Alexander Welle

Adding spatial control to click chemistry: phototriggered Diels-Alder surface (bio)functionalization at ambient temperature.

A photoconjugation strategy based on light-triggered Diels–Alder addition of o-quinodimethanes is compatible with biomolecules and proceeds rapidly at ambient temperature without the need of a catalyst. Spatial control was confirmed by photopatterning of a small-molecule ATRP initiator, a polymer, and a peptide in a time-of-flight secondary-ion mass spectrometry investigation.

UV‐Triggered Dopamine Polymerization: Control of Polymerization, Surface Coating, and Photopatterning

UV irradiation is demonstrated to initiate dopamine polymerization and deposition on different surfaces under both acidic and basic pH. The observed acceleration of the dopamine polymerization is explained by the UV-induced formation of reactive oxygen species that trigger dopamine polymerization. The UV-induced dopamine polymerization leads to a better control over polydopamine deposition and formation of functional polydopamine micropatterns.

Photo‐Patterning of Non‐Fouling Polymers and Biomolecules on Paper

Functional cellulose substrates with tetrazole moieties are generated to serve as universal platforms for the spatio-temporal immobilization of synthetic ultra-low fouling polymer brushes and protein species via a nitrile imine-mediated tetrazole-ene cycloaddition (NITEC)-based protocol. Poly(carboxybetaine acrylamide) brushes are grafted from initiators photo-patterned by NITEC utilizing single electron transfer living radical polymerization. Streptavidin is photo-immobilized with remarkable efficiency, opening the possibility to generate new materials for biomedical and biosensing applications.

UV-Based Patterning of Polymeric Substrates for Cell Culture Applications

We studied the physico/chemical effects of deep UV irradiation of polystyrene, polymethylmethacrylate and polycarbonate with respect to cell adhesion in vitro. Photochemical modifications of the polymer surfaces yielded active peroxide compounds which allowed graft coupling of acrylic monomers (acrylamide, acrylic acid) together with oxidized chemical groups which were identified by X-ray photoelectron spectroscopy, dye binding and contact angle titrations as being mainly carboxylic groups. It was observed that hepatocytes (HepG2, human hepatoma cell line) and fibroblasts (L929, murine cell line) exhibit strong adhesion on the irradiated polymer surfaces. Masked irradiations opened a simple, fast, and economical route to obtain chemically patterned polymeric substrates for structured cell adhesion. This is more advantageous as compared to silane based patterning techniques on glass or thiol based patterning on gold due to the elimination of any chemical treatment, the clean room compatibility and the small size of achieved structures. The described cell patterning technique may become a useful tool for the study of a variety of defined co-cultures (for example hepatocytes and fibroblasts), neuronal networks, intercellular communication, organogenesis and for applications like biosensors or engineered highly functional tissues and implants as bioartificial organs.

Micropatterned superhydrophobic structures for the simultaneous culture of multiple cell types and the study of cell–cell communication

The ability to control spatial arrangement and geometry of different cell types while keeping them separated and in close proximity for a long time is crucial to mimic and study variety of biological processes in vitro. Although the existing cell patterning technologies allow co-culturing of different cell types, they are usually limited to relatively simple geometry. The methods used for obtaining complex geometries are usually applicable for patterning only one or two cell types. Here we introduce a convenient method for creating patterns of multiple (up to twenty) different cell types on one substrate. The method virtually allows any complexity of cell pattern geometry. Cell positioning on the substrate is realized by a parallel formation of multiple cell-containing microreservoirs confined to the geometry of highly hydrophilic regions surrounded by superhydrophobic borders built-in a fine nanoporous polymer film. As a case study we showed the cross-talk between two cell populations via Wnt signaling molecules propagation during co-culture in a mutual culture medium.

Hyaluronic acid/Chitosan nanoparticles as delivery vehicles for VEGF and PDGF-BB.

The development of a vascular network in tissue-engineered constructs is a fundamental bottleneck of bioregenerative medicine, particularly when the size of the implant exceeds a certain limit given by diffusion lengths and/or if the host tissue shows a very active metabolism. One of the approaches to achieve the vascularization of tissue constructs is generating a sustained release of proangiogenic factors from the ischemic site. This work describes the formation and characterization of hyaluronic acid-chitosan (HA/CS) nanoparticles for the delivery of two pro-angiogenic growth factors: vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF-BB). These nanoparticles were prepared by an ionic gelification technique, and different formulations were developed by encapsulating the growth factors in association with two stabilizing agents: bovine serum albumin or heparin sodium salt. These carriers were characterized with regard to their physicochemical properties, their stability in biological media, and their cytotoxicity in the C3a hepatoma cell line. The results show that nanoparticles around 200 nm can be prepared by this method. HA/CS nanoparticles were stable when incubated in EMEM cell culture medium or in water at 37°C for 24 h. Cell culture tests confirmed that HA/CS nanoparticles are not cytotoxic within the concentration range used for growth factor delivery. Moreover, HA/CS nanoparticles were able to entrap efficiently both growth factors, reaching association values of 94% and 54% for VEGF and PDGF, respectively. In vitro release studies confirm that PDGF-BB is released from HA/CS nanoparticles in a sustained manner over ∼ 1 week. On the other hand, VEGF is completely released within the first 24 h.

Nanoparticles based on PLGA: Poloxamer blends for the delivery of proangiogenic growth factors

New blood vessel formation is a critical requirement for treating many vascular and ischemia related diseases, as well as for many tissue engineering applications. Angiogenesis and vasculogenesis, in fact, represent crucial processes for the functional regeneration of complex tissues through tissue engineering strategies. Several growth factors (GFs) and signaling molecules involved in blood vessels formation have been identified, but their application to the clinical setting is still strongly limited by their extremely short half-life in the body. To overcome these limitations, we have developed a new injectable controlled release device based on polymeric nanoparticles for the delivery of two natural proangiogenic GFs: platelet derived growth factor (PDGF-BB) and fibroblast growth factor (FGF-2). The nanoparticle system was prepared by a modified solvent diffusion technique, encapsulating the GF both in presence and in the absence of two stabilizing agents: bovine serum albumin (BSA) and heparin sodium salt (Hp). The developed nanocarriers were characterized for morphology, size, encapsulation efficiency, release kinetics in vitro and GF activity in cell cultures. The results have indicated that the coencapsulation of stabilizing agents can preserve the GF active structure and, in addition, increase their encapsulation efficiency into nanoparticles. Through this optimization process, we were able to raise the encapsulation efficiency of FGF-2 to 63%, and that of PDGF-BB to 87%. These PLGA:poloxamer blend nanoparticles loaded with GFs were able to release PDGF-BB and FGF-2 in a sustained fashion for more than a month. This work also confirms other positive features of PLGA:poloxamer nanoparticles. Namely, they are able to maintain their stability in simulated biological medium, and they are also nontoxic to cell culture models. Incubation of nanoparticles loaded with FGF-2 or PDGF-BB with endothelial cell culture models has confirmed that GFs are released in a bioactive form. Altogether, these results underline the interest of PLGA:poloxamer nanoparticles for the controlled delivery of GFs and substantiate their potential for the treatment of ischemic diseases and for tissue engineering applications.

Reactive superhydrophobic surface and its photoinduced disulfide-ene and thiol-ene (bio)functionalization

Reactive superhydrophobic surfaces are highly promising for biotechnological, analytical, sensor, or diagnostic applications but are difficult to realize due to their chemical inertness. In this communication, we report on a photoactive, inscribable, nonwettable, and transparent surface (PAINTS), prepared by polycondensation of trichlorovinylsilane to form thin transparent reactive porous nanofilament on a solid substrate. The PAINTS shows superhydrophobicity and can be conveniently functionalized with the photoclick thiol-ene reaction. In addition, we show for the first time that the PAINTS bearing vinyl groups can be easily modified with disulfides under UV irradiation. The effect of superhydrophobicity of PAINTS on the formation of high-resolution surface patterns has been investigated. The developed reactive superhydrophobic coating can find applications for surface biofunctionalization using abundant thiol or disulfide bearing biomolecules, such as peptides, proteins, or antibodies.

Benzylguanine thiol self-assembled monolayers for the immobilization of SNAP-tag proteins on microcontact-printed surface structures.

The site-selective, oriented, covalent immobilization of proteins on surfaces is an important issue in the establishment of microarrays, biosensors, biocatalysts, and cell assays. Here we describe the preparation of self-assembled monolayers consisting of benzylguanine thiols (BGT) to which SNAP-tag fusion proteins can be covalently linked. The SNAP-tag, a modified O(6)-alkylguanine-DNA alkyltransferase (AGT), reacts with the headgroup of BGT and becomes covalently bound upon the release of guanine. Bacterially produced recombinant His-tag-SNAP-tag-GFP was used to demonstrate the site-specific immobilization on BGT surface patterns created by microcontact printing (microCP). With this versatile method, any SNAP-tag protein can be coupled to a surface.

Photon Upconversion at Crystalline Organic-Organic Heterojunctions

Triplet transfer across a surface-anchored metal-organic-framework heterojunction is demonstrated by the observation of triplet-triplet annihilation photon -upconversion in a sensitizer-emitter heterostructure. Upconversion thresholds under 1 mW cm-2 are achieved. In the broader context, the double-electron-exchange mechanism of triplet transfer indicates that the heterojunction quality is sufficient for electrons to move between layers in this solution-processed crystalline heterostructure.