Agarose Stearate-Carbomer940 as Stabilizer and Rheology Modifier for Surfactant-Free Cosmetic Formulations.

Some commonly used surfactants in cosmetic products raise concerns due to their skin-irritating effects and environmental contamination. Multifunctional, high-performance polymers are good alternatives to overcome these problems. In this study, agarose stearate (AS) with emulsifying, thickening, and gel properties was synthesized. Surfactant-free cosmetic formulations were successfully prepared from AS and carbomer940 (CBM940) mixed systems. The correlation of rheological parameter with skin feeling was determined to study the usability of the mixed systems in cosmetics. Based on rheological analysis, the surfactant-free cosmetic cream (SFC) stabilized by AS-carbomer940 showed shear-thinning behavior and strongly synergistic action. The SFC exhibited a gel-like behavior and had rheological properties similar to commercial cosmetic creams. Scanning electron microscope images proved that the AS-CBM940 network played an important role in SFC's stability. Oil content could reinforce the elastic characteristics of the AS-CBM940 matrix. The SFCs showed a good appearance and sensation during and after rubbing into skin. The knowledge gained from this study may be useful for designing surfactant-free cosmetic cream with rheological properties that can be tailored for particular commercial cosmetic applications. They may also be useful for producing medicine products with highly viscous or gel-like textures, such as some ointments and wound dressings.

Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins.

Cap-binding proteins have been routinely isolated using m7GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m7GTP. Here, we report the synthesis of new affinity resins, m7GpCH2pp- and m7GpCH2ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m7GpppA-Sepharose, bind recombinant mouse eIF4E²8⁻²¹7 specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m7GpCH2pp- and m7GpCH2ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m7GpCH2ppA-Sepharose. Our data prove the applicability of these novel resins, especially m7GpCH2ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.

Development of resin adsorbents for blood purification at Nankai University in China.

Various types of porous resin adsorbents based on polystyrene, agarose, and cellulose as matrixes coupling with DNA, amino acids and other biological active molecules as ligands were extensively studied in China. Molecular recognition between the ligand and pathogenic molecule was investigated. Several commercialized products are now widely used in hospitals all over China. Whole blood hemoperfusion is used to treat patients suffering from autoimmune diseases, uremia acute intoxication, and hyperbilirubinemia. Clinical performances of hundreds and thousands of patients treated by whole blood sorption therapy show that the therapy is safe, efficient, and cost-effective.

Agarose oligosaccharide- silver nanoparticle- antimicrobial peptide- composite for wound dressing.

Marine polysaccharides or oligosaccharides have potential to promote wound healing due to their biocompatibility and physicochemical properties. However, microbial infection delays wound healing process, and novel antimicrobial wound dressings are urgently needed. Here, agarose oligosaccharides (AGO) obtained from marine red algae were used as a reducing and stabilizer for green synthesis of silver nanoparticles (AgNPs), and further successfully connected with odorranain A (OA), one of antimicrobial peptides (AMPs), to obtain a novel composite nanomaterial (AGO-AgNPs-OA). Transmission electron microscopy (TEM) and Malvern particle size analyzer showed that AGO-AgNPs-OA was spherical or elliptic with average size of about 100 nm. Circular dichroism (CD) spectroscopy showed that AGO-AgNPs stabilized the α-helical structure of OA. AGO-AgNPs-OA showed stronger anti-bacterial activities than AGO-AgNPs, and had good biocompatibility and significant promoting effect on wound healing. Our data suggest that AMPs conjugated marine oligosaccharides and AgNPs may be effective and safe antibacterial materials for wound therapy.

Preparation, characterization, and emulsification properties of agarose fatty acid derivatives with different hydrophobic chains.

Two types of fatty acid derivatives were used to synthesize agarose fatty acid esters in a heterogeneous medium. Agarose esters with low degree of substitution were synthesized with succinic anhydride, octenyl succinic anhydride, dodecyl succinic anhydride as esterifying agents. Agarose esters with high degree of substitution were synthesized with lauroyl chloride, palmitoyl chloride, and stearoyl chloride as esterifying agents. Scanning electron microscopy revealed that agarose anhydride modification mostly occurred at the surface of the particles, whereas chloride modification occurred at both the surface and interior of the particles. Fourier transform infrared spectroscopy and nuclear magnetic resonance analyses indicated that hydrophobic groups were successfully introduced in agarose, and the hydroxyl group in the C-2 of D-galactose was the preferred location for esterification. The results also showed that agarose esters with long-chain fatty acids and high substitution degree showed higher emulsifying ability and low interfacial tension property than derivatives with short-chain fatty acids and low substitution degree. Compared with commonly used food emulsifiers, such as Tween, sucrose fatty acid ester, and glycerin monostearate, agarose esters were slightly deficient in emulsifying ability but presented high emulsion stability in oil-in-water emulsion.

Evaluation of dispersive liquid-liquid microextraction by coupling with green-based agarose gel-electromembrane extraction: An efficient method to the tandem extraction of basic drugs from biological fluids.

Nowadays, developing new methods for the effective extraction/separation of drugs (present at trace levels) from complicated matrices (as biological fluids) is certainly a great challenge for many operators. In this regard, green-based agarose gel electromembrane extraction (G-EME) was for the first time combined with dispersive liquid-liquid microextraction (DLLME) toward G-EME/DLLME methodology (i.e., tandem extraction approach). Two basic drugs such as trimipramine (TRI) and clomipramine (CLO) extracted from the urine samples, were used as model compounds. Regarding method workflow, analytes were extracted from the 5 mL sample, through a synthesized agarose gel membrane, to the 700 µL aqueous acceptor phase under the optimized conditions (pH of acceptor phase: 2.0; pH of gel membrane: 2.0; pH of donor phase: 4.0, voltage value: 30 V, and extraction time: 25 min). In the next step, acceptor solution was poured to a conic vial and mixed with 100 µL alkaline solution (NaOH, 1 M). Afterwards, DLLME procedure took place again at optimal conditions, i.e., extraction solvent was carbon tetrachloride (10 µL), and dispersive solvent was acetone (100 µL). Ultimately, gas chromatography (GC) was applied for the detection and quantification of drugs. Such G-EME/DLLME configuration has brought two main advantages. Firstly, interferences such as proteins and other large biological molecules were eliminated from biological fluids via G-EME. Further, high enrichment factors (EFs of 260-370 refer to extraction recoveries of 52-74%) were obtained using DLLME with acceptable detection limits (1.0-3.0 ng mL-1). Finally, the suggested approach was successfully utilized to determine drugs at trace levels in urine samples.

Cyclodextrin functionalized agarose gel with low gelling temperature for controlled drug delivery systems.

Conventional agaroses with high gelling temperature are limited to apply to the field of drug delivery. In this study, ß-cyclodextrin (ßCD) functionalized agarose (CFA) with low gelling temperature was successfully prepared from ethylenediamine-functionalized agarose using mono-succinyl ßCD. The gelling temperature of CFA dramatically decreased to 26.7 °C from 65 °C and the melting temperature declined from 95 °C to 66.1 °C. Upon drug loading, CFA can be used at 30 °C because of its low gelling temperature compared to agarose. CFA gel could be used both for bovine serum albumin as a full release, and for the doxorubicin (DOX) for sustained release, via inclusion complexation of ßCD. Furthermore, cytotoxicity tests revealed that CFA was noncytotoxic. DOX in the CFA gel could retain the anti-cancer activity. Newly synthesized CFA with low gelling temperature offer a new means for the development of hydrogel-based delivery systems for a variety of therapeutic drugs.

Rapid monoclonal antibody adsorption on dextran-grafted agarose media for ion-exchange chromatography.

The binding capacity and adsorption kinetics of a monoclonal antibody (mAb) are measured for experimental cation exchangers obtained by grafting dextran polymers to agarose beads and compared with measurements for two commercial agarose-based cation exchangers with and without dextran grafts. Introduction of charged dextran polymers results in enhanced adsorption kinetics despite a dramatic reduction of the accessible pore size as determined by inverse size-exclusion chromatography. Incorporation of neutral dextran polymers in a charged agarose bead results instead in substantially lower binding capacities. The effective pore diffusivities obtained from batch uptake curves increase substantially as the protein concentration is reduced for the resins containing charged dextran grafts, but are much less dependent on protein concentration for the resins with no dextran or uncharged dextran grafts. The batch uptake results are corroborated by microscopic observations of transient adsorption in individual particles. In all cases studied, the adsorption kinetics is characterized by a sharp adsorption front consistent with a shell-progressive, diffusion limited mechanism. Greatly enhanced transport rates are obtained with an experimental resin containing charged dextran grafts with effective pore diffusivities that are 1-9 times larger than the free solution diffusivity and adsorption capacity approaching 300 mg/cm3 of particle volume.

5-Aminoindole, a new ligand for hydrophobic charge induction chromatography.

Hydrophobic charge induction chromatography (HCIC) is a mixed-mode chromatography that achieves high adsorption capacity by hydrophobic interaction and facile elution by pH-induced charge repulsion between the solute and ligand. This article reports a new medium, 5-aminoindole-modified Sepharose (AI-Sepharose) for HCIC. The adsorption equilibrium and kinetics of lysozyme and bovine serum albumin (BSA) to AI-Sepharose were determined by batch adsorption experiments at different conditions to provide insight into the adsorption properties of the medium. The influence of salt type on protein adsorption to AI-Sepharose corresponded with the trend for other hydrophobicity-related properties in literature. Both ligand density and salt concentration had positive influences on the adsorption of the two proteins investigated. The adsorption capacity of lysozyme, a basic protein, decreased rapidly when pH decreased from 7 to 3 due to the increase of electrostatic repulsion, while BSA, an acidic protein, achieved maximum adsorption capacity around its isoelectric point. Dynamic adsorption experiments showed that the effective pore diffusion coefficient of lysozyme remained constant at different salt concentrations, while that of BSA decreased with increased salt concentration due to its greater steric hindrance in pore diffusion. High protein recovery by adsorption at pH 7.10 elution at pH 3.0 was obtained at a number of NaCl concentrations, indicating that the adsorbent has typical characteristics of HCIC and potentials for applications in protein purification.

Large-Scale Preparation of Yeast Agarose Plugs to Isolate Yeast Artificial Chromosome DNA.

The microinjection of DNA directly into the pronuclei of fertilized zygotes is the most extensively used method of gene transfer in the mouse. The injection of very large pieces of DNA, including bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), has become increasingly popular because their size normally accommodates all of the regulatory elements that are needed for a given expression domain to function adequately in an ectopic genomic location. This protocol describes how to prepare large-scale yeast agarose plugs to isolate YAC DNA. The quality of the YAC DNA preparation is the most important consideration in optimizing transgenic mouse production.

Preparation of uniform-sized agarose beads by microporous membrane emulsification technique.

Uniform-sized agarose beads were prepared by membrane emulsification technique in this study. Agarose was dissolved in boiling water (containing 0.9% sodium chloride) and used as water phase. A mixture of liquid paraffin and petroleum ether containing 4 wt% of hexaglycerin penta ester (PO-500) emulsifier was used as oil phase. At 55 degrees C, the water phase permeated through uniform pores of microporous membrane into the oil phase by a pressure of nitrogen gas to form uniform W/O emulsion. Then the emulsion was cooled down to room temperature under gentle agitation to form gel beads. The effect of oil phase, emulsifier, especially temperature on the uniformity of the beads were investigated and interpreted from interfacial tension between water phase and oil phase. Under optimized condition, the coefficient variation (C.V.) showing the size distribution of the beads was under 15%. This was the first report to prepare uniform agarose beads by membrane emulsification, and to investigate the effect of temperature on the size distribution of the droplets and beads. The beads with different size can be prepared by using membranes with different pore size, and the result showed that there was a linear relationship between the average diameter of beads and pore size of the membranes; beads with diameter from 15 to 60 microm were able to obtain in this study.

Mechanical characterisation of agarose-based chromatography resins for biopharmaceutical manufacture.

Mechanical characterisation of agarose-based resins is an important factor in ensuring robust chromatographic performance in the manufacture of biopharmaceuticals. Pressure-flow profiles are most commonly used to characterise these properties. There are a number of drawbacks with this method, including the potential need for several re-packs to achieve the desired packing quality, the impact of wall effects on experimental set up and the quantities of chromatography media and buffers required. To address these issues, we have developed a dynamic mechanical analysis (DMA) technique that characterises the mechanical properties of resins based on the viscoelasticity of a 1ml sample of slurry. This technique was conducted on seven resins with varying degrees of mechanical robustness and the results were compared to pressure-flow test results on the same resins. Results show a strong correlation between the two techniques. The most mechanically robust resin (Capto Q) had a critical velocity 3.3 times higher than the weakest (Sepharose CL-4B), whilst the DMA technique showed Capto Q to have a slurry deformation rate 8.3 times lower than Sepharose CL-4B. To ascertain whether polymer structure is indicative of mechanical strength, scanning electron microscopy images were also used to study the structural properties of each resin. Results indicate that DMA can be used as a small volume, complementary technique for the mechanical characterisation of chromatography media.

Agarose based large molecular systems: Synthesis of fluorescent aromatic agarose amino acid nano-conjugates - their pH-stimulated structural variations and interactions with BSA.

Two new nano-sized fluorescent 6-amino agarose naphthalic acid half ester derivatives were synthesized (ca.60% yields) employing 1,8- and 2,3-naphthalic acid anhydrides (1,8-AANE, and 2,3-AANE respectively). These large nano molecular frameworks (DLS 3 & 100 nm, and 3 & 152 nm respectively) contains amino, naphthalate half-ester carboxyl groups at the C-6 positions of the 1,3-ß-D-galactopyranose moieties of the agarose backbone (overall DS 0.94). Structures were characterized (FT-IR, and 13C &1H NMR spectrometries). These materials mimicked a large protein conjugate (GPC 123, and 108 kDa) exhibiting pH-responsive conformational variations (optical rotatory dispersion), offering a mixed solubility pattern like a soluble random coil (pH 4-10), and precipitate (pH 2) formation. With bovine serum albumin 1,8- and 2,3-AANE showed complexation, and decomplexation at pH 5.2, and 6.8 respectively. However, they showed decomplexation, and complexation respectively at pH 10 (circular dichroism). These fluorogenic systems may be of prospective utility as chiral sensors and in the realms demanding the virtues of preferential protein bindings.

Synthesis of bio-based aldehyde from seaweed polysaccharide and its interaction with bovine serum albumin.

Here, we demonstrate a successful synthesis of bio-based aldehyde namely dialdehyde-carboxymethylagarose (DCMA) using carboxymethyagarose (CMA). Further reaction parameters (i.e. reaction temperature, pH and periodate concentration) were optimized to achieve maximum aldehyde content and product yield. The synthesis of DCMA was confirmed by employing FTIR, (1)H NMR, XRD, SEM, AFM, TGA, DSC, EA and GPC techniques. To investigate the aldehyde functionality, DCMA was allowed to interact with BSA and obtained results were found to be comparable with that of synthetic aldehyde (Formaldehyde). Further interaction of DCMA with BSA was confirmed by using UV-vis, FTIR, fluorescent spectroscopy, CD and DLS analysis. Results of this study revealed that bio-based aldehyde behaves like formaldehyde. This study adds value to abundant marine biopolymers and opens the new research area for polymer researchers.

Agarose functionalization: Synthesis of PEG-agarose amino acid nano-conjugate - its structural ramifications and interactions with BSA in a varying pH regime.

In a rapid one-step method protein-mimicking large agarose amino acid framework (AAE; GPC 156.7kDa) was conjugated with polyethylene glycol (PEG 9kDa) affording nano-sized PEGylated amphoteric agarose (PEG-AAE; <10nm; DLS) containing amino, carboxyl and ester groups [overall degree of substitution (DS) 0.91]. The PEG groups were at the residual free carboxylic acid groups of succinate half-ester moiety at C-6 positions of the 1, 3 ß-d-galactopyranose moieties of AAE. This new nano-sized PEG-AAE performed like a giant protein conjugate (GPC 331.2kDa) and exhibited pH-responsive interconversion between the triple helix and single-stranded random structures (optical rotatory dispersion) presenting a mixed solubility pattern like random coil (soluble), helical (soluble) and aggregate (precipitation) formations. Circular dichroism studies showed its pH-dependent complexation and decomplexation with bovine serum albumin (BSA). Such pH-responsive PEG-conjugate may be of pronounced therapeutic potential in the area of pharmacology as well as in sensing applications.

Isonitrile derivatives of polysaccharides as supports for the covalent fixation of proteins and other ligands.

A method for the introduction of side chains containing isonitrile (isocyanide, functional group) on the backbone of polysaccharides and other hydroxylic polymers was developed. The method was based on (a) ionization of some of the hydroxyl groups on the polymer by treatment with a strong base (tert-butoxide) in a polar aprotic solvent (dimethylsulfoxide), and (b) introduction of side chains containing isonitrile groups by nucleophilic attack of the polymeric alkoxide ions on a low molecular weight isonitrile containing a good leaving group in the omega-position, (1-tosyl-3-isocyanopropane). By this method, the side chains containing the-NC functional groups are attached to the polymeric backbone via stable ether bonds. The isonitrile derivatives of cellulose, linear and cross-linked dextran and cross-linked agarose utilized for the covalent fixation of high and low molecular weight ligands by four-component reactions carried out in aqueous medium, at neutral pH.

Sepharose-stearate as substrate for rat liver long-chain fatty acyl-CoA synthetase.

Stearic acid coupled covalently to Sepharose 6B serves as substrate for thioesterification catalyzed by rat liver long-chain fatty acyl-CoA synthetase (ATP-forming) (EC 6.2.1.3). Availability as substrate is dependent upon the conservation of the free omega-terminal in addition to that of the free carboxyl function. The enzymatic overall formation of matrix-acyl-CoA in the presence of ATP and CoA as cosubstrates conforms to the stoichiometry reported for thioesterification of the free long-chain fatty acyl substrate. The preformed matrix-acyl-CoA serves as substrate for the backward synthetase reaction in the presence of AMP and PPi. The apparent Km values for ATP and CoA in the presence of the acyl matrix are similar to the respective Km values observed in the presence of the free acid substrate. The apparent Km for the acyl matrix is 10-fold higher (0.5 mM) than the apparent Km value for the free acid. The feasibility of enzymatic thioesterification of bound long-chain fatty acids implies that the exact nature of the bulky chain situated between the carboxy and omega-terminal plays a secondary role in defining the fatty acyl substrate specificity for long-chain fatty acyl-CoA synthetase. Also, dissociation of bound long-chain fatty acids does not constitute an obligatory preliminary step to fatty acid thioesterification.

Immobilization of phospholipid vesicles and protein-lipid vesicles containing red cell membrane proteins on octyl derivatives of large-pore gels.

For improved immobilization of phospholipid vesicles and protein-lipid vesicles (cf. Sandberg, M., Lundahl, P., Greijer, E. and Belew, M. (1987) Biochim. Biophys. Acta 924, 185-192) and for chromatographic experiments with vesicles containing membrane protein, we have prepared octyl sulfide derivatives of the large-pore gels Sephacryl S-1000 and Sepharose 2B with ligand concentrations up to 14 and 5 mumol/ml gel, respectively. The Sephacryl derivatives allowed higher flow rates, gave higher rates of adsorption and showed equally high or higher capacities than the Sepharose adsorbents. 'Small', 'medium' and 'large' vesicles of radii approx. 20, 50 and 100 nm showed distribution coefficients on Sephacryl S-1000 of 0.7, 0.5 and 0.05, respectively and could be immobilized on octyl sulfide-Sephacryl S-1000 in amounts corresponding to 110, 40 and 20 mumol of phospholipids per ml gel, respectively. 'Small' vesicles became absorbed onto this gel at a rate of 1.5 mumol of phospholipids per min per ml gel until 60 mumol of phospholipids had become immobilized, whereas the initial adsorption rate was about 0.4 mumol.min-1.ml-1 on octyl sulfide-Sepharose 4B (see reference above) and on octyl sulfide-Sepharose 2B. Lower ligand concentrations gave lower capacities for 'small' vesicles. When vesicles entrapping calcein were immobilized on octyl sulfide-Sephacryl S-1000 some calcein was released during the adsorption process. For 'small' and 'medium' vesicles, respectively, the leakage was 75 and 25% at a ligand concentration of 14 mumol/ml but only 3 and 2% at 5 mumol/ml. The internal volumes of immobilized 'small' and 'medium' vesicles were estimated at 0.97 and 2.9 microliters per mumol of phospholipid by determination of entrapped calcein, which could indicate vesicle radii 20 and 50 nm, respectively. The total volumes of immobilized 'medium' lipid vesicles and 'medium' protein-lipid vesicles containing integral membrane proteins from human red cells, were estimated at 2.9 and 2.0 microliters/mumol, respectively, by chromatography of D- and L-[14C]glucose and calcein on the octyl sulfide-Sephacryl S-1000 column before and after immobilization. These volumes are roughly consistent with the internal volume of the vesicles. A zone of D-glucose eluted 90 microliters later than a zone of L-glucose on a 4- or 5-ml column of octyl sulfide-Sephacryl S-1000 with immobilized 'medium' protein-lipid vesicles containing the glucose transporter from human red cells, probably since part of the internal vesicle volume was accessible to the D-glucose but not to the L-glucose. This indicates that the glucose transporter was active in the immobilized vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Facile synthesis of nano-sized agarose based amino acid-Its pH-dependent protein-like behavior and interactions with bovine serum albumin.

In a facile synthesis agarose was amphoterically functionalized to afford nano-sized agarose amino acids, aminoagarose succinate half-esters (AAE) containing one pendant carboxyl group. Nano-sized AAEs (<10 nm; DLS) were characterized and they had three various degrees of substitution [overall DSs 0.88, 0.89 and 0.96], both the amino and half-ester groups were placed on C-6 positions of the 1,3 beta-d-galactopyranose moieties of agarose backbone ((13)C NMR). AAEs performed like large protein molecules exhibiting pH-responsive structural variations (optical rotatory dispersion), presenting a mixed solubility pattern like random coil (soluble) and aggregate (precipitation) formations. Circular dichroism studies showed their pH-dependent associative interactions with bovine serum albumin, which indicated complexation at acidic and basic pHs, and decomplexation at pH 6.8 with AAE (DS 0.96). Thus, these nano-sized AAE based systems may be of potential utility in the domains demanding the merits of preferential protein bindings e.g. pH-responsive cationic/anionic drug carrier, separations or chiral sensing applications.

Synthesis of a yohimbine-agarose matrix useful for large-scale and micropurification of multiple alpha 2-receptor subtypes.

We have provided a detailed protocol for the synthesis of a yohimbine-agarose matrix that has been shown to be effective for isolation of the alpha 2A-adrenergic receptor from human platelet and purification of the alpha 2A-adrenergic receptor to apparent homogeneity from porcine brain cortex using chromatography on only two sequential yohimbine-agarose columns. In addition, this affinity matrix also interacts with alpha 2 receptors of the alpha 2B subtype extracted from cultured NG108-15 cells. Finally, this affinity matrix has proven useful for monitoring posttranslational modifications of the receptor in digitonin extracts of metabolically labeled cells. Thus, this affinity matrix can be exploited for the purification of multiple alpha 2-adrenergic receptor subtypes on both a macro- and microscale and should be of value to any laboratory exploring the molecular basis for alpha 2-adrenergic functions.