Effect of Noise on Determining Ultrathin-Film Parameters from QCM-D Data with the Viscoelastic Model.

Quartz crystal microbalance with dissipation monitoring (QCM-D) is a well-established technique for studying soft films. It can provide gravimetric as well as nongravimetric information about a film, such as its thickness and mechanical properties. The interpretation of sets of overtone-normalized frequency shifts, ∆f/n, and overtone-normalized shifts in half-bandwidth, ΔΓ/n, provided by QCM-D relies on a model that, in general, contains five independent parameters that are needed to describe film thickness and frequency-dependent viscoelastic properties. Here, we examine how noise inherent in experimental data affects the determination of these parameters. There are certain conditions where noise prevents the reliable determination of film thickness and the loss tangent. On the other hand, we show that there are conditions where it is possible to determine all five parameters. We relate these conditions to the mathematical properties of the model in terms of simple conceptual diagrams that can help users understand the model's behavior. Finally, we present new open source software for QCM-D data analysis written in Python, PyQTM.

When a transmembrane channel isn't, or how biophysics and biochemistry (mis)communicate.

Annexins are a family of soluble proteins that bind to acidic phospholipids such as phosphatidylserine in a calcium-dependent manner. The archetypical member of the annexin family is annexin A5. For many years, its function remained unknown despite the availability of a high-resolution structure. This, combined with the observations of specific ion conductance in annexin-bound membranes, fueled speculations about the possible membrane-spanning forms of annexins that functioned as ion channels. The channel hypothesis remained controversial and did not gather sufficient evidence to become accepted. Yet, it continues to draw attention as a framework for interpreting indirect (e.g., biochemical) data. The goal of the mini-review is to examine the data on annexin-lipid interactions from the last ~30 years from the point of view of the controversy between the two lines of inquiry: the well-characterized peripheral assembly of the annexins at membranes vs. their putative transmembrane insertion. In particular, the potential role of lipid rearrangements induced by annexin binding is highlighted.

Platelet Immobilization on Supported Phospholipid Bilayers for Single Platelet Studies.

The worldwide cardiovascular disease (CVD) epidemic is of grave concern. A major role in the etiology of CVDs is played by the platelets (thrombocytes). Platelets are anuclear cell fragments circulating in the blood. Their primary function is to catalyze clot formation, limiting traumatic blood loss in the case of injury. The same process leads to thrombosis in the case of CVDs, which are commonly managed with antiplatelet therapy. Platelets also have other, nonhemostatic functions in wound healing, inflammation, and tissue regeneration. They play a role in the early stages of atherosclerosis and the spread of cancer through metastases. Much remains to be learned about the regulation of these diverse platelet functions under physiological and pathological conditions. Breakthroughs in this regard are expected to come from single platelet studies and systems approaches. The immobilization of platelets at surfaces is advantageous for developing such approaches, but platelets are activated when they come in contact with foreign surfaces. In this work, we develop and validate a protocol for immobilizing platelets on supported lipid bilayers without activation due to immobilization. Our protocol can therefore be used for studying platelets with a wide variety of surface-sensitive techniques.

Lipid phase behavior studied with a quartz crystal microbalance: A technique for biophysical studies with applications in screening.

Quartz crystal microbalance (QCM) is emerging as a versatile tool for studying lipid phase behavior. The technique is attractive for fundamental biophysical studies as well applications because of its simplicity, flexibility, and ability to work with very small amounts of material crucial for biomedical studies. Further progress hinges on the understanding of the mechanism, by which a surface-acoustic technique such as QCM, senses lipid phase changes. Here, we use a custom-built instrument with improved sensitivity to investigate phase behavior in solid-supported lipid systems of different geometries (adsorbed liposomes and bilayers). We show that we can detect a model anesthetic (ethanol) through its effect on the lipid phase behavior. Further, through the analysis of the overtone dependence of the phase transition parameters, we show that hydrodynamic effects are important in the case of adsorbed liposomes, and viscoelasticity is significant in supported bilayers, while layer thickness changes make up the strongest contribution in both systems.

The sweeter aspects of platelet activation: A lectin-based assay reveals agonist-specific glycosylation patterns.

BACKGROUND: The diversity of platelet functions implies multiple activation states arising in response to different stimuli. Distinguishing between these states has been challenging. METHODS: We used fluorescently labelled carbohydrate binding proteins lectins to investigate agonist-induced changes in platelet surface glycosylation. RESULTS: Each of the seven agonists we used caused a unique set of changes in platelet surface glycosylation, eliciting a unique functional state. Some of these changes could be correlated with the expression of granule-specific markers CD62P and CD63, but lectins proved much more sensitive to differences between agonists than antibodies against those markers. This sensitivity appears to arise from the relation between the surface glycosylation changes and the signalling pathways through which various agonists act. In this context it is interesting that the effects of calcium ionophore were significantly different from those of other agonists. We also found that that P-selectin (CD62P) contains haptens for lectins VFA and PTII, because these lectins compete with the anti-CD62P antibody binding and vice a versa. CONCLUSIONS: We report for the first time that changes in platelet surface glycosylation are agonist-specific and can be distinguished using lectin-binding assays. Lectin fingerprinting represents a new research and diagnostic tool for studying platelet activation. GENERAL SIGNIFICANCE: The observation of agonist-specific platelet surface glycosylation changes is interesting in the context of the diversity of platelet function, because surface glycans mediate contact interactions between platelets and other cells and serve as binding sites for some of the agonists (galectins).

Label-free characterization of biomembranes: from structure to dynamics.

We review recent progress in the study of the structure and dynamics of phospholipid membranes and associated proteins, using novel label-free analytical tools. We describe these techniques and illustrate them with examples highlighting current capabilities and limitations. Recent advances in applying such techniques to biological and model membranes for biophysical studies and biosensing applications are presented, and future prospects are discussed.

Quartz crystal microbalance in soft and biological interfaces.

Applications of quartz crystal microbalance with dissipation to studying soft and biological interfaces are reviewed. The focus is primarily on data analysis through viscoelastic modeling and a model-free approach focusing on the acoustic ratio. Current challenges and future research and development directions are discussed.

Nanoscale departures: excess lipid leaving the surface during supported lipid bilayer formation.

The behavior of small liposomes on surfaces of inorganic oxides remains enigmatic. Under appropriate conditions it results in the formation of supported lipid bilayers (SLBs). During this process, some lipids leave the surface (desorb). We were able to visualize this by a combination of time-resolved fluorescence microscopy and fluorescence recovery after photobleaching studies. Our observations also allowed us to analyze the kinetics of bilayer patch growth during the late stages of SLB formation. We found that it entails a balance between desorption of excess lipids and further adsorption of liposomes from solution. These studies were performed with liposomes containing zwitterionic phospholipids (dioleoylphosphatidylcholine alone or a mixture of dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and cholesterol) on TiO2 in the presence of Ca(2+) but in the absence of other salts.

Unraveling supported lipid bilayer formation kinetics: osmotic effects.

Solid-supported lipid bilayers are used as cell membrane models and form the basis of biomimetic and biosensor platforms. The mechanism of their formation from adsorbed liposomes is not well-understood. Using membrane-permeable solute glycerol, impermeable solutes sucrose and dextran, and a pore forming peptide melittin, we studied experimentally how osmotic effects affect the kinetics of the adsorbed liposome-to-bilayer transition. We find that its rate is enhanced if adsorbed liposomes are made permeable but is not significantly retarded by impermeable solutes. The results are explained in terms of adsorbed liposome deformation and formation of transmembrane pores.

Time-of-flight secondary ion mass spectrometry with principal component analysis of titania-blood plasma interfaces.

Treatment of osseoimplant surfaces with autologous platelet-rich plasma prepared according to the plasma rich in growth factors (PRGF-Endoret) protocol prior to implantation yields promising results in the clinic. Our objective is to understand the organization of complex interfaces between blood plasma preparations of various compositions and model titania surfaces. Here we present the results of the morphological and chemical characterization of TiO(2) surfaces incubated with four types of blood plasma preparations devoid of leukocytes and red blood cells: either enriched in platelets (PRGF-Endoret) or platelet-depleted, and either activated with CaCl(2) to induce clotting, or not. Chemical characterization was done by time-of-flight secondary ion mass spectrometry with principal component analysis (ToF-SIMS/PCA). The interface morphology was studied with scanning electron and atomic force microscopy. Immunofluorescence microscopy was used to identify platelets and infer their activation state. We observe clear differences among the four types of interfaces by ToF-SIMS/PCA. Some of these could be straightforwardly related to the differences in the sample morphology and known effects of platelet activation, but others are more subtle. Strikingly, it was possible to differentiate between these samples by ToF-SIMS/PCA of the protein species alone. This clearly indicates that the composition, orientation, and/or conformation of the proteins in these specimens depend both on the platelets' presence and on their activation. The ToF-SIMS imaging functionality furthermore provides unique insight into the distribution of phospholipid species in these samples.

Adsorbed liposome deformation studied with quartz crystal microbalance.

Deformation of surface-adsorbed liposomes is an important parameter that governs the kinetics of their transformations, but one that is very difficult to measure in the case of nm-size liposomes. We investigate the deformation of dimyristoyl phosphatidyl choline liposomes by quartz crystal microbalance (QCM) as a function of temperature and show that it follows the dependence of this lipid's bending modulus on temperature, as expected from theoretical considerations. To corroborate our approach, we model QCM response from adsorbed liposomes by explicitly considering their shape and mechanical properties.

Hearing what you cannot see and visualizing what you hear: interpreting quartz crystal microbalance data from solvated interfaces.

Over the last 2 decades, the quartz crystal microbalance (QCM or QCM-D) has emerged as a versatile tool for investigating soft and solvated interfaces between solid surfaces and bulk liquids because it can provide a wealth of information about key structural and functional parameters of these interfaces. In this Feature, we offer QCM users a set of guidelines for interpretation and quantitative analysis of QCM data based on a synthesis of well-established concepts rooted in rheological research of the last century and of new results obtained in the last several years.

A Multichannel Microfluidic Sensing Cartridge for Bioanalytical Applications of Monolithic Quartz Crystal Microbalance.

Integrating acoustic wave sensors into lab-on-a-chip (LoC) devices is a well-known challenge. We address this challenge by designing a microfluidic device housing a monolithic array of 24 high-fundamental frequency quartz crystal microbalance with dissipation (HFF-QCMD) sensors. The device features six 6-µL channels of four sensors each for low-volume parallel measurements, a sealing mechanism that provides appropriate pressure control while assuring liquid confinement and maintaining good stability, and provides a mechanical, electrical, and thermal interface with the characterization electronics. We validate the device by measuring the response of the HFF-QCMD sensors to the air-to-liquid transition, for which the robust Kanazawa-Gordon-Mason theory exists, and then by studying the adsorption of model bioanalytes (neutravidin and biotinylated albumin). With these experiments, we show how the effects of the protein-surface interactions propagate within adsorbed protein multilayers, offering essentially new insight into the design of affinity-based bioanalytical sensors.

Dissipation in films of adsorbed nanospheres studied by quartz crystal microbalance (QCM).

The quartz crystal microbalance (QCM) has become a popular method to study the formation of surface-confined films that consist of discrete biomolecular objects--such as proteins, phospholipid vesicles, virus particles--in liquids. The quantitative interpretation of QCM data--frequency and bandwidth (or, equivalently, dissipation) shifts--obtained with such films is limited by the lack of understanding of the energy dissipation mechanisms that operate in these films as they are sheared at megahertz frequencies during the QCM experiment. Here, we investigate dissipation mechanisms in such films experimentally and by finite-element method (FEM) calculations. Experimentally, we study the adsorption of globular proteins and virus particles to surfaces with various attachment geometries: direct adsorption to the surface, attachment via multiple anchors, or attachment via a single anchor. We find that the extent of dissipation caused by the film and the evolution of dissipation as a function of surface coverage is not dependent on the internal properties of these particles but rather on the geometry of their attachment to the surface. FEM calculations reproduce the experimentally observed behavior of the dissipation. In particular, a transient maximum in dissipation that is observed experimentally is reproduced by the FEM calculations, provided that the contact zone between the sphere and the surface is narrow and sufficiently soft. Both a small-angle rotation of the sphere in the flow field of the background fluid (rocking) and a small-amplitude slippage (sliding) contribute to the dissipation. At high coverage, lateral hydrodynamic interactions between neighboring spheres counteract these modes of dissipation, which results in a maximum in dissipation at intermediate adsorption times. These results highlight that, in many scenarios of biomolecular adsorption, the dissipation is not primarily determined by the adsorbate itself, but rather by the link by which it is bound to the substrate.

An ultrafast quartz crystal microbalance based on a frequency comb approach delivers sub-millisecond time resolution.

Quartz crystal microbalance with dissipation monitoring (QCMD) is a simple and versatile sensing technique with applications in a wide variety of academic and industrial fields, most notably electrochemistry, biophysics, quality control, and environmental monitoring. QCMD is limited by a relatively poor time resolution, which is of the order of seconds with conventional instrument designs at the noise level usually required. In this work, we present a design of an ultrafast QCMD with submillisecond time resolution. It is based on a frequency comb approach applied to a high-fundamental-frequency (HFF) resonator through a multifrequency lock-in amplifier. The combination allows us to reach data acquisition rates >10 kHz. We illustrate the method using a toy model of a glass sphere dropped on the resonator surfaces, bare or coated with liposomes, in liquid. We discuss some interesting features of the results obtained with the dropped spheres, such as bending of the HFF resonators due to the impact, sphere bouncing (or the absence of it), and contact aging.

BloodSurf 2017: News from the blood-biomaterial frontier.

From stents and large-diameter vascular grafts, to mechanical heart valves and blood pumps, blood-contacting devices are enjoying significant clinical success owing to the application of systemic antiplatelet and anticoagulation therapies. On the contrary, research into material and device hemocompatibility aimed at alleviating the need for systemic therapies has suffered a decline. This research area is undergoing a renaissance fueled by recent fundamental insights into coagulation and inflammation that are offering new avenues of investigation, the growing recognition of the limitations facing existing therapeutic approaches, and the severity of the cardiovascular disorders epidemic. This Opinion article discusses clinical needs for hemocompatible materials and the emerging research directions for fulfilling those needs. Based on the 2017 BloodSurf conference that brought together clinicians, scientists, and engineers from academia, industry, and regulatory bodies, its purpose is to draw the attention of the wider clinical and scientific community to stimulate further growth. STATEMENT OF SIGNIFICANCE: The article highlights recent fundamental insights into coagulation, inflammation, and blood-biomaterial interactions that are fueling a renaissance in the field of material hemocompatibility. It will be useful for clinicians, scientists, engineers, representatives of industry and regulatory bodies working on the problem of developing hemocompatible materials and devices for treating cardiovascular disorders.

Pegylated poly(anhydride) nanoparticles for oral delivery of docetaxel.

The aim of this work was to investigate the potential of pegylated poly(anhydride) nanoparticles to enhance the oral bioavailability of docetaxel (DTX). Nanoparticles were prepared after the incubation between the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN), poly(ethylene glycol) (PEG2000 or PEG6000) and docetaxel (DTX). The oral administration of a single dose of pegylated nanoparticles to mice provided sustained and prolonged therapeutic plasma levels of docetaxel for up 48-72 h. In addition, the relative oral bioavailability of docetaxel was around 32%. The organ distribution studies revealed that docetaxel underwent a similar distribution when orally administered encapsulated in nanoparticles as when intravenously as Taxotere®. This observation, with the fact that the clearance of docetaxel when loaded into the oral pegylated nanoparticles was found to be similar to that of intravenous formulation, suggests that docetaxel would be released at the epithelium surface and then absorbed to the circulation.

Stirred, shaken, or stagnant: What goes on at the blood-biomaterial interface.

There is a widely recognized need to improve the performance of vascular implants and external medical devices that come into contact with blood by reducing adverse reactions they cause, such as thrombosis and inflammation. These reactions lead to major adverse cardiovascular events such as heart attacks and strokes. Currently, they are managed therapeutically. This need remains unmet by the biomaterials research community. Recognized stagnation of the blood-biomaterial interface research translates into waning interest from clinicians, funding agencies, and practitioners of adjacent fields. The purpose of this contribution is to stir things up. It follows the 2014 BloodSurf meeting (74th International IUVSTA Workshop on Blood-Biomaterial Interactions), offers reflections on the situation in the field, and a three-pronged strategy integrating different perspectives on the biological mechanisms underlying blood-biomaterial interactions. The success of this strategy depends on reengaging clinicians and on the renewed cooperation of the funding agencies to support long-term efforts.

Subpopulations in purified platelets adhering on glass.

Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process.

Differences in intracellular calcium dynamics cause differences in α-granule secretion and phosphatidylserine expression in platelets adhering on glass and TiO2.

In this study, the activation of purified human platelets due to their adhesion on glass and TiO2 in the absence of extracellular calcium was investigated. Differences in α-granule secretion between platelets adhering on the two surfaces were detected by examining the expression and secretion of the α-granule markers P-selectin (CD62P) and ß-thromboglobulin. Similarly, differences in the expression of phosphatidylserine (PS), and in the activation of the major integrin GPIIb/IIIa, on the surfaces of the adhering platelets, were also observed. While all of these activation markers were expressed in platelets adhering on glass, the surface markers were not expressed in platelets adhering on TiO2, and ß-thromboglobulin secretion levels were substantially reduced. Differences in marker expression and secretion correlated with differences in the intracellular calcium dynamics. Calcium ionophore treatment triggered α-granule secretion and PS expression in TiO2-adhering platelets but had no effect on the activation of GPIIb/IIIa. These results demonstrate specificity in the way surfaces of artificial materials activate platelets, link differences in the intracellular calcium dynamics observed in the platelets adhering on the two surfaces to the differences in some of the platelet responses (α-granule secretion and PS expression), but also highlight the involvement of synergistic, calcium-independent pathways in platelet activation. The ability to control activation in surface-adhering platelets makes this an attractive model system for studying platelet signaling pathways and for tissue engineering applications.