Phenol-soluble modulins form amyloids in contact with multiple surface chemistries.

Functional amyloids are commonly produced by many microorganisms and their biological functions are numerous. Staphylococcus aureus can secrete a group of peptides named phenol-soluble modulins (PSMs) in their biofilm extracellular matrix. PSMs have been found inside biofilms both in their soluble form and assembled into amyloid structures. Yet, the actual biological function of these amyloids has been highly debated. Here, we assessed the ability of PSMs to form amyloids in contact with different abiotic surfaces to unravel a potential unknown bioadhesive and/or biofilm stabilization function. We combined surface plasmon resonance imaging, fluorescence aggregation kinetics, and FTIR spectroscopy in order to evaluate the PSM adsorption as well as amyloid formation properties in the presence of various surface chemistries. Overall, PSMs adsorb even on low-binding surfaces, making them highly adaptable adsorbants in the context of bioadhesion. Moreover, the PSM aggregation potential to form amyloid aggregates is not impacted by the presence of the surface chemistries tested. This versatility regarding adsorption and amyloid formation may imply a possible role of PSMs in biofilm adhesion and/or structure integrity.

Insulin aggregation starts at dynamic triple interfaces, originating from solution agitation.

The consequences of agitation on protein stability are particularly relevant to therapeutic proteins. However, the precise contribution of the different effects induced by agitation in pathways leading to protein denaturation and aggregation at interfaces is not entirely understood. In particular, the contribution of a moving triple line, induced by the sweeping of a solution meniscus on a container wall upon agitation, has only been rarely assessed. In this article, we therefore designed experimental setups to analyze how mixing, shear stress, and dynamic triple interfaces influence insulin aggregation in physiological conditions. This has been achieved by controlling agitation speed, shear stress, and the extension of triple interfaces in order to shed light on the contribution of different agitation-induced effects on insulin aggregation in physiological conditions. We demonstrate that strong agitation is necessary for the onset of insulin aggregation, while the growth of the aggregates is sustained even under weak agitation. Kinetic insulin aggregation studies in conditions of intermittent wetting show that the aggregation rate correlates with the amount of dynamic triple interfaces that the proteins are exposed to. Finally, we demonstrate that the triple line, where the protein solution, the air, and a hydrophobic surface meet constitutes a preferential early aggregation site.

Interplay between integrins and cadherins to control bone differentiation upon BMP-2 stimulation.

Introduction: Upon BMP-2 stimulation, the osteoblastic lineage commitment in C2C12 myoblasts is associated with a microenvironmental change that occurs over several days. How does BMP-2 operate a switch in adhesive machinery to adapt to the new microenvironment and to drive bone cell fate is not well understood. Here, we addressed this question for BMP-2 delivered either in solution or physically bound of a biomimetic film, to mimic its presentation to cells via the extracellular matrix (ECM). Methods: Biommetics films were prepared using a recently developed automated method that enable high content studies of cellular processes. Comparative gene expressions were done using RNA sequencing from the encyclopedia of the regulatory elements (ENCODE). Gene expressions of transcription factors, beta chain (1, 3, 5) integrins and cadherins (M, N, and Cad11) were studied using quantitative PCR. ECM proteins and adhesion receptor expressions were also quantified by Western blots and dot blots. Their spatial organization in and around cells was studied using immuno-stainings. The individual effect of each receptor on osteogenic transcription factors and alkaline phosphatase expression were studied using silencing RNA of each integrin and cadherin receptor. The organization of fibronectin was studied using immuno-staining and quantitative microscopic analysis. Results: Our findings highlight a switch of integrin and cadherin expression during muscle to bone transdifferentiation upon BMP-2 stimulation. This switch occurs no matter the presentation mode, for BMP-2 presented in solution or via the biomimetic film. While C2C12 muscle cells express M-cadherin and Laminin-specific integrins, the BMP-2-induced transdifferentiation into bone cells is associated with an increase in the expression of cadherin-11 and collagen-specific integrins. Biomimetic films presenting matrix-bound BMP-2 enable the revelation of specific roles of the adhesive receptors depending on the transcription factor. Discussion: While ß3 integrin and cadherin-11 work in concert to control early pSMAD1,5,9 signaling, ß1 integrin and Cadherin-11 control RunX2, ALP activity and fibronectin organization around the cells. In contrast, while ß1 integrin is also important for osterix transcriptional activity, Cadherin-11 and ß5 integrin act as negative osterix regulators. In addition, ß5 integrin negatively regulates RunX2. Our results show that biomimetic films can be used to delinate the specific events associated with BMP-2-mediated muscle to bone transdifferentiation. Our study reveals how integrins and cadherins work together, while exerting distinct functions to drive osteogenic programming. Different sets of integrins and cadherins have complementary mechanical roles during the time window of this transdifferentiation.

Surfactant Protection Efficacy at Surfaces Varies with the Nature of Hydrophobic Materials.

OBJECTIVE: Monoclonal antibodies are in contact with many different materials throughout their life cycle from production to patient administration. Plastic surfaces are commonly found in single use bags, syringes, perfusion bags and tubing and their hydrophobic nature makes them particularly prone for adsorption of therapeutic proteins. The addition of surfactants in therapeutic formulations aims at minimizing surface and interface adsorption of the active molecules. However, their protection efficacy related to the nature of the plastic material is still poorly investigated. METHODS: We use real-time surface-sensitive techniques and immunosorbent assays, to quantify surfactant and monoclonal antibody adsorption on hydrophobic model surfaces and different plastic polymers to analyse the effect of material surface properties on the level of surfactant protection. RESULTS: We show that Polysorbate 80 protects monoclonal antibodies significantly better from adsorption on a polystyrene surface than on a hexadecane self-assembled monolayer, used as a model surface with similar hydrophobicity. This enhanced protective effect on polystyrene is observed for different antibodies and also other surfactants, and its extent depends on the surfactant concentration for a given antibody concentration. A comparative adsorption study allows ranking different in-use plastics and highlights the dependence of Polysorbate 80 protection efficacy on the nature of the plastic material. CONCLUSION: This study demonstrates that, beyond hydrophobicity, the nature of plastic polymer surfaces affects surfactant adsorption and thereby impacts their protection efficacy in therapeutic antibody formulations.

Adsorption rate constants of therapeutic proteins and surfactants for material surfaces.

Adsorption of therapeutic proteins to material surfaces can be a pivotal issue in drug development, especially for low concentration products. Surfactants are used to limit adsorption losses. For each formulation component, surface adsorption is the result of a combination of its diffusion and surface adsorption rates. The latter are difficult to measure accurately because a depletion layer forms rapidly in the bulk solution above a bare surface, slowing down adsorption. Adapting flow conditions and local surface chemistry, we are able to minimize depletion limitations and measure apparent adsorption rate constants of three monoclonal antibodies, other proteins and surfactants with a hydrophobic surface. We show that surface adsorption rates scale with the molecular mass of the molecule, with polysorbates therefore showing thousand times slower rates than antibodies. Moreover, we observed that the desorption dynamic of polysorbates from a given hydrophobic surface depends on surface coverage, whereas this is not the case for Poloxamer 188. These novel contributions to surface adsorption dynamics enable a new perspective on the evaluation of drug surface compatibility and can, together with diffusion rates, be used to predict the protective potential of surfactants in given conditions.

Surface modification of polyurethane with eptifibatide-loaded degradable nanoparticles reducing risk of blood coagulation.

The main purpose of the work was to develop a drug releasing coatings on the surface of medical devices exposed to blood flow, what should enable effective inhibition of blood coagulation process. As a part of the work, the process of encapsulating the anticoagulant drug eptifibatide (EPT) in poly(DL-lactic-co-glycolic acid) (PLGA) nanoparticles was developed. EPT encapsulation efficiency was 29.1 ± 2.1%, while the EPT loading percentage in the nanoparticles was 4.2 ± 0.3%. The PLGA nanoparticles were suspended in a polyanion solution (hyaluronic acid (HA)) and deposited on the surface-treated thermoplastic polyurethane (TPU) by a layer-by-layer method. As a polycation poly-L-lysine (PLL) was used. The influence of released EPT on the activation of the coagulation system was analyzed using dynamic blood tester. Performed experiments show an effective delivery of the drug to the bloodstream and low risk of platelets (membrane receptor) activation. The dynamic blood test process, including its physical phenomenon, was described using numerical methods, i.e. a finite volume cone-and-plate test model as well as non-Newtonian blood models. The values of shear stress and blood flow velocity under the fast-rotating cone were computed applying boundary conditions of cylinder wall imitating blood-nanomaterial interaction. Implementing boundary conditions as initial shear stress values of bottom cylinder wall resulted in the increase of shear stress in blood under rotating cone. The developed system combining drug eluting polymeric nanoparticles with the polyelectrolyte "layer-by-layer" coating can be easily introduced to medical implants of various shape, with the advantages of resorbable drug carriers allowing for local and controllable delivery of anti-thrombogenic drugs.

Functions of the Dictyostelium LIMP-2 and CD36 homologues in bacteria uptake, phagolysosome biogenesis and host cell defence.

Phagocytic cells take up, kill and digest microbes by a process called phagocytosis. To this end, these cells bind the particle, rearrange their actin cytoskeleton, and orchestrate transport of digestive factors to the particle-containing phagosome. The mammalian lysosomal membrane protein LIMP-2 (also known as SCARB2) and CD36, members of the class B of scavenger receptors, play a crucial role in lysosomal enzyme trafficking and uptake of mycobacteria, respectively, and generally in host cell defences against intracellular pathogens. Here, we show that the Dictyostelium discoideum LIMP-2 homologue LmpA regulates phagocytosis and phagolysosome biogenesis. The lmpA knockdown mutant is highly affected in actin-dependent processes, such as particle uptake, cellular spreading and motility. Additionally, the cells are severely impaired in phagosomal acidification and proteolysis, likely explaining the higher susceptibility to infection with the pathogenic bacterium Mycobacterium marinum, a close cousin of the human pathogen Mycobacterium tuberculosis Furthermore, we bring evidence that LmpB is a functional homologue of CD36 and specifically mediates uptake of mycobacteria. Altogether, these data indicate a role for LmpA and LmpB, ancestors of the family of which LIMP-2 and CD36 are members, in lysosome biogenesis and host cell defence.

Visible light-induced insulin aggregation on surfaces via photoexcitation of bound thioflavin T.

Insulin is known to form amyloid aggregates when agitated in a hydrophobic container. Amyloid aggregation is routinely measured by the fluorescence of the conformational dye thioflavin T, which, when incorporated into amyloid fibers, fluoresces at 480 nm. The kinetics of amyloid aggregation in general is characterized by an initial lag-phase, during which aggregative nuclei form on the hydrophobic surface. These nuclei then lead to the formation of fibrils presenting a rapid growth during the elongation phase. Here we describe a novel mechanism of insulin amyloid aggregation which is surprisingly devoid of a lag-time for nucleation. The excitation of thioflavin T by visible light at 440 nm induces the aggregation of thioflavin T-positive insulin fibrils on hydrophobic surfaces in the presence of strong agitation and at physiological pH. This process is material surface-induced and depends on the fact that surface-adsorbed insulin can bind thioflavin T. Light-induced insulin aggregation kinetics is thioflavin T-mediated and is based on an energy transfer from visible light to the protein via thioflavin T. It relies on a constant supply of thioflavin T and insulin from the solution to the aggregate. The growth rate increases with the irradiance and with the concentration of thioflavin T. The supply of insulin seems to be the limiting factor of aggregate growth. This light-induced aggregation process allows the formation of local surface-bound aggregation patterns.

Rapid immunoassay exploiting nanoparticles and micromagnets: proof-of-concept using ovalbumin model.

AIM: We present a fast magnetic immunoassay, combining magnetic nanoparticles and micromagnets. High magnetic field gradients from micromagnets are used to develop a new approach to the standard ELISA. Materials & methods/results: A proof-of-concept based on colorimetric quantification of antiovalbumin antibody in buffer is performed and compared with an ELISA. After optimization, the magnetic immunoassay exhibits a limit of detection (40 ng/ml) and a dynamic range (40-2500 ng/ml) similar to that of ELISAs developed using same biochemical tools. CONCLUSION: Micromagnets can be fully integrated in multiwell plates at low cost to allow the efficient capture of immunocomplexes carried by magnetic nanoparticles. The method is generic and permits to perform magnetic ELISA in 30 min.

Insulin Aggregation at a Dynamic Solid-Liquid-Air Triple Interface.

Therapeutic proteins are privileged in drug development because of their exquisite specificity, which is due to their three-dimensional conformation in solution. During their manufacture, storage, and delivery, interactions with material surfaces and air interfaces are known to affect their stability. The growing use of automated devices for handling and injection of therapeutics increases their exposure to protocols involving intermittent wetting, during which the solid-liquid and liquid-air interfaces meet at a triple contact line, which is often dynamic. Using a microfluidic setup, we analyze the effect of a moving triple interface on insulin aggregation in real time over a hydrophobic surface. We combine thioflavin T fluorescence and reflection interference microscopy to concomitantly monitor insulin aggregation and the morphology of the liquid as it dewets the surface. We demonstrate that insulin aggregates in the region of a moving triple interface and not in regions submitted to hydrodynamic shear stress alone, induced by the moving liquid. During dewetting, liquid droplets form on the surface anchored by adsorbed proteins, and the accumulation of amyloid aggregates is observed exclusively as fluorescent rings growing eccentrically around these droplets. The fluorescent rings expand until the entire channel surface sweeped by the triple interface is covered by amyloid fibers. On the basis of our experimental results, we propose a model describing the growth mechanism of insulin amyloid fibers at a moving triple contact line, where proteins adsorbed at a hydrophobic surface are exposed to the liquid-air interface.

Role of SpdA in Cell Spreading and Phagocytosis in Dictyostelium.

Dictyostelium discoideum is a widely used model to study molecular mechanisms controlling cell adhesion, cell spreading on a surface, and phagocytosis. In this study we isolated and characterize a new mutant created by insertion of a mutagenic vector in the heretofore uncharacterized spdA gene. SpdA-ins mutant cells produce an altered, slightly shortened version of the SpdA protein. They spread more efficiently than WT cells when allowed to adhere to a glass substrate, and phagocytose particles more efficiently. On the contrary, a functional spdA knockout mutant where a large segment of the gene was deleted phagocytosed less efficiently and spread less efficiently on a substrate. These phenotypes were highly dependent on the cellular density, and were most visible at high cell densities, where secreted quorum-sensing factors inhibiting cell motility, spreading and phagocytosis are most active. These results identify the involvement of SpdA in the control of cell spreading and phagocytosis. The underlying molecular mechanisms, as well as the exact link between SpdA and cell spreading, remain to be established.

Dual Effect of (LK)nL Peptides on the Onset of Insulin Amyloid Fiber Formation at Hydrophobic Surfaces.

Soluble proteins are constantly in contact with material or cellular surfaces, which can trigger their aggregation and therefore have a serious impact on the development of stable therapeutic proteins. In contact with hydrophobic material surfaces, human insulin aggregates readily into amyloid fibers. The kinetics of this aggregation can be accelerated by small peptides, forming stable beta-sheets on hydrophobic surfaces. Using a series of (LK)nL peptides with varying length, we show that these peptides, at low, substoichiometric concentrations, have a positive, cooperative effect on insulin aggregation. This effect is based on a cooperative adsorption of (LK)nL peptides at hydrophobic surfaces, where they form complexes that help the formation of aggregation nuclei. At higher concentrations, they interfere with the formation of an aggregative nucleus. These effects are strictly dependent on the their adsorption on hydrophobic material surfaces and highlight the importance of the impact of materials on protein stability. (LK)nL peptides prove to be valuable tools to investigate the mechanism of HI aggregation nuclei formation on hydrophobic surfaces.

The effect of delivering the chemokine SDF-1α in a matrix-bound manner on myogenesis.

Several chemokines are important in muscle myogenesis and in the recruitment of muscle precursors during muscle regeneration. Among these, the SDF-1α chemokine (CXCL12) is a potent chemoattractant known to be involved in muscle repair. SDF-1α was loaded in polyelectrolyte multilayer films made of poly(L-lysine) and hyaluronan to be delivered locally to myoblast cells in a matrix-bound manner. The adsorbed amounts of SDF-1α were tuned over a large range from 100 ng/cm(2) to 5 µg/cm(2), depending on the initial concentration of SDF-1α in solution, its pH, and on the film crosslinking extent. Matrix-bound SDF-1α induced a striking increase in myoblast spreading, which was revealed when it was delivered from weakly crosslinked films. It also significantly enhanced cell migration in a dose-dependent manner, which again depended on its presentation by the biopolymeric film. The low-crosslinked film was the most efficient in boosting cell migration. Furthermore, matrix-bound SDF-1α also increased the expression of myogenic markers but the fusion index decreased in a dose-dependent manner with the adsorbed amount of SDF-1α. At high adsorbed amounts of SDF-1α, a large number of Troponin T-positive cells had only one nucleus. Overall, this work reveals the importance of the presentation mode of SDF-1α to emphasize its effect on myogenic processes. These films may be further used to provide insight into the role of SDF-1α presented by a biomaterial in physiological or pathological processes.

Peptides that form ß-sheets on hydrophobic surfaces accelerate surface-induced insulin amyloidal aggregation.

Interactions between proteins and material or cellular surfaces are able to trigger protein aggregation in vitro and in vivo. The human insulin peptide segment LVEALYL is able to accelerate insulin aggregation in the presence of hydrophobic surfaces. We show that this peptide needs to be previously adsorbed on a hydrophobic surface to induce insulin aggregation. Moreover, the study of different mutant peptides proves that its sequence is less important than the secondary structure of the adsorbed peptide on the surface. Indeed, these pro-aggregative peptides act by providing stable ß-sheets to incoming insulin molecules, thereby accelerating insulin adsorption locally and facilitating the conformational changes required for insulin aggregation. Conversely, a peptide known to form α-helices on hydrophobic surfaces delays insulin aggregation.

Human insulin adsorption kinetics, conformational changes and amyloidal aggregate formation on hydrophobic surfaces.

The formation of insulin amyloidal aggregates on material surfaces is a well-known phenomenon with important pharmaceutical and medical implications. Using surface plasmon resonance imaging, we monitor insulin adsorption on model hydrophobic surfaces in real time. Insulin adsorbs in two phases: first, a very fast phase (less than 1 min), where a protein monolayer forms, followed by a slower one that can last for at least 1h, where multilayered protein aggregates are present. The dissociation kinetics reveals the presence of two insulin populations that slowly interconvert: a rapidly dissociating pool and a pool of strongly bound insulin aggregates. After 1h of contact between the protein solution and the surface, the adsorbed insulin has practically stopped dissociating from the surface. The conformation of adsorbed insulin is probed by attenuated total reflection-Fourier transform infrared spectroscopy. Characteristic shifts in the amide A and amide II' bands are associated with insulin adsorption. The amide I band is also distinct from that of soluble or aggregated insulin, and it slowly evolves in time. A 1708 cm⁻¹ peak is observed, which characterizes insulin adsorbed for times longer than 30 min. Finally, Thioflavin T, a marker of extended ß-sheet structures present in amyloid fibers, binds to adsorbed insulin after 30-40 min. Altogether, these results reveal that the conformational change induced in insulin upon binding to hydrophobic surfaces allows further insulin binding from the solution. Adsorbed insulin is thus an intermediate along the α-to-ß structural transition that results in the formation of amyloidal fibers on these material surfaces.

Surface chemistry at the nanometer scale influences insulin aggregation.

We synthesized surfaces with different hydrophobicities and roughness by forming self-assembled monolayers (SAMs) of mixed amine and octyl silanes. Insulin aggregation kinetics in the presence of the above surfaces is characterized by a typical lag phase and growth rate. We show that the lag time but not the growth rate varies as a function of the amine fraction on the surface. The amount of adsorbed protein and the adsorption rate during the aggregation process also vary with the amine fraction on the surface and are maximal for equal parts of amine and octyl groups. For all surfaces, the growth phase starts for identical amounts of adsorbed insulin. The initial surface roughness determines the rate at which protein adsorption occurs and hence the time to accumulate enough protein to form aggregation nuclei. In addition, the surface chemistry and topography influence the morphology of aggregates adsorbed on the material surface and the secondary structures of final aggregates released in solution.

Hemocompatibility of Inorganic Physical Vapor Deposition (PVD) Coatings on Thermoplastic Polyurethane Polymers.

Biocompatibility improvements for blood contacting materials are of increasing interest for implanted devices and interventional tools. The current study focuses on inorganic (titanium, titanium nitride, titanium oxide) as well as diamond-like carbon (DLC) coating materials on polymer surfaces (thermoplastic polyurethane), deposited by magnetron sputtering und pulsed laser deposition at room temperature. DLC was used pure (a-C:H) as well as doped with silicon, titanium, and nitrogen + titanium (a-C:H:Si, a-C:H:Ti, a-C:H:N:Ti). In-vitro testing of the hemocompatibility requires mandatory dynamic test conditions to simulate in-vivo conditions, e.g., realized by a cone-and-plate analyzer. In such tests, titanium- and nitrogen-doped DLC and titanium nitride were found to be optimally anti-thrombotic and better than state-of-the-art polyurethane polymers. This is mainly due to the low tendency to platelet microparticle formation, a high content of remaining platelets in the whole blood after testing and low concentration of platelet activation and aggregation markers. Comparing this result to shear-flow induced cell motility tests with e.g., Dictostelium discoideum cell model organism reveals similar tendencies for the investigated materials.

Diamagnetically trapped arrays of living cells above micromagnets.

Cell arrays are of foremost importance for many applications in pharmaceutical research or fundamental biology. Although arraying techniques have been widely investigated for adherent cells, organization of cells in suspension has been rarely considered. The arraying of non-adherent cells using the diamagnetic repulsive force is presented. A planar arrangement of Jurkat cells is achieved at the microscale above high quality microfabricated permanent magnets with remanent magnetization of J(r)≈ 1 T, in the presence of a paramagnetic contrast agent. The cytotoxicity of three Gd based contrast agents, Gd-DOTA, Gd-BOPTA and Gd-HP-DO3A, is studied. Among them, Gd-HP-DO3A appears to be the most biocompatible toward Jurkat cells. In close agreement with analytical simulations, diamagnetically 'suspended' cells have been successfully arrayed above square and honeycomb-like micromagnet arrays, which act as a "diamagnetophobic" surface. Living cell trapping is achieved in a simple manner using concentrations of Gd-HP-DO3A as low as 1.5 mM.

Synchronization of Dictyostelium discoideum adhesion and spreading using electrostatic forces.

Synchronization of cell spreading is valuable for the study of molecular events involved in the formation of adhesive contacts with the substrate. At a low ionic concentration (0.17 mM) Dictyostelium discoideum cells levitate over negatively charged surfaces due to electrostatic repulsion. First, a two-chamber device, divided by a porous membrane, allows to quickly increase the ionic concentration around the levitating cells. In this way, a good synchronization was obtained, the onsets of cell spreading being separated by less than 5 s. Secondly applying a high potential pulse (2.5 V/Ref, 0.1s) to an Indium Tin Oxide surface attracts the cells toward the surface where they synchronously spread as monitored by LimE(Deltacoil)-GFP. During spreading, actin polymerizes in series of active spots. On average, the first spot appears 8-11s after the electric pulse and the next ones appear regularly, separated by about 10s. Synchronized actin-polymerization activity continues for 40s. Using an electric pulse to control the exact time point at which cells contact the surface has allowed for the first time to quantify the cellular response time for actin polymerization. Electrochemical synchronization is therefore a valuable tool to study intracellular responses to contact.

Hemocompatible, pulsed laser deposited coatings on polymers.

State-of-the-art non-thrombogenic blood contacting surfaces are based on heparin and struggle with the problem of bleeding. However, appropriate blood flow characteristics are essential for clinical application. Thus, there is increasing demand to develop new coating materials for improved human body acceptance. Materials deposited by vacuum coating techniques would be an excellent alternative if the coating temperatures can be kept low because of the applied substrate materials of low temperature resistance (polymers). Most of the recently used plasma-based deposition techniques cannot fulfill this demand. However, adequate film structure and high adhesion can be reached by the pulsed laser deposition at room temperature, which was developed to an industrial-scaled process at Laser Center Leoben. Here, this process is described in detail and the resulting structural film properties are shown for titanium, titanium nitride, titanium carbonitride, and diamond-like carbon on polyurethane, titanium and silicon substrates. Additionally, we present the biological response of blood cells and the kinetic mechanism of eukaryote cell attachment. In conclusion, high biological acceptance and distinct differences for the critical delamination shear stress were found for the coatings, indicating higher adhesion at higher carbon contents.