Fluorescein-labeled Bovine Serum Albumin Research Articles

Effect of Viscosity on Fouling: Investigation into Pre‐Treatment of Saliva Samples for a Point‐of‐care SARS‐CoV‐2 Device

Since the emergence of COVID‐19, the focus has been on curing, vaccinating against, and detecting the virus that spread into virtually every community. With that effort came the need for not just increased testing but also cheap and fast testing. One possible solution is a point‐of‐care device. Similar to a pregnancy test, a device in which users could deposit a saliva sample and receive a measurable result could be accessible to millions all over the world. However, building such a device requires a lot of forethought, including any pre‐treatment of the sample. The goal of pre‐treatment, in this case, is to lessen the variability and complexity of the saliva for testing. Egg white and glycerol solutions were prepared to mimic saliva and determine if viscosity played a role in flux or possible membrane fouling using a custom flux setup designed in the lab for a method to ready samples for a rapid SARS‐CoV‐2 test. By comparing flux rate of fluorescein‐labeled bovine serum albumin (BSA) (0.1 g/L) in phosphate‐buffered saline (PBS) (pH 7.4, 15mM), buffer/glycerol solutions, and egg white solutions through a membrane, the effect of viscosity on fouling of black polycarbonate track‐etched (BPCTE) membranes was observed. Fluorescence imaging of BPCTE membranes allowed for further observation of fouling. Higher viscosity solutions showed a decrease in the measured flux, and no fouling occurred for any solutions. However, egg whites and imaging indicated alternate implications of pre‐treatment for saliva‐based rapid tests.

The impact of calcium phosphate on FITC-BSA loading of sonochemically prepared PLGA nanoparticles for inner ear drug delivery elucidated by two different fluorimetric quantification methods

Although therapeutically active proteins are highly efficacious, their content in protective nanoparticles is often too low to elicit adequate plasma levels. A strategy to increase protein loading is the in-situ generation of calcium phosphate as a protein adsorbent. To verify this approach, a highly sensitive and reliable fluorimetric method for quantification of incorporated fluorescein-labelled bovine serum albumin (FITC-BSA) as a model protein drug was developed. Dequenching the fluorescein label by pronase E, which digests the protein backbone, and dissolving the nanoparticle matrix in acetonitrile enabled FITC-BSA quantification in the nanogram per milliliter range. This test was confirmed by a second assay involving alkaline hydrolysis of FITC-BSA and the matrix. Nanoparticles prepared with calcium phosphate contained 40 µg FITC-BSA/mg and nanoparticles without calcium phosphate only 15 µg FITC-BSA/mg, representing a 2.7-fold increase in model protein loading. In this work the nanoparticle preparation procedure was optimized in terms of size for administration in the inner ear, but the range of applications is not limited.

Controlled Release of Therapeutics from Thermoresponsive Nanogels: A Thermal Magnetic Resonance Feasibility Study.

Thermal magnetic resonance (ThermalMR) accommodates radio frequency (RF)-induced temperature modulation, thermometry, anatomic and functional imaging, and (nano)molecular probing in an integrated RF applicator. This study examines the feasibility of ThermalMR for the controlled release of a model therapeutics from thermoresponsive nanogels using a 7.0-tesla whole-body MR scanner en route to local drug-delivery-based anticancer treatments. The capacity of ThermalMR is demonstrated in a model system involving the release of fluorescein-labeled bovine serum albumin (BSA-FITC, a model therapeutic) from nanometer-scale polymeric networks. These networks contain thermoresponsive polymers that bestow environmental responsiveness to physiologically relevant changes in temperature. The release profile obtained for the reference data derived from a water bath setup used for temperature stimulation is in accordance with the release kinetics deduced from the ThermalMR setup. In conclusion, ThermalMR adds a thermal intervention dimension to an MRI device and provides an ideal testbed for the study of the temperature-induced release of drugs, magnetic resonance (MR) probes, and other agents from thermoresponsive carriers. Integrating diagnostic imaging, temperature intervention, and temperature response control, ThermalMR is conceptually appealing for the study of the role of temperature in biology and disease and for the pursuit of personalized therapeutic drug delivery approaches for better patient care.

Fabrication and In Situ Cross-Linking of Carboxylic-Acid-Functionalized Poly(Ester Amide) Scaffolds for Tissue Engineering

Three-dimensional (3D) scaffolds are important tools for tissue engineering and should ideally provide both biochemical cues and biomechanical support for cells. Poly(ester amide)s (PEAs) have emerged as promising materials for the preparation of tissue engineering scaffolds, and the pendant side chains of residues, such as l-lysine and l-aspartic acid, can provide sites for the conjugation of biochemical signals. However, it has been challenging to combine scaffold morphological stability with the presentation of reactive groups on PEA scaffolds. We describe here a new approach involving the functionalization of a l-lysine-containing PEA with maleic anhydride to simultaneously introduce cross-linkable alkenes and carboxylic acid conjugation sites. Maleic-acid-functionalized PEA was processed to form 3D scaffolds using a salt leaching method and the scaffolds were cross-linked in situ using a poly(ethylene glycol) dimethacrylate cross-linking agent by thermal free radical curing. Microcomputed tomography analysis indicated that the cross-linked scaffolds had higher polymer volume fraction, lower porosity, and smaller pore size than the non-cross-linked scaffolds, but both scaffolds exhibited high morphological stability and negligible mass loss upon incubation in phosphate-buffered saline for 5 days. The Young’s moduli of the cross-linked and non-cross-linked scaffolds were 28 and 9 kPa, respectively. Fluorescein-labeled bovine serum albumin was successfully conjugated to the scaffolds using a carbodiimide-based coupling. Finally, it was shown that the scaffolds supported the attachment and proliferation of mouse embryonic mesenchymal multipotent cells, showing their promise as platforms for tissue engineering applications.

Biomimetic Delivery Strategies at the Urothelium: Targeted Cytoinvasion in Bladder Cancer Cells via Lectin Bioconjugates

Urothelial cells, including bladder cancer (BCa) cells, represent a highly valuable but challenging target for localized antineoplastic therapy. This study describes a novel, biomimetic approach to improve intravesical drug delivery, based on glycan-specific targeting. In direct analogy to the invasion mechanism used by uropathogenic bacteria, we evaluate the potential of lectin bioconjugates to facilitate binding and uptake of large payload molecules at this penetration-hostile barrier. Wheat germ agglutinin (WGA) served as a targeting ligand and was covalently coupled to fluorescein-labeled bovine serum albumin (fBSA), yielding multivalent protein bioconjugates. Cytoadhesion, uptake and intracellular processing were characterized on a panel of urothelial cell lines of non-malignant and malignant origin. Conjugation to WGA rendered the fBSA payload protein strongly cytoadhesive, with a clear preference in binding to cancerous cells. The highly specific, lectin-mediated recognition process was followed by rapid internalization, and extensive but non-exclusive accumulation in acid and LAMP-2-positive compartments. Stage of malignancy and mechano-structural cell configuration were important determinants for the sorting between different processing pathways. Lectin-bioconjugates allow for triggering endogenous uptake routes and influencing the intracellular distribution in BCa cells. They hold considerable promise for enhancing the delivery of small molecule drugs and complex biomolecules in intravesical therapy.

Lectin bioconjugates trigger urothelial cytoinvasion – A glycotargeted approach for improved intravesical drug delivery

A unique structural and functional configuration renders the human urothelium, one of the hardest to overcome biological barriers, and accounts for critical shortcomings in the adjuvant localized therapy of bladder cancer and other severe medical conditions. Strategies to improve intravesical drug absorption are urgently sought, but so far have hardly adopted biorecognitive delivery vectors that are more specifically tailored to the natural characteristics of the target site. The efficient cytoinvasion of uropathogenic bacteria, mediated via a mannose-directed FimH lectin adhesin, and malignancy-dependent differences in bladder cell glycosylation point to considerable unrealized potential of lectins as targeting vectors on the molecular/functional and recognitive level. Here, we outline the ability of wheat germ agglutinin (WGA) to induce endocytosis of conjugated payload in human urothelial SV-HUC-1 cells after stable adhesion to internalizing receptors. A panel of model bioconjugates was prepared by covalently coupling one to six WGA units to fluorescein-labeled bovine serum albumin (fBSA). Cytoadhesive capacity was found to directly correlate to the degree of modification up to a critical threshold of on average three targeting ligands per conjugate. The highly specific, glycan-triggered interaction proved essential for endosomal sorting and was followed by rapid (<60min) and extensive (>40%) internalization. fBSA/WGA bioconjugates were processed analogously to the free lectin, irrespective of the significantly higher molecular weight (100–300kD). Durable entrapment of conjugates in acidic, perinuclear compartments without kiss-and-run recycling to the plasma membrane was found in both single cells and monolayers. Our results assign promising potential to glycotargeted delivery concepts in the intravesical setting and offer new perspectives for the application of complex biologicals in the urinary tract.

Star-Like Oligo-Arginyl-Maltotriosyl Derivatives as Novel Cell-Penetrating Enhancers for the Intracellular Delivery of Colloidal Therapeutic Systems

A novel nonpeptide, multiarmed oligo-arginyl derivative was engineered as a cell-penetration enhancer for the delivery of bioactive macromolecules and colloidal drug systems. Hepta-arginyl-maltotriosylamido-N-acetyl-dodecanoyl acid (Arg(7)-Malt-NAcC(12) acid) was synthesized through a carefully designed multistep chemical protocol, as follows: (1) maltotriose derivatization with 12-amino-dodecanoic acid and acetylation of the free amino group; (2) esterification of the maltotriosyl hydroxyl groups with 2-bromo-isobutyryl bromide; and (3) synthesis of star-like oligomer bearing multiple copies of arginine moieties under atom transfer radical polymerization (ATRP) conditions. The intermediates and final product were characterized by (1)H NMR, IR, mass spectrometry, colorimetric assays, and elemental analysis. Cytotoxicity studies on the final polymeric material showed that this novel cell-penetrating enhancer does not have significant toxic effects on MCF-7 and MC3T3-E1 cell lines. The IC(50) was greater than 100 μM with both cell lines, while the polyethylenimine with similar average molecular mass (M(n)) that was used as a reference showed an IC(50) of 30 and 40 μM, for MCF-7 and MC3T3-E1, respectively. The biological properties of the novel bioconjugate were investigated using a fluorescein-labeled bovine serum albumin (FITC-BSA) as a hydrophilic cargo model. MCF-7 and MC3T3-E1 cells were incubated for 60 min with the Arg(7)-Malt-NAcC(12)-conjugated FITC-BSA [(Arg(7)-Malt-NAcC(12))(2)-FITC-BSA] or FITC-BSA, and the intracellular fluorescence level was analyzed by spectrofluorimetric analysis of cell lysate, cytofluorimetry, and confocal microscopy. The fluorescence of the lysate of MCF-7 and MC3T3-E1 cells that were incubated with (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA at 37 °C was approximately 4.5 times higher than the fluorescence obtained with cells incubated with FITC-BSA. At 4 °C, the cell uptake of (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA was only 2 times higher than that of FITC-BSA. Cytofluorimetric studies showed that, after (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA treatment, over 80% of MCF-7 cells and over 95% of MC3T3-E1 cells displayed enhanced fluorescence. Confocal investigations showed punctuated fluorescence within the cytosol in both cell lines, indicating that (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA was confined to endosomes, with no fluorescence observed in the nucleus.

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith's surface are envisaged for the future development of monoliths with improved enrichment characteristics.

Diffusion of rhodamine B and bovine serum albumin in fibrin gels seeded with primary endothelial cells

Scaffolds with adequate mass transport properties are needed in many tissue engineering applications. Fibrin is considered a good biological material to fabricate such scaffolds. However, very little is known about mass transport in fibrin. Therefore, a method based on the analysis of fluorescence intensity for measuring the apparent diffusion coefficient of rhodamine B and fluorescein-labelled bovine serum albumin (FITC-BSA) is described. The experiments are performed in fibrin gels with and without human umbilical vein endothelial cells (HUVEC). The apparent diffusion coefficients of rhodamine B and FITC-BSA in fibrin (fibrinogen concentration of 4mg/mL) with different cell densities are reported. A LIVE/DEAD® assay is performed to confirm the viability of HUVEC seeded at high densities. Diffusion coefficients for rhodamine B remain more or less constant up to 5×105cells/mL and correlate well with literature values measured by other methods in water systems. This indicates that the presence of HUVEC in the fibrin gels (up to 5×105cells/mL) has almost no effect on the diffusion coefficients. Higher cell densities (>5×105cells/mL) result in a decrease of the diffusion coefficients. Diffusion coefficients of rhodamine B and FITC-BSA obtained by this method agree with diffusion coefficients in water predicted by the Stokes–Einstein equation. The experimental design used in this study can be applied to measure diffusion coefficients in different types of gels seeded or not with living cells.

Application of epifluorescence scanning for monitoring the efficacy of protein removal by RF gas–plasma decontamination

The development of methods for measuring the efficiency of gas–plasma decontamination has lagged far behind application. An approach to measuring the efficiency of protein removal from solid surfaces using fluorescein-labelled bovine serum albumin and epifluorescence scanning (EFSCAN) is described. A method for fluorescently labelling proteins, which are adsorbed and denatured on metal surfaces, has been developed. Both approaches have been used to evaluate the efficiency of radio frequency (RF) gas–plasma decontamination protocols. Examples with ‘real’ surgical instruments demonstrate that an argon–oxygen RF gas–plasma treatment can routinely reduce the protein load by about three orders of magnitude beyond that achieved by current decontamination methods.

Targeting of the β 2-adrenoceptor increases nonviral gene delivery to pulmonary epithelial cells in vitro and lungs in vivo

Coupling of targeting ligands to polyethylenimine (PEI) has been previously used to improve transfection efficiency of PEI gene vectors. Here, we show that the β 2-adrenoceptor (β 2-AR) agonist, clenbuterol (Clen), can be used to improve gene transfer efficiency of PEI gene vectors on alveolar epithelial cells in vitro and in the lungs of mice in vivo. Clenbuterol conjugated to fluorescein-labeled bovine serum albumin resulted in clenbuterol-specific cellular uptake predominantly into alveolar but not bronchial epithelial cells. Clen- g-PEI (4/1) conjugates were combined with increasing molar ratios of PEI for transfection. At optimized PEI- g-Clen/PEI composition, transfection efficiency on alveolar epithelial cells was up to 14-fold higher than for unmodified PEI and could be inhibited by an excess of free clenbuterol. No increase of transfection efficiency was observed on bronchial epithelial cells. Increasing the PEI- g-Clen/PEI molar ratio resulted in an increase of gene vector size, decrease of the zeta potential and cytotoxicity. Aerosol delivery of optimized PEI- g-Clen/PEI (1/5) gene vectors resulted in a significant 3-fold increase of gene expression in the lungs of mice compared with unmodified PEI gene vectors. We suggest that coupling of β 2-adrenoceptor ligands to nonviral gene vectors represents a promising approach to improve gene delivery to the lungs.

Development of Immunoaffinity Restricted Access Media for Rapid Extractions of Low-Mass Analytes

Restricted access media using antibodies as immobilized ligands were developed for the rapid and selective capture of small analytes by immunoextraction, giving rise to materials referred to as immunoaffinity restricted access media (IA-RAM). To make such a material, intact antibodies for the desired target were first immobilized onto porous silica, with antibodies at or near the outer surface of the support then being treated with papain (or a related agent) to release and remove their binding domains. The result was a support in which only antibodies deep within the pores remained intact and able to bind to the target. Items evaluated in the development of such media included the immobilization method used for the antibodies, the pore size of the support, and the amount of papain and time that were used for support treatment. A theoretical model was also developed to describe the extent of binding domain removal based on the measured polypeptide content of the IA-RAM support before and after treatment with papain. The final optimized conditions for making the IA-RAM supports were used to prepare columns that contained antifluorescein antibodies. Injections of fluorescein and fluorescein-labeled bovine serum albumin onto these IA-RAM columns gave selective and quantitative extraction of fluorescein in 1-2 s. This approach can be used with other antibodies and low-mass targets and should be valuable for such applications as the rapid separation of drugs from drug-protein complexes or the isolation of labeled/modified peptides from intact proteins that contain the same modification or label.

Improved method for fluorophore deposition atop a polyelectrolyte spacer for quantitativestudy of distance-dependent plasmon-assisted luminescence

A layer-by-layer deposited polyelectrolyte spacer is used to attach CdSe nanocrystals(NCs), dye molecules and fluorescein-labelled bovine serum albumin (BSA-FITC) to flatglass and rough silver island surfaces in order to study the effect of spacer thickness onhomogeneity and surface concentration of fluorophore coverage. Three different methods offluorophore deposition atop the polyelectrolyte spacer are examined using steady-statespectroscopy, fluorescent microscopy and statistical analysis. The best homogeneouscovering with nanocrystals at a controllable concentration was found for deposition from asolution of NCs which are electrostatically bound to polyelectrolyte macromolecules. Thisfluorophore deposition method allows one to avoid artefacts and to evaluate thefluorescence enhancement factor of BSA-FITC adsorbed on silver island films.

Increased permeability of intestinal epithelial monolayers mediated by electroporation

This study assessed whether electroporation enhances transport across intact intestinal epithelial monolayers that mimic the intestinal epithelium. Confluent Caco-2 monolayers were exposed to electroporation pulses and then monitored over time for transepithelial transport of calcein, a small fluorescent tracer, or fluorescein-labeled bovine serum albumin, a large protein. Cumulative transport of both molecules across the monolayers increased significantly (up to 34-fold) after electroporation and depended on electroporation voltage and pulse length and on molecular size. Increased transport was accompanied by a decrease in the transepithelial electrical resistance of the monolayers. Further analysis of these results suggests that the increase in transport observed after electroporation is due, at least in part, to the killing of a small fraction of cells, which increased transport across “leaky” dead cells that remained adherent and increased transport through small, temporary holes left by dead cells that detached, but appeared to reseal within minutes by monolayer restitution. These findings could form the basis for the development of electroporation as a clinical tool to increase intestinal permeability and, thereby, increase the absorption of poorly absorbed drugs.

Direct Determination of Effective Interfacial Optical Constants by Nonlinear Optical Null Ellipsometry of Chiral Films

Nonlinear optical null ellipsometry (NONE) measurements of chiral interfaces allowed direct experimental measurement of the linear interfacial optical constants in surface second harmonic generation (SHG) measurements. Since phase information is retained in NONE measurements, the real and imaginary components of the interfacial refractive index (n and k, respectively) were uniquely obtained from the measured chiral chi((2)) tensor elements of a fluorescein-labeled bovine serum albumin film. The sensitivity of the calculated chi((2)) tensor elements on the assumed values of the interfacial optical constants allowed measurements of n and k to four significant figures with no additional adjustable parameters and independent of molecular symmetry. The optical constants measured by SHG agreed within a relative error of 0.8% with values predicted independently using a simple effective medium approximation, also with no adjustable parameters. Additionally, those same optical constants produced relationships between the achiral chi((2)) tensor elements in excellent agreement with predictions for systems exhibiting weak orientational order. This study suggests that the far-field intensity and polarization state of the nonlinear optical beam may be largely independent of the near-field optical constants within the interfacial layer in the limit of a film thickness much less than the wavelength of light.

Electroporation-mediated delivery of molecules to model intestinal epithelia

This study was conducted to determine if electroporation can deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. In addition, the long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. Caco-2 and T84 cells were electroporated as monolayers using calcein and fluorescein-labeled bovine serum albumin as marker molecules for measuring delivery into cells. Confocal microscopy and flow cytometry were used, respectively, to visualize and quantify uptake of these molecules. Transepithelial resistance was used as a measure of physiologic barrier function. We found that intracellular uptake of calcein and bovine serum albumin occurred uniformly throughout both types of model epithelia and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. We also observed that monolayers exposed to electroporation that induced uptake of up to 10 6 molecules/cell were able to recover normal barrier function within one day. These findings suggest that electroporation may be useful for intracellular delivery into monolayers to study epithelial biology and, possibly, for drug delivery to intestinal epithelium.

A hydrogel prepared by in situ cross-linking of a thiol-containing poly(ethylene glycol)-based copolymer: a new biomaterial for protein drug delivery

A new poly(ethylene glycol)-based copolymer containing multiple thiol (–SH) groups was cross-linked in situ to form a polymer hydrogel under mild conditions. No organic solvent, elevated temperature, or harsh pH is required in the formulation or patient administration processes, making it particularly useful for delivery of fragile therapeutics, such as proteins. The in vitro release of fluorescein-labeled bovine serum albumin and the in vivo release of the model proteins, erythropoietin, RANTES and three PEG-conjugated RANTES derivatives showed sustained release for 2–4 weeks and demonstrated prolonged biological activity of the released proteins in animals.

Lectin-mediated drug delivery: binding and uptake of BSA-WGA conjugates using the Caco-2 model

To examine whether the dietary lectin wheat germ agglutinin (WGA) can facilitate binding and uptake of protein drugs due to its cytoadhesive and cytoinvasive properties, conjugates were prepared by covalent coupling of fluorescein-labeled bovine serum albumin (F-BSA) to WGA using divinylsulfone for crosslinking. Increasing the molar ratio of F-BSA/WGA resulted in 2.6–8.7 times higher Caco-2 binding as compared with glycyl-F-BSA. About 75% of F-BSA-WGA were bound specifically to Caco-2 cells according to inhibition studies in presence of the complementary carbohydrate. The Caco-2 association of F-BSA-WGA was temperature-dependent indicating active uptake of membrane bound conjugate, which was confirmed by confocal microscopy. The conjugate accumulated within lysosomal compartments followed by proteolytic degradation of F-BSA-WGA 1–4 h after conjugate loading as observed by equilibrating the intracellular pH with monensin. Finally low molecular weight degradation products of the proteinaceous prodrug appear in the extracellular medium. Contrary to Caco-2 single cells, a minor part of the conjugate is degraded by brush border proteases already 30 min after exposure to Caco-2 monolayers. But most of the conjugate is taken up into differentiated cells and processed as in single cells. Though the enzymic barrier remains to be surmounted, WGA-mediated drug delivery is a promising strategy for peroral delivery of even high molecular weight drugs to overcome the mucosal barrier.

Azithromycin, a lysosomotropic antibiotic, impairs fluid-phase pinocytosis in cultured fibroblasts

The dicationic macrolide antibiotic azithromycin inhibits the uptake of horseradish peroxidase (HRP) by fluid-phase pinocytosis in fibroblasts in a time- and concentration-dependent fashion without affecting its decay (regurgitation and/or degradation). The azithromycin effect is additive to that of nocodazole, known to impair endocytic uptake and transport of solutes along the endocytic pathway. Cytochemistry (light and electron microscopy) shows a major reduction by azithromycin in the number of HRP-labeled endocytic vesicles at 5 min (endosomes) and 2 h (lysosomes). Within 3 h of exposure, azithromycin also causes the appearance of large and light-lucentlelectron-lucent vacuoles, most of which can be labeled by lucifer yellow when this tracer is added to culture prior to azithromycin exposure. Three days of treatment with azithromycin result in the accumulation of very large vesicles filled with pleiomorphic content, consistent with phospholipidosis. These vesicles are accessible to fluorescein-labeled bovine serum albumin (FITC-BSA) and intensively stained with filipin, indicating a mixed storage with cholesterol. The impairment of HRP pinocytosis directly correlates with the amount of azithromycin accumulated by the cells, but not with the phospholipidosis induced by the drug. The proton ionophore monensin, which completely suppresses azithromycin accumulation, also prevents inhibition of HRP uptake. Erythromycylamine, another dicationic macrolide, also inhibits HRP pinocytosis in direct correlation with its cellular accumulation and is as potent as azithromycin at equimolar cellular concentrations. We suggest that dicationic macrolides inhibit fluid-phase pinocytosis by impairing the formation of pinocytic vacuoles and endosomes.

Development of sandwich HPLC microcolumns for analyte adsorption on the millisecond time scale.

A new class of columns is reported that uses only microgram quantities of active support and that provides for the retention of biological compounds and other analytes on the millisecond time scale. This was accomplished by packing standard HPLC supports into layers as small as 60 microm in length and using only 90 microg of support material. This provided columns with effective residence times in the millisecond time range when routine HPLC flow rates and pressures were used. The retention of analytes by such columns was examined under both adsorption- and diffusion-limited conditions. The RPLC adsorption of hemoglobin (a system with diffusion-limited retention) was found to give 95% binding in as little as 4 ms. The adsorption of fluorescein by an anti-fluorescein antibody column (an adsorption-limited system) gave 95% retention in 100-120 ms. One application examined for these columns was their use in a chromatographic-based competitive binding immunoassay. This used bovine serum albumin (BSA) as the model analyte, and fluorescein-labeled BSA was used for detection. The resulting approach had a contact time of 180 ms between the sample and an anti-BSA immunoaffinity microcolumn and provided a signal within 5-25 s after sample injection. The columns developed in this work should also be useful in other situations that involve a small amount of a stationary phase or that require short column residence times.