A dynamic light scattering study of the influence of the phospholipid concentration in a model perfluorocarbon emulsion

Perfluorohexyl iodide in water emulsions stabilised by phospholipids were prepared by microfluidisation. Photon correlation spectroscopy revealed that the particle size distributions of these emulsions were bimodal. Centrifugation experiments indicated that the larger mode was caused by the emulsion droplets, whereas the smaller mode was due to phospholipid vesicles formed from the excess amount of phospholipid emulsifier. Comparing the particle size distributions of perfluorocarbon emulsions containing different amounts of phospholipids, it could be concluded that emulsions with a phospholipid/fluorocarbon ratio of 2% at the most were emulsifier limited, whereas those with a ratio of at least 5% were energy input limited.     

Properties of various phospholipid mixtures as emulsifiers or dispersing agents in nanoparticle drug carrier preparations

The characteristics of mixed phospholipids were examined when used as dispersing agents and emulsifiers. Synthesized phospholipids were mixed to investigate the potential effects of different hydrophilic or lipophilic groups on emulsification and dispersion. To examine the effects of the hydrophilic polar head group on the dispersing or emulsifying potency of phospholipids, l--phosphatidylcholine dimyristoyl (DMPC) and l--phosphatidylethanolamine dimyristoyl (DMPE) were mixed in various ratios. Moreover, all combinations of two kinds of phosphatidylcholines (PCs) out of l--phosphatidylcholine dilauroyl (DLPC), DMPC, l--phosphatidylcholine dipalmitoyl (DPPC) and l--phosphatidylcholine distearoyl (DSPC) were tested (50:50, w/w) to examine the effects of the hydrophobic carbon chains on the dispersing or emulsifying potency of phospholipids. Mean diameters of vesicles and O/W emulsions prepared by sonication were measured. Vesicles prepared with DMPC–DMPE mixtures gave larger particle sizes than those of DMPC alone. Particle sizes of vesicles prepared with a mixture of two kinds of PCs increased when adding a PC with a longer carbon chain, while particle sizes in a mixture with a PC having a shorter carbon chain was comparable to those in pure PC. In vesicles that were generated by hydration of phospholipids and had a bilayer form, the physical form of the phospholipids consisting of bilayers was thought to be an important factor influencing particle sizes. Among the emulsions, DMPC–DMPE mixtures gave a similar droplet size to DMPC alone. Droplet size in emulsions prepared with a mixture of two kinds of PCs had a strong positive correlation with the total number of carbons, which corresponds to hydrophilic–lipophilic balance (HLB). In O/W emulsions, in which phospholipids were absorbed at water–oil interfaces and which have a single layer form, HLB was thought to be a major factor in the determination of particle size; likewise with non-ionic emulsifiers.     

The effects of coexisting lipids on the action of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C toward liposomal substrate.

The hydrolytic activity of phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) from Bacillus thuringiensis was studied in detail toward mixed liposomes consisting of PI and one of other phospholipids and cholesterol. Among PI-liposomes, small unilamellar vesicles (SUV) were the most sensitive to PI-PLC; the enzymatic hydrolysis of PI in SUV was not less than 10-fold that in large unilamellar vesicles (LUV) or in multilamellar vesicles (MLV). Thus, in a survey of the effects of coexisting lipids on PI-PLC activity, PI-SUV was used. Phosphatidylcholine (PC) was stimulative for the enzyme activity toward PI-SUV at any molar ratio of PC to PI. Also, the effects of the addition of sphingomyelin (SM), phosphatidylethanolamine (PE) and cholesterol on the enzymatic hydrolysis of PI were studied in detail on the basis of concentration of total lipids or PI.     

Double emulsion templated monodisperse phospholipid vesicles

We present a novel approach for fabricating monodisperse phospholipid vesicles with high encapsulation efficiency using controlled double emulsions as templates. Glass-capillary microfluidics is used to generate monodisperse double emulsion templates. We show that the high uniformity in size and shape of the templates are maintained in the final phospholipid vesicles after a solvent removal step. Our simple and versatile technique is applicable to a wide range of phospholipids.     

The radiation resistance of the bioburden from medical devices

An extensive study of the radiation resistance of microbial species constituting the bioburden of a number of different medical devices obtained from Japanese medical device manufacturers has been carried out. A standard protocol for determining radiation resistance was used and validated at the fourteen centres involved in the study. Individual microbial isolates from the bioburden obtained from seven different devices manufactured in these centres were studied. A total of 3742 unselected isolates were obtained, of which 197 failed to survive long enough for subsequent radiation resistance studies. The remainder were subjected to an initial screen test to identify those organisms that were sensitive to the lethal effects of radiation with a D₁₀ of < 1.5kGy. The 465 isolates that survived the screen doses were then tested for survival in an incremental series of radiation doses using methods similar to those of Whitby (1979) and Yan and Tallentire(1995). The isolates from “dry” devices were more resistant than those obtained from the one water filled (“wet”) device studied. The overall distribution of radiation resistance among the isolates was considered to be similar to that forming the “Standard Distribution of Resistance” (SDR) included in the ISO International Standard 11137 “Sterilization of Health Care Products — Requirements for validation and routine control — Radiation sterilization”.     

Stability of phospholipid vesicles studied by asymmetrical flow field-flow fractionation and capillary electrophoresis

The stability of zwitterionic phosphatidylcholine vesicles in the presence of 20 mol% phosphatidyl serine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI), and diacylphosphatidyl glycerol (PG) phospholipid vesicles, and cholesterol or calcium chloride was investigated by asymmetrical flow field-flow fractionation (AsFlFFF). Large unilamellar vesicles (LUV, diameter 100 nm) prepared by extrusion at 25 °C were used. Phospholipid vesicles (liposomes) were stored at +4 and −18 °C over an extended period of time. Extruded egg yolk phosphatidylcholine (EPC) particle diameters at peak maximum and mean measured by AsFlFFF were 101 ± 3 nm and 122 ± 5 nm, respectively. No significant change in diameter was observed after storage at +4 °C for about 5 months. When the storage period was extended to about 8 months (250 days) larger destabilized aggregates were formed (172 and 215 nm at peak maximum and mean diameters, respectively). When EPC was stored at −18 °C, large particles with diameters of 700–800 nm were formed as a result of dehydration, aggregation, and fusion processes. In the presence of calcium chloride, EPC alone did not form large aggregates. Addition of 20 mol% of negatively charged phospholipids (PS, PA, PI, or PG) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles increased the electrostatic interactions between calcium ion and the vesicles and large aggregates were formed. In the presence of cholesterol, large aggregates of about 250–350 nm appeared during storage at +4 and −18 °C for more than 1 day.

The effect of liposome storage temperature on phospholipid coatings applied in capillary electrophoresis (CE) was studied by measuring the electroosmotic flow (EOF). EPC coatings with and without cholesterol, PS, or calcium chloride, prepared from liposomes stored at +25, +4, and −18 °C, were studied at 25 °C. The performances of the coatings were further evaluated with three uncharged compounds. Only minor differences were observed between the same phospholipid coatings, showing that phospholipid coatings in CE are relatively insensitive to storage at +25, +4 °C or −18 °C.     

Solid-stabilized emulsions

The comprehension of bulk properties of solid-stabilized emulsions (stability, compressibility, elasticity) in relation with interfacial properties has progressed. The association of oil, water and particles allows a large set of materials to be obtained, where emulsions are used either as intermediate or end products. The efficiency of some stimulus-responsive particles to stabilize or destabilize emulsions “on demand” has been experimentally evidenced.     

The behaviour of some selenoglycollic acid derivatives in aqueous solutions and spectrophotometric determination of constants of the system Pd(II)/ethylene-bis- selenoglycollic acid

Interactions between ethylselenoglycollic, selenoglycollic and ethylene-bis- selenoglycollic acids and some “soft”, “borderline” and “hard” metallic ions have been studied. The interactions of [PdCl₄]_aq²⁻ with the three ligands were verified conductometrically and spectrophotometrically. The stability constants β₁ and β₂ for the system [PdCl₄]^2-- ethylene-bis-selenoglycollic acid have been determined at 25°C at ionic strength 3.0 M (NaCl). The stoichiometric ionization constant of the mentioned acid was also studied.     

Cosurfactant effect of a semifluorinated alkane at a fluorocarbon/water interface: impact on the stabilization of fluorocarbon-in-water emulsions

Previous work has demonstrated that semifluorinated alkanes CnF2n+1CmH2m+1 (FnHm diblocks), when used in conjunction with phospholipids, strongly stabilize fluorocarbon (FC)-in-water emulsions destined to be used as oxygen carriers. Although the presence of FnHm diblocks in the emulsion's interfacial phospholipid film was suggested to account for the observed stabilization, no direct proof of the diblock's location has been provided so far. We now report definite experimental evidence of the diblock's presence at the interfacial film, both on a macroscopic level by investigating the FC/water interface using the pendant drop method and directly on emulsions by monitoring their stability for various phospholipid chain lengths. We first establish that F8H16 has a strong cosurfactant effect with phospholipids [dimyristoylphosphatidylcholine (DMPC), dilaurylphosphatidylcholine (DLPC), dioctanoylphosphatidylcholine (PCL8)] at a perfluorooctyl bromide (PFOB)/water interface, as evidenced by a dramatic F8H16-concentration-dependent decrease of the interfacial tension. Where FC emulsions are concerned, we show that the stabilization effect, which consists of a decrease of the rate of molecular diffusion of the FC, depends strongly on the length of the phospholipid's fatty chain as compared to the length of the hydrocarbon segment, Hm, of the diblock. Stabilization is maximized when the Hm length is similar to that of the phospholipid's fatty chains. A strong mismatch between Hm and the phospholipid chain length can actually destabilize the emulsion. A different destabilization mechanism is then at work: coalescence. The presence of F8H16 at the interfacial film is further supported by the fact that perfluorodecyl bromide, a heavy analogue of PFOB that stabilizes PFOB emulsions by lowering the solubility and diffusibility of the emulsion's dispersed FC phase, exercises its stabilizing effect similarly for all the phospholipids investigated.     

Zur nomenklatur der phane—II

The new term “Phanes” has been clearly defined and a nomenclature system tentatively developed. This system is comprehensible and of general application and at the same time relatively simple. The notations “nucleus”, “bridge”, “number of bridge members” and “number of ring members” are defined. In order to get a definite characterisation of the phanes which contain a carbocyclic nucleus, a carbocyclic and heterocyclic bridge the following terms: “carbophanes”, “carba-phanes” and “hetera-phanes” have been newly introduced. The prefix “hetera-” has been proposed as a general expression and as a representative term for the syllables “aza-”, “oxa-”, “thia-” and so on. The so called “a-nomenclature” is clearly called “hetera-nomenclature”. The new expressions “heteralogous” and “substitulogous” are explained. As the various examples will show, the “Phane-Nomenclature” can also be applied to the naming of complicated metallocenophanes.     

Electro-osmotic studies of endothelial cell surface

The surface properties of human umbilical vein endothelial cells (HUVEC), which form the inner walls of blood vessels, have been studied by electro-osmosis measurements. The data are analyzed on the basis of an electrophoresis theory of “soft” particles (that is, particles covered by polyelectrolyte layers). This theory utilizes two parameters, i.e. the density of fixed charges in the cell surface layer and a “softness” parameter (1/λ) which corresponds to the reciprocal of the coefficient of the frictional forces exerted by the surface layer on the liquid flow in the surface layer. It is found that the “softness” parameter for HUVEC is very large, i.e. the surface of HUVEC is very soft compared with those of other biological cells such as erythrocytes or human promyelocytic leukemia cell lines (HL-60RG cells) which we have studied before. The obtained value of 1/λ is comparable to that observed for a poly (NIPAAm) hydrogel layer in a swollen state. The charge density in the surface layer of HUVEC, however, is found to be similar to those for other biological cells.     

A differential microcalorimetric study of whey proteins and their behaviour in oil-in-water emulsions

Oil-in-water emulsions (20% soya oil, 1% protein) have been prepared containing lysozyme or isolates of -lactalbumin and β-lactoglobulin from whey protein. The structural characteristics of these proteins adsorbed at an oil/water interface were determined by following their thermal transitions using differential scanning microcalorimetry. Thermograms of the proteins in the adsorbed state differed markedly from the corresponding transitions seen for the proteins in solution. This suggests that the proteins underwent substantial changes in secondary and tertiary structure upon adsorption. In general, for all the proteins studied, a net decrease in the total energy absorbed during denaturation was found when the proteins were in an adsorbed state. Both lysozyme and -lactalbumin were in part “surface denatured”, and they showed a certain degree of reversibility between solution and the adsorbed state. β-Lactoglobulin showed the highest degree of denaturation upon adsorption and the conformational change was irreversible.     

Aggregation and fusion of phospholipid vesicles

Membrane fusion and aggregation of phospholipid vesicles are reviewed and discussed. The fusion process is viewed as consisting of several stages: aggregation and close apposition of the particles, destabilization, and finally, merging of the bilayers. A procedure is presented which yields both the rate constant of the dimerization (C₁₁) and the rate constant for fusion of the dimers (f₁₁), which is a direct measure of the probability that two apposed vesicles will fuse. Experimental methods used in the study of membrane fusion are reviewed, primarily with respect to their capacity to monitor the kinetics of vesicle fusion. A few kinetic studies on the mixing of aqueous contents, leakage and increase in size of fusing vesicles are presented in detail.The range of C₁₁ values for Ca²⁺-induced aggregation and fusion of small unilamellar vesicles (SUV, ～ 125 Å radius) composed of phosphatidylserine (PS) is 10⁶ to 5 × 10⁷ M^-1 in the presence of Ca²⁺ concentrations from 1.15 to 2 mM, respectively. For larger PS vesicles (LUV, ～ 500 Å radius) C₁₁ = 6.5 × 10⁷ M^-1s^-1 in the presence of 5 mM Ca²⁺. These values are in good agreement with theoretical calculations based on van der Waals and electrostatic interactions, in which binding of cations is explicitly taken into account. The rate constants of fusion, f₁₁, are 5 s^-1 for PS SUV and 0.08 s^-1 for LUV in the presence of 2 mM and 5 mM Ca²⁺, respectively. The significance of these fusion rate constants to the duration of the fusion event is discussed.Factors affecting fusion such as cations, temperature, membrane composition vesicle concentration and size are reviewed and analyzed. Di- or tri-valent cations induce fusion of acidic phospholipid vesicles (except for phosphatidylinositol) in either pure or mixed form. Among the neutral phospholipids, phosphatidylcholine (PC) inhibits but phosphatidylethanolamine (PE) sustains or enhances the fusion capacity of acidic phospholipid vesicles. Monovalent cations induce reversible aggregation of negatively charged vesicles, but they inhibit the fusion induced by divalent cations such as Ca²⁺ or Mg²⁺. Fusion of neutral phospholipid vesicles, and it occurs the cation-induced fusion of acidic phospholipid vesicles, and it occurs only at temperatures below the gel to liquid crystalline phase transition temperature Tc. This is in contrast to the acidic phospholipid vesicle fusion which is greatly enhanced when the temperature is above the Tc of the phospholipid.     

Pharmaceutical microcalorimetry: recent advances in the study of solid state materials

This paper discusses the theory and experimental application of microcalorimetry to the study of solid state reactions of pharmaceutical importance. The practical example selected is that of formulated products containing benzoyl peroxide – a treatment for acne and athlete’s foot. A newly developed procedure for data analysis is outlined and preliminary results from chemometric-based analysis of complex solid state reaction schemes is presented. Finally, the microcalorimetric requirements for such stability studies are contrasted with the newly emerging multi-channel “chip calorimeters” that operate in the nano range (watts, material, concentration) with high throughput potential.     

Rheological properties of deionized Chinese ink

Rheological properties for Chinese ink in exhaustively deionized aqueous media were carefully examined. In the steady shear measurement, the shear viscosities of the ink could be well explained by considering the “effective” volume fraction of the particles in the ink including the electrical double layers and by using Einstein's equation for dilute suspension viscosity, when the particle volume fraction was substantially low. In the case that the volume fraction was higher, the shear viscosities showed extremely higher than those from Einstein's prediction, though the ink remained a Newtonian liquid. In the stress-strain measurement, the shear moduli were observed at strain smaller than 0.2. The “weak” aggregation among the particles in the ink under no shear or low shear rates was supported. It should be noted that the glue in the suspension plays an important role for the good liquidity of the ink and for the “weak” bridges among the particles resulting its good dispersion stability.     

Novel photochemical surface functionalization of polysulfone ultrafiltration membranes for covalent immobilization of biomolecules

The major objective of the work was to develop a heterogeneous modification method for attachment of reactive groups, suitable for covalent immobilization of active biomolecules on the surface of polysulfone ultrafilters without loss of membrane selectivity. For applying a polymer specific activation chemistry, the materials of commercial “polysulfone” UF membranes were identified using elemental analysis along with ¹H NMR, FTIR-ATR and UV spectroscopy. Heterogeneous photoinitiated graft polymerization was realized using acrylic acid (AA) as model monomer and as carrier of reactive groups. Polymer structure (polysulfone, PSf, or polyethersulfone, PESf), coating with photoinitiator (benzophenone, BP, or benzoylbenzoic acid, BPC) and UV excitation energy (λ_exc220> 300 or 350 nm) were the major parameters. Grafted polyAA (g-PAA) could be obtained under almost all conditions but with largely varying yields (DG). However, only with λ_exc350 nm, polymer and pore degradation could be excluded. A new selective initiation of graft polymerization onto PSf, H-abstraction by photoexcited BP derivatives from the methyl side groups, thus avoiding polymer chain scission, was proved indirectly. Modified structures were characterized spectroscopically, including visualization with SFM of laterally patterned surfaces generated by UV irradiation through a mask. UF tests of PSf-g-PAA and PESf-g-PAA UF membranes (DG 100…150 μg/cm²), prepared under “mildly degrading” conditions (λ_exc300 nm), indicated only slight permeability and selectivity changes compared with unmodified samples. Selective PSf functionalization (BPC coating, λ_exc350 nm; DG 5 μg/cm²) caused flux reductions and dextran selectivity increases by factors of 1.3. Covalent immobilization onto g-PAA-functionalized and carbodiimide-activated PSf or PESf membrane surfaces was studied with a protein (BSA), an enzyme (invertase, INV), an antibody-enzyme (IgG-POD) conjugate, and a peptide (“PC1”) as specific antigen of a monoclonal antibody. High binding capacities, up to 40 fold compared with a flat unmodified surface, were detected either directly (BSA) or indirectly via specific activity/binding assays (INV, IgG-POD, “PC1”). This indicated an increased outer membrane surface area due to multifunctional reactive and hydrophilic grafted polymer chains.     

EVALUATION OF A SERIES OF PERFLUOROALKYLATED SURFACTANTS DERIVED FROM TERTIARY AMINES: SURFACE PROPERTIES AND EMULSIFICATION OF FLUOROCARBONS

In view of their easy preparation, the physicochemical properties of a series of highly fluorinated cationic (ammonium salts), zwitterionic (carboxybetaines) or nonionic (amine oxide) surfactants derived from perfluoroalkylated tertiary amines were investigated. Their solubility in water, interfacial properties and the exceptionally low hemolytic activity of the zwitterionic series mark them out as potential surfactants or co-surfactants for the preparation of fluorocarbon emulsions and other preparations for biomedical use. Fine and moderately concentrated fluorocarbon emulsions (50 w/w% in fluorocarbon) could be obtained in some cases when they were used as the sole surfactant. However, none was found suitable for preparing concentrated emulsions (90 w/v% in fluorocarbon) and no appreciable stabilization was found when they were used as co-surfactants with egg yolk phospholipids.     

Colloidal adhesion of phospholipid vesicles: high-resolution reflection interference contrast microscopy and theory

High-resolution reflection interference contrast microscopy (HR-RICM) was developed for probing the deformation and adhesion of phospholipid vesicles induced by colloidal forces on solid surfaces. The new technique raised the upper limit of the measured membrane–substrate separation from 1 to 4.5 μm and improved the spatial resolution of the heterogeneous contact zones. It was applied to elucidate the effects of wall thickness, pH and osmotic stress on the non-specific adhesion of giant unilamellar vesicles (ULV) and multilamellar vesicles (MLV) on fused silica substrates. By simultaneous cross-polarization light microscopy and HR-RICM measurements, it was observed that ULV with the wall thickness of a single bilayer would be significantly deformed in its equilibrium state on the substrate as the dimension of its adhesive–cohesive zone was 29% higher than the theoretical value of a rigid sphere with the same diameter. Besides, electrostatic interaction was shown as a significant driving force for vesicle adhesions since the reduction in pH significantly increased the degree of deformation of adhering ULV and heterogeneity of the adhesion discs. The degree of MLV deformation on the solid surfaces was significantly less than that of ULV. When the wall thickness of vesicle increased, the dimension of contact zone was reduced dramatically due to the increase of membrane bending modulus. Most important, the adhesion strength of colloidal adhesion approached that of specific adhesion. Finally, the increase of osmotic stress led to the collapse of adhering vesicles on the non-deformable substrate and raised the area of adhesive contact zone. To interpret these results better, the equilibrium deformation of adhering vesicle was modeled as a truncated sphere and the adhesion energy was calculated with a new theory.     

Optical potential theory of diatom-diatom scattering. II. Indistinguishable molecules

The theory of vibro-rotational energy exchange in “indistinguishable” diatom-diatom scattering is formulated in terms of effective potentials. Due the necessity of considering both “symmetric” and “antisymmetric” molecular two-particle states, the formalism has a characteristic (two-by-two matrix structure. A mathematical generalization of Schwinger's theory of sources allows a compact derivation of exact and approximate expressions for t “optical” and “transition” potentials of elastic and inelastic processes, respectively. Finally, considerations based on a partial wave analysi of the working equations, suggest that the present theory should be more readily amenable to numerical implementations than the close coupling approach     

Synthesis and photochromism of polymer-bound phenoxyquinone derivatives

Acrylate- and styrene-derived polymers having pendant phenoxyquinones for photochromism were prepared by 2,2′-azoisobutyronitrile (AIBN)-initiated radical polymerization. Synthesis of the monomers were straightforward and the polymers were obtained in high yields in spite of the quinone moieties presented in the monomers, which usually can function as radical scavengers and/or catalysts poison. Photo-induced rearrangement from the “trans”-quinone forms to the “ana”-quinone forms readily occurred when the polymer films were irradiated with UV light.