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.

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.

Nanocapsules: lipid-coated aggregates of cisplatin with high cytotoxicity.

Cisplatin is one of the most widely used agents in the treatment of solid tumors, but its clinical utility is limited by toxicity. The development of less toxic, liposomal formulations of cisplatin has been hampered by the low water solubility and low lipophilicity of cisplatin, resulting in very low encapsulation efficiencies. We describe a novel method allowing the efficient encapsulation of cisplatin in a lipid formulation; it is based on repeated freezing and thawing of a concentrated solution of cisplatin in the presence of negatively charged phospholipids. The method is unique in that it generates nanocapsules, which are small aggregates of cisplatin covered by a single lipid bilayer. The nanocapsules have an unprecedented drug-to-lipid ratio and an in vitro cytotoxicity up to 1000-fold higher than the free drug. Analysis of the mechanism of nanocapsule formation suggests that the method may be generalized to other drugs showing low water solubility and lipophilicity.

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.

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.

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.

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.

Recurrent intraocular inflammation after implantation of the Artiflex phakic intraocular lens for the correction of high myopia.

We describe a 54-year old man who developed a severe cell deposition 1 week after implantation of a foldable Artiflex phakic intraocular lens (pIOL) (Ophtec B.V.) with a silicone optic and poly(methyl methacrylate) (PMMA) haptics in the left eye for the correction of high myopia. Nine months after implantation, many cell deposits remained visible on the posterior surface of the IOL, causing severe glare, especially during daylight conditions. We explanted the Artiflex pIOL and exchanged it for a PMMA Artisan pIOL. One month after the exchange, the uncorrected visual acuity was 20/20 and the patient's glare complaints had disappeared. Slitlamp examination showed no signs of inflammation in the anterior segment or cell deposits on the PMMA Artisan pIOL. Scanning electron microscopy demonstrated multiple cell deposits on the explanted Artiflex pIOL.

Different interactions of egg-yolk phosphatidylcholine and sphingomyelin with detergent bile salts.

To examine physical-chemical aspects of bile salt-phospholipid interactions that could contribute to preferential phosphatidylcholine (PC) secretion into bile, we have compared transitions between vesicles and micelles in model systems containing taurocholate (TC) and either egg-yolk PC (EYPC), egg-yolk sphingomyelin (EYSM), buttermilk SM (BMSM) or dipalmitoyl PC (DPPC). Phase transitions from micelles to vesicles were observed at 4-fold dilution of serially diluted EYPC/TC systems, but not earlier than at 16-fold dilution of SM/TC or DPPC/TC systems, indicating lower concentrations of the detergent required for micellization in the case of SM or DPPC. Cryo-transmission electron microscopy of phase transitions initiated by addition of TC to phospholipid vesicles revealed extremely long SM-containing intermediate structures, but shorter EYPC-containing intermediate structures. Again, larger amounts of bile salt were required to induce phase transitions in the case of EYPC compared to SM. Sizes of TC-phospholipid micelles increased progressively upon increasing phospholipid contents in the rank order: DPPC-TC

Cryoelectron microscopy of liposomes.

A thin aqueous film of suspended lipid vesicles?micelles is the object of choice for vitrification and subsequent study by cryoelectron microscopy. Just prior to vitrification, a thin film (compare with a soap film) is vulnerable to heat and mass exchange. Preparation of thin films in a temperature- and humidity-controlled environment is essential to prevent osmotic and temperature-induced alterations of the lipid structure, as will be explained in this chapter. Further automation of the preparative procedure by automatic blotting and PC control over the timing of critical steps (including vitrification) may further assist in the reproducible throughput of high-quality specimens. By cryotomography, taking a tilt series under low-dose conditions, a three-dimensional reconstruction of the specimen can be analyzed.

Morphological control and molecular recognition by bis-urea hydrogen bonding in micelles of amphiphilic tri-block copolymers.

Hydrogen bonding between urea groups of amphiphilic tri-block copolymers considerably affects their self-assembly in water, which results in a strong modification of morphology and viscosity of aqueous solutions; the hydrogen bonding motif in these amphiphilic copolymers allows molecular recognition of small molecules with complementary hydrogen bonding units.

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.

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.

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.