Surface Of Nanomaterials Research Articles

In situ oxidative polymerization of platinum(iv) prodrugs in pore-confined spaces of CaCO3 nanoparticles for cancer chemoimmunotherapy.

Drug resistance and metastases are the leading causes of death in clinics. To overcome this limitation, there is an urgent need for new therapeutic agents and drug formulations that are able to therapeutically intervene by non-traditional mechanisms. Herein, the physical adsorption and oxidative polymerization of Pt(iv) prodrugs in pore-confined spaces of CaCO3 nanoparticles is presented, and the nanomaterial surface was coated with DSPE-PEG2000-Biotin to improve aqueous solubility and tumor targeting. While the nanoparticle scaffold remained stable in an aqueous solution, it quickly degraded into Ca2+ in the presence of acid and into cisplatin in the presence of GSH. The nanoparticles were found to interact in cisplatin-resistant non-small lung cancer cells by a multimodal mechanism of action involving mitochondrial Ca2+ overload, dual depletion of GSH, nuclear DNA platination, and amplification of ROS and lipid peroxide generation, resulting in triggering cell death by a combination of apoptosis, ferroptosis and immunogenic cell death in vitro and in vivo. This study could present a novel strategy for the treatment of drug-resistant and metastatic tumors and therefore overcome the limitations of currently used therapeutic agents in the clinics.

Molecular properties controlling chirality transfer to halide perovskite: computational insights

Computationally investigate chirality transfer effect from chiral ligands bound to the surface of a lead-halide perovskite nanomaterial. By changing the molecular polarity of the chiral molecules, the chirality of the system can be tuned.

In Ukrainian

Metal composites modified with various heteroatoms, such as N, B, Si, are used to obtain matrix composites with specified parameters with the strongest adhesive-cohesive bonds between metal atoms and a carbon nanoparticle. Such carbon nanoparticles functionalized with heteroatoms are promising for many metal composites. One of the interesting and promising metals as a matrix for such research work is iron. To predict the specifics of the interaction of iron with the surface of carbon nanomaterials supplemented with heteroatoms of different chemical structure, it is advisable to model such processes using quantum chemistry methods. The aim of the work was to find out the effect of temperature on the chemical interaction of iron clusters with native, boron-, silicon-, and nitrogen-containing graphene-like planes (GLP). The results of the calculations show that the highest value of the energy effect of the chemical interaction for the native graphene-like plane is +204.3 kJ/mol, in the case of calculations both by the B3LYP/6-31G(d,p) method and by the MP2/6-31G(d, p) (+370.7 kJ/mol). The lower value of the energy effect is found in the presence of nitrogen atoms in the composition of the graphene-like plane. This value is even lower for the interaction of iron dimers with a silicon-containing carbon nanocluster. The lowest values of the energy effect, calculated by both methods, are characteristic of the boron-containing graphene-like plane. In particular, for the B3LYP/6-31G(d,p) method, the value of the energy effect of the reaction is ‑210.5 kJ/mol, and for the MP2/6-31G(d,p) method this value is +16.6 kJ/mol. The presence of boron atoms in the composition of the nanocarbon matrix best contributes to the interaction with the iron nanocluster, regardless of the chosen research method. The dependence curves of the Gibbs free energy of the interaction of iron dimers with a graphene-like plane and its derivatives in all cases qualitatively correlate with similar energy effects. In addition, in all cases, the values of the Gibbs free energy increase with increasing temperature.

In English

It is known that the addition of a small amount of carbon nanomaterials significantly improves the mechanical properties of composites with a metal matrix. One of the most important, promising and available metals as a matrix for such modification is aluminum. However, at the interface between the carbon material and Al, aluminum carbides of different composition are formed, which are brittle and have the main disadvantage - solubility in water. Therefore, the appearance of aluminum carbide is a serious problem, since it contributes to the formation of defects, which, when the composite is deformed, leads to cracking of the composite due to the presence of microneedles. In this regard, in order to predict the features of the interaction of aluminum itself with the surface of carbon nanomaterials, it is advisable to model such processes using quantum chemistry methods. The aim of the work was to reveal the effect of temperature on the chemical interaction of aluminum clusters with native, boron-, silicon-, and nitrogen-containing graphene-like planes (GLP). All the calculated by three methods (B3LYP/6-31G(d,p), MP2/6-31G(d,p) and PВЕ0/6-31G(d,p)) values of the dependence of the Gibbs free energy on temperature for different cluster sizes of aluminum and graphene-like clusters are the highest for native graphene-like planes. In all cases, the values of the Gibbs free energy increase with temperature. The lowest values of the temperature dependence of the Gibbs free energy vary as dependent on the size of the reactant models and research methods, this is especially characteristic of the presence of boron and silicon atoms in the graphene-like clusters. Therefore, the absence of heteroatoms in the composition of the nanocarbon matrix contributes to the fact that aluminum carbide islands should not be formed in the carbon-containing nanocomposite with aluminum, which negatively affects the physical and chemical characteristics of the resulting nanocomposite.

Assembled Porphyrin Nanofiber on the Surface of g-C3N4 Nanomaterials for Enhanced Photocatalytic Degradation of Organic Dyes

In this work, a g-C3N4/porphyrin nanocomposite was fabricated through the self-assembling of monomeric Tetrakis (4-carboxyphenyl) porphyrin (TCPP) molecules with g-C3N4 nanomaterials. The characterizing results showed a good distribution of TCPP nanofibers with a diameter of < 100 nm and several micrometers in length on the g-C3N4 nanoflakes’ surfaces. The prepared g-C3N4/porphyrin nanocomposite had two bandgap energies of 2.38 and 2.7 eV, which could harvest a wide range of photon energy in the light spectrum, particularly in visible light. The obtained C3N4/TCPP nanocomposite revealed a remarkable photodegradation efficiency toward rhodamine B dyes, with a RhB removing rate of 3.3 × 10−2 min−1. The plausible mechanism for the photocatalytic performance of the g-C3N4/porphyrin photocatalyst for the RhB dye’s degradation was also studied and discussed.

Effective Antibacterial/Photocatalytic Activity of ZnO Nanomaterials Synthesized under Low Temperature and Alkaline Conditions.

The purpose of this study was to evaluate the surface properties of ZnO nanomaterials based on their ability to photodegrade methyl blue dye (MB) and to show their antibacterial properties against different types of Gram-positive bacteria (Bacillus manliponensis, Micrococcus luteus, Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). In this study, ZnO nanomaterials were synthesized rapidly and easily in the presence of 1-4 M NaOH at a low temperature of 40 °C within 4 h. It was found that the ZnO nanomaterials obtained from the 1.0 M (ZnO-1M) and 2.0 M (ZnO-2M) aqueous solutions of NaOH had spherical and needle-shaped forms, respectively. As the concentration of NaOH increased, needle thickness increased and the particles became rod-like. Although the ZnO nanomaterial shapes were different, the bandgap size remained almost unchanged. However, as the NaOH concentration increased, the energy position of the conduction band shifted upward. Photo current curves and photoluminescence intensities suggested that the recombination between photoexcited electrons and holes was low in the ZnO-4M materials prepared in 4.0 M NaOH solution; however, charge transfer was easy. ∙O2- radicals were generated more than ∙OH radicals in ZnO-4M particles, showing stronger antibacterial activity against both Gram-positive and Gram-negative bacteria and stronger decomposition ability on MB dye. The results of this study suggest that on the ZnO nanomaterial surface, ∙O2- radicals generated are more critical for antibacterial activity than particle shape.

SnO2 nanostructured materials used as gas sensors for the detection of hazardous and flammable gases: A review

SnO 2 has been extensively used in the detection of various gases. As a gas sensing material, SnO 2 has excellent physical-chemical properties, high reliability, and short adsorption-desorption time. The application of the traditional SnO 2 gas sensor is limited due to its higher work-temperature, low gas response, and poor selectivity. Nanomaterials can significantly impact gas-sensitive properties due to the quantum size, surface, and small size effects of nanomaterials. By applying nanotechnology to the preparation of SnO 2 , the SnO 2 nanomaterial-based sensors could show better performance, which greatly expands the application of SnO 2 gas sensors. In this review, the preparation method of the SnO 2 nanostructure, the types of gas detected, and the improvements of SnO 2 gas-sensing performances via elemental modification are introduced as well as the future development of SnO 2 is discussed.

Construction of polyaniline/MnO2 core-shell nanocomposites in carbonized wood tracheids for high-performance all-solid-state asymmetric supercapacitors

Polyaniline (PANI) nanomaterials or carbon nanotubes (CNTs) are first synthesized on the tracheid wall of activated wood carbon (AWC) to obtain PANI@AWC and CNT@AWC, respectively. Then MnO2 nanosheets are electrodeposited on the outer surface of the PANI nanomaterials to form a core-shell structure. The introduction of PANI in wood tracheids not only increases the loading of MnO2, but also improves the electrical conductivity and structural stability, and the core-shell structure can synergistically store energy. Then using the MnO2/PANI@AWC composite electrode as an anode and using CNT@AWC as a cathode, a high-performance all-solid-state asymmetric supercapacitor with high energy density and high cycling stability is successfully constructed. This supercapacitor has a wide working voltage of 0–1.8 V and a high energy density of 0.875 mWh cm−2 at 9 mW cm−2. The capacitance retention rate is as high as 93.21 % after 10,000 charge-discharge cycles. Therefore, wood-based composites with aligned tracheids have great potential in the development of high-power and high-energy-density energy storage devices.

Rare earth fluorescent nanoprobes with minimal side effects enable tumor microenvironment activation for chemotherapy

Chemotherapy, the use of antitumor drugs to kill cancer cells, is currently one of the most effective treatments for cancer. However, serious toxic side effects caused by long-term drug accumulation can cause significant damage to the body, which limits the clinical application of antitumor drugs. In this study, a novel RENPs@DOX-Fe nanoprobe (NP) was constructed by coating the surface of rare earth nanomaterials (NaLuF4:Yb,Er) with a complex formed by doxorubicin (DOX) and iron ion (III). Due to the low toxicity of anthracycline-metal complexes, the damage to normal cells is reduced. The unique acidic microenvironment in tumor cells facilitates the decomposition and gradual release of DOX from the DOX–Fe complex. In addition, the DOX–Fe complex can convert near-infrared (NIR) light into heat energy, which promotes the decomposition of the complex, further enhancing the release of DOX in the tumor environment. The change of ratio fluorescence of rare earth nanomaterials at 660 and 1550 nm after DOX release enables visual monitoring of drug release, which can potentially improve the chemotherapeutic effect. In vitro experiments established that RENPs@DOX-Fe NPs with NIR illumination had good therapeutic efficacy in tumors. This work provides new insights into designing tumor microenvironment-responsive nanoprobes for chemotherapy with minimal side effects.

Muscle cytotoxicity and immuno-reactivity analysis of the porous carbon nanospheres fabricated by high temperature calcination

Carbon-based nanomaterials have a high specific surface area, biocompatibility, and controlled mesopore structures. These characteristics make carbon nanospheres excellent carriers for drugs, biological dyes, photosensitizers, etc. Nevertheless, little is known about the impact of topological features on the surface of carbon nanomaterials on their in vivo immunoreactivity. In this study, we fabricated mesoporous carbon nanoparticles (MCNs) and solvent-processable carbon vesicles (CVs) by high-temperature calcination. The hematoxylin and eosin (H&E) staining suggested CVs' relatively poor dispersion capacity compared to MCNs and carbon precursors (CPs), leading to more severe muscle inflammation and necrosis. Immunostaining and Fluorescence Activated Cell Sorter (FACS) analysis further showed that both MCNs and CVs triggered a transient immune response in transplanted muscle and muscle-draining lymph nodes, but did not alter muscle resistance to exogenous viruses. In conclusion, this study provides insights into how carbon nanoparticles modulate the activation of immune responses in vivo.

The Antibacterial Properties of Nanocomposites Based on Carbon Nanotubes and Metal Oxides Functionalized with Azithromycin and Ciprofloxacin.

Different microorganisms are present in nature, some of which are assumed to be hazardous to the human body. It is crucial to control their continuing growth to improve human life. Nanomaterial surface functionalization represents a current topic in continuous evolution that supports the development of new materials with multiple applications in biology, medicine, and the environment. This study focused on the antibacterial activity of different nanocomposites based on functionalized multi-walled carbon nanotubes against four common bacterial strains. Two metal oxides (CuO and NiO) and two antibiotics (azithromycin and ciprofloxacin) were selected for the present study to obtain the following nanocomposites: MWCNT-COOH/Antibiotic, MWCNT-COOH/Fe3O4/Antibiotic, and MWCNT-COOH/Fe3O4/MO/Antibiotic. The present study included two Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Ciprofloxacin (Cip) functionalized materials (MWCNT-COOH/Fe3O4/Cip) were most efficient against all tested bacterial strains; therefore, we conclude that Cu and Ni reduce the effects of Cip. The obtained results indicate that the nanocomposites functionalized with Cip are more effective against selected bacteria strains compared to azithromycin (Azi) functionalized nanocomposites. The current work determined the antibacterial activities of different nanocomposites and gave fresh insights into their manufacture for future research regarding environmental depollution.

Highly permeable thin film nanocomposite membrane utilizing a MoS2@NH2-UiO-66 interlayer for forward osmosis removal of Co2+, Sr2+ and Cs+ nuclide ions

Novel MoS2@NH2-UiO-66 thin-film nanocomposites with interlayer (TFNi) membranes were developed to improve forward osmosis (FO) membrane performance. The prepared TFNi membrane shows a high water permeability of 38.13 ± 0.8 L m−2h−1 and low reverse salt flux (JS/JV) of 0.038 when using 2.0 mol/L sodium chloride as the draw solution (DS) and deionized water as the feed solution (FS) under AL-FS mode. This is attributed to MoS2@NH2-UiO-66 modifications of substrate properties, which fix m-phenylenediamine (MPD) on the nanomaterial surface, increase the polyamide (PA) layer crosslinking degree on the material surface, and allow trimesoyl chloride (TMC) to stay on the substrate surface during interfacial polymerization (IP) and prevent accumulation in the pores of the substrate. Therefore, the trade-off between permeability and selectivity of TFN membranes is overcome. The MoS2@NH2-UiO-66 TFNi membrane effectively removes Co2+, Sr2+ and Cs+ from aqueous solution via FO retention. When Co2+, Sr2+ and Cs+, nuclide ions with small hydration radii, are present in the FS at 20 mg/L, the retention rates are maintained at 99.6%, 99.7% and 97%, respectively, which are higher than those of existing commercial membranes. These results indicate the potential of MoS2@NH2-UiO-66-TFNi membranes for treatment of nuclide ions in wastewater.

Effect of surface and internal Bi0 on the performance of the Bi2WO6 photocatalyst

In recent years, nonprecious metallic Bi with the surface plasmon resonance (SPR) effect has attracted the attention of researchers in the field of photocatalysis and has been loaded onto the surface of various photocatalytic materials to study its effect on photocatalytic activity. In this work, the internal and surface of Bi2WO6 nanomaterials were modified by cleverly using the chelating and reducing properties of ethylene glycol (EG) to obtain photocatalytic materials with different metallic Bi modifications. A comprehensive analysis showed that modified Bi-BWO with a uniform distribution of metallic Bi was obtained by in situ etching on the surface of Bi2WO6 nanosheets, and the SPR effect of metallic Bi can be used to expand the light absorption range and improve the separation and migration efficiency of electrons and holes. Under simulated sunlight irradiation, the Bi-BWO sample achieved 100 % degradation of a Rhodamine B (RhB) solution within 30 min, which was 25 % higher than that of the unmodified sample. Bi0 inside the crystal structure caused lattice defects and served as a complex center for photogenerated carriers, which decreased the photocatalytic efficiency. This study investigated the modification of different parts of photocatalytic materials with metallic Bi to introduce contrasting effects, developed a solution for in situ etching on the surface of modified bismuth-based photocatalytic materials, and provided a new idea to improve photocatalytic materials with cavities as the main active species.

Recent Development of Nano-Carbon Material in Pharmaceutical Application: A Review.

Carbon nanomaterials have attracted researchers in pharmaceutical applications due to their outstanding properties and flexible dimensional structures. Carbon nanomaterials (CNMs) have electrical properties, high thermal surface area, and high cellular internalization, making them suitable for drug and gene delivery, antioxidants, bioimaging, biosensing, and tissue engineering applications. There are various types of carbon nanomaterials including graphene, carbon nanotubes, fullerenes, nanodiamond, quantum dots and many more that have interesting applications in the future. The functionalization of the carbon nanomaterial surface could modify its chemical and physical properties, as well as improve drug loading capacity, biocompatibility, suppress immune response and have the ability to direct drug delivery to the targeted site. Carbon nanomaterials could also be fabricated into composites with proteins and drugs to reduce toxicity and increase effectiveness in the pharmaceutical field. Thus, carbon nanomaterials are very effective for applications in pharmaceutical or biomedical systems. This review will demonstrate the extraordinary properties of nanocarbon materials that can be used in pharmaceutical applications.

Anti-HSV Activity of Metallic Nanoparticles Functionalized with Sulfonates vs. Polyphenols.

Metallic nanoparticles exhibit broad-spectrum activity against bacteria, fungi, and viruses. The antiviral activity of nanoparticles results from the multivalent interactions of nanoparticles with viral surface components, which result from the nanometer size of the material and the presence of functional compounds adsorbed on the nanomaterial surface. A critical step in the virus infection process is docking and entry of the virus into the host cell. This stage of the infection can be influenced by functional nanomaterials that exhibit high affinity to the virus surface and hence can disrupt the infection process. The affinity of the virus to the nanomaterial surface can be tuned by the specific surface functionalization of the nanomaterial. The main purpose of this work was to determine the influence of the ligand type present on nanomaterial on the antiviral properties against herpes simplex virus type 1 and 2. We investigated the metallic nanoparticles (gold and silver) with different sizes (5 nm and 30 nm), coated either with polyphenol (tannic acid) or sulfonates (ligands with terminated sulfonate groups). We found that the antiviral activity of nano-conjugates depends significantly on the ligand type present on the nanoparticle surface.

Advances in design of polymer brush functionalized inorganic nanomaterials and their applications in biomedical arena.

Grafting of polymer brush (assembly of polymer chains tethered to the substrate by one end) is emerging as one of the most viable approach to alter the surface of inorganic nanomaterials. Inorganic nanomaterials despite their intrinsic functional superiority, their applications remain restricted due to their incompatibility with organic or biological moieties vis-à-vis agglomeration issues. To overcome such a shortcoming, polymer brush modified surfaces of inorganic nanomaterials have lately proved to be of immense potential. For example, polymer brush-modified inorganic nanomaterials can act as efficient substrates/platforms in biomedical applications, ranging from drug-delivery to protein-array due to their integrated advantages such as amphiphilicity, stimuli responsiveness, enhanced biocompatibility, and so on. In this review, the current state of the art related to polymer brush-modified inorganic nanomaterials focusing, not only, on their synthetic strategies and applications in biomedical field but also the architectural influence of polymer brushes on the responsiveness properties of modified nanomaterials have comprehensively been discussed and its associated future perspective is also presented. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Successive cytotoxicity control by evolutionary surface decorated electronic push-pull green ZnCr-LDH nanostructures: Drug delivery enlargement for targeted breast cancer chemotherapy

The reason for the increasing bioavailability and biocompatibility of the porous nanomaterials in the presence of different (bio)molecules is still unknown. The role of difference functional groups and their interactions with the potential bioavailability and biocompatibility is of great importance. To investigate the potential contribution of the electronic effects (especially on the surface of the porous nanomaterials) on their biomedical behavior, a series of surface-decorated green ZnCr-layered double hydroxide (LDH) porous nanocarriers is developed as a non-viral vector. Different conjugations investigated these porous LDHs for optimizing and minimizing the cytotoxicity for targeted breast cancer therapy. Quick low-temperature synthesized ZnCr-LDH nanocarriers method with enlarged drug delivery windows decorated with leaf extracts and benzamide-like molecules revealed a push-pull electronic synergistic effect on cytotoxicity and enhanced cell viability, biocompatibility, aggregations, and interactions with the cell membranes. The pre-defined model drug, doxorubicin (DOX), unraveled chemotherapy performance in response to MCF-7 cell lines, with ≈ 60% drug payload contributed from functional groups of leaf extracts. Moreover, electron-poor and electron-rich benzamide-like molecules attached to the ZnCr-LDH surface enhanced the relative cell viability up to 29% and 32%, respectively. The in vivo experiments on breast cancer of treated mice (H&E) revealed unaggregated cellular arrays and antibacterial activity against E. coli (gram-negative) and S. aureus (gram-positive) bacteria. Benzamide-like ZnCr-LDH nanocarriers showed a suitable zone of inhibition beyond 10 mm, compared to the standard. Cytotoxicity control achieved herein seems promising for the future ahead of nanomedicine.

In Ukrainian

A brief literature review proves that nanosized fluorescent carbon materials are widely used. In particular, they are promising in biomedicine (due to biocompatibility – for example, for biovisualization); optoelectronics; as chemical fluorescent sensors for measuring the concentration of metals, pH, anions, organic substances and biomolecules; as markers for fingerprinting. This paper investigates carbon materials obtained by oxidation of activated carbon, which are similar in their optical characteristics to carbon nanotubes. The aim of this work was the synthesis of nanocarbon material from available chemical raw materials. As a prototype, the synthesis is based on the method of obtaining carbon weakly acid cation-exchange resin. The nanocarbon material is easily dispersed in water, forming stable colloidal solutions that exhibit luminescence in the blue-green region of the visible spectrum. According to the results of thermogravimetric analysis, the thermal destruction of surface functional groups was found. The nature of the functional groups on the surface of the carbon nanomaterial was based on the obtained data of infrared spectra. The purity of the samples was monitored by X-ray diffraction analysis of the powder. For the pure sample, only the amorphous carbon spectrum was observed, and for the crude, NaCl reflexes were observed. In the region of MALDI positive ions, clusters of molecular mass have been obtained, which may belong to fullerene-like carbon structures. We believe that the high signal intensity at m/z 44 indicates a significant number of carboxyl groups. For aqueous solutions, the luminescence spectrum was measured, on which blue-green fluorescence was observed. Excitation by radiation with a wavelength was chosen based on the results of preliminary measurements of the dependence of the emission intensity on the length of the excitatory radiation. The fluorescence spectrum shows a wide maximum at 450 nm, which is slightly shifted to the long-wavelength region after centrifugation of the sample and precipitation of large fractions. The method of dynamic light scattering shows that particles with a wide range of sizes are present in the solution, the maximum distribution occurs in relatively large units.

Design principles of bioinspired interfaces for biomedical applications in therapeutics and imaging.

In the past two decades, we have witnessed rapid developments in nanotechnology, especially in biomedical applications such as drug delivery, biosensing, and bioimaging. The most commonly used nanomaterials in biomedical applications are nanoparticles, which serve as carriers for various therapeutic and contrast reagents. Since nanomaterials are in direct contact with biological samples, biocompatibility is one of the most important issues for the fabrication and synthesis of nanomaterials for biomedical applications. To achieve specific recognition of biomolecules for targeted delivery and biomolecular sensing, it is common practice to engineer the surfaces of nanomaterials with recognition moieties. This mini-review summarizes different approaches for engineering the interfaces of nanomaterials to improve their biocompatibility and specific recognition properties. We also focus on design strategies that mimic biological systems such as cell membranes of red blood cells, leukocytes, platelets, cancer cells, and bacteria.

Nanoparticle and Nanostructure Synthesis and Controlled Growth Methods.

Nanomaterials are materials with one or more nanoscale dimensions (internal or external) (i.e., 1 to 100 nm). The nanomaterial shape, size, porosity, surface chemistry, and composition are controlled at the nanoscale, and this offers interesting properties compared with bulk materials. This review describes how nanomaterials are classified, their fabrication, functionalization techniques, and growth-controlled mechanisms. First, the history of nanomaterials is summarized and then the different classification methods, based on their dimensionality (0–3D), composition (carbon, inorganic, organic, and hybrids), origin (natural, incidental, engineered, bioinspired), crystal phase (single phase, multiphase), and dispersion state (dispersed or aggregated), are presented. Then, the synthesis methods are discussed and classified in function of the starting material (bottom-up and top-down), reaction phase (gas, plasma, liquid, and solid), and nature of the dispersing forces (mechanical, physical, chemical, physicochemical, and biological). Finally, the challenges in synthesizing nanomaterials for research and commercial use are highlighted.