Cell‐Derived Vesicles for Single‐Molecule Imaging of Membrane Proteins

A new approach is presented for the application of single‐molecule imaging to membrane receptors through the use of vesicles derived from cells expressing fluorescently labeled receptors. During the isolation of vesicles, receptors remain embedded in the membrane of the resultant vesicles, thus allowing these vesicles to serve as nanocontainers for single‐molecule measurements. Cell‐derived vesicles maintain the structural integrity of transmembrane receptors by keeping them in their physiological membrane. It was demonstrated that receptors isolated in these vesicles can be studied with solution‐based fluorescence correlation spectroscopy (FCS) and can be isolated on a solid substrate for single‐molecule studies. This technique was applied to determine the stoichiometry of α3β4 nicotinic receptors. The method provides the capability to extend single‐molecule studies to previously inaccessible classes of receptors.     

Organization of the glycoprotein (GP) IIb/IIIa heterodimer on resting human platelets studied by flow cytometric energy transfer.

Glycoprotein IIb/IIIa is a heterodimer of glycoproteins IIb and IIIa which serves as the inducible receptor for fibrinogen and other adhesive proteins at the surface of platelets. Although a model of the quaternary structure of the GPIIb/IIIa molecule has been constructed in solution by Calvete et al. [Biochem. J. 282 (1992) 523], a corresponding model at the surface of intact platelets is still missing. In the present work conformation and lateral distribution of the GPIIb/IIIa heterodimer were studied at a nanometer resolution on the surface of resting human platelets under physiological conditions. The experiments were based on dual wavelength flow cytometric detection of fluorescence resonance energy transfer and application of a panel of monoclonal antibodies raised against well described binding sites. Monodisperse distribution of the GPIIb/IIIa heterodimer has been observed and a detailed three-dimensional proximity map of antibody binding sites was constructed on the platelet membrane, under physiological conditions, for the first time. Our data support the view that the GPIIb subunit is in a bent conformation. A detailed analysis of the K(d)-values and the number of binding sites for a set of monoclonal antibodies was also carried out giving supplementary data for the topology of the binding sites. Our results provide a refinement of the membrane-topology of the GPIIb/IIIa heterodimer.     

The synthesis,configuration, and conformation of cis- and trans-3-amino-3,4-dihydro-1-hydroxy-4-methylcarbostyrils and other configurationally related compounds

The cis- and trans-3-amino-3,4-dihydro-1-hydroxy-4-methylcarbostyrils (Ia and Ib) were synthesized by catalytic hydrogenation of erythro- and threo-α-amino-β-(o-nitrophenyl)butyric acid hydrochlorides, IIIa and IIIb, respectively, under acidic conditions. The free bases of IIIa and IIIb were catalytically hydrogenated under neutral conditions to yield the erythro- and threo-α-amino-β-(o-aminophenyl)butyric acids (VIa and VIb), which were converted by acidification to their corresponding lactams, cis- and trans-3-amino-3,4-dihydro-4-methylcarbostyrils, IIa and IIb. The erythro and threo isomers of α-amino-β-(o-nitrophenyl)-butyric acid were prepared and separated by liquid chromatography via a diastereomeric mixture of (V) of methyl α-acetamido-β-(o-nitrophenyl)butyrates. The configurations and conformational assignments of the cyclic hydroxamic acids Ia and Ib were first established by analysis of the proton nmr spectra. In turn, the configurations of the o-nitroaromatic amino acids IIIa and IIIb were assigned as well as the other structurally related compounds (VIa, VIb, IIa and IIb) derived therefrom.     

Stability and Drug Loading of Spontaneous Vesicles of Comb‐Like PEG Derivates

A novel comb‐like derivative CPEG‐g‐cholesterol was prepared by the reaction of cholesteryl chloroformate with hydroxyl groups of CPEG. The TEM and SEM results showed that CPEG‐cholesterol spontaneously aggregated vesicles with the membrane thickness of 4.27 ± 0.48 nm. Compared with the vesicles formed by comb‐like PEG (CPEG), the derivation of cholesteryl chloroformate increased the thickness of vesicle membrane and developed corrugations. The hydrophobic doxorubicin (Dox) was added into the solution of CPEG and CPEG‐g‐cholesterol to test their vesicle stability. The drug‐loaded vesicles of CPEG‐g‐cholesterol still existed but those of CPEG disappeared, which indicated that stability of vesicles was enhanced by the derived cholesteryl chloroformate. The vesicles were further cross‐linked by the reaction between divinyl sulfone (DVS) and the hydroxy groups in the side chains of the CPEG and CPEG‐g‐cholesterol. Both cross‐linked vesicles of CPEG and CPEG‐g‐cholesterol entrapped considerable hydrophobic Dox in the vesicles membrane. The spontaneous vesicles of CPEG‐g‐cholesterol and the crosslinked vesicles of CPEG and CPEG‐g‐cholesterol might have great potential as a cargo of the hydrophobic drug.

     

Hemostatic Ability of Chitosan‐Phosphate Inspired by Coagulation Mechanisms of Platelet Polyphosphates

Hemostatic materials have been studied to minimize bleeding time. Recently, polyphosphate (polyP) have received attention as potential hemostatic compounds, which are released from activated platelets. Long polyP chains are essential to form thick fibrin clots. Herein, chitosan is functionalized by covalently tethering phosphate groups to mimic polyP. It is hypothesized that utilizing a known hemostatic polysaccharide, chitosan, and tethering phosphate groups to mimic polyP's functionality show synergistic effect in hemostasis. Five different phosphorylated chitosan conjugates (Chi‐Ps), s‐Chi‐7P, s‐Chi‐28P, s‐Chi‐74P, is‐Chi‐29P, and is‐Chi‐56P are prepared, where “s” indicates water soluble Chi‐Ps and “is” represents water insoluble Chi‐Ps. Unexpectedly, an important carbon in D‐glucosamine is found, which determines chitosan solubility. Phosphate groups conjugated to C6 carbon resulted in water soluble Chi‐P, but conjugation to C3 group exhibited water insoluble behavior. Hemostasis capability showed a positive correlation with the degree of phosphate conjugations regardless of water solubility of Chi‐P.     

Optimization of fibrin gelation for enhanced cell seeding and proliferation in regenerative medicine applications

In order to improve the cell seeding efficiency and cell compatibility inside porous tissue scaffolds, a method of fibrin gel‐mediated cell encapsulation inside the scaffold was optimized. Disc‐type poly(d ,l ‐glycolic‐co‐lactic acid) (PLGA) scaffolds without a dense surface skin layer were fabricated using an established solvent casting and particulate leaching method as a model porous scaffold, which showed high porosity ranging from 90 ± 2% to 96 ± 2%. The thrombin and fibrinogen concentration as precursors of fibrin gel was varied to control the gelation kinetics as measured by rheology analysis, and optimized conditions were developed for a uniform fibrin gel formation with the target cells inside the porous PLGA scaffold. The fibroblast cell seeding accompanied by a uniform fibrin gel formation at an optimized gelation condition inside the PLGA scaffold resulted in an increase in cell seeding efficiency, a better cell proliferation, and an increase in final cell density inside the scaffold. Scanning electron microscopy images revealed that cells were better spread and grown by fibrin gel encapsulation inside scaffold compared with the case of bare PLGA scaffold. Copyright © 2016 John Wiley & Sons, Ltd.     

Evaluation of affinity interaction between small molecules and platelets by open tubular affinity capillary electrochromatography

In this paper, an open tubular affinity capillary electrochromatography (OT‐ACEC) was developed by physical adsorption of rabbit platelets on the inner surface of capillary. The interactions between small molecules include adenosine diphosphate (ADP) (positive control), protocatechuic acid (negative control) and seven natural products (salvianolic acid B, salvianic acid A sodium, hydroxysafflor yellow A, ferulic acid, chlorogenic acid, sinapic acid, caffeic acid) and platelets were evaluated by their retention factors and binding constants obtained based on peak‐shift assay. Then, the activities of anti‐platelet aggregation induced by thrombin (THR), ADP and arachidonic acid (AA) for those small molecules (except ADP) were evaluated by turbidimetric method. The results indicate that: (i) ADP, a platelet aggregation inducer, had strong interaction with platelet, while protocatechuic acid that had no inhibition on platelet aggregation behaved no specific interaction; (ii) there was a positive correlation between the anti‐platelet aggregation activities of small molecules and their interactions with platelet, generally those compounds with higher binding constants with platelet exhibited higher activities. Therefore, the OT‐ACEC method developed in the present study can be a potential method to evaluate affinity interactions between small molecules and platelets, so as to predict the biological activities such as anti‐platelet aggregation for the small molecules.     

Nanoscale Distribution of Transforming Growth Factor Receptor on Post‐Golgi Vesicle Revealed by Super‐resolution Microscopy

Transforming growth factor‐β (TGF‐β) type II receptor (TβRII) plays a critical role in the initiation of TGF‐β signaling pathway; therefore, the study of its synthesis and transport processes is of great important. In this work, we achieved super‐resolution imaging of a new type of TβRII‐containing post‐Golgi vesicle by our home‐built stimulated emission depletion (STED) microscope. We visualized the ring‐shaped structure of these vesicles containing newly synthesized TβRII in the cytoplasm and characterized their size distribution from 300 to 1000 nm. These vesicles could be swollen by chloroquine treatment. Further investigation revealed that TβRII formed clusters on the outer ring of the post‐Golgi vesicles. This study offers new information on the intracellular transportation of TGF‐β receptors for better understanding its signaling process.     

Universal Approach Towards r‐Hirudin Derivatives with High Anti‐Thrombin Activity Based on Chemical Differentiation of Primary Amino Groups

Chemical modification of recombinant hirudin (r‐hirudin) is necessary whenever surface‐confinement to a biomaterial or biotinylation for subsequent conjugation with carriers is intended. Here, we report a modification strategy that permits chemical discrimination between r‐hirudin's amino groups and preserves its thrombin inhibitor activity. By reaction with Msc‐ONSu, protective groups were successively introduced in r‐hirudin yielding four derivatives (Msc)_x‐hirudin (1 ≤ x ≤ 4) and pure fractions were isolated by ion exchange chromatography. Structure–function relationships were studied for all derivatives and revealed a decrease in activity of more than 90% as compared to unprotected r‐hirudin. MALDI‐TOF MS was used to determine the locations of the Msc groups. Furthermore, evidence was provided that r‐hirudin's N‐terminal amino group is highly important for its anti‐thrombin activity. Selective modification of the lysine residues which maintained the free N‐terminal amino group preserved the anti‐thrombin activity of r‐hirudin even after biotinylation and subsequent linkage to streptavidin or confinement to a polymer surface.     

Structure-based virtual screening of small-molecule antagonists of platelet integrin αIIbβ3 that do not prime the receptor to bind ligand

Integrin αIIbβ3 has emerged as an important therapeutic target for thrombotic vascular diseases owing to its pivotal role in mediating platelet aggregation through interaction with adhesive ligands. In the search for effective anti-thrombotic agents that can be administered orally without inducing the high-affinity ligand binding state, we recently discovered via high-throughput screening of 33,264 compounds a novel, αIIbβ3-selective inhibitor (RUC-1) of adenosine-5′-diphosphate (ADP) -induced platelet aggregation that exhibits a different chemical scaffold and mode of binding with respect to classical Arg-Gly-Asp (RGD)-mimicking αIIbβ3 antagonists. Most importantly, RUC-1 and its higher-affinity derivative, RUC-2, do not induce major conformational changes in the protein β3 subunit or prime the receptor to bind ligand. To identify additional αIIbβ3-selective chemotypes that inhibit platelet aggregation through similar mechanisms, we screened in silico over 2.5 million commercially available, ‘lead-like’ small molecules based on complementarity to the predicted binding mode of RUC-2 into the RUC-1-αIIbβ3 crystal structure. This first reported structure-based virtual screening application to the αIIbβ3 integrin led to the identification of 2 αIIbβ3-selective antagonists out of 4 tested, which compares favorably with the 0.003?% “hit rate” of our previous high-throughput chemical screening study. The newly identified compounds, like RUC-1 and RUC-2, showed specificity for αIIbβ3 compared to αVβ3 and did not prime the receptor to bind ligand. They thus may hold promise as αIIbβ3 antagonist therapeutic scaffolds.     

Metadynamics Simulations Rationalise the Conformational Effects Induced by N‐Methylation of RGD Cyclic Hexapeptides

We combined metadynamics, docking and molecular mechanics/generalised born surface area (MM/GBSA) re‐scoring methods to investigate the impact of single and multiple N‐methylation on a set of RGD cyclopeptides displaying different affinity for integrin αIIbβ3. We rationalised the conformational effects induced by N‐methylation and its interplay with receptor affinity, obtaining good agreement with experimental data. This approach can be exploited before entering time‐consuming and expensive synthesis and binding experiments.     

Sugar‐Decorated Sugar Vesicles: Lectin–Carbohydrate Recognition at the Surface of Cyclodextrin Vesicles

An artificial glycocalix self‐assembles when unilamellar bilayer vesicles of amphiphilic β‐cyclodextrins are decorated with maltose and lactose by host–guest interactions. To this end, maltose and lactose were conjugated with adamantane through a tetra(ethyleneglycol) spacer. Both carbohydrate–adamantane conjugates strongly bind to β‐cyclodextrin (K_a≈4×10⁴ M ^?1). The maltose‐decorated vesicles readily agglutinate (aggregate) in the presence of the lectin concanavalin A, whereas the lactose‐decorated vesicles agglutinate in the presence of peanut agglutinin. The orthogonal multivalent interaction in the ternary system of host vesicles, guest carbohydrates, and lectins was investigated by using isothermal titration calorimetry, dynamic light scattering, UV/Vis spectroscopy, and cryogenic transmission electron microscopy. It was shown that agglutination is reversible, and the noncovalent interaction can be suppressed and eliminated by the addition of competitive inhibitors, such as D ‐glucose or β‐cyclodextrin. Also, it was shown that agglutination depends on the surface coverage of carbohydrates on the vesicles.     

Preparation and pharmacological evaluation of novel glycoprotein (Gp) IIb/IIIa antagonists. 1. The selection of naphthalene derivatives

The synthesis and design using molecular modeling techniques for non-peptide, low molecular weight novel fibrinogen receptor (glycoprotein IIb/IIIa: Gp IIb/IIIa) antagonists, is reported. We used a highly potent serine protease inhibitor, Nafamostat, having an amidinonaphthyl unit as the starting compound. The compounds 4-(6-amidino-2-naphthylaminocarbonyl)phenoxyacetic acid (5a) and 4-(6-amidino-2-naphthalenecarboxamido)phenoxyacetic acid (5b) inhibited adenosin-5'-diphospate (ADP)-induced aggregation of human platelet-rich plasma (PRP) with IC50 values of 0.05 and 0.07 microM, respectively, and had lost their ability to inhibit a variety of serine proteases, including thrombin, factor Xa, plasmin and trypsin.     

Spontaneous Vesicle Formation in Aqueous Solutions of Comb‐Like PEG

Summary: A novel comb‐like poly(ethylene glycol) (CPEG), with dominant water‐soluble PEG, is found to spontaneously aggregate into vesicles above a certain concentration in water. The hollow, three‐dimensional structure of the vesicles is proven by TEM, SEM, and AFM. The diameters of the vesicles are from 200 to 500 nm with 50 nm walls. The spontaneously formed vesicles can be further cross‐linked by the reaction between divinyl sulfone (DVS) and the hydroxy groups in the side chains of the CPEG. The spontaneously formed vesicles with dense reactive hydroxy groups will have great potential in both applications and research.

SEM image of the uncross‐linked vesicles.     

Lyophilised Platelet-Rich Fibrin: Physical and Biological Characterisation

Background: Platelet-rich fibrin (PRF) has gained popularity in craniofacial surgery, as it provides an excellent reservoir of autologous growth factors (GFs) that are essential for bone regeneration. However, the low elastic modulus, short-term clinical application, poor storage potential and limitations in emergency therapy use restrict its more widespread clinical application. This study fabricates lyophilised PRF (Ly-PRF), evaluates its physical and biological properties, and explores its application for craniofacial tissue engineering purposes. Material and methods: A lyophilisation method was applied, and the outcome was evaluated and compared with traditionally prepared PRF. We investigated how lyophilisation affected PRF’s physical characteristics and biological properties by determining: (1) the physical and morphological architecture of Ly-PRF using SEM, and (2) the kinetic release of PDGF-AB using ELISA. Results: Ly-PRF exhibited a dense and homogeneous interconnected 3D fibrin network. Moreover, clusters of morphologically consistent cells of platelets and leukocytes were apparent within Ly-PRF, along with evidence of PDGF-AB release in accordance with previously reports. Conclusions: The protocol established in this study for Ly-PRF preparation demonstrated versatility, and provides a biomaterial with growth factor release for potential use as a craniofacial bioscaffold.     

Folding Up of Gold Nanoparticle Strings into Plasmonic Vesicles for Enhanced Photoacoustic Imaging

The stepwise self‐assembly of hollow plasmonic vesicles with vesicular membranes containing strings of gold nanoparticles (NPs) is reported. The formation of chain vesicles can be controlled by tuning the density of the polymer ligands on the surface of the gold NPs. The strong absorption of the chain vesicles in the near‐infrared (NIR) region leads to a much higher efficiency in photoacoustic (PA) imaging than for non‐chain vesicles. The chain vesicles were further employed for the encapsulation of drugs and the NIR light triggered release of payloads. This work not only offers a new platform for controlling the hierarchical self‐assembly of NPs, but also demonstrates that the physical properties of the materials can be tailored by controlling the spatial arrangement of NPs within assemblies to achieve a better performance in biomedical applications.     

Probing the interaction of a glycoprotein IIb/IIIa receptor antagonist with bound platelets using electrochemical impedance

A label-free assay is described to monitor the interaction of abciximab, a glycoprotein IIb/IIIa receptor antagonist (ReoPro), with platelets bound to a fibrinogen-functionalised electrode surface. Firstly, fibrinogen is deposited in a defined pattern onto a gold electrode using microcontact printing, and then platelets from whole blood are captured on the patterned surface. Patterning influences the spreading of platelets, which is strikingly different to that observed on homogeneously coated surfaces. The drug–platelet interaction has been investigated using AC impedance on uniform and patterned fibrinogen-modified surfaces. The results demonstrate that patterned fibrinogen surfaces can provide deep insights into the interaction of abciximab with different platelet sub-populations. The key advantages of this approach are that it is rapid, label free and does not require pre-processing of patient blood samples.     

Thiol‐ene photopolymerization for efficient curing of vinyl esters

Monomers for radical photopolymerization based on vinyl esters (VEs) have recently been identified as suitable alternatives to (meth)acrylates on account of their low irritancy and cytotoxicity. The drawback of most VEs with abstractable hydrogens is their relatively low reactivity compared with (meth)acrylates. Within this article, we proved by photo‐differential scanning calorimetry measurements and real‐time Fourier transform infrared spectroscopy that the thiol‐ene concept is able to improve the photoreactivity of these VEs to a large extent to a level between those of acrylates and methacrylates. Other VEs have now a reactivity of at least the level of similar acrylates. Mechanical properties as determined by Dynamic Mechanical Analysis and Charpy impact tests showed significant toughening of these materials. Furthermore, we were able to confirm low toxicity of all components by osteoblast cell culture experiments. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Targeted Antithrombotic Protein Micelles

Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site‐specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one‐pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single‐chain antibody (scFv), which binds to the ligand‐induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events.     

Small‐Angle Neutron Scattering (SANS) Study of Magneto‐Vesicles

Small‐angle neutron scattering from magneto‐vesicles (MVs) prepared by extrusion was studied. Contrast variation allowed the determination of structure and sizes of the vesicles and the encapsulated magnetic nanoparticles, respectively. The results from MVs synthesized with a 0.3% volume fraction of citrate‐coated magnetic nanoparticles are compared to those of similarly prepared vesicles of the neutral lipid 1,2‐Dioleoyl‐sn‐Glycero‐3‐Phosphocholine (DOPC) (without magnetic particles), and magnetic particles not encapsulated in vesicles. It is observed that the bilayers of the as‐prepared MVs, and the encapsulated nanoparticles retain their structural properties, highlighting the suitability of the MVs for applications.