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The invention refers to a new process for preparing coated particles and hollow shells by coating colloidal particles with alternating layers of oppositely charged nanoparticles and polyelectrolytes and optionally removing the colloidal cores.
1. A process for preparing coated particles comprising the steps:
(a) providing template particles and
(b) coating said template particles with a multilayer comprising (i) alternating layers of oppositely charged nanoparticles and polyelectrolytes and/or (ii) alternating layers of oppositely charged nanoparticles.
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16. A coated particle having a core which is a template particle and a multilayer shell comprising a member selected from the group consisting of alternating layers of (i) oppositely charged nanoparticles and polyelectrolytes, and (ii) oppositely charged nanoparticles.
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18. A hollow shell obtained by disintegrating the template particle of the coated particle of
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The invention refers to a new process for preparing coated capsules and hollow shells by coating colloidal particles with alternating layers of oppositely charged nanoparticles and polyelectrolytes.
The area of thin film fabrication, in which ordered, functional supramolecular structures are the chief goal, has been greatly impacted by the recent introduction of the layer-by-layer (LbL) self-assembly technique (Decher, Science 1997, 277, 1232). The LbL method permits the fabrication of multilayer thin film assemblies on solid supports by the spontaneous sequential adsorption of oppositely charged species from dilute aqueous solutions onto charged substrates. The driving force for the multilayer film build-up is primarily due to the electrostatic attraction and complex formation between the charged species deposited. The LbL approach was initially employed to construct multilayer films of polyelectrolytes (Decher, Science 1997, 277, 1232), and subsequently extended to include proteins (Lvov et al, J. Am. Chem. Soc. 1995, 117, 6117; Onda et al, T. Biotech. Bioeng. 1996, 51, 163; Caruso et al, Langmuir 1997, 13, 3427), nucleic acids (Decher et al, J. Biosens. Bioelectron. 1994, 9, 677; Sukhorukov et al, Thin Solid Films 1996, 284/285, 220; Caruso et al, Anal. Chem. 1997, 69, 2043), dyes (Araki et al, Langmuir 1996, 12, 5393; Yoo et al, Synthetic Metals 1997, 85, 1425; Ariga et al, J. Am. Chem. Soc. 1997, 119, 2224), dendrimers (Tsukruk et al, Langmuir 1997,13, 2171), and various inorganic nanoparticles (Kleinfeld et al, Science 1994, 265, 370; Keller et al, J. Am. Chem. Soc. 1994, 116, 8817; Kotov et al, J. Am. Chem. Soc. 1997, 119, 6821; Kotov et al, J. Phys. Chem. 1995, 99, 13065; Feldheim et al, J. Am. Chem. Soc. 1996, 118, 7640; Schmitt et al, Adv. Mater 1997, 9, 61; Lvov et al, Langmuir 1997, 13, 6195) in polyelectrolyte multilayer assemblies by replacing one of the polyions by a similarly charged species.
The vast majority of studies concerning the LbL technique have employed macroscopically flat charged surfaces as substrates for multilayer film formation. For example, U.S. Pat. No. 5,716,709 describes multilayered nanostructures comprising alternating organic and inorganic ionic layers on a flat substrate, such as a silicon wafer. Recently, Keller et al reported the preparation of alternating composite multilayers of exfoliated zirconium phosphate sheets and charged redox polymers on (3-aminopropyl)-triethoxysilane-modified silica particles (Keller et al, J. Am. Chem. Soc. 1995, 117, 12879).
In more recent studies (Caruso et al, J. Phys. Chem. B. 1998, 102, 2011; Sukhorukov et al., Colloids Surf. A: Physicochem.Eng.Aspects 1998, 137, 253), the LbL approach was successfully applied to utilise submicron- and micron-sized charged colloidal particles as the adsorbing substrates to produce colloid-supported polyelectrolyte multilayer films: regular step-wise polyelectrolyte multilayer growth was observed on the colloids.
Considerable scientific effort has focussed on the fabrication of composite micro- and nanoparticles that consist of either organic or inorganic cores coated with shells of different chemical composition (Kawahashi and Matijevic, J. Colloid Interface Sci. 1991, 143, 103; Garg and Matijevic, J. Colloid Interface Sci. 1988, 126; Kawahashi and Matijevic, J. Colloid Interface Sci. 1990, 138, 534; Ohmori and Matijevic, J. Colloid Interface Sci. 1992, 150, 594; Giersig et al., Adv. Mater. 1997, 9, 570; Liz-Marzan et al., Langmuir 1996, 12, 4329; Liz-Marzan et al., J. Chem.Soc.Chem.Commun. 1996, 731; Giersig et al., Ber.Bunsenges.Phys.Chem. 1997, 101, 1617; Correa-Duarte et al., Chem.Phys.Lett. 1998, 286, 497; Bamnolker et al., J.Mater.Sci.Lett. 1997, 16, 1412; Margel and Weisel, J.Polym.Sci.Chem.Ed. 1984, 22, 145; Philipse et al., Langmuir 1994, 10, 92). These core-shell particles often exhibit properties which are significantly different to those of the templated core (e.g. different surface chemical composition, increased stability, higher surface area, as well as different magnetic and optical properties), thus making them attractive both from a scientific and technological viewpoint. Applications for such particles are diverse, ranging from capsule agents for drug delivery, catalysis, coatings, composite materials, as well as for protecting sensitive agents such as enzymes and proteins. Previous investigations have demonstrated that polymeric microparticles and inorganic cores can be coated with uniform layers of various materials, including silica, yttrium basic carbonate, zirconium hydrous oxide, either by controlled surface precipitation reactions on the core particles, or by direct surface reactions.
U.S. Pat. No. 5,705,222 discloses a process for preparing composite particle dispersions wherein a plurality of core particles is dispersed in a first solution wherein the core particles do not irreversibly self-flocculate, an amount of polymer is added to the dispersion of core particles, wherein the polymer has an affinity for the dispersed core particles and the excess polymer is removed by a solid/liquid separation process, i.e. centrifugation or decanting.
An important extension of core-shell particles is the subsequent removal of the core, resulting in hollow particles or shells. Removal of the templated core has previously been achieved by calcining the coated particles at elevated temperatures or by chemical reactions causing dissolution of the core material. Hollow, submicron sized shells of yttrium compounds have been produced (Kawahashi and Matijevic, 1991, supra) by coating cationic polystyrene latex with yttrium basic carbonate and subsequently calcining. More recently, silica shells were generated by seeded polymerization of tetraethoxysilane on the surface of polystyrene particles, followed by calcination (Bamnolker et al., 1997, supra). Using a similar method, monodisperse, hollow silica nanoparticles have been produced by silicacoating gold nanoparticles, and by chemically dissolving the cores (Giersig et al., Ber.Bunsenges.Phys.Chem., 1997, supra). Hollow particles represent a special class of materials: their lower density and optical properties make them of interest in the fields of medicine, pharmaceutics, materials science and the paint industry.
Conventional methods for the preparation of coated nanoparticles or hollow nanoshells, however, have several disadvantages, since in many cases the formation of uniform and smooth layer structures having sufficient particle coverage as well as control of thickness is very difficult to achieve.
Further, it was suggested (DE 198 12 083.4) that the use of soluble colloidal cores as templates for the sequential deposition of polyelectrolytes can be used to fabricate novel three-dimensional hollow polymer shells.
Herein we report the construction of composite multilayers of nanoparticles and an oppositely charged polyelectrolyte on submicron-sized colloidal particles via the sequential electrostatic adsorption of nanoparticles and polyelectrolyte from dilute solution. Alternating nanoparticle-polyelectrolyte multilayers with various thicknesses have been fabricated. Further, a novel and yet simple method for the fabrication of submicron-sized, hollow, inorganic or composite organic-inorganic particles via colloid templated electrostatic LBL self-assembly of nanoparticle-polymer multilayers, followed by removal of the templated core and optionally the polymer used in the assembly process is presented.
Thus, a first aspect of the present invention is a process for preparing coated particles comprising the steps:
(a) providing template particles and
(b) coating said template particles with a multilayer comprising (i) alternating layers of oppositely charged nanoparticles and polyelectrolytes and/or (ii) alternating layers of oppositely charged nanoparticles.
Preferably, the template particles have an average diameter of up to 10 μm, more preferably≦5 μm, and most preferably≦2 μm. The minimum diameter of the template particles is preferably 10 nm, more preferably 100 nm, and most preferably 200 nm.
Suitable template particles may be selected from organic particles, inorganic particles, or any combination thereof. For example, the template particles may be inorganic particles including inorganic structures. In a preferred embodiment, the template particles are selected from organic polymer latices, such as polystyrene or styrene copolymer latices. On the other hand, also partially cross-linked melamine-formaldehyde template particles can be used which can be disintegrated under mild conditions, e.g. by adjusting the pH value to an acid value of e.g. ≦1.5, by dissolving in mild organic solvents such as DMSO or by chemical reactions, e.g. sulfonation with alkali sulfites, alkali hydrogen sulfites etc. Regarding the preparation of partially cross-linked melamine-formaldehyde template particles reference is made to DE 198 12 083.4, particularly Example 1, where it is described that in the polycondensation of melamine-formaldehyde precondensates (cf. DD 224 602) the polycondensation process can be interrupted after some time, e.g. 1 min to 1 h after start of the reaction, so that dissoluble, partially cross-linked melamine-formaldehyde template particles are obtained.
Other organic template particles, e.g. polystyrene latices, can be disintegrated by dissolving in appropriate organic solvents such as THF or by heating, e.g. to temperatures of 500° C. or greater.
The process according to the present invention comprises coating the template particles with alternating coatings of polyelectrolyte molecules and nanoparticles. The polyelectrolytes are usually polymers having ionically dissociable groups which may be a component or substituent of the polymer chain. Preferably, linear or/and water-soluble polyelectrolytes are used. Depending on the type of dissociable group, polyelectrolytes are subdivided into polyacids and polybases. Upon dissociation polyacids separate protons to give polyanions. Examples of polyacids are polyphosphoric acid, polyvinyl or polystyrene sulphuric acid, polyvinyl or polystyrene sulfonic acid, polyvinyl or polystyrene phosphonic acid and polyacrylic acid. Examples of the respective salts, which are also called polysalts, are polyphosphate, polysulphate, polysulfonate, polyphosphonate and polyacrylate. If the polyelectrolyte is a polycation, the nanoparticles preferably have an overall anionic charge.
Polybases contain groups which are capable of accepting protons, e.g. by reacting with acids to give salts. Examples of polybases are polyamines, such as polyethylene amine, polyvinyl amine and polyvinyl pyridine or poly(ammonium salts), such as poly (diallyl dimethylammonium chloride). Polybases form polycations by accepting protons. Preferably, polybases (i.e. polycations) are used as polyelectrolyte. If the polyelectrolyte is a polyanion, the nanoparticles preferably have an overall cationic charge.
The nanoparticles are preferably inorganic materials and may be selected from ceramic particles, e.g. oxidic ceramic particles, such as silicon dioxide, titanium dioxide, zirconium dioxide optionally doped with other metal oxides, magnetic particles such as iron oxide-containing particles such as Fe3O4 , magneto-optical particles, nitridic ceramic particles, e.g. Si3N4, carbidic ceramic particles, metallic particles, e.g. gold, silver, palladium and sulfur or selene-containing particles such as cadmium sulfide, cadmium selenide etc. Especially preferred are oxidic ceramic particles such as silicon dioxide. It should be noted, however, that also organic or biological nanoparticles are suitable for performing the present invention, e.g. macromolecules, such as polypeptides, proteins, nucleic acids, etc. Especially preferred biological nanoparticles are proteins, e.g. immunologically reactive proteins such as antigens and antibodies, e.g. IgG, which may be deposited on the template particle alternating with polyelectrolyte molecules. The biological nanoparticles may be deposited as conjugates containing a labelling group, e.g. a fluorescent group such as fluorescein. The resulting particles are suitable for analytical, e.g. immunological, detection methods. Further, an immobilization of enzymes, single enzymes or a plurality of enzymes, e.g. members of an enzymatic cascade, in single layers or different layers is of special interest because of the ability to increase the catalysis efficiency. Substrates could readily diffuse through the film and react with the immobilized enzyme, thereby producing product.
For the preparation of the coated particles according to the present application preferably an aqueous dispersion of template particles of suitable size is provided. The aqueous dispersion may contain a salt, e.g. NaCl in a concentration which preferably ranges from 50 mmole/l to 1 mole/l. Alternating layers of oppositely charged components, i.e. polyelectrolyte molecules and nanoparticles, different types of nanoparticles or combinations thereof are then deposited on said template particles. The pH of the aqueous dispersion is adjusted in such a way that the molecules in each alternating layer, e.g. polyelectrolyte molecules and nanoparticles, each have opposite total charges. The thickness of the coating which is determined by the number of layers is preferably 2 to 1000 nm, with 2 to 40 and particularly 2 to 20, e.g. 3 to 10 coatings being applied. Suitably, each layer may be comprised of a single species of polyelectrolyte or nanoparticle or of a mixture comprising at least two polyelectrolyte or nanoparticle species. Further, for each layer different polyelectrolyte or nanoparticle species may be used.
After application of each layer the excessive molecules (e.g. polyelectrolyte or nanoparticle) which have not contributed to forming the layer are preferably separated off before the next layer is applied. Such separation can be done according to any known method, particularly centrifugation, filtration or/and dialysis.
In a preferred embodiment of the invention the template particles are at first coated with several layers of oppositely charged cationic and anionic polyelectrolytes before the alternating layers of nanoparticles and polyelectrolyte or the alternating nanoparticle layers are applied. The coating of template particles with several layers of oppositely charged polyelectrolytes is described in DE 198 12 083.4 to which express reference is made. Preferably, the template particles are coated with at least two and up to six layers of oppositely charged cationic and anionic polyelectrolytes, e.g. with three layers. The outermost polyelectrolyte layer is preferably oppositely charged with regard to the nanoparticle to be deposited.
The template particles can be of any shape whatsoever, e.g. they can be spherical or rod-shaped. They can have any regular or irregular structure including crystal structures. Furthermore, the template particles can also be composed of several small sub-particles.
The thickness of the shell walls around the template particles can be readily controlled by varying the number of deposition cycles, whilst the shell size and shape are predetermined by the dimensions of the template particle employed. The thickness of the shell can vary in a wide range, e.g. from 2 to 1000 nm, particularly from 5 to 250 nm.
Preferably, the template particles are at least partially disintegrated after the coating has been completed. They can be dissolved in appropriate solvents or thermally (e.g. by calcination to temperatures of at least 500° C.) or—e.g. if partially cross-linked melamine-formaldehyde template particles are used—by mild chemical methods, e.g. in DMSO, or a change in the pH value. After dissolution of the template particles hollow shells remain which are composed of the nanoparticle material and optionally the polyelectrolyte material. The resulting hollow shells may be inorganic or organic shells or composite inorganic-organic or composite inorganic-inorganic shells depending on the method of core removal. For example, when thermal treatment (calcination) is used, all organic matter is removed, hence only inorganic shells are obtained. Exposure to solvent or low-pH solutions to remove the core results in composite hollow particles in which the core is removed but the polyelectrolyte assembled between the nanoparticle layers remains in the shell.
The hollow shells may be characterized by any known methods, e.g. by scanning and transmission electron microscopy and atomic force microscopy. Preferably, the shells are uniform layers of regular thickness and can find applications in numerous areas, such as medicine, pharmaceutics, catalysis, optics, magnetics, separation and sensing methods. In the hollow shells of the present invention active agents, e.g. inorganic or/and organic substances can be encapsulated. Examples of active agents are pharmaceutic agents so that the shells can be used as drug delivery systems in order to transport the active agents to the desired place in the organism. Further, also contrasting agents can be enclosed in the shells, e.g. to improve the quality of images obtained by ultrasonic examination. It is also possible that the hollow shells themselves are used as contrasting agents.
Further possible applications are fillers or pigments in paints, toners in printing or coatings. A still further field of application is the use as high surface area materials for catalysis , e.g. SiO2 or TiO2 shells, for solar energy applications, e.g. TiO2 shells, wherein, if necessary, further active agents may be applied to the inner and/or outer side of the shells. Still another application is the preparation of high-temperature ceramics, e.g. using zirconia nanoparticles or zirconia nanoparticles doped with other metal oxides. Moreover, the nanoparticles can also be used for the slow release of chemical substances, including pesticides, herbicides etc., as magnetic shells, e.g. for medical applications, or for separation and sensing procedures. Finally, the shells may be used as microreactors, e.g. to produce encapsulated colloidal particles, e.g. metal particles such as gold or silver particles, ceramic particles, magnetic particles or semiconductor particles.
Of particular importance for the use of shells is the permeability of the shell wall. The permeability of the shell wall can be influenced by the selection of the polyelectrolytes used for the shell, the wall thickness and the ambient conditions. It is thus possible to selectively determine and change the permeability properties.
The permeability properties can be further modified by pores in at least one of the layers. Such pores can be formed by the polyelectrolytes or nanoparticles themselves if suitably chosen. By incorporating selective transport systems such as carriers or channels into the polyelectrolyte shell the transversal transport properties of the shell can be adapted to the respective application. The pores or channels of the shell wall can be selectively opened and closed, respectively, by chemically modifying and/or changing the ambient conditions. A high salt concentration of the medium used for the deposition of the polyelectrolyte results in a low packing density and a high permeability of the shell wall. On the other hand, a high salt concentration of the medium used for the deposition of the nanoparticles (SiO2) results in a high packing density of the silica particles. Thus, by adjusting the salt concentrations in the deposition medium, the permeability of the shell can be controlled, as desired. Further, the permeability properties of the shell may be modified by selecting the conditions for decomposing the core, e.g. by selecting the temperature and heating conditions in a calcination procedure.
A further aspect of the present invention is a coated particle having a core which is a template particle and a multilayer shell comprising alternating layers of (i) oppositely charged inorganic nanoparticles and polyelectrolytes or (ii) oppositely charged nanoparticles. Preferably, the average diameter of the coated particle is 15 μm or less, more preferably 100 nm to 1 μm.
Still a further aspect of the present invention is a hollow shell obtainable by disintegrating the template particles of the coated particle as described above. The hollow shell may be an inorganic structure or a composite organic-inorganic structure depending on the method used to remove the core.
Preferably, the shell contains an active agent which may be selected from pharmaceuticals, contrast agents, herbicides, pesticides, catalysts and pigments.
The shell may be used as a system for slow and/or targeted release of active substances such as pharmaceuticals, herbicides, pesticides etc. Further, the shell may be used for high surface area applications, e.g. as a carrier for catalysts or photovoltaic materials or as a catalyst itself.
It has been demonstrated that the method of the present invention when applied to produce composite nanoparticle-polymer or nanoparticle-nanoparticle multilayers on colloidal template cores, coupled with removal of the core, provides a successful pathway to fabricate novel hollow shells. Important advantages pertaining to this method are: (i) the thickness of the shell walls can be readily controlled by varying the number of deposition cycles; (ii) the shell size and shape are predetermined by the dimensions of the templating colloid employed; (iii) the method is generally applicable to a wide variety of charged nanoparticles, thereby making possible the production of various inorganic, composite-inorganic (e.g. magnetic nanoparticle and SiO2 or TiO2) and composite inorganic-organic shell structures by the simple solution adsorption of charged particles in alteration with polymer; (iv) the method is a suitable alternative of those currently used (e.g. surface precipitation reactions) to produce hollow shells, with the added advantage of eliminating the necessity of more complex preparation procedures; and (v) it can be applied to systems for which the current methods are not suitable.
Further, the invention is illustrated by the following figures and examples:
FIG. 1 is a schematic illustration of the assembly of composite multilayers on colloid latices and the subsequent colloid and polyelectrolyte (optional) removal, resulting in hollow inorganic or composite shells. The first stage involves the sequential adsorption of oppositely charged polyelectrolytes, e.g. a Pr3 coating [poly(diallyldimethylammonium chloride) (PDADMAC)/poly(styrene sulfonate), sodium salt (PSS)/PDADMAC] (step 1) in order to produce a smooth and uniformly positively charged outer surface to facilitate the adsorption of negatively charged SiO2 nanoparticles. Subsequent alternate adsorption of SiO2 (step 2) and PDADMAC (step 3) results in SiO2-PDADMAC multilayers being formed on the latices. The latices may be decomposed by calcination or exposure to low pH or solvent, whereby hollow inorganic or composite inorganic-organic shells are obtained.
FIG. 2 is a normalized light scattering intensity distribution of Pr3-coated PS latices (squares) and PS latices coated with Pr3/(SiO2/ PDADMAC)N wherein N=1 (circles), N=2 (triangles) and N=4 (diamonds). Multilayer growth is confirmed by the systematic shift in the SPLS intensity distributions.
FIG. 3 are transmission electron micrographs (TEM) of uncoated PS latices (a) and PS latices coated with Pr3/(SiO2/PDADMAC)N wherein N=1 (b), N=2 (c) and N=4 (d). Regular growth of the SiO2-PDADMAC multilayers is seen by an increase in diameter of the coated PS latices. The scale bar corresponds to all four TEM images shown.
FIG. 4 shows scanning electron micrographs (SEM) of PS latices coated with one SiO2/PDADMAC multilayer (a) before and (b) after calcination. Uniform and smooth multilayer coatings are apparent in (a). The calcination process results in the PS latex core being removed (b): the thickness of the shell wall with one SiO2 layer is not always sufficient to maintain the original sphericity of the PS latices (breakage of the hollow shells could also be caused by the vacuum in the SEM). Some intact shells were also observed with one SiO2 layer.
FIG. 5 shows SEM micrographs of PS latices coated with three SiO2/PDADMAC multilayers (a) before and (b) after calcination. Homogenous coatings are produced on the PS latices (a). Both complete, intact and broken shells are seen in (b): intact spheres are observed for the calcined sample with 3 SiO2 layers; some of the shells are broken as a result of applying a force to them. The thickness of the silica wall is also significantly increased from that shown in FIG. 4 (image a) as a result of successive SiO2/PDADMAC depositions.
FIG. 6 is a higher magnification of FIG. 5b.
FIG. 7 is an atomic force (AFM) image of a broken sphere wall.
FIG. 8 shows a TEM micrograph of hollow silica spheres produced by calcination of PS latices coated with two SiO2/PDADMAC multilayers.
FIG. 9 shows a TEM micrograph of a cross section of silica hollow capsules obtained after calcination of PS latices coated with three SiO2/PDADMAC multilayers. The calcined samples were embedded in a resin prior to imaging.
FIG. 10 shows an SEM image of hollow magnetite spheres. These hollow magnetic spheres are formed by depositing four layers of magnetite (Fe3O4) nanoparticles in alternation with PDADMAC on 640 nm PS latices and then calcining the sample.
FIG. 11 shows a TEM micrograph of a 3 μm melamine-formaldehyde particle coated with three SiO2/PDADMAC layer pairs followed by exposure to a solution of pH=1.
FIG. 12 shows the relationship of Zeta-potential and electrophoretic mobility as a function of polyelectrolyte layer number for FITC-BSA/PDADMAC (open squares) and IgG/PSS (filled squares) multilayers on polyelectrolyte-modified PS latex particles. FITC-BSA multilayers were formed on PDADMAC/PSS/PDADMAC-coated PS latex particles and IgG multilayers on (PAH/PSS)2-coated particles. The odd layer numbers correspond to protein adsorption and the even layer numbers to polyelectrolyte deposition.
FIG. 13 shows Zeta-potential and electrophoretic mobility as a function of pH for a monomolecular IgG layer adsorbed on (PAH/PSS)2coated PS latex particles.
FIG. 14 shows fluorescence spectra of FITC-BSA in multilayer films of FITC-BSA/PDADMAC assembled onto PDADMAC/PSS/PDADMAC-precoated PS latex particles. The solid line corresponds to the spectrum of the multilayer film when FITC-BSA forms the outer layer, and the dashed line to that of the same film with PDADMAC deposited on top.
FIG. 15 shows normalised single particle light scattering (SLPS) intensity distributions of PDADMAC/PSS/PDADMAC-coated PS latex particles (a) and the same particles with one (b) and (c) three multilayers of FITC-BSA/PDADMAC. The final multilayer film structures on the colloids are: [PDADMAC/PSS/PDADMAC/(FITC-BSA/PDADMAC)N] wherein (a) N=0, (b) N=1, and (c) N=3.
FIG. 16 shows FITC-BSA layer thickness (determined from SPLS) as function of protein layer number for FITC-BSA multilayers assembled on PDADMAC/PSS/PDADMAC-coated PS latex particles. The FITC-BSA multilayers were deposited in alternation with PDADMAC.
FIG. 17 shows the total film thickness (determined from SPLS) as a function of layer numbers for IgG/PSS multilayers assembled onto (PAH/PSS)2-coated PS latex particles. The odd and even layer numbers correspond to protein and polyelectrolyte deposition, respectively.
FIG. 18 shows IgG layer thickness (determined from SPLS) as a function of protein layer number for IgG multilayers deposited in alternation with PSS on (PAH/PSS)2-coated PS latex particles.
FIG. 19 shows TEM micrographs of PDADMAC/PSS/PDADMAC-coated PS latex particles (a and c) and the same particles additionally coated with [(FITC-BSA/PDADMAC)2/FITC-BSA] (b and d).
FIG. 20 shows TEM micrographs of IgG multilayers assembled onto (PAH/PSS)2-coated PS latex particles. The final multilayer film structure on the particles is [(PAH/PSS)2/(IgG/PSS)2/IgG]. Image (b) is at a higher magnification than (a).
Alternating SiO2-poly(diallyldimethylammonium chloride) (PDADMAC) multilayers were prepared by first assembling a precursor three-layer PDADMAC and poly(styrene sulfonate) sodium salt (PSS) film (Pr3) onto negatively charged 640 nm diameter polystyrene (PS) latices and then SiO2-PDADMAC multilayers on the Pr3-coated PS latices.
The Pr3 film (PDADMAC/PSS/PDADMAC) was formed by the alternate adsorption of PDADMAC (Aldrich, Mw<200,000) and PSS (Aldrich, Mw 70,000) from aqueous solutions: 0.5 mL of 1 mg mL−1 aqueous polyelectrolyte solution (containing 0.5 M NaCl) was added to the PS latices (1010 particles in 0.5 mL H2O), 20 min allowed for adsorption, and excess polyelectrolyte removed by 4 repeated centrifugation (13500×g) /wash/redispersion cycles. (SPLS measurements reveal that about 0.5% of the PS particles are lost at each wash/centrifugation/redispersion step). The Pr3-coated PS latices exhibit a positive surface charge, as determined from electrophoretic mobility (EPM) measurements. Negatively charged sulfate-stabilized PS latices were prepared as described in Furusawa et al, Kolloid-Z. u. Z. Polymere 1972, 250, 908.
SiO2-PDADMAC multilayers on the PS latices were formed by adding 50 μL of an aqueous 40 wt % SiO2 suspension (Ludox TM, DuPont) to the Pr3coated PS latices dispersed in 0.1 M NaCl (larger amounts of SiO2 adsorb when the adsorbing solution contains NaCl), allowing 15 min for SiO2 adsorption, removing excess SiO2 by 4 repeated centrifugation (13500×g)/wash/redispersion cycles, and subsequently depositing PDADMAC (1 mg mL−1/0.5 M NaCl). The isoelectric point of the SiO2 particles is 3, therefore SiO2 is negatively charged under the conditions of adsorption (pH 5-6).
Electrophoretic mobility (EPM) measurements using a Malvern Zetasizer 4 show that the surface charge of the multilayer-coated particles alternates from negative to positive with each adsorption of SiO2 and PDADMAC, respectively. This qualitatively demonstrates that the composite SiO2-PDADMAC multilayers are formed by the step-wise adsorption of SiO2 and PDADMAC.
The growth of the SiO2-PDADMAC multilayers on the PS latices was first followed by the method of single particle light scattering (SPLS) (Lichtenfeld et al, Progr. Colloid Polym. Sci. 1997, 104, 148). Normalised SPLS intensity distributions for the Pr3-coated PS latices and those coated with 1, 2 and 4 SiO2-PDADMAC multilayers are shown in FIG. 2. Deposition of the SiO2-PDADMAC multilayers onto the PS latices is manifested as a shift (in the x-axis direction) in the SPLS intensity distributions, confirming multilayer growth. Using the SPLS technique, it is also possible to distinguish between singlets, doublets and triplets (Lichtenfeld et al, Progr. Colloid Polym. Sci. 1997, 104, 148): no aggregation of the multilayer-coated particles is observed, as no intensity peaks are observed at higher intensities.
Using the Raleigh-Debye-Gans theory (Kerker, The Scattering of Light and Other Electromagnetic Radiation: Academic Press: New York, London, 1969) and an estimated refractive index (n) of 1.40 for the adsorbed layer(s), the average thickness calculated for each SiO2-PDADMAC layer pair for PS/Pr3/(SiO2/PDADMAC)N multilayers with N=1-5 is 30±6 nm. This value closely corresponds to the mean diameter of SiO2 particles (26±4 nm, determined from TEM), and suggests that on the average approximately a monolayer of SiO2 is deposited with each SiO2 adsorption. The layer thickness increases linearly with the number of SiO2 or SiO2-PDADMAC layers deposited onto the PS latices.
Direct observation of the multilayer growth process was provided by transmission electron microscopy (TEM) using a Philips CM12 microscope operating at 120 kV: representative TEM images of uncoated PS latices and Pr3-modified PS latices coated with SiO2-PDADMAC multilayers are displayed in FIG. 3. The uncoated PS latices (a) exhibit a smooth surface. TEM images obtained for PS latices coated with Pr3 are essentially identical to those of the uncoated PS latices: the thickness increase (ca. 4 nm, determined from SPLS experiments) is not discernible. The presence of SiO2-PDADMAC multilayers on the PS latices results in both an increase in surface roughness and an increase in the diameter of the PS latices (b-d). The increase in surface roughness is due to adsorbed SiO2. It was found that adsorption of PDADMAC onto an outermost layer of SiO2 reduces the surface roughness of the multilayer. This finding is corroborated by preliminary scanning electron microscopy (SEM) measurements.
The increase in diameter with increasing SiO2-PDADMAC multilayer number of the coated PS latices (relative to uncoated PS latices, a) is approximately 60 nm (b), 140 nm (c) and 250 nm (d), for 1, 2 and 4 SiO2-PDADMAC multilayers, respectively. Evaluation of the TEM data for PS/Pr3/(SiO2/PDADMAC)N multilayers with N=1-5 yields an average diameter increment of 65±5 nm, corresponding to a layer thickness of ca. 32 nm, for the SiO2-PDADMAC layer pair.
To remove the PS latex the solution was initially dried at room temperature and then placed into an oven under N2 and heated to 500° C. at a rate of 5 K/min. After 4 h at this temperature the gas was changed to O2 and the sample remained at 500° C. for another 8 h. The sample was then cooled to room temperature under O2. The samples were then examined by SEM using a Zeiss DSM instrument operated at an accelerating voltage of 20 kV.
FIGS. 4 and 5 compare SiO2/PDADMAC multilayer coated latex particles having different shell thicknesses before and after core removal. FIG. 6 is a magnified picture of a hollow shell. It can be gathered from these results that intact hollow shells may be obtained which have sufficient permeability for the contents of the decomposed core to be expelled. FIG. 7 shows an image of a broken sphere wall taken by atomic force microscopy (AFM), showing the wall is comprised of silica nanoparticles. The TEM micrograph of hollow silica spheres produced by calcination of PS latices coated with two SiO2/PDADMAC in FIG. 8 shows the uniformity of the wall thickness. Upon careful examination two layers of silica nanoparticles can be observed. The uniformity of the shell wall thickness can also be seen from the TEM micrograph of resin-embedded silica hollow capsules as shown in FIG. 9. The similar contrast both inside and outside the hollow spheres indicates that the spheres are permeable to the resin.
According to the procedure described in Example 1 alternating Fe3O4-PDADMAC multilayers were deposited on 640 nm PS latex particles. After calcining the sample hollow magnetic spheres were formed. An SEM image of these spheres is shown in FIG. 10.
According to the procedure described in Example 1 alternating SiO2-PDADMAC multilayers were deposited on 3 μm partially cross-linked melamine formaldehyde particles as described in DE 198 12 083.4. The melamine formaldehyde particles were dissolved by exposure to a solution of pH=1. A TEM micrograph of a resulting hollow particle is shown in FIG. 11. The diameter of the hollow composite sphere is significantly larger than the size of the MF colloid template due to drying of the sample on a solid substrate. The rough surface texture is due to the presence of SiO2 nanoparticles embedded between polymer layers.
Alternating protein-polyelectrolyte multilayers were deposited on colloid particles.
4.1 Materials
Fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) and sheep immunoglobulin G (IgG) were obtained from Sigma. Poly(allylamine hydrochloride)(PAH), Mw 8,000-1,000 or 50,000-65,000, poly(diallyldimethyl-ammonium chloride) (PDADMAC), Mw<200,000, and poly(sodium 4-styrenesulfonate) (PSS), Mw70,000, were purchased from Aldrich. All proteins and polyelectrolytes were used as received, except for the 70,000 Mw PSS which was dialyzed against water (Mw cut-off 14,000) and lyophilized before use. The negatively charged sulfate-stabilized polystyrene (PS) latex particles (diameter 640 nm) were prepared as described in Example 1.
4.2 Assembly of Protein Multilayers onto Colloids
Precursor polyelectrolyte multilayer films consisting of PAH and PSS or PDADMAC and PSS were first deposited in order to provide a uniformily charged surface and to facilitate subsequent protein adsorption. The precursor films were formed as described in Example 1.
Protein multilayers were fabricated by exposure of the polyelectrolyte-coated PS latex particles to protein solution under conditions where the protein and particles bear opposite charges, followed by alternate adsorptions of polyelectrolyte and protein. FITC-BSA was deposited onto (PDADMAC/PSS/PDADMAC)-precoated PS latex particles, and IgG onto (PAH/PSS)2-precoated particles. The particles have a positive surface charge when PAH or PDADMAC form the outermost layers, and a negative charge when PSS is the outermost layer. FITC-BSA multilayers were formed by the alternate adsorption of FITC-BSA (0.5 mg/ml in PBS buffer at pH 7.0, 30 min adsorption, or 1 mg/ml in water at pH≈5.6, 20 min adsorption) and PDADMAC (1 mg ml/0.5 M NaCl, 20 min) onto the coated PS latex particles (5×109 particles). IgG multilayers were prepared by successive adsorptions of IgG (1 mg/ml in 0.05 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer at pH 6.0 pH adjusted using NaOH, 45 min adsorption) and PSS (Mw 8,000-11,000, 1 mg/ml/0.5 M NaCl, 20 min) layers onto the coated PS latex particles (6×109 particles). (In all cases the concentration of the protein is approximately ten times that required for saturation adsorption of the particle surface). After each deposition of protein or polyelectrolyte layer, the samples were centrifuged at ca. 5000×g for 10 min, the supernatant removed, and at least three water washings performed.
4.3 Electrophoretic Mobility (EPM) Measurements
Electrophoretic mobilities of the bare and coated PS latex particles were measured using a Malvern Zetasizer 4 as described in Example 1. All Zeta-potential measurements were performed on coated PS latex particles re-dispersed in air-equilibrated pure water (pH≈5.6).
4.4 Single Particle Light Scattering (SPLS) Experiments
Details of the SPLS experimental system and measurement principle were as described in Example 1. Briefly, the dispersion (of either uncoated or coated PS latex particles) is passed through a capillary with a 0.1 mm diameter orifice at the end. Hydrodynamic focussing is applied so that the dispersion stream is directed through a laser beam which is focussed to allow only a single particle or aggregate in focus at a particular time. This requires particle concentrations of less than 3×108 particles ml−1. The light scattered by the particles moving through the laser focus is recorded in the angular region of 5-10° in the forward direction. The intensity distributions, obtained with a resolution of 0.5%, are collected by a multi-channel analyser and then stored on a PC.
4.5 Transmission Electron Microscopy (TEM)
TEM measurements were performed on a Philips CM12 microscope operating at 120 kV. Samples for TEM were prepared by deposition of aqueous solutions of the coated PS latex particles upon a carbon-coated copper grid. The mixtures were allowed to air dry for one minute and the extra solution was then blotted-off.
4.6 Steady-state Fluorescence Measurements
Fluorescence spectra were recorded using a Spex Fluorolog 1680 spectrometer with excitation and emission bandwidths set at 1.0 nm. Typically, ca. 50-100 μl of the protein multilayer-coated PS latex suspension (ca. 109 particles ml−1) was pipetted into 3 ml of water in a fluorimeter cell and the dispersion agitated for 0.5 min. The fluorescence spectrum of this dispersion was then recorded.
4.7 Results
The assembly of protein multilayers was first followed by EPM measurements. Prior to the formation of protein multilayers, a precursor three or four layer polyelectrolyte multilayer film was deposited onto the PS latex particles. The precursor film not only provides a uniformly charged surface, which facilitates subsequent protein adsorption, but it also allows the surface charge to be altered (depending on whether the polycation or polyanion forms the outermost layer) so that the protein can be deposited under conditions where it is oppositely charged to the adsorbing surface.
A three layer (PDADMAC/PSS/PDADMAC) film was assembled onto the negatively charged PS latices prior to deposition of FITC-BSA, whilst a four layer (PDADMAC/PSS)2 film was deposited onto the particles before IgG adsorption. The negatively charged (uncoated) PS latex particles have a Zeta-potential of about −65 mV in water. The Zeta-potential of the PDADMAC/PSS/PDADMAC-coated particles is ca. +50 mV, in accordance with the outermost layer being a polycation. For the (PDADMAC/PSS)2-coated PS latex particles, the Zeta-potential is ca. −40 mV, consistent with the outermost layer being the polyanion PSS. FIG. 12 shows the Zeta-potential as a function of layer number for the polyelectrolyte-modified PS latex coated with FITC-BSA/PDADMAC or IgG/PSS multilayers. The alternate assembly of FITC-BSA and PDADMAC causes a reversal in sign of the Zeta-potential with each deposition up to 10 layers. When FITC-BSA forms the outermost layer, the Zeta-potential of the coated particles is slightly negative (−10 to −20 mV). Subsequent adsorptions of PDADMAC and FITC-BSA produce positive and negative Zeta-potentials, respectively. For the IgG/PSS multilayer system, Zeta-potential values close to 0 or slightly negative are observed when IgG is the outermost layer. Thus, a reversal of surface charge is not required in the build-up of monoparticle/monoelectrolyte multilayers, e.g. IgG/PSS.
EPM measurements where the Zeta-potential was recorded as a function of pH were conducted on (PAH/PSS)2-modified PS latex particles coated with one IgG layer (FIG. 13). The results show an isoelectric point of approximately 5.5.
Further evidence for the growth of FITC-BSA/PDADMAC multilayers was provided by fluorescent measurements. The fluorescence spectra for FITC-BSA/PDADMAC multilayers, for the cases where FITC-BSA or PDADMAC form the outermost layer, are shown in FIG. 14. The emission maximum occurs at 515 nm when the outer layer is FITC-BSA. When PDADMAC is the outer layer, this maximum red-shifts by about 6-7 nm to 521-522 nm. Reproducible, oscillating maxima were observed for each deposition of FITC-BSA and PDADMAC.
In order to obtain quantitative evidence of step-wise protein multilayer growth, the technique of SPLS was employed. SPLS is a sensitive optical technique which enables determination of the thickness of layers assembled onto colloids, as well as the state and degree of the coated colloids with respect to aggregation. By passing a dispersion of the coated particles through a capillary and hydrodynamically focusing the dispersion, the light scattered from one particle at a given moment in time is recorded. Repeating this process allows a histogram of particle number versus scattering intensity to be obtained. FIG. 15 shows the normalized SPLS intensity distributions for (PDADMAC/PSS/PDADMAC)-modified PS latex particles (a), and the same particles coated with one (b) and three (c) FITC-BSA/PDADMAC multilayers. There is a systematic shift in the SPLS intensity distribution (in the x-axis direction) with increasing multilayer layer number, confirming the growth of FITC-BSA/PDADMAC multilayers on PS latex particles. Similar SPLS intensity distributions were obtained for the IgG/PSS multilayers on PS particles. The peaks seen in the SPLS curves shown in FIG. 15 correspond to singlets, i.e. unaggregated protein multilayer-coated particles. Analysis of the data revealed that the coated particles exist predominantly as singlets, with less than 20% of the particles as doublets (an aggregate of two particles). The fraction of doublets was considerably reduced (<5%) when the polyelectrolyte was outermost layer. This indicates that adsorption of the polyelectrolyte separates some of the weakly and reversibly flocculated protein multilayer-coated particles.
By using the Rayleigh-Debye-Gans theory, and refractive indices (n) of 1.43 and 1.47 for the protein and polyelectrolyte layers, respectively, the average thickness of the protein/polyelectrolyte multilayers on the PS latex particles (d) can be determined. For the FITC-BSA multilayers, the layer thickness increases linearly with the number of protein layers deposited (FIG. 16). The calculated average layer thickness increment for the FITC-BSA layers is 3.3±1.1 nm when FITC-BSA is adsorbed from pure water, and 5.8±2.5 nm when adsorbed from PBS. The difference in thickness is attributed to the different conditions under which the protein was deposited. These data clearly show that BSA multilayers can be grown by the step-wise adsorption of protein and PDADMAC onto PS latex particles.
FIG. 17 shows the layer thickness of IgG/PSS multilayers assembled on (PAH/PSS)2-coated PS latex particles as a function of layer number. Regular, step-wise multilayer growth is observed. The IgG multilayer film growth is linear after the first deposition step, as shown in FIG. 18. The thickness of the first IgG layer deposited is approximately 11 nm. The IgG average thickness increment after the first deposition cycle (of IgG and PSS) is 37±7 nm.
Direct visualization of the protein multilayer growth process is provided by TEM. FIG. 19 shows the TEM micrographs of PDADMAC/PSS/PDADMAC-modified PS latex particles (a and c) and the same particles coated with (FITC-BSA/PDADMAC)2/FITC-BSA multilayers (b and d). The polyelectrolyte-coated PS latex particles closely resemble uncoated PS latices in appearance: they exhibit a smooth surface. The thickness of the polyelectrolyte coating (PDADMAC/PSS/PDADMAC) is approximately 3-4 nm (from SPLS experiments). The presence of FITC-BSA multilayers on the PS latices produces both an increase in surface roughness and an increase in the diameter of the polyelectrolyte-coated PS latices (b and d). The increase in surface roughness is most notable at higher magnification (compare images a and d). The diameter increase of the particles with (FITC-BSA/PDADMAC)2/FITC-BSA layers is approximately 20 nm, corresponding to a layer thickness increase of about 10 nm. This value is in close agreement with the SPLS thickness for the same multilayer (11 nm). The TEM images for FITC-BSA multilayer-coated PS latex particles confirm that a uniform coating of the particles is obtained with deposition of FITC-BSA multilayers (FIG. 19b and 19 d).
The TEM micrographs for the IgG/PSS multilayer-coated PS latex particles (FIG. 20) clearly demonstrate a regular layering of the particle surface by the protein multilayers. The diameter of the IgG multilayer particles is significantly larger than those onto which the multilayers were formed. The deposition of one, three and five IgG layers resulted in an increase in diameter of approximately 16, 164 and 296 nm, respectively. These values correspond to layer thickness increases of 8, 82 and 148 nm, and are in excellent agreement with those calculated from SPLS measurements (11, 90 and 160 nm for 1, 3 and 5 IgG layers, respectively).
PS latex particles (640 nm in diameter) were used as substrate for the adsorption of polyelectrolyte and enzyme layers. The layering procedure started with the adsorption of four alternating PAH (Mw 50-65 kD) and PSS (Mw about 70 kD) layers onto the PS latex resulting in negatively charged particles with PSS at the outermost coating. Polyelectrolytes were allowed to adsorb within 20 min. Each layering step was followed by four washing cycles with deionized water. After every layering and washing step the mixtures were centrifuged at 7000 rpm for 8 min and the supernatants were removed. Onto the last PSS layer βglucosidase from Caldocellum saccharolyticum obtained from Sigma (EC 220.127.116.11) was adsorbed in one hour. The enzyme was used as 1.4 mg/ml solution in 0.1 M acetate buffer pH=4.8. The described procedure was repeated until four enzyme layers each separated by one PSS layer were assembled on the particles. Samples were taken after each layering step yielding samples with 1 to 4 enzyme layers having either enzyme or PSS as the outermost coating. These samples were used as catalysts in enzymatic glucosidations.
Glucosidations were carried out in 2 ml tubes which were shaken (200 min−1) at 40° C. Enzyme modified latex particles were suspended in 100 μl phosphate buffer pH=6.8 solution. In a typical experiment 413 mg (2.22 mmoles) n-dodecanol (Fluka), 600 μl acetonitrile (Merck), and 100 mg (0.55 mmoles) anhydrous glucose (Merck) were added. The mixtures were shaken thoroughly to homogenize and then kept at 40° C. for 72 hours with shaking. The mixtures were then centrifuged at 5000 rpm for 10 min. The supernatants were removed and the remainders were washed with 500 μl acetonitrile. From the combined organic phases of each sample the solvent was distilled off. The concentrated crude products were subjected to a preparative chromatographic separation to remove excess dodecanol.
Preparative liquid chromatography was done with a Büchi-680 HPLC equipment consisting of chromatography pump, gradient former, fraction collector, and a Knauer polar monitor detector. The column was a Kronlab bio-cart (20 mm×120 mm) packed with 10 g of 15 μm spherical silica. By collecting 10 ml fractions with an ethyl acetate/methanol gradient program (0 to 100% methanol, 34 min) all excess dodecanol could be separated from the crude product. The remaining fractions were combined, concentrated and then fixed to a volume of 1 ml by adding a water/propane-2-ol (1:33 v/v) mixture. To determine the amount of formed dodecyl glucoside aliquots of these stock solutions were analyzed by HPLC on a JASCO chromatograph equipped with a light scattering detector and silica column. For the quantitative determination of dodecyl glucoside calibration curves were constructed.
In the case of particles having enzyme or PSS as outermost layers dodecyl glucoside could be detected. Thus, the enzyme is not inactivated by the coating procedure. In the case of particles having PSS as the outermost layer an increasing yield of dodecyl glucoside with increasing numbers of deposited enzyme layers on the particles was observed. This shows that also layers underneath take part in the catalytic process.
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Thank you so much for your all your contributions! Through your donations, we have been able to carry out a series of workshops on Identity: Girls vs Boys. We are so grateful for your support and wanted to share with you how we worked with girls in South Africa on re-shaping traditional gender norms and ways in which to challenge these roles.
In South Africa, patriarchy still reigns and men are seen as dominant figures, whether in a household, a community or in an institutional setting. Girls and women face stereotypes; often encouraged by those identifying them as caretakers or as domestic figures. By motivating girls to trust in their capacities outside of these pre established gender roles, they are able to explore other, perhaps more productive, options in their community and in their future.
As a result, Girls & Football SA carried out a series of workshops that encourage participants to understand that physical differences in female versus male bodies doesn't mean boys are more important, more intelligent or 'better' at sport. The participants learn that girls and boys should be treated equally, and that being strong and enjoying games or work traditionally allocated to boys, doesn't make them any "less of a girl". To ensure a gender balance, the workshop also highlights that boys should be able to perform in traditionally female roles.
We carried out a series of workshops at an all-girls' tournament in Pretoria, South Africa. The tournament was held at Prestige College in Hammanskraal Gauteng. There were 3 local club teams if approximately of 15 players per team, in addition to a visiting team from Sweden. The age group ranged from 11-19. For the first time, we introduced role play activities that encouraged the girls to explore gender roles on a more in-depth level.
Through these role plays, we worked to encourage girls to understand that the different stereotypes created by society can always be broken, and consequently by breaking them, the workshop ensures girls have the courage to further their dreams of becoming a professional soccer player, an engineer or a doctor; roles traditionally filled by men. Opening the discussion around gender identity allows girls to understand there are no set rules and that they can explore their own boundaries.
Without your support, we would not have been able to carry out this work. We are immensely grateful for all your donations! Thank you for your support, and we wish you a fantastic year-end.
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22nd April 2014
Human Rights Watch: Louisiana voting to keep sodomy law is 'state-endorsed homophobia'
10:23 PM — The LGBT director of Human Rights Watch has said the decision by the Louisiana House to keep a sodomy law is 'state-endorsed homophobia'.
India: Supreme Court agrees to hear plea against anti-gay law 3
2:45 PM — The Indian Supreme Court on Tuesday agreed to hear a plea by gay rights activists against a recently reinstated law banning gay sex.
20th April 2014
Rachel Maddow: Louisiana voting to keep sodomy law is like chicken running round with its head cut off 1
1:30 PM — News anchor Rachel Maddow has ridiculed Louisiana's state legislature for voting to keep an unconstitutional sodomy law.
16th April 2014
US: Louisiana House votes to keep unconstitutional sodomy law 5
7:14 PM — The Louisiana House of Representatives has overwhelmingly voted to keep a sodomy law in the state's penal code.
12th April 2014
US: Louisiana committee votes 9-6 to remove sodomy law from penal code 5
7:04 PM — A House of Representatives committee in Louisiana has finally voted to remove a sodomy law from the state's penal code, eleven years after it was declared unconstitutional.
2nd April 2014
Video: Ugandan teen arrested over gay sex with 22-year-old father 18
1:42 PM — Two men have been arrested in Uganda for allegedly having gay sex, Ugandan police have said.
25th March 2014
UAE: Court upholds men's 6-month jail term for 'sodomy' 10
8:29 PM — A court in the United Arab Emirates has upheld a six month jail term handed to two men who are alleged to have had sex.
17th March 2014
Homophobic pastor claims Ugandan President will join procession in celebration of anti-gay law 4
6:46 PM — Ugandan President Yoweri Museveni, is set to join a procession celebrating the implementation of a law harshening punishments for those found guilty of homosexuality, an adamantly homophobic pastor has claimed.
7th March 2014
Malaysia: Ex-Deputy Prime Minister jailed for five years on sodomy charges 4
7:20 PM — Malaysia's former Deputy Prime Minister Anwar Ibrahim has been sentenced to five years in jail on sodomy charges.
7th January 2014
Uganda: Former national football team coach charged with sodomy
2:10 PM — The former head of Uganda's national football team has been charged with "sodomising both male and female youths" – although campaigners claim it could be just the latest example of a homophobic witch hunt.
17th December 2013
US: In lawsuit based on sodomy ruling, judge rules that Utah's polygamy law is unconstitutional 12
1:07 PM — A federal judge has ruled that a law banning polygamy in the state of Utah is unconstitutional.
11th December 2013
Protesters take a stand against 'strange and regressive' Indian Supreme Court ban on gay sex 2
5:30 PM — Protesters have made a stand against today's Supreme Court decision in India to reinstate a ban on gay sex.
9th December 2013
Christian Concern: Tom Daley came out because his father died so Jamaica should keep its buggery law 89
4:30 PM — Christian conservatives in Jamaica are being urged by activists from the US and UK to resist against calls for the country to repeal its buggery law.
6th November 2013
Anti-gay Attorney General who tried to ban oral and anal sex loses Virginia governor race 21
1:34 PM — Virginia Attorney General Ken Cuccinelli, who unsuccessfully tried to maintain a ban on oral and anal sex and believes "homosexuality is wrong", has been defeated in the race to become state governor.
2nd October 2013
Singapore High Court dismisses challenge to anti-sodomy law 9
2:04 PM — The High Court of Singapore on Wednesday dismissed a constitutional challenge against a law which criminalises consensual sex between men.
28th July 2013
US: Police use invalid anti-sodomy law to arrest gay men for 'crimes against nature' 55
8:49 PM — Police officers from the Sheriff's office in Baton Rouge, Louisiana have made at least a dozen arrests of gay men since 2011 using an anti-sodomy law which was struck down ten years ago.
11th June 2013
Zambia: Gay couple facing sodomy allegations go on trial 5
11:23 AM — A gay couple "charged with the offence of sodomy or having sex against the order of nature contrary to the laws of Zambia" have gone on trial.
20th May 2013
Human Rights Watch urges Zambia to drop charges in sodomy case
4:53 PM — The Human Rights Watch has urged authorities in Zambia to release two men who were arrested and charged on allegations of sodomy earlier in May.
16th May 2013
Belize: Attorney General wraps up in sodomy law Supreme Court case 1
2:57 PM — The Attorney General wrapped up in the case challenging Belize's anti-sodomy law, during which the Supreme Court heard arguments from attorneys pushing for the country to remove the law, citing its unconstitutionality.
9th May 2013
Belize: Supreme Court hears closing arguments from attorneys urging strike down of anti-sodomy law 3
2:04 AM — At the end of a second day of hearings around the country's anti-sodomy law, Belize's Supreme Court heard closing arguments from attorneys pushing for the country to remove the law, citing its unconstitutionality.


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Ask a garden pro: Plumeria
CORPUS CHRISTI - QUESTION: When can I bring out my plumeria?
ANSWER: If your plant is in a pot, I would go ahead and bring it out. The key is to avoid night temperatures in the 30s. It looks like most of the freezing weather for the winter might be over, especially for the coastal areas. Inland areas probably should wait another week or two to make sure that a late season frost doesn't hurt your plumeria plants.
When you bring your plumeria out, remember that it has been dormant for several months and the soil is probably extremely dry. Make sure you water it multiple times to rehydrate the soil. Only after your soil is moist all the way through should you consider applying fertilizer to get your plant off to a good start.
Michael Womack is a horticulturist and executive director of the South Texas Botanical Gardens & Nature Center. Contact him at email@example.com.
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From 1° May 2012 the Municipality of Vicenza will apply the residence tax " The price of tourist tax of stay and 1,00 € for a maximum of 5 nights.
What travellers think
Very nice Place very Nice and helpfull People We van Warmluft recommend
Sat 06 Jun 2015
Very good location close to the city center. The hostel is old but clean. The dorm rooms are small. I was glad we were not 4 persons in the room. The breakfast is very basic, please use fresh bread. Overall nice stay, and the manager is a nice person.
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V3 question about buffer
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V3 question about buffer
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Can you tell me how long does it take (in seconds) to clear the buffer when shooting continuous ? Let's say I saw something interesting so I press down the shutter and hold, camera takes a serie but moment later I see next great thing so how long do I have to wait to get camera operational again ?
BTW : Is there a way to zone focus with 1 system ?
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Tuesday, August 28, 2007
© Copyright 2015 Gwinnett Daily Post
ATLANTA - A mobile dental practice serving 64,000 Georgia children, mostly from low-income families, is back in good graces with one of two HMO-like companies that had been poised to terminate its contract.Officials with WellCare of Georgia announced Monday that the company will continue covering Medicaid and PeachCare enrollees served by Help a Child Smile, which operates 12 dental vans out of its office in Conyers. The extension will cover 60 days beyond the expiration of the current contract Friday. "Help a Child Smile is very excited we will be able to continue providing quality dental services at school sites throughout the state of Georgia without interruption and looks forward to reaching a long-term agreement with WellCare," Darryl Smith, the dental practice's group administrator, said in a prepared statement. Early this month, WellCare and Peach State Health Plan, a second managed-care company hired by the state to run Georgia's Medicaid program, notified Help a Child Smile and another dental chain that serves mostly children that they would be terminating their contracts effective Friday. Officials with Atlanta-based Kool Smiles held a news conference to complain that the move threatened to leave more than 70,000 of their 100,000 patients across the state without a dentist. Then last week, 13 parents of children who are patients at Kool Smiles or Help a Child Smile filed a federal lawsuit challenging the terminations. The insurers at first characterized the moves strictly as business decisions. But last week, the state Department of Community Health, responding to the lawsuit, announced that the state is investigating complaints against Kool Smiles from parents and other dentists that it has been overutilizing some services and making too frequent use of patient restraints. Kool Smiles officials shot back, accusing the state of trying to direct attention away from the terminations. The decision by WellCare to extend its contract with Help a Child Smile will let 21,600 patients continue receiving dental coverage, Smith said. He confirmed that Help a Child Smile also is working with representatives of Peach State to extend that contract as well, which would continue coverage for another 15,000 patients. "We're cautiously optimistic," Smith said. "We certainly left the door open to them," added David Bear, a spokesman for Peach State Health Plan. "They said, 'We'd like to try and find a way to continue this.' ... We told them we'd be willing to listen." Meanwhile, WellCare released a statement Monday indicating that it has hundreds of dentists ready to accept patients who would be left without coverage by the termination of Kool Smiles. "We have assured the state ... and all of our members that we have many times more dentists than needed near Kool Smiles offices who are ready, willing and able to treat our WellCare members," said Michael Cotton, chief operating officer of WellCare of Georgia. One of the exhibits introduced in the lawsuit was a statement from an economic consultant that an "immediate and serious shortage" of dental care for children would occur in Georgia if Kool Smiles' contracts with the two insurers were terminated.
More like this story
- Dentists going to court over Medicaid cutoff ( August 23, 2007 )
- PeachCare managers dropping major children's dental firm ( August 7, 2007 )
- Medicaid managed care under fire at 1-year mark ( August 26, 2007 )
- Doctors rally against Medicaid managed care ( August 17, 2006 )
- Spa dentist offices treat patients like royalty ( February 19, 2006 )


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The film is based on Tove Jansson's original comic strips written for London Evening News in 1955. The film premiered in London Film Festival in October 2014 and has been sold to over 40 countries for theatrical distribution. The film was received extremely well in Finland during the autumn 2014, it was just released theatrically in Switzerland, in France and in Japan. The English are to launch the film in May with 250 copies. Hanna Hemila is the producer, co-director and co-writer of the film. The director of the film is French Xavier Picard, who is also the co-director and one of the writers. Other writers are Leslie Stewart, Annina Enckell and Beata Harju.
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NYWIFT programs, screenings and events are supported, in part, by grants from New York City Department of Cultural Affairs in partnership with the City Council, and New York State Council on the Arts with the support of Governor Andrew Cuomo and the New York State Legislature.


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User Reviews & Ratings - lisinopril oral
Read user comments about the side effects, benefits, and effectiveness of lisinopril oral.
Overall User Ratings4068 Total User Reviews
User Reviews
Learn about
User Reviews
Reviewer: peetee, 45-54 Female on Treatment for 1 to 6 months (Patient)
Ease of Use
My blood pressure has decreased over time and I feel better.
Reviewer: Helen, 55-64 Female on Treatment for 2 to less than 5 years (Patient)
Ease of Use
I did not realize that Lisinopril was responsible for my hair loss immediately. But I now know that it absolutely has made me lose my hair! I am so upset. My hair is falling out in clumps on my pillow. I just thought I was not moisturizing my hair adequately. Until I purchased some products that totally moisturized my hair. When my hair continued to fall out. I then realized it had to be the Lisi ... Show Full Comment
Reviewer: 45-54 Female on Treatment for 1 to 6 months (Patient)
Ease of Use
Hae.hate.hate this medication. Even though my doctor told me I would get a dry cough, it turned out to be much worst. The phlegm was horrible and I would cough so much I would get a head-ache.Coughing in front of my students is not good and makes them and myself worry. If you are on this medication and coughing is a problem, ask to be switched to something different
found this review helpful.
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Reviewer: ita, 65-74 on Treatment for less than 1 month (Patient)
Ease of Use
The cough I have been getting has to do with the lisinopril oral. Was not aware of it until I read the other comments that were shown on this medication. This has been the only new medication that I have started taking recently. Will stop!!
Reviewer: 55-64 Female on Treatment for less than 1 month (Patient)
Ease of Use
Lisinopril caused the worst cough I've ever experienced in my lifetime--to the point of vomiting. After two weeks of utter misery while taking this drug, I contacted my PCP and she immediately changed me to something else. The cough took about another 1.5 weeks to subside after discontinuing this med. I have friends who have taken it for years without incident, however. If you're prescribe ... Show Full Comment
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The Global FlyFisher - The Largest and Best Place to go for Online Fly Fishing and Fly Tying
kayak loops
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Viewed 2426 times.
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More pictures of Casting and fishing Gear Landscapes, mountains Plants and flowers
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|More great articles on The Global FlyFisher
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Fishy photographer: Henry HegemanHenry Hegeman got the crazy idea that maybe he could make some extra money to help support his hunting and fishing endeavors by writing articles and illustrating them with his photos
May 13th 2012


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Maryland Resources
Contact JGSMD
Other Area JGS Websites
Greater Washington
Greater Philadelphia
New York
Jewish Genealogy Society of Maryland
Lara Diamond is President of JGSMD and has been researching her family for 25 years, since she was too young to have a driver's license and had to rely on her mother to drive her to the National Archives. She has traced all branches of her family back to Europe and most multiple generations back using Russian Empire-era records. Most of her research is in modern-day Ukraine, with a smattering of Belarus and Poland. Her genealogy blog is at http://larasgenealogy.blogspot.com.
See our past programs here.
Do you have a genealogy brick wall that you'd like help breaking down? Click here to find out how to have our membership help.
JGS Maryland has been reconstituted!
JGSMD had its initial meeting on Sunday August 18, 2013 to see if there was interest in the group's reconstituting. The turnout was overwhelming, with 31 people attending with only a few days' notice. Look forward to new programming over the next year!
Some of the attendees:


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Picnic Plus Double Bottle Carrier
Item # 3135144
|Eligible for $20 Off Your Order of $100 Or More! (see promotion details)
|Eligible for Free Shipping, No Minimum! (see promotion details)
|Members of The League get 5% back when you spend $100! (see promotion details)
Product Details
The Picnic Plus double bottle carrier is a soft, supple tote that holds 2 bottles with a fully insulated interior to maintain the beverage temperature. It includes a bottle stopper and a corkscrew/bottle opener/knife.
- Dimensions: W 9 in; D 5 in; H 14 in
- Soft, supple tote
- Holds 2 bottles
- Adjustable shoulder strap
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- Bottle stopper
- Corkscrew/bottle opener/knife


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Search here for the books we have available to read online.
You can use a computer or device and pick up where you left off anytime.
If we don't have the book you want, make a request by clicking here.
We'll do our best to get it to you the same day!
If you have question, ask LMC staff or
Open Hours: 7:15 am to 4:30 pm Monday - Friday


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This poll is to see how violent the fans think the live action He-Man should be.
He-Man will leave you unharmed. Filmation violence.
He-Man will punch foes out, but not kill. MYP violence.
He-Man should only kill in extreme situations.
He-Man should kill in battle, but is merciful to surrendering foes.
He-Man should dutifully slay every threat to Eternia.
He-Man is bloodthirsty and lusts for battle.
This poll is to see how violent the fans think the live action He-Man should be.
The Blonde Teela Thread
The Cloak and Dagger Evil-Lyn Thread
Sagitar (Jetlag) Horde Wraiths Azdar Belzar Chazdar Cloak and Dagger Evil-Lyn 200X Teenage Prince Adam 200X Teenage Teela 200X Sorceress General Rattlor (MYP) 200X King Randor and Queen Marlena 200X Battle Armor King Randor and Battle Armor Queen Marlena Ice Armor He-Man WMD Roboto


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Spicy Mesquite Chicken Fettuccine
27 Apr 2010
|Uncooked fettuccine
|8 Ounce
|Chili powder
|1 Tablespoon
|Ground cumin
|1 Teaspoon
|1 Teaspoon
|Ground red pepper
|1⁄4 Teaspoon
|Vegetable oil
|2 Teaspoon
|Mesquite marinated chicken breasts
|1 Pound, cut into bite size pieces
|Mesquite marinated chicken breasts
|1 Pound, cut into bite-size pieces
Cook pasta according to package directions, omitting salt.
Drain; set aside.
Combine chili powder, cumin, paprika and red pepper in small bowl; set aside.
Heat oil in large nonstick skillet over medium-high heat until hot.
Add chili powder mixture; cook 30 seconds, stirring constantly.
Add chicken; cook and stir 5 to 6 minutes or until cooked through and lightly browned.
Add pasta to skillet; stir.
Cook 1 to 2 minutes or until heated through.
Sprinkle with additional chili powder, if desired.
Rate It
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Spicy Mesquite Chicken Fettuccine Recipe


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Speed Works
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Children's Summer Reading Program: Nancy the Turtle Lady - Morning Program
Nancy will be back for this year's Summer Reading Program! We can't wait to see all of her reptiles. 11:00 a.m. (Preschool - 2nd grade) and 2:00 p.m. (3rd grade and up). Checkout her Facebook Page!
Wednesday, July 1, 2015 - 11:00am
Event & Program Ages:
Events & Programs Locations:


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Originally written on Patriots Life | Last updated 1/5/13
Two key players for the Patriots, Aaron Hernandez and Chandler Jones, have not been playing as well as they usually do.The Patriots have never have had a problem with their tight ends, but recently with Aaron Hernandez (who usually plays superbly) they might have to worry. Hernandez dropped 6 passes in the last three games of the regular season. Hernandez usually performs extremely well and is one of Tom Brady's go-to guys to get the football down the field, but it's very scary when am important player like Hernandez isn't doing as well as he usually does. @inthistweet agility, speed, actually catching the ball and not dropping 6 passes in past 3 games— Greg A. Bedard (@GregABedard) January 2, 2013 Chandler Jones (defensive end, #95) has been in a slump as well. He was being seriously considered to become a candidate for Defensive Rookie of the Year earlier this season, but an ankle injury in November caused his numbers to fall. He has gotten 1 quarterback hit since the end of Octob...
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The CCNY-MSKCC Partnership for Cancer Research, Training, and Community Outreach presents our third annual research symposium. This year's symposium is focused on translational research, with part of the day dedicated to the bench to bedside aspect of translation and part of the day focused on bringing clinical practice to the community.
This free program is open all.


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
42 year old woman | Miami Shores, FL, USA | Seeking men 37-48 in United States
Let us have a good time :)
Very outgoing woman, strong personality, proffessional driven, caring, smart. Love dogs and the outside type of activities. Not very sporty but love water and swimming as well as extreme sports like sky diving. One of my favorite things is spending time with my beautiful daughter, but I always find time for my friends. Love a nice dinner and drinks night out, even just a movie, or maybe some dancing :).
Coffee and conversation , Business networking , Dining out , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Nightclubs/Dancing , Performing arts , Travel/Sightseeing
Auto racing / Motorcross, Bowling, Dancing, Soccer, Swimming, VolleyBall, Walking / Hiking, Weights / Machines, Yoga
I exercise 1-2 times per week.
I have Dogs
Miami, Key West, London, San Francisco, NYC The beach and the mountains
Love Asian, Indian, Spanish, Italian, and of course Colombian food. My favorite movies: Gladiator, The Notebook, and all movies base on real events My favorite books: Jonathan Livingston Seagull, Love in the Time of Cholera
Love a nice dinner and drinks night out or even just a movie, or maybe some dancing :).
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
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|5'7" (170cms)
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|Wants kids:
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|Latino / Hispanic
|Latino / Hispanic, White / Caucasian
|Christian / Catholic
|Christian / Catholic, Jewish, Spiritual but not religious
|English, Spanish
|English, Spanish
|Graduate degree
|Bachelors degree, Graduate degree


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Green light: Langley goes lowest in Round 1
Catch the action from the opening round of the 2015 Greenbrier Classic. Watch Golf Central 6PM ET.
Keegs: 'Bummer' finish but Shaq was 'awesome'
Keegan Bradley talks about his opening round at the 2015 Greenbrier Classic and what it was like to play alongside his pro-am partner Shaquille O'Neal.
Golfing World Slice: Bubba milks his Travelers win
The latest Golfing World Slice takes a look at what Bubba Watson did with his latest trophy, Martin Kaymer and his powers of recovery, and another of the most stupid golfers of all time.
Tiger's game 'very close' at Greenbrier
George Savaricas catches up with Tiger Woods after his opening round 66 at the 2015 Greenbrier Classic.
2015 Greenbrier Classic
2015 Greenbrier Classic
2015 LPGA winners
2015 LPGA winners
Quotes of the Week: June 28, 2015
Quotes of the Week: June 28, 2015
2014-15 PGA Tour winners photos
2014-15 PGA Tour winners photos
Fowler, girlfriend back on vacation in Bahamas
Life for Rickie Fowler these days looks like one big trip to the Bahamas, with the occasional stop on the PGA Tour thrown in.
Golf Channel Report: Tiger shoots 66 at Greenbrier
Get caught up with Tiger Woods' opening round at the 2015 Greenbrier Classic. Hear from Tiger as he talks about getting his 'feel' back.Hi there, I'm Brian with your golf channel report. Tiger Woods more rounds in the 80s than the 60s in 2015. That would change opening round of the Greenbrier Classic. Is Tiger Woods fire his best round of the season on the PGA tour With a four under 66. And that would include a double bogie at four. But Tiger, who started on the back nine
Golf Central: July 2, 2015
Tiger Woods shows progress with an opening round 66 at the 2015 Greenbrier Classic. Watch Golf Central 6PM ET.and well get into golf center Ryan broke apparent PGA tour winners Arron Oberholser and didn't. Gallagher and junior. Guys Tiger Woods is back in back in the sixties it seems like a long time ago that tiger to break seventy Guinea pigs is
Bubba to paint over Confederate flag on General Lee
Bubba Watson tweeted Thursday that he will be removing the Confederate flag's image from the roof of the General Lee, the iconic car from the "Dukes of Hazzard" TV series
Tiger pulls off 'hat trick,' finishes with 3 birdies
Tiger Woods talks with the media about his progress and how he felt during his opening round at the 2015 Greenbrier Classic.guys are all gonna be somewhere up there. The nature of this golf course is soft and the guys are going to go get it. You've been playing better than you've been scoring. So, what doeson my front nine, or the back nine a couple nice little tee shots that I thought I hit out there But overall, I can't really say I hit any great shots, but I hit a lot of good ones. I agree. How important for your confidence is it to get a good start? It's more important because this golf course , everyone is gonna go low. That's more important for that. I mean, everyone's gonna go low. Everyone's gonna shoot under par here,
Night School: Swing like Sam Snead
School of Golf's Martin Hall and Sara Brown teach a drill to improve the rhythm of your swing. Watch School of Golf Tuesdays 8PM ET.Thanks for joining us on Golf Channel Academy. This is School of Golf. I'm Sara Brown alongside Professor Martin Hall, and we're here to get you playing your best golf. We're actually in the 18th at the Greenbrier, so of course we're thinking Sam Snead . And this would be a sort of rhythm timing drill from Sam Snead . I don't think he did this, but this is what we could learn. Wonderful regimen, his golf swing . He had a little forward press to get him going, and I do with players is I'll put a shaft Between theirSo let's put a little one, two, three in this, young lady. Sam Snead in this swing. One, two, three. You did that absolutely beautifully, and have you. Well, it's just going to be on the level level there. Yeah. But it's a very good shot. But you can feel it gives you a starting point for the swing. Yeah. Some people have trouble getting the backswing going and this would be a good one. A little one, two, three. I think it's pretty good. And if we can hit anything like Sam Snead , I'll take it. In our dreams Yeah. Thanks for joining us on Golf Channel Academy for the best in golf instruction. For more great tips like this, make sure to watch School of Golf.
Safe house: Bubba opens with 67 in home game
Bubba Watson cards an opening round 67 at the 2015 Greenbrier Classic. Watch Golf Central 6PM ET.and well get into golf center Ryan broke apparent PGA tour winners Arron Oberholser and didn't. Gallagher and junior. Guys Tiger Woods is back in back in the sixties it seems like a long time ago that tiger to break seventy Guinea pigs is
Process progress: Tiger fires 66 at Greenbrier
Catch the action from Tiger Woods' opening round at the 2015 Greenbrier Classic.fairways we have said that. And it long long time once again Arron Oberholser Jim Gallagher junior. And JR I'll start with the U. We just haven't seen this with tiger the more it was a
Bubba: Finish 'will make dinner taste really good'
Hear from Bubba Watson as he recaps his opening round at the 2015 Greenbrier Classic. Watch Golf Central 6PM ET.the Travelers. Obviously a long, grueling week to get your eighth career PGA Tour victory . How much did fatigue play a factor, at least leading up to the start of tournament play? Yeah, wanting to practice, it was kind of sluggish. Sluggish. Didn't really wanna get out here and practice because my energy level. But when you step on the first tee out here at a golf tournament , you get energized real fast. Or you better. And, so I have full energy today I was ready to go today. I
Last 3 major winners grouped at U.S. Women's Open
Michelle Wie is out to defend her U.S. Women's Open title, while the last three major champions - Inbee Park, Brittany Lincicome and Hyo-Joo Kim - have been grouped together.


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When you say "Barack Obama, a resolutely anti-torture president," whom are you referring to exactly? Obama has done absolutely nothing to change the institutionalized practices of human rights violations that were left in place when Bush left office. None of the illegal kidnappings ("renditions"), habeus corpus violations, indefinite imprisonment, drone strikes etc. have been substantially altered.
Tibor Koos
Newark, NJ
Sep 1 2011 - 2:21pm


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Kitchen Recipe Box
Citrus Floral
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Activating Side Heated Mirrors & Radio Traffic Warning Sign removal?
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Dodge Ram Forum Senior Member
Join Date: Jul 2010
Posts: 258
Gender: Male
Vehicle: 2010 RAM SLT REG. CABX2
Trim Level: SLT
Color: CRYSTAL BLACK PEARL
Engine: 2009-2010 345ci (5.7L) Hemi V8 390hp 407lb/ft
Rep Power:
@ Rootbeer, wow that was a quick response...thanks. The 3rd knob in the manual states it's for the rear window defrost..but as I mentioned I did notice the defrost emblems were on both mirrors so I figured that may do the trick for all 3...thanks for the confirmation...later in the year I'm sure i'll be using that option.
As far as the mute...turning the unit off is a one button thing so no biggie...was just curious if there was an option there...now I know there isn't...thanks again.
And lastly...as far as the message thing being on all gps'....there's no way at all to remove it? I know there are ways to do things like remove the seat belt chime..etc....so I thought there may be a way to remove that warning notice. Because it goes away after a few seconds, it's really no big deal. If it stayed up for 30 seconds or you had to hit accept every single time, that could get annoying.....but it's pretty quick so I was just curious...if there's no way to remove it...at least it's only there a few seconds each time. Thanks for the quick reply.
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PRLog - Jan. 27, 2014 - NEW YORK -- New York{http://en.wikipedia.org/
Skilled carpenters from the company are trained to prepare joints, wall forms, wall and floor frames. The services are available around the clock and can fulfill any kind of request in the given deadline. Availing professional handyman services not only saves time, but also helps save future maintenance expenditure too.
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· Furniture making, fine woodwork, parquetry, fine woodwork
· Molding and trimming of doors and window casings
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· Stud, post and beam work.
· Shuttering and false work
About Hays NYC
Based in New York, Hays NYC {http://www.hays-
ban verdi
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text:
Product DescriptionGreenPan is a range of beautifully designed, technologically advanced bakeware pieces with a unique environmental message; No PFOA is used in the manufacture of GreenPan.
The ceramic non stick coating contains no PTFE. Less energy used in the manufacturing process and Environmentally friendly materials used as much as possible; The ceramic non stick coating is hard wearing and has excellent food release properties.
No more rubbing and scrubbing, clean-up has become a breeze! GreenPan is a Belgian Cookware and bakeware manufacturer which is the first to launch PTFE free ceramic non stick coatings.
The Milan collection features the Thermolon ceramic non-stick interior coating; Oven safe rivetted metal handle; Suitable for all hobs including induction; Oven safe to 250 degC (Gas Mark 9); Lifetime guarantee; 20cm; The ceramic non-stick coating works with the perfectly smooth surface to provide excellent food release plus uses less oil and easy to clean.
- Aluminium
- Non stick coating
- Suitable for all hob types, including induction
- Handwash recommended
- Not microwave safe
- Oven safe
- Lifetime guarantee
- Depth 8.5 cm
- Diameter 20 cm
- Length 40 cm
- Width 21 cm
Buy & Collect - FREE (7 days a week)
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(For Belfast and Dundrum please allow up to 48hrs for delivery.)
Standard - £3 (Monday to Friday)
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Demi Lovato at the 2012 MTV Video Music Awards at Staples Center on September 6, 2012 in Los Angeles.
Australian hunk Chris Hemsworth and his Spanish wife Elsa Pataky have some fun while vacationing off the Island of Corsica and paddleboarding in gorgeous blue water on the Fourth of July.
Is there anything hotter than Matt Bomer, shirtless, on a beach? We don't think so! That's why we've rounded up his best shirtless moments.
Elizabeth Olsen totally had a Marilyn Monroe moment. …