Particle Sizing of Liposomal Dispersions: A Critical Evaluation of Some Quasi-Elastic Light-Scattering Data-Analysis Software Programs

Abstract

The particle size of four liposomal dispersions was estimated by quasi-elastic light scattering. The data were analyzed by three different software programs. Comparing the estimated values for the average particle size and the particle size distribution, some differences became obvious. First, it was observed that the average values obtained by different calculation methods were hardly comparable, since some systematic deviations occurred. Comparing the estimated particle size distributions, the Malvern Automeasure software was shown to produce overly smoothed, broad monomodal distributions. On the other hand, the results of both the CONTIN and the Brookhaven ISDA package agreed much better with the particle size distributions estimated by image analysis of electron microscopic photographs, revealing that all samples studied were bimodal, containing small liposomes with a rather constant diameter as well as much bigger aggregates that were gradually broken down during sonication. Concerning the ISDA software in detail, more accurate results were obtained by the NNLS-based histogram method compared to the exponential sampling method. Overall, CONTIN seemed to yield the most reliable distributions, since the smaller liposomes were detected even in the unsonicated sample.     

Determination of the size distribution of liposomes by SEC fractionation,and PCS analysis and enzymatic assay of lipid content

In this study, small liposomes obtained by high-pressure homogenization were fractionated according to their particle sizes by size exclusion chromatography (SEC). The subfractions were analyzed by photon correlation spectroscopy (PCS) as well as enzymatic phosphatidylcholine (PC) assay for their particle sizes and lipid contents, respectively. For small egg PC-liposomes, a size range of 15 nm to 60 nm was found, with 80% of the vesicles being smaller than 30 nm in size. This is in contradiction to a mean size of 85±32 nm as indicated by PCS without fractionation. The PCS technique appears to underestimate very small particles below 30 nm if (few) bigger particles are present. The PCS particle size analysis of unfractionated hydrogenated egg PC/cholesterol-liposomes (2:1, mole/mole) by PCS did not yield any significant results. On fractionation, however, a particle size range of 40 nm to 120 nm was determined in a reproducible manner. Our results indicate that the combination of size exclusion fractionation with subsequent photon correlation spectroscopic particle size analysis and enzymatic PC assay can give both more detailed and more reliable insight into the particle size distribution of small liposomes than PCS alone. Published: May 15, 2002.     

Tip size of ion-exchanger based K+-selective microelectrodes. II. Effects on measurement of evoked [K+]0 transients

Double-barreled ion-exchanger based K+-selective microelectrodes (K+ ISMs) of a variety of tip diameters were used to provide an experimental test of predictions that microelectrode tip induced tissue damage influences the magnitude of measured [K+]0 increases. The measured magnitude of stimulation induced [K+]0 rises in the rat optic nerve depended on the tip diameter of the measuring K+ ISM; smaller tipped K+ ISMs tended to measure larger rises of [K+]0 than bigger tipped K+ ISMs. When stimulated [K+]0 increases were simultaneously recorded with 2 K+ ISMs of different tip size, the smaller tipped K+ ISM recorded [K+]0 increases that were, on average, 14% larger than the increases measured by the bigger tipped K+ ISM. In view of these results, and those presented in the previous paper, investigators should standardize the tip sizes of the ISMs used in their experiments.     

Size distributions of chylomicrons from human lymph from dynamic light scattering measurements

Chylomicrons, the vehicles for the transport of exogeneous triglycerides and cholesterol in the lymph and the blood, were characterized by their size from dynamic light scattering measurements. To achieve an appropriate resolution, correlation data were collected over several hours. Analysis was performed with an extended version of the regularization method CONTIN, and special attention was given to errors in the experimental baseline and to randomness of the residuals. The solutions selected by means of Fisher's F-test by CONTIN agreed with those obtained with the stability plot of Schnablegger and Glatter, when in the case of data of lower statistical accuracy the solution was taken from the lower part of the confidence interval of the F-test. The intensity-weighted size distributions indicated two classes of particle, their mean diameters being 100–140 nm and 330–350 nm. The ability to resolve two peaks of such a size ratio is demonstrated. The numbers of particles associated with the two peaks were estimated by means of the scattering properties of the particles, which showed that the overwhelming majority were small ones. This estimation also suggested that the mean size of the first peak of the number distribution is significantly smaller than the typical size of chylomicrons. This was consistent with the finding that the sample contained not only apolipoprotein B-48 but also a similar amount of apolipoprotein B-100, which is associated with lipoproteins of smaller size. The larger particles of the second peak are probably dietary triglyceride-rich chylomicrons. Received: 12 October 1997 / Revised version: 26 June 1998 / Accepted: 7 August 1998     

Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters

Filter-extrusion is a widely used technique for down-sizing of phospholipid vesicles. In order to gain a detailed insight into size and size distributions of filter-extruded vesicles composed of egg phosphatidyl-choline (with varying fractions of cholesterol) – in relation to extrusion-parameters (pore-size, number of filter passages, and flow-rate), flow field-flow fractionation in conjunction with multi-angle laser light scattering (AF4-MALLS, Wyatt Technology Corp., Santa Barbara, CA) was employed. Liposome size-distributions determined by AF4-MALLS were compared with those of dynamic light scattering and correlated with cryo-transmission electron microscopy and ³¹P-NMR-analysis of lamellarity. Both the mean size of liposome and the width of size distribution were found to decrease with sequential extrusion through smaller pore size filters, starting at a size range of ≈70–415?nm upon repeated extrusion through 400?nm pore-filters, eventually ending with a size range from ≈30 to 85?nm upon extrusion through 30?nm pore size filters. While for small pores sizes (50?nm), increased flow rates resulted in smaller vesicles, no significant influence of flow rate on mean vesicle size was seen with larger pores. Cholesterol at increasing mol fractions up to 0.45 yielded bigger vesicles (at identical process conditions). For a cholesterol mol fraction of 0.5 in combination with small filter pore size, a bimodal size distribution was seen indicating cholesterol micro-crystallites. Finally, a protocol is suggested to prepare large (～?300?nm) liposomes with rather narrow size distribution, based on the filter extrusion at defined flow-rates in combination with freeze-/thaw-cycling and bench-top centrifugation.     

Reducing Liposome Size with Ultrasound: Bimodal Size Distributions

Sonication is a simple method for reducing the size of liposomes. We report the size distributions of liposomes as a function of sonication time using three different techniques. Liposomes, mildly sonicated for just 30 sec, had bimodal distributions when surface-weighted with modes at about 140 and 750 nm. With extended sonication, the size distribution remains bimodal but the average diameter of each population decreases and the smaller population becomes more numerous. Independent measurements of liposome size using Dynamic Light Scattering (DLS), transmission electron microscopy (TEM), and the nystatin/ergosterol fusion assay all gave consistent results. The bimodal distribution (even when number-weighted) differs from the Weibull distribution commonly observed for liposomes sonicated at high powers over long periods of time and suggests that a different mechanism may be involved in mild sonication. The observations are consistent with the following mechanism for decreasing liposome size. During ultrasonic irradiation, cavitation, caused by oscillating microbubbles, produces shear fields. Large liposomes that enter these fields form long tube-like appendages that can pinch-off into smaller liposomes. This proposed mechanism is consistent with colloidal theory and the observed behavior of liposomes in shear fields.     

Application of photon correlation spectroscopy as a technique for detecting culture contamination

The application of photon correlation spectroscopy (PCS) to detect culture contamination in chemostats was studied. It was found that the presence of a given particle size in a population of particles of a different size could be detected, but this ability was strongly dependent on particles of a different size could be detected, but this ability was strongly dependent on particle size difference and was most sensitive when contaminants are larger than the host. The inherent polydisparity of actively growing and dividing microbial cells negates any advantage in the use of multi-angle PCS to detect contaminants.     

Solution properties of starch nanoparticles in water and DMSO as studied by dynamic light scattering

Solution properties of starch nanoparticles dispersed in DMSO and in water were studied using dynamic light scattering. The particle size distribution had two main peaks in both solvents at all scattering angles studied. They were at around 40 and 300 nm, ascribed to isolated starch nanoparticles and their aggregates, respectively. From the excess scattering intensity by the 40-nm particles, the molecular weight of the nanoparticle was estimated as 2.2–2.6×10⁶ g/mol. When the concentration was increased, another peak appeared at around 1 μm. Raising the temperature from 25 to 65 °C did not change the distribution, indicating a purely entropic process in dynamic equilibrium of the aggregation. In DMSO, an oscillatory behavior was observed in the autocorrelation function at high temperatures.     

On the sensitivity of forward scattering patterns from bacterial colonies to media composition

Morphology of colonies is important for taxonomy and diagnostics in microbiology where the response to environmental factors is sensitive enough to support discrimination. In this research, we analyzed the forward scattering patterns of individual Escherichia coli K12 colonies when agar hardness and nutrition levels were varied from the control sample. As the agar concentration increased from 1.2% to 1.8%, the diameter of the forward scattering patterns also increased for the same experimental condition which reflects that the colony thickness at the apex is greater for increased agar concentrations. Regarding nutrition, increasing dextrose resulted in smaller mean colony diameters while the mean diameters of the colonies were proportional to the yeast extract concentration up to 0.5%. The result reveals that ±0.3% agar concentration from the control sample is sufficient to create variations in the scattering patterns. For nutrition –0.25% of yeast extract showed significant variations while +0.25% from control sample showed minimal variations. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Double orientation tuning of neurons in the primary cortex of cat at different levels of alertness

Orientation tuning (OT) of 225 cat neurons of the primary visual cortex (field 17) to the flashing of a light bar in the discharge centers of their receptive field (RFs) were investigated. It was found that 43% of the cells investigated were monomodally tuned, i.e., were primarily detecting horizontal and vertical orientations. The remaining 57% of the neurons exhibited double OT, i.e, exhibited, in addition to a main preferred orientation (PO), an additional preferred orientation (aPO) at a right or acute angle to the main orientation (the mean angle between the two OT maxima equalled 71.4±2.4°). In bimodal cells, the additional maximum of OT was comparable in magnitude to the main maximum (averaging 0.7±0.03 of the PO) in half the cases. The orientational properties of the main and additional maxima were almost indistinguishable. Under light or moderate anesthesia, approximately half the neurons with double OT became monomodal; at the same time, a small fraction of monomodal cells (12%) manifested double OT. Under anesthesia, the angle between two the preferred orientations decreased, while the ratio of amplitude characteristics remained unchanged. Monomodal neurons frequently exhibited simple RFs and OTs unaffected by anesthesia. Neurons with double OT, on the other hand, exhibited simple and complex types of RFs just as often and their OT changed under the influence of anesthesia. It is suggested that neurons with double OT can function as detectors of angles and angles of intersecting lines; such angles, together with line orientation, are important attributes of images. In contrast, monomodal neurons may provide a benchmark for a stable reference system of orientation coordinates. The interaction of the two neuronal systems mentioned may allow effective analysis of image attributes at the level of the primary visual cortex.Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences Moscow. Translated from Neirofiziologiya, Vol. 24, No. 3, pp. 260–269, May–June, 1992.     

Rheology and dynamic light scattering of octa-ethyleneglycol-monododecylether/chitosan solutions

In this work we used rheometry and DLS to probe relaxation phenomena in solutions of chitosan and octa-ethyleneglycol-monododecylether. The dispersions had a marked pseudoplastic behavior, which became less evident, as surfactant concentration was increased. Arrhenius plots showed that systems with surfactant presented a characteristic temperature at which apparent enthalpy of activation (varying from 3 to 40 kJ mol⁻¹) changed: this change was correlated to a possible transition of colloidal aggregates to a wormlike configuration. DLS intensity correlation functions were described by KWW equation: pure chitosan solutions had relaxation rate distributions centered at a characteristic relaxation rate around 4.6 × 10⁻⁶ μS⁻¹; as surfactant was added, a new component, with a faster characteristic relaxation rate with a magnitude order of 10⁻³ μs⁻¹, appeared. It was shown that the dependence between these relaxation rates and surfactant concentration could be used to describe DLS-related relaxation phenomena as an Arrhenius-activated process, agreeing with results obtained using rheometry.     

Formation of unilamellar liposomes from total polar lipid extracts of methanogens.

Unilamellar liposomes were formed by controlled detergent dialysis of mixed micelles consisting of acetone-insoluble total polar lipids extracted from various methanogens and the detergent n-octyl-beta-D-glucopyranoside. The final liposome populations were studied by dynamic light scattering and electron microscopy. Unilamellar liposomes with mean diameters smaller than 100 nm were obtained with lipid extracts of Methanococcus voltae, Methanosarcina mazei, Methanosaeta concilii, and Methanococcus jannaschii (grown at 50 degrees C), whereas larger (greater than 100-nm) unilamellar liposomes were obtained with lipid extracts of M. jannaschii grown at 65 degrees C. These liposomes were shown to be closed intact vesicles capable of retaining entrapped [14C]sucrose for extended periods of time. With the exception of Methanospirillum hungatei liposomes, all size distributions of the different liposome populations were fairly homogeneous.     

黄酒中酒液粒度与混浊沉淀的关系

对煎酒前后酒液品质进行了分析,并应用９０°光散射实验考查煎酒前后,储存阶段酒液平均粒度和酒液沉淀物的平均粒度,发现酒液中沉淀物的出现和酒液平均粒径的一定大小存在耦合,当酒液平均径达到一定水平,沉淀过程就逐渐开始。     

Multi-scale analysis of the effect of nano-filler particle diameter on the physical properties of CAD/CAM composite resin blocks

The objective of this study was to assess the effect of silica nano-filler particle diameters in a computer-aided design/manufacturing (CAD/CAM) composite resin (CR) block on physical properties at the multi-scale in silico. CAD/CAM CR blocks were modeled, consisting of silica nano-filler particles (20, 40, 60, 80, and 100 nm) and matrix (Bis-GMA/TEGDMA), with filler volume contents of 55.161%. Calculation of Young’s moduli and Poisson’s ratios for the block at macro-scale were analyzed by homogenization. Macro-scale CAD/CAM CR blocks (3 × 3 × 3 mm) were modeled and compressive strengths were defined when the fracture loads exceeded 6075 N. MPS values of the nano-scale models were compared by localization analysis. As the filler size decreased, Young’s moduli and compressive strength increased, while Poisson’s ratios and MPS decreased. All parameters were significantly correlated with the diameters of the filler particles (Pearson’s correlation test, r = ?0.949, 0.943, ?0.951, 0.976, p < 0.05). The in silico multi-scale model established in this study demonstrates that the Young’s moduli, Poisson’s ratios, and compressive strengths of CAD/CAM CR blocks can be enhanced by loading silica nanofiller particles of smaller diameter. CAD/CAM CR blocks by using smaller silica nano-filler particles have a potential to increase fracture resistance.     

On the relationship between mass and diameter distributions in tree communities

It has been suggested that frequency distributions of individual tree masses in natural stands are characterized by power-law distributions with exponents near -3/4, and that therefore tree communities exhibit energetic equivalence among size classes. Because the mass of trees is not measured directly, but estimated from diameter, this supposition is based on the fact that the observed distribution of tree diameters is approximately characterized by a power-law with an exponent approximately -2. Here we show that diameter distributions of this form are not equivalent to mass distributions with exponents of -3/4, but actually to mass distributions with exponents of -11/8. We discuss the implications of this result for the metabolic theory of ecology and for understanding energetic equivalence and the processes structuring tree communities.     

Use of the electronic particle counter to investigate phosphorus on particles in streams

A simple and rapid method is described for the measurement by electronic particle counter of the relative proportions of sediment particles, of diameters 1 to 40 µm, which could enter the stream suspended load. The method is useful to further investigations into nutrients borne upon such material because the amounts of phosphate per unit weight of particles varies through orders of magnitude depending upon the particle size. An example is given to illustrate the application of the method.     

Structural and functional properties of apolipoprotein A-I mutants containing disulfide-linked cysteines at positions 124 or 232

Recombinant Cys mutants of apolipoprotein A-I (apoA-I) (A124C and A232C) have been prepared in disulfide-linked forms in order to assess the effects of unnatural covalent constraints on the folding of apoA-I in solution, its ability to bind lipids, form HDL-like particles, activate LCAT, and undergo structural adaptations to changing lipid contents. Both mutants, in dimer form, were shown to fold similarly to plasma apoA-I in solution, but had a slightly decreased alpha-helix content and no evidence of intermonomer interactions. All forms of the mutants bound to and disrupted dimyristoylphosphatidylcholine (DMPC) liposomes with similar kinetics and efficiency to plasma apoA-I, and formed reconstituted HDL (rHDL) particles with palmitoyloleoylphosphatidylcholine (POPC) in high yields at three different ratios of lipid/protein. While the monomeric mutants produced identical rHDL to plasma apoA-I, the disulfide-linked dimers had distinct particle distributions from each other and from native apoA-I. The A124C-dimer formed rHDL with diameters of 86 and 78 A, while the A232C-dimer predominantly formed 96 A rHDL. These particles, and particles containing plasma apoA-I (96 and 78 A), were purified prior to structural and functional analyses. The structural properties of particles with similar diameters were comparable, as were their reactivities with LCAT; however, their ability to undergo structural rearrangements differed. The larger rHDL particles (96 and 86 A) containing native apoA-I or A124C-dimer, rearranged into smaller 78 A particles, while the 96 A particles containing A232C-dimer were resistant to rearrangement and did not form 78 A particles. From the results, it is concluded that synthetic, random disulfide-linked dimers of apoA-I have many properties analogous to those of the naturally occurring Cys mutants, apoA-I-Milano and apoA-I-Paris, which are thought to have antiatherogenic effects in vivo. Also, the results have implications for current models of rHDL structure.     

Measurement of resuspended aerosol in the Chernobyl area

Size distribution measurements of particulate radionuclides were performed at two sites in the Chernobyl 30-km exclusion zone using several cascade impactors. The results obtained in the period September 1986 till June 1993 were discussed with regard to the general assumption of a log-normal activity size distribution in inhalation dose assessment. At Zapolie (a site 14 km from the Chernobyl reactor) a bimodal distribution was observed in 91% of all measured distributions. In most cases the medians were about 4 µm and in the range 20–30 µm. According to soil granulometric data this finding was explained by superimposing two processes: local resuspension and advective transport of radioactive aerosol from highly contaminated territories. The mean air concentration showed an increasing proportion of inhalable particles over the years since the accident. In 1993 the inhalable fraction was about 48% of the total concentration. At Pripyat, a site situated within a highly contaminated area, unimodal types of size distributions were predominant with the median diameters in the range 5–10 µm for ¹³⁷Cs. For the three nuclides ¹³⁷Cs, ¹⁴⁴Ce and ¹⁰⁶Ru, very similar types of distribution were observed. Apparently, the radioactive aerosol was of fuel origin. During a forest fire at a distance of 17 km, the majority of the radioactivity was associated with submicrometer particles with median diameters in the range 0.28–0.50 µm.     

Energy deposition by protons and alpha particles in spherical sites of nanometer to micrometer diameter

Summary Monte Carlo simulated proton- and alpha-particle tracks in water vapor were used to develop an analytical function for calculating number distributions of ionizations induced in spherical sites. For charged particles crossing the site, Fermi-like functions were used to approximate the ionization distributions. Ionization event distributions due to particles passing outside the site were approximated with an exponentially decreasing function. The function parameters were calculated for protons and alpha particles in the energy range 0.3–5.0 MeV/ amu and for site diameters of 1 to 1000 nm. The quality of fit obtained is very good for the particles, energy range and site diameters considered.On leave from the Institute of Nuclear Physics, ul. Radzikowskiego 152, PL-31342 Kraków, Poland     

Reliability of muscle fibre conduction velocity distribution estimation from surface EMG

Most of the neuromuscular diseases induce changes in muscle fibre characteristics. For example, Duchenne dystrophy is characterized by a specific loss of fast fibres, and an increase in small diameter fibres. These morphological changes may lead to large modifications in the distribution of fibre diameters, possibly producing bimodal distributions. It has already been shown that it is possible to reveal these morphological modifications through the distribution of muscle fibre conduction velocity (MFCV) as estimated from needle electromyography (EMG) recordings. In this paper, we investigate whether such changes can be extracted from surface EMG signals.

Simulation allows generation of surface EMG signals in which features are well described especially at a morphological level. Therefore, we generated a database of simulated signals both in voluntary and electrically elicited contraction conditions using a bimodal distribution of muscle fibre diameters. MFCV distributions were computed using two short-term methods based on cross-correlation and peak-to-peak techniques for voluntary contraction signals, and using a deconvolution method in time domain for electrically elicited signals. MFCV distributions were compared with true ones, as generated from modelling.

This work reveals that estimating MFCV distribution through these methods does not appear yet as precise and robust enough to accurately characterize changes in redistribution of various muscle fibre diameters. However, it opens to new experimental protocols that can be explored in order to improve the robustness of MFCV distribution estimation for the follow-up of patients suffering from neuromuscular disorders.