Alcohol ethoxylates mediate their bacteriostatic effect by altering the cell membrane of Escherichia coli NCTC 8196.

The minimum inhibitory concentration (MIC) of a homologous series of alcohol ethoxylates with the same head group size (E6) but differing in the number of carbon atoms in their 'tail group' from 10 to 16 was determined for Staphylococcus aureus NCTC 4163 and Escherichia coli NCTC 8196 using a turbidimetric assay. All the surfactants tested demonstrated bacteriostatic activity against both organisms. A tetrazolium assay showed that C14E6 and C16E6 had little effect on the membrane-bound dehydrogenase enzyme activity of E. coli NCTC 8196 compared with C10E6 and C12E6. C10E6 caused leakage both of K(+) and nucleotides in a concentration-dependent manner above its MIC of 0.2 mM. C12E6 caused some leakage at concentrations below its MIC (0.12 mM).

SPR analysis of the total reduction of protein adsorption to surfaces coated with mixtures of long- and short-chain polyethylene oxide block copolymers.

PEO/PPO/PEO triblock copolymers have previously been shown to reduce the binding of proteins to a variety of surfaces. In this study, mixtures of long- and short-chain copolymers have been shown to adhere to gold substrate surface plasmon resonance slides. The mixtures have been shown to significantly reduce the binding of BSA to gold surfaces, compared to the more commonly used long chain PEO copolymers. These mixtures have been shown to be more effective, than either short, or long-chain copolymers used individually, complementing a published theoretical treatise of PEO surfactant behaviour towards protein interaction with surfaces.

Surface modification of albumin microspheres.

Submicron sized hydrophobic and hydrophilic albumin microspheres (MS) were prepared using a chemical crosslinking technique. Spermine was linked to the surface of the hydrophilic MS. The degree of hydrophobicity for these three types of MS was investigated using a novel technique of sedimentation volume. The surface tension of the hydrophobic MS was 31 mN m-1. The ST of the hydrophilic MS was 68 mN m-1, whereas the surface tension of spermine-linked MS corresponded to 62, 65.5, 69 and 71 mN m-1 indicating heterogeneous hydrophilic characteristics. Ligands can be successfully linked to MS using a water-soluble carbodiimide.

ac Polarography for tetracycline analysis.

The electrode processes for the reduction of several tetracyclines by ac polarography were examined. In pH 4.0 Walpole acetic acid-acetate buffer, two main waves occurred; the first was quasireversible and the second was reversible. Results showed that the first wave can readily be used for quantitative work. The second wave would also be suitable provided that there was no interference from other electroreducible substances.

Dielectric relaxation spectroscopy and some applications in the pharmaceutical sciences.

With a few exceptions, dielectric relaxation spectroscopy (DRS) has been largely neglected by pharmaceutical scientists, despite the potential for this technique as a noninvasive and rapid method for the structural characterization and quality control of pharmaceutical materials. DRS determines both the magnitude and time dependency of electrical polarization (i.e. the separation of localized charge distributions) by either measuring the ability of the material to pass alternating current (frequency domain DRS) or by investigating the current that flows on application of a step voltage (time domain DRS). DRS is thus (i) sensitive to molecular mobility and structure, (ii) non-invasive, and (iii) employs only mild stresses (a weak electromagnetic field) in order to measure the sample properties. The technique covers a broad-band frequency window (from 10(-5) to 10(11) Hz) and therefore enables the investigation of a diverse range of processes, from slow and hindered macromolecular vibrations and restricted charge transfer processes (such as proton conductivity in nearly dry systems) to the relatively fast reorientations of small molecules or side chain groups. The dielectric response provides information on (i) structural characteristics of polymers, gels, proteins, and emulsions, (ii) the interfacial properties of molecular films, (iii) membrane properties, (iv) water content and states of water (and the effects of water as a plasticizer), and (v) lyophilization of biomolecules. This review article details the basis of dielectric theory and the principles of measuring dielectric properties (including a comprehensive account of measurement artifacts), and gives some applications of DRS to the pharmaceutical sciences.

Effects of plasma irradiation on the wettability and dissolution of compacts of griseofulvin.

In this study, the use of plasma irradiation was investigated as a possible technique for increasing the dissolution rate of the poorly soluble drug griseofulvin. Plasma is a partially ionised gas consisting of ions, electrons and neutral species. Oxygen plasma was used to treat griseofulvin compacts as this would lead to the formation of oxygen containing functional groups on the surface of the compact thus increasing the wettability. Compacts containing 300 mg of the drug were prepared using a stainless steel punch and die assembly and plasma treated. The effect of the length and power of the plasma treatment upon the dissolution rate of griseofulvin was investigated. Dissolution experiments of griseofulvin were carried out using the paddle method using 0.1 M HCl and 0.1 M HCl with 2% sodium dodecyl sulphate (SDS) as the dissolution media. The wettability was assessed by contact angle measurements using the sessile drop technique with the contact angle being measured every second for a period of ten seconds using pure water (to European Pharmacopoeia standards). Plasma treated and untreated samples were also analysed by scanning electron microscopy. Although plasma treatment was found to increase the wettability of griseofulvin it was not found to increase the dissolution rate as the treatment caused surface fusion of the material.

Application of the quartz crystal microbalance to the monitoring of Staphylococcus epidermidis antigen-antibody agglutination.

The change in solution properties due to the agglutination of an antigen with its specific antibody has previously been used as a marker of infection. This method has been modified to allow the binding activity between species to be followed using the frequency response of a quartz crystal microbalance (QCM). The Bayston agglutination plate assay for Staphylococcus epidermidis has been modified to allow the electrode of a QCM to act as a direct sensor for the change in solution properties as agglutination occurs. Antibody and antigen were introduced to the crystal surface and the agglutination process was followed as a change in crystal resonant frequency. Serum, known to be infected with the organism, gave a titre of 3.9x10(-2)% v/v (-118 Hz, +/-12 SD, N = 9) matching that given by triplicate plate assay. Uninfected serum gave no frequency changes at this concentration, yielding a titre of 2.5x10(-2)% v/v again matching the plate titre (N = 3). Infected serum gave responses 40 times faster then those of the uninfected serum. The piezoelectric quartz crystal method gave a positive or negative diagnosis in <15 min compared with the 24 h required for the plate assay.

A study of modified betaines as cryoprotective additives.

Glycinebetaine and N-modified betaines have been previously shown to be effective at reducing leakage from liposomes on freeze-thaw procedures. This study involved the preparation of a series of other modified betaines and the comparison of their abilities to reduce leakage from frozen multilamellar liposomes. All the compounds investigated, with the exception of the octyl ester of betaine, reduced the degree of leakage on freezing and thawing with additive concentrations up to 0.6 M. The betaine esters were less effective than betaine as cryoprotective additives and caused an increase in the leakage from unfrozen liposomes. Taurinebetaine, a sulphobetaine, was also less effective at reducing leakage on freezing than betaine and again increased leakage from unfrozen liposomes. Increasing the number of methylene groups between the carboxylate group and the nitrogen improved the ability to reduce leakage, particularly at lower additive concentrations.

Factors influencing cryoprotective activity and drug leakage from liposomes after freezing.

The effects of freezing and thawing conditions and cryoprotective additives on release of streptomycin from lecithin liposomes following freeze-thaw cycles have been investigated. Drug retention was maximized by slow cooling (approx. 1 degree C min-1). At temperatures between 0 degree and -20 degrees C, the extent of drug loss was time-dependent indicating incomplete freezing; below -45 degrees C this effect was abolished and the system was stable. Osmotic gradients across the liposome membrane during freezing were found to have little effect on drug loss. Marked cryoprotective activity was shown by dimethylsulphoxide, glycerol, alanine and glycinebetaine at concentrations of 3% w/v or less. At this concentration sucrose and mannitol had little activity.

A comparative investigation of glycinebetaine and dimethylsulphoxide as liposome cryoprotectants.

The release of streptomycin from lecithin liposomes following a freeze-thaw cycle was used to measure the cryoprotective activities of glycinebetaine and dimethylsulphoxide (DMSO). At concentrations between 4 and 8% w/v in the external solution, glycinebetaine was superior to DMSO at freezing rates faster than 50 degrees C min-1. At lower rates their activities were similar, and drug loss ranged between 10 and 20% depending upon freezing rate and cryoprotectant concentration. The pattern of streptomycin loss when the concentrations of cryoprotectants inside and outside the liposome were varied indicated that glycinebetaine, in contrast to DMSO, does not diffuse across the liposome membrane. The activity of glycinebetaine was not impaired by the presence in the membrane of cholesterol or charged lipids.

The retention of entrapped molecules within erythrocyte ghosts during cryopreservation.

In view of the interest in erythrocyte ghosts and carrier erythrocytes as potential drug delivery systems, this work was undertaken to determine conditions facilitating the retention of entrapped molecules during cryopreservation. Upon freeze-thaw treatment intact erythrocytes and erythrocyte ghosts displayed different damage profiles with respect to cryoprotectant concentration. Non-penetrating cryoprotectants showed optimum protection of intact cells at 0.4-0.5 M; this optimum was not observed with ghosts, in which damage decreased with concentration up to 1.0 M. The concentration optimum for intact cells was not abolished by oxidative or reductive treatments suggesting that its absence in ghosts is not due to altered protein-protein or protein-lipid interactions. The extent of freeze-thaw damage to ghosts was influenced by the qualitative ionic composition of a cryoprotectant-free suspending medium, with 10-12% haemolysis observed in the presence of Li+ and Mg2+ but greater than 60% for Na+, Cs+, K+ and NH4+ with increasing loss following that order. The release on freezing of entrapped haemoglobin, insulin and sucrose was found to be inversely proportional to their molecular weights.

Effects of oxygen plasma treatment on the surface wettability and dissolution of furosemide compacts.

The plasma irradiation of furosemide (frusemide) was investigated as a possible technique for increasing the dissolution rate of this drug. Oxygen plasma was used to generate oxygen-containing functional groups on the surface of the compact to increase the wettability of the surface and the dissolution rate of the drug. Compacts of furosemide (300 mg) were produced using a stainless steel die and punch assembly, which was placed into a KBr press. The time of the plasma treatment was varied to assess the effect if any upon the dissolution rate and the wettability of the drug. Dissolution experiments of the plasma-treated and untreated compacts were carried out using the paddle apparatus method. Dissolution was carried out at 37 degrees C using 1 L of 0.1 M HCl and phosphate buffer (pH 6). The wettability was assessed by contact angle measurements using the sessile drop technique. Untreated and plasma-treated samples were analysed by scanning electron microscopy at x 5000 magnification. Plasma treatment was found to lower the equilibrium contact angle from approximately 50 to 35 degrees but the dissolution rate was not significantly affected. This was attributed to fusion of the surface by the plasma treatment.

A comparison of glycine, sarcosine, N,N-dimethylglycine, glycinebetaine and N-modified betaines as liposome cryoprotectants.

Glycinebetaine has previously been shown to be effective at reducing leakage from liposomes which are frozen then thawed. This study involved the preparation of a series of N-modified betaines and the comparison of their cryoprotective activities with those of glycine, sarcosine, N,N-dimethylglycine and glycinebetaine. All the compounds investigated, with the exception of (dimethyloctylammonio)acetate, reduced the degree of leakage, after freezing and thawing, with additive concentrations up to 0.6 M. Reducing the degree of N-terminal methylation of glycinebetaine appeared to increase the leakage from liposomes at additive concentrations between 0.2 and 0.6 M. (Dimethylethylammonio)acetate, (dimethylisopropylammonio)acetate and (N,N,N',N'-tetramethylethylenediammonio)-N,N'-diacetate appeared to be no more effective than glycinebetaine, whereas improved protection was afforded by (triethylammonio)acetate and (diethylmethylammonio)acetate at most concentrations. This study demonstrates that the cryoprotective activity of glycinebetaine may be improved with modifications to the N-terminal.

A novel image-analysis technique for measurement of bacterial cell surface tension.

Cell-surface hydrophobicity is different for Staphylococcus epidermidis cells grown under different environmental conditions; this might influence attachment and colonization of surfaces. Although a wide variety of techniques has been employed to measure bacterial surface hydrophobicity, including contact angle determinations, adherence to hydrocarbons, hydrophobic-interaction chromatography and salt aggregation, many of these either require large numbers of cells or do not yield comparable quantitative data. This study describes a novel, quantitative method for the determination of bacterial surface tension on the basis of image analysis of cell-cell interactions. S. epidermidis (strains 900 and 901) were suspended in different concentrations of propanol of known surface tension and examined by bright-field microscopy linked via a charge-couple device (CCD) camera to an image analyser. Frames were chosen randomly and the data recorded as a ratio of count/percentage coverage for each frame. The results showed that for strains 900 and 901 this ratio was maximum at surface tensions of 67 and 61 mN m(-1) respectively. At these values of minimal interaction the surface tension of the liquid was equal to the bacterial cell surface tension. The results were in close agreement with those obtained from contact angles. The advantage of surface tension measurements is that, irrespective of the method used, the results generated are quantitative values and are therefore directly comparable. The method reported is reliable, reproducible and is of particular value because the number of cells required is, typically, at least two orders of magnitude lower than is required for commonly used alternative methods.

Reversed-phase chromatographic behaviour of beta-endorphin: evidence of conformational change.

The effect of alteration in isocratic mobile phase constituents, composition of sample solution, flow-rate and column temperature on the reversed-phase chromatographic behaviour of beta-endorphin was investigated. Beta-Endorphin was shown to be particularly sensitive to the concentration of organic modifier within the mobile phase. The relative contact area of beta-endorphin was demonstrated to be less than that of the much smaller molecule, gamma-endorphin, indicating that beta-endorphin is in a folded form under the mobile phase conditions utilised. Buffer molarity and pH were implicated in the conformational transition of beta-endorphin. In addition, the micro-environment of beta-endorphin prior to its injection onto the high-performance liquid chromatographic (HPLC) column is crucial to its chromatographic behaviour. Manipulation of the sample solvent environment produced reversible conformational modifications ultimately resulting in asymmetric and even split peaks. This phenomenon was more clearly seen when altering HPLC flow-rate. Elevation of HPLC column temperature provided additional evidence of structural change in beta-endorphin, with further conformational forms of this molecule being observed at higher temperatures. This work suggests that the chromatography of beta-endorphin involves a complex mechanism of separation which cannot be adequately explained by the two-state model of kinetic processes.

Measurement of beta-endorphin in human plasma by high-performance liquid chromatography with electrochemical detection: validation of a method employing the simultaneous purification and concentration of beta-endorphin.

The simultaneous purification and concentration of synthetic human beta-endorphin from plasma is described, which when used together with an appropriate isocratic high-performance liquid chromatographic-electrochemical detection (HPLC-ED) system allows the determination of elevated physiological levels of beta-endorphin. Purification of plasma was gained by flash-freezing in liquid nitrogen, acidifying with 100 microliters of trifluoroacetic acid (10%, v/v) per ml of plasma, thawing at 4 degrees C and centrifuging to remove any precipitate. Solid-phase extraction with silica sorbent was utilised, which allowed further isolation of the analyte, a method of concentration and a procedure whereby beta-endorphin could be transferred to the HPLC mobile phase. Silica sorbent demonstrated greater selectivity than C18 for synthetic human beta-endorphin and, in addition, provided improved recovery of this analyte when utilising elution volumes of 500 microliters or less. Proteolytic degradation and heparin-induced high-affinity binding in plasma were shown not to effect the recovery of beta-endorphin if blood was rapidly chilled and plasma quickly obtained, frozen and acidified. Validation of this purification/concentration method using [125I]beta-endorphin demonstrated a recovery of 85.6% which was not jeopardised when concentrating the sample twenty-fold. This provided an increase in the sensitivity of detection, when used in conjunction with HPLC-ED, from 5 ng/ml to 250 pg/ml.