Assessment of the applicability of HLB values for natural phospholipid emulsifiers for preparation of stable emulsions.

Natural phospholipid (PL) excipients are native, biocompatible and relatively inexpensive alternatives to synthetic emulsifiers. A well-known PL excipient is lecithin, which primarily contains phosphatidylcholine (PC) and (depending on the purity grade) also contains a well-defined mixture of other PLs with a fatty acid composition, which reflects their natural source. Since all of these lipid species are emulsifiers, natural PLs can be considered as a mixture of (co-) emulsifiers. Many different HLB values for lecithins are given in the literature, which is why this needs to be clarified. To assess this, HLB values of thirteen different plant derived PLs differing in PC content were determined using a centrifugation stress method to determine the relative stability of an emulsion with the respective emulsifier and different oil phases. It could be shown that the studied PLs can be characterized by a broad HLB range, which may be linked to the composition of the PLs and the oil used. In order to emphasize the results of the HLB determination, it could be demonstrated that stable emulsions could be prepared with a wide range of oils using the plant-based PLs and that the preparation method of the emulsions is important in order to obtain stable emulsions. Therefore, assigning a specific exact HLB value to natural PLs instead of a wider range is inappropriate. The broad HLB ranges indicate the suitability of the studied PL emulsifiers for the preparation of oil-in-water emulsions for a wide range of oils: It is recommended to experimentally evaluate the suitability of these natural emulsifiers for the preparation of stable emulsions and to benefit from the wide range of HLB values of these emulsifiers instead of relying on inaccurate and confusing HLB values in the literature.

The use of fluorescence resonance energy transfer to study the disintegration kinetics of liposomes containing lysolecithin and oleic acid in rat plasma.

PURPOSE: To validate Fluorescence Resonance Energy Transfer (RET) as method to monitor disintegration of fluorescently labeled liposomes varying in lysolecithin/oleic acid (equimolar) content, lysolecithin fatty acid composition and vesicle size in rat blood plasma and buffer. METHODS: NBD-PE and Rho-PE were used for RET. The measurements were performed on a Perkin Elmer LS-50 spectrofluorimeter. Liposomes were prepared by the extrusion method. RESULTS: Analysis of the RET data was optimised using a fitting procedure to correct for fluorescence interference by plasma. The disintegration patterns of liposomes could be described by a biexponential decay model. Disintegration rate increased at increasing lysolecithin/oleic acid content and decreasing size. In contrast, all liposomes showed no disintegration in buffer. CONCLUSIONS: RET is a suitable method to monitor liposome disintegration in non-diluted plasma. Rate and extent of liposome disintegration increases at decreasing liposome size and increasing lysolecithin/oleic acid content.

Advantages of Artificial Neural Networks (ANNs) as alternative modelling technique for data sets showing non-linear relationships using data from a galenical study on a solid dosage form.

Artificial Neural Networks (ANN) methodology was used to assess experimental data from a tablet compression study showing highly non-linear relationships (i.e. measurements of ejection forces) and compared to classical modelling technique (i.e. Response Surface Methodology, RSM). These kinds of relationships are known to be difficult to model using classical methods. The aim of this investigation was to quantitatively describe the achieved degree of data fitting and predicting abilities of the developed models. The comparison between the ANN and RSM was carried out both graphically and numerically. For comparing the goodness of fit, all data were used, whereas for the goodness of prediction the data were split into a learning and a validation data set. Better results were achieved for the model using ANN methodology with regard to data fitting and predicting ability. All determined ejection properties were mainly influenced by the concentration of magnesium stearate and silica aerogel, whereas the other factors showed very much lower effects. Important relationships could be recognised from the ANN model only, whereas the RSM model ignored them. The ANN methodology represents a useful alternative to classical modelling techniques when applied to variable data sets presenting non-linear relationships.

Pitfalls of artificial neural networks (ANN) modelling technique for data sets containing outlier measurements using a study on mixture properties of a direct compressed dosage form.

An application of the Artificial Neural Networks (ANN) methodology was investigated using experimental data from a mixture properties study and compared to classical modelling technique (i.e. Response Surface Methodology, RSM) both graphically and numerically. The aim of this investigation was to quantitatively describe the achieved degree of data fitting and robustness of the developed models. For comparing the goodness of fit, the R2 coefficient was used, whereas for the robustness of the models an outlier measurement was integrated in the data set. Comparable results were achieved for both ANN- and RSM methodologies for data fitting. The robustness of the models towards outliers was clearly better for the RSM methodology. All determined mixture properties were mainly influenced by the concentration of silica aerogel, whereas the other factors showed very much lower effects. For that reason the physical properties of this excipient (e.g. its specific surface area) are of importance for the behaviour of the mixtures.

Comparison of artificial neural networks (ANN) with classical modelling techniques using different experimental designs and data from a galenical study on a solid dosage form.

Artificial Neural Networks (ANN) methodology was used to analyse experimental data from a tabletting study and compared both graphically and numerically to classical modelling techniques (i.e. Response surface methodology, RSM). The aim of this investigation was to describe quantitatively the degree of data fitting achieved and the robustness of the developed models using two types of experimental design (i.e. a statistical, highly organised design and a randomised design). To compare goodness of fit, the R(2) coefficient was used, whereas for the robustness of the models the R(2) coefficient of an independent validation data set was computed. Comparable results were achieved for both ANN and RSM methodology when using the statistical plan. However, the robustness of the models when developed based on a randomised plan was clearly better for the ANN methodology. Based on the results of this study, it appears that the ANN methodology is much less sensitive to the organisational level of a trial design and is therefore better adapted to the data analysis of the results of historical or poorly organised trials. All tablet properties determined were largely influenced by the dwell time during compression as well as by concentration of silica aerogel and magnesium stearate, whereas the other factors showed very much weaker effects.

Preparation of liposomes encapsulating water-soluble compounds using supercritical carbon dioxide.

In this paper the development of a new preparation method of liposomes containing a water soluble marker (fluorescein isothiocyanate-dextran (FITC-dextran) or zinc phthalocyanine tetrasulfonic acid (TSZnPc) using supercritical carbon dioxide (called "the supercritical liposome method") is described. The apparatus used consisted of two main parts: the high-pressure part, in which the lipid components 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and cholesterol (Chol) (7:3 molar ratio) were dissolved under pressure in supercritical carbon dioxide, and a low-pressure part, in which the homogeneous supercritical solution is expanded and simultaneously mixed with the aqueous phase to yield liposomes encapsulating the water soluble marker. Addition of 7% absolute ethanol to carbon dioxide at 25 MPa and 60 degrees C and the use of a high-pressure recycling system during 30 min form the homogeneous solution with high reproducibility of both lipid components and resulted in an equal expansion profile (recovery after expansion versus time) of POPC and Chol. Incubation of the lipid components during 60 min at the above mentioned conditions generated only 3% degradation. The average size of the liposomes was about 200 nm and could not be influenced by the experimental conditions used. Optimal values for encapsulated volume (1.25 L/mol) and efficiency (20%) of the liposomes were obtained using statistical experimental design by using the water soluble marker TSZnPc and an encapsulation capillary with 5.0 cm length and 0.5 mm inner diameter. The total amount of ethanol used to obtain an encapsulation efficiency of 20% was 15-fold reduced compared to the ethanol injection method of Batzri and Korn (Biochim. Biophys. Acta 1973, 298, 1015-1019).

Basic concepts of artificial neural networks (ANN) modeling in the application to pharmaceutical development.

Artificial neural networks (ANN) methodology is a new modeling method that has not been broadly applied to pharmaceutical sciences up to now. The aim of this paper is to give a detailed description of the associating networks as well as a description of less well-known networks (i.e., feature-extracting and nonadaptive networks) and their scope of application in pharmaceutical sciences. The descriptions include the historical origin and the basic concepts behind the computing. ANN are based on the attempt to model the neural networks of the brain. Learning algorithms for associating ANN use mathematical procedures usually derived from the gradient descent method whereas feature-extracting ANN map multidimensional input data sets onto two-dimensional spaces. Nonadaptive ANN map data sets and are able to reconstruct their patterns when presented with corrupted or noisy samples. Associating networks can typically be applied in the pharmaceutical field as an alternative to traditional response surface methodology, feature-extracting networks as alternative to principal component analysis, and nonadaptive networks for image recognition. Based on these abilities, the potential application fields of the ANN methodology in the pharmaceutical sciences is broad, ranging from clinical pharmacy through biopharmacy, drug and dosage form design, to interpretation of analytical data. The few applications presented in the pharmaceutical technology area seem promising and should be investigated in more detail.

Application of artificial neural networks (ANN) in the development of solid dosage forms.

The application of ANN in pharmaceutical development has been assessed using theoretical as well as typical pharmaceutical technology examples. The aim was to quantitatively describe the achieved data fitting and predicting abilities of the models developed with a view to using ANN in the development of solid dosage forms. The comparison between the ANN and a traditional statistical (i.e., response surface methodology, RSM) modeling technique was carried out using the squared correlation coefficient R2. Using a highly nonlinear arbitrary function the ANN models showed better fitting (R2 = 0.931 vs. R2 = 0.424) as well as predicting (R2 = 0.810 vs. R2 = 0.547) abilities. Experimental data from a tablet compression study were fitted using two types of ANN models (i.e., multilayer perceptrons and a hybrid network composed of a self-organising feature map joined to a multilayer perception). The achieved data fitting was comparable for the three methods (MLP R2 = 0.911, SOFM-MLP R2 = 0.850, and RSM R2 = 0.897). ANN methodology represents a promising modeling technique when applied to pharmaceutical technology data sets.

Analysis of drug/plasma protein interactions by means of asymmetrical flow field-flow fractionation.

PURPOSE: The applicability of Asymmetrical Flow Field-Flow Fractionation (Asymmetrical Flow FFF) as an alternative tool to examine the distribution of a lipophilic drug (N-Benzoyl-staurosporine) within human plasma protein fractions was investigated with respect to high separation speed and loss of material on surfaces due to adsorption. METHODS: Field-Flow Fractionation is defined as a group of pseudochromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicochemical properties. This method was used to separate human plasma in its protein fractions. The drug distribution in the fractions was investigated by monitoring the fractionated eluate for drug content by fluorescence spectroscopy. RESULTS: Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), alpha 2-macroglobulin and low density lipoprotein (LDL) fractions in less than ten minutes. Calibration of the system and identification of the individual fractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composition on the absolute recovery of N-Benzoyl-staurosporine and fluorescein-isothiocyanate-albumin (FITC-albumin) was found to be quite significant. Both factors were optimized during the course of the investigations. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA. CONCLUSIONS: If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorption at the separation membrane, Asymmetrical Flow FFF shows high recoveries and fast separation of human plasma proteins, and can be a reliable tool to characterize drug/plasma protein interactions. For analytical purposes it has the potential to rival established technologies like ultracentrifugation in terms of ease-of-use, precision, and separation time.

Pharmacokinetics and body distribution of liposomal zinc phthalocyanine in tumor-bearing mice: influence of aggregation state, particle size, and composition.

The pharmacokinetics and body distribution of zinc phthalocyanine (ZnPc) intravenously administered in liposomes composed of ZnPc, 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and 1,2-dioleoylphosphatidylserine (OOPS) (1:90:10 or 1:70:30 w/w) to tumor (Meth A sarcoma) bearing mice were studied. It was found that aggregation of ZnPc in the liposomes (i) increases the clearance rate of the dye from plasma, (ii) lowers the maximal dye concentration in tumor tissue, and (iii) increases the maximal dye concentration in the liver. In addition, aggregated dye is hardly eliminated from the liver and monomeric dye is eventually completely eliminated from this organ. Liposomes in the size range of 48-123 nm, containing the dye with the same aggregation state, showed the same pharmacokinetics and body distribution of the dye. The PS-content of the ZnPc liposomes (POPC alone versus POPC/OOPS 7:3) did not influence tumor, liver, and plasma pharmacokinetics during the studied time intervals. Free flow electrophoretic analysis showed in lyophilisates of ZnPc liposomes containing aggregated ZnPc the presence of two distinct populations differing in size, aggregation state of the dye, and PC/PS and ZnPc/phospholipid ratio. The liposomal formulation with monomeric ZnPc has a compositional homogeneity and demonstrated selectivity and reached high uptake in tumors, 48 h after intravenous administration and appears promising for photodynamic therapy.

MF59. Design and evaluation of a safe and potent adjuvant for human vaccines.

MF59 is a safe, practical, and potent adjuvant for use with human vaccines. The formulation is easily manufactured, may be sterilized by filtration, and is both compatible and efficacious with all antigens tested to date. MF59 has been shown to be a potent stimulator of cellular and humoral responses to subunit antigens in both animal models and clinical studies. Toxicology studies in animal models and Phase I-III studies in humans have demonstrated the safety of MF59 with HSV, HIV, and influenza vaccines.

Large-scale production of liposomes containing monomeric zinc phthalocyanine by controlled dilution of organic solvents.

This work describes the development of an organic solvent dilution method suitable for the large scale manufacturing of small unilamellar liposomes containing the water-insoluble photosensitizer zinc phthalocyanine in the monomeric state. N-Methyl pyrrolidone (NMP)/tert-butyl alcohol was selected as water miscible organic phase in which the phospholipids 1-palmitoyl, 2-oleoylphosphatidylcholine (POPC), and 1,2-dioeloylphosphatidylserine (OOPS) and the dye were dissolved. This organic phase was mixed with an excess of a water phase using a dynamic mixing device, yielding reproducibly unilamellar liposomes with a mean size of 50-150 nm as measured with quasielastic light scattering. After concentration, the organic solvents were efficiently removed by cross-flow filtration. The liposomes were then sterile filtered and freeze-dried. A method to measure the aggregation state of the dye in the liposomes was developed. A stable lyophilized formulation containing monomeric liposomal ZnPc could be obtained by using a solution of ZnPc in NMP (2 mg/mL) and ZnPc/phospholipid (1:100 w/w ratio) and performing concentration and dialysis at 4 degrees C and lyophilization in presence of a mixture of lactose and phospholipid (5:1 w/w ratio).

Comparative pharmacokinetics of free muramyl tripeptide phosphatidyl ethanolamine (MTP-PE) and liposomal MTP-PE.

The comparative pharmacokinetics of free MTP-PE (muramyl tripeptide phosphatidyl ethanolamine) and MTP-PE entrapped in negatively charged multilamellar liposomes (liposomal MPT-PE) was evaluated in rats at a bolus intravenous (i.v.) dose of 0.2 mg/kg and in dogs at a bolus i.v. dose of 0.1 mg/kg. Additional studies were performed with the free form in rats (1.4 mg/kg, bolus i.v.) and dogs (1 mg/kg, bolus i.v.) and with the liposomal form in dogs (0.5 mg/kg, bolus i.v.). Plasma samples were obtained at various times up to 48 h postinjection and assayed for the drug by a chemiluminescence immunoassay. The pharmacokinetic data regarding liposomal MTP-PE describe the distribution of free drug released from liposomes and total drug concentrations. The present studies demonstrate that the distribution characteristics of MTP-PE changed dramatically depending on the dosage form. The elimination kinetics of free MTP-PE from blood is substantially slower than that of the liposomal drug. For liposomal MTP-PE, free drug levels in plasma are very low compared with free MTP-PE. In rats at a dose of 0.2 mg/kg, 96% of MTP-PE contained in liposomes is removed from the plasma compartment 10 min after injection, and in dogs at a dose of 0.1 mg/kg, 100% of MTP-PE contained in liposomes is removed in the same time period. This rapid phase of liposome clearance is followed by a slower rate of clearance for the remainder of the liposomes in rats at a dose of 0.2 mg/kg and in dogs at a dose of 0.5 mg/kg.

Optimization of the liposomes encapsulating a new lipopeptide CGP 31362 for efficient activation of tumoricidal properties in monocytes and macrophages.

The purpose of this study was to optimize a suitable liposomal carrier for CGP 31362, a new synthetic lipopeptide analogue of gram-negative bacterial cell walls. CGP 31362 was inserted into the membranes of different liposomes with different phospholipid composition. We determined the ability of these liposomes to activate tumoricidal properties in mouse peritoneal and bone marrow macrophages, and in human monocytes. The ideal liposome carrier for CGP 31362 consisted of phosphatidylcholine and phosphatidylserine in a 7:3 molar ratio. Subsequent to efficient binding and endocytosis, CGP 31362 in liposomes of this composition rendered mouse macrophages and human monocytes highly tumoricidal. Moreover, even in the absence of interferon-gamma, human monocytes released significant levels of tumor necrosis factor and interleukin-1. These data show that in a suitable liposomal carrier, the new synthetic lipopeptide in liposomes is a potent activator of tumoricidal properties in macrophages.

Comparative efficacy of liposomes containing synthetic bacterial cell wall analogues for tumoricidal activation of monocytes and macrophages.

We examined the activation to the tumoricidal state of normal mouse peritoneal exudate macrophages, bone marrow macrophages, and human blood monocytes by liposomes containing either lipophilic muramyl tripeptide (CGP 19,835) or a new synthetic analogue of lipoprotein from gram-negative bacteria outer wall, CGP 31,362, or combinations of the two. The superiority of liposomes containing the synthetic lipopeptide over liposomes containing lipophilic muramyl tripeptide for in vitro activation of monocytes and macrophages was demonstrated in several experiments. First, liposome-CGP-19,835 activated monocytes only in the presence of interferon-gamma, whereas activation with liposome-CGP 31,362 was interferon-independent. Second, activation of both mouse macrophages and human blood monocytes by liposome-CGP 31,362 occurred at a lower liposomal concentration than that by liposome-CGP 19,835. Third, monocytes incubated with liposome-CGP 31,362 released both tumor necrosis factor (TNF) and interleukin-1 activities, whereas monocytes treated with liposome-CGP 19,835 (in the absence of interferon-gamma) released only TNF activity. These data suggest that liposomes containing the synthetic lipopeptide CGP 31,362 are superior to liposomes containing CGP 19,835 for systemic activation of macrophages.

Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles.

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.

The influence of lipid composition and lectin-glycophorin interaction on the rotational diffusion of glycophorin in vesicles, as measured by time-resolved phosphorescence depolarization.

The rotational mobility of glycophorin in various lipid vesicles was studied, using time-resolved measurements of the depolarization of laser flash excited phosphorescence of glycophorin labelled with the triplet probe erythrosin. With the exception of dimyristoylphosphatidylcholine at the phase transition no phosphorescence depolarization decays were observed in the 1-300 microseconds time interval following the laser flash. Instead, a constant anisotropy level was observed, with two distinct values depending on the experimental system. In liquid-crystalline bilayers of dioleoylphosphatidylcholine, bovine brain phosphatidylserine and dimyristolyphosphatidylcholine, the anisotropy was 0.01. This was increased to 0.03 upon addition of wheat germ agglutinin which aggregates glycophorin. In the case of gel state dimyristoylphosphatidylcholine and liquid-crystalline dioleoylphosphatidylethanolamine the anisotropy also amounted to 0.03. Experiments with glycerol to vary the viscosity of the medium, and theoretical considerations, exclude the possibility that these different anisotropy levels are related to differences in motional properties of the entire protein/lipid vesicles. These results strongly suggest that the anisotropy level of 0.03 corresponds to slowly rotating glycophorin (rotational relaxation time greater than 0.3 ms) while the anisotropy level of 0.01 corresponds to fast rotating glycophorin (rotational correlation time less than 1 microseconds). The difference in glycophorin mobility is discussed in terms of aggregation state of the protein, lipid composition of the vesicle bilayer and membrane viscosity. The observed differences in rotational mobility of glycophorin in glycophorin/dioleoylphosphatidylcholine vesicles, glycophorin/bovine heart phosphatidylserine vesicles as compared to glycophorin/dioleoylphosphatidylethanolamine vesicles are not in quantitative agreement with the relative size of the intramembrane particles in these systems as revealed by freeze-fracture electron microscopy.

The influence of lipid composition on the barrier properties of band 3-containing lipid vesicles.

Band 3 protein has been incorporated into lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine or total erythrocyte lipids by means of a Triton X-100 Bio-Beads method, with an additional sonication step prior to the removal of the detergent. This methods results, for both types of band 3 lipid vesicles, in rather homogeneous vesicles with comparable protein content and vesicle trap. Freeze-fracture electron microscopy revealed that band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles have considerably more intramembrane particles as compared to the band 3-erythrocyte lipid vesicles. The dimensions of the nonspecific permeation pathways present in the band 3-lipid vesicles were measured using an influx assay procedure for nonelectrolytes of different size, in which the vesicles were sampled and subsequently freed from nonenclosed labeled permeant by means of gel-filtration. The band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles have nonspecific permeation pathways (pores), with diameters of up to 60 A. In contrast, the band 3-total erythrocyte lipid vesicles are more homogeneous and show much smaller nonspecific permeation pathways, having a diameter of about 12 A. These results suggest that the nonspecific permeability of the band 3-lipid vesicles is strongly lipid-dependent. Increase in specific anion permeability expected as a consequence of the presence of band 3 in the erythrocyte lipid vesicles was found to be very limited. However, stereospecific, phloretin-inhibitable D-glucose permeability could clearly be demonstrated in these vesicles. The difference of the nonspecific permeability of the band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles and band 3-erythrocyte lipid vesicles, is discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce formation of pores.

The influence of lipid composition on glycophorin-induced bilayer permeability.

Glycophorin was incorporated into large unilamellar vesicles and the bilayer permeability was measured as a function of the lipid composition. In agreement with previous data (Van der Steen, A.T.M., De Kruijff, B. and De Gier, J. (1982) Biochim. Biophys. Acta 691, 13-23) it was found that glycophorin greatly increased the bilayer permeability of DOPC vesicles. This effect was observed for a large variety of phosphatidylcholines, differing in their fatty acid composition and homogeneity. In sharp contrast, it was observed that variations in the polar headgroups by incorporation of DOPE, DOPS and, to a lesser extent, cholesterol, into the DOPC/glycophorin vesicles restored the barrier function. These results are compared to the size of the particles, revealed by freeze-fracture electron microscopy on the glycophorin-containing bilayer and are discussed in the light of various types of lipid-protein interactions and protein aggregation state.

The anion permeability of vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane.

Band 3 protein was reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 2500:1 phospholipid:protein molar ratio by means of a Triton X-100/beads method. The SO2-4 permeability of the resulting vesicles was measured using an influx assay procedure in which the vesicles were sampled and subsequently eluted over Sephadex columns at appropriate time intervals. The accuracy of the assay was greatly increased by using an internal standard in order to correct for vesicle recovery. In agreement with previous work, it could be demonstrated that incorporation of band 3 in the vesicles caused an increase in SO2-4 permeability, which could be (partially) inhibited by high concentrations of DIDS or a competitive anion such as thiocyanate. However, the magnitude of the increased SO2-4 permeability was highly variable, even when vesicles were reconstituted using band 3 isolated from one batch of ghosts. In addition, the SO2-4 influx curves showed complex kinetics. These results are related to the existence of vesicle heterogeneity with respect to protein content and vesicle size as revealed by stractan density gradient centrifugation and freeze-fracture electron microscopy. Band 3 incorporation also increased the L-glucose permeability of the vesicles which could also be inhibited by DIDS. Glycophorin, which has no known transport function, reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 400:1 phospholipid:protein molar ration increased the bilayer permeability towards SO2-4 as well as towards L-glucose. Surprisingly, the SO2-4 permeability in the vesicles could also be inhibited by DIDS and thiocyanate. It is concluded that the use of DIDS and a competitive anion, thiocyanate, in order to prove that band 3 is functionally reconstituted, is highly questionable. The increased SO2-4 and L-glucose permeability of band 3-lipid as well as glycophorin-lipid vesicles and the inhibitory action of DIDS are discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce the formation of pores. Since the band 3-lipid vesicles are more permeable for SO2-4 than for L-glucose, in contrast to the glycophorin-containing vesicles, it is suggested that some anion specificity of the increased bilayer permeability in the band 3-lipid vesicles is still preserved.