Thermally-induced aggregation and fusion of protein-free lipid vesicles.

Membrane fusion is an important phenomenon in cell biology and pathology. This phenomenon can be modeled using vesicles of defined size and lipid composition. Up to now fusion models typically required the use of chemical (polyethyleneglycol, cations) or enzymatic catalysts (phospholipases). We present here a model of lipid vesicle fusion induced by heat. Large unilamellar vesicles consisting of a phospholipid (dioleoylphosphatidylcholine), cholesterol and diacylglycerol in a 43:57:3 mol ratio were employed. In this simple system, fusion was the result of thermal fluctuations, above 60 °C. A similar system containing phospholipid and cholesterol but no diacylglycerol was observed to aggregate at and above 60 °C, in the absence of fusion. Vesicle fusion occurred under our experimental conditions only when (31)P NMR and cryo-transmission electron microscopy of the lipid mixtures used in vesicle preparation showed non-lamellar lipid phase formation (hexagonal and cubic). Non-lamellar structures are probably the result of lipid reassembly of the products of individual fusion events, or of fusion intermediates. A temperature-triggered mechanism of lipid reassembly might have occurred at various stages of protocellular evolution.

Ion-association complexes unite classical and non-classical theories for the biomimetic nucleation of calcium phosphate.

Despite its importance in many industrial, geological and biological processes, the mechanism of crystallization from supersaturated solutions remains a matter of debate. Recent discoveries show that in many solution systems nanometre-sized structural units are already present before nucleation. Still little is known about the structure and role of these so-called pre-nucleation clusters. Here we present a combination of in situ investigations, which show that for the crystallization of calcium phosphate these nanometre-sized units are in fact calcium triphosphate complexes. Under conditions in which apatite forms from an amorphous calcium phosphate precursor, these complexes aggregate and take up an extra calcium ion to form amorphous calcium phosphate, which is a fractal of Ca(2)(HPO(4))(3)(2-) clusters. The calcium triphosphate complex also forms the basis of the crystal structure of octacalcium phosphate and apatite. Finally, we demonstrate how the existence of these complexes lowers the energy barrier to nucleation and unites classical and non-classical nucleation theories.

The role of prenucleation clusters in surface-induced calcium phosphate crystallization.

Unravelling the processes of calcium phosphate formation is important in our understanding of both bone and tooth formation, and also of pathological mineralization, for example in cardiovascular disease. Serum is a metastable solution from which calcium phosphate precipitates in the presence of calcifiable templates such as collagen, elastin and cell debris. A pathological deficiency of inhibitors leads to the uncontrolled deposition of calcium phosphate. In bone and teeth the formation of apatite crystals is preceded by an amorphous calcium phosphate (ACP) precursor phase. ACP formation is thought to proceed through prenucleation clusters--stable clusters that are present in solution already before nucleation--as was recently demonstrated for CaCO(3) (refs 15,16). However, the role of such nanometre-sized clusters as building blocks for ACP has been debated for many years. Here we demonstrate that the surface-induced formation of apatite from simulated body fluid starts with the aggregation of prenucleation clusters leading to the nucleation of ACP before the development of oriented apatite crystals.

Imaging of self-assembled structures: interpretation of TEM and cryo-TEM images.

The investigation of solution-borne nanostructures by transmission electron microscopy (TEM) is a frequently used analytical method in materials chemistry. In many cases, the preparation of the TEM sample involves drying and staining steps, and the collection of images leads to the interaction of the specimen with the electron beam. Both aspects call for cautious interpretation of the resulting electron micrographs. Alternatively, a near-native solvated state can be preserved by cryogenic vitrification and subsequent imaging by low-dose cryogenic TEM. In this Minireview, we provide a critical analysis of sample preparation, and more importantly, of the acquisition and interpretation of electron micrographs. This overview should provide a framework for the application of (cryo)-TEM as a powerful and reliable tool for the analysis of colloidal and self-assembled structures with nanoscopic dimensions.

The development of morphology and structure in hexagonal vaterite.

Inspired by the remarkable shapes and properties of CaCO(3) biominerals, many studies have investigated biomimetic routes aiming at synthetic equivalents with similar morphological and structural complexity. Control over the morphology of CaCO(3) crystals has been demonstrated, among other methods, by the use of additives that selectively allow the development of specific crystal faces, while inhibiting others. Both for biogenic and biomimetic CaCO(3), the crystalline state is often preceded by an amorphous precursor phase, but still limited information is available on the details of the amorphous-to-crystalline transition. By using a combination of cryoTEM techniques (bright field imaging, cryo-tomography, low dose electron diffraction and cryo-darkfield imaging), we show for the first time the details of this transition during the formation of hexagonal vaterite crystals grown in the presence of NH(4)(+) ions. The formation of hexagonal plate-like vaterite occurs via an amorphous precursor phase. This amorphous phase converts into the crystalline state through a solid state transformation in which order and morphology develop simultaneously. The mineral initially develops as polycrystalline vaterite which transforms into a single crystal directed by an NH(4)(+)-induced crystal plane that acts as a templating surface.

Transfection efficiency of lipoplexes for site-directed delivery.

Targeted gene delivery is a promising strategy to cure disease on its basic level at the site of interest. The ultrastructure, internalization, and transfection efficiency of lipoplexes was investigated. We found that at a charge ratio (rho) of 4.0 lipoplexes had optimum characteristics for gene delivery in vitro. To decrease the size of lipoplexes, we used a method of continuous-flow microfluidics. PEGylation of lipoplexes did not hinder internalization, but was found to hamper transfection. To discriminate between uptake and transfection efficiency of lipoplexes, we used fluorescence-based approaches: microscopy and FACS. To this end, GFP plasmid was labeled with Alexa 594, and, in parallel experiments, GFP plasmid was combined with rhodamine-labeled lipid. Our studies confirm that cellular uptake does not imply transfection efficiency, and that hurdles in cellular processing have to be taken before targeted gene delivery becomes an established therapeutic option.

End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.

A phospholipase C/sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties; the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230-3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme.

Environment-sensitive stabilisation of silver nanoparticles in aqueous solutions.

We describe the preparation and characterisation of inorganic-organic hybrid block copolymer silver nanoparticles via the preparation of spherical multi-responsive polymeric micelles of poly(N-methyl-2-vinyl pyridinium iodide)-block-poly(ethylene oxide), P2MVP(38)-b-PEO(211) and poly(acrylic acid)-block-poly(isopropyl acrylamide), PAA(55)-b-PNIPAAm(88) in the presence of AgNO(3). Hence, the P2MVP and PAA segments were employed to fix Ag(+) ions within the micellar core (25 degrees C) or shell (60 degrees C), while the PEO segments ensured spontaneous reduction of Ag(+) ions into metallic Ag, as well as colloidal stabilisation. Spherical and elongated composite core-shell(-corona) nanoparticles (CNPs) were formed containing several small, spherical silver nanoparticles within the micellar core or shell. As the co-assembly of the oppositely charged copolymers into micelles is electrostatically driven, the CNPs can be destabilised by, for example, addition of simple salts, i.e., the CNPs are stimuli responsive. CNP size and morphology control can be achieved via the preparation protocol. For example, heating to 60 degrees C, i.e., above the PNIPAAm LCST, results in core-shell-corona CNPs with the Ag-NPs situated in the aggregate shell.

Thin-walled microvessels in human coronary atherosclerotic plaques show incomplete endothelial junctions relevance of compromised structural integrity for intraplaque microvascular leakage.

OBJECTIVES: This study sought to examine the ultrastructure of microvessels in normal and atherosclerotic coronary arteries and its association with plaque phenotype. BACKGROUND: Microvessels in atherosclerotic plaques are an entry point for inflammatory and red blood cells; yet, there are limited data on the ultrastructural integrity of microvessels in human atherosclerosis. METHODS: Microvessel density (MVD) and ultrastructural morphology were determined in the adventitia, intima-media border, and atherosclerotic plaque of 28 coronary arteries using immunohistochemistry for endothelial cells (Ulex europeaus, CD31/CD34), basement membrane (laminin, collagen IV), and mural cells (desmin, alpha-smooth muscle [SM] actin, smoothelin, SM1, SM2, SMemb). Ultrastructural characterization of microvessel morphology was performed by electron microscopy. RESULTS: The MVD was increased in advanced plaques compared with early plaques, which correlated with lesion morphology. Adventitial MVD was higher than intraplaque MVD in normal arteries and early plaques, but adventitial and intraplaque MVD were similar in advanced plaques. Although microvessel basement membranes were intact, the percentage of thin-walled microvessels was similarly low in normal and atherosclerotic adventitia, in the adventitia and the plaque, and in all plaque types. Intraplaque microvascular endothelial cells (ECs) were abnormal, with membrane blebs, intracytoplasmic vacuoles, open EC-EC junctions, and basement membrane detachment. Leukocyte infiltration was frequently observed by electron microscopy, and confirmed by CD45RO and CD68 immunohistochemistry. CONCLUSIONS: The MVD was associated with coronary plaque progression and morphology. Microvessels were thin-walled in normal and atherosclerotic arteries, and the compromised structural integrity of microvascular endothelium may explain the microvascular leakage responsible for intraplaque hemorrhage in advanced human coronary atherosclerosis.

The initial stages of template-controlled CaCO3 formation revealed by cryo-TEM.

Biogenic calcium carbonate forms the inorganic component of seashells, otoliths, and many marine skeletons, and its formation is directed by an ordered template of macromolecules. Classical nucleation theory considers crystal formation to occur from a critical nucleus formed by the assembly of ions from solution. Using cryotransmission electron microscopy, we found that template-directed calcium carbonate formation starts with the formation of prenucleation clusters. Their aggregation leads to the nucleation of amorphous nanoparticles in solution. These nanoparticles assemble at the template and, after reaching a critical size, develop dynamic crystalline domains, one of which is selectively stabilized by the template. Our findings have implications for template-directed mineral formation in biological as well as in synthetic systems.

Internalization of annexin A5-functionalized iron oxide particles by apoptotic Jurkat cells.

Apoptosis plays an important role in the etiology of various diseases. Several studies have reported on the use of annexin A5-functionalized iron oxide particles for the detection of apoptosis with MRI, both in vitro and in vivo. The protein annexin A5 binds with high affinity to the phospholipid phosphatidylserine, which is exposed in the outer leaflet of the apoptotic cell membrane. When co-exposed to apoptotic stimuli, this protein was shown to internalize into endocytic vesicles. Therefore in the present study we investigated the possible internalization of commercially available annexin A5-functionalized iron oxide particles (r1 = 34.0 +/- 2.1 and r2 = 205.0 +/- 10.4 mm(-1) s(-1) at 20 MHz), and the effects of their spatial distribution on relaxation rates R2*, R2 and R1. Two different incubation procedures were performed, where (1) Jurkat cells were either incubated with the contrast agent after induction of apoptosis or (2) Jurkat cells were simultaneously incubated with the apoptotic stimulus and the contrast agent. Transmission electron microscopy images and relaxation rates showed that the first incubation strategy mainly resulted in binding of the annexin A5-iron oxide particles to the cell membrane, whereas the second procedure allowed extensive membrane-association as well as a small amount of internalization. Owing to the small extent of internalization, only minor differences were observed between the DeltaR2*/DeltaR2 and DeltaR2/DeltaR1 ratios of cell pellets with membrane-associated or internalized annexin A5 particles. Only the increase in R1 (DeltaR1) appeared to be diminished by the internalization. Internalization of annexin A5-iron oxide particles is also expected to occur in vivo, where the apoptotic stimulus and the contrast agent are simultaneously present. Where the extent of internalization in vivo is similar to that observed in the present study, both T2- and T2*-weighted MR sequences are considered suitable for the detection of these particles in vivo.

Histologic evaluation of human posterior lamellar discs for femtosecond laser Descemet's stripping endothelial keratoplasty.

PURPOSE: To evaluate the histologic changes in corneal structure after femtosecond laser preparation of posterior lamellar discs, more specifically, the smoothness of the stromal bed and the accuracy of the predicted depth of the horizontal lamellar cut. MATERIALS AND METHODS: Nineteen human donor eyes unsuitable for transplantation were used. Femtosecond laser was used to prepare a horizontal lamellar cut in donor corneas at a depth of 400 microm. Transmission electron microscopy images were used to evaluate the changes in the corneal structure and to measure the damage zone. Scanning electron microscopy images were used to determine the relative depth of the horizontal lamellar cut, and the stromal bed was examined to determine the smoothness of the surface. RESULTS: Transmission electron microscopy images showed a mean damage zone of 6.8 +/- 3.1 microm, which consisted of irregularly oriented collagen fibrils and electron-dense granular material. The collagen lamellae, both anteriorly and posteriorly of the damaged zone, showed a regular parallel configuration. The relative depth of the horizontal lamellar cut as percentage of the total corneal thickness in the center and periphery was 70.4% +/- 4.5% and 55.6% +/- 5.9%. Scanning electron microscopy images of the stromal bed showed a relatively smooth surface. CONCLUSION: The femtosecond laser is effective to prepare a deep horizontal lamellar cut in a standardized method. The stromal bed is smooth and without extensive adjacent tissue damage. The is thinner in the center and thicker at the edges, which may produce a mild hyperopic shift after femtosecond laser-assisted Descemet's stripping endothelial keratoplasty.

Electron tomography shows molecular anchoring within a layer-by-layer film.

The layer-by-layer self-assembly of thin films consisting of alternating layers of DNA and bis-urea nanoribbons prevents diffusion of the components within the film and allows the anchoring of biotinylated molecules through molecular recognition in a predetermined layer of the film. Electron tomography demonstrates with nanometer precision the location of gold-labeled streptavidin bound to the incorporated biotinylated molecules.

Functionalized-quantum-dot-liposome hybrids as multimodal nanoparticles for cancer.

Functionalized-quantum-dot-liposome (f-QD-L) hybrid nanoparticles are engineered by encapsulating poly(ethylene glycol)-coated QD in the internal aqueous phase of different lipid bilayer vesicles. f-QD-L maintain the QD fluorescence characteristics as confirmed by fluorescence spectroscopy, agarose gel electrophoresis, and confocal laser scanning microscopy. Cationic f-QD-L hybrids lead to dramatic improvements in cellular binding and internalization in tumor-cell monolayer cultures. Deeper penetration into three-dimensional multicellular spheroids is obtained for f-QD-L by modifying the lipid bilayer characteristics of the hybrid system. f-QD-L are injected intratumorally into solid tumor models leading to extensive fluorescent staining of tumor cells compared to injections of the f-QD alone. f-QD-L hybrid nanoparticles constitute a versatile tool for very efficient labeling of cells ex vivo and in vivo, particularly when long-term imaging and tracking of cells is sought. Moreover, f-QD-L offer many opportunities for the development of combinatory therapeutic and imaging (theranostic) modalities by incorporating both drug molecules and QD within the different compartments of a single vesicle.

The development of a glove-box/Vitrobot combination: air-water interface events visualized by cryo-TEM.

Aqueous interfaces are of paramount importance in the study of biological systems as well as in the biomedical sciences. To study these interfaces at the nanometer level it is of interest to develop methods that allow their observation with cryogenic transmission electron microscopy (cryo-TEM). Prevention of dehydration to preserve the "native" state during sample preparation prior to vitrification is often one of the most important parameters to control in cryo-TEM experiments. For the preparation of these types of samples, we felt the need for an extended workspace with temperature and humidity control; a 'glove-box' that seamlessly connects to the vitrification instrument, the Vitrobot. In this paper we describe the use of the glove-box in the 2D and 3D cryo-TEM study of DNA adsorption and calcium carbonate mineralization to Langmuir films. The data presented illustrates the necessity of a humidity-controlled environment to preserve the original "native" state of the monolayer system.

A quasi-time-resolved CryoTEM study of the nucleation of CaCO3 under langmuir monolayers.

Calcium carbonate biomineralization uses complex assemblies of macromolecules that control the nucleation, growth, and positioning of the mineral with great detail. To investigate the mechanisms involved in these processes, for many years Langmuir monolayers have been used as model systems. Here, we descibe the use of cryogenic transmission electron microscopy in combination with selected area electron diffraction as a quasi-time-resolved technique to study the very early stages of this process. In this way, we assess the evolution of morphology, polymorphic type, and crystallographic orientation of the calcium carbonate formed. For this, we used a self-assembled Langmuir monolayer of a valine-based bisureido surfactant (1) spread on a CaCl2-containing subphase and deposited on a holey carbon TEM grid. In a controlled environment, the grid is exposed to an atmosphere containing NH3 and CO2 (the (NH4)2CO3 diffusion method) for precisely determined periods of time (reaction times 30-1800 s) before it was plunged into melting ethane. This procedure allows us to observe amorphous calcium carbonate (ACC) particles growing from a few tens of nanometers to hundreds of nanometers and then crystallizing to form [00.1] oriented vaterite. The vaterite in turn transforms to yield [10.0] oriented calcite. We also performed the reaction in the absence of monolayer or in the presence of a nondirective monolayer of surfactant containing an oligo(ethylene oxide) 2 head group. Both experiments also showed the formation of a transient amorphous phase followed by a direct conversion into randomly oriented calcite crystals. These results imply the specific though temporary stabilization of the (00.1) vaterite by the monolayer. However, experiments performed at higher CaCl2 concentrations show the direct conversion of ACC into [10.0] oriented calcite. Moreover, prolonged exposure to the electron beam shows that this transformation can take place as a topotactic process. The formation of the (100) calcite as final product under different conditions shows that the surfactant is very effective in directing the formation of this crystal plane. In addition, we present evidence that more than one type of ACC is involved in the processes described.

Lipid-quantum dot bilayer vesicles enhance tumor cell uptake and retention in vitro and in vivo.

We report the construction of lipid-quantum dot (L-QD) bilayer vesicles by incorporation of the smallest (2 nm core size) commercially available CdSe/ZnS QD within zwitterionic dioleoylphosphatidylcholine and cationic 1,2-dioleoyl-3-trimethylammonium-propane lipid bilayers, self-assembling into small unilamellar vesicles. The incorporation of QD in the acyl environment of the lipid bilayer led to significant enhancement of their optical stability during storage and exposure to UV irradiation compared to that of QD alone in toluene. Moreover, structural characterization of L-QD hybrid bilayer vesicles using cryogenic electron microscopy revealed that the incorporation of QD takes place by hydrophobic self-association within the biomembranes. The L-QD vesicles bound and internalized in human epithelial lung cells (A549), and confocal laser scanning microscopy studies indicated that the L-QD were able to intracellularly traffick inside the cells. Moreover, cationic L-QD vesicles were injected in vivo intratumorally, leading to enhanced retention within human cervical carcinoma (C33a) xenografts. The hybrid L-QD bilayer vesicles presented here are thought to constitute a novel delivery system that offers the potential for transport of combinatory therapeutic and diagnostic modalities to cancer cells in vitro and in vivo.

Optical and magnetic resonance imaging of cell death and platelet activation using annexin a5-functionalized quantum dots.

A quantum-dot-based nanoparticle is presented, allowing visualization of cell death and activated platelets with fluorescence imaging and MRI. The particle exhibits intense fluorescence and a large MR relaxivity (r1) of 3000-4500 mM-1 s-1 per nanoparticle due to a newly designed construct increasing the gadolinium-DTPA load. The nanoparticle is suitable for both anatomic and subcellular imaging of structures in the vessel wall and is a promising bimodal contrast agent for future in vivo imaging studies.