Improvement of the HPLC analysis of anthocyanins in red wines by use of recently developed columns

Comparison of the separation performance of five newly developed pH-stable HPLC columns is described for the analysis of anthocyanins in red wines. Separation of twenty anthocyanins in a single run is described using the most appropriate method.     

Novel anisotropic supramolecular hydrogel with high stability over a wide pH range

The hydrolysis of the carboxylic ester bond, by a base or catalyzed by an enzyme under weak basic conditions, serves as the only path to obtain a novel anisotropic supramolecular hydrogel that is stable over a wide pH range. This result not only expands the molecular scope of supramolecular hydrogelators but also illustrates the design principles for creating pH-stable supramolecular soft materials.     

Fluorous affinity purification of oligonucleotides

[structure: see text] Nucleoside phosphoramidites bearing a fluorous dimethoxytrityl (FDMT) group were used to synthesize fluorous-tagged oligonucleotides, which were subjected to solid-phase extraction using a pH-stable fluorinated adsorbent. On-column detritylation afforded the purified oligonucleotides. The fluorous affinity purification method offers one-pass loading without ammonia removal, high selectivity for the removal of failure sequences, high recoveries (typically 70-100%), and the ability to purify long oligonucleotides (e.g., 50-100-mers).     

Micellar electrokinetic capillary chromatography using polymeric hollow fibers

Summary In this paper, polymeric hollow fibers prepared from pH-stable polypropylene were used as columns for micellar electrokinetic capillary chromatography (MECC). The electroosmotic flow (EOF) for polypropylene hollow fibers was evaluated in the pH range of 5.0–12.0. With untreated polypropylene hollow fibers a stabilized but enhanced EOF was achieved when SDS was used in the buffer, decreasing the separation window for uncharged substances in MECC to impractical levels. Uncharged acrylamide and charged 2-acryloylamido-2-methylpropane sulfonic acid surface modifications were used to lower the strength of the EOF, increase the separation window and prevent local overheating that could melt the column wall.     

In-channel atom-transfer radical polymerization of thermoset polyester microfluidic devices for bioanalytical applications

A new technique for polymer microchannel surface modification, called in-channel atom-transfer radical polymerization, has been developed and applied in the surface derivatization of thermoset polyester (TPE) microdevices with poly(ethylene glycol) (PEG). X-ray photoelectron spectroscopy, electroosmotic flow (EOF), and contact angle measurements indicate that PEG has been grafted on the TPE surface. Moreover, PEG-modified microchannels have much lower and more pH-stable EOF, more hydrophilic surfaces and reduced nonspecific protein adsorption. Capillary electrophoresis separation of amino acid and peptide mixtures in these PEG-modified TPE microchips had good reproducibility. Phosducin-like protein and phosphorylated phosducin-like protein were also separated to measure the phosphorylation efficiency. Our results indicate that PEG-grafted TPE microchips have broad potential application in biomolecular analysis.     

Controlled microfluidic encapsulation of cells, proteins, and microbeads in lipid vesicles

Cells have been encapsulated inside lipid vesicles by using a new microfluidic lipid vesicle formulation technique. Lipid vesicles are formulated within minutes without using toxic lipid solvents. The encapsulation efficiency inside the vesicles is controlled by the microfluidic flows. Green fluorescent proteins (GFP), carcinoma cells, and bead encapsulated vesicles have mean diameters of 27.2 mum, 62.4 mum, and 55.9 mum, respectively. The variations of vesicle sizes are approximately 20% for the GFP and cell encapsulated vesicles and approximately 10% for the bead encapsulated vesicles.     

Surfactant vesicles for high-efficiency capture and separation of charged organic solutes

We demonstrate the unique ability of catanionic vesicles, formed by mixing single-tailed cationic and anionic surfactants, to capture ionic solutes with remarkable efficiency. In an initial study (Wang, X.; Danoff, E. J.; Sinkov, N. A.; Lee, J.-H.; Raghavan, S. R.; English, D. S. Langmuir 2006, 22, 6461) with vesicles formed from cetyl trimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS), we showed that CTAT-rich (cationic) vesicles could capture the anionic solute carboxyfluorescein with high efficiency (22%) and that the solute was retained by the vesicles for very long times (t1/2 = 84 days). Here we expand on these findings by investigating the interactions of both anionic and cationic solutes, including the chemotherapeutic agent doxorubicin, with both CTAT-rich and SDBS-rich vesicles. The ability of these vesicles to capture and hold dyes is extremely efficient (>20%) when the excess charge of the vesicle bilayer is opposite that of the solute (i.e., for anionic solutes in CTAT-rich vesicles and for cationic solutes in SDBS-rich vesicles). This charge-dependent effect is strong enough to enable the use of vesicles to selectively capture and separate an oppositely charged solute from a mixture of solutes. Our results suggest that catanionic surfactant vesicles could be useful for a variety of separation and drug delivery applications because of their unique properties and long-term stability.     

Exosome-Mimetic Supramolecular Vesicles with Reversible and Controllable Fusion and Fission**

The fusion and fission behaviors of exosomes are essential for the cell-to-cell communication. Developing exosome-mimetic vesicles with such behaviors is of vital importance, but still remains a big challenge. Presented herein is an artificial supramolecular vesicle that exhibits redox-modulated reversible fusion-fission functions. These vesicles tend to fuse together and form large-sized vesicles upon oxidation, undergo a fission process and then return to small-sized vesicles through reduction. Noteworthy, the aggregation-induced emission (AIE) characteristics of the supramolecular building blocks enable the molecular configuration during vesicular transformation to be monitored by fluorescence technology. Moreover, the presented vesicles are excellent nanocarrier candidates to transfer siRNA into cancer cells.     

Rigid polymerics: the future of oligonucleotide analysis and purification

A family of rigid macroporous HPLC materials, reversed phase and anion exchange, has been evaluated for the analysis and purification of a range of de-protected, dimethoxytrityl-off, oligonucleotides. A 25-base pair (bp) double-stranded DNA ladder was used to determine the resolving range for the four pore sizes of reversed-phase media. The 100 A pore size resolves up to 50-75 bp, the 300 A up to 250-300 bp, the 1000 A up to 400-450 bp and the 4000 A pore size is capable of resolving in excess of 500 bp. The dynamic capacity of these four pore sizes was also determined using a synthetic oligonucleotide with two ion-pairing agents at ambient and 60 degrees C. The dynamic capacity was shown to decrease with increasing pore size and that with the triethylammonium acetate ion-pairing agent there was negligible temperature dependency. The dynamic capacity was higher when tetrabutylammonium bromide was used at elevated temperature. A strong anion-exchange functionality on a pH-stable polymeric particle was used to investigate the selectivity and resolution of the technique. Using a poly-T-oligonucleotide size standard, resolution of full length oligonucleotide (n) from the truncated species due to coupling failure (n-1, n-2, etc.) was demonstrated up to at least the 30mer. Resolution of a phospho diester contaminant from a phospho thioate oligonucleotide and a truncated sequence was demonstrated using anion-exchange HPLC at high pH.     

Polymerization of oriented monomers. IX. Further studies on the polymerization and vesicular properties of allyl and diallyl based monomeric and polymerized quaternary ammonium salts

The polymerization of the previously reported allyl and diallyl quaternary ammonium salts, which from vesicles in water, to their polymerized analogs was further investigated. The structure of the polymer derived from the diallyl derivative, for which some controversy existed, was elucidated by ¹³C-NMR spectroscopy. Mixed vesicles, based on the monoallyl monomer and incorporating certain vesicle forming quaternary ammonium salts, cholesterol, or cholesteryl acrylate were also formed. These mixed vesicles were characterized primarily as far as their stability is concerned. It was found that the incorporation of the additives does not enhance the stability of the mixed vesicles compared to that exhibited by monoallyl simple vesicles. In addition, polymerization of mixed monoallyl-cholesteryl acrylate vesicles results in the destruction of their structure. On the contrary, simple polymerized vesicles, resulting from the diallyl monomer, showed excellent long-term stability. Specifically, samples prepared two years ago are still dispersed and, very possibly, they will remain so for a longer period since they are not showing signs of precipitation.     

Permeabilities and stabilities of large dihexadecylphosphate and dioctadecyldimethylammonium chloride vesicles

Sodium dihexadecylphosphate (DHP) vesicles, with a mean external diameter of 0.27 μm, were obtained by chloroform vaporization followed by gel filtration. The relative volume of the internal aqueous compartment, estimated measuring entrapment of [¹⁴C] sucrose, was 13 liters/mol. DHP and dioctadecyldimethyl ammonium chloride (DODAC) large vesicles were stable up to 20 days at room temperature. In contrast, small sonicated DHP and DODAC vesicles were stable for some hours after preparation. Both DHP and DODAC large vesicles were impermeable toward most solutes tested. Large DHP vesicles responded as ideal osmometers toward gradients of sucrose, NaCl, or NaOH. The behavior of large vesicles of synthetic amphiphiles and of phospholipids was found to be analogous in the gel state.     

Network formation of catanionic vesicles and oppositely charged polyelectrolytes. Effect of polymer charge density and hydrophobic modification

In nonequimolar solutions of a cationic and an anionic surfactant, vesicles bearing a net charge can be spontaneously formed and apparently exist as thermodynamically stable aggregates. These vesicles can associate strongly with polymers in solution by means of hydrophobic and/or electrostatic interactions. In the current work, we have investigated the rheological and microstructural properties of mixtures of cationic polyelectrolytes and net anionic sodium dodecyl sulfate/didodecyldimethylammonium bromide vesicles. The polyelectrolytes consist of two cationic cellulose derivatives with different charge densities; the lowest charge density polymer contains also hydrophobic grafts, with the number of charges equal to the number of grafts. For both systems, polymer-vesicle association leads to a major increase in viscosity and to gel-like behavior, but the viscosity effects are more pronounced for the less charged, hydrophobically modified polymer. Evaluation of the frequency dependence of the storage and loss moduli for the two systems shows further differences in behavior: while the more long-lived cross-links occur for the more highly charged hydrophilic polymer, the number of cross-links is higher for the hydrophobically modified polymer. Microstructure studies by cryogenic transmission electron microscopy indicate that the two polymers affect the vesicle stability in different ways. With the hydrophobically modified polymer, the aggregates remain largely in the form of globular vesicles and faceted vesicles (polygon-shaped vesicles with largely planar regions). For the hydrophilic polycation, on the other hand, the surfactant aggregate structure is more extensively modified: first, the vesicles change from a globular to a faceted shape; second, there is opening of the bilayers leading to holey vesicles and ultimately to considerable vesicle disruption leading to planar bilayer, disklike aggregates. The faceted shape is tentatively attributed to a crystallization of the surfactant film in the vesicles. It is inferred that a hydrophobically modified polyion with relatively low charge density can better stabilize vesicles due to formation of molecularly mixed aggregates, while a hydrophilic polyion with relatively high charge density associates so strongly to the surfactant films, due to strong electrostatic interactions, that the vesicles are more perturbed and even disrupted.     

Rapid and Sensitive Hplc Determination of Ranitidine in Plasma. Application to Pharmacokinetics Study

Abstract

A rapid and sensitive reversed-phase high-performance liquid chrormatography (RP-HPLC) method for the separation and quantification of the H₂-receptor antagonist drug ranitidine in human plasma is described.

The extraction of ranitidine from plasma by an organic solvent was eliminated in this method. Instead, the pre-chromatography isolation of the drug was done by adding approximately 50 mg of zinc sulfate and 200 μL of acetonitrile in 1.0 mL of plasma. A short column packed with pH-stable (1–13) reversed phase PLRP-S^TM particles was used with an isocratic elution of 5.0mM dibasic potassium phosphate plus 0.50mM tetraethyl ammonium hydroxide/ acetonitrile, 80:20 (v/v). The ranitidine was monitored at 315 nm and 0.20 to 0.002 absorption units full scale (AUFS). The completion time of the assay was less than 15 minutes and had a limit of detection of 1.0 ng/mL for a 100-μL injection volume.

After an oral dose of 150 mg of ranitidine, plasma samples were collected at several time points and were analyzed by using this method to determine various pharmacokinetic parameters.     

Stability and Size Control of Reverse Vesicles

The stability and size control of reverse vesicles have been investigated for a sucrose monoalkanoate/hexaethylene glycol hexadecyl ether/decane/water system. The stability is highly dependent on the surfactant mixing ratio, amount of added water, and vesicle size. The size distribution of reverse vesicles produced by simple mixing is very large, but larger vesicles can be removed by means of the extrusion method and reasonably homogeneously size-distributed reverse vesicles can be obtained. If a probe-type ultrasonicator is used, the reverse vesicles obtained are homogeneous and of very small size (50-70 nm in diameter) and they are considered to be of the unilamellar type.     

Vesicles in Nature and the Laboratory: Elucidation of Their Biological Properties and Synthesis of Increasingly Complex Synthetic Vesicles

The important role of vesicles in many aspects of cell function is well‐recognized, but only recently have sophisticated imaging techniques begun to reveal their ubiquity in nature. While we further our understanding of the biological properties of vesicles and their physiological functions, increasingly elegant artificial vesicles are being developed for a wide range of technological applications and basic research. Herein, we examine the state of the art of biological and synthetic vesicles and place their biological features in the context of recent synthetic developments, thus providing a unique overview of these complex and rapidly developing fields. The challenges and opportunities associated with future biological and synthetic studies of vesicles are also presented.     

Flow cytometric characterization of interactions between U-937 human macrophages and positively charged catanionic vesicles

Catanionic vesicles are considered a potential alternative to liposomes for drug delivery systems because of their greater stability and lower cost. Before using catanionic vesicles in vivo, their interactions with macrophages must be fully understood because they are primarily removed from circulation by the macrophages of the mononuclear phagocyte system. Using flow cytometry, we examined the intracellular responses-reactive oxygen species (ROS) content, mitochondrial membrane potential, cell size and complexity, and cell cycle profiles-in U-937 human macrophages treated with positively charged catanionic vesicles. Kinetic hydrogen peroxide production initially increased at lower concentrations (4-10nM) but declined at higher concentrations (40 nM and 80 nM) over the entire incubation period. Superoxide content generation, however, increased over the entire concentration range and incubation period. Catanionic vesicles decreased mitochondrial membrane potential for every concentration after 4h of incubation but caused a significant fluctuation in mitochondrial membrane potential at 6h. After 6h of incubation, catanionic vesicles produced more changes in cell size and complexity than after 4h. The increase in the subG1 population of cells treated with catanionic vesicles at higher doses indicated that apoptosis progressed. Positively charged catanionic vesicles induced different activated patterns of ROS generation and changes in mitochondrial membrane potential than did cationic liposomes. The nature of the interactions between macrophages and catanionic vesicles is of great importance for the design of safer and more effective delivery systems for macrophages. Our findings contribute to a better understanding of the molecular action of catanionic vesicles in the cellular system.     

Bioinspired vesicle restraint and mobilization using a biopolymer scaffold

Biology employs vesicles to package molecules (e.g., neurotransmitters) for their targeted delivery in response to specific spatiotemporal stimuli. Biology is also capable of employing localized stimuli to exert an additional control on vesicle trafficking; intact vesicles can be restrained (or mobilized) by association with (or release from) a cytoskeletal scaffold. We mimic these capabilities by tethering vesicles to a biopolymer scaffold that can undergo (i) stimuli-responsive network formation (for vesicle restraint) and (ii) enzyme-catalyzed network cleavage (for vesicle mobilization). Specifically, we use the aminopolysaccharide chitosan as our scaffold and graft a small number of hydrophobic moieties onto its backbone. These grafted hydrophobes can insert into the bilayer to tether vesicles to the scaffold. Under acidic conditions, the vesicles are not restrained by the hydrophobically modified chitosan (hm-chitosan) because this scaffold is soluble. Increasing the pH to neutral or basic conditions allows chitosan to form interpolymer associations that yield a strong, insoluble restraining network. Enzymatic hydrolysis of this scaffold by chitosanase cleaves the network and mobilizes intact vesicles. Potentially, this approach will provide a controllable means to store and liberate vesicle-based reagents/therapeutics for microfluidic/medical applications.     

Shape and size of giant unilamellar phospholipid vesicles containing cardiolipin

The effect of cardiolipin content on the shape and size of giant palmitoyloleylphosphatidylcholine/cardiolipin vesicles was studied. Unilamellar vesicles were prepared in sugar solution by the method of electroformation, from mixtures containing up to 50% weight ratio of cardiolipin. At room temperature the vesicles containing cardiolipin exhibited abrupt changes in the curvature of the vesicle contour indicating regions of phase separation. The deviations from the spherical shape were larger if vesicles were made from mixtures with a higher content of cardiolipin. Numerous vesicles with soft fluctuating walls were observed. The estimated size of the vesicles containing cardiolipin was found to be smaller than the size of pure palmitoyloleylphosphatidylcholine vesicles.     

Giant vesicles formed by gentle hydration and electroformation: a comparison by fluorescence microscopy

Giant unilamellar vesicles (diameter of a few tens of micrometers) are commonly produced by hydration of a dried lipidic film. After addition of the aqueous solution, two major protocols are used: (i) the gentle hydration method where the vesicles spontaneously form and (ii) the electroformation method where an ac electric field is applied. Electroformation is known to improve the rate of unilamellarity of the vesicles though it imposes more restricting conditions for the lipidic composition of the vesicles. Here we further characterize these methods by using fluorescence microscopy. It enables not only a sensitive detection of the defects but also an evaluation of the quantity of lipids in these defects. A classification of the defects is proposed and statistics of their relative importance in regard to both methods and lipid composition are presented: it shows for example that 80% of the vesicles obtained by electroformation from 98% 1,2-dioleoyl-sn-glycero-3-phosphocholine are devoid of significant defects against only 40% of the vesicles with the gentle hydration method. It is also shown that the presence of too many negatively charged lipids does not favor the formation of unilamellar vesicles with both methods. For the gentle hydration, we checked if the negatively charged lipids were inserted in the vesicles membrane in the same proportion as that of the lipid mixture from which they are formed. The constant incorporation of a negatively charged labeled lipid despite an increasing presence of negatively charged 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] tends to confirm that the composition of vesicles is indeed close to that of the initial mixture.