Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction.

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.

Heat-activated liposome targeting to streptavidin-coated surfaces.

There is a great need of improved anticancer drugs and corresponding drug carriers. In particular, liposomal drug carriers with heat-activated release and targeting functions are being developed for combined hyperthermia and chemotherapy treatments of tumors. The aim of this study is to demonstrate the heat-activation of liposome targeting to biotinylated surfaces, in model experiments where streptavidin is used as a pretargeting protein. The design of the heat-activated liposomes is based on liposomes assembled in an asymmetric structure and with a defined phase transition temperature. Asymmetry between the inside and the outside of the liposome membrane was generated through the enzymatic action of phospholipase D, where lipid head groups in the outer membrane leaflet, i.e. exposed to the enzyme, were hydrolyzed. The enzymatically treated and purified liposomes did not bind to streptavidin-modified surfaces. When activation heat was applied, starting from 22°C, binding of the liposomes occurred once the temperature approached 33±0.5°C. Moreover, it was observed that the asymmetric structure remained stable for at least 2 weeks. These results show the potential of asymmetric liposomes for the targeted binding to cell membranes in response to (external) temperature stimulus. By using pretargeting proteins, this approach can be further developed for personalized medicine, where tumor-specific antibodies can be selected for the conjugation of pretargeting agents.

Synthesis and Evaluation of Astatinated N-[2-(Maleimido)ethyl]-3-(trimethylstannyl)benzamide Immunoconjugates.

Effective treatment of metastasis is a great challenge in the treatment of different types of cancers. Targeted alpha therapy utilizes the short tissue range (50-100 µm) of α particles, making the method suitable for treatment of disseminated occult cancers in the form of microtumors or even single cancer cells. A promising radioactive nuclide for this type of therapy is astatine-211. Astatine-211 attached to tumor-specific antibodies as carrier molecules is a system currently under investigation for use in targeted alpha therapy. In the common radiolabeling procedure, astatine is coupled to the antibody arbitrarily on lysine residues. By instead coupling astatine to disulfide bridges in the antibody structure, the immunoreactivity of the antibody conjugates could possibly be increased. Here, the disulfide-based conjugation was performed using a new coupling reagent, maleimidoethyl 3-(trimethylstannyl)benzamide (MSB), and evaluated for chemical stability in vitro. The immunoconjugates were subsequently astatinated, resulting in both high radiochemical yield and high specific activity. The MSB-conjugate was shown to be stable with a long shelf life prior to the astatination. In a comparison of the in vivo distribution of the new immunoconjugate with other tin-based immunoconjugates in tumor-bearing mice, the MSB conjugation method was found to be a viable option for successful astatine labeling of different monoclonal antibodies.

Graphene Oxide and Lipid Membranes: Size-Dependent Interactions.

We have investigated the interaction of graphene oxide (GO) sheets with supported lipid membranes with focus on how the interaction depends on GO sheet size (three samples in the range of 90-5000 nm) and how it differs between small and large liposomes. The layer-by-layer assembly of these materials into multilamellar structures, as discovered in our previous research, is now further explored. The interaction processes were monitored by two complementary, real time, surface-sensitive analytical techniques: quartz crystal microbalance with dissipation monitoring (QCM-D, electroacoustic sensing) and indirect nanoplasmonic sensing (INPS, optical sensing). The results show that the sizes of each of the two components, graphene oxide and liposomes, are important parameters affecting the resulting multilayer structures. Spontaneous liposome rupture onto graphene oxide is obtained for large lateral dimensions of the graphene oxide sheets.

Effects of Al(3+) on Phosphocholine and Phosphoglycerol Containing Solid Supported Lipid Bilayers.

Aluminum has attracted great attention recently as it has been suggested by several studies to be associated with increased risks for Alzheimer's and Parkinson's disease. The toxicity of the trivalent ion is assumed to derive from structural changes induced in lipid bilayers upon binding, though the mechanism of this process is still not well understood. In the present study we elucidate the effect of Al(3+) on supported lipid bilayers (SLBs) using fluorescence microscopy, the quartz crystal microbalance with dissipation (QCM-D) technique, dual-polarization interferometry (DPI), and molecular dynamics (MD) simulations. Results from these techniques show that binding of Al(3+) to SLBs containing negatively charged and neutral phospholipids induces irreversible changes such as domain formation. The measured variations in SLB thickness, birefringence, and density indicate a phase transition from a disordered to a densely packed ordered phase.

Construction and modeling of concatemeric DNA multilayers on a planar surface as monitored by QCM-D and SPR.

The sequential hybridization of a 534 base pair DNA concatemer layer was monitored by QCM-D and SPR, and the QCM-D data were analyzed by Voigt viscoelastic models. The results show that Voigt-based modeling gives a good description of the experimental data but only if shear viscosity and elasticity are allowed to depend on the shear frequency. The derived layer thickness, shear viscosity and elasticity of the growing film give a representation of the DNA film in agreement with known bulk properties of DNA, and reveal a maximum in film viscosity when the molecules in the layer contain 75 base pairs. The experimental data during construction of a 3084 bp DNA concatemer layer were compared to predictions of the QCM-D response of a 1 µm thick film of rod-like polymers. A predicted nonmonotonous variation of dissipation with frequency (added mass) is in qualitative agreement with the experiments, but with a quantitative disagreement which likely reflects that the flexibility of such long DNA molecules is not included in the model.

Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

Formation of supported lipid bilayers on silica: relation to lipid phase transition temperature and liposome size.

DPPC liposomes ranging from 90 nm to 160 nm in diameter were prepared and used for studies of the formation of supported lipid membranes on silica (SiO2) at temperatures below and above the gel to liquid-crystalline phase transition temperature (Tm = 41 °C), and by applying temperature gradients through Tm. The main method was the quartz crystal microbalance with dissipation (QCM-D) technique. It was found that liposomes smaller than 100 nm spontaneously rupture on the silica surface when deposited at a temperature above Tm and at a critical surface coverage, following a well-established pathway. In contrast, DPPC liposomes larger than 160 nm do not rupture on the surface when adsorbed at 22 °C or at 50 °C. However, when liposomes of this size are first adsorbed at 22 °C and at a high enough surface coverage, after which they are subject to a constant temperature gradient up to 50 °C, they rupture and fuse to a bilayer, a process that is initiated around Tm. The results are discussed and interpreted considering a combination of effects derived from liposome-surface and liposome-liposome interactions, different softness/stiffness and shape of liposomes below and above Tm, the dynamics and thermal activation of the bilayers occurring around Tm and (for liposomes containing 33% of NaCl) osmotic pressure. These findings are valuable both for preparation of supported lipid bilayer cell membrane mimics and for designing temperature-responsive material coatings.

Acoustical sensing of cardiomyocyte cluster beating.

Spontaneously beating human pluripotent stem cell-derived cardiomyocytes clusters (CMCs) represent an excellent in vitro tool for studies of human cardiomyocyte function and for pharmacological cardiac safety assessment. Such testing typically requires highly trained operators, precision plating, or large cell quantities, and there is a demand for real-time, label-free monitoring of small cell quantities, especially rare cells and tissue-like structures. Array formats based on sensing of electrical or optical properties of cells are being developed and in use by the pharmaceutical industry. A potential alternative to these techniques is represented by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, which is an acoustic surface sensitive technique that measures changes in mass and viscoelastic properties close to the sensor surface (from nm to µm). There is an increasing number of studies where QCM-D has successfully been applied to monitor properties of cells and cellular processes. In the present study, we show that spontaneous beating of CMCs on QCM-D sensors can be clearly detected, both in the frequency and the dissipation signals. Beating rates in the range of 66-168 bpm for CMCs were detected and confirmed by simultaneous light microscopy. The QCM-D beating profile was found to provide individual fingerprints of the hPS-CMCs. The presented results point towards acoustical assays for evaluation cardiotoxicity.

Acoustic detection of melanosome transport in Xenopus laevis melanophores.

Organelle transport studies are often performed using melanophores from lower vertebrates due to the ease of inducing movements of pigment granules (melanosomes) and visualizing them by optical microscopy. Here, we present a novel methodology to monitor melanosome translocation (which is a light-sensitive process) in the dark using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. This acoustic sensing method was used to study dispersion and aggregation of melanosomes in Xenopus laevis melanophores. Reversible sensor responses, correlated to optical reflectance measurements, were obtained by alternating addition and removal of melatonin (leading to melanosome aggregation) and melanocyte-stimulating hormone (MSH) (leading to melanosome dispersion). By confocal microscopy, it was shown that a vertical redistribution of melanosomes occurred during the dispersion/aggregation processes. Furthermore, the transport process was studied in the presence of cytoskeleton-perturbing agents disrupting either actin filaments (latrunculin) or microtubules (nocodazole). Taken together, these experiments suggest that the acoustic responses mainly originate from melanosome transport along actin filaments (located close to the cell membrane), as expected based on the penetration depth of the QCM-D technique. The results clearly indicate the potential of QCM-D for studies of intracellular transport processes in melanophores.

Effect of molecular composition of heparin and cellulose sulfate on multilayer formation and cell response.

Here, the layer-by-layer method was applied to assemble films from chitosan paired with either heparin or a semisynthetic cellulose sulfate (CS) that possessed a higher sulfation degree than heparin. Ion pairing was exploited during multilayer formation at pH 4, while hydrogen bonding is likely to occur at pH 9. Effects of polyanions and pH value during layer formation on multilayers properties were studied by surface plasmon resonance ("dry layer mass"), quartz crystal microbalance with dissipation monitoring ("wet layer mass"), water contact angle, and zeta potential measurements. Bioactivity of multilayers was studied regarding fibronectin adsorption and adhesion/proliferation of C2C12 myoblast cells. Layer growth and dry mass were higher for both polyanions at pH 4 when ion pairing occurred, while it decreased significantly with heparin at pH 9. By contrast, CS as polyanion resulted also in high layer growth and mass at pH 9, indicating a much stronger effect of hydrogen bonding between chitosan and CS. Water contact angle and zeta potential measurements indicated a more separated structure of multilayers from chitosan and heparin at pH 4, while CS led to a more fuzzy intermingled structure at both pH values. Cell behavior was highly dependent on pH during multilayer formation with heparin as polyanion and was closely related to fibronectin adsorption. By contrast, CS and chitosan did not show such dependency on pH value, where adhesion and growth of cells was high. Results of this study show that CS is an attractive candidate for multilayer formation that does not depend so strongly on pH during multilayer formation. In addition, such multilayer system also represents a good substrate for cell interactions despite the rather soft structure. As previous studies have shown specific interaction of CS with growth factors, multilayers from chitosan and CS may be of great interest for different biomedical applications.

Graphene oxide and lipid membranes: interactions and nanocomposite structures.

We have investigated the interaction between graphene oxide and lipid membranes, using both supported lipid membranes and supported liposomes. Also, the reverse situation, where a surface coated with graphene oxide was exposed to liposomes in solution, was studied. We discovered graphene oxide-induced rupture of preadsorbed liposomes and the formation of a nanocomposite, bio-nonbio multilayer structure, consisting of alternating graphene oxide monolayers and lipid membranes. The assembly process was monitored in real time by two complementary surface analytical techniques (the quartz crystal microbalance with dissipation monitoring technique (QCM-D) and dual polarization interferometry (DPI)), and the formed structures were imaged with atomic force microscopy (AFM). From a basic science point of view, the results point toward the importance of electrostatic interactions between graphene oxide and lipid headgroups. Implications from a more practical point of view concern structure-activity relationship for biological health/safety aspects of graphene oxide and the potential of the nanocomposite, multilayer structure as scaffolds for advanced biomolecular functions and sensing applications.

The inflammation-associated protein TSG-6 cross-links hyaluronan via hyaluronan-induced TSG-6 oligomers.

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).

Reversible hybridization of DNA anchored to a lipid membrane via porphyrin.

The binding of zinc-porphyrin-anchored linear DNA to supported lipid membranes was studied using quartz crystal microbalance with dissipation monitoring (QCM-D). The hydrophobic anchor is positioned at the ninth base of 39-base-pair-long DNA sequences, ensuring that the DNA is positioned parallel to the membrane surface when bound, an important prerequisite for using this type of construct for the creation of two-dimensional (2D) DNA patterns on the surface. The anchor consists of a porphyrin group linked to the DNA via two or three phenylethynylene moieties. Double-stranded DNA where one of the strands was modified with either of these anchors displayed irreversible binding, although binding to the membrane was faster for the derivatives with the short anchor. The binding and subsequent hybridization of single-stranded constructs on the surface was demonstrated at 60 °C, for both anchors, revealing a coverage-dependent behavior. At low coverage, hybridization results in an increase in mass (as measured by QCM-D) by a factor of ~1.5, accompanied by a slight increase in the rigidity of the DNA layer. At high coverage, hybridization expels molecules from the membrane, associated with an initial increase, followed by a decrease in DNA mass (as detected both by QCM-D and by an optical technique). Melting of the DNA on the surface was performed, followed by rehybridization of the single-stranded species left on the surface with their complementary strand, demonstrating the reversibility inherent in using DNA for the formation of membrane-confined nanopatterns.

Well-defined lipid interfaces for protein adsorption studies.

The biomolecule-artificial lipid membrane interface has been investigated by QCM-D, SPR, and FRAP techniques, to study the adsorption process of ferritin on supported lipid bilayers (SLBs) of different composition and charge. Results point out to the predominant role of electrostatics in triggering the interaction of ferritin with SLBs.

Kinetics of ligand binding to membrane receptors from equilibrium fluctuation analysis of single binding events.

Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.

Pore spanning lipid bilayers on mesoporous silica having varying pore size.

Synthetic lipid bilayers have similar properties as cell membranes and have been shown to be of great use in the development of novel biomimicry devices. In this study, lipid bilayer formation on mesoporous silica of varying pore size, 2, 4, and 6 nm, has been investigated using quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescent recovery after photo bleaching (FRAP), and atomic force microscopy (AFM). The results show that pore-spanning lipid bilayers were successfully formed regardless of pore size. However, the mechanism of the bilayer formation was dependent on the pore size, and lower surface coverages of adsorbed lipid vesicles were required on the surface having the smallest pores. A similar trend was observed for the lateral diffusion coefficient (D) of fluorescently labeled lipid molecules in the membrane, which was lowest on the surface having the smallest pores and increased with the pore size. All of the pore size dependent observations are suggested to be due to the hydrophilicity of the surface, which decreases with increased pore size.

Chemical modifications of Au/SiO2 template substrates for patterned biofunctional surfaces.

The aim of this work was to create patterned surfaces for localized and specific biochemical recognition. For this purpose, we have developed a protocol for orthogonal and material-selective surface modifications of microfabricated patterned surfaces composed of SiO(2) areas (100 µm diameter) surrounded by Au. The SiO(2) spots were chemically modified by a sequence of reactions (silanization using an amine-terminated silane (APTES), followed by amine coupling of a biotin analogue and biospecific recognition) to achieve efficient immobilization of streptavidin in a functional form. The surrounding Au was rendered inert to protein adsorption by modification by HS(CH(2))(10)CONH(CH(2))(2)(OCH(2)CH(2))(7)OH (thiol-OEG). The surface modification protocol was developed by testing separately homogeneous SiO(2) and Au surfaces, to obtain the two following results: (i) SiO(2) surfaces which allowed the grafting of streptavidin, and subsequent immobilization of biotinylated antibodies, and (ii) Au surfaces showing almost no affinity for the same streptavidin and antibody solutions. The surface interactions were monitored by quartz crystal microbalance with dissipation monitoring (QCM-D), and chemical analyses were performed by polarization modulation-reflexion absorption infrared spectroscopy (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS) to assess the validity of the initial orthogonal assembly of APTES and thiol-OEG. Eventually, microscopy imaging of the modified Au/SiO(2) patterned substrates validated the specific binding of streptavidin on the SiO(2)/APTES areas, as well as the subsequent binding of biotinylated anti-rIgG and further detection of fluorescent rIgG on the functionalized SiO(2) areas. These results demonstrate a successful protocol for the preparation of patterned biofunctional surfaces, based on microfabricated Au/SiO(2) templates and supported by careful surface analysis. The strong immobilization of the biomolecules resulting from the described protocol is advantageous in particular for micropatterned substrates for cell-surface interactions.

Viscoelastic sensing of conformational changes in plasminogen induced upon binding of low molecular weight compounds.

Plasminogen is a precursor to the fibrinolytic enzyme plasmin and is known to undergo large conformational changes when subjected to low molecular lysine analogues such as tranexamic acid (TA) or ε-amino-n-caproic acid (EACA). Here, we demonstrate how well-controlled surface immobilization of biotinylated plasminogen allows for monitoring of the interaction between TA and EACA with plasminogen. The interaction was studied by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique as well as by surface plasmon resonance (SPR) based sensing. QCM-D measures changes in acoustically coupled mass (by detection of changes in the resonance frequency of the crystal, Δf) and is sensitive to changes in mass adsorbed on the sensor surface including how liquid medium is associated with this material. Through the dissipation factor (i.e., changes in the energy dissipation of the crystal oscillation, ΔD), QCM-D is also sensitive to the viscoelastic properties of material adsorbed to the sensor surface. Upon binding of TA or EACA, changes in the plasminogen structure were recorded as distinct, although small, ΔD responses which were used to determine affinity constants. By comparing native and truncated plasminogen, we conclude that the observed dissipation shifts were caused by conformational changes in the proteins leading to changes in the viscoelastic properties of the protein layer on the surface. These results demonstrate a novel application of the QCM-D technique, paving the way for a whole new approach to screening of this target for novel lead structures.

Combined QCM-D and EIS study of supported lipid bilayer formation and interaction with pore-forming peptides.

A novel set-up combining the quartz crystal microbalance with dissipation monitoring technique (QCM-D) and electrochemical impedance spectroscopy (EIS) under flow conditions was successfully used to follow supported lipid bilayer (SLB) formation on SiO(2). This study demonstrates the simultaneous detection, in real time, of both the electrical and the structural properties of the SLB. The combination of the two techniques provided novel insights regarding the mechanism of SLB formation: we found indications for an annealing process of the lipid alkyl chains after the mass corresponding to complete bilayer coverage had been deposited. Moreover, the interaction of the SLB with the pore-forming toxin, gramicidin D (grD) was studied for grD concentrations ranging from 0.05 to 40 mg L(-1). Membrane properties were altered depending on the toxin concentration. For low grD concentrations, the electrical properties of the SLB changed upon insertion of active ion channels. For higher concentrations, the QCM-D data showed dramatic changes in the viscoelastic properties of the membrane while the EIS spectra did not change. AFM confirmed significant structural changes of the membrane at higher grD concentrations. Thus, the application of combined QCM-D and EIS detection provides complementary information about the system under study. This information will be particularly important for the continued detailed investigation of interactions at model membrane surfaces.