Relaxivity of liposomal paramagnetic MRI contrast agents.

Paramagnetic liposomes, spherical particles formed by a lipid bilayer, are able to accommodate a high payload of Gd-containing lipid and therefore can serve as a highly potent magnetic resonance imaging contrast agent. In this paper the relaxation properties of paramagnetic liposomes were studied as a function of composition, temperature and magnetic field strength. The pegylated liposomes with a diameter of approximately 100 nm were designed for favorable pharmacokinetic properties in vivo. The proton relaxivity, i.e. the T1 relaxation rate per mmol of Gd(III) ions, of liposomes with unsaturated DOPC phospholipids was higher than those with saturated DSPC lipids. Addition of cholesterol was essential to obtain monodisperse liposomes and led to a further, although smaller, increase of the relaxivity. Nuclear magnetic relaxation dispersion measurements showed that the relaxivity was limited by water exchange. These results show that these paramagnetic liposomes are very effective contrast agents, making them excellent candidates for many applications in magnetic resonance imaging.

Controlled heterocoagulation of platelets and spheres.

We report the controlled heterocoagulation of platelets and spheres, leading to the formation of colloidally stable, anisotropic hybrid particles. Anionically charged, nanosized polymer latex spherical particles were heterocoagulated on the surface of cationically charged hexagonal gibbsite platelets via the adsorption of a single layer of spheres onto both sides of the hexagonal platelets. The latex particles were annealed at a temperature above the Tg of the latex polymer, resulting in a thin polymer layer covering the gibbsite platelets. This heterocoagulation approach enabled the encapsulation of hydrophilic inorganic particles with polymer latexes and the formation of anisotropic hybrid particles.

Improved glucose homeostasis in mice overexpressing human UCP3: a role for AMP-kinase?

OBJECTIVE: An unexplained phenotype of mice overexpressing human UCP3 is their improved glucose homeostasis. Since overexpression of UCP3 might affect the energy charge of the cell, we investigated whether these mice have an increased AMP-activated protein kinase (AMPK) activity. METHODS: Mitochondrial localisation of UCP3 was determined by immunoelectronmicroscopy and AMPK activity was measured in medial gastrocnemius of control mice and mice overexpressing human UCP3. RESULTS: Mice overexpressing human UCP3 had 5.8 fold higher levels of UCP3 protein, for which mitochondrial localisation was confirmed by immunoelectronmicroscopy. The ATP/AMP ratio was significantly lower in mice over-expressing UCP3 compared to the wild-type (10.9+/-1.6 vs 20.4+/-1.9 AU, P=0.03). Over-expression of UCP3 resulted in increased AMPK alpha1 activity (1.23+/-0.05 vs 1.00+/-0.06 normalized values, P=0.004) and a tendency towards increased AMPK alpha2 activity (1.18+/-0.08 vs 1.00+/-0.10 normalized values, P=0.08). CONCLUSION: Increased AMPK activity provides a plausible explanation for the improved glucose tolerance characteristic for these mice.

The observation of intact hepatic endothelial cells by cryo-electron microscopy.

Liver sinusoidal endothelial cells (LSECs) can optimally be imaged by whole mount transmission electron microscopy (TEM). However, TEM allows only investigation of vacuum-resistant specimens and this usually implies the study of chemically fixed and dried specimens. Cryo-electron microscopy (cryo-EM) can be used as a good alternative for imaging samples as whole mounts. Cryo-EM offers the opportunity to study intact, living cells while avoiding fixation, dehydration and drying, at the same time preserving all solubles and water as vitrified ice. Therefore, we compared the different results obtained when LSECs were vitrified using different vitrification conditions. We collected evidence that manual blotting at ambient conditions and vitrification by the guided drop method results in the production of artefacts in LSECs, such as the loss of fenestrae, formation of gaps and lack of structural details in the cytoplasm. We attribute these artefacts to temperature and osmotic effects during sample preparation just prior to vitrification. By contrast, by using an environmentally controlled glove box and a vitrification robot (37 degrees C and 100% relative humidity), these specific structural artefacts were nearly absent, illustrating the importance of controlled sample preparation. Moreover, data on glutaraldehyde-fixed cells and obtained by using different vitrification methods suggested that chemical prefixation is not essential when vitrification is performed under controlled conditions. Conditioned vitrification therefore equals chemical fixation in preserving and imaging cellular fine structure. Unfixed, vitrified LSECs show fenestrae and fenestrae-associated cytoskeleton rings, indicating that these structures are not artefacts resulting from chemical fixation.

Accumulation of ultrasmall superparamagnetic particles of iron oxide in human atherosclerotic plaques can be detected by in vivo magnetic resonance imaging.

BACKGROUND: One of the features of high-risk atherosclerotic plaques is a preponderance of macrophages. Experimental studies with hyperlipidemic rabbits have shown that ultrasmall superparamagnetic particles of iron oxide (USPIOs) accumulate in plaques with a high macrophage content and that this induces magnetic resonance (MR) signal changes. The purpose of our study was to investigate whether USPIO-enhanced MRI can also be used for in vivo detection of macrophages in human plaques. METHODS AND RESULTS: MRI was performed on 11 symptomatic patients scheduled for carotid endarterectomy before and 24 (n=11) and 72 (n=5) hours after administration of USPIOs (Sinerem) at a dose of 2.6 mg Fe/kg. Histological and electron microscopical analyses of the plaques showed USPIOs primarily in macrophages within the plaques in 10 of 11 patients. Histological analysis showed USPIOs in 27 of 36 (75%) of the ruptured and rupture-prone lesions and 1 of 14 (7%) of the stable lesions. Of the patients with USPIO uptake, signal changes in the post-USPIO MRI were observed by 2 observers in the vessel wall in 67 of 123 (54%) and 19 of 55 (35%) quadrants of the T2*-weighted MR images acquired after 24 and 72 hours, respectively. For those quadrants with changes, there was a significant signal decrease of 24% (95% CI, 33% to 15%) in regions of interest in the images acquired after 24 hours, whereas no significant signal change was found after 72 hours. CONCLUSIONS: Accumulation of USPIOs in macrophages in predominantly ruptured and rupture-prone human atherosclerotic lesions caused signal decreases in the in vivo MR images.

Direct observation of dipolar chains in iron ferrofluids by cryogenic electron microscopy.

A key issue in research on ferrofluids (dispersions of magnetic colloids) is the effect of dipolar interactions on their structure and phase behaviour, which is not only important for practical applications but gives fundamental insight in dipolar fluids in general. In 1970, de Gennes and Pincus predicted a Van der Waals-like phase diagram and the presence of linear chains of particles in ferrofluids in zero magnetic field. Despite many experimental studies, no direct evidence of the existence of linear chains of dipoles has been reported in the absence of magnetic field, although simulations clearly show the presence of chain-like structures. Here, we show in situ linear dipolar structures in ferrofluids in zero field, visualized on the particle level by electron cryo-microscopy on thin, vitrified films of organic dispersions of monodisperse metallic iron particles. On systematically increasing the particle size, we find an abrupt transition from separate particles to randomly oriented linear aggregates and branched chains or networks. When vitrified in a permanent magnetic field, these chains align and form thick elongated structures, indicating lateral attraction between parallel dipole chains. These findings show that the experimental model used is well suited to study the structural properties of dipolar particle systems.

Liposome-mediated DNA vaccination: the effect of vesicle composition.

Liposome-entrapped DNA has been shown to enhance the potency of DNA vaccines, possibly by facilitating uptake of the plasmid by antigen-presenting cells (APC). In this paper, we have investigated the influence of the liposomal composition and surface charge on such potency. Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen was entrapped within cationic liposomes of various compositions and surface charges with high efficiency (88-97% of the amount used) by the dehydration-rehydration method that generates dehydration-rehydration vesicles (DRV). Cryo-electron microscopy revealed that DNA-containing DRV (DRV(DNA)) were multilamellar. In immunisation studies, female Balb/c mice were given two to four intramuscular injections of 10 microg naked or liposome-entrapped pRc/CMV HBS and bled at time intervals. Results indicate that the lipid composition of the DRV(DNA) influences the strength of the humoural response (immunoglobulin (Ig)G subclasses) with inclusion of dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylethanolamine (PE) in the liposomal structure contributing to greater responses. DRV(DNA) in which the DOPE or PE were omitted or substituted with cholesterol led to significant reduction of humoural responses against the encoded antigen. Replacing phosphatidylcholine (PC) in the DRV(DNA) with the high-melting distearoyl phosphatidylcholine also contributed to lower responses. In other experiments, IgG responses were monitored in mice immunised with pRc/CMV HBS entrapped in DRV composed of PC and DOPE as before but incorporating increasing amounts of DOTAP (1-16 micromol). Maximal IgG responses were observed at 10 weeks after the first of four injections and suggested a trend of higher responses when 4 or 8 micromol DOTAP was present in the DRV(DNA) formulation. Cell-mediated immunity (measured in terms of endogenous antigen-specific splenic interferon-gamma) in mice immunised with pRc/CMV HBS entrapped in liposomes composed of PC, DOPE and DOTAP (16:8:4 molar ratio) was much greater than in animals treated with naked plasmid. These results indicate that liposome-mediated DNA immunisation is more effective than the use of naked DNA, and also suggest that the presence of fusogenic phosphatidylethanolamine in DRV in conjunction with a low-melting phosphatidylcholine and an appropriate content of cationic lipid might contribute to more effective liposomal DNA vaccines. The notion that liposomes improve immune responses to the plasmid-encoded vaccine by facilitating the latter's uptake by APC was supported by the observation that in Balb/c mice injected intramuscularly with liposome-entrapped pCMV. Enhanced green fluorescent protein, expression of the gene in terms of fluorescence intensity in the draining lymph nodes, was much greater than in animals treated with the naked plasmid.

Spontaneous vesiculation.

The thermodynamics of vesicle formation was analyzed by using the elastic bending energy approach. Several different possibilities of spontaneous vesiculation, due to soft bilayers, non-zero spontaneous curvature and Gaussian curvature, respectively, were presented and discussed. Intermediate structures in the closed vesicle-disklike mixed micelle phase transition could be either cup-like particles or open bilayers partially rolled into lipid tubules.

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer.

Annexin V belongs to the family of calcium-dependent phospholipid binding proteins and binds almost solely to phosphatidylserine (PS). When annexin V is used to detect loss of membrane asymmetry in cellular systems, the binding properties under physiological conditions are of importance. Most biochemical studies use optimized binding conditions, conditions that are often far from physiological. For the interpretation of flow cytometric studies that use fluorescent annexin V to probe PS exposure, it is important to know what mixture of lipid species exposed in the outer leaflet of a membrane can evoke a positive annexin V signal. The lipid species is important in this respect as well as the concentration that just evokes a positive signal (detection level). Furthermore, the influence of the composition of the lipid matrix (cholesterol content, other phospholipid species) was investigated, as well as the influence of the calcium concentration on annexin V binding. In this study, we report on the binding of annexin V to phospholipid bilayers (adsorbed to glass beads) as measured by flow cytometry at physiological conditions. Annexin V binding was found to increase rapidly, with increasing PS concentrations up to a certain level (attained at 6 mol% PS). Further increase of the PS concentration resulted only in a slight increase of annexin V binding. Calcium concentrations below 3 mM were found to reduce the sensitivity of the binding assay. Phosphatidylethanolamine incorporated in the phospholipid bilayer resulted in a lower threshold for the binding assay, whereas sphingomyelin had no influence on the binding of annexin V and cholesterol reduces binding of annexin V to lipid bilayers. These data may help in the interpretation of results obtained from binding of annexin V to cell membranes (e.g., involved in apoptosis).

Surface exposure of phosphatidylserine during apoptosis of rat thymocytes precedes nuclear changes.

Cell surface exposure of phosphatidylserine (PS) during apoptosis serves recognition and removal of the dying cell by phagocytes. Loss of phospholipid asymmetry and PS exposure is investigated by immunocytochemistry and related to morphological changes. Loss of membrane asymmetry was determined on dexamethasone-treated rat thymocytes using the PS specific probe annexin V. Thymocytes incubated in the presence of dexamethasone were studied in time series during the execution of the apoptotic program. Thymocytes first start to expose PS at their cell surface. At this initial stage the barrier function of the plasma membrane remains intact. At a later stage the plasma membrane becomes leaky for compounds like propidium iodide and subsequently the cell disintegrates into apoptotic bodies. Microscopical evaluation of dexamethasone-treated thymocytes showed that the cells with an apoptotic morphology all bound annexin V. The cells with a normal viable morphology lacked annexin V binding except for those cells that started to shed small vesicles. These vesicles were positive for annexin V, indicating a local disturbance of the phospholipid asymmetry. The local exposure of PS is considered to be a very early event of apoptosis, preceding the full sequence of morphological changes at the ultrastructural level.

Morphological observations on liposomes bearing covalently bound protein: studies with freeze-fracture and cryo electron microscopy and small angle X-ray scattering techniques.

The appearance of protein bound to the surface of intact and microfluidized liposomes and its possible influence on their morphology was examined by freeze-fracture electron microscopy, cryo electron microscopy and small angle X-ray scattering (SAXS) techniques. Results obtained by the two microscopy techniques were in agreement with one another in terms of vesicle size and localization of protein (tetanus toxoid or immunoglobulin G) on the surface of vesicles. Surface-bound protein was observed as particles (10-12 nm diameter) by freeze-fracture electron microscopy and was confirmed by immunogold cryo microscopy. SAXS was shown to be a suitable means to further characterize liposomes with, or without bound protein.

Reevaluation of the effect of lysoyzme on Escherichia coli employing ultrarapid freezing followed by cryoelectronmicroscopy or freeze substitution.

Lysozyme is able to lyse Gram-positive bacteria acting as muramidase on the peptidoglycan polymer. Gram-negative bacteria in vitro are not lysed by lysozyme. It was assumed that the peptido-glycan is protected by the outer membrane and thus that Gram-negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane. Accidentally, Pellegrini et al. [(1992) J. Appl. Bacteriol., 72:180-187] found that lysozyme per se is able to kill some Gram-negative bacteria. On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram-negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down. In an attempt to clarify the action of lysozyme on E. coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling. Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact. Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds. However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium. After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent. Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane. The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration. Disintegration is accomplished by changes of the environment starting at the cell membranes. The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified.

Improved DNA: liposome complexes for increased systemic delivery and gene expression.

To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.

Pixe microanalysis of trace elements of normoxic, ischemic and reperfused rat hearts.

With a 3 x 3 mu m(2) proton microbeam spatial distributions of Na, Mg, P, S, K, Ca and Fe were measured via PIXE in 50 x 50 mu m(2) areas of rat heart, sliced into 10-15 mu m thick cryosections. The isolated rat hearts were subjected to normal perfusion, ischemia and reperfusion. Substantial changes in the elemental distribution were found in tissue after 40 min. of reperfusion, particularly indicated by locally elevated Ca and decreased K levels. Electron microscopic examination was used for assessment of artefacts due to sample preparation and handling. Results of stained cryosections analyzed via STIM demonstrated that this latter technique can be employed prior to PIXE analysis to localize individual cells in freeze-dried cryosections.

Transmembrane gradient driven phase transitions within vesicles: lessons for drug delivery.

Phase transitions in closed vesicles, i.e., microenvironments defined by the size of the vesicle, its contents, and permeability of its membrane are becoming increasingly important in several scientific disciplines including catalysis, growth of small crystals, cell function studies, and drug delivery. The membrane composed from lipid bilayer is in general impermeable to ions and larger hydrophilic ions. Ion transport can be regulated by ionophores while permeation of neutral and weakly hydrophobic molecules can be controlled by concentration gradients. Some weak acids or bases, however, can be transported through the membrane due to various gradients, such as electrical, ionic (pH) or specific salt (chemical potential) gradients. Upon permeation of appropriate species and reaction with the encapsulated species precipitation may occur in the vesicle interior. Alternatively, these molecules can also associate with the leaflets of the bilayer according to the transmembrane potential. Efficient liposomal therapeutics require high drug to lipid ratios and drug molecules should have, especially when associated with long circulating liposomes, low leakage rates. In this article we present very efficient encapsulation of two drugs via their intraliposomal precipitation, characterize the state of encapsulated drug within the liposome and try to fit the experimental data with a recently developed theoretical model. Nice agreement between a model which is based on chemical potential equilibration of membrane permeable species with experimental data was observed. The high loading efficiencies, however are only necessary but not sufficient condition for effective therapies. If adequate drug retention within liposomes, especially in the case of long-circulating ones, is not achieved, the therapeutic index decreases substantially. Anticancer drug doxorubicin precipitates in the liposome interior in a form of gel with low solubility product and practically does not leak out in blood circulation in the scale of days. With an antibiotic, ciprofloxacin, the high loading efficacy and test tube stability is not reproduced in in vitro plasma leakage assays and in vivo. We believe that the reasons are higher solubility product of precipitated drug in the liposome, larger fraction of neutral molecules due closer pK values of the drug with the pH conditions in the solutions and high membrane permeability of this molecule. High resolution cryoEM shows that encapsulated anticancer agent doxorubicin is precipitated in the form of bundles of parallel fibers while antibiotic ciprofloxacin shows globular precipitate. Doxorubicin gelatin also causes the change of vesicle shape.

Ultrastructural detection of surface exposed phosphatidylserine on activated blood platelets.

Phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane of cells (including blood platelets). Upon cell activation PS may become exposed to the outer surface of the cell. Cell membranes with surface exposed PS at the outside form a catalytic surface for coagulation reactions. When platelets are activated with ionophore or with thrombin in combination with thapsigargin, calcium induced scrambling of phospholipids takes place, resulting in PS exposure. Concomitant with PS exposition structural changes take place. On resting and activated platelets we combined the immunocytochemical detection of surface exposed PS with (ultra)structural information. Blood platelets were activated in the presence of annexin V, a protein which binds to PS in the presence of Ca2+. Annexin V was found to bind to lipid bilayers containing more than 5 mole % PS as estimated by binding of fluorescent-labelled annexin V to liposomes with varying PS concentrations. After vitrification, freeze-substitution and embedding of the platelets, annexin V was located on ultra thin sections, as detected by an anti-annexin V antibody and gold labelled protein A. Upon activation, the platelets show two different forms; irregular platelets with unchanged cytoplasm and round cells with apparently diluted cytoplasm. Activation with ionophore initially resulted in both forms, but after ten minutes only round platelets with diluted cytoplasm were observed. Both forms of these platelets as well as the microvesicles were found to be annexin V positive. However upon activation with thrombin in combination with thapsigargin, only the round cells with diluted cytoplasm and microvesicles were annexin V positive, whereas platelets with unchanged cytoplasm, even when microvesicles are present, are negative for annexin V.

CMV induced vascular injury: an electron-microscopic study in the rat.

There is evidence in support of a viral etiology for atherosclerosis. In our study, we demonstrated that cytomegalovirus (CMV) infection in rats caused morphological alterations of the endothelium and subendothelial space of the large vessels and that these alterations are similar to those which are induced by hypercholesterolemia. These alterations consisted of swollen endothelial cells with a surface showing bleb and microvilli formation. In addition, adhesion of leukocytes to the endothelium and the presence of leukocytes (lymphocytes and macrophages) in the subendothelium were found. Lipid accumulation occurred in the endothelium and in the subendothelial space, especially in hypercholesterolemic animals. This lipid accumulation in the subendothelial space consisted of extracellular lipid deposition and of subendothelial located "foam cells". A characteristic phenomenon of the effect of CMV infection of rats was a loosening of the endothelial cells from the basement membrane. The space between the basement membrane and endothelium was expanded and was filed with an increased amount of reticular basal lamina-like material. These observations show that CMV infection is associated with a non-deniding aortic endothelial injury which is consistent with the early events in the process of atherosclerosis.

Aggregation of phospholipid vesicles by a chimeric protein with the N-terminus of annexin I and the core of annexin V.

A chimeric protein was produced with the N-terminal domain (amino acids 1-45) of annexin I and the core of annexin V (amino acids 19-320). This protein, annexin IN-VC, has a similar Ca2+ requirement for binding to phospholipid bilayers of 20% phosphatidylserine (PS)/80% phosphatidylcholine (PC) as annexin V. In contrast to annexin V, this protein has a strong potency to aggregate phospholipid vesicles as is shown by turbidimetric measurements and cryo-electron microscopy. Ellipsometry was employed to study quantitatively the phenomenon of phospholipid vesicle adhesion to annexin IN-VC bound to a planar phospholipid bilayer. The amount of phospholipid vesicles bound by annexin IN-VC on the planar bilayer is proportional to its surface coverage and can be inhibited by coadsorption of annexin V on the planar bilayer or by shielding the phospholipid surface of the vesicles with blood coagulation factor Va. Annexin IN-VC, like annexin V, does not bind to pure PC bilayers, but its adsorption on anionic phospholipid bilayers brings about the capacity to bind pure PC vesicles. This suggests that annexin IN-VC generates or exposes after binding to anionic phospholipids another phospholipid binding site, that differs from the annexin V phospholipid binding site. Collectively, the data suggest that two-dimensional cluster formation of annexin IN-VC on a bilayer with anionic phospholipids is involved in vesicle adherence.