Nanocapsule

Last updated

A nanocapsule is a nanoscale shell made from a nontoxic polymer. They are vesicular systems made of a polymeric membrane which encapsulates an inner liquid core at the nanoscale. Nanocapsules have many uses, including promising medical applications for drug delivery, food enhancement, nutraceuticals, and for self-healing materials. The benefits of encapsulation methods are for protection of these substances to protect in the adverse environment, for controlled release, and for precision targeting. [1] Nanocapsules can potentially be used as MRI-guided nanorobots or nanobots, although challenges remain. [2]

Contents

IUPAC definition

Hollow nanoparticle composed of a solid shell that surrounds a core-forming
space available to entrap substances. [3]

Structure

The typical size of the nanocapsule used for various applications ranges from 10-1000 nm. However, depending on the preparation and use of the nanocapsule, the size will be more specific. [4]

Nanocapsule structure consists of nanovesicular system that is formed in a core-shell arrangement. The shell of a typical nanocapsule is made of a polymeric membrane or coating. The type of polymers used is of biodegradable polyester, as nanocapsules are often used in biological systems. Poly-e-caprolactone (PCL), poly(lactide) (PLA), and poly(lactide-co-glicolide) (PLGA) are typical polymers used in nanocapsule formation. [5] Other polymers include thiolated poly(methacrylic acid) and poly(N-vinyl Pyrrolidone). [6] As synthetic polymers have proven to be more pure and reproducible when compared naturally occurring polymers, they are often preferred for the construction nanocapsules. However, some natural occurring polymers such as chitosan, gelatin, sodium alginate, and albumin are used in some drug delivering nanocapsules. [4] Other nanocapsule shells include liposomes, [7] along with polysaccharides and saccharides. Polysaccharides and saccharides are used due to their non-toxicity and biodegradability. They are attractive to use as they resemble biological membranes. [8]

The core of a nanocapsule is composed of an oil surfactant that is specifically selected to coordinate with the selected drug within the polymeric membrane. The specific oil used must be highly soluble with the drug, and non-toxic when used in a biological environment. The oil-drug emulsion must have low solubility with the polymer membrane to ensure that the drug will be carried throughout the system properly and be released at the proper time and location. When the proper emulsion is obtained, the drug should be uniformly dispersed throughout the entire internal cavity of the polymeric membrane. [4]

Processing

The encapsulation method depends on the requirements for any given drug or substance. These processes depend on the physiochemical properties of the core material, the wall material, and the required size. [1] The most common ways to produce nanocapsules are nanoprecipitation, [9] emulsion-diffusion, and solvent-evaporation.

In the nanoprecipitation method, also termed solvent displacement method, nanocapsules are formed by creating a colloidal suspension between two separate phases. The organic phase consists of a solution and a mixture of organic solvents. The aqueous phase consists of a mixture of non-solvents that forms a surface film. The organic phase is slowly injected in the aqueous phase which then is agitated to form the colloidal suspension. Once the colloidal suspension is formed it will be agitated until nanocapsules begin to form. The size and shape of the nanocapsule depend on the rate of injection along with the rate of agitation. [5]

Another common way to prepare nanocapsules is the emulsion diffusion method. This method consists of three phases: organic, aqueous, and dilution phase. In this method the organic phase is added to the aqueous phase under conditions of high agitation which form an emulsion. During this process water is added to the emulsion which causes the solvent to diffuse. The result of this emulsion-diffusion is nanocapsule formation. [5]

Solvent evaporation is another effective method to prepare nanocapsules. In this process, single or double emulsions are formed from solvents and are used to formulate a nanoparticle suspension. High speed homogenization or ultrasonication is used to form small particle size in the nanoparticle suspension. Once the suspension is stable, the solvents are evaporated using either continuous magnetic stirring at room temperature, or by reducing the ambient pressure. [4]

The table below displays how nanocapsules exhibit different traits based on the method by which they were prepared. Nanocapsule types vary by size, drug concentration, and active substance release time.[ citation needed ]

Mean size (nm)[ dubious – discuss ]Drug concentration in diluted dispersion (mg/ml) [5] Drug concentration in concentrated dispersion (mg/ml) [5] Active substance release time (90%) (min) [5]
Nanoprecipitation2500.002–0.090.15–6.5750
Emulsion-diffusion425~0.25060
Double emulsification4002–520–5045
Emulsification coacervation300~0.2412>2000

Processing issues and solutions

Nanocapsules tend to aggregate and become unstable. Thus, substances within capsules can leak. To control the instability, nanocapsules can be dried either through spray drying or freeze-drying (lyophilization [10] ). [1]

Spray drying – Solutions are sprayed into a drying medium. This method is more widely used in the food industry and used for encapsulation of many food products as flavors, minerals, colors, and vitamins. This method makes nanocapsules more stable, and increases shelf-life of foods. [1]

Freeze-drying – This process involves dehydration of materials that are heat-sensitive. Unlike spray drying, water is removed through the sublimation process without changing the structure or shape of the nanoparticles. Freeze-drying involves four states: freezing, primary drying, secondary drying, and storage. Because of the multiple stages involved, this method is considered to demand more energy and time. [1]

Properties

Absorbability

Aspect ratio affects the ability of the nanocapsule to penetrate tumor cells. Low aspect ratios (spherical capsules) tend to penetrate cells more easily than high aspect ratios (rod-shaped capsules). [6]

Structure

The nano-sized structure of nanocapsules allows permeating through basal membranes, which makes them effective carriers of medicine in biological systems. The specific processing of nanocapsules gives them unique properties in how they release drugs in certain situations. Generally, there are three physico-chemical release mechanisms that are used to release the drug or medicine from the polymeric shell of the nanocapsule. [4]

Delivery

  1. Hydration and diffusion – In this release mechanism the nanocapsule will swell due to the effects of hydration. Once the nanocapsule has swollen to a point where it stretches, the polymeric membrane will allow for diffusion of the drug through the polymeric membrane and into the biological system. [4]
  2. Enzymatic reaction – The polymer shell must be first selected to coordinate with the enzymes produced by the human body to produce and enzymatic reaction. This reaction will cause a rupture in the polymeric membrane which allows the drug to be dispersed into the system. [4]
  3. Dissociation of the drug – The drug dissociates from the swelled nanocapsule and diffuses out into the rest of the cell. [4]
Other delivery methods: substance delivery in medical use

Near-infrared light: Drug release is triggered from heat. The infrared technology can be absorbed deep in the body, turn to heat. The heat-sensitive material, particularly a polymer shell that swells upon heating, collapses. The action of deflating is what releases the drug. [7]

Magnetic fields: Magnetic bars of millimeter-scale are embedded in poly(vinyl alcohol). The magnetic field within the bars is alternated, which results in the change of shape and ultimate collapse of the nanocapsules. The change in the structure then triggers the drug release. [7]

Ultrasound: Another option of drug release is through ultrasound, which is a "longitudinal pressure wave". [7] The ultrasound can either be low-frequency, or LFUS, (between ~20 and ~100 kHz) or high-frequency, HFUS, (>1 MHz). Transdermal delivery (sonophoresis) is enhanced through LFUS, which then further allows the drug to be released. Since the wave of HFUS is higher, success of drug delivery has been demonstrated through the form of bubbles. The bubbles with in the capsule are formed and collapsed due to the higher temperatures of the wave. [7]

Some other ways include oral, which is the most active, nasal, transdermal, and through the lung. Oral is the most common, and the most challenging. Demands for consistent release persist, although developments are being made for drugs to bioadhere to the intestinal tract. Bioadhesion is also being considered for nasal delivery, to prolong the life of the drug within the nose. Drugs can also be transferred through the skin (transdermal). Inhalers are also of interest, as for example, asthma drugs consist of macromolecules. Currently, the inhalation systems are undesirable to patients, and it is hoped that there will be advances in this delivery system at some time. [7]

Applications

Cancer

Water-soluble polymer shells are being created to deliver a protein, apoptin, [11] into cancer cells. The protein goes into the nucleus of the cancer cells while leaving healthy cells alone, unlike other conventional therapies as gene therapies and chemotherapy. [12] The capsules are 100 nm in size. [12]

Active targeting of cancer cells is also being researched. Through active targeting, the nanocapsules form ligands that bind to malignant cells for cell delivery. This method is especially beneficial for those drugs that are not as permeable through the cell membrane, and where tissues are diseased, the nanoparticles are able to bond easier with the malignant cells. [7]

Food usage

Nanoencapuslation in foods involves the changing of textures, flavorings, colorings, and stability in shelf-life. [1]

Nutraceuticals

Nutraceuticals (from nutrient + pharmaceutical ) are substances that are placed in food to enhance nutrition. The increased bioavailability of these substances is relative to the size of the nanocarrier. The smaller the nanocarrier, the better the delivery properties and the solubility of the nutraceuticals; the nanocarrier is able to enter the bloodstream easier if smaller. [1]

Lipid or polymer-based (natural biodegradable) are used for encapsulation for nutraceuticals. Types of polymers used include collagen, gelatin, and albumin. [1]

Ethyl alcohol absorption

Relatively new research involves the encapsulation of digestive enzymes within a non-toxic polymer shell. The enzyme filled nanoshell has been proven in lab mice to absorb ethyl alcohol from the bloodstream, therefore resulting in reduced blood alcohol levels. It has been concluded that the particles act as organelles, which proposes other benefits to enzyme therapies. This discovery is introducing other studies, such as encapsulation methods for hair loss. [13]

Self-healing materials

For materials such as components in microelectronics, polymeric coatings, and adhesives, nanocapsules can reduce damage caused by high loads. The healing of cracks within these materials is alleviated by dispersing nanocapsules within the polymer. The healing substances include dicyclopentadiene (DCPD), which is prepared on site within the material by sonication. The nanoencapsulated material is first emulsified within the host material by creating an oil-in-water self-healing epoxy. The emulsified material is then agitated within the host material to form particles which then bond to the host material. [14]

Concerns of use

As of 2016, it is unknown what the impacts of nano-sized materials are to human health and the environment. It is only via chemical risk and toxic assessments over time can affirm any effects. The measures for testing are currently insufficient, and the approval for the use of nanoparticles, especially in food, is ambiguous. [1]

Related Research Articles

<span class="mw-page-title-main">Liposome</span> Composite structures made of phospholipids and may contain small amounts of other molecules

A liposome is a small artificial vesicle, spherical in shape, having at least one lipid bilayer. Due to their hydrophobicity and/or hydrophilicity, biocompatibility, particle size and many other properties, liposomes can be used as drug delivery vehicles for administration of pharmaceutical drugs and nutrients, such as lipid nanoparticles in mRNA vaccines, and DNA vaccines. Liposomes can be prepared by disrupting biological membranes.

Microencapsulation is a process in which tiny particles or droplets are surrounded by a coating to give small capsules, with useful properties. In general, it is used to incorporate food ingredients, enzymes, cells or other materials on a micro metric scale. Microencapsulation can also be used to enclose solids, liquids, or gases inside a micrometric wall made of hard or soft soluble film, in order to reduce dosing frequency and prevent the degradation of pharmaceuticals.

<span class="mw-page-title-main">Dendrimer</span>

Dendrimers are highly ordered, branched polymeric molecules. Synonymous terms for dendrimer include arborols and cascade molecules. Typically, dendrimers are symmetric about the core, and often adopt a spherical three-dimensional morphology. The word dendron is also encountered frequently. A dendron usually contains a single chemically addressable group called the focal point or core. The difference between dendrons and dendrimers is illustrated in the top figure, but the terms are typically encountered interchangeably.

<span class="mw-page-title-main">Electrospinning</span>

Electrospinning is a fiber production method that uses electric force to draw charged threads of polymer solutions or polymer melts up to fiber diameters in the order of some hundred nanometers. Electrospinning shares characteristics of both electrospraying and conventional solution dry spinning of fibers. The process does not require the use of coagulation chemistry or high temperatures to produce solid threads from solution. This makes the process particularly suited to the production of fibers using large and complex molecules. Electrospinning from molten precursors is also practiced; this method ensures that no solvent can be carried over into the final product.

An artificial cell, synthetic cell or minimal cell is an engineered particle that mimics one or many functions of a biological cell. Often, artificial cells are biological or polymeric membranes which enclose biologically active materials. As such, liposomes, polymersomes, nanoparticles, microcapsules and a number of other particles can qualify as artificial cells.

Microparticles are particles between 0.1 and 100 μm in size. Commercially available microparticles are available in a wide variety of materials, including ceramics, glass, polymers, and metals. Microparticles encountered in daily life include pollen, sand, dust, flour, and powdered sugar.

Targeted drug delivery, sometimes called smart drug delivery, is a method of delivering medication to a patient in a manner that increases the concentration of the medication in some parts of the body relative to others. This means of delivery is largely founded on nanomedicine, which plans to employ nanoparticle-mediated drug delivery in order to combat the downfalls of conventional drug delivery. These nanoparticles would be loaded with drugs and targeted to specific parts of the body where there is solely diseased tissue, thereby avoiding interaction with healthy tissue. The goal of a targeted drug delivery system is to prolong, localize, target and have a protected drug interaction with the diseased tissue. The conventional drug delivery system is the absorption of the drug across a biological membrane, whereas the targeted release system releases the drug in a dosage form. The advantages to the targeted release system is the reduction in the frequency of the dosages taken by the patient, having a more uniform effect of the drug, reduction of drug side-effects, and reduced fluctuation in circulating drug levels. The disadvantage of the system is high cost, which makes productivity more difficult, and the reduced ability to adjust the dosages.

Pharmaceutical formulation, in pharmaceutics, is the process in which different chemical substances, including the active drug, are combined to produce a final medicinal product. The word formulation is often used in a way that includes dosage form.

<span class="mw-page-title-main">Ouzo effect</span> Phenomenon observed in drink mixing

The ouzo effect is a milky oil-in-water emulsion that is formed when water is added to ouzo and other anise-flavored liqueurs and spirits, such as pastis, rakı, arak, sambuca and absinthe. Such emulsions occur with only minimal mixing and are highly stable.

<span class="mw-page-title-main">Janus particles</span> Type of nanoparticle or microparticle

Janus particles are special types of nanoparticles or microparticles whose surfaces have two or more distinct physical properties. This unique surface of Janus particles allows two different types of chemistry to occur on the same particle. The simplest case of a Janus particle is achieved by dividing the particle into two distinct parts, each of them either made of a different material, or bearing different functional groups. For example, a Janus particle may have one-half of its surface composed of hydrophilic groups and the other half hydrophobic groups, the particles might have two surfaces of different color, fluorescence, or magnetic properties. This gives these particles unique properties related to their asymmetric structure and/or functionalization.

A nanogel is a polymer-based, crosslinked hydrogel particle on the sub-micron scale. These complex networks of polymers present a unique opportunity in the field of drug delivery at the intersection of nanoparticles and hydrogel synthesis. Nanogels can be natural, synthetic, or a combination of the two and have a high degree of tunability in terms of their size, shape, surface functionalization, and degradation mechanisms. Given these inherent characteristics in addition to their biocompatibility and capacity to encapsulate small drugs and molecules, nanogels are a promising strategy to treat disease and dysfunction by serving as delivery vehicles capable of navigating across challenging physiological barriers within the body. 

Nano-scaffolding is a medical process used to regrow tissue and bone, including limbs and organs. The nano-scaffold is a three-dimensional structure composed of polymer fibers very small that are scaled from a Nanometer scale. Developed by the American military, the medical technology uses a microscopic apparatus made of fine polymer fibers called a scaffold. Damaged cells grip to the scaffold and begin to rebuild missing bone and tissue through tiny holes in the scaffold. As tissue grows, the scaffold is absorbed into the body and disappears completely.

In polymer chemistry, in situ polymerization is a preparation method that occurs "in the polymerization mixture" and is used to develop polymer nanocomposites from nanoparticles. There are numerous unstable oligomers (molecules) which must be synthesized in situ for use in various processes. The in situ polymerization process consists of an initiation step followed by a series of polymerization steps, which results in the formation of a hybrid between polymer molecules and nanoparticles. Nanoparticles are initially spread out in a liquid monomer or a precursor of relatively low molecular weight. Upon the formation of a homogeneous mixture, initiation of the polymerization reaction is carried out by addition of an adequate initiator, which is exposed to a source of heat, radiation, etc. After the polymerization mechanism is completed, a nanocomposite is produced, which consists of polymer molecules bound to nanoparticles.

<span class="mw-page-title-main">Nano spray dryer</span>

Nano spray dryers refer to using spray drying to create particles in the nanometer range. Spray drying is a gentle method for producing powders with a defined particle size out of solutions, dispersions, and emulsions which is widely used for pharmaceuticals, food, biotechnology, and other industrial materials synthesis.

<span class="mw-page-title-main">Cell encapsulation</span>

Cell encapsulation is a possible solution to graft rejection in tissue engineering applications. Cell microencapsulation technology involves immobilization of cells within a polymeric semi-permeable membrane. It permits the bidirectional diffusion of molecules such as the influx of oxygen, nutrients, growth factors etc. essential for cell metabolism and the outward diffusion of waste products and therapeutic proteins. At the same time, the semi-permeable nature of the membrane prevents immune cells and antibodies from destroying the encapsulated cells, regarding them as foreign invaders.

Nanoparticles for drug delivery to the brain is a method for transporting drug molecules across the blood–brain barrier (BBB) using nanoparticles. These drugs cross the BBB and deliver pharmaceuticals to the brain for therapeutic treatment of neurological disorders. These disorders include Parkinson's disease, Alzheimer's disease, schizophrenia, depression, and brain tumors. Part of the difficulty in finding cures for these central nervous system (CNS) disorders is that there is yet no truly efficient delivery method for drugs to cross the BBB. Antibiotics, antineoplastic agents, and a variety of CNS-active drugs, especially neuropeptides, are a few examples of molecules that cannot pass the BBB alone. With the aid of nanoparticle delivery systems, however, studies have shown that some drugs can now cross the BBB, and even exhibit lower toxicity and decrease adverse effects throughout the body. Toxicity is an important concept for pharmacology because high toxicity levels in the body could be detrimental to the patient by affecting other organs and disrupting their function. Further, the BBB is not the only physiological barrier for drug delivery to the brain. Other biological factors influence how drugs are transported throughout the body and how they target specific locations for action. Some of these pathophysiological factors include blood flow alterations, edema and increased intracranial pressure, metabolic perturbations, and altered gene expression and protein synthesis. Though there exist many obstacles that make developing a robust delivery system difficult, nanoparticles provide a promising mechanism for drug transport to the CNS.

<span class="mw-page-title-main">Interfacial polymerization</span>

Interfacial polymerization is a type of step-growth polymerization in which polymerization occurs at the interface between two immiscible phases, resulting in a polymer that is constrained to the interface. There are several variations of interfacial polymerization, which result in several types of polymer topologies, such as ultra-thin films, nanocapsules, and nanofibers, to name just a few.

Nanocomposite hydrogels are nanomaterial-filled, hydrated, polymeric networks that exhibit higher elasticity and strength relative to traditionally made hydrogels. A range of natural and synthetic polymers are used to design nanocomposite network. By controlling the interactions between nanoparticles and polymer chains, a range of physical, chemical, and biological properties can be engineered. The combination of organic (polymer) and inorganic (clay) structure gives these hydrogels improved physical, chemical, electrical, biological, and swelling/de-swelling properties that cannot be achieved by either material alone. Inspired by flexible biological tissues, researchers incorporate carbon-based, polymeric, ceramic and/or metallic nanomaterials to give these hydrogels superior characteristics like optical properties and stimulus-sensitivity which can potentially be very helpful to medical and mechanical fields.

Nanoparticle drug delivery systems are engineered technologies that use nanoparticles for the targeted delivery and controlled release of therapeutic agents. The modern form of a drug delivery system should minimize side-effects and reduce both dosage and dosage frequency. Recently, nanoparticles have aroused attention due to their potential application for effective drug delivery.

<span class="mw-page-title-main">Polystyrene (drug delivery)</span> Polystyrene in drug delivery

Polystyrene is a synthetic hydrocarbon polymer that is widely adaptive and can be used for a variety of purposes in drug delivery. These methods include polystyrene microspheres, nanoparticles, and solid foams. In the biomedical engineering field, these methods assist researchers in drug delivery, diagnostics, and imaging strategies.

References

  1. 1 2 3 4 5 6 7 8 9 Ezhilarasi, P. N.; Karthik, P.; Chhanwal, N.; Anandharamakrishnan, C. (2012). "Nanoencapsulation Techniques for Food Bioactive Components: A Review". Food and Bioprocess Technology. 6 (3): 628–47. doi:10.1007/s11947-012-0944-0. S2CID   98381808.
  2. ↑ Vartholomeos, P.; Fruchard, M.; Ferreira, A.; Mavroidis, C. (2011). "MRI-Guided Nanorobotic Systems for Therapeutic and Diagnostic Applications". Annu Rev Biomed Eng. 13: 157–84. doi:10.1146/annurev-bioeng-071910-124724. PMID   21529162. S2CID   32852758.
  3. ↑ Vert, Michel; Doi, Yoshiharu; Hellwich, Karl-Heinz; Hess, Michael; Hodge, Philip; Kubisa, Przemyslaw; Rinaudo, Marguerite; Schué, François (2012). "Terminology for biorelated polymers and applications (IUPAC Recommendations 2012)" (PDF). Pure and Applied Chemistry . 84 (2): 377–410. doi:10.1351/PAC-REC-10-12-04. S2CID   98107080.
  4. 1 2 3 4 5 6 7 8 Nagavarma, B V N; Yadav, Hemant K S; Ayaz, A; Vasudha, L S; Shivakumar, H G (2012). "Different Techniques for Preparation of Polymeric Nanoparticles – A Review" (PDF). Asian Journal of Pharmaceutical and Clinical Research. 5 (Suppl 3): 16–23.
  5. 1 2 3 4 5 6 Mora-Huertas, C.E.; Fessi, H.; Elaissari, A. (2010). "Polymer-based nanocapsules for drug delivery". International Journal of Pharmaceutics. 385 (1–2): 113–42. doi:10.1016/j.ijpharm.2009.10.018. PMID   19825408.
  6. 1 2 Shimoni, Olga; Yan, Yan; Wang, Yajun; Caruso, Frank (2013). "Shape-Dependent Cellular Processing of Polyelectrolyte Capsules". ACS Nano. 7 (1): 522–30. doi: 10.1021/nn3046117 . PMID   23234433.
  7. 1 2 3 4 5 6 7 Timko, Brian P.; Whitehead, Kathryn; Gao, Weiwei; Kohane, Daniel S.; Farokhzad, Omid; Anderson, Daniel; Langer, Robert (2011). "Advances in Drug Delivery". Annual Review of Materials Research . 41: 1–20. Bibcode:2011AnRMS..41....1T. doi:10.1146/annurev-matsci-062910-100359.
  8. ↑ Long, Li-xia; Yuan, Xu-bo; Chang, Jiang; Zhang, Zhi-hua; Gu, Ming-qi; Song, Tian-Tian; Xing, Ying; Yuan, Xiao-yan; et al. (2012). "Self-assembly of polylactic acid and cholesterol-modified dextran into hollow nanocapsules". Carbohydrate Polymers. 87 (4): 2630–7. doi:10.1016/j.carbpol.2011.11.032.
  9. ↑ "Dictionary of nanotechnology - Nanoprecipitation".
  10. ↑ http://www.rpi.edu/dept/chem-eng/Biotech-Environ/LYO/%5B%5D
  11. ↑ Pietersen, Alexandra; Noteborn, Mathieu H. M. (2000). "Apoptin". Cancer Gene Therapy: Past Achievements and Future Challenges. Advances in Experimental Medicine and Biology. Vol. 465. pp. 153–61. doi:10.1007/0-306-46817-4_14. ISBN   978-0-306-46817-9. PMID   10810623.
  12. 1 2 Zhao, Muxun; Hu, Biliang; Gu, Zhen; Joo, Kye-Il; Wang, Pin; Tang, Yi (2013). "Degradable polymeric nanocapsule for efficient intracellular delivery of a high molecular weight tumor-selective protein complex". Nano Today. 8: 11–20. doi:10.1016/j.nantod.2012.12.003.
  13. ↑ Liu, Yang; Du, Juanjuan; Yan, Ming; Lau, Mo Yin; Hu, Jay; Han, Hui; Yang, Otto O.; Liang, Sheng; et al. (2013). "Biomimetic enzyme nanocomplexes and their use as antidotes and preventive measures for alcohol intoxication". Nature Nanotechnology. 8 (3): 187–92. Bibcode:2013NatNa...8..187L. doi:10.1038/nnano.2012.264. PMC   3670615 . PMID   23416793.
  14. ↑ Blaiszik, B.J.; Sottos, N.R.; White, S.R. (2008). "Nanocapsules for self-healing materials". Composites Science and Technology. 68 (3–4): 978–86. doi:10.1016/j.compscitech.2007.07.021.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.