Hydrophobic Ion Pairing Research Articles

Encapsulation and controlled release of lysozyme from electrospun poly(ε-caprolactone)/poly(ethylene glycol) non-woven membranes by formation of lysozyme–oleate complexes

In this study, the concept of hydrophobic ion pairing was adopted for incorporating lysozyme into electrospun poly(epsilon-caprolactone) (PCL)/poly(ethylene glycol) (PEG) non-woven membranes. The solubility of lysozyme in organic solvent was enhanced through the formation of lysozyme-oleate complexes, which could be directly loaded into PCL/PEG membranes using electrospinning technique. The resultant PCL/PEG nanofibers have a compact structure with an average diameter ranged from about 0.4 microm to 0.9 microm. The addition of PEG into the PCL nanofibers not only improved the hydrophilicity of the membrane, but also played an important role on in vitro lysozyme release rate. It was found that the release rate of lysozyme was enhanced with the increase of PEG content. In addition, the increase of salt concentration in the release medium accelerated lysozyme release. It has also been shown that the released lysozyme retained most of its enzymatic activity.

Organic Nanoparticles of Cyanine Dye in Aqueous Solution

Abstract Ion-based organic dye nanoparticles ranging from tens to hundreds of nanometers have been prepared in aqueous solution. The synthetic approach is based on “ion-association” (hydrophobic ion-pair formation) in water between the dye cation pseudoisocyanine (PIC) and the anion tetraphenylborate (TPB) or tetrakis(4-fluorophenyl)borate (TFPB) ion. Size tuning of spherical and amorphous PIC nanoparticles was accomplished by varying the molar ratio (ρ) of the loaded anion to the dye cation. Electrophoretic characterization revealed that the increase in surface adsorption of anions onto PIC particles brings about an increase in the negative surface charge density, causing a reduction in the surface tension and the mean size of nanoparticles. We found that the optical absorption properties of the PIC nanoparticles exhibited (i) matrix dependence, that is, the 0–0 band position of the PIC chromophore within similar-sized nanoparticles was dependent on the type of counter anions used, and (ii) size dependence, i.e., the 0–0 band position of the PIC chromophore was dependent on the mean nanoparticle diameter when the particles were prepared using the same counter anion.

Spectrofluorometric Determination of Tiotropium Bromide by Ion Pair Extraction Using 9, 10 Dimethoxyanthracene-2-sulphonate Sodium

A rapid, simple and specific fluorometric procedure is described for the analysis of the anticholinergic drug, tiotropium bromide (TIO) in bulk and in capsule formulation. The method is sensitive and is based on the reaction of this drug as a quaternary ammonium compound with the fluorogenic reagent 9, 10-dimethoxyanthracene-2-sulphonate (DMOAS) as counter ion in acidic medium and the proposal of the reaction pathway was postulated. The hydrophobic ion-pair formed was extracted in dichloroethane and measured spectrofluorometrically at 450 nm after excitation at 385 nm. The fluorescence intensity-concentration plot was rectilinear over the ranges of 7-30 ng ml-1 y=mx+b, r2=0, 9991 (m =12.054; b =156.27) . The detection limit was 0.0326 ng ml-1 based upon United States Pharmacopeia (USP 2002) . The different parameters affecting the reaction pathway were studied and optimized. The proposed method was successfully applied to the analysis of tiotropium capsules. No interference was observed from pharmaceutical excepients such as lactose.

Characterization of physiochemical and biological properties of an insulin/lauryl sulfate complex formed by hydrophobic ion pairing

An insulin/lauryl sulfate complex was prepared by hydrophobic ion pairing (HIP). The physiochemical and biological properties of the HIP complex were characterized using octanol/water partition measurement, isothermal titration calorimetry (ITC), ultraviolet-circular dichroism (UV-CD) and Fourier transform infrared spectroscopy (FTIR). Sodium dodecyl sulfate (SDS) bound to the insulin in a stoichiometric manner. The formed complex exhibited lipophilicity, and its insulin retained its native structure integrity. The in vivo bioactivity of the complex insulin was evaluated in rats by monitoring the plasma glucose level after intravenous (i.v.) injection, and the glucose level was compared with that for free insulin. The pharmacodynamic study result in rats showed that the complex insulin had in vivo bioactivity comparable to free insulin.

Hydrophobic ion pairing to enhance encapsulation of water-soluble additives into CO 2-swollen polymer microparticles

Ionic dyes were successfully encapsulated in colloidal polymer particles by a CO 2-based microencapsulation technique. The water-soluble dyes were made hydrophobic by forming ion pairs with several types of alkyl quaternary ammonium cations. The hydrophobic ion pairs were then encapsulated in preexisting size monodisperse polystyrene particles dispersed in water. High-pressure carbon dioxide swelled and plasticized polystyrene and thus facilitated mass transport of the dye into the particles. The results show that the particles maintain their size and morphology after exposure to CO 2, and that ion-paired dyes have significantly higher loading in the polymer particles than the original dyes. The effects of cosolvent and surfactant are also discussed.

Solubility of an acid dye in supercritical carbon dioxide by ion-pairing with dodecyltrimethylammonium bromide

In this study, solubility of Acid Red 57 (AR57), which is an anionic dye, in supercritical CO 2 was investigated using an ion-pairing with dodecyltrimethylammonium (DTMA) bromide. The measurements of AR57 and AR57-DTMA in supercritical CO 2 without/with methanol as a modifier solvent were carried out at temperatures from 45 to 100 °C and pressures from 250 to 325 bar. Although AR57 is insoluble in supercritical CO 2 even with methanol as a modifier solvent, AR57-DTMA can dissolve in methanol modified supercritical CO 2. The hydrophobic ion-pairing provides a possibility for the solubility of hydrophilic dye in supercritical CO 2. The solubilities of AR57-DTMA were measured in methanol modified supercritical CO 2 and the results were correlated by using a semi-empirical equation.

Development and validation of a stability-indicating analytical method for the quantitation of oxytocin in pharmaceutical dosage forms

A single stability-indicating assay for oxytocin (OT) in pharmaceutical dosage forms using gradient elution over 21 min has been reported in the literature. Furthermore, published and compendial methods for the analysis of OT containing dosage forms also involve using HPLC with gradient elution and complicated mobile phases that include hydrophobic ion pairing agents. A simple isocratic and stability-indicating assay was developed and validated. The conditions are as follows, column: Phenomenex ® C 18 Hypersil, 5 μm packing, 4.6 mm × 150 mm with acetonitrile–phosphate buffer (pH 5; 0.08 M) (20:80) as the mobile phase with UV detection at 220 nm The method was found to be specific for OT in the presence of degradation products and chlorbutol (preservative) with an overall analytical run time of 16 min. Accuracy was determined to be 0.77–1.18% bias for all samples tested. Intra-assay precision (repeatability) was found to be 0.22–1.04%R.S.D. while the inter-day precision (intermediate precision) was found to be 1.27–1.68%R.S.D. for the samples studied. The calibration curve was found to be linear with the equation y = 1.81 x + 0.02 and a linear regression coefficient of 0.9991 over the range 0.4–12.0 IU/ml. The LOD and the LOQ were determined to be 0.1 and 0.4 IU/ml, respectively. Syntocinon ®, a commercially available dosage form of OT was assayed resulting in 100.5–106.6% recovery of the label claim and an average of 10.04 IU/ml.

Counterion‐dye staining for DNA in electrophoresed gels using indoine blue and methyl orange

In this study, we describe a sensitive staining method for DNA in agarose and polyacrylamide gels using organic visible dyes, indoine blue (IB) and methyl orange (MO). The counterion-dye staining method uses two oppositely charged dyes to form a hydrophobic ion pair complex in the staining solution. A decrease in the number of free forms of dyes in staining solution can enhance the selectivity of binding between the dye and DNA, and can reduce nonspecific background staining. As a result, the sensitivity of counterion-dye staining was significantly improved compared with other dye-based staining. This method uses a staining solution consisting of 0.008% IB, 0.002% MO, 10% ethanol and 0.2 M sodium acetate at pH 4.7, and can detect 5 ng of lambda DNA/HindIII within 60 min in agarose gels and 10 ng of PhiX174 DNA/HaeIII within 20 min in polyacrylamide gels.

Nanoprecipitation versus emulsion-based techniques for the encapsulation of proteins into biodegradable nanoparticles and process-related stability issues

The goal of this study was to investigate the entrapment of 3 different model proteins (tetanus toxoid, lysozyme, and insulin) into poly(D,L-lactic acid) and poly(D,L-lactic-co-glycolic acid) nanoparticles and to address process-related stability issues. For that purpose, a modified nanoprecipitation method as well as 2 emulsion-based encapsulation techniques (ie, a solid-in oil-in water (s/o/w) and a double emulsion (w(1)/o/w(2)) method) were used. The main modification of nanoprecipitation involved the use of a wide range of miscible organic solvents such as dimethylsulfoxide and ethanol instead of the common acetone and water. The results obtained showed that tetanus toxoid and lysozyme were efficiently incorporated by the double emulsion procedure when ethyl acetate was used as solvent (>80% entrapment efficiency), whereas it was necessary to use methylene chloride to achieve high insulin entrapment efficiencies. The use of the s/o/w method or the formation of a more hydrophobic protein-surfactant ion pair did not improve protein loading. The nanoprecipitation method led to a homogenous population of small nanoparticles (with size ranging from approximately 130 to 560 nm) and in some cases also improved experimental drug loadings, especially for lysozyme (entrapment efficiency > 90%). With respect to drug content determination, a simple and quick matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method provided results very close to those obtained by reverse phase-high-performance liquid chromatography. With respect to protein stability, the duration and intensity of sonication were not a concern for tetanus toxoid, which retained more than 95% of its antigenicity after treatment for 1 minute. Only a high methylene chloride:water ratio was shown to slightly decrease toxoid antigenicity. Finally, no more than 3.3% of A21 desamido insulin and only traces of covalent insulin dimer were detected in nanoparticles. In conclusion, both the double emulsion and nanoprecipitation methods allowed efficient protein encapsulation. MALDI-TOF MS allowed accurate drug content determination. The manufacturing processes evaluated did not damage the primary structure of insulin.

Structure of ATP-dependent phosphoenolpyruvate carboxykinase fromThermus thermophilusHB8 showing the structural basis of induced fit and thermostability

In order to understand the induced fit and the thermostabilization mechanisms of ATP-dependent phosphoenolpyruvate carboxykinase, the crystal structure of the enzyme from the extreme thermophile Thermus thermophilus HB8 (TtPEPCK) was determined and compared with those of orthologues of known structure from two mesophilic organisms. The protomer structures in these orthologues, which exhibit open/closed interdomain conformations, are similar. Isomorphous crystals of unliganded and ATP-bound TtPEPCK were obtained. The asymmetric units of both crystal forms contain two protomers A and B with closed and open conformations, respectively. ATP was only observed in the interdomain cleft of the closed protomer, suggesting that the induced fit of TtPEPCK agrees with the so-called ;conformational selection' mechanism where ligand binding is not essential for domain closure although its binding leads to the stabilization of the closed state. A bound calcium observed in the N-terminal domain of TtPEPCK probably contributes to the thermal stability. A combination of hydrophobic effects, ion pairs and entropic effects might also contribute to the thermostability of TtPEPCK.

Measurement and correlation of solubility of Acid Red 57 in supercritical carbon dioxide by ion-pairing with hexadecyltrimethylammonium bromide

Solubility of C.I. Acid Red 57 (AR57) in supercritical carbon dioxide was measured by ion-pairing with hexadecyltrimethylammonium (HDTMA) bromide. The solubility measurements of AR57 and AR57-HDTMA in supercritical carbon dioxide without/with methanol as a modifier solvent were carried out at the temperature range from 35 to 75 °C and for pressures from 250 to 325 bar. The solubility of AR57 and AR57-HDTMA was examined in terms of pressure and temperature of supercritical carbon dioxide. Even though Acid Red 57 is insoluble both in supercritical carbon dioxide and methanol-modified supercritical carbon dioxide, AR57-HDTMA can easily dissolve in methanol-modified supercritical carbon dioxide. The hydrophobic ion-pairing of HDTMA provides a possibility to dissolve a hydrophilic dye in supercritical carbon dioxide. A semi-empirical equation was used to correlate the obtained experimental solubilities of AR57-HDTMA by means of the density of carbon dioxide in methanol-modified supercritical carbon dioxide.

Extended thermodynamic approach to ion interaction chromatography. Influence of the chain length of the solute ion: a chromatographic method for the determination of ion-pairing constants

The influence of the chain length in Ion Interaction Chromatography (IIC), previously named Ion Pair Chromatography, was investigated. The system examined is a C18 stationary phase and a variable, salt-controlled, mixture of a phosphate buffer pH 2.1 in methanol containing tetrabutylammonium bromide as Ion Interaction Reagent. IIC proves to be a good alternative to the well established spectrophotometric and conductometric methods of obtaining thermodynamic ion-pairing constants. The latter increase with increasing analyte chain length and decrease with increasing methanol concentration in the eluent: both results support a physical chemical description of the hydrophobic ion-pairing process at variance with the classical, Bjerrum-type modelling of the purely electrostatic interaction between inorganic ions. The calculated deltaG(o) are reliable because they compare to non-chromatographic estimates of deltaG(o) for similar systems. The present extended thermodynamic approach is able to predict and explain why the purely electrostatic approach works better for lower analyte lipophilicity or higher organic modifier concentration.

Degradation kinetics of high molecular weight poly(L-lactide) microspheres and release mechanism of lipid:DNA complexes

Plasmid DNA encoding the green lantern protein was ion-paired with 1,2-dioleoyl, 3-trimethylammonium propane (DOTAP) at a (+/−) charge ratio of (1:1) to form a hydrophobic ion-pair (HIP) complex using the Bligh and Dyer method, and transferred into methylene chloride. Precipitation with a compressed antisolvent (PCA) was then employed to encapsulate plasmid DNA into poly(L-lactide) (PLLA) microspheres. The hydrophobicity of DOTAP:DNA complexes allowed consistently high encapsulation efficiencies (>70%) to be achieved. Release of the DOTAP:DNA complex from PLLA microspheres exhibitedminimal burst and a short (ca. 1 week) lag phase, followed by sustained release over a 20 week period. Release kinetics were consistent with a simple Fickian diffusion model. No correlation was identified between release rate of soluble poly(L-lactide) species (≤10 lactate units) from PLLA and the DNA release kinetics. Only ~12% of the polymer was degraded into soluble poly(L-lactide) over the time frame where ~90% of the plasmid load had been released.

Improved potency of cisplatin by hydrophobic ion pairing

Cisplatin is one of the most widely used and effective chemotherapeutic drugs ever discovered against certain forms of cancer. However, its use is limited by toxicity. A more potent form might allow lower doses to be used and would diminish the toxicity. A new analog of cisplatin has been synthesized by stoichiometric replacement of the chloride ligands with the anionic surfactant, Aerosol OT (AOT). The new compound has a very low aqueous solubility (about 2 mg/l) and a log P value of 2.17, which is more than 4 log units higher than cisplatin itself, indicating a dramatic increase in hydrophobicity. While hydrophobic cisplatin analogs have been synthesized previously, this is the first one with readily dissociable ligands replacing the chlorides. The resultant AOT complex is able to penetrate cellular membranes more efficiently, resulting in a threefold to fivefold increase in intracellular platinum levels. These increased intracellular concentrations correlate with lower IC50 values in a number of cancer and normal cell lines. These findings suggest that further development of the AOT complex as a chemotherapeutic agent is warranted, given its marked increase in potency over the parent compound.

Kinetics of degradation and oil solubility of ester prodrugs of a model dipeptide (Gly-Phe).

Oil-based depot formulations may constitute a future delivery method for small peptides. Thus, a requirement is attainment of sufficient oil solubility for such active compounds. A model dipeptide (Gly-Phe) has been converted into lipophilic prodrugs by esterification at the C-terminal carboxylic acid group. The decomposition kinetics of octyl ester of Gly-Phe (IV) has been investigated at pH 7.4 (37 degrees C) and IV was shown to degrade by first-order kinetics via two parallel pathways (1) intramolecular aminolysis resulting in formation of a 2,5-diketopiperazine and (2) hydrolysis of the ester bond producing the dipeptide. The cyclisation reaction was dominating in the decomposition of methyl (II) butyl (III) octyl (IV) decyl (V) and dodecyl (VI) esters of Gly-Phe at pH 7.4. However, this degradation pathway was almost negligible for pH below 6. During degradation of the dipeptide esters in 80% human plasma pH 7.4 (37 degrees C) a minimal amount of cyclo(-Gly-Phe) was formed. A faster degradation of the esters in 80% human plasma pH 7.4 compared to those in aqueous solution pH 7.4 was suggested to be due to fast cleavage of the peptide bond. Low oil solubilities for Gly-Phe and the hydrochlorides of the dipeptide esters III and VI were observed. Although the solubility of Gly-Phe in oil solutions was enhanced by hydrophobic ion pairing with sodium decyl sulfonate the oil solubility was still less than 1 mg Gly-Phe/ml. By addition of a solubiliser, 10% N,N-dimethylacetamide (DMA), to Viscoleo the solubility of the HIP complexes increased significantly. The present study indicates that sufficient oil solubility might only be obtained for relatively small peptides by using the prodrug approach in combination with solubility enhancing organic solvents like DMA.

Correlation between Electric Potential and Interfacial Tension Oscillations in a Water−Oil−Water System

Oscillations of the electric potential and of the interfacial tension were simultaneously recorded in a water−oil−water system (cetyltrimethylammonium bromide (CTAB) in water/picric acid in dichloromethane/water). Both types of oscillations were perfectly correlated in time and shape. In each oscillatory cycle, the fast jumps in the electric potential are related to a rapid adsorption of the surfactant at the interface while the slow motion relaxations correspond to the liberation of the interface from the surfactant by the formation of hydrophobic cetyltrimethylammonium picrate ion pairs that diffuse into the CH2Cl2 phase. The fast inflow of CTAB to the interface is probably caused by periodically occurring convective Marangoni instabilities. The transitions to the convective regime are assumed to be controlled by the fractional surfactant coverage of the interface.

Solution structure of the 30 kDa polysulfide-sulfur transferase homodimer from Wolinella succinogenes.

The periplasmic polysulfide-sulfur transferase (Sud) protein encoded by Wolinella succinogenes is involved in oxidative phosphorylation with polysulfide-sulfur as a terminal electron acceptor. The polysulfide-sulfur is covalently bound to the catalytic Cys residue of the Sud protein and transferred to the active site of the membranous polysulfide reductase. The solution structure of the homodimeric Sud protein has been determined using heteronuclear multidimensional NMR techniques. The structure is based on NOE-derived distance restraints, backbone hydrogen bonds, and torsion angle restraints as well as residual dipolar coupling restraints for a refinement of the relative orientation of the monomer units. The monomer structure consists of a five-stranded parallel beta-sheet enclosing a hydrophobic core, a two-stranded antiparallel beta-sheet, and six alpha-helices. The dimer fold is stabilized by hydrophobic residues and ion pairs found in the contact area between the two monomers. Similar to rhodanese enzymes, Sud catalyzes the transfer of the polysulfide-sulfur to the artificial acceptor cyanide. Despite their similar functions and active sites, the amino acid sequences and structures of these proteins are quite different.

Submicrometric lipobead-based fluorescence sensors for chloride ion measurements in aqueous solution.

This paper describes the preparation and optimization of the analytical properties of fluorescence-based submicrometric chloride ion sensing lipobeads. Fluorescence sensing lipobeads are polystyrene nanoparticles that are coated with a phospholipid membrane that contains a fluorescent indicator for a targeted analyte. In this study, the halide-specific fluorescence dye, lucigenin, was immobilized into the phospholipid membrane of the lipobeads to enable chloride ion detection. The fluorescence intensity of lucigenin decreases with increasing chloride ion concentration due to dynamic quenching. Lipobeads that contained only lucigenin were ineffective as chloride ion sensors due to poor partition of the water-soluble lucigenin molecules into the phospholipid membrane and high leakage rate of immobilized lucigenin molecules to the aqueous solution. To stabilize the chloride ion sensing lipobeads we coimmobilized hexadecanesulfonate molecules into the phospholipid membrane. The formation of ion pairs between hexadecanesulfonate and lucigenin decreased the hydrophilicity of the dye, increased its partition rate into the membrane, increased the brightness of the particles, and significantly decreased the leakage rate of the hydrophobic ion pair from the membrane to the solution. To further improve their chloride ion sensitivity, we also immobilized the chloride ionophore [9] mercuracarborand-3 into the lipobead membrane. The study resulted in a unique submicrometric chloride ion sensor, which is suitable for chloride ion measurements in biological fluids.

Solubilization of an indicator dye for optical fiber chemical sensors in supercritical CO 2 by ion pairing

Bromothymol blue (BTB), the indicator dye for optical fiber chemical sensors was lipophilized by ion pairing with quaternary ammoniums, dodceyltrimethylammonium, myristyltrimethylammonium, hexadecyltrimethyammonium and tetrahexylammonium. The dissolution of BTB and the four ion pairs in supercritical CO 2 without/with ethanol as the co-solvent were experimentally investigated. Although BTB is insoluble in supercritical carbon dioxide even with ethanol as the co-solvent, the four hydrophobic ion pairs formed can dissolve in ethanol modified supercritical carbon dioxide. It was found that pressure, temperature, the co-solvent concentration in CO 2, and the chemical structure of the quaternary ammonium, influenced the dissolution of ion pairs in supercritical CO 2. The hydrophobic ion pairing provides the possibilities for solubilization of hydrophilic indicators in supercritical CO 2 and applications of supercritical fluid techniques to chemical optical sensors.

A spot test for aqueous phosphate by color band formation.

Phosphate is generally analyzed by color intensity of phosphoantimonylmolybdenum blue (PAMB): especially by a spot test based on spectrophotometric or visual determination. Visual determination is one of the simplest and most inexpensive methods; however, it is believed to be of low precision. In this work, we have developed an alternative phosphate spot test, where phosphate concentration is measured by the color band length of PAMB formed in a detection tube similar to a gas detection tube. The color band is formed by the entrapment of the hydrophobic ion pair of PAMB and quaternary ammonium ions in the column; we demonstrated that this color band length correlates quantitatively with the phosphate concentration. In order to optimize the measurement performance, systematic investigations have been carried out for various experimental parameters such as phosphate concentration, preparation conditions of the column packing material, the type of support material (either silica-gel or PVC), the solution feeding conditions (filtration or penetration), and volume of the sample solution. The effects from some co-existing components were also studied for the measurement of real samples. Determination of phosphate concentrations in actual wastewaters was successfully carried out by this method, indicating that the quantification range achievable was of 3-18 mg PO(4) L(-1) without dilution prior to the analysis. No interference was observed from suspended solid (SS) and organic pollutants (COD, TOC) during the wastewater analysis. Because of the simplicity and rapid processing of the samples (not requiring any special instruments) this newly developed method can be applied to the on-site analysis of wastewater.