Biodegradation-resistant multilayers coated with gold nanoparticles. Toward a tailor-made artificial extracellular matrix

Polymer multicomponent coatings such as multilayers mimic extracellular matrix (ECM) that attracts significant attention for their use as functional supports for advanced cell culture and tissue engineering. Herein, biodegradation and molecular transport in hyaluronan/polylysine multilayers coated with gold nanoparticles was described. Nanoparticle coating acts as semipermeable barrier that governs molecular transport into/from the multilayers and makes them biodegradation resistant. Model protein lysozyme (mimics of ECM soluble signals) diffuses in the multilayers as fast and slow diffusing populations existing in an equilibrium. Such composite system may have high potential to be exploited as degradation-resistant drug delivery platforms suitable for cell-based applications. The extracellular matrix (ECM) provides not only a structural support for cell-based applications

Thermoresponsive Microgel Coatings as Versatile Functional Compounds for Novel Cell Manipulation Tools

Uhlig K, Wegener T, Hertle Y, et al. Thermoresponsive Microgel Coatings as Versatile Functional Compounds for Novel Cell Manipulation Tools. POLYMERS. 2018;10(6): 656.For the effective use of live cells in biomedicine as in vitro test systems or in biotechnology, non-invasive cell processing and characterisation are key elements. Thermoresponsive polymer coatings have been demonstrated to be highly beneficial for controlling the interaction of adherent cells through their cultivation support. However, the widespread application of these coatings is hampered by limitations in their adaptability to different cell types and because the full range of applications has not yet been fully explored. In the work presented here, we address these issues by focusing on three different aspects. With regard to the first aspect, by using well-defined laminar flow in a microchannel, a highly controllable and reproducible shear force can be applied to adherent cells. Employing this tool, we demonstrate that cells can be non-invasively detached from a support using a defined shear flow. The second aspect relates to the recent development of simple methods for patterning thermoresponsive coatings. Here, we show how such patterned coatings can be used for improving the handling and reliability of a wound-healing assay. Two pattern geometries are tested using mouse fibroblasts and CHO cells. In terms of the third aspect, the adhesiveness of cells depends on the cell type. Standard thermoresponsive coatings are not functional for all types of cells. By coadsorbing charged nanoparticles and thermoresponsive microgels, it is demonstrated that the adhesion and detachment behaviour of cells on such coatings can be modulated

Multi-fractional analysis of molecular diffusion in polymer multilayers by FRAP: a new simulation-based approach

Comprehensive analysis of the multifractional molecular diffusion provides a deeper understanding of the diffusion phenomenon in the fields of material science, molecular and cell biology, advanced biomaterials, etc. Fluorescence recovery after photobleaching (FRAP) is commonly employed to probe the molecular diffusion. Despite FRAP being a very popular method, it is not easy to assess multifractional molecular diffusion due to limited possibilities of approaches for analysis. Here we present a novel simulation-optimization-based approach (S-approach) that significantly broadens possibilities of the analysis. In the S-approach, possible fluorescence recovery scenarios are primarily simulated and afterward compared with a real measurement while optimizing parameters of a model until a sufficient match is achieved. This makes it possible to reveal multifractional molecular diffusion. Fluorescent latex particles of different size and fluorescein isothiocyanate in an aqueous medium were utilized as test systems. Finally, the S-approach has been used to evaluate diffusion of cytochrome c loaded into multilayers made of hyaluronan and polylysine. Software for evaluation of multifractional molecular diffusion by S-approach has been developed aiming to offer maximal versatility and user-friendly way for analysis

Temperature-induced molecular transport through polymer multilayers coated with PNIPAM microgels

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Polyelectrolyte multilayers serve as effective reservoirs for bioactive molecules which are stored and released from the multilayers for cellular applications. However, control over the release without significantly affecting the multilayers and biomolecules is still a challenge. On the other hand, externally stimulated release would make the multilayers promising for the development of stimuli-sensitive planar carriers with release performance switched on demand. In this study soft composite films are designed by coating hyaluronic acid/poly-L-lysine (HA/PLL) multilayers with temperature responsive poly( N-isopropylacrylamide) (PNIPAM) microgels. Microgels are flattened and immersed into the multilayers to maximize the number of contacts with the surrounding polyelectrolytes (HA and PLL). The microgel coating serves as an efficient switchable barrier for the PLL transport into the multilayers. PLL diffusion into the film is significantly hindered at room temperature but is dramatically enhanced at 40 degrees C above the volume phase transition temperature (VPTT) of PNIPAM at 32 degrees C associated with microgel shrinkage. Scanning force microscopy micrographs show that the mechanism of volume phase transition on soft surfaces cannot be directly deduced from the processes taking place at solid substrates

Design, construction and testing of a COC 3D flow-over flow-through bioreactor for hepatic cell culture

In this poster, we present the joint development efforts for a 3D microfluidic bioreactor for hepatic cell cultures. Cyclic Olefin Copolymer (COC) was selected for constructing the bioreactor, since the material has good chemical resistance, low adsorption and good optical properties, including low auto-fluorescence. A downside of COC is that it is much more difficult to structure than more traditional microfluidic materials, such as PDMS, PMMA, … Two parallel approaches were developed for structuring the COC. In a first approach, mechanical micro-milling of the channels allows for extremely fast manufacturing of new design variations, at the expense of difficulties in scalability to mass-production and a channel surface that requires post-processing to achieve sufficient optical quality. In a second approach, hot embossing using epoxy molds allows for direct structuring of optical grade channels and is scalable to mass production, at the expense of longer cycle time in the development of new channel designs. To facilitate the handling of the bioreactor, a holder was designed to provide the fluidic connections to a pump,ensuring medium exchange and sampling to down-stream sensors connected to the outlets. The design of the bioreactor was intended to maintain and expose pre-formed hepatic co-culture spheroids to toxicants for more than a week. Once seeded, spheroids rest on a polycarbonate membrane with 12 µm pore size, allowing the medium to flow-through, while flow-over is maintained to avoid an excess pressure on the cells. In a single bioreactor, 9 wells are connected to a common inlet to provide the cells with fresh culture medium or test compounds. On a first cell culture trial, it was possible to visually detect the spheroids in the wells after seeding, however, after 1 week of culture there was no possibility to accurately detect the presence and viability of the cells. In the framework of HeMiBio, significant progress has been made towards producing a 3D COC-based bioreactor for hepatic cell culture, and most technological hurdles in producing prototype reactors have been overcome. Further testing is needed to see which improvements to the reactor or the flow conditions should be made to ensure cell viability

Mobility of lysozyme in poly(L-lysine)/hyaluronic acid multilayer films

The spatial and temporal control over presentation of protein-based biomolecules such as growth factors and hormones is crucial for in vitro applications to mimic the complex in vivo environment. We investigated the interaction of a model protein lysozyme (Lys) with poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films. We focused on Lys diffusion as well as adsorption and retention within the film as a function of the film deposition conditions and post-treatment. Additionally, an effect of Lys concentration on its mobility was probed. A combination of confocal fluorescence microscopy, fluorescence recovery after photobleaching, and microfluidics was employed for this investigation. Our main finding is that adsorption of PLL and HA after protein loading induces acceleration and reduction of Lys mobility, respectively. These results suggest that a charge balance in the film to a high extent governs the protein–film interaction. We believe that control over protein mobility is a key to reach the full potential of the PLL/HA films as reservoirs for biomolecules depending on the application demand

Addressing nanomaterial immunosafety by evaluating innate immunity across living species

The interaction of a living organism with external foreign agents is a central issue for its survival and adaptation to the environment. Nanosafety should be considered within this perspective, and it should be examined that how different organisms interact with engineered nanomaterials (NM) by either mounting a defensive response or by physiologically adapting to them. Herein, the interaction of NM with one of the major biological systems deputed to recognition of and response to foreign challenges, i.e., the immune system, is specifically addressed. The main focus is innate immunity, the only type of immunity in plants, invertebrates, and lower vertebrates, and that coexists with adaptive immunity in higher vertebrates. Because of their presence in the majority of eukaryotic living organisms, innate immune responses can be viewed in a comparative context. In the majority of cases, the interaction of NM with living organisms results in innate immune reactions that eliminate the possible danger with mechanisms that do not lead to damage. While in some cases such interaction may lead to pathological consequences, in some other cases beneficial effects can be identified

Probing the immune responses to nanoparticles across environmental species. A perspective of the EU Horizon 2020 project PANDORA

Understanding how engineered nanomaterials affect immune responses of living organisms requires a strong collaborative effort between immunologists, toxicologists, ecologists, physiologists, inorganic chemists, nanomaterial scientists and experts in law and risk management. This perspective aims to provide a new viewpoint on the interaction between engineered nanomaterials and the immune defensive systems across living species, gained within the EU Horizon 2020 project PANDORA. We consider the effects of nanoparticle exposure on immune functions in plants, marine and terrestrial invertebrates and their relation to the current state of knowledge for vertebrates (in particular humans). These studies can shed light on the broader perspective of defensive and homeostatic mechanisms (immunity, inflammation, stress responses, microbiota, stem cell differentiation) suggesting ways to: i) perform a comparative analysis of the nanoparticle impact on immunity across model organisms; ii) inspire best practices in experimental methodologies for nanosafety/nanotoxicity studies; iii) regroup and harmonise fragmented research activities; iv) improve knowledge transfer strategies and nano-security; v) propose innovative tools and realistic solutions, thereby helping in identifying future research needs and tackling their challenges

A new class of thiolipids for the attachment of lipid bilayers on gold surfaces

A new class of lipid mols. is synthesized, based on two dipalmitoylphosphatidic mols., each extended at the lipid phosphate by a hydrophilic spacer chain of ethoxy groups of variable length, which are then coupled as a bilipid via a terminal disulfide group at the hydrophilic spacer. These anchor-bearing \"thiolipids\" can attach to gold substrates by forming stable gold-sulfur bonds. In this way, the authors can couple lipid bilayers to gold surfaces, with the possibility of preserving a water layer between the support and the first monolayer. The thiolipid mols. are characterized on a Langmuir film balance using fluorescence microscopy. The mol. areas of the thiolipids on the water surface are 80-90 .ANG.2 at a fully compressed state. The thiolipid monolayers show a typical first-order phase transition on the water surface with regular, starlike domains. The formation of thiolipid-attached mono- and bilayers on gold surfaces was studied by surface plasmon resonance (SPR), impedance measurements, and cyclic voltammetry. Four different supported membrane systems are studied in detail: (1) pure thiolipid layers; (2) mixed lipid bilayers contg. a first pure thiolipid monolayer and a second one of conventional phospholipids; (3) bilayers, where the first gold-attached monolayer is composed of a mixt. of thio- and conventional phospholipids with another second phospholipid layer on top; (4) monolayers of pure 1-hexadecanethiol and layers with a second phospholipid film on top of the 1-hexadecanethiol. The electrochem. expts. reveal elec. blocking layers for all lipid systems investigated with specific resistances of 104-105 W cm2. The capacitance values for pure thiolipid bilayers are in the range of 0.5-0.7 mF/cm2 for the pure thiolipid bilayers and 0.7-0.8 mF/cm2 for the mixed thiolipid/phospholipid bilayers, which is comparable to the values found for unsupported, so-called black lipid membranes. SPR measurements confirm qual. the results of the electrochem. expts. [on SciFinder (R)

A miniaturized monolayer trough with variable surface area in the square-millimeter range

A new simple concept for a miniaturized monolayer trough is described. The overall monolayer area in the expanded state is approx. 150 mm2 and can be reduced by a factor of 2. The surface area is a function of the shape of the meniscus formed by the subphase and is controlled by the amt. of water in the monolayer trough. The controlled compression of monolayers to a desired area per mol. with simultaneous observation of the lateral distribution of fluorescently labeled mols. is shown. A biol. reaction between a specific antibody and lipid anchored peptide demonstrates the feasibility of monolayer expts., which require only very small quantities of substance (in the pmol range). This trough might also be a valuable tool for the 2D crystn. of proteins at lipid layers via specific binding sites such as metal chelators. [on SciFinder (R)