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.

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.

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.

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.

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.

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.

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.

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.

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.

Preparation and validation of low cost microfluidic chips using a shrinking approach.

The present paper describes the production of microfluidic chips using an approach based on shrinkable biocompatible polymers (i.e. agarose) for the production of size controlled microfluidic channels. In addition, all steps of chip production were carried out using an inexpensive approach that uses low cost chemicals and equipment. The produced chips were then validated by producing monodisperse polymeric microparticles for drug delivery and hydrogel microfibers for cell embedding.

Preparation, optimization and application of affinity absorbent with a polysaccharide YCP as the ligand.

YCP, an α-glucan from the mycelium of marine filamentous fungus Phoma herbarum YS4108, has great antitumor potential via enhancement of host immune through Toll-like receptor (TLR) 2 and TLR4 signaling. In the current study, YCP was coupled to EAH Sepharose 4B agarose beads to prepare the YCP-Sepharose affinity absorbent using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as the activating agent. An orthogonal experiment L9 (3)(4) was applied to optimize the coupling procedure, giving the optimal parameters as follows: molar ratio of CDAP to YCP of 1:2, CDAP-activation time of 5 min, gel volume of 0.1 mL, and gel-incubation time of 72 h, respectively. Scanning electron microscopy analysis indicated successfully preparation of YCP immobilized sepharose beads, while these beads essentially maintained biological properties of free YCP since they can interact with TLR2 and TLR4 specifically at comparable level. Collectively, our findings provide an alternative approach to immobilize carbohydrate-based molecules for studying the carbohydrate-protein interaction.

Self-assembly of model graft copolymers of agarose and weak polyelectrolyte-based amphiphilic diblock copolymers: controlled drug release and degradation.

Polysaccharide-based copolymers are promising biomaterials due to their biocompatibility and biodegradability. For potential biomedical applications the copolymer as a whole and all the degraded species must be biocompatible and easily removable from the system. In this regards, new model pH-responsive seaweed agarose (Agr) grafted with weak polyelectrolyte-based well-defined amphiphilic block copolymers ca. poly[(methyl methacrylate)-b-(2-dimethylamino)ethyl methacrylate)] (PMMA-b-PDMA) were designed and synthesized to study the self-assembly, degradation, and in vitro hydrophobic/hydrophilic drug release behavior. The graft copolymer solutions display extremely low critical micelle concentration (CMC) and form pH responsive stable micelles. The degradation study of the graft copolymer reveals that the entire degraded components are well soluble/dispersible in water due to formation of mixed micelles. The micelles are also strongly adsorbed on the mica surface owing to electrostatic interaction. One application of the graft copolymer micelles is that it can entrap both hydrophilic and poorly water soluble hydrophobic drugs effectively and exhibit slow release kinetics. The release kinetics of both the hydrophilic and poorly water soluble hydrophobic drugs change with pH as well as with the composition of the graft copolymer.

One-pot synthesis of fluorescent polysaccharides: adenine grafted agarose and carrageenan.

New fluorescent polysaccharides were synthesized by grafting the nucleobase adenine on to the backbones of agarose and κ-carrageenan, which were characterized by FT-IR, (13)C NMR, TGA, XRD, UV, and fluorescence properties. The synthesis involved a rapid water based potassium persulfate (KPS) initiated method under microwave irradiation. The emission spectra of adenine grafted agarose and κ-carrageenan were recorded in aqueous (5×10(-5) M) solution, exhibiting λ(em,max) 347 nm by excitation at 261 nm, affording ca. 30% and 40% enhanced emission intensities, respectively compared to that of pure adenine solution in the same concentration. Similar emission intensity was recorded in the pure adenine solution at its molar equivalent concentrations present in the 5×10(-5) M solution of the agarose and carrageenan grafted products, that is, 3.28×10(-5) M and 4.5×10(-5) M respectively. These fluorescent adenine grafted products may have potential utility in various sensor applications.

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.

Studies on the cationization of agarose.

Cationized agaroses with different degrees of substitution (0.04-0.77) were synthesized, employing 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC). The influence of different reaction parameters on the substitution degree and molecular weight was evaluated. The investigated parameters were concentration of reagents, temperature, time, and addition of NaBH(4). The products were characterized by means of scanning electronic microscopy, infrared spectroscopy, viscosimetry, and NMR spectroscopy. Methanolysis products were studied by electrospray ionization mass spectrometry. The higher the concentration of CHPTAC employed, a higher degree of substitution was obtained, if the optimum concentration of NaOH in each case was employed. Insufficient quantities of NaOH reduced epoxide formation and the reacting alkoxides of the polysaccharide, whereas an excess of NaOH favored degradation of the epoxide and decrease in the molecular weight of the product. A reaction time of 2h was sufficient to obtain products with the maximum degree of substitution for each case. The addition of NaBH(4) gave products with a slightly higher molecular weight, but the extra cost involved should not justify its use for large-scale application.

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.

Surface-modification-directed controlled adsorption of serum albumin onto magnetite nanocuboids synthesized in a gel-diffusion technique.

Magnetite nanocuboids have been synthesized via gel-diffusion technique in agarose gel. Here, the agarose gel matrix has been used as an organic template for formation and growth modification of magnetite. Gel mineralization mimics the membrane-based biomineralization, controls the diffusion process and gives the micro/nano environment for the crystal growth. We also attempt to understand the influence of different surface modifications of synthesized magnetite nanocuboids on protein interaction. For this purpose, magnetite particles were coated with trimesic acid (benzene-1,3,5-tricarboxylic acid) and stearic acid, which generates a hydrophilic and a hydrophobic modified surface, respectively. We report controlled adsorption behavior of bovine serum albumin (BSA) by surface modification of magnetite nanocuboids with different functional groups. The adsorption capacity of BSA increases on trimesic acid-coated surfaces compared to bare magnetite surfaces, while it decreases on stearic acid-coated surfaces. In situ fluorescence spectroscopy has been used to analyze the tertiary protein structure in the adsorbed state on these three surfaces. Partial unfolding in the tertiary structure of BSA was observed upon adsorption onto bare magnetite surfaces. On trimesic acid-coated surfaces, tertiary unfolding of BSA was greater than on bare magnetite surfaces, while BSA undergoes minor tertiary structural change on stearic acid-coated surfaces.

Spleen-specific suppression of TNF-alpha by cationic hydrogel-delivered antisense nucleotides for the prevention of arthritis in animal models.

This study developed a transplantable platform based on cationic hydrogels to deliver antisense oligodeoxynucleotides (ASOs) targeting the mRNA of TNF-alpha. Cationic agarose (c-agarose) was obtained by conjugating ethylenediamine to agarose via an N,N'-carbonyldiimidazole (CDI)-activation method. ASO-c-agarose system was constructed by mixing ASO in cationic agarose gel of proper concentration and gelation temperature. In vivo assessment of ASO distribution suggested that the system specifically target to spleen, wherein the c-agarose-delivered ASO had a concentration remarkably 50-fold higher than that of the naked ASO. The distribution of c-agarose-delivered ASO was scarcely detectable in liver and kidney. Next, three types of animal models were setup to evaluate the therapeutic efficacies of ASO-Gel, including the adjuvant-induced arthritis (AA), carrageen/lipopolysaccharide (LPS)-induced arthritis (CLA) and collagen-induced arthritis (CIA) models. The effects of ASO-c-agarose in alleviating inflammation and tissue destruction were evidenced in more than 90% of the testing animals, with decrease of main inflammatory cytokines, lightening of joint swelling and tissue damage, as well as increase in their body weights. All these findings suggest that this highly operable devise for the conveyance of antisense nucleotides together with its spleen-targeting property, could become a useful means of antisense-based therapeutics against rheumatoid arthritis and other diseases.