INTRODUCTION:

There are two major misunderstandings in the literature regarding nano-emulsions. One arises from their similarities to microemulsions. Nano-emulsions are emulsions with an extremely small droplet size ¹⁶which can overlap those of micro-emulsions. The definition of emulsions by the International Union of Pure and Applied Chemistry (IUPAC) states: “In an emulsion, liquid droplets and/or liquid crystals are dispersed in a liquid”^17.Obviously, microemulsions are excluded from this definition if the word “dispersed” is interpreted as non-equilibrium and opposite to “solubilized”, a term that can be applied to microemulsions and micellar systems. Therefore, there is a fundamental difference between microemulsions and nano-emulsions: microemulsions are equilibrium systems (i.e. thermodynamically stable), while nano-emulsions are non-equilibrium systems with a spontaneous tendency to separate into the constituent phases. Nevertheless, nano-emulsions may possess a relatively high kinetic stability, even for several years ¹⁶.

The increasing interest in the area of nanoemulsions is caused by growing number of their promising applications in material science, medicine, pharmacology or agriculture. In order to understand behavior of nanoemulsions and thus extend their application potential, studies on model systems are beneficial. By definition, nanoemulsions are transparent or bluish, kinetically Stable, two-phase systems with a typical particle size range of 20-200 nm ^[1,2]. Emulsions with droplet size in the nanometric scale (typically in the range 20–200 nm) are often referred to in the literature as miniemulsions [3], nanoemulsions ^{[4, 5],} ultrafine emulsions [^4], submicron emulsions ^[6], etc. The term nanoemulsions ^[1] is preferred because in addition to give an idea of the nano-scale size range of the droplets it is concise and it avoids misinterpretation with the term microemulsion (which are thermodynamically stable systems). Due to their characteristic size, nano-emulsions appear transparent or translucent to the naked eye and possess stability against sedimentation or creaming. These properties make nanoemulsions of interest for fundamental studies and for practical applications.

Nanoemulsions are very fragile systems by nature. As they are transparent and usually very fluid, the slightest sign of destabilization easily appears. They become opaque and creaming may be visible. We identified two major sources of instability in these systems that are discussed in this paper: one caused by the nature of the oil phase and another originating from the addition of polymers to thicken or to gel the nanoemulsions. The very small size of the droplets (D; 50 nm) gives them characteristic properties, which can be easily valued related to freshness, purity, simplicity, water they are easily absorbed by the skin. They can be sterilized by filtration. They lead to a large variety of products from water-like fluids to ringing gels^.[7].

Nano-emulsions are attractive for application in personal care and cosmetics as well as in health care due to the following advantages: ^[22]

1. The very small droplet size causes a large reduction in the gravity force and Brownian motion may be sufficient to overcome gravity. This means that no creaming or sedimentation occurs on storage.

2. The small droplet size also prevents any flocculation of the droplets. Weak flocculation is prevented and this enables the system to remain dispersed with no separation.

3. The small droplets size also prevents their coalescence, since these droplets are non-deformable and hence surface fluctuations are prevented. In addition, the significant surfactant film thickness (relative to droplet radius) prevents any thinning or disruption of the liquid film between the droplets.

4. Nano-emulsions are suitable for efficient delivery of active ingredients through the skin. The large surface area of the emulsion system allows rapid penetration of actives.

5. Due to their small size, nano-emulsions can penetrate through the ‘‘rough’’ skin surface and this enhances penetration of actives.

6. The transparent nature of the system, their fluidity (at reasonable oil concentrations) as well as the absence of any thickeners may give them a pleasant aesthetic character and skin feel.

7. Unlike microemulsions (which require a high surfactant concentration, usually in the region of 20% and higher), nano-emulsions can be prepared using reason-able surfactant concentrations. For a 20% O/W nano-emulsion, a surfactant concentration in the region of 5–10% may be sufficient.

8. The small size of the droplets allow them to deposit uniformly on substrates wetting, spreading and penetration may be also enhanced because of the low surface tension of the whole system and the low interfacial tension of the O/W drop-lets.

9. Nano-emulsions can be applied for delivery of fragrant, which may be incorporated in many personal care products. This could also be applied in perfumes, which are desirable to be formulated alcohol free.

10. Nano-emulsions may be applied as a substitute for liposomes and vesicles (which are much less stable) and it is possible in some cases to build lamellar liquid crystalline phases around the nano-emulsion droplets

Despite the above advantages, nano-emulsions have only attracted interest in recent years because:

1. Their preparation requires, in many cases, special application techniques such as the use of high-pressure homogenizers as well as ultrasonic’s. Such equipment (such as the Microfluidiser) has become available only in recent years.

2. There is a perception in the Personal Care and Cosmetic Industry that nano-emulsions are expensive to produce. Expensive equipment is required as well as the use of high concentrations of emulsifiers.

3. Lack of understanding of the mechanism of production of submicron droplets and the role of surfactants and co-surfactants.

4. Lack of demonstration of the benefits that can be obtained from using nano-emulsions when compared with classical macroemulsion systems.

5. Lack of understanding of the interfacial chemistry involved in production of nano-emulsions. For example, few formulation chemists are aware of the use of the phase inversion temperature (PIT) concept and how this can be usefully applied for the production of small emulsion droplets.

6. Lack of knowledge on the mechanism of Ostwald ripening, which is perhaps the most serious instability problem with nano-emulsions.

7. Lack of knowledge of the ingredients that may be incorporated to overcome Ostwald ripening. For example, addition of a second oil phase with very low solubility and/or incorporation of polymeric surfactants that strongly adsorb at the O/W interface (which are also insoluble in the aqueous medium).

8. Fear of introduction of new systems without full evaluation of the cost and benefits ^[20-21]

Method of preparation of nanoemulsions: There are two primary methods to prepare nanoemulsions ⁶:

1. Persuasion and;

2. Brute force

1.1 by Persuasion:

(1) Phase Transition from Near-Optimum State via Change in Single Variable: This method involves change in one formulation variable such as salinity or temperature for a system near optimal (HLD (hydrophilic Lipophilic deviation) near 0), such as applying a higher temperature to a microemulsion.

(2) Phase Transition from Near-Optimum State via Change in Multiple Variables: This method involves change in more than one formulation variable, such as applying higher temperature and inclusion of additional salt in a microemulsion.

(3) Catastrophic Inversion: This method involves causing a low internal phase emulsion to invert such that the internal phase becomes the external phase.

(4) Phase Transition Stabilized by Liquid Crystal Formation: This method involves stabilization of nano droplets by liquid crystal formation from a state near HLD=0.

2. By Brute Force: This method may involve the use of a high speed mixer, a high pressure homogenizer, a high frequency ultra-sonic device, a small pore membrane, etc. Formation of O/W and W/O nanoemulsions by dispersion or high-energy emulsification methods is apparently fairly common, while nanoemulsion formation by condensation or “low-energy” emulsification methods, take advantage of the physicochemical properties of these systems based on the phase transition that takes place during the emulsification process. It can be carried out by operating in particular areas of the phase diagram with a very low interfacial tension, which are areas of liquid crystals and microemulsions; at the end of the emulsification process, nanoemulsions formed. Properties of nanoemulsions, such as small droplet size, relative high kinetic stability and optical transparency seem to depend not only on composition variables but also on preparation variables such as emulsifying path, degree of mixing energy input and emulsification time

2. Nano-emulsion formation.

There are primary methods to prepare a nanoemulsion ^[1]:

1) High energy method.

2) Low energy method.

3) Microfluidization

4) Jet disperser.

5) Spontaneous Emulsification

6) Solvent Evaporation Technique

7) Hydrogel Method

8) Solvent Displacement Method

2.1) High energy method.

Nano-emulsions, being non-equilibrium systems, cannot be formed spontaneously. Consequently, energy input generally from mechanical devices or from the chemical potential of the components, is required. Nano-emulsion formation by the so-called dispersion or high-energy emulsification methods is generally achieved using high shear stirring, high-pressure homogenizers and ultrasound generators. It has been shown that the apparatus supplying the available energy in the shortest time and having the most homogeneous flow produces the smallest sizes ^[8]. High-pressure homogenizers meet these requirements. Therefore, they are the most widely used emulsifying machines to prepare nano-emulsions. Generally, the conventional high-pressure homogenizers work in a range of pressures between 50 and 100 MPa. Pressures as high as 350 MPa have been achieved in a recently developed instrument ^[9], although O/W nano-emulsions with methylcellulose as emulsifier could only be stabilized by selecting a homogenization pressure lower than 150 MPa because of the very strong elongation flow produced at higher pressures that promoted the irreversible degradation of long chain molecules. Ultrasonic emulsification is also very efficient in reducing droplet size but it is only appropriate for small batches ^[8]. A recent study ^[10] on the preparation of polymerizable nano-emulsions has shown that the efficiency of the dispersion process is strongly dependent on the ultrasonication time at different amplitudes and that the more hydrophobic the monomer is, the longer the sonication time required. It is well known that, by taking advantage of the physicochemical properties of the system, dispersions can be produced almost spontaneously ^[11,12]. This is the case with the condensation or low-energy emulsification methods that make use of the phase transitions taking place during the emulsification process. The phase inversion temperature (PIT) method, introduced by Shinoda and Saito ^[11]is, among these methods, the most widely used in industry ^[13].It is based on the changes in solubility of polyoxy ethylene-type nonionic surfactants with temperature. These types of surfactants become Lipophilic with increasing temperature because of dehydration of the polyoxy ethylene chains.

2) Low energy method.

These methods make use of the phase transitions that take place during the emulsification process. The so-called phase inversion temperature (PIT) method is widely used in industry. This method, introduced by Shinoda and Saito [18] is based on the changes in solubility of polyoxy-ethylene type nonionic surfactants with temperature. These types of surfactants become Lipophilic with increasing temperature because of dehydration of the polyoxy-ethylene chains. At low temperature, the surfactant monolayer has a large positive spontaneous curvature forming oil-swollen micellar solution phases (or O/W microemulsions), which may coexist with an excess oil phase. At high temperatures, the spontaneous curvature becomes negative and water-swollen reverse micelles (or W/O microemulsions) coexist with excess water phase. At intermediate temperatures, the hydrophile-lipophile balance (HLB) temperature, the spontaneous curvature becomes close to zero and abicontinuous, D phase, microemulsion containing comparable amounts of water and oil phases coexists with both excess water and oil phases. Because a transition from O/W to W/O emulsions takes place at this intermediate temperature, it is also designated as the phase inversion temperature, PIT ^[18-20].

2.3) Microfluidization^[26]:

Microfluidization is a mixing technique, which makes use of a device called Microfluidiser. This device uses a high-pressure positive displacement pump (500 to 20000psi), which forces the product through the interaction chamber, which consists of small channels called ‘micro-channels’. The product flows through the micro channels on to an impingement area resulting in very fine particles of submicron range. The two solutions (aqueous phase and oily phase) are combined together and processed in an inline homogenizer to yield a coarse emulsion. The coarse emulsion is into a micro-fluidizer where it is further processed to obtain a stable nanoemulsion. The coarse emulsion is passed through the interaction chamber micro-fluidizer repeatedly until desired particle size is obtained. The bulk emulsion is then filtered through a filter under nitrogen to remove large droplets resulting in a uniform nanoemulsion.

2.4) JET DISPERSER^[26]

Forcing the flow stream by high pressure through micro channels towards an impregnated area creates a tremendous shearing action, which can provide an exceptionally fine emulsion. In general, initial forces in turbulent flow along with cavitations are predominantly responsible for droplet disruption in Microfluidiser. Disruption in laminar elongational flow is also possible, especially when emulsion has high viscosity. In the jet disperser two or more jets of crude emulsion each from opposing bores collide with one another but at a different design than Microfluidiser, the diameter of the bores injet dispersers are typically 0.3-0.5mm. Finally an “orifice plate” is the simplest construction form for a homogenizing nozzle. The diameter of orifice bore is of same order of magnitude as the jet dispersers and inlet head diameter of orifice plate is typically 10-60nm, in jet dispersers and orifice plates, droplets are disrupted predominantly due to laminar elongational flow ahead of the bores. Unlike radial diffusers, the nozzle is microfluidizers; jet dispersers and orifice plate contain no moving parts, so they can be used at high pressures up to 300-400 Mpa26

2.5) Spontaneous Emulsification^[14]:

It involves three main steps

a. Preparation of homogeneous organic solution composed of oil and Lipophilic surfactant in water miscible solvent and hydrophilic surfactant.

b. The organic phase was injected in the aqueous phase under magnetic stirring the o/w emulsion was formed.

c. The water-miscible solvent was removed by evaporation under reduced pressure

2.6) Hydrogel Method^[15]:

It is similar to solvent evaporation method. The only difference between the two methods is that the drug solvent is miscible with the drug anti-solvent. Higher shear force prevent crystal growth and Ostwald ripening. Other method used for Nanoemulsion preparation is the phase inversion temperature technique.

2.7) Solvent Evaporation Technique^[15]:

This technique involves preparing a solution of drug followed by its emulsification in another liquid that is non -solvent for the drug. Evaporation of the solvent leads to precipitation of the drug. Crystal growth and particle aggregation can be controlled by creating high shear forces using a high-speed stirrer.

2.8 ) Solvent Displacement Method

The solvent displacement method for spontaneous fabrication of nanoemulsion has been adopted from the nano- precipitation method used for polymeric nanoparticles. In this method, oily phase is dissolved in water-miscible organic solvents, such as acetone, ethanol and ethyl methyl ketone. The organic phase is poured into an aqueous phase containing surfactant to yield spontaneous nanoemulsion by rapid diffusion of organic solvent. The organic solvent is removed from the nanoemulsion by a suitable means, such as vacuum evaporation. Spontaneous nanoemulsification has also been reported when solution of organic solvents containing a small percentage of oil is poured into aqueous phase without any surfactant. Solvent displacement methods can yield nanoemulsions at room temperature and require simple stirring for the fabrication. Hence, researchers in pharmaceutical sciences are employing this technique for fabricating nanoemulsions mainly for parenteral use. However, the major drawback of this method is the use of organic sol- vents, such as acetone, which require additional inputs for their removal from nanoemulsion. Furthermore, a high ratio of solvent to oil is required to obtain a nanoemulsion with a desirable droplet size. This may be a limiting factor in certain cases. In addition, the process of solvent removal may appear simple at laboratory scale but can pose several difficulties during scale-up^[43].

3.) Characterization and evaluation of nanoemulsion:

Different characterization parameters for nanoemulsion include transmission electron microscopy, nanoemulsion droplet size analysis, viscosity, refractive index, thermodynamic stability studies, and surface characteristics. Particle size and size distribution, Zeta potential, Turbidity, Emulsion stability affected by environmental factors (pH, ionic strength (NaCl), and thermal treatment), skin permeation studies and these characteristics are measured by the various techniques discuss below.

3.1) Polydispersity Index:

The average diameters and polydispersity index of samples were measured by photon correlation spectroscopy. The measurements were performed at 25^oC using a He–Ne laser^[27].

3.2) Viscosity Determination:

The viscosity of the formulations was determined as such without dilution using a Brookfield DV III ultra V6.0 RV cone and plate rheometer using spindle. Viscosity will be measured to ensure the better delivery of the formulation^[14,15].

3.3) Refractive Index^[27]:

The refractive index, n, of a medium is defined as the ration f the speed, c, of a wave such as light or sound in a reference medium to the phase speed, v p , of the wave in the medium. n=c/v p It was determined using an Abbes type refractrometer (Nirmal International) at 25 ± 0.5°C.

3.4) pH:

The apparent pH of the formulation was measured by pH meter^[15].

3.5) Droplet size measurements:

Size analysis of nanoemulsion was carried out by dynamic light scattering with zeta sizerhsa 3000 (Malvern instruments ltd., Malvern, U.K). Samples were placed in square glass cuvettes and droplet size analysis was carried out at Temperature 25⁰C, for 80 second duration^[27-30].

3.6) Zeta potential measurements:

Zeta potential for nanoemulsion was determined using zetasizer has 3000 (Malvern instrument ltd., UK). Samples were placed in clear disposable zeta cells and results were recorded. Before putting the fresh sample, cuvettes were washed with the methanol and rinsed using the sample to be measured before each experiment^[28].

3.7) Transmission Electronic Microscopy (TEM):

Morphology and structure of the nanoemulsion were studied using Transmission Electron Microscopy To perform the TEM observations, samples were placed on a form var carbon-coated copper grid (200 mesh in-1) and then stained with 1% phosphotungstic acid. The excess phosphotungstic acid on the sample was gently wiped off using filter paper and examined after drying for about half an hour at room temperature. Observations was performed as, a drop of the nanoemulsion was directly deposited on the holey film grid and observed after drying^[29-30].

3.8) Measurement of particle size distribution:

Particle size and particle size distribution of nanoemulsions were determined by photon correlation spectroscopy (PCS) in terms of z-average diameter using a Zetasizer Nano ZS instrument (Malvern Instruments, UK) at 25 °C.^[14,30].

3.9) Emulsion stability:

Stability of emulsions was evaluated by following a phase separation both visually and by microscopy as well as by following the particle size and distribution by PCS. For stability evaluation, the emulsions were stored at three different temperatures of 4 °C, 25 °C and 35 °C and observed at regular time intervals. Microscopic observation was performed using a microscope OLYMPUS CX^[14] .

3.10) Thermodynamic Stability Studies:

During the thermodynamic stability of Nanoemulsions following stress tests as reported.^[14,30].

Heating cooling cycle:

Nanoemulsion formulations were subjected to six cycles between refrigerator temperature (4°C) and 45°C .Stable formulations were then subjected to centrifugation test.

Centrifugation:

Nanoemulsion formulations were centrifuged at 3500 rpm and those that did not show any phase separation were taken for the freeze thaw stress test.

Freeze thaw cycle:

In this the formulation were subjected to three freeze thaw cycles between 21°C and +25°C kept under standard laboratory conditions. These studies were performed for the Period of 3 months. Three batches of formulations were kept at accelerated temperature of 30°C, 40°C, 50°C and 60°C at ambient humidity. The samples were withdrawn at regular intervals of 0, 1, 2 and 3 months and were analyzed for drug content by stability-indicating HPLC method.

3.11) Morphology of Nanoemulsions:

The morphology of nanoemulsions can be determined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). SEM gives a three-dimensional image of the globules. The samples are examined at suitable accelerating voltage, usually 20 kV, at different magnifications. A good analysis of surface morphology of disperse phase in the formulation is obtained through SEM. Image analysis software, (e.g., Leica Imaging systems, Cambridge, UK), may be employed to obtain an automatic analysis result of the shape and surface morphology ^[30].

4). Application of nanoemulsion.

Nanoemulsions have found increasing use in many different applications. The advantages of nanoemulsions over conventional emulsions (or macroemulsions) are a consequence of their characteristic properties, namely small droplet size, high kinetic stability, and optical transparency. In addition, nanoemulsions offer the possibility of using microemulsion-like dispersions without the need for high surfactant concentrations.

4.1) Applications of Nanoemulsions in Cosmetics & personal care.

Nanoemulsions have recently become increasingly important as potential vehicles for the controlled delivery of cosmetics and for the optimized dispersion of active ingredients in particular skin layers. Due to their lipophilic interior, Nanoemulsions are more suitable for the transport of lipophilic compounds than liposomes. Similar to liposomes, they support the skin penetration of active ingredients and thus increase their concentration in the skin. Another advantage is the small sized droplet with its high surface area allowing effective transport of the active to the skin. Furthermore, Nanoemulsions gain increasing interest due to their own bioactive effects. This may reduce the trans-epidermal water loss (TEWL), indicating that the barrier function of the skin is strengthened. Nanoemulsions are acceptable in cosmetics because there is no inherent creaming, sedimentation, flocculation, or coalescence that is observed with macroemulsions. The incorporation of potentially irritating surfactants can often be avoided by using high energy equipment during manufacturing. Nanoemulsion can be applied for delivery of fragrance that often is incorporated in personal care product. Additionally, fragrances such as ester, aldehydes, and keton which are alcohol-free can be used in nanoemulsion formulation^[25-30].

4.2) Nanoemulsions in Biotechnology:

Many enzymatic and biocatalytic reactions are conducted in pure organic or aqua-organic media. Biphasic media are also used for these types of reactions. The use of pure a polar media causes the denaturation of biocatalysts. The use of waterproof media is relatively advantageous.

Enzymes in low water content display and have –

• Increased solubility in non-polar reactants.

• Possibility of shifting thermodynamic equilibria in favour of condensations.

• Improvement of thermal stability of the enzymes, enabling reactions to be carried out at higher temperatures.

Many enzymes, including lipases, esterases, dehydrogenases and oxidases often function in the cells in microenvironments that are hydrophobic in nature. In biological systems many enzymes operate at the interface between hydrophobic and hydrophilic domains and these usuall interfaces are stabilized by polar lipids and other natural amphiphiles. Enzymatic catalysis in Nanoemulsions has been used for a variety of reactions, such as synthesis of esters, peptides and sugar acetals transesterification; various hydrolysis reactions and steroid transformation. The most widely used class of enzymes in microemulsion-based reactions is of lipase^[30].

4.3) Nanoemulsions in Food Applications:

Nanoemulsion production for encapsulation and delivery of functional compounds is one of the emerging fields of nanotechnology applied to food industry. Application examples are given below. NutraLease, a technology start-up company established by a scientific team, is working to improve the bioavailability of functional compounds. Beverages containing encapsulated functional compounds such as coenzyme Q10, lycopene, lutein, β-carotene, omega-3, vitamins A, D3 and E, phytosterols and isoflavones are available (NutraLease 2011a). Their technology is based on self assembled nanoemulsions where a better encapsulation rate as well as an improved bioavailability in the human body can be achieved (Halliday 2007; NutraLease 2011b). NutraLease nanoemulsions can protect flavor compounds from manufacturing conditions and throughout the beverages’ shelf-life. It is claimed that nanoemulsions can capture the flavor and protect it from temperature, oxidation, enzymatic reactions and hydrolysis and are thermodynamically stable at a wide range of pH values (NutraLease 2011c). Other applications of nanoemulsions into the food industry include antimicrobial nanoemulsions for decontamination of food equipment, packaging or food (Center for Biological Nanotechnology 2001; Gruère et al. 2011). Also being evaluated is the possibility of delivering hydrophilic or hydrophobic compounds (functional compounds), in order to improve their solubility and bioavailability (Robinson and Morrison 2009). Despite of nanotechnology already being applied to the food industry, there is still a major gap in the regulatory framework, and most countries are still relying on existing legislation to regulate nanomaterials (Gruère et al. 2011). Improving the actual legislation framework is a crucial step to prevent consumers’ misinformation regarding nanotechnology applied to foods^[18].

4.4) Antimicrobial Nanoemulsions:

Antimicrobial nanoemulsions are oil-in-water droplets that range from 200-600 nm. They are composed of oil and water and are stabilized by surfactants and alcohol. The Nanoemulsion has a broad spectrum activity against bacteria (eg E.coli, Salmonella, and S.aureus), enveloped viruses (eg HIV, Herpes simplex), fungi (eg Candida, Dermatophytes) and spores (eg anthrax). The Nanoemulsion particles are thermodynamically driven to fuse with lipidcontaining organisms. This fusion is enhanced by the electrostatic attraction between the cationic charge of the emulsion and the anionic change of the pathogen. When enough nano particles fuse with the pathogens, the released part of the energy trapped within the emulsion. Both the active ingredient and the energy released destabilize the pathogen lipoid membrane, resulting in cell lysis and death^[31]. In the case of spores, additional germination enhancers are incorporated in to the emulsion. Once initiation of germination takes place, the germinating spores become susceptible to the antimicrobial action of the nanoemulsion. A unique aspect of the nanoemulsion is their selective toxicity to microbes at concentrations that are nonirritating to skin or mucous membrane. The safety margin of the Nanoemulsions is due to the low level of detergent in each droplet, yet when acting in concert, these droplets have sufficient energy and surfactant to destabilize the targeted microbes without damaging healthy cells. As a result the Nanoemulsions can achieve a level of topical antimicrobial activity that has only been previously achieved by systemic antibiotics. The nanoemulsion technology can be formulated into a cream, foam, liquid or spray to decontaminate a variety of materials. Marketed as NANOSTAT (Nano bio Corp).

4.5) Nanoemulsion as Non-Toxic Disinfectant Cleaner:

A breakthrough nontoxic disinfectant cleaner for use in commercial markets that include healthcare, hospitality, travel, food processing and military applications has been developed by Envirosystems, Inc. kills tuberculosis and a wide spectrum of viruses, bacteria and fungi in five to ten minutes without any of the hazards posed by other categories of disinfectants. The product needs no warning labels. It does not irritate eyes and can be absorbed through the skin, inhaled or swallowed without harmful effects.. The disinfectant formulation is made up of Nanospores of oil droplets <=106m which are suspended in water to create a nanoemulsion requiring only miniscule amounts of the active ingredient, PCMX (parachlorometaxylenol). The Nano spheres carry surface charges that efficiently penetrate the surface charges on microorganism’s membranes – much like breaking through an electric fence. Rather than ‘drowning’ cells, the formulation allows PCMX to target and penetrate cell walls. As s result, PCMX is effective at concentration levels one-to-two orders of magnitude lower than those of other disinfectants; hence there are no toxic effects on people, animals or the environment. Other microbial disinfectants require large doses of their respective active ingredients to surround pathogen cell wall, which cause them to disintegrate, fundamentally ‘drowning’ them in the disinfectant solution. The disinfectant is nonflammable and therefore safe to store most anywhere and also to use in unstable conditions. It is non-oxidizing, nonacidic & nonionic. It does not corrode plastic, metals or acrylic, making the product ideal for use on equipment and instruments. It is environmentally safe hence the costs and health risks associated with hazardous chemicals disposal are eliminated. The formulation is a broad-spectrum disinfectant cleaners that can be applied to any hard surface, including equipment, counters, walls, fixtures and floors. One product can now take the place of many, reducing product inventories and saving valuable storage space. Chemicals disposal costs can be eliminated and cleanings costs can be reduced. Marketed as EcoTru (Envirosystems, Inc)^[32].

4.6) Nanoemulsions in Cell Culture Technology:

Cell cultures are used for in vitro assays or to produce biological compounds, such as antibodies or recombinant proteins. To optimize cell growth, the culture medium can be supplemented with a number of defined molecules or with blood stream. Up to now, it has been very difficult to supplement the media with oil-soluble substances that are available to the cells, and only small amounts of these lipophilic compounds could be absorbed by the cells. Nanoemulsions are new method for the delivery of oil soluble substances to mammalian cell cultures. The delivery system is based on a nanoemulsion, which is stabilized by phospholipids. The nanoemulsions are transparent and can be passed through 0.1-1m filters for sterilization. Nanoemulsion droplets are easily taken up by the cells. The encapsulated oil-soluble substances therefore have a high bioavailability to cells in culture. The advantages of using nanoemulsions in cell culture technology are ^[32].

1) Better uptake of oil-soluble supplements in cell cultures.

2) Improve growth and vitality of cultured cells.

3) Allows toxicity studies of oil-soluble drugs in cell cultures

4.7) prophylactic In Bio-Terrorism Attack ^[33].

Based on their antimicrobial activity, research has began on use of nanoemulsions as a prophylactic medication, a human protective treatment, to protect people exposed to bio-attack pathogens such as Anthrax and Ebola. A broad-spectrum nanoemulsion was tested on surfaces by the US Army (RestOps) in Dec 1999 for decontamination of Anthrax spore surrogates. It was tested again by RestOps in March 2001 as a chemical decontamination agent. All tests were successful. The technology has been tested on gangrene and clostridium botulism spores and can even be used on contaminated wounds to salvage limbs. The nanoemulsion technology can be formulated into a cream, foam, liquid or spray to decontaminate a variety of materials. Marketed as NANOSTAT™ (Nanobio Corp.)

4.8 ) Nanoemulsions in fuel–emulsion :

Nano-Emulsion fuel is a special mixture of fuel with water combined with emulsifying and stabilizing additives, using nanotechnology. This technology creates homogeneously dispersed nano-sized water particles enclosed within a drop of oil, which when used in a combustion system, creates a water vapors explosion that disperses fuel particles into the superheated steam, thereby generating water-gas reaction. As the oxidized particles of the superheated vapors are very small, the reaction takes place instantaneously and smoothly. As a result, combustion is more efficient compared to conventional systems which deploy micro-sized water particles. This reduces fuel consumption as well as the amount of nitrogen dioxide and carbon dioxide produced into the air, without compromising the output of the existing engines, dynamos, boilers, and so on^[34].

4.9). Chemical Applications: Polymerization

One of the earliest applications of nano-emulsions was in the preparation of polymer latexes Ugelstad et al. ^[41], who introduced the term miniemulsions to designate this type of emulsion, found that the mechanism involved in miniemulsions polymerization was quite different from that of macroemulsion polymerization. They suggested that the main locus of nucleation was the monomer droplets instead of micelles^[41].The so-called miniemulsions polymerization is a broad term that is used to designate all polymerization processes performed in nano-emulsion (miniemulsions) media. However, it is also used in a more restrictive sense refer-ring to the polymerization of nano-emulsion droplets giving the same number of polymer particles with particle size distributions equal to those of the droplets. Several advantages of miniemulsions polymerization over conventional emulsion polymerization have been reported^[42]. It is considered to be a process more insensitive to variations in the composition or to the presence of impurities. The wide variations in the conversion rate and particle size obtained in a continuous macroemulsion polymerization process are highly reduced when performing continuous nanoemulsion polymerization ^[42]

4.10) Pharmaceutical Applications

The use of nanoemulsion in the Pharmaceutical industry, because aviabilty of the instrument to produce nanoemulsion. The characteristic properties of nano-emulsions (kinetic stability, small and controlled droplet size, etc.) make them interesting systems for pharmaceutical applications. Indeed, nano-emulsions are used as drug delivery systems for administration through various systemic routes. There are numerous publications on nano-emulsions as drug delivery systems for parenteral ^[39], oral , 40and topical administration, which includes the administration of formulations to the external surfaces of the body skin ^[36] and to the body cavities nasal ^[34,] as well as ocular administration ^[35,]. Moreover, many patents concerning pharmaceutical applications of nano-emulsions have been registered.

An application of nano-emulsions in this field has been in the development of vaccines ^[30]. Nano-emulsions are also interesting candidates for the delivery of drugs through the skin (topical administration). Positively and negatively charged submicrometer emulsions containing antifungal drugs (econazole nitrate and miconazole nitrate) have been described ^[36]. The positively charged submicrometer emulsions were more effective in terms of skin penetration of econazole or miconazole nitrate than negatively charged emulsions. Other nano-emulsions described for topical administration contain diazepam ^[37] as well as steroidal and nonsteroidal anti-inflammatory drugs ^[38].

5) Limitation of nanoemulsion

Although this formulation provide great advantages as a delivery system for the consumers but sometimes the reduced size of droplets are responsible for the limited use of nanoemulsion formulation. Some limitations of nanoemulsion are as follows ^[43].

• The manufacturing of nanoemulsion formulation is an expensive process because size reduction of droplets is very difficult as it required a special kind of instruments and process methods. For example, homogenizer (instrument required for the nanoemulsion formulation) arrangement is an expensive process. Again microfludization and ultrasonication (manufacturing process) require high amount of financial support.

• Stability of nanoemulsion is quite unacceptable and creates a big problem during the storage of formulation for the longer time period. Ostwald ripening is the main factor associated with unacceptability of nanoemulsion formulations. This is due to the high rate of curvature of small droplet show greater solubility as compared to large drop with a low radius of curvature.

• Less availability of surfactant and cosurfactant required for the manufacturing of nanoemulsion is another factor which marks as a limitation to nanoemulsion manufacturing.

6.) Commercial Nanoemulsions

In spite of some difficulties, certain nanoemulsion formulations have been translated into commercial products, available in the market for use. Some commercial nanoemulsion formulations are listed in Table [1] ^[43].

CONCLUSION:

It can be concluded that the application of nanoemulsions are burgeoning in the industry, due to. There are various techniques available to produce and characterize nanoemulsions; some of them have shown to be more suitable than others. In short, HPLC can be used for quantification of functional compounds, DLS may quickly determine the hydrodynamic diameter of nanoparticles in a nanoemulsion, zeta potential can indicate the stability of the nanoemulsions and TEM may be used to confirm the hydrodynamic diameter given by DLS technique and to have a general image of the nanoemulsion structure. Possible application of nanoemulsion formulation has been discussed in the paper, but these applications are limited by the instability of nanoemulsion. Stability of formulation may be enhanced by controlling various factors such as type and concentration of surfactant and co-surfactant, type of oil phase, methods used, process variables and addition of additives over the interfaces of nanoemulsion formulation. Overall nanoemulsion formulation may be considered as effective, safe, and patient compliance formulation.

REFERENCE

1. Tadros t., izquierdo p., esquena j., solans c., formation and stability of nanoemulsions, Colloid Interface Sci., 108, 2004, pp. 303-318.

2. Sadtler V., Rondon-Gonzalez M, Acrement A., Choplin A., Marie E., Peo-covered nanoparticles by emulsion inversion point (eip) method, Macromol. Rapid Commun., 31, 2010, pp. 998-1002.

3. M.S. El-Aasser, E.d. Sudol, Miniemulsions: Overview of research and applications, jct. Res. 1 (1) (2004) 21–31.

4. H. Nakajima, Microemulsions in cosmetics, in: C. Solans, H. Kunieda (Eds.), Industrial Applications of microemulsions, Marcel Dekker, New York, 1997, pp. 175– 197.

5. O. Sonneville-Aubrun, J.T. Simonnet, F. L’alloret, nanoemulsions a new vehicle for skincare products, adv. Colloid Interface Sci. 108– 109 (2004) 145–149.

6. S. Amselem, D. Friedman, Submicron emulsions as drug carriers for topical administration, in: s. Benita (ed.), submicron emulsions in drug targeting and delivery, Harwood Academic Publishers, London, 1998, pp. 153– 173.

7. O. Sonneville-Aubrun, J.-T. Simonnet, F. L’alloret, Nanoemulsions: a new vehicle for skincare products advances in Colloid and Interface Science 108 –109 (2004) 145–149.

8. P. Walstra, Emulsion stability, in: P. Becher (ed.), Encyclopedia of Emulsion Technology, Marcel Dekker, New York, 1996, pp. 1 – 62.

9. J. Floury, A. Desrumaux, M.A.V. Axelos, J. Legrand, Effect of high pressure homogenisation on methycellulose as food emulsifier, J. Food. Eng. 58 (2003) 227– 238.

10. K. Landfester, J. Eisenbla¨tter, R. Rothe, Preparation of polymerizable miniemulsions by ultrasonication, Jct Res. 1 (2004) 65– 68.

11. K. Shinoda, H. Saito, The effect of temperature on the phase equilibria and the types of dispersion of the ternary system composed of water, cyclohexane, and nonionic surfactant, J. Colloid Interface Sci. 26 (1968) 70– 74.

12. M.J. Rang, C.A. Miller, Spontaneous emulsification of oils containing hydrocarbon, nonionic surfactant, and oleyl alcohol, J. Colloid Interface Sci. 209 (1999) 179– 192.

13. T. Forster, W.V. Rybinski, applications of emulsions, in: B.P. Binks (ed.), Modern aspects of emulsion science, the royal society of chemistry, Cambridge, 1998, pp. 395–426.

14. Ahuja A Ali J, Baboota S, Faisal M.S, Shakeell F and Shafiq S: Stability evaluation of celecoxib nanoemulsion containing tween 80. Thai J. Pharm. Sci. 2008;32 :4-9.

15. Ali Javed Ahuja Alka, Baboota S, Shakeel F, Shafiq S: Design development and evaluation of novel nanoemulsion formulations for transdermal potential of celecoxib. Acta Pharm.2007; 57 315–33210.2478/v10007-007-0025-5.

16. Solans C, Esquena J, Forgiarini AM, Usón N, Morales D, Izquierdo P, Azemar N, Garcia-Celma MJ. Nano-emulsions: formation, properties and applications. Surfactant science series 2003; 109:525–54. First review on nano-emulsions obtained by low energy methods.

17. International union of pure and applied chemistry; Manual of Colloid Science. London: Butterworth; 1972.

18. Hélder Daniel Silva, Miguel Ângelo Cerqueira , António A. Vicente, Nanoemulsions for Food Applications: Development and Characterization Food Bioprocess Technol (2012) 5:854–867

19. K Shinoda, H Kunieda. J Colloid Interface Sci 42:381, 1973.

20. K Shinoda, H Kunieda. In: P Becher, ed. Encyclopedia of emulsion technology. Vol. 1. New York: Marcel Dekker, 1983, pp 337-367.

21. S. Benita, M. Y. Levy,J. Pharm. Sci.,1993, 82, 1069.

22. Tharwat F. Tadros Applied surfactants: principles and applications. ISBN: 3-527-30629-3 page 285-86.

23. Chen Huabing , Du Danrong , Mao Chengwen Mou Dongsheng, Wan Jiangling, Xu Huibi, Yang Xiangliang: Hydrogel-thickened nanoemulsion system for topical delivery of lipophilic drugs. International Journal of Pharmaceutics 2008; 353:272.

24. Pavankumar VK, Nanoemulasions, Pharmainfo.net; 2008.

25. Thompson.W ,Kelin L, On the equilibrium of vapour at curved surface of liquid,1871 Phil.Marg,42-448

26. SL Hari Kumar, Vishal Singh , Nanoemulsification - a novel targeted drug delivery tool, Journal of Drug Delivery & therapeutics; 2012, 2(4), 40-45.

27. Wang, L., R. Tabor, J. Eastoe, X. Li, K. Richard, et al., 2004. Formation and stability of nonoemulsion with mixed ionic-nonionic surfactants. Colloid and Interface Science, 108: 303-318.

28. Bhatt, P. and S. Madhav,. A detailed. Review on nanoemulsion drug delivery system. International Journal Pharmacy Science and Research,2011 2(9): 2292-2298.

29. Kamat, P.V., S.K. Pansare, S.R. Parakh, S.C. Jagdale, R.A. Patil, et al.. Pharmaceutical nanotechnology novel nanoemulsion-high energy concept of emulsification preparation, application. The Pharma Research, 2010,3: 117-138.

30. R.B. Desi Reddy, Ch.T. Lalitha Kumari, G. Naga Sowjanya, S.L. Sindhuri, P. Bandhavi, Nanoemulsions an emerging trend: A review ,IJPRD, 2011; Vol 4(06): August-2012 (137 – 152).

31. Bourne Dwa and Dittert LW. Chapter 3 in modern pharmaceutics 3rd ed., Banker GS and Rhodes.CT. Ed. Dekker. New York, 1996.

32. R. Rajalakshmi, K. Mahesh, C.K. Ashok Kumar , A critical review on nano emulsions, International Journal of Innovative Drug Discovery/ vol 1/ issue 1/ 2011/ 1-8.

33. S.C. kothekar, J.T. Waghmare, S.A. Momin, Rationalizing and producing nanoemulsion for personal care, Cosmetics & Toiletries July 2006 vol.121no.7

34. Gh Lowell, RW Kaminski, TC Vancott, B Silkie, K Kersey, E Zawoznik, L Loomis-Price, G Smith, DL Birx. J Infect Dis 175:292-301, 1997.

35. P Calvo, MJ Alonso, J Vila-Jato. J Pharm Sci 85:530-536, 1996.

36. MP Youenang Piemi, D Komer, S Benita, JP Marty. J Controlled Release 58(2):177-187, 1999

37. JS Schwartz, MR Weisspapir, DI Friedman. Pharm Res 12:687-692, 1995.

38. DI Friedman, JS Schwarz, MR Weisspapir. J Pharm Sci 84:324-329, 1995

39. Sharma, N., M. Bansal and S. Visht, 2010 Nanoemulsion: a new Concept of Delivery System, 1(2): 2-6.

40. Patel, P.D., G.J. Patel, P.D. Bharadia, V.M. Pandya and D.A. Modi, 2011. Nanoemulsion: an advance concept of dosage form. Journal and Cosmetology, 5(1): 122-133.

41. J Ugelstad, MS El-Aasser, JW Vanderhoff. J Polym Sci Polym Lett 11:503, 1973.

42. (a) FJ Schork, GW Poehlein, S Wang, J Reimers, J Rodrigues, C Samer. Colloids Surf a 153:39-45, 1999. (b) DT Barnette, FJ Schork. Chem Eng Prog 83:25, 1987.

43. Charles Lovelyn, Anthony A. Attama, Current State of nanoemulsions in Drug Delivery, Journal of Biomaterials and Nanobiotechnology, 2011, 2, 626-639.

Received on 25.02.2013 Accepted on 11.04.2013

Res. J. Topical and Cosmetic Sci. 4(1): Jan. –June 2013 page 32-40

Formulations. Drug/Bioactive	Brand Name	Manufacturer	Indication
Palmitate alprostadil	Liple	Mitsubishi Pharmaceutical, Japan	Vasodilator, platelet inhibitor
Dexamethason	Limethason	Mitsubishi Pharmaceutical, Japan	Steroid
Propofol	Diprivan	Astra Zaneca	Anaesthetic
Flurbiprofenaxtil	Ropion	Kaken Pharmaceutical, Japan	NSAID
Vitamins A, D, E and K	Vitalipid	Fresenius Kabi Europe	Parenteral nutrition