Solid lipid nanoparticles and nanoemulsions with solid shell: Physical and thermal stability.

HYPOTHESIS: Nanoemulsions (NE) and solid lipid nanoparticles (SLN) used for drug delivery should have a solid shell to be stable during long shelf life and become liquid at human body temperature. The core components of lipid nanoparticles can be partially incorporated into the shell and affect the physical and thermal stability. EXPERIMENTS: We prepared NE and SLN by the phase inversion temperature (PIT) method. Solidification of the surfactants Tween60 and Span 60 on the surface of NE droplets with paraffin oil resulted in the formation of the solid shell. SLN contained stearic acid in the core and the same surfactants in the solid shell. The size, structure and stability of the NE and SLN were studied by DLS and cryo-TEM. Their crystallization and melting were analyzed using DSC. FINDINGS: The lipid nanoparticles were resistant to aggregation and sedimentation and hold up to at least two cycles of heating to 50-60 °C and subsequent cooling to 5 °C, even though the upper temperatures were higher than the melting point of the surfactant shell. The expected liquid core/solid shell morphology of NE was confirmed. SLN were composed of a semi-liquid core of supercooled stearic acid melt and coated with a solid surfactant shell, so they can be treated as NE. Stearic acid molecules penetrated the shell, leading to an increase in its melting point.

Mother-to-child transmission of extended-spectrum-beta-lactamase-producing Enterobacteriaceae.

BACKGROUND: Preterm infants are at high risk for extended-spectrum-beta-lactamase-producing Enterobacteriaceae (ESBL-E) sepsis and neonatal intensive care unit (NICU) outbreaks. Maternal colonization with ESBL-E may be precursory to mother-to-child transmission. However, there is no consensus regarding surveillance of pregnant women for ESBL-E colonization. AIM: To identify pairs of mothers and infants harbouring same-strain ESBL-E colonization and to determine whether maternal transmission may play a role in increasing ESBL-E carriage in preterm infants. METHODS: This was a one-year analysis from an ongoing, prospective ESBL-E surveillance of mothers of premature infants and their offspring. Mother-infant pairs colonized with the same bacteria underwent strain analysis using pulsed-field gel electrophoresis (PFGE). Clinical parameters were collected from the hospital computerized records. FINDINGS: Between January 2015 and January 2016, 313/409 (76.5%) mothers and all 478 (100%) infants were screened for ESBL-E colonization; carriage rates were 21.5% and 14.8%, respectively. Four (5.6%) colonized infants developed late-onset sepsis and two (2.8%) died. Twenty-five mother-infant pairs colonized with the same bacterial strain were identified; a subgroup of 10 pairs of isolates underwent PFGE, and 70% displayed an identical PFGE fingerprint. No similarities were found between isolates recovered from unrelated neonates and mothers. ESBL-E colonization was found significantly earlier in infants of mothers colonized at birth (P<0.001) compared with infants of non-colonized mothers. CONCLUSIONS: ESBL-E carriage rates in mothers and NICU infants with non-negligible maternal-neonatal ESBL-E transmission in the study region indicate that maternal colonization surveillance and/or further infection control interventions should be considered.

Real-time genomic investigation underlying the public health response to a Shiga toxin-producing Escherichia coli O26:H11 outbreak in a nursery.

Shiga toxin-producing Escherichia coli (STEC) is a significant cause of gastrointestinal infection and the haemolytic-uremic syndrome (HUS). STEC outbreaks are commonly associated with food but animal contact is increasingly being implicated in its transmission. We report an outbreak of STEC affecting young infants at a nursery in a rural community (three HUS cases, one definite case, one probable case, three possible cases and five carriers, based on the combination of clinical, epidemiological and laboratory data) identified using culture-based and molecular techniques. The investigation identified repeated animal contact (animal farming and petting) as a likely source of STEC introduction followed by horizontal transmission. Whole genome sequencing (WGS) was used for real-time investigation of the incident and revealed a unique strain of STEC O26:H11 carrying stx2a and intimin. Following a public health intervention, no additional cases have occurred. This is the first STEC outbreak reported from Israel. WGS proved as a useful tool for rapid laboratory characterization and typing of the outbreak strain and informed the public health response at an early stage of this unusual outbreak.

Poly(glycoamidoamine) brush nanomaterials for systemic siRNA delivery in vivo.

Delivery is the key challenge for siRNA based therapeutics. Here, we report the development of new poly(glycoamidoamine) brush nanomaterials for efficient siRNA delivery. GluN4C10 polymer brush nanoparticles, a lead material, demonstrated significantly improved delivery efficiency for siRNA against factor VII (FVII) in mice compared to poly(glycoamidoamine) brush nanomaterials reported previously.

Viscoelastic micellar water/CTAB/NaNO(3) solutions: rheology, SANS and cryo-TEM analysis.

Aqueous micellar solutions of the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) and sodium nitrate (NaNO(3)) were examined using steady and dynamic rheology, small-angle neutron scattering (SANS) and cryogenic-transmission electron microscopy (cryo-TEM). Upon addition of NaNO(3), the CTAB spherical micelles transform into long, flexible wormlike micelles, conveying viscoelastic properties to the solutions. The zero-shear viscosity (eta(0)) versus NaNO(3) concentration curve exhibits a well-defined maximum. Likewise, upon increase in temperature, the viscosity decreases. Dynamic rheological data of the entangled micellar solutions can be well described by the Maxwell model. Changes in the structural parameters of the micelles with addition of NaNO(3) were inferred from SANS measurements. The intensity of scattered neutrons at the low q region was found to increase with increasing NaNO(3) concentration. This suggests an increase in size of the micelles and/or decrease of intermicellar interactions with increasing salt concentration. Analysis of the SANS data using prolate ellipsoidal structure and Yukawa form of interaction potential between micelles indicates that addition of NaNO(3) leads to a decrease in the surface charge of the ellipsoidal micelles and consequently an increase in their length. The structural transition from spherical to entangled threadlike micelles, induced by the addition of NaNO(3) to CTAB micelles is further confirmed by cryo-TEM.

Inhibition of cholesterol transport into skin cells in cultures by phytosterol-loaded microemulsion.

Cholesterol and plant phytosterols are lipophilic compounds solubilized by intestinal micelles in a competitive manner. In this work, we used radioactive cholesterol- and phytosterol-loaded oil-in-water microemulsions to follow their incorporation and mutual competition in HaCaT keratinocytes, SZ95 sebocytes, and skin pieces in cultures. Dynamic light scattering showed homogenous nanostructures of 10.5+/-1.5 nm diameter and cryo-transmission electron microscopy confirmed the presence of uniform spherical droplets of 7.0+/-1.0 nm diameter. Up to 320 nmol/ml of cholesterol can be solubilized and transported into cells with minimal toxic effect by 0.5 wt% nanodroplets in a cell medium. Phytosterols inhibit incorporation of cholesterol into cells, in vitro, at molar ratios (phytosterols/cholesterol) of 4 and above. The loaded nanodroplets accumulate in intracellular vesicles (presumably endosomes). No metabolic conversion of cholesterol or phytosterols was found in these cells, in vitro, after 24 h, at 37 degrees C.

Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity.

Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic lipid-DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride, N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl)ammonium bromide) and polycationic (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate) lipid-based assemblies, with or without neutral lipid (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, cholesterol) were used to prepare lipoplexes of different L(+)/DNA(-) charge ratios. Circular dichroism, cryogenic-transmission electron microscopy, and static light scattering were used for lipoplex characterization, whereas expression of human growth hormone or green fluorescent protein was used to quantify transfection efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, cholesterol) induced appearance of Psi(-) DNA, a chiral tertiary DNA structure. The formation of Psi(-) DNA was also dependent on cationic lipid-DNA charge ratio. On the other hand, monocationic lipids either alone or with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine as helper lipid, or polycationic 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate-based assemblies, neither of which promotes a lipid-DNA hexagonal phase, did not induce the formation of Psi(-) DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of Psi(-) DNA structure, correlate with optimal transfection.

Dynamin family of mechanoenzymes.

The dynamin family of proteins is continually growing, and in recent years members have been localized to areas of mitochondrial fission, plant phragmoplasts and chloroplasts, and viral ribonucleoprotein complexes. All the dynamin-like proteins examined to-date appear to assemble into oligomers, such as rings or spirals; however, it remains to be determined if a global mechanism of action exists. Even the role of dynamin in vesicle formation remains controversial as to whether it behaves as a molecular switch or as a mechanochemical enzyme.

Formation of complement-activating particles in aqueous solutions of Taxol: possible role in hypersensitivity reactions.

We reported earlier that the anticancer drug paclitaxel (Taxol) activated the complement (C) system in human serum in vitro, raising the possibility that C activation might play a role in the ill-understood hypersensitivity reactions (HSRs) to this drug [J. Natl. Cancer Inst. 90 (1998) 300]. In pursuing the mechanism of C activation by Taxol, the present study provided evidence that dilution of the injection concentrate in aqueous solvents led to the formation of micelles and needle-like structures, both of which caused C activation in vitro. Micelles were formed mainly from Cremophor EL (CrEL), the nonionic emulsifier vehicle of paclitaxel, whose level in Taxol infusion exceeded its critical micelle concentration by at least 400-fold. CrEL micelles were shown by quasi-elastic light scattering and cryo-transmission electron microscopy (cryo-TEM) to be spherical with diameters in the 8-22 nm range; however, de novo formation of 50-300 nm microdroplets following incubation with human plasma suggested further fundamental structural transformation in blood. The needle-like structures extended to the multimicron range and were shown by electron diffraction to be crystalline paclitaxel. Taxol-induced C activation was manifested in varying rises of serum C3a-desarg, iC3b and SC5b-9. The causal role of CrEL micelles in C activation was demonstrated by the fact that filtration of aqueous solutions of Taxol or pure CrEL via 30-kDa cutoff filters eliminated, while the filter retentate restored C activation. C activation by Taxol was also inhibited by 10 mg/ml human immunoglobulin (IVIG). If proven clinically, HSRs to Taxol may represent a hitherto vaguely classified adverse drug reaction recently called C activation-related pseudoallergy (CARPA) [Circulation 99 (1999) 2302].

Microstructural evolution of lipid aggregates in nucleating model and human biles visualized by cryogenic transmission electron microscopy.

Obtaining reliable information on the physical state and ultrastructure of bile is difficult because of its mixed aqueous-lipid composition and thermodynamic metastability. We have used time-lapse cryogenic transmission electron microscopy (cryo-TEM) combined with video-enhanced light microscopy (VELM) to study microstructural evolution in nucleating bile. A well-characterized model bile and gallbladder biles from cholesterol and pigment gallstone patients were studied sequentially during cholesterol nucleation and precipitation. In model bile, cholesterol crystallization was preceded by the appearance of the following distinct microstructures: spheroidal micelles (3-5 nm), discoidal membrane patches (50-150 nm) often in multiple layers (2-10), discs (50-100 nm), and unilamellar (50-200 nm) and larger multilamellar vesicles (MLVs). The membrane patches and discs appeared to be short-lived intermediates in a micelle-to-vesicle transition. Vesicular structures formed by growth and closure of patches as well as by budding off from vesicles with fewer bilayers. MLVs became more abundant, uniform, and concentric as a function of time. In native bile, all the above microstructures, except discoidal membrane patches, were observed. However, native MLVs were more uniform and concentric from the beginning. When cholesterol crystals appeared by light microscopy, MLVs were always detected by cryo-TEM. Edges of early cholesterol crystals were lined up with micelles and MLVs in a way suggesting an active role in feeding crystal growth from these microstructures. These findings, for the first time documented by cryo-TEM in human bile, provide a microstructural framework that can serve as a basis for investigation of specific factors that influence biliary cholesterol nucleation and crystal formation.

Lyotropic Liquid Crystalline Phases from Symmetric Double-Tailed Surfactants: Sodium 1'-(6)-Undecylbenzenesulfonate, 1'-(7)-Tridecylbenzenesulfonate, and 1'-(8)- Pentadecylbenzenesulfonate in Water.

Phase diagrams of binary symmetric double-tailed alkylbenzenesulfonate surfactant/water systems, as well as the structure of the phases, were determined using crossed polars, polarized light microscopy, 2H NMR spectroscopy, Nomarski differential interference contrast optics, and cryo-transmission electron microscopy. The isotropic phase, the lamellar phase, and a high-viscosity solution representing an intermediate between the isotropic phase and the mesophase, which refers to the beginning of the formation of vesicles, were found. As may be expected, the isotropic phases at higher concentrations are formed mostly at higher temperatures. Isotropic regions are followed by regions characterized as the isotropic solutions containing vesicles; the optically isotropic phase contains a lamellar dispersion of vesicles in solution. The viscosity of these phases is found to be high near the borderline of the two-phase regions. At the same concentrations two-phase regions were found to contain the isotropic + lamellar phases at the higher temperature and the isotropic + crystalline phase at the lower temperature. Molecular-mechanical and semiempirical quantum-mechanical calculation searches for the global minimum in the conformational space of alkylbenzenesulfonate ions were conducted to examine the influence of monomer molecular structure on the micellar shape. Copyright 1998 Academic Press.

Aggregation and Microstructure in Aqueous Solutions of the Nonionic Surfactant C12E8

Mixtures of water and of the nonionic surfactant C12E8 (octaoxyethyleneglycol monododecylether) have been investigated by time-resolved fluorescence quenching (TRFQ) and transmission electron microscopy at cryogenic temperature (cryo-TEM) to obtain information on the size and shape of the surfactant aggregates and on the microstructure of the mixtures. The studies were performed at surfactant contents and temperatures where the micelles grew significantly and also where the systems undergo phase transitions from micellar-to-cubic upon increasing surfactant content or decreasing temperature and from cubic-to-hexagonal-to-micellar upon increasing temperature. No rapid change or discontinuity was observed in the variation of the aggregation number with the surfactant content or temperature upon crossing the micellar-to-cubic phase boundary nor at the approach of the hexagonal phase from the cubic phase. The results confirmed that the cubic phase consists of spheroidal micelles forming a three-dimensional array. The aggregation numbers at high surfactant content or temperature can be much larger than that of the minimum spherical micelle for a surfactant with a dodecyl chain, suggesting that the micelles should be anisotropic. However, cryo-TEM showed that in the micellar phase the micelles are always spheroidal. These apparently inconsistent results are explained in terms of a partial mixing of the surfactant dodecyl chains and octaoxyethylene head groups which allows for spheroidal micelles of aggregation number much larger than for a surfactant with a dodecyl chain. The results show extensive exchange of material at 40°C, taking place most likely via temporary merging of micelles in micelle clusters then present in the system.

Cryo-TEM and NMR studies of a micelle-forming phosphoglucolipid from membranes of Acholeplasma laidlawii A and B.

The chemical structure of a phosphoglucolipid from the membrane of the bacterium Acholeplasma laidlawii strain B-PG9 has been determined by high resolution NMR to be 1,2-diacyl-3-O-[glycerophosphoryl-6-O-(alpha-D-glucopyranosyl-(1 -->2)-O-alpha-D-glucopyranosyl)]-sn-glycerol (GPDGlcDAG). It was concluded that this lipid has exactly the same structure as one of the phosphoglucolipids from A. laidlawii strain A-EF22. By cryo transmission electron microscopy (cryo-TEM) and NMR diffusion techniques it was shown that, in highly diluted aqueous solutions, this membrane lipid forms long thread-like micelles in equilibrium with lipid vesicles. The cause of the occurrence of these different aggregates is discussed in terms of the varying molecular shapes of the lipid because of a heterogeneous composition of the acyl chains. A second membrane phosphoglucolipid from the bacterium, namely 1,2-diacyl-3-O-[glycerophosphoryl-6-O-(alpha-D- glucopyranosyl-(1 -->2)-monoacylglycerophosphoryl-6-O-alpha-D-glucopyranosyl)]-sn-gl ycerol (MABGPDGlcDAG), was found to form only a lamellar liquid crystalline phase coexisting with water.

Vesicle-to-Micelle Transformation in Systems Containing Dimeric Surfactants

The vesicle-to-micelle transformation has been investigated thus far in lipid + surfactant systems where the vesicle-forming lipid is chemically very different from the micelle-forming surfactant. The dimeric surfactants, alkanediyl-alpha, omega-bis(dimethyldodecylammonium bromide), are known to form vesicles when the alkanediyl spacer is long enough (for instance, spacer = eicosanediyl, referred to as 12-20-12) and spheroidal micelles for shorter spacers (spacer = decanediyl, referred to as 12-10-12). These surfactants together with the conventional surfactant dodecyltrimethylammonium bromide (DTAB) permitted us to study the transformation of the 12-20-12 vesicles into micelles on addition of the chemically similar micelle-forming DTAB and 12-10-12. Spectrophotometry (light absorbance measurements), video-enhanced light microscopy, and transmission electron microscopy at cryogenic temperatures (cryo-TEM) were used to study the transformation at different scales of aggregate size. Electrical conductivity, which probes the system at the atomic scale (free counterions), was also used. Absorbance measurements showed the transformation to occur between 1.8 and 2.8 wt% added surfactant at a constant 12-20-12 concentration of 1.4 wt%. Light microscopy showed the progressive solubilization of the larger vesicles. Cryo-TEM showed that the initial effect of DTAB addition was to reduce the size of the vesicles, whereas 12-10-12 addition resulted in the formation of multilamellar vesicles. Further additions of either surfactant reduced the size of the vesicles, then brought about the formation of spheroidal micelles until complete solubilization of the vesicles. The giant threadlike micelles seen in previous studies of vesicle-to-micelle transformation in lipid/surfactant systems were never observed with the systems investigated. The conductivity results also revealed differences in behavior on additions of DTAB and 12-10-12.

Branched threadlike micelles in an aqueous solution of a trimeric surfactant.

Very long threadlike micelles observed in aqueous solutions of some surfactants have attracted much attention because of the peculiar rheological properties of these systems. Molecular dynamics simulations have suggested that branched threadlike micelles should exist in concentrated solutions of dimeric surfactants. Here experimental evidence, obtained from transmission electron microscopy at cryogenic temperature, is presented of branched threadlike micelles in aqueous solutions of a triquaternary ammonium (trimeric) surfactant made up of three amphiphilic moieties connected at the level of the head-groups by two propanediyl spacers.