Development of encapsulation technique for curcumin loaded O/W emulsion using chitosan based cryotropic gelation

Cryogel based encapsulation of curcumin, an herbal extract, was successfully carried out with a ternary system of colloidal chitosan, κ-carrageenan, and carboxymethylcellulose sodium salt. The effects of chitosan concentration, κ-carrageenan/CMC ratio of the polymer suspension and molecular weight of chitosan on the sol–gel formation were investigated. The effects of cooling rate during freeze-drying and oil phase composition on the encapsulation yield and the release behavior of curcumin from the hydrogel were determined. And so were the effects of pH of the phosphate-buffered media and oil phase composition on the swelling of the specimens. The microstructure of the resulting specimens revealed core-shell nanoparticles (i.e. oil droplet for core and cryogel membrane for shell) entrapped in the cryogel matrix. The encapsulation yield for two types of suspensions was in a range of 83.9 to 99.6% when a high-MW chitosan was used. Controlled release of the encapsulated curcumin in an aqueous system could be maintained for 4 days, and the releasable amount of curcumin was in a range of 41.1 to 59.9%. The encapsulation yield as well as the released pattern and releasable amount of curcumin were significantly influenced by the cooling protocol used during freezing. Irrespective of the introduced oil phase composition, controlled release of curcumin was achievable when the cooling rate was sufficiently high at ? 2.0 °C/min and, interestingly, either a burst release or a first order release could simply be achieved by changing the freezing condition.     

Engineering of poly(ε-caprolactone) microcarriers to modulate protein encapsulation capability and release kinetic

Drug delivery applications using biodegradable polymeric microspheres are becoming an important means of delivering therapeutic agents. The aim of this work was to modulate the microporosity of poly(ε-caprolactone) (PCL) microcarriers to control protein loading capability and release profile. PCL microparticles loaded with BSA (bovine serum albumin) have been de novo synthesized with double emulsion solvent evaporation technique transferred and adapted for different polymer concentrations (1.7 and 3% w/v) and stabilizer present in the inner aqueous phase (0.05, 0.5 and 1% w/v). SEM (scanning electron microscope) and CLSM (confocal laser scanning microscope) analysis map the drug distribution in homogeneously distributed cavities inside the microspheres with dimensions that can be modulated by varying double emulsion process parameters. The inner structure of BSA-loaded microspheres is greatly affected by the surfactant concentration in the internal aqueous phase, while a slight influence of polymer concentration in the oil phase was observed. The surfactant concentration mainly determines microspheres morphology, as well as drug release kinetics, as confirmed by our in-vitro BSA release study. Moreover, the entrapped protein remained unaltered during the protein encapsulation process, retaining its bio-activity and structure, as shown through a dedicated gel chromatographic analytical method.     

Synthesis of monodisperse spherical nanometer ZrO2 (Y2O3) powders via the coupling route of w/o emulsion with urea homogenous precipitation

Using xylol as the oil phase, span-80 as the surfactant, and an aqueous solution containing zirconium (3 mol% Y₂O₃) and urea as the water phase, tetragonal phase ZrO₂ nano-powders have been prepared via the coupling route of w/o emulsion with urea homogenous precipitation. The effects of the zirconium concentration, the reaction temperature and the urea content on the average size of the products have been examined. The as-prepared ZrO₂ powders and the precursor powders were characterized by TGA–DTA, XRD, TEM and BET. Experimental results indicate that ZrO₂ powders prepared via the coupling route of w/o emulsion with urea homogenous precipitation possess some excellent characteristics, such as well-rounded spherical shape and excellent dispersing.     

Effect of sonication parameters on the mechanical properties of multi-walled carbon nanotube/epoxy composites

In this study, the effects of duration and output power of sonication on the dispersion state of 0.5 wt.% multi-walled carbon nanotube (MWNT) in epoxy matrix were investigated. To disperse the MWNT in the polymer matrix, sonication powers of 25, 50 and 100 W and sonication times of 15, 45 and 135 min were used. Dynamic mechanical thermal analysis (DMTA) and tensile test were performed under different dispersion states of MWNT. The results indicated that with increase in the sonication time, there was an initial increase in Young’s modulus values followed by a drop in values at longer sonication times. The highest Young modulus was gained for the sonication power of 50 W and sonication times of 45 min. Also the highest tensile strength was obtained for the sonication power of 25 W and sonication time of 45 min. Also sonication at 50 W for 15 min was the most effective dispersion for achieving the highest glass transition temperature (T_g). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the dispersion state of MWNT. Well dispersion was observed as the power and duration of sonication were increased.     

Successful factorial design for the optimization of methylprednisolone encapsulation in biodegradable nanoparticles

Abstract

Due to their crystalline nature, the encapsulation of hydrophobic corticosteroids within polymeric nanoparticles by o/w solvent evaporation method is often difficult to achieve. The aim of this study was to evaluate the effect of both process and formulation parameters on the encapsulation of a model corticosteroid: methylprednisolone (MP). For this purpose, a 3²factorial design was performed evaluating the effects of the concentration of emulsifiers and sonication time on the manufactured nanoparticles, followed by a multiresponse optimization. The study also included the evaluation of other parameters such as the type of organic solvent used, polymer characteristics and the initial mass of drug. The optimal nanoparticle formulation using 0.25% (w/v) of emulsifying agent (Polyvinyl-alcohol, PVA) and 5 min of sonication was then characterized. The highest encapsulation was obtained with an organic phase consisting of acetone: dichloromethane (1:1), polyD,L-lactide-co-glycolide (PLGA) 50:50 as polymer and an initial mass of 6.6 mg of methylprednisolone. Nanoparticles size and ζ potential of optimized formulation were respectively around 230 nm and ?14 mV. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) demonstrated that the drug was molecularly dispersed within the nanoparticles. Release study showed that MP-loaded nanoparticles sustained drug release for up to 120 h. This study reflects the importance of factorial design to optimize the manufacture of nanoparticles encapsulating hydrophobic drugs.     

Selective dispersive liquid–liquid microextraction and preconcentration of Ni(II) into a micro droplet followed by ETAAS determination using a yellow Schiff's base bisazanyl derivative

A simple, rapid and sensitive method was developed for the selective separation and preconcentration of Ni(II) using dispersive liquid–liquid microextraction, by a yellow Schiff's base bisazanyl derivative, as a selective complexing agent. In this method, a mixture of 45 μL chloroform (extraction solvent) and 450 μL tetrahydrofuran (dispersive solvent) is rapidly injected by syringe into a 5 mL aqueous sample containing 3% (w/v) sodium chloride and an appropriate amount of the Schiff's base. As a result, a cloudy solution is formed by entire dispersion of the extraction solvent into the aqueous phase. After centrifuging for 5 min at 5000 rpm, the sedimented phase is directly injected into the electrothermal atomic absorption spectrometry for Ni(II) determination. Some important parameters, such as kind and volume of extraction and dispersive solvents, extraction time, salt effect, pH and concentration of the chelating agent have been optimized. Under the optimum conditions, the enrichment factor for the presented method is 138. The calibration curve was linear over a nickel concentration range of 10–50 ng mL^? 1. The detection limit and relative standard deviation were 0.04 ng mL^? 1 and 2.1%, respectively. The method was successfully applied to the extraction and determination of Ni(II) in different water samples.     

Lovastatin release from polycaprolactone coated β-tricalcium phosphate: Effects of pH,concentration and drug–polymer interactions

The approach of local drug delivery from polymeric coating is currently getting significant attention for both soft and hard tissue engineering applications for sustained and controlled release. The chemistry of the polymer and the drug, and their interactions influence the release kinetics to a great extent. Here, we examine lovastatin release behaviour from polycaprolactone (PCL) coating on β-tricalcium phosphate (β-TCP). Lovastatin was incorporated into biodegradable water insoluble PCL coating. A burst and uncontrolled lovastatin release was observed from bare β-TCP, whereas controlled and sustained release was observed from PCL coating. A higher lovastatin release was observed pH 7.4 as compared to pH 5.0. Effect of PCL concentration on lovastatin release was opposite at pH 7.4 and 5.0. At pH 5.0 lovastatin release was decreased with increasing PCL concentration, whereas release was increased with increasing PCL concentration at pH 7.4. High Ca^2 + ion concentration due to high solubility of β-TCP and degradation of PCL coating were observed at pH 5.0 compared to no detectable Ca^2 + ion release and visible degradation of PCL coating at pH 7.4. The hydrophilic–hydrophobic and hydrophobic–hydrophobic interactions between lovastatin and PCL were found to be the key factors controlling the diffusion dominated release kinetics of lovastatin from PCL coating over dissolution and degradation processes. Understanding the lovastatin release chemistry from PCL will be beneficial for designing drug delivery devices from polymeric coating or scaffolds.     

Capture and release of genomic DNA by PEI modified Fe3O4/Au nanoparticles

Polyethylenimine (PEI) modified Fe₃O₄/Au nanoparticles were synthesized in aqueous solution and characterized by photo correlation spectroscopy (PCS) and vibrating sample magnetometer (VSM). The so-obtained Fe₃O₄/Au-PEI nanoparticles were capable of efficient electrostatic capture of DNA. The maximum amount of genomic DNA captured on 1.0 mg Fe₃O₄/Au-PEI nanoparticles was 90 μg. The DNA release behavior was studied and the DNA recovery from Fe₃O₄/Au-PEI nanoparticles approached 100% under optimal conditions. DNA extraction from mammalian cells using Fe₃O₄/Au-PEI nanoparticles was successfully performed. Up to approximately 43.1 μg of high-purity (OD₂₆₀/OD₂₈₀ ratio = 1.81) genomic DNA was extracted from 10 mg of liver tissue. The results indicated that the prepared Fe₃O₄/Au-PEI nanoparticles could be successfully used for DNA capture and release.     

Effect of processing methods on physicochemical properties of titania nanoparticles produced from natural rutile sand

Titania (TiO₂) nanoparticles were produced from natural rutile sand using different approaches such as sol–gel, sonication and spray pyrolysis. The inexpensive titanium sulphate precursor was extracted from rutile sand by employing simple chemical method and used for the production of TiO₂ nanoparticles. Particle size, crystalline structure, surface area, morphology and band gap of the produced nanoparticles are discussed and compared with the different production methods such as sol–gel, sonication and spray pyrolysis. Mean size distribution (d₅₀) of obtained particles is 76 ± 3, 68 ± 3 and 38 ± 3 nm, respectively, for sol–gel, sonication and spray pyrolysis techniques. The band gap (3.168 < 3.215 < 3.240 eV) and surface area (36 < 60 < 103 m² g^?1) of particles are increased with decreasing particle size (76 > 68 > 38 nm), when the process methodology is changed from sol–gel to sonication and sonication to the spray pyrolysis. Among the three methods, spray pyrolysis yields high-surface particles with active semiconductor bandgap energy. The effects of concentration of the precursor, pressure and working temperature are less significant for large-scale production of TiO₂ nanoparticles from natural minerals.     

Optimized Formulation of High-Payload PLGA Nanoparticles Containing Insulin–Lauryl Sulfate Complex

A novel poly(lactic acid-co-glycolic acid) nanoparticle loaded with insulin–lauryl sulfate complex was prepared by spontaneous emulsion solvent diffusion method. The effects of key parameters such as agitation speed, poly(vinyl alcohol) concentration, solvent composition, polymer concentration, and the volume of external aqueous phase on the properties of the nanoparticles were investigated. To enhance the drug recovery and drug content simultaneously, a response surface methodology with five-level, two-factor central composite design was employed. The weight ratio of polymer to drug and volume ratio of external aqueous phase to solvent phase were selected as controlled factors on account of their interactions found in the monofactorial investigations. The experimental datum allowed the development of quadratic models (p < .05) describing the inter-relationships between the dependent and independent variables. By solving the regression equation, and graphic analyzing the response surface contour and plots, the optimum values of the two factors were determined as 20/1 and 10/1. The optimized conditions led to 89.6% of drug recovery and 4.57% of drug content during nanoparticle preparation.     

Preparation of hydroxypropyl methyl cellulose phthalate nanoparticles with mixed solvent using supercritical antisolvent process and its application in co-precipitation of insulin

To obtain hydroxypropyl methyl cellulose phthalate (HPMCP)/insulin nanospheres by supercritical antisolvent process, the formation of HPMCP nanoparticles was first investigated. The effects of ratio of the mixed solvent, pressure, temperature, concentration, flow rate of CO₂ and solution on forming HPMCP nanoparticles are discussed. It was found that different morphologies of HPMCP could be produced by varying the ratio of DMSO to acetone in the solvent. The operating parameters were optimized for making HPMCP nanoparticles. Formation of HPMCP/insulin nanospheres was further inspected. The nanospheres with the size ranging from 138 nm to 342 nm were obtained. The loading of insulin in the nanospheres ranged from 10.76% to 16.04% and the encapsulation efficiency reached 100%. The release of insulin is also discussed.     

Sunitinib release from biodegradable films of poly(l-lactide-co-caprolactone)

The aim of this study was to prepare sunitinib-loaded biodegradable films using poly(l-lactide-co-?-caprolactone) (PLCL) for anti-tumor drug delivery. Sunitinib-loaded PLCL film has a rough surface, while empty film has a smooth surface. PLCL film loaded with 5% (w/w) sunitinib showed an absence of a crystalline peak of sunitinib, while sharp peaks were observed at 10% (w/w) loading, indicating that sunitinib was molecularly distributed in the polymer matrix at 5% (w/w). A drug release study revealed an initial burst during the first 2 h, followed by continuous release until 24 h. Since weight loss of film was <10% for 1 week, drug release mechanism was dominantly dependent on the diffusion-mediated release of drugs to the medium. Sunitinib has a dose-dependent anti-proliferation effect against HuCC-T1 human cholangiocarcinoma cells in vitro. These results indicate that sunitinib-loaded PLCL film is a appropriate candidate as a vehicle for anti-tumor drug delivery.     

Synthesis of hydroxyapatite crystals using carboxymethyl inulin for use as a delivery of ibuprofen

In the present study biodegradable, environmentally friendly polysaccharide-based polycarboxylate, carboxymethyl inulin (CMI), was used to produce hydroxyapatite (HAP) particles by wet chemical synthesis under controlled temperature, pH, and atmospheric conditions. The morphology and microstructure of the HAP nanoparticles were investigated by XRD, SEM, DTA–TGA and FTIR. CMI affects morphology, surface area, dimension and particle size distribution of the crystals. The reduction in size is greater in the direction of the c-axis. The increase in the polymer concentration to 7.5 g/L resulted in the mixture of nanoparticles with particle sizes of less than 100 nm. The SEM micrograph shows the formation of well-crystallized, agglomerated small particles of HAP. X-ray analysis has shown that the resulting particles have high thermal stability.The obtained crystals were used to produce tablets by direct compression of HAP. The influence of sample's CMI concentration on drug release profiles was investigated by using ibuprofen (C₁₃H₁₈O₂) as a model drug. The model was used to determine the effective diffusion coefficient of the drug from the tablets. A good agreement between experimental data and model predictions was obtained as calculated in the present work. The values of the diffusion coefficients range from 1.62 × 10^? 7 to 4.72 × 10^? 7 m² h^? 1.     

Effects of Au/Fe and Fe nanoparticles on Serratia bacterial growth and production of biosurfactant

The overall objective of this study was to compare the effects of Au/Fe and Fe nanoparticles on the growth and performance of Serratia Jl0300. The nanoparticle effect was quantified not only by the bacterial growth on agar plate after 1 hour interaction with the nanoparticles, but also by its production of a biosurfactant from used vegetable oil. The nanoparticles were prepared using the foam method. The concentrations of the nanoparticles used for the bacterial interaction study were varied from 1 mg/L to 1 g/L. The test results showed that the effect of nanoparticles on the bacterial growth and biosurfactant production varied with nanoparticle type, concentrations, and interaction time with the bacteria. Au/Fe nanoparticles didn't show toxicity to Serratia after short time (1 h) exposure, while during 8 days fermentation Au/Fe nanoparticles inhibited the growth of Serratia as well as the biosurfactant production when the concentration of the nanoparticles was higher than 10 mg/L. Fe nanoparticles showed inhibition effects to bacterial growth both after short time and long time interaction with Serratia, as well as to biosurfactant production when its concentration was higher than 100 mg/L. Based on the trends observed in this study, analytical models have been developed to predict the bacterial growth and biosurfactant production with varying concentrations of nanoparticles.     

Fabrication and evaluation of a sustained-release chitosan-based scaffold embedded with PLGA microspheres

Nutrient depletion within three-dimensional (3D) scaffolds is one of the major hurdles in the use of this technology to grow cells for applications in tissue engineering. In order to help in addressing it, we herein propose to use the controlled release of encapsulated nutrients within polymer microspheres into chitosan-based 3D scaffolds, wherein the microspheres are embedded. This method has allowed maintaining a stable concentration of nutrients within the scaffolds over the long term. The polymer microspheres were prepared using multiple emulsions (w/o/w), in which bovine serum albumin (BSA) and poly (lactic-co-glycolic) acid (PLGA) were regarded as the protein pattern and the exoperidium material, respectively. These were then mixed with a chitosan solution in order to form the scaffolds by cryo-desiccation. The release of BSA, entrapped within the embedded microspheres, was monitored with time using a BCA kit. The morphology and structure of the PLGA microspheres containing BSA before and after embedding within the scaffold were observed under a scanning electron microscope (SEM). These had a round shape with diameters in the range of 27–55 μm, whereas the chitosan-based scaffolds had a uniform porous structure with the microspheres uniformly dispersed within their 3D structure and without any morphological change. In addition, the porosity, water absorption and degradation rate at 37 °C in an aqueous environment of 1% chitosan-based scaffolds were (92.99 ± 2.51) %, (89.66 ± 0.66) % and (73.77 ± 3.21) %, respectively. The studies of BSA release from the embedded microspheres have shown a sustained and cumulative tendency with little initial burst, with (20.24 ± 0.83) % of the initial amount released after 168 h (an average rate of 0.12%/h). The protein concentration within the chitosan-based scaffolds after 168 h was found to be (11.44 ± 1.81) × 10^? 2 mg/mL. This novel chitosan-based scaffold embedded with PLGA microspheres has proven to be a promising technique for the development of new and improved tissue engineering scaffolds.     

New strategy to prepare platinum salts by electrochemical methods and subsequent synthesis of platinum nanoparticles

As shown in the literature, most of Pt nanoparticles (NPs) were synthesized from precursors of commercial Pt salts. However, the impurity of the commercial Pt salts is a concerned issue. In this work, we report a new pathway based on electrochemical methods to prepare Pt-containing complexes with high purity in aqueous solutions from bulk Pt substrates. Experimental results indicate that Pt complexes with higher concentration can be obtained in 0.1 N HCl by using square-wave oxidation–reduction cycles (ORCs) under a frequency of 8 Hz with a step potential of 6.3 mV. Moreover, concentrations of other heavy metals of Hg and Cr in 65 ppm Pt complexes-containing solution are just 0.65 and 0.78 ppb, respectively. These Pt complexes were further reduced to Pt NPs by using NaBH₄ and poly(vinylpyrrolidone) (PVP) as reducing agent and stabilizer, respectively. The concentration and the particle size of synthesized Pt (1 1 1) NPs are ca. 60 ppm and smaller than 5 nm, respectively.     

Structural and optical properties of sol gel derived Cu2ZnSnS4 nanoparticles

The spherical Cu₂ZnSnS₄ nanoparticles with the average diameters (～8–10 nm) have been synthesized by sol gel method. The effects of solvents and reaction temperatures on the properties of the as-synthesized nanoparticles were investigated. The X-ray diffraction shows as grown Cu₂ZnSnS₄ nanoparticles exhibit kesterite crystal structure along preferential orientation (1 1 2) plane. The crystalline nature of nanoparticles was improved in ethylene glycol solvent with the increase in reaction temperature. Rietveld refinement study was performed and structural parameters were determined for the Cu₂ZnSnS₄ nanoparticles. The Raman spectra show the main characteristic peak of A₁ vibrational mode which confirmed the formation of Cu₂ZnSnS₄ phase in all the samples. Scanning electron micrographs depict the irregular aggregate formation of nanoparticles in methanol solvent and uniformly distributed aggregates of nanoparticles with ethylene glycol solvent. Transmission electron microscopy results show the synthesis of polycrystalline porous nanostructures and uniform spherical nanoparticles in methanol and ethylene glycol solvents respectively at the temperature of 250 °C. UV–vis absorption spectra indicated the broad absorption in visible range and the band gap of the nanoparticles was found to 1.38 and 1.45 eV which is suitable for absorbing the solar radiation. The obtained results revealed ethylene glycol as a suitable solvent and 250 °C as the favorable synthesis temperature.     

Free-standing,roll-able,and transparent silicone polymer film prepared by using nanoparticles as cross-linking agents

A free-standing, roll-able, and transparent silicone-based polymer film with a tensile modulus of ca. 7.8 MPa and strain at the break point of 0.76% was successfully prepared by reaction between a reactive silicone oligomer with methyl- and methoxy-side groups and hydrophilic SiO₂ nanoparticles. First, SiO₂ nanoparticles were grafted with silicone chains by a controlled wet chemical sol–gel-type reaction with the reactive oligomers. The solvent of the resulting solution was evaporated to form a viscous suspension, casted into a film, and finally heat-treated at 100 °C and 150 °C. A hydrolysis and condensation reaction among silicone-grafted SiO₂ nanoparticles and free silicone oligomers in the final heat treatment resulted to produce free-standing, roll-able, and transparent silicone-based polymer film. The fact that the silicone film cannot be synthesized without the presence of SiO₂ nanoparticles suggests that these nanoparticles act as cross-linking agents of silicone components providing the improved mechanical properties to the composite film. The rate-controlled mixing and heating of the SiO₂ aqueous/alcohol suspension and the silicone oligomer/alcohol solution was found to be the key step in the synthesis of the free-standing transparent film. While rapid addition/mixing resulted in a fragile and opaque film, a transparent material was achieved when those solutions were slowly mixed. The effect of the synthesis process on the macroscopic and microscopic properties of the prepared films is discussed along with their formation mechanism.     

Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m²/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 μg/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA.     

Towards sustained delivery of small molecular drugs using hydroxyapatite microspheres as the vehicle

The aim of this work is to explore the possibilities of using hydroxyapatite microspheres (HAP-MS) and polymer coated HAP-MS as the vehicles for the sustained release of small molecular drugs. The adsorption/desorption behaviors of model drug, doxycycline hydrochloride (Dox·HCl), on HAP-MS were systemically studied. Drug loaded HAP-MS was encapsulated by biodegradable PLGA using S/O/W emulsion–solvent evaporation method, and the in vitro drug release was tested. The adsorption kinetics of Dox·HCl onto HAP-MS fitted well to Freundlich model at lower drug concentrations, but when the HAP-MS was incubated in concentrated drug solutions higher than a critical concentration, precipitation of drug from solutions occurred. Rapid desorption or release of Dox·HCl from HAP-MS was observed. While, the release profile of Dox·HCl from PLGA coated microspheres showed steady slow drug release lasted for at least 7 days without obvious burst release. PLGA coated HAP-MS may provide a novel, injectable carrier for loading and long-period sustained release of small molecular, water-soluble drugs.