INTRODUCTION:

Nano-emulsions are transparent or translucent systems mostly covering the droplet size in the nanometric scale (typically in the range 20–200 nm). Nanoemulsions consist of mixture of immiscible liquids, where one of the liquids is dispersed in the form of nanometric scale droplets into the other one¹. Because of their small droplet sizes nanoemulsion appears transparent and is kinetically stable against sedimentation or creaming². The long-term physical stability of nano-emulsions (with no apparent flocculation or coalescence) make them unique and they are sometimes referred to as ‘Approaching Thermodynamic Stability’ .steric stabilization of nanoemulsions is affected by the ratio of the adsorbed layer thickness to droplet radius from which the stability can be understood. Unless adequately prepared and stabilized against Ostwald ripening nano-emulsions may lose their transparency with time as a result of increase in droplet size. Nanoemulsion properties depend not only on thermodynamic condition, preparation methods and order of addition of components³.

Three methods are used to prepare nano-emulsions (covering the droplet radius size range 50–200 nm): use of high-pressure homogenizers (aided by the appropriate choice of surfactants and cosurfactants), use of the low-energy emulsification method at constant temperature or application of the phase inversion temperature (PIT) concept⁴. Nanoemulsions have increased attention as drug delivery systems for poorly water soluble drugs because of their drug targeting effect ⁵.

Advantages:

1. Nano-emulsions can be prepared using lower surfactant concentration (3–10 %)

2. Nano-emulsions are suitable for efficient delivery of active ingredients through the skin,⁶ the large surface area, the low surface tension and the low interfacial tension of the O/W droplets allow enhancing penetration of actives agents.

3. Due to their small size, nano-emulsions can penetrate through the “rough” skin surface and this enhances penetration of actives.

4. The fluidity nature of the system (at low oil concentrations) as well as the absence of any thickeners may give them a pleasant aesthetic character and skin feel.

5. 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.

6. Nano-emulsions constitutes the primary step in nanocapsules and nanospheres synthesis using nanoprecipitation⁷ and the interfacial polycondensation combined with spontaneous emulsification^8,9

The attraction of nanoemulsions for application in pharmaceuticals is due to the following advantages:¹⁰

1. The very small droplet size causes a large reduction in the gravity force and the Brownian motion may be sufficient for overcoming 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 also prevent 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. Nanoemulsions are suitable for efficient delivery of active ingredients through the skin. The large surface area of the emulsion system allows rapid penetration of actives. Due to their small size, nano-emulsions can penetrate through the ‘rough’ skin surface and this enhances penetration of actives.

5. 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.

6. Unlike micro emulsions (which require a high surfactant concentration, usually in the region of 20 % and higher), nanoemulsions can be prepared using reasonable surfactant concentration. For a 20 % O/W nanoemulsion, a surfactant concentration in the region of 5–10 % may be sufficient.

7. The small size of the droplets allows them to deposit uniformly on substrates. Wetting, spreading and penetration may be also enhanced as a result of the low surface tension of the whole system and the low interfacial tension of the O/W droplets.

8. 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.

9. 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.

Components used in Nano-emulsion of formulation.

Choice of oil¹¹:

The influence of the oil nature on the emulsion size was studied using different kinds of oil in the organic phase. There is a wide difference in the average size of the emulsion drops according to the nature of the oil used. Nevertheless the high viscosity of oil is not a sufficient condition to obtain emulsions with small drops size, since hexyl laurate presents the lower viscosity (4.5–7.5 mPa s at 20ºC) and allowed to obtain Nano-emulsion with a small mean size (310 ±14 nm).

Choice of surfactants:¹¹

All surfactants cited in the literature have theoretical values of HLB from 1 to approximately 50. The more hydrophilic emulsifiers have HLB values greater than 10, while the more lipophilic emulsifiers have HLB values from 1 to 10.

X_a and X_b represent the weight proportions of lipophilic and hydrophilic surfactant, respectively. m_a and m_b representing the weight of lipophilic and hydrophilic surfactant, respectively,

Nano-emulsion size varied widely according to the surfactant system. The mean particle size decreased when the HLB value of the system increased with an o/w emulsion. Examples of liposoluble surfactants are lipoid S75, Span 80, span 85 and hydro soluble surfactants are pluronic F68, Tween 80 and Tween 20.

Solvent optimization:

Solvents which are totally or partially miscible with water were selected on the European Pharmacopoeia¹². There is a wide variety of solvents used in pharmaceutical processing. These solvents have been classified according to their toxicity on three classes.

Class I: Solvents to be avoided.

Class II: Solvents to be limited.

Class III: Solvents with low toxic potential.

For safety reasons, solvents of the Class III were chosen.

Table1: Water miscibility of Class III solvents according to the European Pharmacopoeia.

Solvents	Water miscibility
Acetone	Miscible
Ethanol	Miscible
Tetrahydrofuran	Very soluble
Methyl ethyl ketone	Very soluble
Methyl acetate	Very soluble
Ethyl acetate	Partially miscible

Types of emulsions:¹³

Single emulsions:

Emulsions are dispersed, multiphase systems consisting of at least two insoluble liquids. The dispersed phase is present in the form of droplets in a continuous phase. Depending on the emulsification process, the diameter of the droplets lies between 0.1µm and 0.1mm.Emulsions of this kind are thermodynamically unstable, which means that there is a tendency to reduce the interface (as a result of a relatively high interfacial tension), causing the droplets to coalesce and therewith decreasing the total amount of interface.

Double emulsions:

A double emulsion is an emulsion in an emulsion. Two main types of double emulsions can be distinguished: water-in-oil-in-water (W/O/W) emulsions, in which a W/O emulsion is dispersed as droplets in an aqueous phase, and oil-in-water-in-oil (O/W/O) emulsions, in which an O/W emulsion is dispersed in an oil phase. W/O/W emulsions are

Fig.1. Types of emulsions and their preparation.

more common than O/W/O emulsions. Double emulsions contain more interfaces and are even more thermodynamically unstable than single emulsions.

Usually double emulsions are prepared in a two-step emulsification process using two surfactants; a hydrophobic one designed to stabilize the interface of the W/O internal emulsion and a hydrophilic one for the external interface of the oil globules (for W/O/W emulsions). The primary W/O emulsion is prepared under high-shear conditions to obtain small droplets while the secondary emulsification step is carried out with less rate of shear to avoid rupture of the internal droplets.

Physical properties of nanoemulsions¹⁴

Nanoemulsions have many interesting physical properties that are different from or are more extreme than those of larger microscale emulsions.

ü The relative transparency of nanoemulsions

ü Their response to mechanical shear or ‘rheology’ and

ü The enhanced shelf stability of nanoemulsions against gravitationally driven creaming.

The relative transparency of nanoemulsions¹⁴

Nanoemulsions appear visibly different from microscale emulsions since the droplets can be much smaller than optical wavelengths of the visible spectrum. Microscale emulsions multiply scatter visible light, and unless the refractive index of the continuous and dispersed phases is matched by specifically altering the composition to achieve this, they appear white. By contrast, nanoemulsions can appear nearly transparent in the visible spectrum and exhibit very little scattering despite significant refractive index contrast. Quantitative measurements of the optical transparency of nanoemulsions in the visible and ultraviolet wavelengths are shown through transmission measurements in Fig.2.

Fig.2. Difference between Micro scale emulsion and Nanoemulsion

Their response to mechanical shear or ‘rheology^’14

Some mechanical shear or ‘rheological’ properties of nanoemulsions are also affected by the nanoscale size of the droplets. In micro scale emulsions, the rheological properties depend strongly on whether the droplets interact repulsively or attractively. For very dilute φ, the shear viscosity of repulsive nanoemulsions resembles that of microscale emulsions or even hard spheres. The rheology of attractive Nanoemulsions systems is only now being explored, so little information is available compared to that of microscopic emulsions.

The enhanced shelf stability of nanoemulsions against gravitationally driven creaming:¹⁴

Nanoemulsions exhibit enhanced shelf stability against gravitationally driven creaming over microscale emulsions at the same φ. Brownian motion, caused by entropic driving forces, keeps the droplets suspended even over very long periods of time.

Mechanism of emulsification:¹⁵

To prepare an emulsions oil, water, surfactant and energy are needed. This can be considered from a consideration of the energy required to expand the interface, ΔAγ (where ΔA is the increase in interfacial area when the bulk oil with area A₁ produces a large number of droplets with area A₂; A₂>> A₁, γ is the interfacial tension). Since g is positive, the energy to expand the interface is large and positive. This energy term cannot be compensated by the small entropy of dispersion TDS (which is also positive) and the total free energy of formation of an emulsion, DG is positive,

ΔG= ΔA γ - T ΔS

Thus, emulsion formation is non-spontaneous and energy is required to produce the droplets. The formation of large droplets (few micrometers) as is the case for macroemulsions is fairly easy and hence high speed stirrers such as the Ultraturrax or Silverson Mixer is sufficient to produce the emulsion. In contrast the formation of small drops (submicron as is the case with nano-emulsions) is difficult and this requires a large amount of surfactant and/or energy. The high energy required for formation of Nanoemulsions can be understood from a consideration of the Laplace pressure p (the difference in pressure between inside and outside the droplet,

P= γ [1/R₁+ 1/R₂]

Where, R₁and R₂are the principal radii of curvature of the drop.

For a spherical drop, R₁=R₂=R and,

Surfactants play major roles in the formation of nanoemulsions: By lowering the interfacial tension, p is reduced and hence the stress needed to break up a drop is reduced. Surfactants prevent coalescence of newly formed drops.

Various processes occurring during emulsification, namely break up of droplets, adsorption of surfactants and droplet collision (which may or may not lead to coalescence occur. To assess nanoemulsion formation, one usually measures the droplet size distribution using dynamic light scattering techniques (photon correlation spectroscopy, PCS). In this technique, one measures the intensity fluctuation of scattered light by the droplets as they undergo Brownian motion.

Methods of emulsification and the role of surfactants:

With nanoemulsions, however, a higher power density is required and this restricts the preparation of nano-emulsions to the use of high pressure homogenisers and ultrasonics.

An important parameter that describes droplet deformation is the Weber number, W, which e gives the ratio of the external stress Gη (where G is the velocity gradient and η is the viscosity) over the Laplace pressure¹⁶

The droplet deformation increases with increase in the Weber number, which means that for producing small droplets one requires high stresses (high shear rates). In other words, the production of nano-emulsions costs more energy than that required to produce macroemulsion¹⁷. Surfactants lower the interfacial tension γ and this causes a reduction in droplet size, latter decrease with decrease in γ. For Turbulent Inertial regime, the droplet diameter is proportional to γ^3/5. The amount of surfactant required to produce the smallest drop size will depend on its activity a (concentration) in the bulk which determines the reduction in g, as given by the Gibbs adsorption equation,

-d γ = RT Гdln a

Where R is the gas constant, T is the absolute temperature and Г is the surface excess (number of moles adsorbed per unit area of the interface).

Another important role of the surfactant is its effect on the interfacial dilational modulus^18,
19

During emulsification an increase in the interfacial area ‘a’ takes place and this causes a reduction in Г.

Apart for their effect on reducing g, surfactants play major roles in deformation and break-up of droplets. Surfactants allow the existence of interfacial tension gradients, which is crucial for formation of stable droplets.

Gibbs–Marangoni effect is the depletion of surfactant in the thin film between approaching drops results in g-gradient without liquid flow being involved. These results in an inward flow of liquid that tends to drive the drops apart. The Gibbs–Marangoni effect also explains the Bancroft rule, which states that the phase in which the surfactant is most soluble form the continuous phase. If the surfactant is in the droplets, a g-gradient cannot develop and the drops would be prone to coalescence. Thus, surfactants with HLB) 7 tend to form O/W emulsions and HLB-7 tend to form W/O emulsions. The Gibbs– Marangoni effect also explains the difference between surfactants and polymers for emulsification. Polymers give larger drops when compared with surfactants Various other factors should also be considered for emulsification, the most important is the disperse phase volume fraction w. Increase in w leads to increase in droplet collision and hence coalescence during emulsification.

Ostwald ripening:

One of the main problems with nano-emulsions is Ostwald ripening which results from the difference in solubility between small and large droplets.

Theoretically, Ostwald ripening should lead to condensation of all droplets into a single drop (i.e. phase separation). This does not occur in practice since the rate of growth decreases with increase of droplet size. For two droplets of radii r1and r₂(where r₁<r₂),

Above equation shows that the larger the difference between r₁and r₂, the higher the rate of Ostwald ripening.

The Lifshitz–Slezov and Wagner (LSW)^{20, 17} theory predicts a linear relationship between the cube of the radius, r3, and time, t, with the slope being the Ostwald ripening rate. The LSW theory assumes that the droplets of the dispersed phase are spherical, the distance between them is higher than the droplet diameter and the kinetics is controlled by molecular diffusion of the dispersed phase in the continuous phase. According to this theory, the Ostwald ripening rate in O/W emulsions is directly proportional to the solubility of the oil in the aqueous phase.

Several methods may be applied to reduce Ostwald ripening:^22-24

(i) Addition of a second disperse phase component, which is insoluble in the continuous phase (e.g. squalene). During Ostwald ripening in two component disperse phase system, equilibrium is established when the difference in chemical potential between different size droplets (which results from curvature effects) is balanced by the difference in chemical potential resulting from partioning of the two components.

(ii) Modification of the interfacial film at the O/W interface: using surfactants, which are strongly adsorbed at the O/W interface (i.e. polymeric surfactants) and which do not desorb during ripening, the rate could be significantly reduced²⁵.

Preparation of nano-emulsions:^{26, 27}

Three methods may be applied for the preparation of Nano-emulsions (covering the droplet radius size range 50–200 nm).

1. Use of high pressure homogenizers:

This technique makes use of high-pressure homogenizer /piston homogenizer to produce nanoemulsions of extremely low particle size (up to 1nm). In a high-pressure homogenizer, the dispersion of two liquids (oily phase and aqueous phase) is achieved by forcing their mixture through a small inlet orifice at very high pressure (500 to 5000 psi), which subjects the product to intense turbulence and hydraulic shear resulting in extremely fine particles of emulsion. Homogenizers of varying design are available for lab scale and industrial scale production of nanoemulsions. This technique has great efficiency, the only disadvantage being high energy consumption and increase in temperature of emulsion during processing.

Fig.3. The production of small droplets (submicron) requires application of high energy.

2. Low energy emulsification methods:²⁸

A study of the phase behaviour of watery oily surfactant systems demonstrated that emulsification can be achieved by three different low energy emulsification methods , as schematically shown in Fig. 1 (a) stepwise addition of oil to a water surfactant mixture; (b) stepwise addition of water to a solution of the surfactant in oil; and (c) mixing all the components in the final composition, pre-equilibrating the samples prior to emulsification Nano-emulsions with droplet sizes of the order of 50 nm were formed only when water was added to mixtures of surfactant and oil (method b).

Fig.4. Low energy emulsification methods.

3. Phase inversion temperature (PIT) principle:²⁹

Phase inversion in emulsions can be one of two types: Transitional inversion induced by changing factors which affect the HLB of the system, e.g. temperature and / or electrolyte concentration. Catastrophic inversion is induced by increasing the volume fraction of the disperse phase. Transitional Inversion can also be induced by changing the HLB number of the surfactant at constant temperature using surfactant mixtures.

When an O/W emulsion is prepared using a non-ionic surfactant of the ethoxylate type is heated, then at a critical temperature (the PIT), the emulsion inverts to a W/O emulsion. At the PIT the droplet size reaches a minimum and the interfacial tension also reaches a minimum. However, the small droplets are unstabless and they coalesce very rapidly. By rapid cooling of the emulsion that is prepared at a temperature near the PIT, very stable and small emulsion droplets could be produced.

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 generators³⁰. It has been shown that the apparatus supplying the available energy in the shortest time and having the most homogeneous flow produces the smallest sizes³¹. 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. Pressure as high as 350 MPa have been achieved in a recently developed instrument ³². Ultrasonic emulsification is also very efficient in reducing droplet size but it is only appropriate for small batches.

By taking advantage of the physicochemical properties of the system, dispersions can be produced almost spontaneously, with the condensation or low-energy emulsification methods that make use of the phase transitions taking place during the emulsification process³³. The phase inversion temperature (PIT) method, introduced by Shinoda and Saito is, among these methods, the most widely used in industry ³⁴. It is based on the changes in solubility of polyoxyethylene- type nonionic surfactants with temperature. These types of surfactants become lipophilic with increasing temperature because of dehydration of the polyoxyethylene chains.

The PIT emulsification method takes advantage of the extremely low interfacial tensions achieved at the HLB temperature, of the order of 10^-2–10^-5mNm^-1, to promote emulsification. At the HLB temperature, although emulsification is favored, the emulsions are very unstable. By rapidly cooling or heating (by about 25–30ºC) the emulsions prepared at the HLB temperature, kinetically stable emulsions (O/W or W/O, respectively) can be produced with very small droplet size and narrow size distribution. If the cooling or heating process is not fast, coalescence predominates and polydisperse coarse emulsions are formed³⁵.

CHARACTERIZATION OF NANOEMULSION^{36, 37}

Droplet size analysis:

Droplet size distribution of the Nanoemulsions was determined by photon correlation spectroscopy (PCS), Light scattering was monitored at 25º C at a scattering angle of 90º.

Viscosity determination:

The viscosity of the Nanoemulsions was determined using Brookfield DV III ultra V Cone and plate rheometer at 25±0.3º C.

Refractive index:

Refractive index of Nanoemulsions formulation was determined using an Abbe’s refractrometer.

Transmission electron microscopy:

The morphology and structure of the Nano-emulsion were studied using transmission electron microscopy (TEM). A Combination of bright Field Imaging at increasing magnification and of Diffraction modes was used to reveal the form and size of the nanoemulsions. To perform the TEM observations, the Nano-emulsion formulation was diluted with water (1/100). A drop of the diluted Nanoemulsions was directly deposited on the holey film grid and observed after drying.

Fig.5. TEM Photograph of Nanoemulsion

Scanning Electron Microscopy (SEM) ⁵

Samples were fixed on an SEM stub using conductive double sided tape and then made electrically conductive by coating in a vaccum with a thin layer of gold or palladium. An accelerating voltage of 15 KV was used.

Fig. 6. SEM photograph of nanoemulsion

Stability studies:

Stability studies on optimized Nanoemulsions were performed by keeping the sample at refrigerator temperature (4ºC) and room temperature (25ºC). These studies were performed for the period of 3 months. The droplet size, viscosity and RI were determined using methods described above during storage. Three batches of formulations were taken in glass vials and were kept at accelerated temperature of 30ºC, 40 ºC, 50ºC and 60ºC at ambient humidity. The samples were withdrawn at regular intervals of 0, 1, 2and 3 months and were analyzed for drug content by stability-indicating HPLC method at a wavelength of 250 μm³⁸. Zero time samples were used as controls. Analysis was carried out at each time interval by taking 50 μl of each formulation and diluting it to 5 ml with methanol and injecting into the HPLC system at 250 nm. In addition, samples of pure oil pure surfactant and cosurfactant were run separately to check there was no interference of the excipients used in the formulations. The amount of drug degraded and the amount remaining at each time interval was calculated. Order of degradation was determined by the graphical method. Degradation rate constant (K) was determined at each temperature. Arrhenius plot was constructed between log K and 1/T to determine the shelf life of optimized Nanoemulsions formulation. The degradation rate constant at 25ºC (K25) was determined by extrapolating the value of 25ºC from Arrhenius plot. The shelf life (T0.9) for each formulation was determined by using the formula.

Direct application of nano-emulsions in final products:³⁹

The main application for nano-emulsions is their stability. The small droplet size makes nano-emulsions break by the Ostwald ripening mechanism⁴⁰ in time periods which pose a great limitation for developing applications different than nanoparticle preparation. In fact, only an extremely low solubility of the dispersed phase, as presented by silicone oils, would give the stability needed for most of the applications. Nano-emulsions with silicone oils as dispersed phase,^41-43, show great stability even for such a high concentration that droplets are deformed to a foam-like structure⁴⁴.

Nano emulsion formulations are studied for their application as nanocarriers which allow the treatment of a variety of diseases. The following are examples of the most recent proposals of drugs solubilized in nano-emulsions for disease treatments: anticonvulsant⁴⁵, antihypertensive⁴⁶, antibiotic⁴⁷, anti-inflammatory applied through skin⁴⁸ There are reports on: drugs solubilized in nano-emulsions for HIV/AIDS therapy⁴⁹; mechanisms of atherogenesis studied with cholesterol nano-emulsions⁵⁰; cancer therapy investigated by solubilizing the drug in a cholesterol rich nano-emulsion⁵¹; intestinal absorption of three model drugs solubilized in nano-emulsions⁵²; efficacy of a schistosomicidal compound solubilized in nano-emulsions⁵³; and application of anthrax vaccine through W/O nanoemulsion⁵⁴.

Special magnetic nano-emulsions are also being studied for medicine applications^55-57. A recent review shows extensively potential multifunctional applications of nanocarriers including nano-emulsions in pharmacy. “Multifunctional nanocarriers⁵⁸ could provide almost unlimited opportunities in producing highly efficient and specialized systems for drugs, genes and diagnostic agents”. Antimicrobial generic activity⁵⁹ of nanoemulsion is also being investigated with W/O nanoemulsions that are diluted in water just before application reverse to O/W emulsions. More specifically, inactivation of Ebola virus⁶⁰ by nano-emulsion was studied with promising results. It was concluded that nano-emulsions could be used as disinfectants.

Nano-emulsions as drug delivery systems:

Nano-emulsions are used as drug delivery systems for administration through various systemic routes. Parenteral (or injectable) administration of Nano-emulsions is employed for a variety of purposes, namely, nutrition (e.g. administration of fats, carbohydrates, vitamins, etc.), controlled drug release and targeting of drugs to specific sites in the body, delivery of vaccines or as gene carriers^{61, 62}. Nano-emulsions are advantageous for intravenous administration, due to the strict requirements of this route of administration, particularly the necessity for a formulation droplet size lower than 1 μm⁶³. The benefit of Nano-emulsions in the oral administration of drugs has been also reported⁶⁴ and the absorption of the emulsion in the gastrointestinal tract has been correlated to their droplet size. Nano-emulsions are also used as ocular delivery systems to sustain the pharmacological effect of drugs in comparison with their respective solutions^{61, 65}. Cationic Nano-emulsions were evaluated as DNA vaccine carriers to be administered by the pulmonary route⁶⁶. They are also interesting candidates for the delivery of drugs or DNA plasmids through the skin after topical administration^{67, 68}

CONCLUSION:

The study of basic and applied aspects of Nanoemulsions is receiving increasing attention in recent years. Dispersion of high energy emulsification methods are traditionally used for Nanoemulsions formation. Nanoemulsions are proposed for numerous applications in pharmacy as drug delivery systems because of their capacity of solubilizing nonpolar active compounds. Thus the use of Nanoemulsions as formulations for active delivery and targeting is also an active and interesting application of nanoemulsion. In the future, we predict that nanoemulsions will become as ubiquitous as many polymer solutions and solid particulate dispersions are today.

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Received on 13.11.2009 Modified on 11.01.2010

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 319-326