Effect of hydrophilic swellable polymers on dissolution enhancement of carbamazepine solid dispersions studied using response surface methodology

The objective of this work was to study dissolution enhancement efficiency and solid dispersion formation ability of hydrophilic swellable polymers such as sodium carboxymethyl cellulose (Na-CMC), sodium starch glycolate (SSG), pregelatinized starch (PGS), and hydroxypropylmethyl cellulose (HPMC) with carbamazepine using 3² full factorial design for each of the polymers. Solid dispersions of carbamazepine were prepared using solvent evaporation method with around 70% solvent recovery. The independent variables were the amount of polymer and organic solvent. The dependent variables assessed were percentage drug dissolved at various time points and dispersion efficiency (ie, in terms of particle size of solid dispersion). Solid dispersions were evaluated for percentage drug dissolved, wettability, differential scanning calorimetry, scanning electron microscopy, and angle of repose. Multiple linear regression of results obtained led to equations, which generated contour plots to relate the dependent variables. Similarity factor and mean dissolution time were used to compare dissolution patterns obtained in distilled water and simulated gastric fluid United States Pharmacopeia (USP) XXVI of pH 1.2. Maximum drug dissolution was obtained with polymer order Na-CMC>SSG>PGS>HPMC. Particle size of drug was reduced ≈ 10–15, 3–5, 5–7, and 10–25 times in Na-CMC, SSG, PGS, and HPMC solid dispersions, respectively; whereas wettability of solid dispersions was found in the order of Na-CMC>HPMC>PGS>SSG. Angle of repose was found to be in the range of 29° to 35° for all solid dispersions, which shows good flowability characteristics. HPMC showed increase in drug dissolution up to an optimized level; however, furthers increase in its concentration decreased drug dissolution. Published: April 6, 2007     

Surface modification of polymers: chemical, biological and surface analytical challenges

Surface modification methods can optimise the biocompatibility or the specificity of biointeraction of a biosensor or medical device. With only the surface modified, the manufacture and implantation protocol remain unchanged. This review article summarises some of the chemical, surface analytical and biological challenges associated with surface modification of biosensors and biomedical devices.     

Effect of unstirred layers on binding and reaction kinetics at a membrane surface

The effects of solution unstirred layers on the time course of chemical reactions and transport processes at a membrane surface are determined. A set of equations which describes non-steady-state diffusion through an unstirred layer coupled with chemical reaction at a membrane surface or transport through a membrane is developed. A numerical solution to the equations is obtained by uncoupling diffusive and chemical processes in an iterative manner. The diffusive process is solved by the Crank-Nicolson method; the chemical process is solved by integrating the differential equations describing the kinetics. Diffusive processes in one dimension, in three dimensions, and in the presence of an arbitrary potential near the membrane surface are solved. General characteristics of the calculated reaction time course are discussed using surface binding and membrane transport examples. Small, neglected, unstirred layers are shown to sometimes yield erroneous values of rate parameters for a surface reaction and to simulate competitive reaction kinetics. Experimental approaches for measuring unstirred layer thickness are reviewed.     

Measurement of specific surface energy of the protein hydration shell using spin label EPR

An approach to measure the microscopic surface tension in a water-protein matrix using spin label EPR is proposed. The approach utilizes the parameters of the viscosity isotherms for the correlation time of the spin labels bound to a protein macromolecule. The pattern of changes in the specific surface energy in the protein hydration shell depending on the protein concentration, structure of reporter group, ligand state of protein, and the presence of salts, water-soluble polymers, and D₂O in solution was studied using serum albumin, hemoglobin, and antibodies as examples. Possible causes underlying the observed changes in surface tension are discussed.     

A preliminary study of modification of gait in real-time using surface electromyography

Modification of abnormal gait was attempted in real-time using a surface electromyography-based protocol to teach recruitment of the anterior tibialis at the correct time in the gait cycle. Two children diagnosed with cerebral palsy were able to learn volitional control of the anterior tibialis as demonstrated by improved clearance of the toe on the swing phase of the gait and newly learned ability to recruit and relax the anterior tibialis. The children were able to walk with the new gait pattern and reproduce the old one at will. Implications for future research in this area are discussed.     

细胞表面的物理化学修饰

移植排斥是生物学中的重大问题,其理论涉及众多生物学科,包括免疫学、生物化学、分子生物学、生理学、细胞生物学、实验血液学等;其实践涉及同种和异种细胞、组织、器官移植,关系到肿瘤、糖尿病、急性放射病、免疫缺陷症、器官衰竭等数以百万计患者的治疗和健康。使用化学材料聚乙二醇、海藻酸钠、壳聚糖、多聚赖氨酸等,发挥化学、物理、生物学科交叉的优势,在移植物细胞表面进行物理化学反应,修饰和改造细胞表面抗原分子,有望为克服移植排斥反应开辟新的途径。     

Insulin receptor‐insulin interaction kinetics using multiplex surface plasmon resonance

Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor‐insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real‐time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR‐A). We used a scaffold approach in which anti‐insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR‐A capture. The multiplex SPR system (ProteOn XPR36?, Bio‐Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two‐site binding pattern, consistent with the literature, with a high‐affinity site (dissociation constant K_D1 = 38.1 ± 0.9 nM) and a low‐affinity site (K_D2 = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants K_D1 = 73.2 ± 1.8 nM and K_D2 = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high‐affinity site was converted to the low‐affinity site. The new SPR assay enables insulin‐eIR‐A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling. Copyright © 2013 John Wiley & Sons, Ltd.     

Studies on interaction of oligopeptides with sodium dodecyl sulfate: Stopped-flow kinetics of chemical modification of tryptophan residues withN-bromosuccinimide

The stopped-flow kinetics of the reaction between oligopeptides containing tryptophan residues andN-bromosuccinimide (NBS) were studied in 50 mM sodium phosphate buffer (pH 7.0) containing sodium dodecyl sulfate (SDS). Decreases in the reaction rates attributable to the interaction between oligopeptides and SDS were observed, and oligopeptides studied were classified into types I and II on the basis of the interaction modes. Type I oligopeptides were dissolved in SDS micelles; type II oligopeptides interacted cooperatively with SDS monomers. The manner of interaction between SDS and oligopeptides of type II could be interpreted by a simple equilibrium relation: oligopeptide+n·(SDS)=oligopeptide·(SDS) _n .     

Evaluation of simple enzyme kinetics by response surface modelling

The activity of a horse liver alcohol dehydrogenase catalysed reduction of cyclohexanone was investigated by using a central composite circumscribed design in which two parameters (pH and cyclohexanone concentration) were varied. By log transformation of the substrate concentration an adequate model could be obtained from which reliable kinetic constants and pH profiles were determined.     

Effect of hydration on the morphology of enzyme powder

We report the first direct images of the hydration of protein powders. Using an environmental scanning electron microscope (ESEM) we have taken a series of micrographs of a region of the enzyme (subtilisin) power whilst hydrating the sample. In addition, the sample has been viewed during exposure to toluene vapors. The ESEM is a remarkable new instrument that will have wide applicability in imaging of biological materials in their native environments.     

Effect of hydration on assessment of early enamel lesion using swept‐source optical coherence tomography

Establishing reproducible methodologies for assessment of early enamel lesions using optical coherence tomography (OCT) appears to be challenging. This in vitro study longitudinally evaluated the subsurface enamel lesion progression after 3, 9 and 15 days by cross‐sectional scanning using 1310 nm centered swept‐source OCT (SS‐OCT) under hydrated and dry conditions. The positive difference between the depth‐integrated OCT signals at dry and hydrated conditions were calculated and adopted as dehydration parameter (DH). A linear regression was found between DH and the square root of demineralization time (R² = 0.99). Significant differences were found in DH between sound and demineralized enamel, and between different periods of demineralization (p < 0.001). Hydration state affects the reflectivity of demineralized porous enamel, and the effect can be potentially used for assessment of early enamel lesion using OCT. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of mineral nutrients on the kinetics of methane utilization by methanotrophs

The effect of different mineral nutrients on the kinetics of methane biodegradation by a mixed culture of methanotrophic bacteria was studied. The substrate factors examined were ammonia, iron, copper, manganese, phosphate, and sulphide. The presence of iron in the growth medium had a strong effect on the yield coefficient. Yield coefficients up to 0.49 mg protein per mg methane were observed when iron was added at concentrations of 0.10–5.0 mg/l. Iron addition also increased the maximum methane utilization rate. The same effect was observed after addition of ammonium to a medium where nitrate was the only nitrogen source. The observed Monod constant for methane utilization increased with increasing concentration of ammonia. This shows that ammonia is a weak competitive inhibitor as observed by other researchers. Relatively high levels of both ammonia (70 mg/l) and copper (300 µg/l) inhibited the methane degradation, probably due to the toxic effect of copper-amine complexes.     

Enzymatic surface modification of cellulose acetate fibre by cutinase-CBM (carbohydrate-binding module) fusion proteins

Abstract

In this paper, two types of bacterial fusion protein, cutinase-CBM_Cel6A and cutinase-CBM_CenA, were used to modify the surface of cellulose acetate fibre. The enzyme binding on cellulose acetate fibres and the hydrolysis of acetyl groups were monitored. Water absorbency and dye uptake were measured to assess the extent of enzymatic modification. The results demonstrated that cutinase-carbohydrate-binding module (CBM) has a greater effect on cellulose acetate fibres than that of cutinase. The use of non-ionic surfactant Triton X-100 could further improve enzymatic modification of cellulose acetate fibres in terms of wettability and dyeability. Scanning electron microscopy confirmed that both cutinase-CBMs could lead to the formation of carving characters on the surface of treated cellulose acetate fibres. Our studies provide a foundation for the potential application of cutinase-CBM in the surface modification of cellulose acetate fibre.     

Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor

The prevalence of aromatic residues in the ligand binding site of the GABA_A receptor, as with other cys‐loop ligand‐gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here, we have examined three conserved tyrosine residues at the GABA binding pocket (β₂Tyr97, β₂Tyr157, and β₂Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK‐293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABA_A receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β₂Tyr157 and β₂Tyr205 are more detrimental than β₂Tyr97 mutations, particularly to the GABA binding rate. Overall, the results suggest that interactions involving multiple tyrosine residues are likely during the binding process.     

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected.     

Effect of fiber type and fabric moisture content on the hydration state of human stratum corneum

1. 1. The purpose of this study was to determine the effect of fiber type and fabric moisture content on SC hydration.

2. 2. Using three similarly constructed fabrics, six fabric type/moisture content combinations were selected.

3. 3. Fabric swatches were placed on both “normal” and “hydrated” volar forearm skin of five subjects for a specified period, then removed.

4. 4. Two minutes after removal, evaporative water loss (EWL) and skin temperature were measured.

5. 5. Data were analyzed using analyses of variance and Bonferroni t-tests.

6. 6. For normal skin, SC hydration generally increased as fabric moisture content increased. SC was significantly drier after being in contact with cotton swatches at regain than at the two moisture content levels above regain, and also under polyester swatches.

7. 7. For hydrated skin, hydration state was significantly lower under the cotton swatch at regain than at 38.6% moisture content or at saturation, but was not significantly different under the polyester swatch at regain or at saturation.

Effect of aminophospholipid glycation on order parameter and hydration of phospholipid bilayer

The effect of aminophospholipid glycation on lipid order and lipid bilayer hydration was investigated using time-resolved fluorescence spectroscopy. The changes of lipid bilayer hydration were estimated both from its effect on the fluorescence lifetime of The 1-[4-(trimethylammonium)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and 1,6-diphenylhexa-1,3,5-triene (DPH) and using solvatochromic shift studies with 1-anilinonaphthalene-8-sulfonic acid. The head-group and acyl chain order were determined from time-resolved fluorescence anisotropy measurements of the TMA-DPH and DPH. The suspensions of small unilamellar vesicles (with phosphatidylethanolamine/phosphatidylcholine molar ratio 1:2.33) were incubated with glyceraldehyde and it was found that aminophospholipids react with glyceraldehyde to form products with the absorbance and the fluorescence properties typical for protein advanced glycation end products. The lipid glycation was accompanied by the progressive oxidative modification of unsaturated fatty acid residues. It was found that aminophospholipid glycation increased the head-group hydration and lipid order in both regions of the membrane. The lipid oxidation accompanying the lipid glycation affected mainly the lipid order, while the effect on the lipid hydration was small. The increase in the lipid order was presumably the result of two effects: (1) the modification of head-groups of phosphatidylethanolamine by glycation; and (2) the degradation of unsaturated fatty acid residues by oxidation.     

Effect of lysine modification on the stability and cellular binding of human amyloidogenic light chains

AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescence microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichroism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.     

Attachment and growth kinetics of anchorage-dependent BHK cells on microcarriers

Anchorage-dependent Baby Hamster Kidney (BHK) cells were cultivated on polyhydroxyethylmethacrylate (PHEMA), polystyrene (PS), and Cytodex microcarriers. Analysis of the experimental data indicated that there were a finite number of sites on the microcarrier surfaces, available for anchorage. The number of these sites was determined by the chemical and physical structure of the surface. A small fraction of these sites were suitable for attachment of the cells before proliferation. A larger fraction of these sites did not support attachment but the cells could proliferate on them by the help of previously attached mother cells. The attachment and proliferation of the BHK cells on these microcarriers were satisfactorily modeled by surface saturation type of mathematical expressions.