**4. Formulation of nanoemulsions**

#### **4.1. Materials used in preparation of nanoemulsions**

Nanoemulsions are prepared using oils, surfactants and co-surfactants and aqueous phase [59]. Oils used in nanoemulsions preparation include Captex 355, Captex 8000, Witepsol, Myritol 318, Isopropyl myristate, Capryol 90, Sefsol-218, triacetin, isopropyl myristate, castor oil, olive oil, etc. Solubility of the drug in the oil phase is an important criterion for the selection of oils. This is particularly important in the case of oral formulation development, as the ability of nanoemulsion to maintain the drug in solubilized form is greatly influenced by the solubility of the drug in the oil phase. While water-in-oil nanoemulsions are better choice for hydrophilic drugs lipophilic drugs are preferably solubilized in oil-in-water nanoemulsions. Drug loading in the formulation is a very critical design factor in the development of nanoemulsions for poorly soluble drugs, which is dependent on the drug solubility in various formulation components. An understanding of factors influencing drug loading capacity while maintain‐ ing the capability of the system to undergo monophasic dilution with water and minimizing the tendency for drug precipitation or crystallization in diluted systems is essential to the design of stable and appropriately low-volume nanoemulsion systems for drug delivery applications [60,61]. Edible oils are not frequently useful due to their poor ability to dissolve large amounts of lipophilic drugs. Moreover, formulation of nanoemulsion with oil of low drug solubility would require incorporation of more oil to incorporate the target drug dose, which in turn would require higher surfactant concentration to achieve oil solubilization, which might increase the toxicity of the system. Novel semi-synthetic medium chain deriva‐ tives (as amphiphilic compounds) having surfactant properties are progressively and effec‐ tively replacing the regular medium chain triglyceride oils [62,63].

Surfactants used for stabilizing nanoemulsions may be non ionic, zwitterionic, cationic and anionic. The surfactants may include Capryol 90, Gelucire 44/14, 50/13, Cremophor RH 40, Imwitor 191, 742, 780 k, 928, 988, Labrafil CS, M, 2125 CS, Lauroglycol 90, PEG MW > 4000, Plurol Oleique CC 497, Poloxamer 124 and 188, Softigen 701, 767, Labrasol, Cremophor EL, Tween 20, Tween 60, and Tween 80, etc. Components of nanoemulsion-based systems are associated with toxicity concerns. Large amounts of surfactants may cause gastrointestinal and skin irritation when administered orally and topically, respectively. Therefore, the proper selection of surfactants is essential. Rational use of the minimum concentration of the surfactant in the formulation is advocated. Nonionic surfactants are relatively less toxic than their ionic counterparts and typically have lower critical micelle concentration (CMCs). Also, o/w nanoemulsion dosage forms for oral or parenteral use based on nonionic surfactants are likely to offer *in vivo* stability [64]. Therefore, proper selection of surfactants is a crucial factor. Another important criterion is the selection of surfactant with proper hydrophile-lipophilebalance (HLB) value. Hydrophilic surfactants and co-surfactants are considered to prefer the interface and to lower the necessary energy to form the nanoemulsions, thereby improving the stability. For instance, the required HLB value to form o/w nanoemulsion is greater than 10 [65]. The right blend of low and high HLB surfactants leads to the formation of a stable nanoemulsion upon dilution with water. The type and nature of the surfactant is also an important factor for consideration; nonionic surfactants are usually selected since they are known to be less affected by pH and changes in ionic strength, are generally regarded as safe, and are biocompatible; ionic surfactants are less commonly used due to toxicological concerns. Solubilization of oil with the surfactant is also an important factor. It is not necessary that the same surfactant that has good solubilizing power for drugs would have equally good affinity for the oil phase. Surfactant–oil miscibility can thus give an initial indication on the possibility of nanoemulsion formation with this system.

Cosurfactants are added to obtain nanoemulsion systems at low surfactant concentration [66]. Short-to medium-chain-length alcohols (C3–C8) are commonly added as cosurfactants, which further reduce the interfacial tension and increase the fluidity of the interface [67,68]. They also increase the mobility of the hydrocarbon tail and allow greater penetration of the oil into this region. Alcohols may also increase the miscibility of the aqueous and oily phases due to its partitioning between these phases. Co-surfactants used in nanoemulsions include Transcutol P, glycerin, ethyleneglycol, ethanol, propanol, ethanol, isopropyl alcohol, n-butanol, PEG 400, Carbitol, and propylene glycol. Nanoemulsion area is often used as the assessment criterion for the evaluation of cosurfactants. The larger the size of the nanoemulsion field, the greater the nanoemulsification efficiency of the system.

Moreover, the most important criterion for selection of all the nanoemulsion components is that all the excipients should be pharmaceutically acceptable for oral administration or topical application, etc., depending upon the requirement and falling under GRAS category.

#### **4.2. Methods of preparation of nanoemulsions**

**•** Use of a large concentration of surfactant and cosurfactant necessary for stabilizing the nano

**•** Nanoemulsion stability is influenced by environmental parameters such as temperature and

**•** Lack of understanding of the mechanism of production of submicron droplets and the role

**•** Lack of demonstration of the benefits that can be obtained from using nanoemulsions when

**•** Lack of understanding of the interfacial chemistry that is involved in production of

Nanoemulsions are prepared using oils, surfactants and co-surfactants and aqueous phase [59]. Oils used in nanoemulsions preparation include Captex 355, Captex 8000, Witepsol, Myritol 318, Isopropyl myristate, Capryol 90, Sefsol-218, triacetin, isopropyl myristate, castor oil, olive oil, etc. Solubility of the drug in the oil phase is an important criterion for the selection of oils. This is particularly important in the case of oral formulation development, as the ability of nanoemulsion to maintain the drug in solubilized form is greatly influenced by the solubility of the drug in the oil phase. While water-in-oil nanoemulsions are better choice for hydrophilic drugs lipophilic drugs are preferably solubilized in oil-in-water nanoemulsions. Drug loading in the formulation is a very critical design factor in the development of nanoemulsions for poorly soluble drugs, which is dependent on the drug solubility in various formulation components. An understanding of factors influencing drug loading capacity while maintain‐ ing the capability of the system to undergo monophasic dilution with water and minimizing the tendency for drug precipitation or crystallization in diluted systems is essential to the design of stable and appropriately low-volume nanoemulsion systems for drug delivery applications [60,61]. Edible oils are not frequently useful due to their poor ability to dissolve large amounts of lipophilic drugs. Moreover, formulation of nanoemulsion with oil of low drug solubility would require incorporation of more oil to incorporate the target drug dose, which in turn would require higher surfactant concentration to achieve oil solubilization, which might increase the toxicity of the system. Novel semi-synthetic medium chain deriva‐ tives (as amphiphilic compounds) having surfactant properties are progressively and effec‐

Surfactants used for stabilizing nanoemulsions may be non ionic, zwitterionic, cationic and anionic. The surfactants may include Capryol 90, Gelucire 44/14, 50/13, Cremophor RH 40, Imwitor 191, 742, 780 k, 928, 988, Labrafil CS, M, 2125 CS, Lauroglycol 90, PEG MW > 4000,

**•** Limited solubility capacity for high melting substances.

compared with the classical macroemulsion systems.

**4.1. Materials used in preparation of nanoemulsions**

tively replacing the regular medium chain triglyceride oils [62,63].

of surfactants and cosurfactants.

90 Application of Nanotechnology in Drug Delivery

**4. Formulation of nanoemulsions**

droplets.

nanoemulsions.

pH.

As nanoemulsions are non-equilibrated systems [69-71], and so their preparation involves the input of a large amount of either energy or surfactants and in some cases a combination of both. As a result, high energy or low energy methods can be used in their formulation [70]. Although high energy emulsification method is traditionally used for the preparation of nanoemulsion formulation but low emulsion emulsification method now create an attraction due to their wide application and advantages as a formulation and stability aspects. Generally, energy is usually required in emulsion formulation because the process may be non-sponta‐ neous. The production of nanoemulsions costs more energy than that required to produce macroemulsions. Presence of surfactants help lower the surface tensions between oil and water. Small molecules such as non-ionic surfactants lower surface tension more than poly‐ meric surfactants such as poly (vinyl alcohol). Another important role of the surfactant is its effect on the interfacial dilatational modulus [72]. During emulsification an increase in the interfacial area takes place and this causes a reduction in surface excess. The equilibrium is restored by adsorption of surfactant from the bulk, but this takes time (shorter times occur at higher surfactant activity). Because of the lack or slowness of equilibrium with polymeric surfactants, dilatational modulus will not be the same for expansion and compression of the interface [72]. In practice, surfactant mixtures are used and these have pronounced effects on surface tension and dilatational modulus. Some specific surfactant mixtures give lower surface tension values than either of the two individual components. Polymer-surfactant mixtures may show some synergistic surface activity. An important role of the emulsifier is to prevent shearinduced coalescence during emulsification. The requirement is that the continuous phase has a significant excess of surfactant. This excess enables new surface area of the nano-scale droplets to be rapidly coated during emulsification, thereby inhibiting shear-induced coales‐ cence. This excess is generally in the form of surfactant micelles in the continuous phase. These micelles dissociate into monomers that rapidly adsorb onto the surfaces of newly created droplets [71].

*4.2.1.1. Phase Inversion Temperature (PIT) method*

This method employs temperature-dependent solubility of non-ionic surfactants, such as polyethoxylated surfactants, to modify their affinities for water and oil as a function of the temperature. It has been observed that polyethoxylated surfactants tend to become lipophilic on heating owing to dehydration of polyoxyethylene groups. This phenomenon forms a basis of nanoemulsion fabrication using the PIT method. In the PIT method, oil, water and nonionic surfactants are mixed together at room temperature. This mixture typically comprises o/w microemulsions coexisting with excess oil, and the surfactant monolayer exhibits positive curvature. When this macroemulsion is heated gradually, the polyethoxylated surfactant becomes lipophilic and at higher temperatures, the surfactant gets completely solubilized in the oily phase and the initial o/w emulsion undergoes phase inversion to w/o emulsion. The surfactant monolayer has negative curvature at this stage [75]. At an intermediate temperature (also termed HLB temperature), the non-ionic surfactant has similar affinity for aqueous and oily phase, and this ternary system has extremely low interfacial tension (in the order of 10−2– 10–5 mNm−1) and spontaneous curvature typically reaches zero [83,84]. The ternary system at this stage typically consists of a D-phase bicontinuous microemulsion or a mixture of a Dphase bicontinuous microemulsion and lamellar liquid crystalline phases. It has been observed that nanoemulsions with very small droplet size and polydispersity index can be generated by rapid cooling of the single-phase or multiphase bicontinuous microemulsions maintained at either PIT or a temperature above PIT (transitional-phase inversion) [76]. Nanoemulsions can also be generated by rapidly diluting the single bicontinuous microemulsions with the aqueous or oil phase (catastrophic phase inversion) to obtain either o/w nanoemulsion or w/o nanoemulsion. It has been observed that the characteristics of the nanoemulsion are mainly dependent on the structure of the surfactant at HLB temperature (bicontinuous or lamellar) and also on the surfactant/oil ratio. Initially, PIT method was believed to be useful for fabricating o/w nanoemulsions. However, in recent years, the application of the PIT method has been established for fabricating w/o emulsions and nanoemulsions. It is important to note that the use of lipophilic polyethoxylated surfactants and appropriate modifications in the

Nanoemulsions — Advances in Formulation, Characterization and Applications in Drug Delivery

http://dx.doi.org/10.5772/15371

93

typical PIT protocol are required for obtaining w/o nanoemulsions [82].

*4.2.1.3. Phase Inversion Composition Method (Self-nanoemulsification 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 [78-81].

This method generates nanoemulsions at room temperature without use of any organic solvent and heat. Forgirani *et al.* observed that kinetically stable nanoemulsions with small droplet size (~50 nm) can be generated by the stepwise addition of water into solution of surfactant in

*4.2.1.2. Solvent displacement method*

#### *4.2.1. Low energy methods*

As the name suggests, low-energy emulsification methods require low energy for the fabrica‐ tion of nanoemulsions. These methods are mainly dependent on modulation of interfacial phenomenon/phase transitions and intrinsic physicochemical properties of the surfactants, coemulsifiers/co-surfactants and oil to yield nano-sized emulsion droplets. The lower energy method, also called the condensation method, is based on the phase transitions taking place during the emulsification process [73,74]. These phase transitions result from changes in the spontaneous curvature of the surfactant and can be achieved (i) at constant composition by changing the spontaneous curvature of non-ionic surfactants with temperature, the wellknown Phase Inversion Temperature, PIT, widely used in industry [75,76] or (ii) at constant temperature by varying the composition of the system by the Emulsion Inversion Point (EIP) method [77-79]. In other words, low-energy emulsification method was developed according to the phase behavior and properties of the constituents, to promote the formation of ultrasmall droplets [80,81]. These low-energy techniques include self-emulsification, phase transition and phase inversion temperature methods [82]. The low energy method is interest‐ ing because it utilizes the stored energy of the system to form small droplets. This emulsifica‐ tion can be brought about by changing the parameters which would affect the hydrophilic lipophilic balance (HLB) of the system like temperature, composition, etc. [83,84]. The limitations include complexity, precise approach required and use of synthetic surfactants. In a nutshell, the most commonly used low-energy emulsification methods include:

#### *4.2.1.1. Phase Inversion Temperature (PIT) method*

due to their wide application and advantages as a formulation and stability aspects. Generally, energy is usually required in emulsion formulation because the process may be non-sponta‐ neous. The production of nanoemulsions costs more energy than that required to produce macroemulsions. Presence of surfactants help lower the surface tensions between oil and water. Small molecules such as non-ionic surfactants lower surface tension more than poly‐ meric surfactants such as poly (vinyl alcohol). Another important role of the surfactant is its effect on the interfacial dilatational modulus [72]. During emulsification an increase in the interfacial area takes place and this causes a reduction in surface excess. The equilibrium is restored by adsorption of surfactant from the bulk, but this takes time (shorter times occur at higher surfactant activity). Because of the lack or slowness of equilibrium with polymeric surfactants, dilatational modulus will not be the same for expansion and compression of the interface [72]. In practice, surfactant mixtures are used and these have pronounced effects on surface tension and dilatational modulus. Some specific surfactant mixtures give lower surface tension values than either of the two individual components. Polymer-surfactant mixtures may show some synergistic surface activity. An important role of the emulsifier is to prevent shearinduced coalescence during emulsification. The requirement is that the continuous phase has a significant excess of surfactant. This excess enables new surface area of the nano-scale droplets to be rapidly coated during emulsification, thereby inhibiting shear-induced coales‐ cence. This excess is generally in the form of surfactant micelles in the continuous phase. These micelles dissociate into monomers that rapidly adsorb onto the surfaces of newly created

As the name suggests, low-energy emulsification methods require low energy for the fabrica‐ tion of nanoemulsions. These methods are mainly dependent on modulation of interfacial phenomenon/phase transitions and intrinsic physicochemical properties of the surfactants, coemulsifiers/co-surfactants and oil to yield nano-sized emulsion droplets. The lower energy method, also called the condensation method, is based on the phase transitions taking place during the emulsification process [73,74]. These phase transitions result from changes in the spontaneous curvature of the surfactant and can be achieved (i) at constant composition by changing the spontaneous curvature of non-ionic surfactants with temperature, the wellknown Phase Inversion Temperature, PIT, widely used in industry [75,76] or (ii) at constant temperature by varying the composition of the system by the Emulsion Inversion Point (EIP) method [77-79]. In other words, low-energy emulsification method was developed according to the phase behavior and properties of the constituents, to promote the formation of ultrasmall droplets [80,81]. These low-energy techniques include self-emulsification, phase transition and phase inversion temperature methods [82]. The low energy method is interest‐ ing because it utilizes the stored energy of the system to form small droplets. This emulsifica‐ tion can be brought about by changing the parameters which would affect the hydrophilic lipophilic balance (HLB) of the system like temperature, composition, etc. [83,84]. The limitations include complexity, precise approach required and use of synthetic surfactants. In

a nutshell, the most commonly used low-energy emulsification methods include:

droplets [71].

*4.2.1. Low energy methods*

92 Application of Nanotechnology in Drug Delivery

This method employs temperature-dependent solubility of non-ionic surfactants, such as polyethoxylated surfactants, to modify their affinities for water and oil as a function of the temperature. It has been observed that polyethoxylated surfactants tend to become lipophilic on heating owing to dehydration of polyoxyethylene groups. This phenomenon forms a basis of nanoemulsion fabrication using the PIT method. In the PIT method, oil, water and nonionic surfactants are mixed together at room temperature. This mixture typically comprises o/w microemulsions coexisting with excess oil, and the surfactant monolayer exhibits positive curvature. When this macroemulsion is heated gradually, the polyethoxylated surfactant becomes lipophilic and at higher temperatures, the surfactant gets completely solubilized in the oily phase and the initial o/w emulsion undergoes phase inversion to w/o emulsion. The surfactant monolayer has negative curvature at this stage [75]. At an intermediate temperature (also termed HLB temperature), the non-ionic surfactant has similar affinity for aqueous and oily phase, and this ternary system has extremely low interfacial tension (in the order of 10−2– 10–5 mNm−1) and spontaneous curvature typically reaches zero [83,84]. The ternary system at this stage typically consists of a D-phase bicontinuous microemulsion or a mixture of a Dphase bicontinuous microemulsion and lamellar liquid crystalline phases. It has been observed that nanoemulsions with very small droplet size and polydispersity index can be generated by rapid cooling of the single-phase or multiphase bicontinuous microemulsions maintained at either PIT or a temperature above PIT (transitional-phase inversion) [76]. Nanoemulsions can also be generated by rapidly diluting the single bicontinuous microemulsions with the aqueous or oil phase (catastrophic phase inversion) to obtain either o/w nanoemulsion or w/o nanoemulsion. It has been observed that the characteristics of the nanoemulsion are mainly dependent on the structure of the surfactant at HLB temperature (bicontinuous or lamellar) and also on the surfactant/oil ratio. Initially, PIT method was believed to be useful for fabricating o/w nanoemulsions. However, in recent years, the application of the PIT method has been established for fabricating w/o emulsions and nanoemulsions. It is important to note that the use of lipophilic polyethoxylated surfactants and appropriate modifications in the typical PIT protocol are required for obtaining w/o nanoemulsions [82].

#### *4.2.1.2. 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 [78-81].

#### *4.2.1.3. Phase Inversion Composition Method (Self-nanoemulsification Method)*

This method generates nanoemulsions at room temperature without use of any organic solvent and heat. Forgirani *et al.* observed that kinetically stable nanoemulsions with small droplet size (~50 nm) can be generated by the stepwise addition of water into solution of surfactant in oil, with gentle stirring and at constant temperature [77]. Although the components used in the aforementioned investigation were not of pharmaceutical grade, the investigation opened doors to design pharmaceutically acceptable nanoemulsions using a similar approach. The spontaneous nano-emulsification has been related to the phase transitions during the emulsi‐ fication process and involves lamellar liquid crystalline phases or D-type bicontinuous microemulsion during the process [77].

**4.3. Formulation factors that affect the stability of nanoemulsions**

formulation for the delivery of pharmaceuticals.

molecules needed to stabilize the nano droplets.

**5. Characterization of nanoemulsions**

others [93]:

Although nanoemulsions enhance the physical as well as chemical stability of drugs, stability of drug product is one of the problems associated with the development of nanoemulsions [88-91]. Stability studies are performed on nanoemulsions by storing them at refrigerator and room temperatures over a number of months. The viscosity, refractive index and droplet size are determined during this period of storage. Insignificant changes in these parameters indicate formulation stability. Accelerated stability studies can also be performed on the nanoemulsions. In this instance, nanoemulsion formulation are kept at accelerated tempera‐ tures and samples withdrawn at regular intervals and analyzed for drug content by stability indicating assay methods. The amount of drug degraded and remaining in nanoemulsion formulation is determined at each time interval [92]. Stability of nanoemulsion formulation may be enhanced by controlling factors such as type and concentration of surfactant and cosurfactant, type of oil phase, methods used, process variables and addition of additives [88-93]. Overall nanoemulsion formulation may be considered as effective, safe and patient compliance

Nanoemulsions — Advances in Formulation, Characterization and Applications in Drug Delivery

http://dx.doi.org/10.5772/15371

95

Factors to be considered during preparation of nanoemulsion include the following among

**a.** The prime requirement in nanoemulsion production is that an ultra low interfacial tension should be attained at the oil water interface, so surfactants must be carefully chosen. **b.** Concentration of surfactant must be high enough to provide the number of surfactant

Characterization of nanoemulsions involves the physical and chemical tests related to oral liquid dosage forms which includes compatibility of the nanoemulsion components, isotro‐ picity of the formulation, assay, uniformity of content, appearance, pH, viscosity, density, conductivity, surface tension, size and zeta potential of the dispersed phase etc. with respect to the effect of the composition on physical parameters [94-104]. Differential scanning calo‐ rimetry (DSC) provides information on the interactions of different components and polari‐ zation microscopy using crossed polarizers is employed to confirm isotropicity of the formulation [99]. The process of self-nanoemulsification can be evaluated by visual assess‐ ment. Its efficiency would be estimated by determining the rate of emulsification and droplet size distribution. Turbidity measurements are carried out to determine the rapid equilibrium reached by the dispersion and reproducibility of this process. The droplet size of the emulsion is a crucial factor in self-nanoemulsification performance because it determines the rate and extent of drug release as well as absorption. Photon correlation spectroscopy (PCS) and light scattering techniques like static light scattering (SLS), dynamic light scattering (DLS) are a useful method for determination of nanoemulsion droplet size [100]. Viscosity, conductivity

**c.** The interface must be flexible to promote the formation of nanoemulsion.

#### *4.2.2. High energy methods*

High-energy emulsification methods make use of devices that use very high mechanical energy to create nanoemulsions with high kinetic energy. The high-energy method utilizes mechanical devices to create intensely disruptive forces which break up the oil and water phases to form nano-sized droplets. This can be achieved with ultrasonicators, microfluidiser and high pressure homogenisers [71,85,86]. Particle size here will depend on the type of instruments employed and their operating conditions like time and temperature along with sample properties and composition [87]. These methods include high-pressure homogeniza‐ tion and ultrasonic emulsification. High-pressure homogenization is the most common method used for the fabrication of nanoemulsions. During high-pressure homogenization, the coarse macroemulsion is passed through a small orifice at an operating pressure in the range of 500 to 5000 psi. During this process, several forces, such as hydraulic shear, intense turbu‐ lence and cavitation, act together to yield nanoemulsions with extremely small droplet size. The resultant product can be re-subjected to high-pressure homogenization until nanoemul‐ sion with desired droplet size and polydispersity index is obtained [71]. Micro-fluidization employs a high-pressure positive displacement pump operating at very high pressures, up to 20,000 psi. This pump forces macroemulsion droplets through the interaction chamber consisting of a series of micro-channels. The macroemulsion flowing through the microchan‐ nels collides with high velocity on to an impingement area resulting in very fine nanoemul‐ sions. The nanoemulsions with desired size range and dispersity can be obtained by varying the operating pressure and the number of passes through interaction chambers like highpressure homogenization. Ultrasonic emulsification uses a probe that emits ultrasonic waves to disintegrate the macroemulsion by means of cavitation forces. By varying the ultrasonic energy input and time, the nanoemulsions with desired properties can be obtained [85-87]. High-energy emulsification methods can be employed to fabricate both o/w and w/o nanoe‐ mulsions. High-pressure homogenization and microfluidization can be used for fabrication of nanoemulsions at laboratory and industrial scale, whereas ultrasonic emulsification is mainly used at laboratory scale. In addition, high-energy methods require sophisticated instruments and extensive energy input, which considerably increases the cost of nanoemulsions fabrica‐ tion. This is particularly significant in the pharmaceutical sciences [85-87]. High energy methods allow for a greater control of particle size and a large choice of composition, which in turn controls the stability, rheology and colour of the emulsion. Although high-energy emulsification methods yield nanoemulsions with desired properties and have industrial scalability, they may not be suitable for thermolabile drugs such as retinoids and macromo‐ lecules, including proteins, enzymes and nucleic acids. Moreover, high energy methods alone normally do not yield oil droplets (<100 nm).

#### **4.3. Formulation factors that affect the stability of nanoemulsions**

oil, with gentle stirring and at constant temperature [77]. Although the components used in the aforementioned investigation were not of pharmaceutical grade, the investigation opened doors to design pharmaceutically acceptable nanoemulsions using a similar approach. The spontaneous nano-emulsification has been related to the phase transitions during the emulsi‐ fication process and involves lamellar liquid crystalline phases or D-type bicontinuous

High-energy emulsification methods make use of devices that use very high mechanical energy to create nanoemulsions with high kinetic energy. The high-energy method utilizes mechanical devices to create intensely disruptive forces which break up the oil and water phases to form nano-sized droplets. This can be achieved with ultrasonicators, microfluidiser and high pressure homogenisers [71,85,86]. Particle size here will depend on the type of instruments employed and their operating conditions like time and temperature along with sample properties and composition [87]. These methods include high-pressure homogeniza‐ tion and ultrasonic emulsification. High-pressure homogenization is the most common method used for the fabrication of nanoemulsions. During high-pressure homogenization, the coarse macroemulsion is passed through a small orifice at an operating pressure in the range of 500 to 5000 psi. During this process, several forces, such as hydraulic shear, intense turbu‐ lence and cavitation, act together to yield nanoemulsions with extremely small droplet size. The resultant product can be re-subjected to high-pressure homogenization until nanoemul‐ sion with desired droplet size and polydispersity index is obtained [71]. Micro-fluidization employs a high-pressure positive displacement pump operating at very high pressures, up to 20,000 psi. This pump forces macroemulsion droplets through the interaction chamber consisting of a series of micro-channels. The macroemulsion flowing through the microchan‐ nels collides with high velocity on to an impingement area resulting in very fine nanoemul‐ sions. The nanoemulsions with desired size range and dispersity can be obtained by varying the operating pressure and the number of passes through interaction chambers like highpressure homogenization. Ultrasonic emulsification uses a probe that emits ultrasonic waves to disintegrate the macroemulsion by means of cavitation forces. By varying the ultrasonic energy input and time, the nanoemulsions with desired properties can be obtained [85-87]. High-energy emulsification methods can be employed to fabricate both o/w and w/o nanoe‐ mulsions. High-pressure homogenization and microfluidization can be used for fabrication of nanoemulsions at laboratory and industrial scale, whereas ultrasonic emulsification is mainly used at laboratory scale. In addition, high-energy methods require sophisticated instruments and extensive energy input, which considerably increases the cost of nanoemulsions fabrica‐ tion. This is particularly significant in the pharmaceutical sciences [85-87]. High energy methods allow for a greater control of particle size and a large choice of composition, which in turn controls the stability, rheology and colour of the emulsion. Although high-energy emulsification methods yield nanoemulsions with desired properties and have industrial scalability, they may not be suitable for thermolabile drugs such as retinoids and macromo‐ lecules, including proteins, enzymes and nucleic acids. Moreover, high energy methods alone

microemulsion during the process [77].

94 Application of Nanotechnology in Drug Delivery

normally do not yield oil droplets (<100 nm).

*4.2.2. High energy methods*

Although nanoemulsions enhance the physical as well as chemical stability of drugs, stability of drug product is one of the problems associated with the development of nanoemulsions [88-91]. Stability studies are performed on nanoemulsions by storing them at refrigerator and room temperatures over a number of months. The viscosity, refractive index and droplet size are determined during this period of storage. Insignificant changes in these parameters indicate formulation stability. Accelerated stability studies can also be performed on the nanoemulsions. In this instance, nanoemulsion formulation are kept at accelerated tempera‐ tures and samples withdrawn at regular intervals and analyzed for drug content by stability indicating assay methods. The amount of drug degraded and remaining in nanoemulsion formulation is determined at each time interval [92]. Stability of nanoemulsion formulation may be enhanced by controlling factors such as type and concentration of surfactant and cosurfactant, type of oil phase, methods used, process variables and addition of additives [88-93]. Overall nanoemulsion formulation may be considered as effective, safe and patient compliance formulation for the delivery of pharmaceuticals.

Factors to be considered during preparation of nanoemulsion include the following among others [93]:

