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Knowledge of the interactions between nanoparticles and immune cells is required for optimal design of nanoparticle-based drug delivery systems, either when aiming to avoid phagocytic clearance of the nanoparticles or promote an immune response by delivering therapeutic agents to specific immune cells. Several studies have suggested that reconstituted high-density lipoproteins (rHDL) are attractive drug delivery vehicles. However, detailed studies of rHDL interactions with circulating leukocytes are limited. Here, we evaluated the association of discoidal rHDL with leukocytes in human whole blood (HWB) using quantitative approaches. We found that while the rHDL of various lipid compositions associated preferentially with monocytes, the degree of association depended on the lipid composition. However, consistent with the long circulation half-life of rHDL, we show that only a minor fraction of the rHDL associated with the leukocytes. Furthermore, we used three-dimensional fluorescence microscopy and imaging flow cytometry to evaluate the possible internalization of rHDL cargo into the cells, and we show increased internalization of rHDL cargo in monocytes relative to granulocytes. The preferential rHDL association with monocytes and the internalization of rHDL cargo could possibly be mediated by the scavenger receptor class B type 1 (SR-BI), which we show is expressed to a higher extent on monocytes than on the other major leukocyte populations. Our work implies that drug-loaded rHDL can deliver its cargo to monocytes in circulation, which could lead to some off-target effects when using rHDL for systemic drug delivery, or it could pave the way for novel immunotherapeutic treatments aiming to target the monocytes

The protein corona is widely recognized as a key concept in contemporary nanomedicine. In recent years, the interest in the protein corona has reached new heights as a number of reports have suggested that a comprehensive protein corona can form around nanomaterials that previously were thought to be resistant to protein binding. For example, PEGylated stealth liposomes were long thought to be protein repellent, but a number of recent studies have found that a significant protein corona forms around such liposomes in the bloodstream. Prompted by these surprising recent findings, we here present an extensive quantitative study of the binding of blood proteins to standard PEGylated stealth liposomes. To make the study relevant for targeted as well as non-targeted drug delivery systems, the liposomes were prepared both with and without a targeting antibody conjugated to their surface. The prepared liposomes were either incubated in vitro in fetal bovine serum or administered in vivo into the bloodstream of mice. Subsequently, the liposomes were recovered and analyzed using a variety of techniques. There was very little protein binding to the liposomes recovered after in vitro incubation. In contrast, there was more protein binding to the liposomes recovered after in vivo circulation, but a deeper analysis estimated that the bound proteins still only covered a very low fraction of the liposomal surface area. Both the liposomes recovered after in vitro incubation and the liposomes recovered after in vivo circulation completely retained their size and receptor targeting characteristics. Taken collectively, our results thus demonstrate that the hard protein corona of both non-targeted and antibody-targeted stealth liposomes is sparse and does not affect the structure and function of the liposomes.

Post-staining of extracellular vesicles (EVs) with lipid-anchored fluorophores (LAFs) such as PKH67 is a widely used strategy for studying EVs but it is associated with several pitfalls. The pitfalls discussed in this commentary are related to LAF labelling of non-EV species due to (1) lipoprotein contamination in EV samples, (2) desorption of the LAF reporters from vesicles into proteins and lipoproteins in blood and serum, and (3) the capability of the amphiphilic LAF compounds to form EV-like particles. Awareness of these challenges and developing solutions to overcome these are important to ensure that we make relevant interpretations when using LAFs to track EVs.

Background: Wnt proteins modulate development, stem cell fate and cancer through interactions with cell surface receptors. Wnts are cysteine-rich, glycosylated, lipid modified, two domain proteins that are prone to aggregation. The culprit responsible for this behavior is a covalently bound palmitoleoyl moiety in the N-terminal domain. Results: By combining murine Wnt3a with phospholipid and apolipoprotein A-I, ternary complexes termed nanodisks (ND) were generated. ND-associated Wnt3a is soluble in the absence of detergent micelles and gel filtration chromatography revealed that Wnt3a co-elutes with ND. In signaling assays, Wnt3a ND induced β-catenin stabilization in mouse fibroblasts as well as hematopoietic stem and progenitor cells (HSPC). Prolonged exposure of HSPC to Wnt3a ND stimulated proliferation and expansion of Lin- Sca-1+ c-Kit+ cells. Surprisingly, ND lacking Wnt3a contributed to Lin- Sca-1+ c-Kit+ cell expansion, an effect that was not mediated through β-catenin. Conclusions: The data indicate Wnt3a ND constitute a water-soluble transport vehicle capable of promoting ex vivo expansion of HSPC.

BACKGROUND: Over the last years the need for a gold standard to determine the sizes of extracellular vesicles including microvesicles by flow cytometry has been emphasized.METHODS: This work suggests to use artificial vesicles as calibrators to ascertain the size of microvesicles from the side scattering (SSC) measured by flow cytometry. We prepared fluorescently labeled liposomes with different maximum sizes defined by the pore size (200, 400, 800, and 1000 nm) of the membrane used for the extrusion. The fluorescence strengths from the largest liposomes pertaining to each pore size enabled us to verify the correlation between the SSC from a liposome and the corresponding size.CONCLUSIONS: We show that artificial vesicles are more accurate size calibrators compared to the commonly used polystyrene calibrator beads illustrated by the SSC from 110 nm polystyrene beads corresponds to the scattering from ~400 nm sized vesicle-like particles. We also show that this method of size assessment based on SSC has a low resolution that is roughly estimated to be between 60 to 200 nm dependent on the vesicle size. This article is protected by copyright. All rights reserved.

Self-assembly of the amphiphilic π-conjugated carbenium ion ATOTA-1(+) in aqueous solution selectively leads to discrete and highly stable nanotubes or nanoribbons and nanorods, depending on the nature of the counterion (Cl(-) vs. PF6(-), respectively). The nanotubes formed by the Cl(-) salt illustrate an exceptional example of a structural well-defined (29±2 nm in outer diameter) unilamellar tubular morphology featuring π-conjugated functionality and high stability and flexibility, in aqueous solution.

Recognition of carbohydrates by proteins is a ubiquitous biochemical process. In legume-rhizobium symbiosis, lipochitin oligosaccharides, also referred to as nodulation (nod) factors, function as primary rhizobial signal molecules to trigger root nodule development. Perception of these signal molecules is receptor mediated, and nod factor receptor 5 (NFR5) from the model legume Lotus japonicus is predicted to contain three LysM domain binding sites. Here we studied the interactions between nod factor and each of the three NFR5 LysM domains, which were chemically synthesized. LysM domain variants (up to 58 amino acids) designed to optimize solubility were chemically assembled by solid-phase peptide synthesis (SPPS) with microwave heating. Their interaction with nod factors and chitin oligosaccharides was studied by isothermal titration calorimetry and circular dichroism (CD) spectroscopy. LysM2 showed a change in folding upon nod factor binding, thus providing direct evidence that the LysM domain of NFR5 recognizes lipochitin oligosaccharides. These results clearly show that the L. japonicus LysM2 domain binds to the nod factor from Mesorhizobium loti, thereby causing a conformational change in the LysM2 domain. The preferential affinity for nod factors over chitin oligosaccharides was demonstrated by a newly developed glycan microarray. Besides the biological implications, our approach shows that carbohydrate binding to a small protein domain can be detected by CD spectroscopy.

Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined. Different variants of the enzymes were isolated by hydrophobic interaction chromatography and showed variations in their glycosylation, N-terminal sequences and activities. Glycosylation at Asn291 and the loss of the first three residues of camel chymosin significantly decreased its activity. Thermal differential scanning calorimetry revealed a slightly higher thermal stability of camel chymosin compared with bovine chymosin. The crystal structure of a doubly glycosylated variant of camel chymosin was determined at a resolution of 1.6 Å and the crystal structure of unglycosylated bovine chymosin was redetermined at a slightly higher resolution (1.8 Å) than previously determined structures. Camel and bovine chymosin share the same overall fold, except for the antiparallel central β-sheet that connects the N-terminal and C-terminal domains. In bovine chymosin the N-terminus forms one of the strands which is lacking in camel chymosin. This difference leads to an increase in the flexibility of the relative orientation of the two domains in the camel enzyme. Variations in the amino acids delineating the substrate-binding cleft suggest a greater flexibility in the ability to accommodate the substrate in camel chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C-terminus of κ-casein. Camel

Phospholipid bilayers host and support the function of membrane proteins and may be stabilized in disc-like nanostructures, allowing for unprecedented solution studies of the assembly, structure, and function of membrane proteins (Bayburt et al. Nano Lett. 2002, 2, 853-856). Based on small-angle neutron scattering in combination with variable-temperature studies of synchrotron small-angle X-ray scattering on nanodiscs in solution, we show that the fundamental nanodisc unit, consisting of a lipid bilayer surrounded by amphiphilic scaffold proteins, possesses intrinsically an elliptical shape. The temperature dependence of the curvature of the nanodiscs prepared with two different phospholipid types (DLPC and POPC) shows that it is the scaffold protein that determines the overall elliptical shape and that the nanodiscs become more circular with increasing temperature. Our data also show that the hydrophobic bilayer thickness is, to a large extent, dictated by the scaffolding protein and adjusted to minimize the hydrophobic mismatch between protein and phospholipid. Our conclusions result from a new comprehensive and molecular-based model of the nanodisc structure and the use of this to analyze the experimental scattering profile from nanodiscs. The model paves the way for future detailed structural studies of functional membrane proteins encapsulated in nanodiscs.

Udgivelsesdato: Mar 17 2009, Three new tris(dialkylamino)trioxatriangulenium (ATOTA(+)) salts rendered amphiphilic by attachment of two (5a center dot PF6 and 5b center dot PF6) or four (5c center dot PF6) n-decyl chains have been synthesized, and their Langmuir films have been studied by grazing incidence X-ray diffraction (GIXD). Compounds 5a center dot PF6 and 5b center dot PF6 both self-assemble into 2D-crystalline Langmuir monolayers, in which the planar triangular shaped carbenium ions form columnar aggregates segregated from the PF6- ions. The column width is found to be close to the width of the triangulenium moiety itself (similar to 17 angstrom), while the repeat distance along the columnar aggregates is only 3.45 angstrom, implicating a near cofacial columnar structure with only a small tilt of the planar carbenium ions relative to the columnar axis. A detailed Bragg rod analysis confirmed an 8-9 degrees tilt and inferred a large anisotropy in the smearing/thermal displacement along the pi-pi stacking and lamellar packing directions. Specular X-ray reflectivity (SXR) was used to confirm the model derived from the GIXD data and elucidate the average position of the disordered PF6- ions, showing that the majority of the anions are accommodated in the ATOTA(+) layer rather than in the water subphase.

We have performed ab initio calculations to examine the potential energy along the normal modes of ground-state HCHO and along the reaction coordinates for loss of H2 and atomic hydrogen, respectively. This exploration showed that there are no specific features that will lead to reaction on the excited-state surfaces for excitations that are relevant to the troposphere and stratosphere. The calculations did however lead to the localization of a conical intersection point through which a specific loss of H2 could take place. However, the conical intersection lies at 5.4 eV relative to the ground state molecule at equilibrium and is thus inaccessible via single photon excitation at tropospheric and stratospheric wavelengths. In addition to the ab initio investigation we have carried out a femtosecond pump–probe experiment using a 266/400 nm excitation. The results show that the timescale for the internal conversion from the initially prepared high-lying Rydberg states is on the order of a picosecond. This process populates the n - p* first excited singlet state which then survives for a substantially longer time before it is depopulated to form hot ground state or triplet-excited molecules that can then decompose.