Visualizing molecular deformation in fibrin networks under tensile loading via FLIM-FRET.

Mapping molecular deformation and forces in protein biomaterials is critical to understanding mechanochemistry. Here we use intramolecular Förster resonance energy transfer (FRET) of dual-labeled fibrin to distinguish molecular conformations of proteins in situ during mechanical loading. The FRET approach offers increased spatial resolution compared to our previous vibrational imaging. By using fluorescence lifetime microscopy (FLIM), we demonstrate that the combination of FRET and FLIM can probe the molecular changes in fibrin with high spatial (nanometer) and temporal (nanosecond) resolution. Our results map changes in fibrin monomer deformation during the macroscopic loading of the fibrin network, paving the way to directly visualizing the biomaterial mechanics and structure in cell-ECM scaffolds for the first time.

Ligand Presentation Inside Protein Crystal Nanopores: Tunable Interfacial Adhesion Noncovalently Modulates Cell Attachment.

Protein crystals with sufficiently large solvent pores can non-covalently adsorb polymers in the pores. In principle, if these polymers contain cell adhesion ligands, the polymer-laden crystals could present ligands to cells with tunable adhesion strength. Moreover, porous protein crystals can store an internal ligand reservoir, so that the surface can be replenished. In this study, we demonstrate that poly(ethylene glycol) terminated with a cyclic cell adhesion ligand peptide (PEG-RGD) can be loaded into porous protein crystals by diffusion. Through atomic force microscopy (AFM), force-distance correlations of the mechanical interactions between activated AFM tips and protein crystals were precisely measured. The activation of AFM tips allows the tips to interact with PEG-RGD that was pre-loaded in the protein crystal nanopores, mimicking how a cell might attach to and pull on the ligand through integrin receptors. The AFM experiments also simultaneously reveal the detailed morphology of the buffer-immersed nanoporous protein crystal surface. We also show that porous protein crystals (without and with loaded PEG-RGD) serve as suitable substrates for attachment and spreading of adipose-derived stem cells. This strategy can be used to design surfaces that non-covalently present multiple different ligands to cells with tunable adhesive strength for each ligand, and with an internal reservoir to replenish the precisely defined crystalline surface.

Designing Non-Textured, All-Solid, Slippery Hydrophilic Surfaces.

Slippery surfaces are sought after due to their wide range of applications in self-cleaning, drag reduction, fouling-resistance, enhanced condensation, biomedical implants etc. Recently, non-textured, all-solid, slippery surfaces have gained significant attention because of their advantages over super-repellent surfaces and lubricant-infused surfaces. Currently, almost all non-textured, all-solid, slippery surfaces are hydrophobic. In this work, we elucidate the systematic design of non-textured, all-solid, slippery hydrophilic (SLIC) surfaces by covalently grafting polyethylene glycol (PEG) brushes to smooth substrates. Furthermore, we postulate a plateau in slipperiness above a critical grafting density, which occurs when the tethered brush size is equal to the inter-tether distance. Our SLIC surfaces demonstrate exceptional performance in condensation and fouling-resistance compared to non-slippery hydrophilic surfaces and slippery hydrophobic surfaces. Based on these results, SLIC surfaces constitute an emerging class of surfaces with the potential to benefit multiple technological landscapes ranging from thermofluidics to biofluidics.

Structural control of fibrin bioactivity by mechanical deformation.

Fibrin is the fibrous protein network that comprises blood clots; it is uniquely capable of bearing very large tensile strains (up to 200%) due to multiscale force accommodation mechanisms. Fibrin is also a biochemical scaffold for numerous enzymes and blood factors. The biomechanics and biochemistry of fibrin have been independently studied. However, comparatively little is known about how fibrin biomechanics and biochemistry are coupled: how does fibrin deformation influence its biochemistry? In this study, we show that mechanically induced protein structural changes in fibrin affect fibrin biochemistry. We find that tensile deformation of fibrin leads to molecular structural transitions of α-helices to ß-sheets, which reduced binding of tissue plasminogen activator (tPA), an enzyme that initiates fibrin lysis. Moreover, binding of tPA and Thioflavin T, a commonly used ß-sheet marker, were mutually exclusive, further demonstrating the mechano-chemical control of fibrin biochemistry. Finally, we demonstrate that structural changes in fibrin suppressed the biological activity of platelets on mechanically strained fibrin due to reduced αIIbß3 integrin binding. Our work shows that mechanical strain regulates fibrin molecular structure and biological activity in an elegant mechano-chemical feedback loop, which possibly extends to other fibrous biopolymers.

Dynamics of long-term protein aggregation on low-fouling surfaces.

Understanding the mechanisms of protein interactions with solid surfaces is critical to predict how proteins affect the performance of materials in biological environments. Low-fouling and ultra-low fouling surfaces are often evaluated in short-term protein adsorption experiments, where 'short-term' is defined as the time required to reach an initial apparent or pseudo-equilibrium, which is usually less than 600 s. However, it has long been recognized that these short-term observations fail to predict protein adsorption behavior in the long-term, characterized by irreversible accumulation of protein on the surface. This important long-term behavior is frequently ignored or attributed to slow changes in surface chemistry over time-such as oxidation-often with little or no experimental evidence. Here, we report experiments measuring protein adsorption on "low-fouling" and "ultralow-fouling" surfaces using single-molecule localization microscopy to directly probe protein adsorption and desorption. The experiments detect protein adsorption for thousands of seconds, enabling direct observation of both short-term (reversible adsorption) and long-term (irreversible adsorption leading to accumulation) protein-surface interactions. By bridging the gap between these two time scales in a single experiment, this work enables us to develop a single mathematical model that predicts behavior in both temporal regimes. The experimental data in combination with the resulting model provide several important insights: (1) short-term measurements of protein adsorption using ensemble-averaging methods may not be sufficient for designing antifouling materials; (2) all investigated surfaces eventually foul when in long-term contact with protein solutions; (3) fouling can occur through surface-induced oligomerization of proteins which may be a distinct step from irreversible adsorption; and (4) surfaces can be designed to reduce oligomerization or the adsorption of oligomers, to prevent or delay fouling.

Blood-Compatible Materials: Vascular Endothelium-Mimetic Surfaces that Mitigate Multiple Cell-Material Interactions.

When flowing whole blood contacts medical device surfaces, the most common blood-material interactions result in coagulation, inflammation, and infection. Many new blood-contacting biomaterials have been proposed based on strategies that address just one of these common modes of failure. This study proposes to mitigate unfavorable biological reactions that occur with blood-contacting medical devices by designing multifunctional surfaces, with features optimized to meet multiple performance criteria. These multifunctional surfaces incorporate the release of the small molecule hormone nitric oxide (NO) with surface chemistry and nanotopography that mimic features of the vascular endothelial glycocalyx. These multifunctional surfaces have features that interact with coagulation components, inflammatory cells, and bacterial cells. While a single surface feature alone may not be sufficient to achieve multiple functions, the release of NO from the surfaces along with their modification to mimic the endothelial glycocalyx synergistically improves platelet-, leukocyte-, and bacteria-surface interactions. This work demonstrates that new blood-compatible materials should be designed with multiple features, to better address the multiple modes of failure of blood-contacting medical devices.

Anomalous protein kinetics on low-fouling surfaces.

In this work, protein-surface interactions were probed in terms of adsorption and desorption of a model protein, bovine serum albumin, on a low-fouling surface with single-molecule localization microscopy. Single-molecule experiments enable precise determination of both adsorption and desorption rates. Strikingly the experimental data show anomalous desorption kinetics, evident as a surface dwell time that exhibits a power-law distribution, i.e. a heavy-tailed rather than the expected exponential distribution. As a direct consequence of this heavy-tailed distribution, the average desorption rate depends upon the time scale of the experiment and the protein surface concentration does not reach equilibrium. Further analysis reveals that the observed anomalous desorption emerges due to the reversible formation of a small fraction of soluble protein multimers (small oligomers), such that each one desorbs from the surface with a different rate. The overall kinetics can be described by a series of elementary reactions, yielding simple scaling relations that predict experimental observations. This work reveals a mechanistic origin for anomalous desorption kinetics that can be employed to interpret observations where low-protein fouling surfaces eventually foul when in long-term contact with protein solutions. The work also provides new insights that can be used to define design principles for non-fouling surfaces and to predict their performance.

Protein adsorption measurements on low fouling and ultralow fouling surfaces: A critical comparison of surface characterization techniques.

Ultralow protein fouling behavior is a common target for new high-performance materials. Ultralow fouling is often defined based on the amount of irreversibly adsorbed protein (< 5 ng cm-2) measured by a surface ensemble averaging method. However, protein adsorption at solid interfaces is a dynamic process involving multiple steps, which may include adsorption, desorption, and irreversible protein denaturation. In order to better optimize the performance of antifouling surfaces, it is imperative to fully understand how proteins interact with surfaces, including kinetics of adsorption and desorption, conformation, stability, and amount of adsorbed proteins. Defining ultralow fouling surfaces based on a measurement at or near the limit of detection of a surface-averaged measurement may not capture all of this behavior. Single-molecule microscopy techniques can resolve individual protein-surface interactions with high temporal and spatial resolution. This information can be used to tune the properties of surfaces to better resist protein adsorption. In this work, we demonstrate how combining surface plasmon resonance, X-ray photoelectron spectroscopy, atomic force microscopy, and single-molecule localization microscopy provides a more complete picture of protein adsorption on low fouling and ultralow fouling polyelectrolyte multilayer and polymer brush surfaces, over different regimes of protein concentration. In this case, comparing the surfaces using surface plasmon resonance alone is insufficient to rank their resistance to protein adsorption. Our results suggest a revision of the accepted definition of ultralow fouling surfaces is timely: with the advent of time-resolved studies of protein adsorption kinetics at the single-molecule level, it is neither necessary nor sufficient to rely on a surface averaging techniques to qualify ultralow fouling surfaces. Since protein adsorption is a dynamic process, understanding how surface properties affect the kinetics of protein adsorption will enable the design of future generations of advanced antifouling materials. STATEMENT OF SIGNIFICANCE: The design of ultralow fouling surfaces is often optimized based on a single surface-averaging technique measuring the amount of irreversibly adsorbed protein. This work provides a critical comparison of alternative techniques for evaluating protein adsorption on low fouling and ultralow fouling surfaces, and demonstrates how additional information about the dynamics of protein-surface interactions at the interface can be obtained by application of single-molecule microscopy. This approach could be used to better elucidate mechanisms of protein resistance and design principles for advanced ultralow fouling materials.

The Preparation and Study of Ethylene Glycol-Modified Graphene Oxide Membranes for Water Purification.

In this work, graphene oxide (GO)/ethylene glycol (EG) membranes were designed by a vacuum filtration method for molecular separation and water purification. The composite membranes were characterized by scanning electron microscopy (SEM), field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The interlayer spacing of GO membranes (0.825 nm) and GO/EG membranes (0.634 nm) are measured by X-ray diffraction (XRD). Using the vacuum filtration method, the membrane thickness can be controlled by selecting the volume of the solution from which the membrane is prepared, to achieve high water permeance and high rejection of Rhodamine B (RhB). The membrane performance was evaluated on a dead-end filtration device. The water permeance and rejection of RhB of the membranes are 103.35 L m-2 h-1 bar-1 and 94.56% (GO), 58.17 L m-2 h-1 bar-1 and 97.13% (GO/EG), respectively. The permeability of GO/EG membrane is about 40 × 10-6 L m-1 h-1 bar-1. Compared with the GO membrane, the GO/EG membrane has better separation performance because of its proper interlayer spacing. In this study, the highest rejection of RhB (99.92%) is achieved. The GO/EG membranes have potential applications in the fields of molecular separation and water purification.

Novel cationic tannin/glycosaminoglycan-based polyelectrolyte multilayers promote stem cells adhesion and proliferation.

Modifying materials with biocompatible surface coatings is an important method for controlling cell responses to biomaterials. In this work, tanfloc (TN), a cationic tannin-derivative polymer was assembled with heparin (HEP) and chondroitin sulfate (CS), using the layer-by-layer (LbL) approach, to build polyelectrolyte multilayers (PEMs) and to design cytocompatible coatings. LbL deposition was monitored through Fourier-transform surface plasmon resonance, and characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and contact angle measurements. The response of human adipose-derived stem cells (ADSCs) was evaluated in vitro. All of the TN-containing PEMs exhibit cytocompatibility and support adhesion, proliferation, and the spreading of ADSCs after 7 days of culture. HEP-TN PEM assembly with 11 layers (HEP-TN11) supports the greatest rate of cell proliferation. When TN is the terminal layer of the PEM, the surfaces promote the spreading of ADSCs, indicating that the surface charge and PEM terminal layer are key determinants of the microenvironmental niche that control cellular response. The promotion of stem cell attachment and proliferation makes these surface coatings potentially useful for biomedical implants and regenerative medicine.

Nanostructured Surfaces That Mimic the Vascular Endothelial Glycocalyx Reduce Blood Protein Adsorption and Prevent Fibrin Network Formation.

Blood-contacting materials are critical in many applications where long-term performance is desired. However, there are currently no engineered materials used in cardiovascular implants and devices that completely prevent clotting when in long-term contact with whole blood. The most common approach to developing next-generation blood-compatible materials is to design surface chemistries and structures that reduce or eliminate protein adsorption to prevent blood clotting. This work proposes a new paradigm for controlling protein-surface interactions by strategically mimicking key features of the glycocalyx lining the interior surfaces of blood vessels: negatively charged glycosaminoglycans organized into a polymer brush with nanoscale domains. The interactions of two important proteins from blood (albumin and fibrinogen) with these new glycocalyx mimics are revealed in detail using surface plasmon resonance and single-molecule microscopy. Surface plasmon resonance shows that these blood proteins interact reversibly with the glycocalyx mimics, but have no irreversible adsorption above the limit of detection. Single-molecule microscopy is used to compare albumin and fibrinogen interactions on surfaces with and without glycocalyx-mimetic nanostructures. Microscopy videos reveal a new mechanism whereby the glycocalyx-mimetic nanostructures eliminate the formation of fibrin networks on the surfaces. This approach shows for the first time that the nanoscale structure and organization of glycosaminoglycans in the glycocalyx are essential to (i) reduce protein adsorption, (ii) reversibly bind fibrin(ogen), and (iii) inhibit fibrin network formation on surfaces. The insights gained from this work suggest new design principles for blood-compatible surfaces. New surfaces developed using these design principles could reduce risk of catastrophic failures of blood-contacting medical devices.

Atomic force microscopy of adsorbed proteoglycan mimetic nanoparticles: Toward new glycocalyx-mimetic model surfaces.

Blood vessels present a dense, non-uniform, polysaccharide-rich layer, called the endothelial glycocalyx. The polysaccharides in the glycocalyx include polyanionic glycosaminoglycans (GAGs). This polysaccharide-rich surface has excellent and unique blood compatibility. We report new methods for preparing and characterizing dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. The GAG-rich surfaces are prepared by adsorbing heparin or chondroitin sulfate-containing polyelectrolyte complex nanoparticles (PCNs) to chitosan-hyaluronan polyelectrolyte multilayers (PEMs). The surfaces are characterized by PeakForce tapping atomic force microscopy, both in air and in aqueous pH 7.4 buffer, and by PeakForce quantitative nanomechanics (PF-QNM) mode with high spatial resolution. These new surfaces provide access to heparin-rich or chondroitin sulfate-rich coatings that mimic both composition and nanoscale structural features of the vascular endothelial glycocalyx.

A Rapid and Sensitive HPLC-Fluorescence Method for Determination of Mirtazapine and Its two Major Metabolites in Human Plasma.

A rapid and sensitive HPLC method has been developed for the quantification of mirtazapine (MRZ), a noradrenergic and specific serotonergic inhibitor antidepressant (NaSSA) and its two major metabolites N-desmethyl mirtazapine (NDM) and 8-hydroxymirtazapine (8-OHM) in human plasma. The separation was achieved using Chromolith C18 column and a mobile phase of acetonitrile: phosphate buffer (pH = 3, 20:80, v/v) in isocratic mode at a flow rate of 2 mL/min. A fluorescence detector was set at 290 and 350 nm for excitation and emission, respectively. Zolpidem was used as the internal standard. Liquid-liquid extraction was applied for sample clean up. All analytes were eluted in less than 5 minutes with LOQ of 1 ng/mL for MRZ and 2 ng/mL for both NDM and 8-OHM. The developed method was successfully applied to quantify MRZ and its metabolites in plasma of a healthy volunteer.