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.

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.

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.

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.

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.

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.

Reversal of cell polarity and actin-myosin cytoskeleton reorganization under mechanical and chemical stimulation.

To study reorganization of the actin system in cells that invert their polarity, we stimulated Dictyostelium cells by mechanical forces from alternating directions. The cells oriented in a fluid flow by establishing a protruding front directed against the flow and a retracting tail. Labels for polymerized actin and filamentous myosin-II marked front and tail. At 2.1 Pa, actin first disassembled at the previous front before it began to polymerize at the newly induced front. In contrast, myosin-II slowly disappeared from the previous tail and continuously redistributed to the new tail. Front specification was myosin-II independent and accumulation of polymerized actin was even more focused in mutants lacking myosin-II heavy chains. We conclude that under mechanical stimulation, the inversion of cell polarity is initiated by a global internal signal that turns down actin polymerization in the entire cell. It is thought to be elicited at the most strongly stimulated site of the cell, the incipient front region, and to be counterbalanced by a slowly generated, short-range signal that locally activates actin polymerization at the front. Similar pattern of front and tail interconversion were observed in cells reorienting in strong gradients of the chemoattractant cyclic AMP.

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.

Influence of glass and polymer coatings on CHO cell morphology and adhesion.

Successful development of cell-on-chip microsystems where living cells are deposited and grown in microfabricated structures is highly dependent on the control of cell/substrate interactions. In this study, several materials of interest were tested for CHO cell growth and morphology: (i) glass, fibronectin-, poly-L-lysine- and 3-aminopropyltriethoxysilane (APTES)--treated glass and UV/O(3)-modified PDMS coating on glass as well as (ii) silicon, poly-L-lysine-, APTES-, O(2) plasma-treated and oxide-coated silicon. In addition, we quantitatively characterized cell adhesion to these substrates using a radial flow detachment assay. Lack of correlation between cell adhesion and cell morphology was systematically observed for all substrates. In particular, we show that PDMS coatings on glass can be finely tuned by UV/O(3) treatment to enhance cell adhesion and induce elongated morphology. Moreover, we observed a low shear stress cell detachment mechanism on silicon oxide coatings on silicon wafers. It is therefore possible with these coatings to selectively influence either cell adhesion or morphology.

Synergistic control of cellular adhesion by transmembrane 9 proteins.

The transmembrane 9 (TM9) family of proteins contains numerous members in eukaryotes. Although their function remains essentially unknown in higher eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently reported to be essential for cellular adhesion and phagocytosis. Herein, the function of Phg1a and of a new divergent member of the TM9 family called Phg1b was further investigated in D. discoideum. The phenotypes of PHG1a, PHG1b, and PHG1a/PHG1b double knockout cells revealed that Phg1a and Phg1b proteins play a synergistic but not redundant role in cellular adhesion, phagocytosis, growth, and development. Complementation analysis supports a synergistic regulatory function rather than a receptor role for Phg1a and Phg1b proteins. Together, these results suggest that Phg1 proteins act as regulators of cellular adhesion, possibly by controlling the intracellular transport in the endocytic pathway and the composition of the cell surface.

High-resolution dissection of phagosome maturation reveals distinct membrane trafficking phases.

Molecular mechanisms of endocytosis in the genetically and biochemically tractable professional phagocyte Dictyostelium discoideum reveal a striking degree of similarity to higher eukaryotic cells. Pulse-chase feeding with latex beads allowed purification of phagosomes at different stages of maturation. Gentle ATP stripping of an actin meshwork entrapping contaminating organelles resulted in a 10-fold increase in yield and purity, as confirmed by electron microscopy. Temporal profiling of signaling, cytoskeletal, and trafficking proteins resulted in a complex molecular fingerprint of phagosome biogenesis and maturation. First, nascent phagosomes were associated with coronin and rapidly received a lysosomal glycoprotein, LmpB. Second, at least two phases of delivery of lysosomal hydrolases (cathepsin D [CatD] and cysteine protease [CPp34]) were accompanied by removal of plasma membrane components (PM4C4 and biotinylated surface proteins). Third, a phase of late maturation, preparing for final exocytosis of undigested material, included quantitative recycling of hydrolases and association with vacuolin. Also, lysosomal glycoproteins of the Lmp family showed distinct trafficking kinetics. The delivery and recycling of CatD was directly visualized by confocal microscopy. This heavy membrane traffic of cargos was precisely accompanied by regulatory proteins such as the Rab7 GTPases and the endosomal SNAREs Vti1 and VAMP7. This initial molecular description of phagocytosis demonstrates the feasibility of a comprehensive analysis of phagosomal lipids and proteins in genetically modified strains.

Phg2, a kinase involved in adhesion and focal site modeling in Dictyostelium.

The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility.

The AP-1 clathrin-adaptor is required for lysosomal enzymes sorting and biogenesis of the contractile vacuole complex in Dictyostelium cells.

Adaptor protein complexes (AP) are major components of the cytoplasmic coat found on clathrin-coated vesicles. Here, we report the molecular and functional characterization of Dictyostelium clathrin-associated AP-1 complex, which in mammalian cells, participates mainly in budding of clathrin-coated vesicles from the trans-Golgi network (TGN). The gamma-adaptin AP-1 subunit was cloned and shown to belong to a Golgi-localized 300-kDa protein complex. Time-lapse analysis of cells expressing gamma-adaptin tagged with the green-fluorescent protein demonstrates the dynamics of AP-1-coated structures leaving the Golgi apparatus and rarely moving toward the TGN. Targeted disruption of the AP-1 medium chain results in viable cells displaying a severe growth defect and a delayed developmental cycle compared with parental cells. Lysosomal enzymes are constitutively secreted as precursors, suggesting that protein transport between the TGN and lysosomes is defective. Although endocytic protein markers are correctly localized to endosomal compartments, morphological and ultrastructural studies reveal the absence of large endosomal vacuoles and an increased number of small vacuoles. In addition, the function of the contractile vacuole complex (CV), an osmoregulatory organelle is impaired and some CV components are not correctly targeted.

Involvement of the AP-1 adaptor complex in early steps of phagocytosis and macropinocytosis.

The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1(-) cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1(-) cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1(-) cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation.

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.

Kinetics of yeast detachment from controlled stainless steel surfaces.

Using a radial flow chamber, we study Saccharomyces cerevisiae kinetics of detachment from stainless steel substrates. Samples of similar surface chemistry, but with different surface topologies are compared: mirror polished and electro-chemically etched. Different grain sizes (20, 40 and 100 microm) and different etching depths (100-650 nm) are tested. Cells are removed from the substrate according to a first-order kinetics defining two macroscopic parameters that depend on the applied stress: the detachment efficiency and the detachment rate constant. Whatever the surface topology, detachment occurs above a threshold and its rate is strongly stimulated by the applied stress. The detachment efficiency is characterized by the shear stress at which half of the cells detach and is independent of surface topology. In contrast, detachment is faster from etched than mirror polished surfaces. Finally, we also show the preferential adhesion of yeast cells to grains of < 001 > crystallographic orientation with respect to the surface.

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.