Inverse Electron Demand Diels-Alder Reactions in the Liposomal Membrane Accelerates Release of the Encapsulated Drugs.

Bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reactions between liposomes containing a tetrazine-based (Tz) compound and 2-norbornene (2-NB) could be a novel trigger for accelerating drug release from the liposomes via temporary membrane destabilization, as shown in our previous report. Herein, we evaluated the in vitro drug release using NB derivatives with carboxyl groups [5-norbornene-2-carboxylic acid (NBCOOH) and 5-norbornene-2,3-dicarboxylic acid (NB(COOH)2)] to investigate the effects of substituents at the NB backbone on the drug release rate. First, POTz-liposome composed of a Tz compound (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) were prepared. The mass spectrometry analysis revealed the binding of NB derivatives to the Tz compound via the IEDDA reaction after the POTz-liposome reacted with the NB derivatives. Indium-111-labeled diethylenetriaminepentaacetic acid (111In-DTPA) was encapsulated inside the liposomes, and the drug release rate was quantified by measuring radioactivity. At 24 h after incubation with 2-NB, NBCOOH, and NB(COOH)2, the release rates of 111In-DTPA from POTz-liposome were 21.0, 80.8, and 23.3%, respectively, which were significantly higher than those of POTz-liposome that was not treated with NB derivatives (4.2%), indicating the involvement of the IEDDA reaction for prompting drug release. Additionally, a thermodynamic evaluation using Langmuir monolayers was conducted to explore the mechanism of the accelerated drug release. An increase in membrane fluidity and a reduction in intermolecular repulsion between POPC and the Tz compound were observed after the reaction with NB derivatives, especially for NBCOOH. Thus, the IEDDA reaction in the liposomal membrane could be a potent trigger for accelerating the release of encapsulated drugs by regulating membrane fluidity and intermolecular repulsion in the liposomal membrane.

Specific interaction restrains structural transitions of an amphiphilic peptide in pulmonary surfactant model systems: an in situ PM-IRRAS investigation.

In situ polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) at the air-water interface has been used to determine secondary structure of the pulmonary surfactant model peptide, Hel 13-5, in the absence and the presence of phospholipid monolayers. Herein, fully saturated phospholipids of DPPC and DPPG are utilized to understand the effect of specific interaction between anionic DPPG and cationic Hel 13-5 on the peptide secondary structure. The spectrum frequency in the amide region (1500-1700cm(-1)) obtained from PM-IRRAS has been confirmed by comparing with that from ATR-FTIR for the corresponding bulk films. The PM-IRRAS spectra of single Hel 13-5 monolayers indicate the alpha-helical contour in the amide region, which coincides with the result from CD measurements in aqueous solutions. In the presence of phospholipid monolayers, however, Hel 13-5 changes its conformation from the alpha-helix to the extended beta-sheet as surface pressure increases upon compression at the interface, and this interconversion is found to be irreversible even during expansion process of monolayers. Furthermore, it is notable that the electrostatic interaction between DPPG and Hel 13-5 inhibits to some extent the interconversion to the beta-sheet during compression. These features are completely different from the bulk behavior, which demonstrates different roles of native proteins in the bulk phase and at the interface for pulmonary functions. In addition, the conformational variation of Hel 13-5 does not indicate close correlation with surface activity, which is common characteristic even for reversible hysteresis curves in pulmonary surfactant systems. This suggests that the secondary structure of native proteins is not strongly related to the surface activity during respiration. This work contributes to secondary structure determination of Hel 13-5 in the phospholipid domains in situ at the air-water interface and will provide insight into the molecular and physiological mechanism for SP-B and SP-C actions across the interface.

Fluorocarbon-hybrid pulmonary surfactants for replacement therapy--a Langmuir monolayer study.

Effective additives to pulmonary surfactant (PS) preparations for therapy of respiratory distress syndrome (RDS) are being intensively sought. We report here the investigation of the effects of partially fluorinated amphiphiles (PFA) on the surface behavior of a model PS formulation. When small amounts of a partially fluorinated alcohol C(8)F(17)C(m)H(2m)OH (F8HmOH, m = 5 and 11) are added to the PS model preparation (a dipalmitoylphosphatidylcholine (DPPC)/Hel 13-5 peptide mixture) considered here, the effectiveness of the latter in in vitro pulmonary functions is enhanced. The mechanism for the improved efficacy depends on the hydrophobic chain length of the added PFA molecules. The shorter PFA, F8H5OH, when incorporated in the monolayer of the PS model preparation, promotes a disordered liquid-expanded (LE) phase upon lateral compression (fluidization). In contrast, the addition of the longer PFA, F8H11OH, reduces the disordered LE/ordered liquid-condensed (LC) phase transition pressure and promotes the growth of ordered domains (solidification). Furthermore, compression-expansion cycles suggest that F8H5OH, when incorporated in the PS model preparation, undergoes an irreversible elimination into the subphase, whereas F8H11OH enhances the squeeze-out phenomenon of the SP-B mimicking peptide, which is important in pulmonary functions and is related to the formation of a solid-like monolayer at the surface and of a surface reservoir just below the surface. F8H11OH particularly reinforces the effectiveness of DPPC in terms of minimum reachable surface tension, and of preservation of the integrated hysteresis area between compression and expansion isotherms, the two latter parameters being generally accepted indices for assessing PS efficacy. We suggest that PFA amphiphiles may be useful potential additives for synthetic PS preparations destined for treatment of RDS in premature infants and in adults.

Water evaporation rates across hydrophobic acid monolayers at equilibrium spreading pressure.

The effect of alkanoic acid [CH(3)(CH(2))(n-2)COOH; HCn] and perfluoroalkanoic acid [CF(3)(CF(2))(n-2)COOH; FCn] monolayers on the water evaporation rate was investigated by thermogravimetry tracing the decrease in amount of water with time. The evaporation rate from the surface covered by a monolayer was measured as a function of temperature and hydrophobic chain length of the acids, where the monolayer was under an equilibrium spreading pressure. From thermal behavior of the crystallized acids, their solid states are C-type in crystalline state over the temperature range from 298.2 to 323.2 K. The dry air was flowed through a furnace tube of a thermogravimetry apparatus at the flow rate of 80 mL min(-1), where the evaporation rate becomes almost constant irrespective of the flow rate. The temperature dependence of the evaporation rate was analyzed kinetically to evaluate the activation energy and thermodynamics values for the activated complex, which demonstrated that these values were almost the same for both alkanoic acids and perfluoroalkanoic acids, although the effect of perfluoroalkanoic acids on the evaporation rate was smaller than that of corresponding hydrogenated fatty acids. The difference in the evaporation rate between FCn and HCn was examined by atomic force microscopy (AFM), Brewster angle microscopy (BAM), surface potential (DeltaV) at equilibrium spreading pressure, and Langmuir curve (pi-A isotherm), and their results were consistent and supported the difference.

Monolayers (Langmuir films) behavior of multi-component systems composed of a bile acid with different sterols and with their 1:1 mixtures.

Different physicochemical properties of Langmuir films (monolayers) composed of 10 mixed systems of a bile acid, deoxycholic acid (DC) with various plant sterols, such as stigmasterol (Stig), beta-sitosterol (Sito) and campesterol (Camp) and a stanol, cholestanol (Chsta) in addition to an animal sterol, cholesterol (Ch) [these sterols and Chsta are abbreviated as St] and DC with 1:1 St mixtures; (Ch+Chsta), (Ch+Stig), (Stig+Chsta), (Ch+Sito) and (Ch+Camp) on the substrate of 5M aqueous NaCl solution (pH 1.2) at 25 degrees C, were investigated in terms of mean surface area per molecule (A(m)), the partial molecular area (PMA), surface excess Gibbs energy (DeltaG((ex))), interaction parameter (I(p)) as well as activity coefficients (f(1) and f(2)) in 2-D phase of each binary (or ternary) component system and elasticity (Cs(-1)) of formed films; these were analyzed on the basis of the respective surface pressure (pi) versus A(m) isotherms as a function of mole fraction of Sts (X(st)) in the DC/St(s) mixtures at discrete surface pressures. Notable findings are: (i) all the binary component systems did form patched film type monolayers consisting of (a) DC-dominant film solubilizing a trace amount of St molecules and (b) St dominant film dissolving a small amount of DC molecules, (ii) DC in 2-D phase exhibited a transition from LE film to LC film at a constant pressure (pi(C)(1)) accompanied by compression and (iii) DeltaG((ex)) as well as I(p) was found to be greatly dependent on (a) the combinations of DC with different St species and (b) to be markedly varied by a difference in mixing ratio of DC to Sts. Compressibility (or elasticity) analyses and fluorescence microscopy images could support the above findings as well as interpretation.

Langmuir monolayer properties of the fluorinated-hydrogenated hybrid amphiphiles with dipalmitoylphosphatidylcholine (DPPC).

Surface pressure-area (pi-A), surface potential-area (DeltaV-A), and dipole moment-area (mu( perpendicular)-A) isotherms were obtained for the Langmuir monolayer of two fluorinated-hydrogenated hybrid amphiphiles (sodium phenyl 1-[(4-perfluorohexyl)-phenyl]-1-hexylphosphate (F6PH5PPhNa) and (sodium phenyl 1-[(4-perfluorooctyl)-phenyl]-1-hexylphosphate (F8PH5PPhNa)), DPPC and their two-component systems at the air/water interface. Monolayers spread on 0.02 M Tris buffer solution (pH 7.4) with 0.13M NaCl at 298.2K were investigated by the Wilhelmy method, ionizing electrode method and fluorescence microscopy. Moreover, the miscibility of two components was examined by plotting the variation of the molecular area and the surface potential as a function of the molar fraction for the fluorinated-hydrogenated hybrid amphiphiles on the basis of the additivity rule. The miscibility of the monolayers was also examined by construction of two-dimensional phase diagrams. Furthermore, assuming the regular surface mixture, the Joos equation for analysis of the collapse pressure of two-component monolayers allowed calculation of the interaction parameter (xi) and the interaction energy (-Deltaepsilon) between the fluorinated-hydrogenated hybrid amphiphiles and DPPC. The observations by a fluorescence microscopy also supported our interpretation as for the miscibility in the monolayer state. Comparing the monolayer behavior between the two binary systems, no remarkable difference was found among various aspects. Among the two combinations, the mole fraction dependence in monolayer properties was commonly classified into two ranges: 0

Properties of two-component Langmuir monolayer of single chain perfluorinated carboxylic acids with dipalmitoylphosphatidylcholine (DPPC).

The surface pressure (pi)- and the surface potential (DeltaV)-area (A) isotherms were obtained for two-component monolayers of four different perfluorocarboxylic acids (FCns; perfluorododecanoic acid: FC12, perfluorotetradecanoic acid: FC14, perfluorohexadecanoic acid: FC16, perfluorooctadecanoic acid: FC18) with dipalmitoylphosphatidylcholine (DPPC) on substrate solution of 0.15 M NaCl (pH 2.0) at 298.2 K as a function of compositions in the mixtures by employing the Wilhelmy method, the ionizing electrode method, the fluorescence microscopy, and the atomic force microscopy. The data for the two-component monolayers on these systems were analyzed in terms of the additivity rule. Assuming a regular surface mixture, the Joos equation which allows one to describe the collapse pressure of a two-component monolayer with miscible components was used to declare the miscibility of the monolayer state, and an interaction parameter and an interaction energy were calculated. The new finding was that FCns and DPPC are miscible or immiscible depending on chain length increment of fluorocarbon part. That is, FC12/DPPC monolayer was perfectly miscible, and FC14/DPPC, and FC16/DPPC (0 < or = X(FC16) < or = 0.3) monolayers were partially miscible. While FC16/DPPC (0.3 < X(FC16) < 1) and FC18/DPPC systems are immiscible in the monolayer state. Furthermore, the mean molecular area, the surface dipole moment, and the phase diagrams enabled us to estimate the molecular orientation of four different perfluorocarboxylic acids/DPPC in the two-component monolayer state. One type of phase diagrams was obtained and classified into the positive azeotropic type. The miscibility of FCns and DPPC in the monolayer was also supported by fluorescence microscopy and atomic force microscopy. FC12/DPPC, FC14/DPPC and FC16/DPPC (0 < or = X(FC16) < or = 0.3) two-component monolayers on 0.15 M NaCl (pH 2) showed that FC12, FC14 and FC16 (0 < or = X(FC16) < or = 0.3) can dissolve or partially dissolve the ordered solid DPPC domains formed upon compression. This indicates that these fluorinated amphiphiles soften or harden the lipid depending on their chain length.

Cerebroside Langmuir monolayers originated from the echinoderms I. Binary systems of cerebrosides and phospholipids.

The surface pressure (pi)-area (A), the surface potential (DeltaV)-A and the dipole moment (mu( perpendicular))-A isotherms were obtained for two-component monolayers of two different cerebrosides (LMC-1 and LMC-2) with phospholipids of dipalmitoylphosphatidylcholine (DPPC) and with dipalmitoylphosphatidylethanolamine (DPPE) on a subphase of 0.5 M sodium chloride solution as a function of phospholipid compositions by employing the Langmuir method, the ionizing electrode method, and the fluorescence microscopy. Surface potentials (DeltaV) of pure components were analyzed using the three-layer model proposed by Demchak and Fort. The contributions of the hydrophilic saccharide group and the head group to the vertical component of the dipole moment (mu( perpendicular)) were estimated. The miscibility of cerebroside and phospholipid in the two-component monolayers was examined by plotting the variation of the molecular area and the surface potential as a function of the phospholipid molar fraction (X(phospholipid)), using the additivity rule. From the A-X(phospholipid) and DeltaV(m)-X(phospholipid) plots, partial molecular surface area (PMA) and apparent partial molecular surface potential (APSP) were determined at the discrete surface pressure. The PMA and APSP with the mole fraction were extensively discussed for the miscible system. Judging from the two-dimensional phase diagrams, these can be classified into two types. The first is a positive azeotropic type; the combinations of cerebrosides with DPPC are miscible with each other. The second is a completely immiscible type: the combination of cerebrosides with DPPE. Furthermore, a regular surface mixture, for which the Joos equation was used for the analysis of the collapse pressure of two-component monolayers, allowed calculation of the interaction parameter (xi) and the interaction energy (-Delta epsilon) between the cerebrosides and DPPC component. The miscibility of cerebroside and phospholipid components in the monolayer state was also supported by fluorescence microscopy.

Cerebroside Langmuir monolayers originated from the echinoderms: II. Binary systems of cerebrosides and steroids.

Two-component Langmuir monolayers formed on a subphase of 0.5M sodium chloride solution were investigated for two different cerebrosides (LMC-1 and LMC-2) with steroids of cholesterol (Ch) and cholesteryl sodium sulfate (Ch-S); i.e. LMC-1/Ch, LMC-1/Ch-S, LMC-2/Ch, and LMC-2/Ch-S were examined in terms of surface pressure (pi), the surface potential (DeltaV) and the dipole moment (mu( perpendicular)) as a function of surface area (A) by employing the Langmuir method, the ionizing electrode method, and the fluorescence microscopy. Surface potentials (DeltaV) of steroids were analyzed using the three-layer model proposed by Demchak and Fort. The miscibility of cerebrosides and steroids in the insoluble monolayers was examined by plotting the variation of the molecular area and the surface potential as a function of the steroid molar fraction (X(steroid)) based upon the additivity rule. From the A-X(steroid) and DeltaV(m)-X(steroid) plots, partial molecular surface area (PMA) and apparent partial molecular surface potential (APSP) were determined at the different surface pressures. The PMA and APSP with the mole fraction were discussed for the miscible system. Judging from the two-dimensional phase diagrams, they can be classified into two types. The first is a completely immiscible type; the combination of cerebrosides with cholesterol. The second is a negative azeotropic type, where cerebrosides and cholesteryl sodium sulfate are completely miscible both in the expanded state and in the condensed state. In addition, a regular surface mixture (the Joos equation for the analysis of the collapse pressure of two-component monolayers) allowed calculation of the interaction parameter (xi) and the interaction energy (-Delta epsilon) between the cerebrosides and Ch-S. The miscibility of cerebroside and steroid components in the monolayer state was also supported by fluorescence microscopy.

New adsorption model -- theory, phenomena and new concept.

According to the conventional Gibbs adsorption model, which is a common assumption about the molecular concentration at surfaces, the adsorbed film of soluble amphiphiles is located at the air/solution interface just like Langmuir monolayer which is illustrated in many physical chemistry text books on "Colloid and Interface Science". According to many proofs of the experimental results here, the newer idea for the surface adsorption is confirmed and explained, which is quite different from the conventional Gibbs surface excess model at the air/solution interface.

Two-component Langmuir monolayers and LB films of DPPC with partially fluorinated alcohol (F8H9OH).

The interaction of (perfluorooctyl)nonanol (F8H9OH) with dipalmitoylphosphatidylcholine (DPPC) was systematically studied in two-component monolayers at air-water interface. The thermodynamic property and phase morphology of the monolayers were investigated by isotherm measurements and several microscopic methods such as Brewster angle microscopy, fluorescence microscopy, and atomic force microscopy (AFM). The AFM topographies for Langmuir-Blodgett films of F8H9OH exhibit the formation of monodispersed surface micelles. In the two-component system, the incorporation of F8H9OH induces condensation (or solidification) of DPPC monolayers. The excess Gibbs free energy and interaction parameter (or energy) of the two components were calculated from the isotherm data. Both the phase transition pressure for the coexistence of ordered and disordered phases and collapse pressure of monolayers vary with the mole fraction of F8H9OH, indicating binary miscibility between F8H9OH and DPPC within a monolayer state. The miscibility is also confirmed visually by in situ and ex situ microscopy at micro- and nanometer scales.

Monolayer compression induces fluidization in binary system of partially fluorinated alcohol (F4H11OH) with DPPC.

A two-component Langmuir monolayer consisting of (perfluorobutyl)undecanol (F4H11OH) and dipalmitoylphosphatidylcholine (DPPC), a major component of pulmonary surfactants in mammals, has been investigated at the air-water interface. The binary monolayer has been systematically examined from both thermodynamic and morphological perspectives. The excess Gibbs free energy of mixing has been calculated from surface pressure (π)-molecular area (A) isotherms, and the results indicate that the miscibility of the two-component system shows a maximum in thermodynamical stability when the mole fraction (X(F4H11OH)) is 0.3. Results from a two-dimensional phase diagram (π vs. X(F4H11OH)) are consistent with these findings and depict the degree of miscibility resulting from the variation in the transition and collapse pressures relative to the concentration of X(F4H11OH). The miscibility is also supported by in situ Brewster angle microscopy and fluorescence microscopy, as well as ex situ atomic force microscopy for the system after transfer onto a mica substrate. Aside from temperature, a known driving force for the fluidization of DPPC monolayers is a change in surface composition caused by the addition of additive molecules. In the present study, however, the fluidization is driven by increasing surface pressures even at constant X(F4H11OH). Such a fluidization is a fascinating property when looked at in context of its potential implications for pulmonary replacement therapy, and hence, this study provides a fundamental insight into designing fluorinated materials for biomedical use.

Langmuir monolayer miscibility of perfluorocarboxylic acids with biomembrane constituents at the air-water interface.

Over the past two or three decades, Langmuir monolayers comprising various fluorinated amphiphiles such as perfluorinated, partially fluorinated, and fluorinated-hydrogenated hybrid molecules at the air-water interface, have been investigated to deepen the understanding of their characteristic phenomena and to explore the mutual interaction of these amphiphiles with biomembrane constituents. Langmuir monolayers of these fluorinated amphiphiles are potentially applicable in the fields of materials, biological and clinical science, where they may be utilized in two-dimensional protein crystallization, microelectronics, and for surfactant replacement in the lung. This review article focuses on the miscibility between perfluorinated long-chain carboxylic acids (CF3(CF2)(n-1)COOH, or FnCOOH) and phospholipids or fatty acids in biomembranes upon lateral compression, and describes how the chain-length mismatch between the fluorinated and the hydrogenated tails relates to the interfacial phase behavior from the thermodynamic and the morphological aspects at the micrometer and nanometer levels.

Miscibility and phase behavior of DPPG and perfluorocarboxylic acids at the air-water interface.

The miscibility and phase behavior of two components of phospholipids and perfluorocarboxylic acids [FCn; perfluorododecanoic acid (FC12), perfluorotetradecanoic acid (FC14), perfluorohexadecanoic acid (FC16), and perfluorooctadecanoic acid (FC18)] have been systematically investigated using Langmuir monolayer technique. Dipalmitoylphosphatidylglycerol (DPPG) is utilized as a phospholipid component in biomembranes. Surface pressure (pi)-molecular area (A) and surface potential (DeltaV)-A isotherms have been measured for the DPPG/FCn systems on 0.15 M NaCl (pH 2.0) at 298.2K. From the isotherm results, two-dimensional phase diagrams are constructed and classified into miscible and immiscible patterns. Furthermore, the phase behavior of the DPPG/FCn systems has been morphologically examined using fluorescence microscopy (FM) and atomic force microscopy (AFM). These images indicate different phases among the four systems. In particular, specific phase morphology is observed in the middle molar fraction range for the DPPG/FC14 system; FC14 is selectively excluded from mixed DPPG-FC14 monolayers to be concentrated in the phase boundary as surface pressure increases. Then DPPG is refined as a patched film. Moreover, the data obtained here are compared to those in the previous systems in which different kinds of phospholipids were treated. Through a series of the miscibility investigations, it is proposed that combinations of hydrophobic chain lengths and of polar headgroups contribute to the monolayer miscibility between phospholipids and perfluorocarboxylic acids.

Two-component Langmuir monolayers of single-chain partially fluorinated amphiphiles with dipalmitoylphosphatidylcholine (DPPC).

The surface pressure (pi)-area (A) and surface potential (DeltaV)-A isotherms were measured for two-component monolayers made of dipalmitoylphosphatidylcholine (DPPC)/single-chain (perfluorooctyl)pentanol (F8C5OH) and DPPC/single-chain (perfluorooctyl)pentylphosphocholine (F8C5PC) on a substrate solution of 0.15 M NaCl at 293.2 K as a function of the composition of the two components. The Langmuir method and the ionizing electrode method were used. The data for these systems were analyzed using an additivity rule. Assuming a regular surface mixture, the Joos equation, which allows description of the collapse pressure of a monolayer made of two miscible components, was used to establish the miscibility within the monolayer. An interaction parameter and an interaction energy were calculated. The two-component DPPC/F8C5OH and DPPC/F8C5PC monolayers were miscible. Furthermore, the mean molecular area, surface potential, and phase diagrams enabled us to determine the molecular orientation of DPPC/F8C5OH and DPPC/F8C5PC in the monolayer. Two types of phase diagrams were obtained and classified into the positive azeotropic and negative azeotropic types. Fluorescence microscopy (FM) and Brewster angle microscopy (BAM) for the DPPC/F8C5OH and DPPC/F8C5PC systems show that both systems can dissolve the ordered micrometer-size solid DPPC domains. However, morphological analyses using atomic force microscopy (AFM) suggest partial miscibility or phase separation for DPPC and the partially fluorinated compounds on the nanometer scale. In particular, triskelion-shaped domains were evidenced for F8C5OH. These results indicate that the partially fluorinated amphiphiles investigated here fluidize the DPPC monolayer upon lateral compression and that these chemicals may be useful to develop their innovative applications in the biomedical field.

Solubilization of n-alkylbenzenes into octaethylene glycol mono-n-tetradecyl ether (C14E8) micelles.

Solubilization of benzene, toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, and n-pentylbenzene into the micelles of octaethylene glycol monotetradecyl ether (C(14)E(8)) was studied, where equilibrium concentrations of all the solubilizates were determined spectrophotometrically at 298.2, 303.2, and 308.2 K. The concentration of the above solubilizates except benzene remained constant below the critical micelle concentration (cmc) and increased linearly with an increase in C(14)E(8) concentration above the cmc, whereas benzene concentration was found to remain constant over the whole concentration range of C(14)E(8). The Gibbs energy change (DeltaG(0)) for their solubilization was evaluated by the partitioning of the solubilizates between the aqueous phase and the micellar phase because of the large aggregation number of the C(14)E(8) micelle. Furthermore, enthalpy and entropy changes for their solubilization were evaluated from the temperature dependence of the DeltaG(0) values. From these thermodynamical parameters and the change in absorption spectra of the solubilizates due to their incorporation into the micelles, the solubilization site was found to move into the inner core of the micelle with increasing alkyl chain length of the solubilizates.