Sulforhodamine Research Articles

Influence of salivary washout on drug delivery to the oral cavity using coated microneedles: An in vitro evaluation

The objective of this study was to determine whether in buccal tissues, after insertion and removal of coated microneedles, the presence of saliva over the insertion site can lead to loss of the deposited drug, and if saliva can influence in vitro permeation of the drug across the tissue. Microneedles were coated with sulforhodamine (SRD), which was used as a model drug, and inserted in to porcine buccal mucosa in vitro. Fluorescence microscopy was used to study microneedle coating quality and the diffusion of SRD through the mucosa. Permeation experiments were conducted for simulated dynamic or static salivary flow by adding 100μL/h or 100, 200 or 300μL of phosphate buffered saline (PBS) in the donor compartment of the Franz diffusion cells, into which buccal tissue after insertion of SRD-coated microneedles was placed. Microscopy showed that microneedles were uniformly coated with SRD and that SRD was successfully delivered in to the mucosa. Some SRD remained in the tissue even after 24h, despite presence of PBS on top of the coated microneedle insertion site. It was found that salivary washout can result in loss of drug that has been deposited in oral cavity mucosal tissues using coated microneedles, and presence of fluid over the coated microneedle insertion site can increase flux across the tissue. Thus, it is advisable to include salivary flow during in vitro studies related to the use of coated microneedles for drug delivery to the oral cavity in order to not obtain misleading results.

Permeation Enhancement via Thiolation: In Vitro and Ex Vivo Evaluation of Hyaluronic Acid-Cysteine Ethyl Ester

It was the aim of this study to evaluate the permeation-enhancing effect of synthesized thiolated hyaluronic acid (HA). HA, a naturally found polysaccharide, was chemically modified with l-cysteine ethyl ether (C) via amide bond formation. In vitro permeation enhancement was tested on Caco-2 cells with two compounds, sulforhodamine (SR) and fluorescein isothiocyanate-dextran (FD4). Cytotoxicity assays as lactate dehydrogenase and thiazolyl blue tetrazolium bromide (MTT) were performed on colon carcinoma cell line. Transepithelial electrical resistance (TEER) measurements were conducted. Ex vivo evaluation was accomplished on rat intestinal mucosa in order to predict the permeation enhancing effect with SR, sodium fluorescein (SF), and FD4, respectively. The MTT as well as lactate dehydrogenase revealed no toxicity over time periods of 3 and 12 h, respectively. The bioconjugate is biocompatible and safe to use. Furthermore, TEER measurements showed the integrity of tight junctions. The in vitro permeation studies on cell studies exhibit 1.28-fold enhancement for SR and 1.47-fold enhancement for FD4 with hyaluronic acid-cysteine ethyl ester (HAC) in comparison to unmodified one. The ex vivo transport studies exhibit 1.9-fold enhancement for SF, 1.31-fold enhancement for Rhodamine123, and 1.3-fold enhancement for FD4 with HAC in comparison to unmodified one, respectively. Thus, the promising results encourage further investigations and exploitation of this versatile polysaccharide.

Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay

BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C(3)(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR-LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.

Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET)

Fluorescently labeled derivatives of a xyloglucan (XG) nonasaccharide Glc 4Xyl 3Gal 2 (XLLG) were used as glycosyl acceptors in assays of xyloglucan endotransglycosylase (XET) from germinated nasturtium ( Tropaeolum majus) seeds. We have investigated how the type of the oligosaccharide label influences the kinetic parameters of the reaction. The fluorescent probes used to label XLLG were anthranilic acid (AA), 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS), fluorescein isothiocyanate (FITC), and sulforhodamine (SR), respectively. The obtained data were compared with those of the reactions where aldose and/or alditol forms of tritium-labeled xyloglucan-derived nonasaccharide served as the respective acceptors. Modification at C-1 of the reducing-end glucose in XLLG by substitution with the fluorophore markedly affected the kinetic parameters of the reaction. The Michaelis constants K m for individual acceptors increased in the order [1- 3H]XLLG < XLLG-SR < [1- 3H]XLLGol < XLLG-FITC < XLLG-ANTS < XLLG-AA, while the turnover numbers characterized by k cat decreased in the order XLLG-FITC > XLLG-SR > XLLG-ANTS > [1- 3H]XLLGol > [1- 3H]XLLG > XLLG-AA. Catalytic efficiency (expressed as k cat/ K m) with XLLG labeled with SR or FITC was 15 and 28 times, respectively, higher than with the tritium-labeled natural substrate [1- 3H]XLLG. Comparison of the kinetic parameters found with acceptors labeled with different types of labels enables to select the most effective substrates for the high-throughput assays of XET.

Chirality of sulforhodamine dye molecules incorporated in DNA thin films

Thin films formed from salmon sperm DNA reacted with a cationic surfactant (CTMA-Cl) included up to 25 wt % fluorescent molecule sulforhodamine (SRh). SRh effect on DNA chirality and vice versa was investigated by circular dichroism (CD) spectroscopy. The CD signals at 250–265 nm indicate that DNA chirality was maintained or enhanced. Induced CD (iCD) signal at 580–610 nm indicates that SRh is chiral in DNA/CTMA. iCD signal from both solutions and thin films generally increases with SRh concentration. The chirality induced in SRh molecules and the absence of significant DNA reduction in chirality are clear indicators of strong binding to DNA/CTMA.

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.

Photoluminescence and lasing from deoxyribonucleic acid (DNA) thin films doped with sulforhodamine

Thin solid films of salmon deoxyribonucleic acid (DNA) have been fabricated by treatment with a surfactant and used as host for the laser dye sulforhodamine (SRh). The DNA films have an absorption peak at approximately 260 nm owing to absorption by the nitrogenous aromatic bases. The SRh molecules in the DNA films have absorption and emission peaks at 578 and 602 nm, respectively. The maximum emission was obtained at approximately 1 wt. % SRh in DNA, equivalent to approximately 100 DNA base pairs per SRh molecule. A distributed feedback grating structure was fabricated on a SiO(2)-Si substrate using interference lithography. The grating period of 437 nm was selected, corresponding to second-order emission at the amplified spontaneous emission wavelength of 650 nm. Lasing was obtained by pumping with a doubled Nd:YAG laser at 532 nm. The lasing threshold was 3 microJ, corresponding to approximately 30 microJ/cm(2) or 4 kW/cm(2). The emission linewidth decreased from approximately 30 nm in the amplified spontaneous emission mode to <0.4 nm (instrument limited) in the lasing mode. The slope efficiency of the lasing was approximately 1.2%.

Screening for hetero-transglycosylating activities in extracts from nasturtium ( Tropaeolum majus)

Using combinations of different polysaccharides as glycosyl donors and of oligosaccharides fluorescently labeled by sulforhodamine (SR) as glycosyl acceptors, we screened for the presence of transglycosylating activities in extracts from nasturtium ( Tropaeolum majus). Besides xyloglucan endotransglycosylase/hydrolase (XTH/XET, EC 2.4.1.207) activity, which transfers xyloglucanosyl residues from xyloglucan (XG) to XG-derived oligosaccharides (XGOs), a glycosyl transfer from XG to SR-labeled cellooligosaccharides and laminarioligosaccharides has been detected. The XGOs also served as acceptors for the glycosyl transfer from soluble cellulose derivatives carboxymethyl cellulose and hydroxyethylcellulose. The effectivity of these polysaccharides as glycosyl donors for transfer to XG-derived octasaccharide [1- 3H]XXLGol decreased in the order XG > HEC > CMC. Isoelectric focusing in polyacrylamide gels showed that bands corresponding to hetero-transglycosylase activities coincided with zones corresponding to XTH/XET. These results can be explained as due either to substrate non-specificity of certain isoenzymes of XTH/XET or to existence of enzymes catalyzing a hetero-transfer, that is the formation of covalent linkages between different types of carbohydrate polymers.

Poly( d, l-lactide) foams modified by poly(ethylene oxide)–block–poly( d, l-lactide) copolymers and a-FGF: in vitro and in vivo evaluation for spinal cord regeneration

The first goal of this study was to examine the influence that poly(ethylene oxide)–block–poly( d, l-lactide) (PELA) copolymer can have on the wettability, the in vitro controlled delivery capability, and the degradation of poly( d, l-lactide) (PDLLA) foams. These foams were prepared by freeze-drying and contain micropores (10 μm) in addition of macropores (100 μm) organized longitudinally. Weight loss, water absorption, changes in molecular weight, polymolecularity ( M w/ M n) and glass transition temperature ( T g) of PDLLA foams mixed with various amounts of PELA were followed with time. It was found that 10 wt% of PELA increased the wettability and the degradation rate of the polymer foams. The release of sulforhodamine (SR) was compared for PDLLA and PDLLA–PELA foams in relation with the foam porosity. An initial burst release was observed only in the case of the 90:10 PDLLA/PELA foam. The ability of the foam of this composition to be integrated and to promote tissue repair and axonal regeneration in the transected rat spinal cord was investigated. After implantation of ca. 20 polymer rods assembled with fibrin-glue, the polymer construct was able to bridge the cord stumps by forming a permissive support for cellular migration, angiogenesis and axonal regrowth.

Wavelength- and Time-Dependent Two-Photon Photoemission Spectroscopy of Dye-Coated Silicon Surface

Two dye molecules, rhodamine B (RB) and sulforhodamine (SR), were adsorbed on doped silicon. The effect of photoexcitation of the adsorbed species on the electronic properties of the surface was investigated applying wavelength- and time-dependent two-photon photoemission spectroscopy (WD-TPPE). Despite the very similar electronic properties of the two molecules, their adsorption affects very differently the electronic properties of the substrate. It was found that while the adsorbed RB interacts weakly with the substrate and hence preserves its spectral properties, SR interacts strongly with the substrate, causes a decrease in the work function, and looses its “molecular” spectral features. It is shown that photoexcitation of a small fraction (0.01%) of the weakly bound RB species causes a significant change in the electronic properties of the substrate.

A new application of Rhodamine 200 B (Sulfo Rhodamine B)

The laser dye Rhodamine 200 B (Sulfo Rhodamine B) (CI Acid Red 52) can be used as a fluorescent counterion for the transfer of various cationic species (e.g. cationic surfactants and crown-complexes of metal ions) into a chloroform phase. The extraction of ionic associates is found to be independent of the acidity of the aqueous phase within the range of pH 2–12. The extractability of the Rhodamine 200 B anion is compared with that of other dye anions.

Intracellular levels of calmodulin are increased in transformed cells

By using Hoechst 33342, rabbit anti calmodulin antibody, FITC-labeled goat anti rabbit IgG and SR101 (sulfo rhodamine 101) simultaneously to stain individual normal and transformed cells, the microspectrophotometric analysis demonstrated that 3 markers which represented the nucleus, calmodulin and total protein respectively, could be recognized in individualj cells without interference. The phase of the cell cycle was determined by DNA content (Hoechst 33342). We found that in transformed cells (NIH3T3) tsRSV-LA90, cultured at 33°C and transformed C3H10T1/2 cells), the ratio of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV−LA90 cells, cultured at 39°C and C3H10T1/2 cells)in every cell cycle phase. This ratio increased obviously only from G1 to S phase in either normal or transformed cells. The results showed that calmodulin really increased during the transformation, and its increase was specific. In the meantime when cells proceeded from G1 to S, the intracellular calmodulin content also increased specifically.

42] Resonance energy transfer microscopy: Visual colocalization of fluorescent lipid probes in liposomes

Publisher Summary This chapter outlines the modifications to a standard epifluorescence microscope for resonance energy transfer (RET) microscopy of one particular donor/acceptor pair, and demonstrates the feasibility of this technique using fluorescent lipid probes in liposomes. Any conventional fluorescence microscope can be modified with appropriate excitation and emission filters to define the three optical channels required for RET microscopy. The optimal filter combinations is used for observing RET on Zeiss equipment when the donor fluorophore is fluorescein and the acceptor fluorophore is sulforhodamine (SRh) or rhodamine. In the donor or nitrobenz-2-oxa-l,3-diazolyl (NBD) channel, the specimen is excited with blue fight and the resulting green NBD fluorescence is observed through a narrow window, which passes only green light. In the transfer channel, the specimen is illuminated with the blue light appropriate for excitation of NBD, but observation of the emitted light is restricted to red wavelengths that are characteristic of SRh fluorescence.

Antibody-bearing liposomes as multicolor immunofluorescence markers for flow cytometry and imaging

Loposomes covalently coupled to monoclonal antibodies retain the specificity of the antibody and bind only to cells bearing the appropriate determinant. As opposed to directly labeled antibodies which generally have fluorochrome to protein ratio of between 2–5, the entrapped space inside liposome can contain several hundred to several thousand molecules of fluorochromes in a space chemically isolated from the outside environment, thus providing the potential for an amplified fluorescence signal. We have prepared small unilamellar liposomes containing the soluble fluorochromes carboxyfluorescein (CF), which fluoresces in the green and sulforhodamine (SR), which fluoresces in the red, and covalently coupled a series of monoclonal antibodies using a heterobifunctional reagent. We were able to detect, on an Epics 753 flow cytometer equipped with an argon ion and a dye laser and by fluorescence microscopy, both single and double labeled mouse spleen lymphocyte subsets, fibroblast L cells and Raji cells. Complete color separation was obtained with CF-labeled cells being detected only by the green photomultiplier and SR-labeled cells by the red photomultiplier. Cells labeled with both were detected by both photomultipliers. Liposomes bearing anti-Ia antibodies bound only to B lymphocytes whereas those with anti-H-2K antibody bound both to T and B lymphocytes. In another system, single and dual color immunofluorescence made possible the simultaneous detection of HLA and H-2K molecules on transfected murine fibroblast L cells. The signal-to-noise ratio was more favorable for the liposome-labeled reagents than reagents labeled with fluorescein isothiocyanate. Cells labeled with antibody-bearing liposomes could be fixed with paraformaldehyde or glutaraldehyde without adversely affecting the original staining patterns. Apart from the two fluorochromes described above, other markers of choice could be encapsulated without any adverse effect on the antibody-liposome coupling procedure or on the specificity of the conjugated antibody. Since the fluorochrome is not directly coupled to the protein, there is no requirement for protein conjugation sites in order for it be usefully encapsulated inside liposomes. Therefore, this system provides new opportunities to exploit different, as yet untapped fluorochromes for use in flow cytometry and imaging.