Detection of Nitric Oxide from Living Cells Using Polymeric Zinc Organic Framework-Derived Zinc Oxide Composite with Conducting Polymer.

Sensitive and selective detection of nitric oxide (NO) in the human body is crucial since it has the vital roles in the physiological and pathological processes. This study reports a new type of electrochemical NO biosensor based on zinc-dithiooxamide framework derived porous ZnO nanoparticles and polyterthiophene-rGO composite. By taking advantage of the synergetic effect between ZnO and poly(TTBA-rGO) (TTBA = 3'-(p-benzoic acid)-2,2':5',2″-terthiophene, rGO = reduced graphene oxide) nanocomposite layer, the poly(TTBA-rGO)/ZnO sensor probe displays excellent electrocatalytic activity and explores to detect NO released from normal and cancer cell lines. The ZnO is immobilized on a composite layer of poly(TTBA-rGO). The highly porous ZnO offers a high electrolyte accessible surface area and high ion-electron transport rates that efficiently catalyze the NO reduction reaction. Amperometry with the modified electrode displays highly sensitive response and wide dynamic range of 0.019-76 × 10-6 m with the detection limit of 7.7 ± 0.43 × 10-9 m. The sensor probe is demonstrated to detect NO released from living cells by drug stimulation. The proposed sensor provides a powerful platform for the low detection limit that is feasible for real-time analysis of NO in a biological system.

Macromolecular Hydrogen Sulfide Donors Trigger Spatiotemporally Confined Changes in Cell Signaling.

Hydrogen sulfide (H2S) is involved in a myriad of cell signaling processes that trigger physiological events ranging from vasodilation to cell proliferation. Moreover, disturbances to H2S signaling have been associated with numerous pathologies. As such, the ability to release H2S in a cellular environment and stimulate signaling events is of considerable interest. Herein we report the synthesis of macromolecular H2S donors capable of stimulating cell signaling pathways in both the cytosol and at the cell membrane. Specifically, copolymers having pendent oligo(ethylene glycol) and benzonitrile groups were synthesized, and the benzonitrile groups were subsequently transformed into primary aryl thioamide groups via thionation using sodium hydrosulfide. These thioamide moieties could be incorporated into a hydrophilic copolymer or a block copolymer (i.e., into either the hydrophilic or hydrophobic domain). An electrochemical sensor was used to demonstrate release of H2S under simulated physiological conditions. Subsequent treatment of HEK293 cells with a macromolecular H2S donor elicited a slow and sustained increase in cytosolic ERK signaling, as monitored using a FRET-based biosensor. The macromolecular donor was also shown to induce a small, fast and sustained increase in plasma membrane-localized PKC activity immediately following addition to cells. Studies using an H2S-selective fluorescent probe in live cells confirmed release of H2S from the macromolecular donor over physiologically relevant time scales consistent with the signaling observations. Taken together, these results demonstrate that by using macromolecular H2S donors it is possible to trigger spatiotemporally confined cell signaling events. Moreover, the localized nature of the observed signaling suggests that macromolecular donor design may provide an approach for selectively stimulating certain cellular biochemical pathways.

Thioamides: versatile bonds to induce directional and cooperative hydrogen bonding in supramolecular polymers.

The amide bond is a versatile functional group and its directional hydrogen-bonding capabilities are widely applied in, for example, supramolecular chemistry. The potential of the thioamide bond, in contrast, is virtually unexplored as a structuring moiety in hydrogen-bonding-based self-assembling systems. We report herein the synthesis and characterisation of a new self-assembling motif comprising thioamides to induce directional hydrogen bonding. N,N',N''-Trialkylbenzene-1,3,5-tris(carbothioamide)s (thioBTAs) with either achiral or chiral side-chains have been readily obtained by treating their amide-based precursors with P2S5. The thioBTAs showed thermotropic liquid crystalline behaviour and a columnar mesophase was assigned. IR spectroscopy revealed that strong, three-fold, intermolecular hydrogen-bonding interactions stabilise the columnar structures. In apolar alkane solutions, thioBTAs self-assemble into one-dimensional, helical supramolecular polymers stabilised by three-fold hydrogen bonding. Concentration- and temperature-dependent self-assembly studies performed by using a combination of UV and CD spectroscopy demonstrated a cooperative supramolecular polymerisation mechanism and a strong amplification of supramolecular chirality. The high dipole moment of the thioamide bond in combination with the anisotropic shape of the resulting cylindrical aggregate gives rise to sufficiently strong depolarised light scattering to enable depolarised dynamic light scattering (DDLS) experiments in dilute alkane solution. The rotational and translational diffusion coefficients, D(trans) and D(rot), were obtained from the DDLS measurements, and the average length, L, and diameter, d, of the thioBTA aggregates were derived (L = 490 nm and d = 3.6 nm). These measured values are in good agreement with the value L(w) = 755 nm obtained from fitting the temperature-dependent CD data by using a recently developed equilibrium model. This experimental verification validates our common practice for determining the length of BTA-based supramolecular polymers from model fits to experimental CD data. The ability of thioamides to induce cooperative supramolecular polymerisation makes them effective and broadly applicable in supramolecular chemistry.

On-resin synthesis of novel arginine-isostere peptides bearing substituted amidine headgroups.

A methodology is presented for the facile synthesis of Arg-containing peptides modified at the guanidine headgroup as substituted amidine cores. This process allows for the iterative construction of these Arg isosteres while the peptide is being built out on the solid support, providing a high potential for diversity in substitution pattern in the resulting peptide. A series of N-Pmc-substituted thioamides were condensed with deprotected δ-N Orn-bearing peptides while attached to the solid support using Mukaiyama's reagent as coupling reagent, yielding isosteric Arg-containing analogs. Peptides were cleaved using trimethylsilyl trifluoromethanesulfonate/TFA and analyzed in their crude form in order to illustrate the amenability of this process toward production of peptide isolates in high crude purity. Arg-containing peptides having a single Arg isostere were utilized to show the general utility of this approach as well as a multiple-Arg-containing construct, illustrating the amenability of this method toward stepwise construction of differently substituted amidine headgroups within the same peptide.

A novel Y-shaped photoiniferter used for the construction of polydimethylsiloxane surfaces with antibacterial and antifouling properties.

The simultaneous introduction of two new functionalities into the same polymeric substrate under mild reaction conditions is an interesting and important topic. Herein, dual-functional polydimethylsiloxane (PDMS) surfaces with antibacterial and antifouling properties were conveniently developed via a novel Y-shaped asymmetric dual-functional photoiniferter (Y-iniferter). The Y-iniferter was initially immobilized onto the PDMS surface by radical coupling under visible light irradiation. Afterwards, poly(2-hydroxyethyl methacrylate) (PHEMA) brushes and antibacterial ionic liquid (IL) fragments were simultaneously immobilized on the Y-iniferter-modified PDMS surfaces by combining the sulfur(VI)-fluoride exchange (SuFEx) click reaction and UV-photoinitiated polymerization. Experiments using E. coli as a model bacterium demonstrated that the modified PDMS surfaces had both the expected antibacterial properties of the IL fragments and the excellent antifouling properties of PHEMA brushes. Furthermore, the cytotoxicity of the modified PDMS surfaces to L929 cells was examined in vitro with a CCK-8 assay, which showed that the modified surfaces maintained excellent cytocompatibility. Briefly, this strategy of constructing an antibacterial and antifouling PDMS surface has the advantages of simplicity and convenience and might inspire the construction of diverse dual-functional surfaces by utilizing PDMS more effectively.

Effect of dentifrices against hydrochloric acid-induced erosion.

PURPOSE: This in vitro investigation assessed whether different dentifrices would be capable of controlling the enamel erosion progression caused by HCl. MATERIALS AND METHODS: Sixty bovine enamel slabs were covered with acid-resistant varnish, except for a 2.5-mm2 circular area on the labial surface. According to a complete block design, the experimental units were immersed in HCl solution (pH 1.2; 0.1M). After storage in artificial saliva for 1 h, specimens (n = 15) were exposed to different dentifrices: Sensodyne Cool Gel (1100 ppm F), Sensodyne ProNamel (1450 ppm F), and PrevDent 5000 (5000 ppm F). The control group was immersed in deionised water. Following five cycles of erosive challenge, the slabs were prepared for porosity evaluation using solutions of copper sulfate and rubeanic acid. RESULTS: ANOVA demonstrated no difference in the enamel porosity as a function of the dentifrice employed (P = 0.5494). CONCLUSION: The damage caused by a simulated intrinsic erosive challenge seems unable to be controlled by fluoridated dentifrices, even when this ion is found in elevated concentrations.

In vitro evaluation of viability, integrity, and inflammation in genital epithelia upon exposure to pharmaceutical excipients and candidate microbicides.

The use of microbicides is a promising approach for the prevention of HIV-1 transmission. Unfortunately, various candidates failed in clinical trials. In some cases, the candidate microbicide even resulted in enhanced virus transmission. Therefore, there is an urgent need to develop more predictive preclinical strategies to anticipate the in vivo efficiency/toxicity rate, including in vitro assays that evaluate effects on epithelial integrity and inflammation. The present study aims to identify potential safety issues concerning the use of microbicides and excipients commonly used in vaginal microbicide preparations. The toxicities of various active pharmaceutical ingredients (APIs; TMC-120, UC-781, tenofovir [PMPA], PRO-2000, and glycerol monolaurate [GML]) and excipients (preservatives, cosolvents, surfactants, and cyclodextrins) were evaluated using an in vitro dual-chamber model and uterine cervical explants. Epithelial viability and permeation of fluorescent virus-sized beads, as well as induction of interleukin-8 (IL-8; as a sensitive marker of an inflammatory response), were assessed. Surprisingly, cell viability and epithelial layer integrity were compromised by most excipients at concentrations near the typical concentration used in vaginal gels, and a significant increase in the production of IL-8 was observed at subtoxic concentrations. Within the APIs, TMC-120, UC-781, and PMPA showed higher selectivity indices than PRO-2000 and GML. In conclusion, identification of safety issues concerning the use of pharmaceutical excipients could help to formulate less toxic vaginal microbicide preparations.

Oxyanion transport across lipid bilayers: direct measurements in large and giant unilamellar vesicles.

A simple di(thioamido)carbazole 1 serves as a potent multispecific transporter for various biologically relevant oxyanions, such as drugs, metabolites and model organic phosphate. The transport kinetics of a wide range of oxyanions can be easily quantified by a modified lucigenin assay in both large and giant unilamellar vesicles.

Direct catalytic asymmetric aldol reactions of thioamides: toward a stereocontrolled synthesis of 1,3-polyols.

A direct catalytic asymmetric aldol reaction of thioamides with a soft Lewis acid/hard Brønsted base cooperative catalytic system comprising (R,R)-Ph-BPE/[Cu(CH(3)CN)(4)]PF(6)/LiOAr is described. Highly chemoselective deprotonative activation of thioamides allows for a direct aldol reaction of alpha-nonbranched aliphatic aldehydes, which are susceptible to self-condensation. Facile reduction of the thioamide functionality and a catalyst-controlled second aldol reaction provides 1,3-diols in a highly stereoselective manner.

Development of a comprehensive human immunodeficiency virus type 1 screening algorithm for discovery and preclinical testing of topical microbicides.

Topical microbicides are self-administered, prophylactic products for protection against sexually transmitted pathogens. A large number of compounds with known anti-human immunodeficiency virus type 1 (HIV-1) inhibitory activity have been proposed as candidate topical microbicides. To identify potential leads, an in vitro screening algorithm was developed to evaluate candidate microbicides in assays that assess inhibition of cell-associated and cell-free HIV-1 transmission, entry, and fusion. The algorithm advances compounds by evaluation in a series of defined assays that generate measurements of relative antiviral potency to determine advancement or failure. Initial testing consists of a dual determination of inhibitory activity in the CD4-dependent CCR5-tropic cell-associated transmission inhibition assay and in the CD4/CCR5-mediated HIV-1 entry assay. The activity is confirmed by repeat testing, and identified actives are advanced to secondary screens to determine their effect on transmission of CXCR4-tropic viruses in the presence or absence of CD4 and their ability to inhibit CXCR4- and CCR5-tropic envelope-mediated cell-to-cell fusion. In addition, confirmed active compounds are also evaluated in the presence of human seminal plasma, in assays incorporating a pH 4 to 7 transition, and for growth inhibition of relevant strains of lactobacilli. Leads may then be advanced for specialized testing, including determinations in human cervical explants and in peripheral blood mononuclear cells against primary HIV subtypes, combination testing with other inhibitors, and additional cytotoxicity assays. PRO 2000 and SPL7013 (the active component of VivaGel), two microbicide products currently being evaluated in human clinical trials, were tested in this in vitro algorithm and were shown to be highly active against CCR5- and CXCR4-tropic HIV-1 infection.

Dissolution enhancement of the anti-HIV drug UC 781 by formulation in a ternary solid dispersion with TPGS 1000 and Eudragit E100.

The present research deals with the improvement of the dissolution properties of the anti-HIV drug UC 781. A ternary solid dispersion consisting of a high amount of TPGS 1000 and exhibiting good powder properties with respect to flowability was developed. Eudragit E100 was selected as a polymer based on supersaturation studies. DSC analysis of solid dispersions containing drug doses from 0 to 80% w/w revealed eutectic phase behaviour of the ternary TPGS 100-Eudragit E100-UC 781 mixture. The release of UC 781 in a medium simulating the gastrointestinal lumen was markedly enhanced, reaching a release of 70% w/w after 4h. XRD results pointed to the presence of crystalline drug in the solid dispersion. The presence of UC 781 in the dispersion had an influence on the TPGS 1000-Eudragit E100 carrier, favoring folding of the polyethylene glycol chains in TPGS 1000. Moreover, the addition of UC 781 to the binary polymer-surfactant mixture was physically expressed by an increase in fluidity of the samples up to a drug load of 50% w/w. NMR was used to investigate this phenomenon, revealing a shielding and/or deshielding effect of the carrier on aromatic C atoms and methyl groups in UC 781. Polyethylene glycol chains present in TPGS 1000 seemed to play a role in this process. In addition, combining UC 781 with the TPGS 1000-Eudragit E100 mixture led to the appearance of TPGS 1000 clusters with a glass transition temperature well below the T(g)'s of the pure compounds.

Free flowing solid dispersions of the anti-HIV drug UC 781 with Poloxamer 407 and a maximum amount of TPGS 1000: investigating the relationship between physicochemical characteristics and dissolution behaviour.

Solid dispersions and physical mixtures made up of the poorly water-soluble drug UC 781, a polymer and a surfactant were prepared to contribute to the understanding of the relationship between physicochemical characteristics and dissolution behaviour. In addition, to facilitate downstream processing while still favouring drug dissolution to a maximum extent, formulation conditions were investigated to obtain a free flowing powder which contains a maximum amount of surfactant. Poloxamer 407, a polyethylene-polypropylene glycol block copolymer, was selected as a suitable polymer based on UC 781 supersaturation results. d-Alpha-tocopheryl polyethyleneglycol succinate 1000 (TPGS 1000) was preferred as a surfactant since it increased UC 781 dissolution when formulated in a self-micro emulsifying drug delivery system (SMEDDS), as compared to TPGS 400, TPGS 4000 and TPGS 6000. Based on flow properties, a TPGS 1000/Poloxamer 407 ratio of 80/20 was used to prepare solid dispersions by spray drying. Pure drugs, physical mixtures and solid dispersions were characterized by differential scanning calorimetry and X-ray powder diffraction. Eutectic phase behaviour was obtained in which the relative distribution of the polyethylene glycol folding was dependent on UC 781 concentration. Drug release was markedly increased when formulated as a solid dispersion with Poloxamer 407 and TPGS 1000. Formulation of solid dispersions did however not further improve the drug dissolution rate compared to that of physical mixtures. Nonetheless, variability of dissolution results was considerably reduced upon solid dispersion formulation.

Formulation of fast disintegrating tablets of ternary solid dispersions consisting of TPGS 1000 and HPMC 2910 or PVPVA 64 to improve the dissolution of the anti-HIV drug UC 781.

Solid dispersion formulations made up of d-alpha-tocopheryl polyethylene glycol succinate 1000 (TPGS 1000) and polyvinyl pyrrolidone co-vinyl acetate 64 (PVPVA 64) or hydroxy propyl methyl cellulose 2910 (HPMC 2910) were developed in order to improve the dissolution of UC 781. UC 781 dissolution rate was markedly improved as compared to the physical mixtures and the pure drug, attaining maximum drug releases of up to 100% after only 5 min in the case of TPGS 1000-UC 781-PVPVA 64 solid dispersions and 30 min in TPGS 1000-UC 781-HPMC 2910. The increased UC 781 dissolution rate could be maintained when formulating UC 781 in PVPVA 64 tablets. The latter disintegrated in only 4 min, reaching drug releases of up to 90% (w/w). In addition, as opposed to the corresponding solid dispersions, no decrease in drug release occurred upon dissolution of PVPVA 64 tablets when the pH was increased to 6.8. Contrary to the PVPVA 64 tablet formulations, HPMC 2910 tablets showed a slow dissolution process due to the gelling nature of the polymer. The drug was slowly released as HPMC 2910 dissolved in the medium, however also in this case 90% (w/w) of the drug was dissolved after 4 h. Both polymers formed compatible blends in combination with the drug. Thermal analysis of the ternary mixtures revealed eutectic behavior exhibiting an extremely fine dispersion of the drug in the carrier. This was confirmed by the fact that no drug crystals could be detected using X-ray diffraction (XRD). As opposed to the physical mixtures, PVPVA 64 and HPMC 2910 solid dispersions did not contain any isolated polymer-rich phases, hence showed improved homogeneity. Amorphous TPGS 1000 clusters occurred in PVPVA 64 and HPMC 2910 formulations upon addition of at least 10% (w/w) UC 781, showing extremely low glass transition temperatures depending of the thermal history of the samples.

Role of seminal plasma in the anti-HIV-1 activity of candidate microbicides.

BACKGROUND: Evaluation of microbicides for prevention of HIV-1 infection in macaque models for vaginal infection has indicated that the concentrations of active compounds needed for protection by far exceed levels sufficient for complete inhibition of infection in vitro. These experiments were done in the absence of seminal plasma (SP), a vehicle for sexual transmission of the virus. To gain insight into the possible effect of SP on the performance of selected microbicides, their anti-HIV-1 activity in the presence, and absence of SP, was determined. METHODS: The inhibitory activity of compounds against the X4 virus, HIV-1 IIIB, and the R5 virus, HIV-1 BaL was determined using TZM-bl indicator cells and quantitated by measuring beta-galactosidase induced by infection. The virucidal properties of cellulose acetate 1,2-benzene-dicarboxylate (CAP), the only microbicide provided in water insoluble, micronized form, in the presence of SP was measured. RESULTS: The HIV-1 inhibitory activity of the polymeric microbicides, poly(naphthalene sulfonate), cellulose sulfate, carrageenan, CAP (in soluble form) and polystyrene sulfonate, respectively, was considerably (range approximately 4 to approximately 73-fold) diminished in the presence of SP (33.3%). Formulations of micronized CAP, providing an acidic buffering system even in the presence of an SP volume excess, effectively inactivated HIV-1 infectivity. CONCLUSION: The data presented here suggest that the in vivo efficacy of polymeric microbicides, acting as HIV-1 entry inhibitors, might become at least partly compromised by the inevitable presence of SP. These possible disadvantages could be overcome by combining the respective polymers with acidic pH buffering systems (built-in for formulations of micronized CAP) or with other anti-HIV-1 compounds, the activity of which is not affected by SP, e.g. reverse transcriptase and zinc finger inhibitors.

Sorbent extraction of rubeanic acid-metal chelates on a new adsorbent: Sepabeads SP70.

A sorbent extraction procedure for lead, iron, cadmium and manganese ions on Sepabeads SP70 adsorption resin has been presented prior to their flame atomic absorption spectrometric determinations. By the passage of aqueous samples including analyte ions-rubeanic acid chelates through Sepabeads SP70 column, metal chelates adsorb quantitatively and almost all matrix elements will pass through the column to drain. The influence of potential interfering ions was also studied. The validation of the method was made though the analysis of LGC 6010 Hard drinking water, SRM 1577b Bovine liver and GBW 07603 Bush branches and leaves standard reference materials (SRM). The method was applied to the determination of analyte ions from various water, wastewater, cow meat and milk, red wine, and tobacco samples with successfully results.

Visualisation of latent fingermarks on polymer banknotes using copper vacuum metal deposition: A preliminary study.

The UK's recent move to polymer banknotes has seen some of the currently used fingermark enhancement techniques for currency potentially become redundant, due to the surface characteristics of the polymer substrates. Possessing a non-porous surface with some semi-porous properties, alternate processes are required for polymer banknotes. This preliminary investigation explored the recovery of fingermarks from polymer notes via vacuum metal deposition using elemental copper. The study successfully demonstrated that fresh latent fingermarks, from an individual donor, could be clearly developed and imaged in the near infrared. By varying the deposition thickness of the copper, the contrast between the fingermark minutiae and the substrate could be readily optimised. Where the deposition thickness was thin enough to be visually indistinguishable, forensic gelatin lifters could be used to lift the fingermarks. These lifts could then be treated with rubeanic acid to produce a visually distinguishable mark. The technique has shown enough promise that it could be effectively utilised on other semi- and non-porous substrates.

Thioacylation reactions for the surface functionalization of phosphorus-containing dendrimers.

The functionalization of phosphorus-containing dendrimers was easily achieved through thioacylation reactions involving new dendrimers capped with dithioester end groups and various functionalized amines. These reactions were successfully applied to the first generation (12 end groups) and the third generation of the dendrimer (48 end groups) and allowed their functionalization by various primary or secondary amines, alcohols, glycols, and azides. [reaction: see text]

Physicochemical characterization of solid dispersions of the antiviral agent UC-781 with polyethylene glycol 6000 and Gelucire 44/14.

The purpose of this study was to prepare and characterize solid dispersions of the antiviral thiocarboxanilide UC-781 with PEG 6000 and Gelucire 44/14 with the intention of improving its dissolution properties. The solid dispersions were prepared by the fusion method. Evaluation of the properties of the dispersions was performed using dissolution studies, differential scanning calorimetry, Fourier-transform infrared spectroscopy and X-ray powder diffraction. To investigate the possible formation of solid solutions of the drug in the carriers, the lattice spacings [d] of PEG 6000 and Gelucire 44/14 were determined in different concentrations of UC-781. The results obtained showed that the rate of dissolution of UC-781 was considerably improved when formulated in solid dispersions with PEG 6000 and Gelucire 44/14 as compared to pure UC-781. From the phase diagrams of PEG 6000 and Gelucire 44/14 it could be noted that up to approximately 25% w/w of the drug was dissolved in the liquid phase in the case of PEG 6000 and Gelucire 44/14. The data from the X-ray diffraction showed that the drug was still detectable in the solid state below a concentration of 5% w/w in the presence of PEG 6000 and Gelucire 44/14, while no significant changes in the lattice spacings of PEG 6000 or Gelucire 44/14 were observed. Therefore, the possibility of UC-781 to form solid solutions with the carriers under investigation was ruled out. The results from infrared spectroscopy together with those from X-ray diffraction and differential scanning calorimetry showed the absence of well-defined drug-polymer interactions.

Physical stability of solid dispersions of the antiviral agent UC-781 with PEG 6000, Gelucire 44/14 and PVP K30.

This paper describes the physical stability of solid dispersions of UC-781 with PEG 6000, Gelucire 44/14 and PVP K30 prepared by the solvent and melting methods. The concentration of the drug in the solid dispersions ranged from 5 to 80% w/w. The solid dispersions were stored at 4-8 and 25 degrees C (25% RH), then their physicochemical properties were analysed by differential scanning calorimetry (DSC), X-ray powder diffraction and dissolution studies as a function of storage time. The DSC curves of solid dispersions of UC-781 with PVP K30 did not show any melting peaks corresponding to UC-781 after storage, indicating no recrystallization of the drug. The DSC data obtained from PEG 6000 and Gelucire 44/14 showed some variations in melting peak temperatures and enthalpy of fusion of the carriers. It was shown that the enthalpy of fusion of PEG 6000 in the dispersions increased after storage; it was more pronounced for samples stored at 25 degrees C compared to those at 4-8 degrees C indicating the reorganization of the crystalline domains of the polymer. Similarly, the enthalpy of fusion of Gelucire 44/14 in the solid dispersions increased as a function of time. Dissolution of UC-781 from all solid dispersions decreased as a function of storage time. While these observations concurred with the DSC data for all solid dispersions, they were not reflected by X-ray powder diffraction data. It was concluded that it is the change of the physical state of the carriers and not that of the drug, which is responsible for the decreased dissolution properties of the solid dispersions investigated.