Assessing the accuracy of routine photon correlation spectroscopy analysis of heterogeneous size distributions

The aim of the current study was to investigate the ability of a fixed-angle routine photon correlation spectrometer (PCS) to resolve bimodal size distributions. The focus was on dispersions consisting of a majority of smaller and a minority of bigger particles. Monodisperse latex beads of sizes from 21 to 269 nm were measured first as single-size dispersions and then with various binary blends. For single-size dispersions, the mean diameters obtained were as indicated by the manufacturer, except for 21- and 34-nm particles, which were somewhat smaller. PCS analysis of blends of 21+102-nm and 34+102-nm particles resulted in bimodal distributions with particle diameters of the 2 peaks in the expected magnitude down to critical blending ratios of 0.002% and 0.08% of bigger particles, respectively. At these ratios, PCS results became inconsistent, and an increased number of monomodal results and/or high residuals were seen. For 21+102-nm blends, at even smaller ratios (0.001%), more consistent results were obtained again with predominantly monomodal distributions in the size range of the smaller particles (ie, the bigger particles were neglected). PCS analysis of blends of 21+269-nm particles yielded bimodal distributions with diameters within the expected magnitude as long as the content of bigger particles did not exceed 0.005%. Above this ratio, predominantly monomodal results with mean diameters in the magnitude of the bigger particles were obtained (ie, the smaller particles were neglected). In conclusion, a routine PCS instrument can resolve bimodal size distributions of colloidal dispersions only at certain ratios of the 2 subpopulations. Both low and high ratios lead to 1 of the 2 subpopulations being neglected. Phone: Not Forwarded; Fax: Not Forwarded;     

Characterization of small unilamellar vesicles using solvatochromic pi* indicators and particle sizing

A suite of small unilamellar vesicles (SUVs) composed of mixtures of phospholipids and cholesterol (CH) or the synthetic surfactant dihexadecyl phosphate (DHP) and cholesterol was investigated using a homologous series of solvatochromic pi* indicators coupled with size exclusion methods and photon correlation spectroscopy (PCS). The solvatochromic method, which is based on the measurement of solvent-dependent shifts in lambda(max) from UV-Vis spectra of solubilized indicators, was used to quantify the dipolarity and polarizability (pi*) of probe solvation environments. The partitioning of the series of individual di-n-alkyl-p-nitroaniline (DNAP) pi* indicators in PG(24)PC(46)Chol(30) and DHP(70)Chol(30) SUVs was examined as a function of the head group structure as well as the method of dye-vesicle preparation. Solubilization of the larger more hydrophobic probes in the bilayer portion of PG(24)PC(46)Chol(30) SUVs was aided through physical entrapment. Such physical methods were not needed for the smaller indicators or for the range of indicators in the DHP(70)Chol(30) dispersions. Extrusion and size-exclusion chromatographic methodologies for the preparation of physically entrapped dopants in SUVs of fixed size range demonstrated that the larger (longer alkyl chain) dopants in the series resided in PG(24)PC(46)Chol(30) liposomes with a wider range of sizes, while the smaller more polar solutes tended to be entrapped in smaller vesicles with a narrower size range.     

Interaction of oligonucleotides with cationic lipids: the relationship between electrostatics, hydration and state of aggregation

Lipoplexes, which are spontaneously formed complexes between oligonucleotide (ODN) and cationic lipid, can be used to deliver ODNs into cells, both in vitro and in vivo. The present study was aimed at characterizing the interactions associated with the formation of lipoplexes, specifically in terms of electrostatics, hydration and particle size. Large unilamellar vesicles (approximately 100 nm diameter), composed of either DOTAP, DOTAP/cholesterol (mole ratio 1:1) or DOTAP/DOPE (mole ratio 1:1) were employed as a model of cationic liposomes. Neutral vesicles ( approximately 100 nm diameter), composed of DOPC/DOPE (mole ratio 1:1), were employed as control liposomes. After ODN addition to vesicles, at different mole ratios, changes in pH and electrical surface potential at the lipid-water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin. In separate 'mirror image' experiments, liposomes were added at different mole ratios to fluorescein isothiocyanate-labeled ODNs, thus yielding data about changes in the pH near the ODN molecules induced by the complexation with the cationic lipid. Particle size distribution and turbidity fluctuations were analyzed by the use of photon correlation spectroscopy and static light-scattering, respectively. In additional fluorescent probe studies, TMADPH was used to quantify membrane defects while laurdan was used to measure the level of hydration at the water-lipid interface. The results indicate that mutual neutralization of cationic lipids by ODNs and vice versa is a spontaneous reaction and that this neutralization is the main driving force for lipoplex generation. When lipid neutralization is partial, induced membrane defects cause the lipoplexes to exhibit increased size instability.     

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.     

Characterization of loaded liposomes by size exclusion chromatography

This review focuses on the use of conventional (SEC) and high performance (HPSEC) size exclusion chromatography for the analysis of liposomes. The suitability of both techniques is examined regarding the field of liposome applications. The potentiality of conventional SEC is strongly improved by using a HPLC system associated to gel columns with a size selectivity range allowing liposome characterization in addition to particle fractionation. Practical aspects of size exclusion chromatography are described and a methodology based on HPSEC coupled to multidetection modes for on-line analysis of liposomes via label or substance encapsulation is presented. Examples of conventional SEC and HPSEC applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. Size exclusion chromatography is a simple and powerful technique for investigation of encapsulation, insertion/interaction of substances from small solutes (ions, surfactants, drugs, etc.) up to large molecules (proteins, peptides and nucleic acids) in liposomes.     

Evaluation of the high-pressure extrusion technique as a method for sizing plasmid DNA-containing cationic liposomes

A promising strategy to improve the immunogenic potential of DNA vaccines is the formulation of plasmid DNA (pDNA) with cationic liposomes. In this respect, particle size may be of crucial importance. This study aimed at the evaluation of high-pressure extrusion as a method for sizing cationic liposomes after entrapment of pDNA. This is a well-known sizing method for liposomes, but so far, it has not been applied for liposomes that are already loaded with pDNA. Liposomes composed of egg PC, DOTAP, and DOPE with entrapped pDNA were prepared by the dehydration-rehydration method and subjected to various extrusion cycles, comparing different membrane pore sizes and extrusion frequencies. At optimized extrusion conditions, liposome diameter (Zave) and polydispersity index (PDI) were reduced from 560 nm and 0.56-150 nm and 0.14 respectively, and 35% of the pDNA was retained. Importantly, gel electrophoresis and transfection experiments with pDNA extracted from these extruded liposomes demonstrated the preservation of the structural and functional integrity of the pDNA. The reduction in size resulted in enhanced transfection of HeLa cells, as detected by functional expression of the fluorescent protein, eGFP. In addition, these liposomes were able to stimulate Toll-like receptor 9, indicating efficient endosomal uptake and release of the included pDNA. In conclusion, high-pressure extrusion is a suitable technique to size cationic liposomes with entrapped pDNA and allows preparation of well-defined nanosized pDNA-liposomes, with preserved pDNA integrity. Their improved transfection efficiency and ability to activate an important pattern-recognition receptor are favorable properties for DNA vaccine delivery vehicles.     

Microfluidic preparation of drug-loaded PEGylated liposomes,and the impact of liposome size on tumour retention and penetration

Understanding the effect of liposome size on tendency for accumulation in tumour tissue requires preparation of defined populations of different sized particles. However, controlling the size distributions without changing the lipid composition is difficult, and differences in compositions itself modify distribution behaviour. Here, a commercial microfluidic format as well as traditional methods was used to prepare doxorubicin-loaded liposomes of different size distributions but with the same lipid composition, and drug retention, biodistribution and localization in tumour tissues were evaluated. The small (～50?nm diameter) liposomes prepared by microfluidics and large (～75?nm diameter) liposomes displayed similar drug retention in in vitro release studies, and similar biodistribution patterns in tumour-bearing mice. However, the extent of extravasation was clearly dependent on size of the liposomes, with the small liposomes showing tissue distribution beyond the vascular area compared to the large liposomes. The use of microfluidics to prepare smaller size distribution liposomes compared to sonication methods is demonstrated, and allowed preparation of different size distribution drug carriers from the same lipid composition to enable new understanding of tissue distribution in compositionally consistent materials is demonstrated.     

Changes in the size of reconstituted high density lipoproteins during incubation with cholesteryl ester transfer protein: the role of apolipoproteins.

It has been reported previously that the particle size distribution of discoidal, reconstituted HDL (r-HDL) changes dramatically during incubation in vitro with cholesteryl ester transfer protein (CETP). The present study was undertaken in order to determine whether these changes are influenced by the apolipoprotein composition of the r-HDL. Two preparations of r-HDL that contained egg phosphatidylcholine (egg PC) and unesterified cholesterol (UC) but differed in their apolipoprotein composition were used for the study. One preparation contained apolipoprotein (apo) A-I only (A-I w/o A-II r-HDL) while the other contained apoA-I and apoA-II (A-I w A-II r-HDL). The Stokes' radius of the major population of particles in the (A-I w/o A-II) and (A-I w A-II) r-HDL was, respectively, 4.8 and 4.9 nm. When the (A-I w/o A-II) r-HDL were incubated with CETP, most of the particles of radius 4.8 nm were converted to populations of smaller and larger particles. The smaller particles had Stokes' radii of 4.3 and 3.9 nm. The radii of the larger particles ranged from 8.2 to 13.7 nm. When the (A-I w A-II) r-HDL were incubated with CETP larger particles (Stokes' radii = 8.4-11.0 nm) appeared but there was minimal conversion to smaller particles. In addition, a significant proportion of the original (A-I w A-II) r-HDL of radius 4.9 nm was still present at the end of the incubation. These results are consistent with apoA-II inhibiting the conversion of r-HDL to small particles. It is concluded that the apolipoprotein content of r-HDL is an important determinant of the sizes of the particles that are formed during incubation with CETP.     

Characterization of Antimicrobial-bearing Liposomes by ζ-Potential, Vesicle Size, and Encapsulation Efficiency

Liposome entrapment may improve activity of protein or polypeptide antimicrobials against a variety of microorganisms. In this study, ability of liposomes to withstand exposure to environmental and chemical stresses typically encountered in foods and food processing operations were tested. Liposomes consisting of distearoylphosphatidylcholine (PC) and distearoylphosphatidylglycerol (PG), with 0, 5, or 10 μg/ml of the antimicrobial peptide nisin entrapped, were exposed to elevated temperatures (25–75 °C) and a range of pH (5.5–11.0). Ability of liposomes to maintain integrity was assessed by measuring the encapsulation efficiency (EE), ζ-potential, and particle size distribution of liposomes. Distearoylphosphatidylcholine, PC/PG 8:2, and PC/PG 6:4 (mole fraction) liposomes retained between ~70–90% EE despite exposure to elevated temperature and alkaline or acidic pH. Particle size of liposomes averaged between 100 and 240 nm depending on liposome preparation. Liposomal surface charge depended primarily on phospholipid composition and changed little with inclusion of nisin. Surface charge was not affected by temperature for PC and PC/PG 8:2 but decreased for PC/PG 6:4 liposomes. Our results suggest that liposomes containing nisin may be suitable for use as antimicrobial-active ingredients in low- or high-pH foods subjected to moderate heat treatments.     

Influence of substrate particle size and wet oxidation on physical surface structures and enzymatic hydrolysis of wheat straw

In the worldwide quest for producing biofuels from lignocellulosic biomass, the importance of the substrate pretreatment is becoming increasingly apparent. This work examined the effects of reducing the substrate particle sizes of wheat straw by grinding prior to wet oxidation and enzymatic hydrolysis. The yields of glucose and xylose were assessed after treatments with a benchmark cellulase system consisting of Celluclast 1.5 L (Trichoderma reesei) and Novozym 188 β‐glucosidase (Aspergillus niger). Both wet oxidized and not wet oxidized wheat straw particles gave increased glucose release with reduced particle size. After wet oxidation, the glucose release from the smallest particles (53–149 μm) reached 90% of the theoretical maximum after 24 h of enzyme treatment. The corresponding glucose release from the wet oxidized reference samples (2–4 cm) was ～65% of the theoretical maximum. The xylose release only increased (by up to 39%) with particle size decrease for the straw particles that had not been wet oxidized. Wet oxidation pretreatment increased the enzymatic xylose release by 5.4 times and the glucose release by 1.8 times across all particle sizes. Comparison of scanning electron microscopy images of the straw particles revealed edged, nonspherical, porous particles with variable surface structures as a result of the grinding. Wet oxidation pretreatment tore up the surface structures of the particles to retain vascular bundles of xylem and phloem. The enzymatic hydrolysis left behind a significant amount of solid, apparently porous structures within all particles size groups of both the not wet oxidized and wet oxidized particles. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.     

Characterization of complexes of egg yolk phosphatidylcholine and apolipoprotein A-II prepared in the absence and presence of sodium cholate

Complexes of apolipoprotein A-II and egg yolk phosphatidylcholine were prepared in mixtures of different composition in the absence and presence of sodium cholate. By gradient gel electrophoresis, complex preparations were polydisperse and particle size distributions were influenced by the composition of the reconstitution mixture. Complexes generally exhibited a discoidal morphology by electron microscopy, but showed increased formation of vesicular complexes at elevated levels of egg yolk PC in the mixtures. By chemical crosslinking, complexes formed in the absence of cholate were shown to consist primarily of discoidal species with three apolipoprotein A-II molecules per particle in the mixtures investigated; complexes formed in the presence of cholate included species ranging from three to five apolipoprotein A-II per particle. The number of apolipoprotein A-II per particle and the sizes of the complexes, prepared in cholate, increased with increase of egg yolk PC in the reconstitution mixture. Relative to the particle size distribution of discoidal complexes formed in the absence of cholate, those prepared in cholate showed a distribution shifted to larger particle sizes. Complexes of similar particle size distribution formed in the presence or absence of cholate showed similar physical-chemical properties. Discoidal complexes with the same number of apolipoprotein A-II per particle but of different size and composition were observed, suggesting the possibility of some conformational adaptation of apolipoprotein A-II leading to stabilization of egg yolk PC bilayers of different diameter. Properties of particle size distributions of discoidal complexes prepared in cholate of apolipoprotein A-II and egg yolk PC were compared with those of complexes of apolipoprotein A-I previously reported (Nichols, A.V., Gong, E.L., Blanche, P.J. and Forte, T.M. (1983) Biochim. Biophys. Acta 750, 353-364).     

Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion

Asymmetrical flow field-flow fractionation (AsFlFFF), a technique that provides direct measurement of particle size and diffusion coefficient, is converted into miniaturized scale. In comparison with conventional AsFlFFF, the separation of proteins in miniaturized AsFlFFF is achieved within shorter time periods, with smaller sample amounts, and with lower mobile phase consumption. Minimization of the overloading and optimization of the separation efficiency are prerequisites to good results. Miniaturized AsFlFFF is applied to the measurement of particle sizes of high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL). The average hydrodynamic diameters at pH 7.4 in 8.5mM phosphate buffer containing 1mM EDTA and 150 mM NaCl are 8.6+/-0.5, 11.2+/-0.2, 22.1+/-0.7, and 48.9+/-7.5 nm for subgroups HDL3, HDL2, LDL, and VLDL, respectively. In addition, the effect of different factors on the aggregation and fusion of LDL particles is studied. LDL particle sizes are unaffected by the addition of up to 300 mM NaCl and by an increase of the carrier solution pH from 3.2 to 7.4, but treatment of LDL with alpha-chymotrypsin, sphingomyelinase, or copper sulfate leads to the formation of aggregated and fused LDL particles.     

Life histories as sampling devices: optimum egg size in pelagic fishes*

In teleosts, egg size is an important determinant of larval size. The latter determines where larvae start feeding in the size spectrum of biomass particles. Egg and larval size also determine the size range of predators exploiting these early ontogenetic stages. Thus, egg size is important in determining the particle size range sampled during ontogeny. However, egg size also largely determines how many can be produced by a female. Optimum egg size for marine, pelagic fishes was estimated using a modification of a model developed by Ware. A numerical analysis using empirical information on size and temperature dependent mortality and growth in pelagic, marine systems suggests that allometry of size-dependent mortality and temperature are significant factors in determining optimum egg size. Allometry of mortality will depend on the frequency of encounters between particles of different sizes. Both empirical and modelling studies of such frequencies in pelagic and other environments are required.     

Liposomes with entrapped doxorubicin exhibit extended blood residence times

The blood residence time of liposomes with entrapped doxorubicin is shown to be significantly longer than for identically prepared empty liposomes. Liposomal doxorubicin systems with a drug-to-lipid ratio of 0.2 (w/w) were administered at a dose of 100 mg lipid/kg. Both doxorubicin and liposomal lipid were quantified in order to assess in vivo stability and blood residence times. For empty vesicles composed of phosphatidylcholine (PC)/cholesterol (55:45, mole ratio) and sized through filters of 100 nm pore size, 15-25% of the administered lipid dose was recovered in the blood 24 h after i.v. injection. The percentage of the dose retained in the circulation at 24 h increased 2-3-fold when the liposomes contain entrapped doxorubicin. For 100 nm distearoyl PC/chol liposomal doxorubicin systems, as much as 80% of the injected dose of lipid and drug remain within the blood compartment 24 h after i.v. administration.     

Role of apolipoproteins in cellular cholesterol efflux

The effects of serum apolipoproteins, particle size and concentration on the effectiveness of phosphatidylcholine (PC)-containing acceptor particles in causing release of cholesterol from cells growing in culture have been investigated. The acceptor particles were prepared by detergent-dialysis procedures and were either egg PC small unilamellar vesicles (SUV) or discoidal complexes of egg PC with apoproteins from human high-density lipoprotein (HDL). Gel filtration chromatography was employed to isolate particles of defined composition and size. The half-times (t 1/2) for the unidirectional efflux of cholesterol from cells prelabeled with [3H]cholesterol were measured as a function of acceptor PC concentration in the extracellular medium. HDL apolipoprotein-egg PC discoidal complexes at 100 micrograms PC/ml gave the following t 1/2 values when incubated with rat Fu5AH hepatoma, human HepG2 hepatoma, human GM3468 skin fibroblast, L-cell and mouse J774 macrophage-tumor cells: 11 +/- 2, 22 +/- 5, 84 +/- 18, 17 +/- 2 and 32 +/- 6 h, respectively. Equivalent experiments using purified apolipoprotein A-I or the total apolipoprotein C fraction to form the egg PC complexes showed that the t 1/2 values for the hepatoma cells were unaltered. However, with the fibroblasts, L-cells and J774 macrophages, the apolipoprotein C complexes gave significantly longer t 1/2 than complexes of egg PC with either apolipoprotein A-I or HDL apolipoprotein which gave the same t 1/2. An analysis based on the theory of fast coagulation of colloid particles to describe collisions between desorbed cholesterol molecules and acceptor particles predicts that the dependence of t 1/2 for cholesterol efflux from a given cell to different acceptors should be normalized when the extracellular level of acceptors is expressed in terms of the product of the radius of the particle times the number concentration of acceptor particles. The decrease in t 1/2 for cholesterol efflux from fibroblasts when the egg PC acceptor was changed from an SUV to an apolipoprotein HDL discoidal complex is consistent with the above concepts. The primary effect of the apolipoproteins in promoting cellular cholesterol efflux seems to be the solubilization of PC so that the PC is present in the extracellular medium as many small particles.     

Basic amphipathic helical peptides induce destabilization and fusion of acidic and neutral liposomes

We have studied the fusion of small unilamellar vesicles composed of egg PC and of a mixture of egg PC plus egg PA using various basic amphipathic peptides. Fusion was monitored by carboxyfluorescein leakage assay, light scattering, membrane intermixing assay, contents mixing assay and electron microscopy. Ac-(L-Leu-L-Ala-L-Arg-L-Leu)3-NHCH3 (peptide 4(3] and Ac-(L-Leu-L-Ala-L-Lys-L-Leu)3-NHCH3 (peptide 4'3), which have high hydrophobic moments, caused transformation of small unilamellar vesicles into larger and relatively homogeneous ones. Ac-(L-Leu-L-Leu-L-Ala-L-Arg-L-Leu)2-NHCH3 (5(2], which has medium hydrophobic moment, induced weak but appreciable fusion, while Ac-(L-Ala-L-Arg-L-Leu)3-NHCH3 (3(3] which has no helical structure did not show any fusion. However, peptides 4(3), 4'3 and 5(2) caused massive leakage of the contents from small unilamellar vesicles. These results indicated that interaction of the peptides with artificial membranes caused extensive perturbation of the lipid bilayer, followed by fusion. The fusogenic capacity of model basic peptides was correlated with the hydrophobic moment of each peptide when the peptides adopted an alpha-helical structure in the presence of acidic liposomes. Peptides 4(3) and 4'3 also showed weak fusogenic ability for neutral liposomes, while 5(2) and 3(3) showed no ability, suggesting that highly amphipathic peptides, such as 4(3), interact weakly but distinctly with neutral liposomes to fuse them.     

Isolation,Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background

Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs.

Methodology

Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to >25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4°C was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes.

Conclusions

ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utility for biotechnological applications.     

Mechanism and size cutoff for steric exclusion from actin-rich cytoplasmic domains.

Subdomains of the cytoplasmic volume in tissue culture cells exclude large tracer particles relative to small. Evidence suggests that exclusion of the large particles is due to molecular sieving by the dense meshwork of microfilaments found in these compartments, but exclusion as a result of the close apposition of the dorsal and ventral plasma membrane of the cell in these regions has not been ruled out conclusively. In principle, these two mechanisms can be distinguished by the dependence of exclusion on tracer particle size. By fluorescence ratio imaging we have measured the partition coefficient (P/PO) into excluding compartments for tracer particles ranging in radius from 1 to 41 nm. The decay of P/PO as a function of particle radius is better fitted by three molecular sieving models than by a slit pore model. The sieving models predict a percolation cutoff radius of the order of 50 nm for partitioning into excluding compartments.     

Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo

The asialoglycoprotein receptor (ASGPr) on hepatocytes plays a role in the clearance of desialylated proteins from the serum. Although its sugar preference (N-acetylgalactosamine (GalNAc) > galactose) and the effects of ligand valency (tetraantennary > triantennary > diantennary > monoantennary) and sugar spacing (20 A 10 A 4 A) are well documented, the effect of particle size on recognition and uptake of ligands by the receptor is poorly defined. In the present study, we assessed the maximum ligand size that still allows effective processing by the ASGPr of mouse hepatocytes in vivo and in vitro. Here too, we synthesized a novel glycolipid, which possesses a highly hydrophobic steroid moiety for stable incorporation into liposomes, and a triantennary GalNAc(3)-terminated cluster glycoside with a high nanomolar affinity (2 nm) for the ASGPr. Incorporation of the glycolipid into small (30 nm) [(3)H]cholesteryl oleate-labeled long circulating liposomes (1-50%, w/w) caused a concentration-dependent increase in particle clearance that was liver-specific (reaching 85 +/- 7% of the injected dose at 30 min after injection) and mediated by the ASGPr on hepatocytes, as shown by competition studies with asialoorosomucoid in vivo. By using glycolipid-laden liposomes of various sizes between 30 and 90 nm, it was demonstrated that particles with a diameter of >70 nm could no longer be recognized and processed by the ASGPr in vivo. This threshold size for effective uptake was not related to the physical barrier raised by the fenestrated sinusoidal endothelium, which shields hepatocytes from the circulation, because similar results were obtained by studying the uptake of liposomes on isolated mouse hepatocytes in vitro. From these data we conclude that in addition to the species, valency, and orientation of sugar residues, size is also an important determinant for effective recognition and processing of substrates by the ASGPr. Therefore, these data have important implications for the design of ASGPr-specific carriers that are aimed at hepatocyte-directed delivery of drugs and genes.