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Efficient and targeted delivery of therapeutic agents remains a bottleneck in modern medicine. Here, biochemical engineering approaches to advance the repurposing of extracellular vesicles (EVs) as drug delivery vehicles are explored. Targeting ligands such as the sugar GalNAc are displayed on the surface of EVs using a HaloTag‐fused to a protein anchor that is enriched on engineered EVs. These EVs are successfully targeted to human primary hepatocytes. In addition, the authors are able to decorate EVs with an antibody that recognizes a GLP1 cell surface receptor by using an Fc and Fab region binding moiety fused to an anchor protein, and they show that this improves EV targeting to cells that overexpress the receptor. The authors also use two different protein‐engineering approaches to improve the loading of Cre recombinase into the EV lumen and demonstrate that functional Cre protein is delivered into cells in the presence of chloroquine, an endosomal escape enhancer. Lastly, engineered EVs are well tolerated upon intravenous injection into mice without detectable signs of liver toxicity. Collectively, the data show that EVs can be engineered to improve cargo loading and specific cell targeting, which will aid their transformation into tailored drug delivery vehicles. [ABSTRACT FROM AUTHOR]

The capabilities of time-of-flight secondary ion mass spectrometry (TOF-SIMS) with regards to limits in lateral resolution for biological samples are examined using supported lipid bilayers and individual lipid vesicles, both being among the most commonly used cell membrane mimics. Using supported 1-palmitoyl-2-oleoyl-sn-giycero3-phosphocholine (POPC) bilayers confined to a Si[O.sub.2] substrate by a chemically modified gold surface, the edge of the lipid bilayer was analyzed by imaging TOF-SIMS to assess the lateral resolution. The results using 80 keV [Bi.sub.3.sup.2] primary ions show that, under optimized conditions, mass spectrometry imaging of specific unlabeled lipid fragments is possible with sub-100 nm lateral resolution. Comparison of the secondary ion yields for the phosphocholine ion (m/z 184) from a POPC bilayer using [C.sub.60] or [Bi.sub.3] primary ions showed similar results, indicating an advantage of [Bi.sub.3] primary ions for high-resolution imaging of lipid membranes, due to their better demonstrated focusing capability. Moreover, using 300 nm vesicles of different lipid composition, the capability to detect and chemically identify individual submicrometer lipid vesicles at separations down to ~1 [micro]m is demonstrated. 10.1021/ac902744u

Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single‐vesicle and single‐molecule level to accurately quantify the efficiency of different EV‐sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV‐sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV‐sorting domains, but quantitative single‐vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti‐tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single‐Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV‐sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high‐resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV‐sorting proteins to be used in EV engineering applications. [ABSTRACT FROM AUTHOR]

Protein–protein interactions (PPIs) of 14‐3‐3 proteins are a model system for studying PPI stabilization. The complex natural product Fusicoccin A stabilizes many 14‐3‐3 PPIs but is not amenable for use in SAR studies, motivating the search for more drug‐like chemical matter. However, drug‐like 14‐3‐3 PPI stabilizers enabling such studies have remained elusive. An X‐ray crystal structure of a PPI in complex with an extremely low potency stabilizer uncovered an unexpected non‐protein interacting, ligand‐chelated Mg2+ leading to the discovery of metal‐ion‐dependent 14‐3‐3 PPI stabilization potency. This originates from a novel chelation‐controlled bioactive conformation stabilization effect. Metal chelation has been associated with pan‐assay interference compounds (PAINS) and frequent hitter behavior, but chelation can evidently also lead to true potency gains and find use as a medicinal chemistry strategy to guide compound optimization. To demonstrate this, we exploited the effect to design the first potent, selective, and drug‐like 14‐3‐3 PPI stabilizers. [ABSTRACT FROM AUTHOR]

Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 in vitro potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR in vitro potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na+/K+ ratio, while eplerenone increased the Na+/K+ ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Inhibition-in-solution assays (ISA) employing surface-based biosensors such as surface plasmon resonance (SPR) are an effective screening approach in drug discovery. However, analysis of potent binders remains a significant hurdle due to limited sensitivity and accompanied depletion of the inhibiting compounds due to high protein concentrations needed for detectable binding signals. To overcome this limitation, we explored a microscopy-based single-molecule ISA compatible with liposome-reconstituted membrane proteins. Using a set of validated small molecule inhibitors against β-secretase 1 (BACEl), the assay was benchmarked with respect to sensitivity and dynamic range against SPR- We demonstrate that the dynamic range of measurable affinities is greatly extended by more than 2 orders of magnitude as compared to SPR, thus facilitating measurements of highly potent (Kd < nM) compounds. [ABSTRACT FROM AUTHOR]

The spatial localization of amyloid-β peptide deposits, the major component of senile plaques in Alzheimer’s disease (AD), was mapped in transgenic AD mouse brains using time-of-flight secondary ion mass spectrometry (ToF-SIMS), simultaneously with several endogenous molecules that cannot be mapped using conventional immunohistochemistry imaging, including phospholipids, cholesterol and sulfatides. Whereas the endogenous lipids were detected directly, the amyloid-β deposits, which cannot be detected as intact entities with ToF-SIMS because of extensive ion-induced fragmentation, were identified by specific binding of deuterated liposomes to antibodies directed against amyloid-β. Comparative investigation of the amyloid-β deposits using conventional immunohistochemistry and fluorescence microscopy suggests similar sensitivity but a more surface-confined identification due to the shallow penetration depth of the ToF-SIMS signal. The recorded ToF-SIMS images thus display the localization of lipids and amyloid-β in a narrow (~10 nm) two-dimensional plane at the tissue surface. As compared to a frozen nontreated tissue sample, the liposome preparation protocol generally increased the signal intensity of endogenous lipids, likely caused by matrix effects associated with the removal of salts, but no severe effects on the tissue integrity and the spatial distribution of lipids were observed with ToF-SIMS or scanning electron microscopy (SEM). This method may provide an important extension to conventional tissue imaging techniques to investigate the complex interplay of different kinds of molecules in neurodegenerative diseases, in the same specimen. However, limitations in target accessibility of the liposomes as well as unspecific binding need further consideration. [ABSTRACT FROM AUTHOR]

We have applied surface-enhanced ellipsometry contrast (SEEC) imaging for time-resolved label-free visualization of biomolecular recognition events on spatially heterogeneous supported lipid bilayers (SLB). Using a conventional inverted microscope equipped with total internal reflection (TIR) illumination, biomolecular binding events were monitored with a lateral resolution near the optical diffraction limit at an acquisition rate of ~1 Hz with a sensitivity in terms of surface coverage of ~1 ng/cm(2). Despite the significant improvement in spatial resolution compared to alternative label-free surface-based imaging technologies, the sensitivity remains competitive with surface plasmon resonance (SPR) imaging and imaging ellipsometry. The potential of the technique to discriminate local differences in protein binding kinetics was demonstrated by time-resolved imaging of anti-GalCer antibodies binding to phase-separated lipid bilayers consisting of phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher antibody binding capacity was observed on the GalCer-diluted fluid region in comparison to the GalCer-rich gel phase domains. This observation is tentatively attributed to differences in the presentation of the GalCer epitope in the two phases, resulting in differences in availability of the ligand for antibody binding. The complementary information obtained by swiftly switching between SEEC and fluorescence (including TIR fluorescence) imaging modes was used to support the data interpretation. The simplicity and generic applicability of the concept is discussed in terms of microfluidic applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Progress with respect to enrichment and separation of native membrane components in complex lipid environments, such as native cell membranes, has so far been very limited. The reason for the slow progress can be related to the lack of efficient means to generate continuous and laterally fluid supported lipid bilayers (SLBs) made from real cell membranes. We show in this work how the edge of a hydrodynamically driven SLB can be used to induce rupture of adsorbed lipid vesicles of compositions that typically prevent spontaneous SLB formation, such as vesicles made of complex lipid compositions, containing high cholesterol content or being derived from real cell membranes. In particular, upon fusion between the moving edge of a preformed SLB and adsorbed vesicles made directly from 3T3 fibroblast cell membranes, the membrane content of the vesicles was shown to be efficiently transferred to the SLB. The molecular transfer was verified using cholera toxin B subunit (CTB) binding to monosialoganglioside receptors (GM1 and GM3), and the preserved lateral mobility was confirmed by spatial manipulation of the GM1/M3—CTB complex using a hydrodynamic flow. Two populations of CTB with markedly different drift velocity could be identified, which from dissociation kinetics data were attributed to CTB bound with different numbers of ganglioside anchors. [ABSTRACT FROM AUTHOR]

The separation of molecules residing in the cell membrane remains a largely unsolved problem in the fields of bioscience and biotechnolo. We demonstrate how hydrodynamic forces can be used to both accumulate and separate membrane-bound proteins in their native state. A supported lipid bilayer (SIB) was formed inside a microfluidic channel with the two proteins streptavidin (SA) and cholera toxin (Cl') coupled to receptors in the lipid bilayer The anchored proteins were first driven toward the edge of the lipid bilayer by hydrodynainic forces from a flowing liquid above the SIB, resulting in the accumulation of protein molecules at the edge of the bilayer. After the concentration process, the bulk flow of liquid in the channel was reversed and the accumulated proteins were driven away from the edge of the bilayer. Each type of protein was found to move at a characteristic drift velocity, determined by the frictional coupling between the protein and the lipid bilayer, as well as the size and shape of the protein molecule. Despite having a similar molecular weight, SA and CF could be separated into monomolecular populations using this approach. The method also revealed heterogeneity among the CF molecules, resulting in three subpopulations with different drift velocities. This was tentatively attributed to multivalent interactions between the protein and the monosialoganglioside GML receptors in the lipid bilayer. [ABSTRACT FROM AUTHOR]

We present Monte Carlo simulations of dissociation of duplexes formed of complementary single-stranded DNAs with one of the strands attached to the surface. To describe the transition from the bound state to the unbound state of two strands located nearby, we use a lattice model taking DNA base-pair interactions and comformational changes into account. The results obtained are employed as a basis for a more coarse-grained model including strand backward association and diffusion resulting in complete dissociation. The distribution of the dissociation time is found to be exponential. This finding indicates that the non-exponential kinetic features observed in the corresponding experiments seem to be related to extrinsic factors, e.g., to the surface heterogeneity. [ABSTRACT FROM AUTHOR]