress has been reported on the use of dendritic polymers for the same purpose. Herein, we demonstrate that horseradish peroxidase (HRP) successfully catalyzes the oxidative cross-linking of a hyperbranched polyglycerol (hPG) functionalized with phenol groups to hydrogels. The tunable cross-linking results in adjustable hydrogel properties. Because the obtained materials are cytocompatible, they have great potential for encapsulating living cells for regenerative therapy. The gel formation can be triggered by glucose and controlled well under various environmental conditions.

Microengineered PEG hydrogels: 3D scaffolds for guided cell growth.

Designing three-dimensional (3D) scaffolds for selective manipulation of cell growth is of high relevance for applications in regenerative medicine. Especially, scaffolds with oriented morphologies bear high potential to guide the restoration of specific tissues. The fabrication of hydrogel scaffolds that support long-term survival, proliferation, and unidirectional growth of embedded cells is presented here. Parallel channel structures are introduced into the bulk hydrogels by uniaxial freezing, providing stable, and uniform porosity suitable for cell invasion (pore diameters of 5-15 µm). In vitro assessment of the scaffolds with murine fibroblasts (NIH L929) shows a remarkable unidirectional movement along the channels, with the cells traveling several millimeters through the hydrogel.

Cell phenotypic changes of mouse connective tissue fibroblasts (L-929) to poly(ethylene glycol)-based gels.

Cellular responses to various gels fabricated by photoinitiated crosslinking using acrylated linear and multi-arm poly(ethylene glycol) (PEG)-based and poly(propylene glycol)-b-poly(ethylene glycol) precursors were investigated. While no protein adsorption and cell adhesion were observed on the hydrophilic PEG-based gels, protein adsorption and cell adhesion did occur on the more hydrophobic gel generated from the block copolymer precursor. Murine fibroblast viability on the poly(ethylene glycol)-based gels was studied in the course of 72 h and the results indicated no cytotoxicity. In a systematic study, extra- and intracellular metabolites of the murine fibroblasts cultured on these PEG-based gels were examined by GC-MS. Distinct intra- and extracellular changes in primary metabolism, namely amino acid metabolism, glycolysis and fatty acid metabolism, were observed. Cells cultured on the polymeric gels induced more intense intracellular changes in the metabolite profile by means of higher metabolite intensities with time in comparison to cells cultured on the reference substrate (tissue culture polystyrene). In contrast, extracellular changes of metabolite intensities were comparable.

Topography-induced cell adhesion to Acr-sP(EO-stat-PO) hydrogels: the role of protein adsorption.

Topographic surface patterning of intrinsically non-adhesive P(EO-stat-PO)-based hydrogels can lead to the adhesion and spreading of fibroblasts. Explanations for this unexpected behavior are discussed, particularly with regard to non-specific protein adsorption from the serum-supplemented culture medium. The presence of serum proteins is shown to be essential for adhesion. Adsorption of plasma and ECM proteins (Fibronectin (FN) and Vitronectin (VN)) to the hydrogels is possible. The effect of VN on initial cell adhesion is analyzed in detail. It appears that VN is the main serum component that is crucial for initial cell adhesion to PEG and that surface topography is essential for further, durable adhesion establishment, and spreading.

Combined influence of substrate stiffness and surface topography on the antiadhesive properties of Acr-sP(EO-stat-PO) hydrogels.

Biomaterials that prevent nonspecific protein adsorption and cell adhesion are of high relevance for diverse applications in tissue engineering and diagnostics. One of the most widely applied materials for this purpose is Poly(ethylene glycol) (PEG). We have investigated how micrometer line topography and substrate elasticity act upon the antiadhesive properties of PEG-based hydrogels. In our studies we apply bulk hydrogel cross-linked from star-shaped poly(ethylene oxide-stat-propylene oxide) macromonomers. Substrate surfaces were topographically patterned via replica molding. Additionally, the mechanical properties were altered by variations in the cross-linking density. Surface patterns with dimensions in the range of the cells' own size, namely 10 µm wide grooves, induced significant cell adhesion and spreading on the Acr-sP(EO-stat-PO) hydrogels. In contrast, there was only little adhesion to smaller and larger pattern sizes and no adhesion at all on the smooth substrates, regardless the rigidity of the gel. The effect of varied substrate stiffness on cell behavior was only manifest in combination with topography. Softer substrates with line patterns lead to significantly higher cell adhesion and spreading than stiff substrates. We conclude that the physical and mechanical surface characteristics can eliminate the nonadhesive properties of PEG-based hydrogels to a large extent. This has to be taken into account when designing surfaces for biomedical application such as scaffolds for tissue engineering which rely on the inertness of PEG.

A hydrophobic perfluoropolyether elastomer as a patternable biomaterial for cell culture and tissue engineering.

We present a systematic study of a perfluoropolyether (PFPE)-based elastomer as a new biomaterial. Besides its excellent long-term stability and inertness, PFPE can be decorated with topographical surface structures by replica molding. Micrometer-sized pillar structures led to considerably different cell morphology of fibroblasts. Although PFPE is a very hydrophobic material we could show that PFPE substrates allow cell adhesion and spreading of primary human fibroblasts (HDF) very similar to that observed on standard cell culture substrates. Less advanced cell spreading was observed for L929 (murine fibroblast cell line) cells during the first 5 h in culture which was accompanied by retarded recruitment of α(v)ß(3)-integrin into focal adhesions (FAs). After 24 h distinct FAs were evident also in L929 cells on PFPE. Furthermore, organization of soluble FN into a fibrillar ECM network was shown for hdF and L929 cells. Based on these results PFPE is believed to be a suitable substrate for several biological applications. On the one hand it is an ideal cell culture substrate for fundamental research of substrate-independent adhesion signaling due to its different characteristics (e.g. wettability, elasticity) compared to glass or TCPS. On the other hand it could be a promising implant material, especially due to its straightforward patternability, which is a tool to direct cell growth and differentiation.

Induction of specific macrophage subtypes by defined micro-patterned structures.

In this study, we investigated the influence of different perfluoropolyether (PFPE) microstructures on the inflammatory response of human macrophages. We generated four different microstructured PFPE surfaces by replica molding from silicon masters. The function-associated surface markers 27E10 and CD163 were monitored using flow cytometry to measure the pro- and anti-inflammatory reactions. Inflammatory mediator expression was measured at the protein and mRNA level. Lipopolysaccharide treatment served as positive control for pro-inflammatory activation. We observed that each micropattern induced a specific morphology, phenotype and mediator profile. A microstructure of regular grooves induced a pro-inflammatory phenotype (M1) which was not accompanied by release of pro-inflammatory mediators. However, the larger cylindrical posts induced an anti-inflammatory phenotype (M2) with a remarkable down-regulation of CXCL10. Smaller posts with a shorter distance exhibited a stronger pro-inflammatory response than those with a longer distance, on the levels of both phenotype and mediator release. Regression analysis suggests that the geometrical parameters of the microstructures, specifically the period of structures, may play an important role in macrophage response. Optimization of such microstructures may provide a method to invoke a predictable response of macrophages to implants and control the mediator release.

Deformation of nanostructures on polymer molds during soft UV nanoimprint lithography.

Soft nanoimprint lithography (soft NIL) relies on a mechanical deformation of a resist by a patterned polymer used as a mold. Here, we report on the investigation of the nanopattern fidelity of the high pressure imprint process based on a perfluorinated polyether (PFPE) soft mold material. The perfluorinated polyether material was found to be well suited to transfer the mold pattern into the resist by a direct imprinting process at low cost. Moderate deformations of the polymer mold structures occurring during the high pressure imprint are systematically studied. Features of decreased size are found to be more sensitive to pattern distortions. An optimized pattern design with increased structure density and constant pattern ratio is developed to minimize deformation effects. Imprints performed on the basis of these design rules result in reduced deformations and repeal their size dependence. The improved pattern transfer, especially for small structural elements, turns the direct and cost-effective soft UV-NIL into an interesting technique also for patterning tasks in the lower nanometer range.

Surface topography induces fibroblast adhesion on intrinsically nonadhesive poly(ethylene glycol) substrates.

Important in developing new biomaterials is the prevention of unspecific protein adsorption and cell interactions that in vivo can lead to a foreign body reaction. On the other hand, the material should support the growth of a specific cell type in a defined way. We investigate the possibility of manipulating cellular behavior on an intrinsically nonadhesive material by topographic patterning without additional surface chemistry modifications. The biomaterial applied is a hydrogel cross-linked from star-shaped poly(ethylene glycol) macromonomers (starPEG). Cell biological studies with a mouse fibroblast cell line (L929) showed that, while substrates with a smooth surface are nonadhesive, as expected, imprinted topography enabled cell adhesion and spreading. The fibroblasts aligned to micrometer groove patterns and were, depending on the respective dimensions, able to span or enter the grooves. Especially substrates with topography dimensions in the cell size range or smaller (<10 microm) lead to an establishment of stable cell-surface contacts (vinculin and actin accumulation). On micrometer post patterns the cells spread on top of the pillars and wrapped around the structures. The strong influence of the topography shows that nonadhesive materials do not necessarily have to be specifically biofunctionalized to enable cell adhesion. Possible explanations for the peculiar cell behavior are discussed in terms of (initial) protein adsorption and geometry-dependent cytoskeletal arrangements.

Nanomolding of PEG-based hydrogels with sub-10-nm resolution.

A simple, soft nanolithographic method is used to fabricate sub-10-nm structures on star polyethylene glycol-based hydrogels and perfluoropolyether-based materials. Very small features, for example, gold nanoparticles of size approximately 8 nm with an interparticle distance of approximately 100 nm, are successfully reproduced from a hard silicon master into both elastomers. Scanning force microscopy is used to investigate the replicas, and the original hexagonal pattern of the nanoparticles is clearly recognized. In addition, both replicas are usable as secondary, soft molds yielding positive copies of the primary, hard master. The results presented here show similar replication capabilities for both elastomers despite the markedly different properties of the precursors. Moreover, the hydrogel material can be easily peeled off from both soft and silicon masters without the need for surface treatment. The procedure allows nanopatterning of a biocompatible material over large areas, which is a useful tool to investigate cellular responses to defined nanotopography.

Microcontact deprinting: a technique to pattern gold nanoparticles.

A simple and general patterning technique for inorganic nanoparticles (NPs, e.g., gold NPs) is demonstrated, consisting of the selective lift-off of metal precursor loaded block copolymer micelles. The procedure works as follows: first, a topographically micropatterned polystyrene (PS) stamp is placed in contact with a substrate covered with hexagonally arranged micelles. Then the assembly is heated above the glass transition temperature (T(g)) of PS, and finally, the PS stamp is peeled off, removing from the substrate the micelles that were in contact with the protrusions of the stamp. As a result, patterns of micelles either exactly identical to the original or with much smaller features down to submicrometer were obtained. In a subsequent step, the organic material can be removed and the metal precursor reduced by plasma treatment, resulting in patterns of NPs. This technique, denoted as "micro-contact deprinting", provides a fast and inexpensive way to obtain hierarchical patterns of NPs on a wide range of substrates. It is demonstrated that it can even be applied on curved surfaces because of the softness of the PS stamp above its T(g).