In vitro and in vivo evaluation of cyclosporine-graphene oxide laden hydrogel contact lenses.

Drug-eluting contact lens can substitute the multiple eye drop therapy. However, loading hydrophobic drug like cyclosporine in the contact lens is very challenging, due to low drug uptake (via soaking method); and alteration in the swelling and optical properties which restricts its clinical application. To address the above issues, graphene oxide (GO, large surface area with oxygen containing functional groups) was incorporated in the contact lenses during fabrication. These GO-laden contact lenses (SM-GO-Cys) as well as blank contact lenses (SM-Cys) were soaked in the solution of cyclosporine. Alternatively, cyclosporine-laden contact lenses (DL-Cys-20) and cyclosporine-GO-laden contact lenses (DL-Cys-20-GO) were fabricated by adding drug and drug-GO (at various level of GO) during fabrication, respectively. Contact angle and swelling data showed increase in water holding capacity of GO laden contact lenses. Optical property was significantly improved due to molecular dispersion of drug on the surface of GO sheets. The drug uptake and in vitro release profile was improved with GO-laden contact lenses by soaking method (SM-GO-Cys-400n) due to hydrophobic interactions between GO and drug. Adding cyclosporine-GO (DL-Cys-20-GO-800n) during fabrication significantly improved drug release kinetics with higher drug leaching (during extraction and sterilization) due to increased swelling, improved dissolution and molecular dispersion of drug on GO sheets. Ocular irritation and histopathological studies demonstrated the safety of GO-contact lens. The in vivo drug release studies in the rabbit eye showed significant improvement in mean residence time (MRT) and area under the curve (AUC) using DL-Cys-20-GO-800n contact lens compared to eye drop solution with reduction in protein adherence value. The study demonstrated that the incorporation of GO into the contact lens can control the release of cyclosporine as well as improved the lens swelling and transmittance properties.

Controlled bimatoprost release from graphene oxide laden contact lenses: In vitro and in vivo studies.

Ocular drug delivery using contact lenses may be able to substitute for eye drop therapy. However, issues with hydrophobic drugs (like bimatoprost that is used to treat glaucoma) such as low drug uptake using a simple soaking method into preformed contact lenses and alteration in the swelling and transmittance of lenses restricts the application for drug delivery. This research uses graphene oxide (GO) to control the release of bimatoprost from contact lenses along with improvements in the drug uptake, and lens swelling and transmittance. GO was loaded into silicone hydrogel contact lenses by adding the GO at the same time as lenses were polymerized. These lenses were soaked in bimatoprost. Alternatively contact lenses, either with or without GO, were produced by adding bimatoprost during lens polymerization. GO improved contact lens swelling due to its water binding capacity and lens transmittance due to the molecular dispersion of bimatoprost on the surface of the GO which prevented the local precipitation of the drug. The bimatoprost uptake was not improved in the presence of GO. However, its in vitro release profile was improved. Adding bimatoprost and GO at the same time as lenses were polymerized (DL-GO-BMT) significantly decreased the loss of drug during extraction and sterilization in comparison to contact lenses (DL-BMT) without GO. As the amount of GO was increased, the DL-GO-BMT lenses showed a significant decrease in the burst and cumulative release of bimatoprost. Ocular irritation and histopathology reports demonstrated the safety of GO contact lens. The in vivo pharmacokinetic studies in the rabbit tear fluid showed significant improvement in mean residence time (MRT) and area under the curve (AUC) with DL-GO-0.2 µg-BMT-100 contact lens in comparison to eye drop solution. The study demonstrated that the addition of GO to contact lenses can control the release of bimatoprost as well as improved the lens swelling and transmittance. However, further optimization is needed to modulate the release of drug within the therapeutic level to manage glaucoma.

Novel Poly(vinylpyrrolidone)-Coated Silicone Contact Lenses to Improve Tear Volume During Lens Wear: In Vitro and In Vivo Studies.

Poly(vinylpyrrolidone) (PVP-K90) is widely used to manage dry eye syndrome (DES). The marketed eye drop solutions (high dose) need frequent instillation, affecting the routine lifestyle of patients. PVP-K90-laden contact lenses can be used to overcome the limitations of eye drop solutions (low bioavailability and frequent instillation). However, the conventional methods of PVP-K90 loading show poor loading capacity and short duration of effect. In the present study, we have developed PVP-K90-coated contact lenses via a short curing approach to increase the PVP-K90 loading capacity with a sustained release profile to manage dry eye syndrome. PVP-K90 was loaded by a soaking method (SM-PVP), direct loading (during fabrication, DL-PVP), a combination of soaking and direct loading (DL-SM-PVP), and a novel coating process (SM-PVP-C and DL-SM-PVP-C). The swelling studies suggested improvement in the water uptake (hydration) property of the contact lenses due to the presence of PVP-K90. The optical transparency was within an acceptable range. The in vitro release of PVP-K90 was in the following order: PVP-coated contact lens (168 h) > DL-SM-PVP (168 h) > DL-PVP (96 h) > SM-PVP (72-96 h). PVP-coated contact lenses showed a high burst effect (lubricating effect) and sustained release (3161-448 ng/h between 24 and 168 h) due to high PVP loading/coating in comparison to the uncoated respective contact lenses (964-113 ng/h between 24 and 96 h). In animal studies, the PVP-K90-coated contact lens showed higher tear volume in comparison to the respective uncoated contact lenses and an eye drop solution. This study demonstrates a novel approach of coating a high amount of PVP-K90 on contact lenses for sustained release to manage several ocular diseases like dry eye syndrome, conjunctivitis, and other ocular injuries.

Multiple drug delivery from the drug-implants-laden silicone contact lens: Addressing the issue of burst drug release.

A fixed combination of bimatoprost/timolol eye drop solution is used to manage the elevated intra-ocular pressure in glaucoma patients, including individuals whose condition is poorly controlled by monotherapy. Eye drop solutions are generally given in high dose, due to poor ocular bioavailability. The high ocular dose of bimatoprost and timolol lead to hyperaemia and systemic cardiac side effects respectively. Here, we introduce multiple implant-laden contact lenses (IM) to passively deliver timolol, bimatoprost and hyaluronic acid at therapeutically relevant doses without high burst release. The drug-loaded implants were individually implanted in the outer periphery of the silicone contact lenses. Atomic force microscopy showed the smooth surface of the implant contact lens, as the implants were inside the contact lens matrix. The implant lens (IM) showed major loss of drugs [timolol = 60.60%, bimatoprost = 61.75% and HA = 46.03%] during the monomer extraction and wet sterilization, while the option of dry radiation sterilization (IM-R lens) and hydration for 24 h prior to use showed relatively lower loss of drugs [timolol = 16.87%, bimatoprost = 47.95% and HA = 24.41%]. The in-vitro drugs release data of IM-R lens, showed sustained release for 72 h, with low burst release in comparison to the soaked (SM) and direct drug-laden contact lenses (DL). The in vivo drug release data in the rabbit tear fluid showed sustained release using IM-R lens in comparison to the SM lens and eye drop therapy. The burst release with the IM-R lens was many folds reduced, which could bypass the side effects associated with multiple eye drop therapy. The in vivo pharmacodynamic study in the rabbit model showed peak and valley profile with multiple eye drop therapy, while IM-R lens showed prolong reduction in intra ocular pressure (IOP) for 120 h. The study demonstrates the application of implantation technology to deliver multiple drug through contact lenses to treat glaucoma.

Tailored gatifloxacin Pluronic® F-68-loaded contact lens: Addressing the issue of transmittance and swelling.

Loading of gatifloxacin in contact lenses affects critical lens properties (optical and swelling) owing to drug precipitation in the contact lens matrix. The presence of Pluronic® F-68 in the packaging solution creates in-situ micelles in the contact lens to dissolve gatifloxacin precipitates and provide sustained drug release. The micelles further improved the drug uptake from the drug-packaging solution to create an equilibrium of drug between the lens matrix and the packaging solution. In this study, we optimized gatifloxacin-pluronic-loaded contact lenses to achieve the desired optical transmittance, swelling, and gatifloxacin loading capacity as well as sustained drug delivery. Optimization of gatifloxacin-pluronic-loaded contact lens was carried out using a 32 factorial design by tailoring the concentration of Pluronic® F-68 in the packaging solution (X1) and the amount of gatifloxacin in the monomer solution (X2) to achieve the desired lens properties. The optimized batch (X1 = 0.3%w/v and X2 = 0.3%w/v) showed an optical transmittance of 92.84%, swelling of 92.36% and gatifloxacin loading capacity of 92.56 µg. The in vitro flux data of the optimized batch (GT-Pl-CL) showed sustained release up to 72 h, whereas soaked contact lenses (SM-CL) and direct gatifloxacin-loaded contact lenses (DL-CL) showed a sustained release up to 48 h. The in vivo gatifloxacin release data for rabbit tear fluid showed sustained release with a high gatifloxacin level for the GT-Pl-CL lens in comparison to the SM-CL and the eye drop solution. This study demonstrates the application of the 32 full factorial design to optimize gatifloxacin-pluronic-loaded contact lenses to achieve the desired optical transmittance, swelling, and drug loading capacity.

Lidocaine tripotassium phosphate complex laden microemulsion for prolonged local anaesthesia: In vitro and in vivo studies.

Lidocaine is widely used as a local anaesthetic in the clinical practice to manage pre- and post-operative pain, skin burns, etc. However, the short duration of action (< 2 h) of marketed dosage forms limit their ability to meet clinical needs. Herein, we prepared a lidocaine-tPP(tri potassium phosphate)-complex loaded microemulsion to achieve greater penetration, followed by destabilization of microemulsion in the skin layer to precipitate oil-complex to produce a depot effect in the skin for prolonging the effects of anaesthesia. The lidocaine-tPP-complex-microemulsion was compared with lidocaine base loaded microemulsion, marketed ointment USP and lidocaine HCl. The pseudo ternary phase diagrams at three Smix ratios (1:2, 1:3 and 1:4; Pluronic F127: PEG 400) were constructed using Capmul MCM C8 EP as oil phase. The Smix at 1:4 ratio showed large microemulsion area in comparison to 1:2 and 1:6 ratio. The lidocaine base (LD-1:4-ME10O45SM and LD-1:4-ME20O45SM) and lidocaine-tPP-complex (LDC-1:4-ME10O45SM and LDC-1:4-ME20O45SM) loaded microemulsion batches (1:4 ratio) were thermodynamically stable. The ex vivo diffusion study showed sustained release up to 12 h with microemulsion batches, in comparison to lidocaine HCl (4 h) and ointment base (7 h). The selected LDC-1:4-ME20O45SM batch was non-irritating on the rabbit skin. In drug retention studies, LD-1:4-ME20O45SM and LDC-1:4-ME20O45SM batches showed 2.68- and 3.93-fold greater lidocaine retention in comparison to ointment USP. The radiant heat tail-flick test showed prolong local anaesthesia using LDC-1:4-ME20O45SM in comparison to ointment USP. The findings suggest that lidocaine-tPP-complex loaded microemulsion could be a potential strategy for providing prolong local anaesthesia.

Plackett-Burman design for screening of critical variables and their effects on the optical transparency and swelling of gatifloxacin-Pluronic-loaded contact lens.

The optical and swelling properties of gatifloxacin-loaded contact lens decrease owing to the precipitation of gatifloxacin (on hydration) in the matrix structure of the contact lens. This paper focuses on the use of Pluronic F68 both inside and outside (in the packaging solution) the contact lens to form micelles to dissolve the gatifloxacin precipitates and not limited to sustain the release of gatifloxacin. The aim of this study was to screen the critical variables affecting the optical and swelling properties of gatifloxacin-loaded contact lens. The independent variables investigated were the concentration of Pluronic F68 incorporated in the monomer solution to fabricate the lens (X1, %w/v), the concentration of Pluronic F68 in the packaging solution (X2, %w/v), the concentration of gatifloxacin incorporated in the monomer solution (X3, %w/v), the concentration of gatifloxacin incorporated in the packaging solution during autoclave (X4, %w/v), the concentration of gatifloxacin incorporated in the packaging solution during extraction (X5, %w/v), the time (stabilization time) after the addition of gatifloxacin and Pluronic F68 to the monomer solution before the fabrication of the lens (X6, h), the pH of the packaging solution (X7), the temperature of the extracted solution (X8, °C), and the curing time for fabricating the contact lens (X9, min). The gatifloxacin-loaded contact lenses were characterized for their optical transmittances after sterilization on day 1 (Y1, %), optical transmittances after 7 days of sterilization (Y2, %) and swelling percentages after 7 days of sterilization (Y3, %). The selected variables showed responses that were in the ranges 53.5% to 97.2%, 51.3% to 92.6%, and 50.3% to 83.7% for Y1, Y2, and Y3, respectively. The data suggest that the presence of Pluronic F68 inside the contact lens (X1) reduced the optical and swelling properties of the contact lens, whereas the presence of Pluronic F68 in the packaging solution (X2) improved them through micelle formation. The other variables (X3 to X9) did not exhibit significant effects on the swelling and transmittance. This study revealed the potential of Plackett-Burman design to screen the selected critical variables that affected the optical and swelling properties of gatifloxacin-loaded contact lens.

Effect of gold nanoparticles on timolol uptake and its release kinetics from contact lenses: In vitro and in vivo evaluation.

Contact lenses are ideally suited for extended drug delivery to the ocular tissues, but incorporation of any particulate system affects the critical properties of the contact lens. Timolol loading by the conventional soaking method does not significantly alter the critical properties of the contact lens. However, there are challenges of low drug loading and high burst release. This research work aimed to investigate the effect of gold nanoparticles (GNPs) on loading and its release kinetics from the contact lens using the soaking method. In one approach, GNPs were loaded into the timolol soaking solution (GNPs-SS), and in another approach, GNPs were incorporated into the contact lenses (GNPs-CL) during fabrication. The contact lenses were soaked at two different concentrations of timolol (i.e., 2 mg/ml and 4 mg/ml). Swelling and optical transmittance were not significantly affected by the presence of GNPs in the contact lenses. A significant uptake/loading of timolol using the GNPs in both the approaches was observed. The in vitro flux data showed no significant improvement in the release rate profiles of timolol when using both approaches. However, the in vivo study in the rabbit tear fluid showed high timolol concentration with the GNPs-laden contact lens at all timepoints in comparison to the soaked contact lenses without GNPs. The in vivo pharmacodynamic study in rabbits showed a 2 mmHg average fall in intraocular pressure (72 h) using the GNPs-laden contact lenses, while the soaked contact lenses without GNPs and eye drops solution (0.5 %w/v) showed 2 mmHg. The drug distribution study in the ocular tissue showed a significant improvement in the drug deposition with the GNPs-laden contact lenses in the ciliary muscle and conjunctiva. This study successfully demonstrated the potential of GNPs to enhance the uptake of drug from the drug soaking solution to treat glaucoma without compromising the critical properties of contact lens. STATEMENT OF SIGNIFICANCE: In this study, we have overcome the limitation of the conventional soaking method of low drug loading and high burst release from the contact lenses. We have investigated the effect of gold nanoparticles (GNPs) on the timolol loading and its release kinetics from the contact lenses. The study revealed the potential of GNPs to enhance the uptake of timolol from the timolol soaking solution to treat glaucoma without compromising the critical lens properties.

Contact lenses with dual drug delivery for the treatment of bacterial conjunctivitis.

Currently, bacterial conjunctivitis is treated by frequent administration of antibiotic eye drop solutions, which is tedious and patient noncompliant. Contact lenses could be ideal medical devices to sustain the release of ophthalmic drugs, but the incorporation of the latter can alter the optical and physical properties of the lenses. In addition, many contact lens users have reported the pink eye syndrome, making them unsuitable as ocular medical devices. In the present study, we have designed a novel type of lenses containing semi-circular rings loaded with moxifloxacin HCl (a broad spectrum antibiotic) and hyaluronic acid (a comfort agent), respectively, in order to treat bacterial conjunctivitis without altering the critical lens properties. The drug loaded rings were implanted separately within the periphery of the contact lenses using the modified cast moulding technology. The atomic force microscopy report showed an average roughness of 22.27 nm for the implant lens, which was significantly lower in comparison to the marketed Freshlook® (116.27 nm) contact lens. The major amount of moxifloxacin HCl was leached (68.16-74.55%) during the monomer extraction and wet sterilization (autoclave) steps; hence the lenses were terminally sterilized by radiation and packaged under dry condition (dehydrated). The in vitro release data showed release for moxifloxacin HCl and hyaluronic acid up to 96 h. The in vivo drug release studies showed significant improvement [>MIC for Staphylococcus aureus] in the drug residence time in comparison to the eye drop therapy. The in vivo efficacy study in the staphylococcus aureus induced conjunctivitis showed equivalent healing effect with the single implant contact lens in comparison to the frequent high dose eye drop therapy. The study demonstrated the successful application of the implantation technology to co-deliver moxifloxacin HCl and hyaluronic acid from the contact lenses for the extended period of time to treat conjunctivitis.

Co-delivery of timolol and hyaluronic acid from semi-circular ring-implanted contact lenses for the treatment of glaucoma: in vitro and in vivo evaluation.

Glaucoma is a chronic disease, which is currently treated using frequent high dose applications of an eye drop solution; this method is tedious, and most of patients are non-compliant to it. Contact lenses are emerging as a convenient option to sustain the release of ophthalmic drugs. However, the incorporation of a drug/formulation changes the optical and physical properties of contact lenses. Contact lens users have also reported pink eye syndrome; this makes contact lenses unsuitable to be accepted as a medical device. The objective of the present study was to design novel timolol and hyaluronic acid (comfort agent)-loaded semi-circular ring-implanted contact lenses that could uphold the release at therapeutic rates without compromising the critical lens properties. The drug-loaded rings were individually implanted within the periphery of the contact lenses using modified cast-moulding technology. Atomic force microscopy showed an average roughness of 12.38 nm for the implanted lens that was significantly lower as compared to that of the Freshlook contact lenses (116.27 nm). A major amount of timolol was leached (from 46.47 to 58.79%) during the monomer extraction and moist sterilization (autoclave) steps; therefore, the lenses were sterilized by radiation and packaged under dry conditions (dehydrated). The in vitro release data showed sustained release of timolol and hyaluronic acid up to 96 h. The in vivo drug release study on rabbit eyes showed the presence of timolol in tear fluid up to 72 h. The in vivo pharmacodynamics studies showed a reduction in IOP till 144 h with a low drug loading (154 µg) as compared to the case of a single instillation eye drop solution (250 µg). This study has demonstrated the successful application of implantation technology to co-deliver timolol and hyaluronic acid from contact lenses for an extended period of time to treat glaucoma.

pH triggered controlled drug delivery from contact lenses: Addressing the challenges of drug leaching during sterilization and storage.

In the present work a novel cyclosporine-loaded Eudragit S100 (pH-sensitive) nanoparticles-laden contact lenses were designed to provide sustained release of cyclosporine at therapeutic rates, without leaching of drug during sterilization and storage period (shelf life). The nanoparticles were prepared by Quasi-emulsion solvent diffusion technique using different weight ratios of cyclosporine to Eudragit S100. The contact lenses with direct drug entrapment were also fabricated (DL-50) for comparison. The percentage swelling and optical transparency of nanoparticles-laden contact lenses were improved in comparison to DL-50 lenses. The nanoparticles-laden contact lenses showed sustained drug release profiles, with inverse relationship to the amount of nanoparticles loaded in the contact lenses. It was interesting to note that nanoparticles form nanochannels/cavities after dissolution of Eudragit S 100 in tear fluid pH=7.4 (in vitro release study). This followed the precipitation of drug in hydrogel matrix of contact lenses. As the amount of nanoparticles loading increased, more number of cavities were formed, which caused the formation of large cavities in contact lens matrix. This in turn precipitated the drug. The nanoparticles-laden contact lenses with 1:1 (drug: Eudragit) weight ratio showed the most promising results of sustaining the drug release up to 156h, without affecting optical and physical properties of contact lenses. Packaging study confirmed that the drug was not leached in packaging solution (buffer, pH=6.5) from nanoparticles-laden lenses during shelf life period. In-vivo study in rabbit tear fluid showed sustained release up to 14days. The study revealed the application of pH-sensitive nanoparticles-laden contact lenses for controlled release of cyclosporine without altering the optical and physical properties of lens material.

Effect of surfactant chain length on drug release kinetics from microemulsion-laden contact lenses.

The effect of surfactant chain lengths [sodium caprylate (C8), Tween 20 (C12), Tween 80 (C18)] and the molecular weight of block copolymers [Pluronic F68 and Pluronic F 127] were studied to determine the stability of the microemulsion and its effect on release kinetics from cyclosporine-loaded microemulsion-laden hydrogel contact lenses in this work. Globule size and dilution tests (transmittance) suggested that the stability of the microemulsion increases with increase in the carbon chain lengths of surfactants and the molecular weight of pluronics. The optical transmittance of direct drug-laden contact lenses [DL-100] was low due to the precipitation of hydrophobic drugs in the lenses, while in microemulsion-laden lenses, the transmittance was improved when stability of the microemulsion was achieved. The results of in vitro release kinetics revealed that drug release was sustained to a greater extent as the stability of microemulsion was improved as well. This was evident in batch PF127-T80, which showed sustained release for 15days in comparison to batch DL-100, which showed release up to 7days. An in vivo drug release study in rabbit tear fluid showed significant increase in mean residence time (MRT) and area under curve (AUC) with PF-127-T80 lenses (stable microemulsion) in comparison to PF-68-SC lenses (unstable microemulsion) and DL-100 lenses. This study revealed the correlation between the stability of microemulsion and the release kinetics of drugs from contact lenses. Thus, it was inferred that the stable microemulsion batches sustained the release of hydrophobic drugs, such as cyclosporine from contact lenses for an extended period of time without altering critical lens properties.

Design and optimization of a novel implantation technology in contact lenses for the treatment of dry eye syndrome: In vitro and in vivo evaluation.

Contact lenses are widely used for ophthalmic drug delivery, but incorporation of drug or formulation in the contact lenses affects its optical and physical property. In the present study, we have designed a novel hyaluronic acid (HA)-laden ring implant contact lenses (modified cast moulding method), to circumvent the changes in critical lens property. The objective was to improve the ocular residence time of HA, by providing sustained ocular HA delivery through implant contact lenses for the treatment of dry eye syndrome. Optimization of HA-implant was carried out using 32 factorial design by tailoring the amount of cross linker and thickness of implant, to achieve sustained HA release with constraint on effective ion diffusivity. The in vivo pharmacokinetic study in rabbit tear fluid showed sustained HA release up to 15days, by fabricating implant (80µgHA loading) with 78.4µm thickness (total thickness of lens=100µm) using 0.925% of cross linker, with effective ion diffusivity>1.5×10-6mm2/min. In vivo efficacy study in benzalkonium chloride induced dry eye syndrome rabbits showed faster healing with implant contact lenses in comparison to positive control group. The study demonstrated the promising potential of implantation technology to deliver hyaluronic acid without compromising optical and physical properties of contact lens. STATEMENT OF SIGNIFICANCE: The limitation of contact lenses to be used as therapeutic device for controlled drug delivery is focused in this study. Incorporation of drug or formulation in the biomaterial affects the optical and physical property of contact lenses. The significance of project was to design a novel hyaluronic acid-laden ring implant contact lenses, to by-pass the changes in critical property of biomaterial.

In vitro and in vivo evaluation of novel implantation technology in hydrogel contact lenses for controlled drug delivery.

Glaucoma is commonly treated using eye drops, which is highly inefficient due to rapid clearance (low residence time) from ocular surface. Contact lenses are ideally suited for controlled drug delivery to cornea, but incorporation of any drug loaded particulate system (formulation) affect the optical and physical property of contact lenses. The objective of the present work was to implant timolol maleate (TM) loaded ethyl cellulose nanoparticle-laden ring in hydrogel contact lenses that could provide controlled drug delivery at therapeutic rates without compromising critical lens properties. TM-implant lenses were developed, by dispersing TM encapsulated ethyl cellulose nanoparticles in acrylate hydrogel (fabricated as ring implant) and implanted the same in hydrogel contact lenses (sandwich system). The TM-ethyl cellulose nanoparticles were prepared by double emulsion method at different ratios of TM to ethyl cellulose. The X-ray diffraction studies revealed the transformation of TM to amorphous state. In vitro release kinetic data showed sustained drug release within the therapeutic window for 168h (NP 1:3 batch) with 150µg loading. Cytotoxicity and ocular irritation study demonstrated the safety of TM-implant contact lenses. In vivo pharmacokinetic studies in rabbit tear fluid showed significant increase in mean residence time (MRT) and area under curve (AUC), with TM-implant contact lenses in comparison to eye drop therapy. In vivo pharmacodynamic data in rabbit model showed sustained reduction in intra ocular pressure for 192h. The study demonstrated the promising potential of implantation technology to treat glaucoma using contact lenses, and could serve as a platform for other ocular diseases.