Sensitizers For Photodynamic Therapy Research Articles

Improved Singlet Oxygen Production of Zinc Phthalocyanine Bearing Pyridine-4-Thiol Groups Using Sonochemistry and Comparison with Photochemistry

The interest in research to enhance the ability of sensitizers to generate singlet oxygen using light and/or ultrasound in cancer treatment has increased significantly in recent years, and studies have attracted considerable interest. In addition, phthalocyanines (Pcs) have become increasingly important as sensitizers in photodynamic therapy (PDT) (stimulation source: light) and/or sonodynamic therapy (SDT) (stimulation source: light and ultrasound). Sonophotodynamic therapy (SPDT), a new technique that gives more effective results than PDT, is now gaining in importance. Although there are published articles on SPDT studies, studies in this field are limited. In this context, we synthesized a zinc(II) phthalocyanine molecule bearing 4-(pyridine-4-ylthio) substituents to determine its potential as a sensitizer in SPDT applications as well as PDT applications, and the structure of the synthesized complex was illuminated using FTIR,1H-NMR, UV-Vis, and MS spectroscopic techniques. When light and ultrasound were combined, the calculated ΦΔ value for zinc (II) phthalocyanine (3) increased to greater than 1, even though it was 0.76 after light excitation. When evaluating the two methods, it was observed that singlet oxygen production was greatly enhanced by the SPDT method. In this way, the research will add to the enhancement of knowledge on the subject of the SPDT method's enhancement of singlet oxygen generation.

Synthesis of non-peripheral phthalocyanine sensitizers and investigation of the effect of ultrasound-light stimulus synergy on singlet oxygen production

The high singlet oxygen production capacity and other unique properties of phthalocyanines allow these compounds to be widely used as sensitizers in photodynamic therapy (PDT) and sonodynamic therapy (SDT) in the treatment of cancer. Sono-photodynamic therapy (SPDT), which uses a combination of light and sound stimulation together and has a very high therapeutic effect compared to PDT, is an increasingly important new method for cancer treatment. Published studies on the application of non-peripherally phthalocyanines in SPDT are limited in the literature. With this in mind, in this study, the synthesis and characterization of novel non-peripherally tetra-substituted (4-tert-butylbenzyl)oxy) metal-free (2), Mg(II) (3), Zn(II) (4), and In(III)CI (5) phthalocyanines and their potential as a sono-photosensitizer in SPDT applications as well as PDT applications were determined. Comparative photochemical and sono-photochemical studies showed that the singlet oxygen quantum yields of the synthesised phthalocyanine compounds were calculated as 0.027, 0.22, 0.29 and 0.32 for 2, 3, 4 and 5 respectively, by photochemical studies using only light irradiation, while these values increased to 0.11, 0.28, 0.58 and 0.64 for 2, 3, 4 and 5 respectively, by sono-photochemical studies after the synergistic effect of light and ultrasound. These results showed that the combined use of ultrasound waves and light as a stimulation method exhibited a more effective therapeutic effect compared to light stimulation alone. According to the singlet oxygen efficiency, it was shown that the presence of a metal ion on the phthalocyanine ring increased the singlet oxygen production in both methods.

Spectral properties of Yb-doped AgBiS2 and AgInS2 nanocrystals

Subject of study. The spectral features of nanocrystals with ternary compositions I-V-VI2 and I-III-VI2 doped with the rare earth metal ytterbium are investigated and described. Purpose of the study. The aim of this work is to develop a novel synthesis method for these nanocrystals to further examine their optical and morphological properties. Method. The average sizes of AgInS2:Yb and AgBiS2:Yb nanocrystals are analyzed using atomic force microscopy and dynamic light scattering, and the results are compared. Spectrophotometry and spectrofluorimetry are employed to record the absorption and photoluminescence spectra, respectively. Additionally, the photoluminescence decay kinetics are recorded using a laser scanning luminescence microscope for a deeper exploration of the electronic structure of the synthesized materials. Main results. Morphological analysis reveals that single-stage synthesis produced nanocrystals based on the AgInS2 matrix are smaller than the reference sample, while two-stage synthesis results in average nanocrystal sizes 1.5 times larger than the reference sample. The absorption spectra of the AgInS2:Yb and AgBiS2:Yb samples, as well as their reference samples, correspond to typical absorption spectra of semiconductor nanocrystals of ternary compounds. The absorption spectrum of AgBiS2:Yb nanocrystals, unlike AgInS2:Yb, spans a broad range from 300 nm to 1300 nm. In the photoluminescence spectra of AgInS2 and AgInS2:Yb nanocrystals, no significant shift of the band maximum is observed, and photoluminescence typical of Yb ions is absent. It is found that the weighted average photoluminescence lifetime in AgInS2:Yb nanocrystals can be modulated by ytterbium doping. Photoluminescence in AgBiS2 and AgBiS2:Yb samples is not detected in the red and near-infrared regions. Practical significance. These materials may be used in the fabrication of absorber layers for solar cells, as well as sensitizers for photodynamic and photothermal therapy. Further studies of isotropic samples of these nanocrystals could not only expand their potential applications but also enhance the physical properties of nanoparticles in the long term.

Lutetium texaphyrin: A photocatalyst that triggers pyroptosis via biomolecular photoredox catalysis

Photon-controlled pyroptosis activation (PhotoPyro) is a promising technique for cancer immunotherapy due to its noninvasive nature, precise control, and ease of operation. Here, we report that biomolecular photoredox catalysis in cells might be an important mechanism underlying PhotoPyro. Our findings reveal that the photocatalyst lutetium texaphyrin (MLu) facilitates rapid and direct photoredox oxidation of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and various amino acids, thereby triggering pyroptosis through the caspase 3/GSDME pathway. This mechanism is distinct from the well-established role of MLu as a photodynamic therapy sensitizer in cells. Two analogs of MLu, bearing different coordinated central metal cations, were also explored as controls. The first control, gadolinium texaphyrin (MGd), is a weak photocatalyst but generates reactive oxygen species (ROS) efficiently. The second control, manganese texaphyrin (MMn), is ineffective as both a photocatalyst and a ROS generator. Neither MGd nor MMn was found to trigger pyroptosis under the conditions where MLu was active. Even in the presence of a ROS scavenger, treating MDA-MB-231 cells with MLu at concentrations as low as 50 nM still allows for pyroptosis photo-activation. The present findings highlight how biomolecular photoredox catalysis could contribute to pyroptosis activation by mechanisms largely independent of ROS.

Porphyrin(2.1.2.1) organopalladium complexes as efficient singlet oxygen sensitizers.

Four new non-planar and non-aromatic porphyrin organopalladium complexes were synthesized. Conformational structures and optical and electronic properties of the obtained organopalladium complexes containing meso-substituted phenyl, p-tert-butylphenyl, or pentafluorophenyl groups were fully investigated. These complexes showed potent capacity for singlet oxygen (1O2) generation under blue-light irradiation, and the 1O2 quantum yields were in the range of 41%-56%, which were comparable to that of Ru(bpy)3Cl2 (57%), and such potency made these organopalladium complexes potential 1O2 photo sensitizers for photodynamic therapy.

The use of nanomaterials in advancing photodynamic therapy (PDT) for deep-seated tumors and synergy with radiotherapy.

Photodynamic therapy (PDT) has been under development for at least 40years. Multiple studies have demonstrated significant anti-tumor efficacy with limited toxicity concerns. PDT was expected to become a major new therapeutic option in treating localized cancer. However, despite a shifting focus in oncology to aggressive local therapies, PDT has not to date gained widespread acceptance as a standard-of-care option. A major factor is the technical challenge of treating deep-seated and large tumors, due to the limited penetration and variability of the activating light in tissue. Poor tumor selectivity of PDT sensitizers has been problematic for many applications. Attempts to mitigate these limitations with the use of multiple interstitial fiberoptic catheters to deliver the light, new generations of photosensitizer with longer-wavelength activation, oxygen independence and better tumor specificity, as well as improved dosimetry and treatment planning are starting to show encouraging results. Nanomaterials used either as photosensitizers per se or to improve delivery of molecular photosensitizers is an emerging area of research. PDT can also benefit radiotherapy patients due to its complementary and potentially synergistic mechanisms-of-action, ability to treat radioresistant tumors and upregulation of anti-tumoral immune effects. Furthermore, recent advances may allow ionizing radiation energy, including high-energy X-rays, to replace external light sources, opening a novel therapeutic strategy (radioPDT), which is facilitated by novel nanomaterials. This may provide the best of both worlds by combining the precise targeting and treatment depth/volume capabilities of radiation therapy with the high therapeutic index and biological advantages of PDT, without increasing toxicities. Achieving this, however, will require novel agents, primarily developed with nanomaterials. This is under active investigation by many research groups using different approaches.

A new approach for BODIPY derivatives: Increasing the singlet oxygen formation by sonophotochemical technique

Research to explore the therapeutic capacities of sensitizers in Photodynamic Therapy (PDT) has been among the topics that have been frequently explored in recent years. In this context, studies that improve the ability of sensitizers to produce singlet oxygen with the help of ultrasound draw so much attention. In addition, the importance of BODIPYs used as sensitizers in photodynamic and/or sonodynamic therapy (SDT) is increasing. Although the positive effect of sonophotodynamic therapy (SPDT) on phthalocyanine derivatives has been shown in both photophysicochemical and in vivo studies, according to the best of our knowledge, the effect of sonophotochemical measurements for BODIPY derivatives has not been reported in the literature yet. Evaluation of the sonophotochemical potentials of BODIPY derivatives and meeting this need in the literature are the biggest impetus of this study.In this context, we have synthesized a new BODIPY compound (BDPY2) with methoxystyryl substituents to evaluate the capability of singlet oxygen production by sonophotochemical method. Synthesized compound was illuminated using different spectroscopic techniques encompassing 1H-NMR, 13C-NMR,19F-NMR, UV-Vis, MS, and elemental analysis. ΦΔ value of BDPY2 was calculated by the photochemical method as 0.33, while ΦΔ value reached 0.68 by applying the sonophotochemical method. The obtained data proved that the singlet oxygen production increased considerably by changing the method from light irradiation to ultrasound/light. This result will be a pioneer study to comprehend the sonophotochemical measurements of BODIPY derivatives for in vivo or in vitro studies in the future.

NMR Spectral Study of the Interaction of Tricationic Chlorin Sensitizer for Photodynamic Therapy with Passive Delivery Vehicles

NMR Spectral Study of the Interaction of Tricationic Chlorin Sensitizer for Photodynamic Therapy with Passive Delivery Vehicles

Relativistic two-component time dependent density functional studies and Hirshfeld surface analysis of halogenated arylidenehydrazinylthiazole derivatives

Crystal structures of compounds 2-(2-chlorobenzylidene)hydrazinylthiazole (2a) 2-(2-bromobenzylidene)hydrazineylthiazole (2b) were analyzed with single crystal XRD. Both compounds possess Monoclinic crystal system and P21/n space group. Intermolecular interactions in single crystals were studied with Hirshfeld surface analysis (HSA). Electronic spectrum of 2a and 2b has been studied with two-component relativistic sf-X2C-S-TD-DFT approach and induction of spin–orbit couplings in organic skeleton by bromine and chlorine atoms has been explored. Magnitude of spin–orbit couplings in 2b at B3LYP-D3/x2c-TZVPPall level of theory promises its use as triplet sensitizer in photodynamic therapy and solar light-based fuel cells.

Metal-Organic Framework (MOF)-A Universal Material for Biomedicine.

Metal-organic frameworks (MOFs) are a very promising platform for applications in various industries. In recent years, a variety of methods have been developed for the preparation and modification of MOFs, providing a wide range of materials for different applications in life science. Despite the wide range of different MOFs in terms of properties/sizes/chemical nature, they have not found wide application in biomedical practices at present. In this review, we look at the main methods for the preparation of MOFs that can ensure biomedical applications. In addition, we also review the available options for tuning the key parameters, such as size, morphology, and porosity, which are crucial for the use of MOFs in biomedical systems. This review also analyses possible applications for MOFs of different natures. Their high porosity allows the use of MOFs as universal carriers for different therapeutic molecules in the human body. The wide range of chemical species involved in the synthesis of MOFs makes it possible to enhance targeting and prolongation, as well as to create delivery systems that are sensitive to various factors. In addition, we also highlight how injectable, oral, and even ocular delivery systems based on MOFs can be used. The possibility of using MOFs as therapeutic agents and sensitizers in photodynamic, photothermal, and sonodynamic therapy was also reviewed. MOFs have demonstrated high selectivity in various diagnostic systems, making them promising for future applications. The present review aims to systematize the main ways of modifying MOFs, as well as the biomedical applications of various systems based on MOFs.

Amino Acid Derivatives of Chlorin-e6-A Review.

Details of the structural elucidation of the clinically useful photodynamic therapy sensitizer NPe6 (15) are presented. NPe6, also designated as Laserphyrin, Talaporfin, and LS-11, is a second-generation photosensitizer derived from chlorophyll-a, currently used in Japan for the treatment of human lung, esophageal, and brain cancers. After the initial misidentification of the structure of this chlorin-e6 aspartic acid conjugate as (13), NMR and other synthetic procedures described herein arrived at the correct structure (15), confirmed using single crystal X-ray crystallography. Interesting new features of chlorin-e6 chemistry (including the intramolecular formation of an anhydride (24)) are reported, allowing chemists to regioselectively conjugate amino acids to each available carboxylic acid on positions 131 (formic), 152 (acetic), and 173 (propionic) of chlorin e6 (14). Cellular investigations of several amino acid conjugates of chlorin-e6 revealed that the 131-aspartylchlorin-e6 derivative is more phototoxic than its 152- and 173-regioisomers, in part due to its nearly linear molecular conformation.

Dissecting the Interactions between Chlorin e6 and Human Serum Albumin.

Chlorin e6 (Ce6) is among the most used sensitizers in photodynamic (PDT) and sonodynamic (SDT) therapy; its low solubility in water, however, hampers its clinical exploitation. Ce6 has a strong tendency to aggregate in physiological environments, reducing its performance as a photo/sono-sensitizer, as well as yielding poor pharmacokinetic and pharmacodynamic properties. The interaction of Ce6 with human serum albumin (HSA) (i) governs its biodistribution and (ii) can be used to improve its water solubility by encapsulation. Here, using ensemble docking and microsecond molecular dynamics simulations, we identified the two Ce6 binding pockets in HSA, i.e., the Sudlow I site and the heme binding pocket, providing an atomistic description of the binding. Comparing the photophysical and photosensitizing properties of Ce6@HSA with respect to the same properties regarding the free Ce6, it was observed that (i) a red-shift occurred in both the absorption and emission spectra, (ii) a maintaining of the fluorescence quantum yield and an increase of the excited state lifetime was detected, and (iii) a switch from the type II to the type I mechanism in a reactive oxygen species (ROS) production, upon irradiation, took place.

Conjugates of Tetrapyrrolic Macrocycles as Potential Anticancer Target-Oriented Photosensitizers

Photodynamic therapy is a minimally invasive treatment of tumors using photosensitizers, light, and reactive oxygen species, which can destroy cellular structures. With the development of photodynamic therapy, significant efforts have been made to create new efficient photosensitizers with improved delivery to cells, stability, and selectivity against cancer tissues. Naturally occurring tetrapyrrolic macrocycles, such as porphyrins and chlorins, are very attractive as photosensitizers, and their structural modification and conjugation with other biologically active molecules are promising approaches for creating new photosensitizers specifically targeting cancer cells. The present review aims to highlight recent developments in the design, preparation, and investigation of complex conjugates of tetrapyrrolic macrocycles, which can potentially be used as sensitizers for target-oriented photodynamic therapy of cancer. In this review, we discuss the structure, photodynamic effect, and anticancer activity of the following conjugates of tetrapyrrolic macrocycles: (1) conjugates obtained by modifying peripheral substituents in porphyrins and chlorins; (2) conjugates of porphyrins and chlorins with lipids, carbohydrates, steroids, and peptides; (3) conjugates of porphyrins and chlorins with anticancer drugs and some other biologically active molecules; (4) metal-containing conjugates. The question of how the conjugate structure affects its specificity, internalization, localization, and photoinduced toxicity within cancer cells is the focus of this review.

A self-reporting photosensitizer for inducing and in-situ monitoring lysosomal damage and cell apoptosis

Photodynamic therapy (PDT) is a desirable method to fight against cancer, because it can achieve selective ablation of tumor tissues via the control of incident light. Self-reporting PDT sensitizers that can in-situ and real-timely monitor cell viability during PDT process are promising tools for PDT. For lysosomal damage would release cathepsins B/D into cytoplasm and efficiently induce cell apoptosis, herein, we have constructed a unique self-reporting PDT sensitizer (NSLN) targeting lysosomes in living cells. NSLN was designed to equip both alkalescent unit and cationic group. And NSLN targeted lysosomes in living cells, and immigrated into nucleus after lysosomal damage and cell death. In this way, the cells viability could be facilely visualized via subcellular localization of NSLN. With the probe, the apoptosis induced by H2O2, rotenone, and paclitaxel has been successfully monitored. Furthermore, NSLN could generate large amount of reactive oxygen species and induce lysosomal damage and cell apoptosis upon light irradiation. The cell damage during PDT procedure could be also monitored with NSLNvia its subcellular immigration. This work provides a novel method for monitoring PDT process and makes it possible to control phototoxicity. We believe that NSLN can be used as a powerful tool to promote the development of PDT.

The role of solvents and concentrations in the properties of oxime bearing A2B corroles.

A comprehensive study on the electronic spectral, photophysical and acid-base properties of phenyl- and methyl-oxime corrole derivatives and of triphenylcorrole (model corrole) has been performed, aiming to shed light on the existing species in the ground and excited states. Solvents and corrole concentration are found to govern the properties of the studied compounds and are determinants of their applicability in in vivo studies. In THF, the neutral corrole has two tautomeric forms (T1 and T2). In DMSO, the deprotonated form shows a characteristic long-wavelength Q band slightly shifted to blue when compared with the T1 tautomer and a higher fluorescence quantum yield. In ACN, with the increase of the corrole concentration formation of an aggregate due to homoconjugation (with dimer characteristics) is observed, and pioneeringly reported using UV-Vis and fluorescence studies and confirmed by carrying out titrations with TFA. The effect of the oxime group on the pK values of a corrole is found to influence the formation of a homoconjugate, namely by precluding its formation (at higher concentrations) when compared with the model corrole. TDDFT electronic quantum calculations support the experimental observations, namely the existence of tautomers and deprotonated species, with their respective electronic spectral features, further allowed proposing a structure for the homoconjugate complex in ACN. The characteristics of the oxime-corroles, namely a pK of ∼ 5, absorption and emission at ca. 650 nm and solvent dependent properties, make them good candidates for their use in biological systems either as probes, sensors, or as new sensitizers for photodynamic therapy.

Rational design of functionalized near-infrared absorbing phthalocyanines by three-component coupling strategy

Near-infrared (NIR) dyes are used in various applications such as organic solar cells and photodynamic therapy sensitizers. One example of a class of dyes, octa-arylthio-substituted phthalocyanines (Pcs), can effectively absorb NIR light above 800 nm. The substitution of the Pc’s peripheral sulfur effectively introduces extra functions without significant perturbation of the optical properties. However, the synthesis of phthalonitrile precursors containing various functionalized arylthio groups has been limited. Herein, we provide the three-component-type coupling reactions for synthesizing phthalonitriles with various functionalized chalcogen-aryl groups. Organometallic reagents were prepared from aryl halides, elemental chalcogen, and 3,6-bis(trifluoromethanesulfonyloxy)phthalonitrile can lead to functionalized phthalonitriles in a one-pot procedure. Commonly used methods were utilized to prepare the corresponding Pcs. This reaction can be extended to other group-16 elements, chemoselective organomagnesium, and organozincate reagents. The NIR absorption and fluorescence properties of Pcs were also revealed and rationalized under substitution effects.

Photochemical synthesis of fluorescent Au16(RGDC)14 and excited state reactivity with molecular oxygen.

Aqueous metal nanoclusters have emerged as effective materials for biomedical imaging and therapy. Among them, gold nanoclusters (AuNCs) have been widely studied due to their unique electronic structures. These nanoclusters are often optically impure, comprising a mixture of fluorescent clusters with different metal/ligand compositions. The polydispersity of nanoclusters makes it challenging to isolate the most stable structure, and poses further risks for eventual clinical applications. Herein, Au16L14 clusters are reported which are optically pure as assessed by fluorescence excitation-emission matrix (EEM) spectroscopy and parallel factor (PARAFAC) analysis. The reactivity of their excited state with molecular oxygen was also probed, demonstrating that the Au16L14 clusters generate type I reactive oxygen species (ROS), which can make them effective sensitizers for photodynamic therapy.

Synergistic effect of ultrasound and light to efficient singlet oxygen formation for photodynamic purposes

As cancer is one of the leading causes of death globally, it is highly desirable to explore the therapeutic potential of new sensitizers in photodynamic therapy (PDT). In recent years, PDT applications performed with the help of ultrasound have attracted attention. Also, the importance of phthalocyanines, which are used as sensitizers in photodynamic and/or sonodynamic therapy (SDT), is increasing. The most important properties of phthalocyanines for therapeutic applications are their ability to generate reactive oxygen species (ROS) at the appropriate wavelength. To improve ROS production by phthalocyanines applying ultrasounds seems to be the reasonable pathway of development for this treatment method. In this context, to evaluate potentials for sonophotodynamic therapy (SPDT), the syntheses of highly soluble non-peripherally substituted metal-free (2), gallium (3), and indium (4) phthalocyanines were completed by substituting 3-methoxybenzyloxy groups. All prepared compounds were elucidated using many different spectroscopic approaches encompassing Fourier transform infrared (FT-IR), ultraviolet–visible (UV–Vis), 1H NMR, and matrix-assisted laser deionization/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) techniques. Additionally, photophysical, photochemical, and sono-photochemical evaluations of the novel phthalocyanines are presented. The combination of ultrasound and light had an enhancing effect on singlet oxygen production in all phthalocyanine derivatives (2–4). Singlet oxygen quantum yields increased between 50% and 80% for metal-free, gallium, and indium phthalocyanines. Density Functional Theory (DFT) and time-dependent (TD-DFT) were used for the description of optical and electronic properties of metal-free, gallium, and indium phthalocyanines. The Spin Orbital Coupling Constant (SOCC) for these molecules was also calculated through the quasi-degenerate perturbation theory method. Considering the singlet and triplet states of lower energy, we have calculated the most probable intersystem spin crossing (ISC) pathways. Our results suggest that all the phthalocyanines have potential application in PDT, however, indium phthalocyanine has greater applicability in PDT.

Development of Meso‐Substituted Heterocyclic BODIPY‐Based Polymeric Nanoparticles for Pathogen Inhibition using Photodynamic Therapy**

AbstractIn this work, a series of novel small fluorophores, meso‐substituted heterocyclic BODIPYs were designed and synthesized as phototherapeutic agents. Boron dipyrromethene (BODIPY) dyes are promising chromophores and currently used as a sensitizers in photodynamic therapy (PDT). However, BODIPY as an organic photosensitizer (PS), suffers from poor hydrophilicity resulting in low efficiency in biological applications. To overcome these problems, biopolymer (gelatin) based nanoparticles are developed to incorporate BODIPYs, to achieve phototherapeutic nanoagents (BODIPY‐GNPs) in the aqueous system. More importantly, BODIPY‐GNPs have better in vitro physicochemical properties in the aqueous medium than native BODIPYs. The BODIPY‐GNPs were further explored for their photo‐inhibition activity towards Gram‐positive and Gram‐negative bacteria and fungi using LED as the light source. These BODIPY molecules also showed the ability to intercalate the DNA in vitro. This study provides a new avenue to design therapeutic agents for biomedical applications in photodynamic therapy.

Theoretical analysis of expanded porphyrins: Aromaticity, stability, and optoelectronic properties

Expanded porphyrin systems are capable of binding a variety of substrates due to their increased cavity size and aromatic nature, holding important applications as magnetic resonance imaging contrast agents and as sensitizers for photodynamic therapy. It is there of fundamental interest to know the photoelectrical properties of expanded porphyrins using quantum chemistry calculations. In this work, we theoretically designed and screened a series of expanded porphyrins by incorporating terthiophene (TTH) and dithienothiophene (DTT) moieties. Our calculations showed that all the designed molecules exhibit excellent optoelectronic performance than the reference molecule. It is suggested that the porphyrin molecule with TTH moiety has better stability than the one with DTT moiety. Finally, we demonstrated that molecule 2 features with TTH moiety and the inverted selenophene ring outperform other molecules because it exhibits increased HOMO-LUMO gap, planar geometry, and strengthened aromaticity. We expect that this work can provide theoretical guidelines for the design of novel porphyrin materials.