Showing papers by "Fredrik Höök published in 2016"

Mucosal Vaccine Development Based on Liposome Technology

Abstract: Immune protection against infectious diseases is most effective if located at the portal of entry of the pathogen. Hence, there is an increasing demand for vaccine formulations that can induce strong protective immunity following oral, respiratory, or genital tract administration. At present, only few mucosal vaccines are found on the market, but recent technological advancements and a better understanding of the principles that govern priming of mucosal immune responses have contributed to a more optimistic view on the future of mucosal vaccines. Compared to live attenuated vaccines, subcomponent vaccines, most often protein-based, are considered safer, more stable, and less complicated to manufacture, but they require the addition of nontoxic and clinically safe adjuvants to be effective. In addition, another limiting factor is the large antigen dose that usually is required for mucosal vaccines. Therefore, the combination of mucosal adjuvants with the recent progress in nanoparticle technology provides an attractive solution to these problems. In particular, the liposome technology is ideal for combining protein antigen and adjuvant into an effective mucosal vaccine. Here, we describe and discuss recent progress in nanoparticle formulations using various types of liposomes that convey strong promise for the successful development of the next generation of mucosal vaccines.

Dual-Wavelength Surface Plasmon Resonance for Determining the Size and Concentration of Sub-Populations of Extracellular Vesicles

Abstract: Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different s...

Quantification of Multivalent Interactions by Tracking Single Biological Nanoparticle Mobility on a Lipid Membrane

Abstract: Macromolecular association commonly occurs via dynamic engagement of multiple weak bonds referred to as multivalent interactions. The distribution of the number of bonds, combined with their strong influence on the residence time, makes it very demanding to quantify this type of interaction. To address this challenge in the context of virology, we mimicked the virion association to a cell membrane by attaching lipid vesicles (100 nm diameter) to a supported lipid bilayer via multiple, identical cholesterol-based DNA linker molecules, each mimicking an individual virion–receptor link. Using total internal reflection microscopy to track single attached vesicles combined with a novel filtering approach, we show that histograms of the vesicle diffusion coefficient D exhibit a spectrum of distinct peaks, which are associated with vesicles differing in the number, n, of linking DNA tethers. These peaks are only observed if vesicles with transient changes in n are excluded from the analysis. D is found to be pro...

Two-dimensional flow nanometry of biological nanoparticles for accurate determination of their size and emission intensity.

Abstract: Biological nanoparticles (BNPs) are of high interest due to their key role in various biological processes and use as biomarkers. BNP size and composition are decisive for their functions, but simultaneous determination of both properties with high accuracy remains challenging. Optical microscopy allows precise determination of fluorescence/scattering intensity, but not the size of individual BNPs. The latter is better determined by tracking their random motion in bulk, but the limited illumination volume for tracking this motion impedes reliable intensity determination. Here, we show that by attaching BNPs to a supported lipid bilayer, subjecting them to hydrodynamic flows and tracking their motion via surface-sensitive optical imaging enable determination of their diffusion coefficients and flow-induced drifts, from which accurate quantification of both BNP size and emission intensity can be made. For vesicles, the accuracy of this approach is demonstrated by resolving the expected radius-squared dependence of their fluorescence intensity for radii down to 15 nm.

Nonspecific Colloidal-Type Interaction Explains Size-Dependent Specific Binding of Membrane-Targeted Nanoparticles

Abstract: Emerging biomedical applications such as molecular imaging and drug delivery often require directed binding of nanoparticles to cell-membrane receptors The specific apparent affinity of such ligand-functionalized particles is size-dependent, an observation so far solely attributed to multivalent receptor–ligand interaction We question the universality of this explanation by demonstrating that the binding kinetics also depends on weak, attractive colloidal-type interaction between nanoparticles and a lipid membrane Applying label-free single-particle imaging, we correlate binding of nanoparticles targeted to a cell-mimetic lipid membrane with the distribution of nontargeted particles freely diffusing close to the membrane interface This analysis shows that already a weak, kBT-scale attraction present between 50 nm gold nanoparticles and the membrane renders these particles an order of magnitude higher avidity compared to 20 nm particles A stronger emphasis on nonspecific particle–membrane interaction

Supported lipid bilayer repair mediated by AH peptide

Abstract: The adsorption and fusion of small unilamellar lipid vesicles on silica-based substrates such as glass is a common method used to fabricate supported lipid bilayers. Successful bilayer formation depends on a number of experimental conditions as well as on the quality of the vesicle preparation. Inevitably, a small fraction of unruptured vesicles always remains in a supported bilayer, and this kind of defect can have devastating influences on the morphological and electrical properties of the supported bilayer when used as a biosensing platform. In this paper, a simple method is reported to improve the completeness of supported bilayers by adding a vesicle rupturing peptide as a final step in the fabrication process. Peptide treatment reduces the fraction of unruptured vesicles to less than 1%, as determined by epifluorescence microscopy and quartz crystal microbalance-dissipation experiments. This step can easily be incorporated into existing procedures for preparing high-quality supported lipid bilayers.

Imaging of amyloid-β in Alzheimer's disease transgenic mouse brains with ToF-SIMS using immunoliposomes

Abstract: Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, the authors present in this work a method developed for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution. The method has been applied to detect and image the distribution of amyloid-β (Aβ), a biologically relevant peptide in Alzheimer's disease (AD), in transgenic mouse braintissue. To ensure specific binding, the immunoliposome binding was verified on a model surface using quartz crystal microbalance with dissipation monitoring. The immunoliposome binding was also investigated on tissue sections with fluorescence microscopy, and compared with conventional immunohistochemistry using primary and secondary antibodies, demonstrating specific binding to Aβ. Using ToF-SIMS imaging, several endogenous lipids, such as cholesterol and sulfatides, were also detected in parallel with the immunoliposome-labeled Aβ deposits, which is an advantage compared to fluorescence microscopy. This method can thus potentially provide further information about lipid–protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.

Hydrodynamic Propulsion of Liposomes Electrostatically Attracted to a Lipid Membrane Reveals Size-Dependent Conformational Changes

Abstract: The efficiency of lipid nanoparticle uptake across cellular membranes is strongly dependent on the very first interaction step. Detailed understanding of this step is in part hampered by the large heterogeneity in the physicochemical properties of lipid nanoparticles, such as liposomes, making conventional ensemble-averaging methods too blunt to address details of this complex process. Here, we contribute a means to explore whether individual liposomes become deformed upon binding to fluid cell-membrane mimics. This was accomplished by using hydrodynamic forces to control the propulsion of nanoscale liposomes electrostatically attracted to a supported lipid bilayer. In this way, the size of individual liposomes could be determined by simultaneously measuring both their individual drift velocity and diffusivity, revealing that for a radius of ∼45 nm, a close agreement with dynamic light scattering data was observed, while larger liposomes (radius ∼75 nm) displayed a significant deformation unless composed ...

Monitoring of single and double lipid membrane formation with high spatiotemporal resolution using evanescent light scattering microscopy

Abstract: Formation and quality of single solid supported lipid membranes and double lipid membranes were investigated with single vesicle resolution using label-free evanescence light scattering microscopy (EvSM). For the formation of double lipid membranes we made use of electrostatic interaction between charged lipids and oppositely charged cations.

Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer

Abstract: Investigations of ligand-binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity-related limitations impaired by low signal-to-noise ratios. Inspired by affinity capturing of water-soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane-protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris-NTA)-biotin conjugate. A polymer-supported lipid bilayer containing His6-tagged β-secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His6-BACE in the Ni2+-NTA-modified region of the substrate resulted in a stationary three-fold increase in surface coverage, and an accompanied increase in ligand-binding response.

A method for determining a hydrodynamic size of an object

Abstract: The disclosure relates to a method for determining a hydrodynamic size of an object, such as a nano-sized object, said method comprising the steps of: - providing a fluid interface, - linking said object to said fluid interface thereby providing a linked object, whereby the movement of said linked object is restricted by virtue of being linked to said fluid interface, - providing and determining a hydrodynamic shear force that acts on said linked object, - tracking the movement of said linked object, and - calculating the hydrodynamic size of the object using the Einstein-Smoluchowski relation.