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).

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.

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.

Real-time monitoring of stain formation and removal on calcium hydroxyapatite surfaces using quartz crystal sensor technology.

Stain formation, stain inhibition and stain removal may be monitored in real-time using a novel method employing a quartz crystal resonance sensor (QCR), based upon quartz crystal microbalance (QCM) technologies. Crystalline hydroxyapatite (HA) surfaces were prepared on phosphate-terminated, polymer-modified gold surfaces of quartz crystal transducers. The resulting sensors were placed in a specially constructed flow cell, and the interaction of adsorbates from the tea stain solution monitored as a function of time. The ability of sodium tripolyphosphate (STP) to remove extrinsic stain and also to inhibit its formation was examined. The adsorption of material from the staining solution passed over the sensor was clearly observable, and once bound, the crystal based real-time data suggest that tea adsorbates were not removed in the absence of an active under conditions of continuous flow. STP was shown to rapidly remove existing stain, and exhibited a clear inhibitory action on stain formation irrespective of whether the HA had been previously exposed to tea chromogens. The continuous data generated by the QCR technique were in good agreement with the results obtained using a discontinuous spectrophotometric method. The presently described quartz crystal model for extrinsic dental stain should provide a valuable tool to aid understanding of the interactions of staining agents with a crystalline HA surface as a model tooth surface, and to evaluate the efficacy and mode of action of STP and putative stain removal agents.

A rapid, non-destructive method for the determination of Staphylococcus epidermidis adhesion to surfaces using quartz crystal resonant sensor technology.

AIMS: To investigate the use of quartz crystal resonant sensor (QCRS) technology to determine the adhesion of Staphylococcus epidermidis to fibronectin-coated surfaces. METHODS AND RESULTS: QCRS sensors (14 MHz) with 4 mm gold electrodes were coated with fibronectin and exposed for 15 min to suspensions of Staph. epidermidis ranging in concentration from 1 x 10(2) to 1 x 10(6) cfu ml(-1). Changes in resonant frequency were recorded and showed a linear relationship with the logarithm of cell concentration over the range tested. CONCLUSIONS: QCRS technology was shown to be a rapid, sensitive and non-destructive method for measuring the adhesion of bacteria to surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This report demonstrates that QCRS technology has the potential to be used for a range of applications requiring measurement of bacteria on surfaces. In particular, it may be used for the real-time monitoring of bacterial biofilm formation.

A quartz crystal resonant sensor (QCRS) study of HSA--drug interactions.

Human serum albumin (HSA) was immobilised on the gold surface of a quartz crystal resonance sensor (QCRS) and exposed to warfarin and diazepam. Distinct decreases in frequency of differing magnitudes were observed upon exposure of the protein to each of the compounds suggesting strongly that a ligand interaction was occurring. Moreover, as sequential exposure in any order was observed to yield distinct repeatable frequency decreases for the ligands indicated, screening for site specific binding may be possible. Identically immobilised bovine serum albumin (BSA) gave no response to either compound.

Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms.

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 x 10(8) purified phage per ml for 24 h at 37 degrees C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10(10) and 10(12) phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.

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.

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.

A standard strain of human ocular keratocytes.

The ability of an injured cornea to regenerate from deep tissue trauma is largely due to wound healing processes mediated by the surviving stromal keratocytes. Despite the importance of the wound healing process, and the ease with which keratocytes can be grown in tissue culture, a standardised strain of the cells has never been made available. Accordingly, this study reports a strain of human embryonic keratocytes, designated EK1.BR as a research tool for the ophthalmic community. EK1.BR has been characterised with respect to life-span, fraction of dividing cells and maintenance of a keratocyte phenotype in culture. It is hoped that these cells will prove useful in the in vitro study of stromal wound healing and the characterisation of keratocyte gene expression.

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.

The development of in vitro biocompatibility tests for the evaluation of intraocular biomaterials.

Recent developments in ocular implant technology require the in vitro evaluation of ocular compatibility in early stage development programs. This requires an understanding and appreciation of the biological interactions which occur in the ocular environment and their relevance with respect to the clinical complications associated with surgical implantation of devices. This paper describes the development of a series of clinically reflective in vitro assays for assessing the potential ocular compatibility of novel intraocular lens materials. Staphylococcus epidermidis attachment, fibrinogen adsorption, mouse embryo fibroblast 3T3 adhesion and proliferation, primary rabbit lens cell adhesion, human peripheral blood macrophage adhesion and granulocyte activation tests were employed to evaluate two widely used intraocular biomaterials poly(methyl methacrylate) (PMMA) and silicone, and a novel biomimetic phosphorylcholine-based coating (PC). The performance of these materials in the in vitro assays was compared to their ability to reduce postoperative inflammation in vivo in a rabbit model. The results demonstrated that the in vitro assays described here are predictive of in vivo ocular compatibility. These assays offer a more relevant means of assessing the ocular compatibility of biomaterials than those presently required by the authorities for regulatory approval of medical devices and implants.

Use of in vitro models of haemofiltration and haemodiafiltration to estimate dosage regimens for critically ill patients prescribed cefpirome.

BACKGROUND: The physico-chemical properties of cefpirome (low protein binding, high water solubility and low molecular weight) suggest that it may be lost readily from the extracorporeal circulation of intensive care unit patients during continuous renal replacement therapy. METHOD: In order to make informed dosage recommendations for patients receiving artificial renal support, cefpirome loss from human blood has been quantified using in vitro models of continuous haemofiltration and haemodiafiltration. Cefpirome clearance was measured using three membrane types at varying ultrafiltrate (UFR) and dialysis flow rates (Qd). RESULTS: During haemofiltration cefpirome was found to cross hollow fibre polyamide (PA) and polyacrylonitrile (PAN) membranes with equal efficiency. The mean sieving coefficients (S) of both PA and PAN membranes were consistently high (> 0.7) when two different ultrafiltration rates were used. Changing the ultrafiltration rate or membrane type had no significant effect on the sieving coefficient of cefpirome but did result in an increase in cefpirome filter clearance (Fcl). Using the haemodiafiltration model, cefpirome penetrated PAN membranes (flat plate AN69S) more efficiently than hollow fibre PA membranes (FH66D). In each case, increasing the dialysis flow rate reduced the S-value. However, although increasing Qd was associated with a greater Fcl of cefpirome when PAN membranes were employed, no such relationship was found for the PA hollow fibre membrane. CONCLUSION: The information generated can be used to estimate a dosing regimen for intensive care patients prescribed cefpirome and receiving continuous renal replacement therapy.

Determination of the surface tension of block copolymer micelles by phagocytosis.

PURPOSE: This work was carried out to determine the surface tension of block copolymer micelles of 14C labelled ABA poly (oxyethylene-bi-isoprene-b-oxyethylene) which have a long circulating half life in animals. METHODS: The method used was that of phagocytosis. The percentage of micelles phagocytosed by human mononuclear cells was determined in solutions of different surface tension. RESULTS: The values obtained were 72 mN/m which may be predicted for a particle with a long circulating half life in animals. The method also gave an estimate of the surface tension for the mononuclear cells. CONCLUSIONS: This technique has the advantage of determining the surface tension of highly hydrated small particles including stable micelles in an environment similar to that in which they normally exist.

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.

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.

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.

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.

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.

Nonmonotonic trends in electrolyte-induced injury to rapidly cooled erythrocytes.

Rapid freeze-thaw injury to erythrocytes and erythrocyte ghosts has been shown to be strongly cation dependent. For the Group I ions this dependence is nonmonotonic in nature with injury increasing in the order Li+ less than Na+ less than Cs+ less than K+. Injury can be reduced by the inclusion in the freezing media of saccharide cryoprotectants or by the substitution with less injurious cations, e.g., Mg2+ or (CH3)4N+. In contrast to the situation observed with cations injury with anions follows Hofmeister lyotropic power series with injury increasing with decreasing hydrated ionic radius. Careful choice of electrolyte species allows injury to be reduced to levels comparable to that afforded by saccharide cryoprotectants. A possible mechanism for the nonmonotonic trends in injury observed with cations is considered.