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

2.1. Self emulsifying formulations (SEFs)

Self-emulsifying formulations (SEFs) are mixtures of oil, surfactant, co-surfactant, and co-solvents (absence of external phase water) and forms a transparent isotropic solution, which emulsify under gentle agitation similar to those which would be encountered in gastro intestinal tract (GIT). It has been recognized that this formulation when administered orally undergo spontaneous emulsification in aqueous GI fluids [6]. This emulsified oil (triglycerides) stimulates bile secretion and drug containing oil droplets are further emulsified by bile salts. Lipid droplets are then metabolized by lipases and co lipases, secreted from the salivary gland, gastric mucosa and pancreas, which also hydrolyze the triglycerides into di-and monoglycerides and free fatty acids. Further, solubilization of these molecules occurs during the passage through the GI tract and eventually forms a range of emulsion droplets, vesicular structures and mixed micelles containing bile salts, phospholipids and cholesterol [6]. Upon mixing with water the system SEFs have an ability to form fine colloidal droplets with very high surface area. In many cases, this accelerates the digestion of the lipid formulation, improves absorption, and reduces food effect and inter-subject variability [7]. Self emulsifying formulations distribute readily in the GI tract, the digestive motility of the stomach and the intestines provides sufficient agitation enough for the spontaneous formation of emulsions [8,9].

SEFs prepared using surfactants of HLB < 12 possess high stability and improved dissolution (for poorly soluble drugs) due to enhancement in surface area on dispersion [6]. Therefore, their absorption is independent of bile secretion and ensures a rapid transport of poorly soluble drugs into the blood [6].

According to Reiss [10], self emulsification occurs when the entropy changes that favor dispersion is greater than the energy required to increase the surface area of the dispersion [10]. The free energy of the conventional emulsion is a direct function of the energy required to create a new surface between the oil and water phase and can be described by the equation:

where, ΔG is the free energy associated with the process, r_i is the radius of droplets, N_i is the number of droplets, σ is the interfacial energy [11]. The two phases of the emulsion tend to separate with time to reduce the interfacial area and thus, minimize the free energy of the system(s). The conventional emulsifying agents stabilize emulsions resulting from aqueous dilution by forming a monolayer around the emulsion droplets, reducing the interfacial energy and forming a barrier to coalescence. On the other hand, emulsification occurs spontaneously with SEDDS, as the free energy required to form the emulsion is low, whether positive or negative [12]. For emulsification to take place, it is vital for the interfacial structure to offer negligible or no resistance against surface shearing [13]. The ease of emulsification has been suggested to be related to the ease of water penetration into various liquid crystals or gel phases formed on the surface of the droplet [14-16]. The interface between the oil and aqueous continuous phases is formed upon addition of a binary mixture (oil/non-ionic surfactant) to water [14]. This is followed by solubilization within the oil phase, as a result of aqueous penetration through the interface. Invariably, this tends to occur until the solubilization limit is attained close to the interphase. Further, aqueous penetration will lead to the formation of the dispersed liquid crystal phase. Ultimately, everything that is in close proximity with the interface will be liquid crystal, the actual amount of which depends upon the emulsifier concentration in the binary mixture. Hence, following gentle agitation of the self-emulsifying system, water rapidly penetrates into the aqueous cores leading to interface disruption and droplet formation [16].

When compared with emulsions, which are sensitive and metastable dispersed forms, SEFs are physically stable formulations that are easy to manufacture. Thus, for lipophilic drugs that exhibit dissolution rate-limited absorption, these systems may offer an improvement in the rate and extent of absorption and result in more reproducible blood time profiles [17]. SEFs have been transformed into solid dosage forms using techniques such as melt granulation, where the lipid excipient acts as a binder and solid granules are produced on cooling. Solvents or supercritical fluids can be used with semisolid excipients, which are solubilized and then the solvent evaporated to produce a waxy powder. Spraying techniques can be used to produce powder formulations. These techniques enable the production of granules or powders that can then be compressed into a tablet form or filled into capsules. In all cases, the lipid excipients used must be semi‐solid at room temperature [18].

3.1. Major challenges

Although nanoemulsions provide great advantages as a delivery system, however they suffer for some major challenges and limitations which include [56-58]:

• The formulation of nanoemulsions is an expensive process due to 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 nanoemulsions formulation) arrangement is an expensive process. More ever micro-fluidization and ultrasonication (manufacturing process) require large amount of financial support.

• One problem associated with nanoemulsion is their stability. Although it is generally accepted that these systems could remain stable even by years, however, due to the small droplet size, it has been reported that the Oswald ripening could damage nanoemulsions, causing their application to be limited. Therefore, in most cases, nanoemulsions are required to be prepared shortly before their use.

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

• Limited solubility capacity for high melting substances.

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

• Lack of understanding of the mechanism of production of submicron droplets and the role of surfactants and cosurfactants.

• Lack of demonstration of the benefits that can be obtained from using nanoemulsions when compared with the classical macroemulsion systems.

• Lack of understanding of the interfacial chemistry that is involved in production 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 maintaining 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 derivatives (as amphiphilic compounds) having surfactant properties are progressively and effectively 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-lipophile-balance (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

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-spontaneous. 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 polymeric 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 shear-induced 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 coalescence. 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].

4.3. Formulation factors that affect the stability of nanoemulsions

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 temperatures 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 co-surfactant, 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]:

1. 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.

2. Concentration of surfactant must be high enough to provide the number of surfactant molecules needed to stabilize the nano droplets.

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

6.1. Applications in cosmetics

Recently importance of nanoemulsions have become increasing as good 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 drug than liposomes Similar to liposomes, nanoemulsions supports the skin penetration of active ingredients and thus increases their concentration in the skin. Another advantage is the small-sized droplet with its high surface area permit effective delivery of the active to the skin. More ever, nanoemulsions gain increasing interest due to their own bioactive effects. This may reduce the trans-epidermal water loss (TEWL), suggesting that the barrier function of the skin is strengthened. Nanoemulsions are acceptable in cosmetics because there is no chance of creaming, sedimentation, flocculation or coalescence, which is observed within microemulsions. The incorporation of potentially irritating surfactants can be avoided by using high-energy equipment during manufacturing process. PEG-free nanoemulsions for cosmetics has also been developed and formulations exhibited good stability [56-58,115,116].

6.2. Antimicrobial nanoemulsions

Antimicrobial nanoemulsions are o/w droplets that range from 200-600 nm. They are made of oil and water and are stabilized by surfactants and alcohol. The nanoemulsions has a broad spectrum of activity against bacteria like E. coli, salmonella, S. aureus; enveloped viruses like HIV, herpes simplex; fungi like candida, dermatophytes, and spores like anthrax. The nanoemulsions particles are thermodynamically driven to fuse with lipid-containing organisms. This fusion is enhanced by the electrostatic attraction between the cationic charge of the emulsion and the anionic charge on the pathogen. When enough nanoparticles fuse with the pathogens, they release part of the energy trapped within the emulsion. Both the active ingredient and the energy released destabilize the pathogen lipid membrane, resulting in cell lysis and death. In the case of spores, additional germination enhancers are added into the emulsion. Once starting of germination takes place, the germinating spores become susceptible to the antimicrobial action of the nanoemulsions. An aspect of the nanoemulsions is their highly selective toxicity to microbes at concentration range that are non-irritating to skin or mucous membrane. The safety range of nanoemulsions is because of the low amount of detergent in each droplet, yet when acting in concert, these droplets have enough energy and surfactant to destabilize targeted microbes without affecting healthy cells. Nanoemulsions can get a level of topical antimicrobial activity, which can only be previously achieved by systemic antibiotics [56-58,115].

6.3. Prophylactic in bio-terrorism attack

Because of their antimicrobial activity, research has begun on use of nanoemulsions as a prophylactic medicated dosage form, a human protective treatment, to prevent the people exposed to bio-attack such as Anthrax and Ebola. The broad-spectrum nanoemulsions were checked on surfaces by the US Army (RestOps) in Dec 1999 for decontamination of Anthrax spore. It was checked again by RestOps in March 2001 as a chemical decontamination agent. This technology has been tested on gangrene and clostridium botulism spores, and can even be used on contaminated wounds to salvage limbs. The nanoemulsions can be formulated into a cream, foam, liquid and spray to decontaminate a large number of materials, which is marketed as NANOSTAT™ (Nanobio Corp.) [56-58,115].

6.4. Nanoemulsions in vaccines delivery

This medication delivery system uses nanotechnology to vaccinate against human immunodeficiency virus (HIV). There is recent evidence that HIV can infect the mucosal immune system. Therefore, developing mucosal immunity through the use of nanoemulsions may become very important in the future fight against HIV [50]. The oil-based emulsion is administered in the nose, as opposed to traditional vaccine routes. Recent research results indicate that genital mucosa immunity may be attained with vaccines that are administered into the nasal mucosa [56-58,115]. Nanoemulsions are being used to transport inactivated organisms to a mucosal surface to produce an immune response. The first applications as vaccine, an influenza vaccine and an HIV vaccine, can proceed to clinical trials. The nanoemulsion causes proteins applied to the mucosal surface to be adjuvant and it help uptake by antigen presenting cells. This results in the significant systemic and mucosal immune response due to that the production of specific IgG and IgA antibody as well as cellular immunity. Work in influenza has shown that animals can be prevented against influenza after a single mucosal exposure to the virus mixed with thenanoemulsions. Research has also show that animals exposed to recombinant gp120 in nanoemulsions on their nasal mucosa create significant responses to HIV, thus giving a basis to use of this material as an HIV vaccine. Additional research has been ongoing to complete the proof of concept in animal trials for other vaccines including Anthrax and Hepatitis B. The University of Michigan has licensed this technology to NanoBio [56].

6.5. Nanoemulsions as non-toxic disinfectant cleaner

Nanemulsions have been employed as a disinfectant cleaner. A nontoxic disinfectant cleaner for use in routine markets that include healthcare, travel, food processing and military applications has been developed by EnviroSystems. They have been found to kill tuberculosis and a large spectrum of viruses, bacteria and fungi within 5 to 10 min without any of the hazards posed by other categories of disinfectants. The product requires no warning labels. It does not irritate eyes and can be absorbed through the skin, inhaled or swallowed with harmless effects. The disinfectant formulation is made up of nanospheres of oil droplets less than 100 μm which are suspended in water to produce a nanoemulsions requiring only small amounts of the active ingredient, parachlorometaxylenol. The nanospheres have surface charges that efficiently penetrate the surface charges on microorganisms’ membranes like breaking through an electric fence. Rather than 'drowning' cells, the formulation allows parachlorometaxylenol to target and penetrate cell walls. So parachlorometaxylenol is applicable at concentration ranges I-2 times lower than those of other disinfectants, so there are no toxic effects on human, animals or the environment [56-58,115].

Other microbial disinfectants need large doses of their respective active ingredients to surround pathogen cell wall, which causes microbe to disintegrate, ideally 'drowning' them in the disinfectant solution. The disinfectant is not flammable and so safe to store anywhere and to use in unstable conditions. It is non oxidizing, non acidic and nonionic. It will not corrode plastic, metals or acrylic, so it makes the product ideal for use on equipment and instruments. It is environmentally safe so the economical cost and health risks associated with hazardous chemical disposal are removed. The preparation is a broad-spectrum disinfectant cleaner that can be applied to any hard surface, including equipment, walls, fixtures, counters, and floors. One product can now take the place of many other, decreasing product inventories and saving valuable storage space. Chemical disposal costs can be removed, and disinfection and cleaning costs can be reduced. Marketed as EcoTru ™ (EnviroSystems) [56-58,115].

6.6. Nanoemulsions in cell culture technology

Cell cultures are used for in vitro assays or to produce biological compounds like an antibodies or recombinant proteins. For optimization of cell growth, the culture medium can be supplemented with a large number of molecules or with blood serum. It has been very difficult to provide the media with oil-soluble substances that are available to the cells, and only few amounts of the lipophilic compounds could be absorbed by the cells. Nanoemulsions are a new method for the delivery of oil-soluble substances to human cell cultures. The system is based on a nanoemulsions that is stabilized by phospholipids. This nanoemulsions is transparent and can be passed through 0.1 mm filters for sterilization. Nanoemulsions oil droplets are very 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 include:

• Better uptake of oil-soluble supplements in cell cultures.

• Improve growth and vitality of cultured cells.

• Allows toxicity studies of oil-soluble drugs in cell cultures [52 – 55].

6.7. Nanoemulsion formulations for improved oral delivery of poorly soluble drugs

Nanoemulsions formulation was developed to increase oral bioavailability of hydrophobic drugs. Paclitaxel was selected as a model hydrophobic drug. The o/w nanoemulsions were made with pine nut oil as the internal oil phase, water as the external phase and egg lecithin as the primary emulsifier. Stearylamine and deoxycholic acid were used to give positive and negative charge to the emulsions, respectively. The formulated nanoemulsions had a particle size range of 100-120 nm and zeta potential ranging from 34 mV to 245 mV. After oral administration of nanoemulsions, a significantly higher concentration of paclitaxel was observed in the systemic circulation compare to control aqueous solution. The results of this study suggest that Nanoemulsions are promising novel formulations which can promote the oral bioavailability of hydrophobic drugs [56-58,115].

6.8. Nanoemulsions in ocular and otic drug delivery

Ophthalmic drug delivery is one of the most interesting and challenging endeavors facing the pharmaceutical scientist [117]. It is a common knowledge that the application of eye drops as conventional ophthalmic delivery systems results in poor bioavailability and therapeutic response because of lacrimal secretion and nasolacrimal drainage in the eye [118,119]. Most of the drug is drained away from the precorneal area in few minutes. As a result, frequent instillation of concentrated solutions is needed to achieve the desired therapeutic effects [120]. But, by the tear drainage, the main part of the administered drug is transported via the nasolacrimal duct to the gastric intestinal tract where it may be absorbed, sometimes causing side effects [121]. In order to increase the effectiveness of the drug, a dosage form should be chosen which increases the contact time of the drug in the eye. This may then increase the bioavailability, reduce systemic absorption, and reduce the need for frequent administration leading to improved patient compliance. Nanoemulsions could be employed to overcome some of these problems. Dilutable nanoemulsions are potent drug delivery vehicles for ophthalmic use due to their numerous advantages as sustained effect and high ability of drug penetration into the deeper layers of the ocular structure and the aqueous humor. Ammar et al. formulated the antiglaucoma drug dorzolamide hydrochloride as ocular nanoemulsion of high therapeutic efficacy and prolonged effect [122]. These nanoemulsions showed acceptable physicochemical properties and exhibited slow drug release. Draize rabbit eye irritation test and histological examination were carried out for those preparations exhibiting superior properties and revealed that they were nonirritant. Biological evaluation of dorzolamide hydrochloride nanoemulsions on normotensive albino rabbits indicated that these products had higher therapeutic efficacy, faster onset of action, and prolonged effect relative to either drug solution or the market product. It was concluded from the study that formulation of dorzolamide hydrochloride in a nanoemulsion form offered a more intensive treatment of glaucoma, a decrease in the number of applications per day, and a better patient compliance compared to conventional eye drops.

6.9. Nanoemulsions as a vehicle for transdermal delivery

Drug delivery through the skin to the systemic circulation is convenient for a number of clinical conditions due to which there has been a considerable interest in this area [123,124]. It offers the advantage of steady state controlled drug delivery over extended period of time, with self administration also being possible, which may not be the case with parenteral route. The drug input can be eliminated at any time by the patient just by removing the transdermal patch. Their transparent nature and fluidity, confers on nanoemulsions a pleasant skin feel. An extra advantage is the total absence of gastrointestinal side effects like irritation and bowel ulcers which are invariably associated with oral delivery. Transdermal drug products have been developed for a number of diseases and disorders including cardiovascular conditions, Parkinsons’ and Alzheimer diseases, anxiety, depression, etc. However, the fundamental disadvantage which limits the use of this mode of administration is the barrier imposed by the skin for effective penetration of the bioactives. The three routes by which drugs can primarily penetrate the skin are through the hair follicles, sweat ducts or directly across stratum corneum, which restricts their absorption to a large extent and limits their bioavailability. For improved drug pharmacokinetics and targeting, the primary skin barriers need to be overcome. Also the locally applied drug redistribution through cutaneous blood and lymph vessel system needs to be controlled. Nano sized emulsions are able to easily penetrate the pores of the skin and reach the systemic circulation thus getting channelized for effective delivery [69]. Caffeine has been used for treatment of different types of cancer by oral delivery. Water-in-oil nanoemulsion formulations of caffeine have been developed for transdermal drug delivery. Comparison of in vitro skin permeation profile between these and aqueous caffeine solutions showed significant increase in permeability parameters for the nanoemulsion loaded drugs [125]. Use of nanoemulsions in transdermal drug delivery represents an important area of research in drug delivery, which enhances the therapeutic efficacy and also the bioavailability of the drugs without any adverse effects. It is also regarded as a promising technique with many advantages including high storage stability, low preparation cost, thermodynamic stability, absence of organic solvents, and good production feasibility. They have also made the plasma concentration profiles and bioavailability of drugs reproducible. These systems are being used currently to provide dermal and surface effects, and for deeper skin penetration [69]. Many studies have shown that nanoemulsion formulations possess improved trans-dermal and dermal delivery properties in vitro [66,126-133], as well as in vivo [134-136]. Nanoemulsions have improved transdermal permeation of many drugs over the conventional topical formulations such as emulsions and gels [137-141].

Barakat et al prepared nanoemulsions by the spontaneous emulsification method for transdermal delivery of indomathacin [142]. A significant increase in the permeability parameters such as steady-state flux, permeability coefficient, and enhancement ratio was observed in nanoemulsion formulations compared with the conventional indomethacin gel. The anti-inflammatory effects of nanoemulsion formulations showed a significant increase in percent inhibition value after 4 hours when compared with conventional indomethacin gel on carrageenan-induced paw edema in rats. Significant increase in permeability parameters was observed in nanoemulsion formulations (P< 0.05). The steady-state flux and permeability coefficient for optimized nanoemulsion formulation (were found to be 22.61±3.45 μg/ cm²/h and 0.22 x 10⁻² ± 0.0003 cm/h, respectively), which were significant compared with conventional indomethacin gel (P< 0.001). Enhancement ratio was found to be 8.939 in optimized formulation compared with indomethacin gel. These results suggested that nanoemulsions can be used as potential vehicles for improved transdermal delivery of indomethacin as an approach to eliminate the side effect of the oral dose.

Singh et al developed nanoemulsion formulation for transdermal delivery of carvedilol to enhance the water solubility as well as bioavailability of drug [143]. O/W nanoemulsions were prepared by the spontaneous emulsification method. Post application plasma carvedilol was increased 6.41 fold to marketed dosage form. The study suggested that nanoemulsion significantly enhanced bioavailability of transdermally applied carvedilol and eliminated the first pass metabolism.

Sajid et al prepared betamethasone valerate nanoemulsions by aqueous phase titration method, using Sefsol, Tween 20, Transcutol P, and distilled water as the oil phase, surfactant, co surfactant and aqueous phase, respectively and evaluated them based on the induction of contact dermatitis in rats using a dispersion of nickel sulfate in solid vaseline at 5%, carrageenan induce inflammation and their irritation study [144]. The optimized nanoemulsion was converted into hydrogel using Carbopol 934. Drug deposition in skin was found to be 58.46 μg/cm2. In vivo anti-inflammatory activity indicated 84.2% and 45.05% inhibition of inflammation in case of developed nanoemulsion gel and marketed cream, respectively. The irritation score was found to be 1.83 which indicates that the optimized nanoemulsion did not cause any irritation. Results of nickel induced dermatitis demonstrate that the nanoemulsion formulation gel did not appear to stimulate an inflammatory or immune response using the contact dermatitis model.

Zhou et al. carried out a study to establish a lecithin nanoemulsion without any synthetic surfactant as a topical delivery vehicle and to evaluate its topical delivery potential [145]. Experimental results demonstrated that an increasing concentration of soybean lecithin and glycerol resulted in a smaller size lecithin nanoemulsion droplet and increasing viscosity, respectively. Lecithin nanoemulsion, incorporated into o/w cream, improved the skin hydration capacity of the cream significantly with about 2.5-fold increase when the concentration of lecithin nanoemulsion reached 10%. Lecithin nanoemulsion was also demonstrated to improve the penetrability of Nile red dye into the dermis layer, when an o/w cream, incorporated with Nile-red-loaded lecithin nanoemulsion, applied on the abdominal skin of rat in vivo. Specifically, the arbitrary unit of fluorescence in the dermis layer that had received the cream with a Nile red-loaded lecithin nanoemulsion was about 9.9-fold higher than the cream with a Nile red-loaded general emulsion. These observations suggest that lecithin nanoemulsion could be used as a promising topical delivery vehicle for lipophilic compounds.

Modi et al investigated the potential of a nanoemulsion formulation for topical delivery of aceclofenac [146]. The in vitro skin permeation profile of optimized formulations was compared with that of aceclofenac conventional gel and nanoemulsion gel. A significant increase in permeability parameters such as steady-state flux, permeability coefficient and enhancement ratio was observed in optimized nanoemulsion formulation consisting of 2 % w/w of aceclofenac, 10 % w/w of Labrafac, 45% w/w surfactant mixture (Cremophor^® EL: Ethanol), and 43 % w/w of distilled water. The anti-inflammatory effects of formulation showed a significant increased percent inhibition value after 24 hours when compared with aceclofenac conventional gel and nanoemulsion gel on carrageenan-induced paw edema in rats. These results suggested that nanoemulsions are potential vehicles for improved transdermal delivery of aceclofenac.

Batoota et al investigated the potential of nanoemulsion formulations for transdermal delivery of celecoxib [91]. The in vitro skin permeation profile of optimized formulations was compared with celecoxib gel and nanoemulsion gel. Significant increase in the steady state flux, permeability coefficient and enhancement ratio was observed in nanoemulsion formulations (p < 0.05). The highest value of these permeability parameters was obtained in the formulation that consisted of 2 % (w/w) of celecoxib, 10 % (w/w) of oil phase (Sefsol 218 and Triacetin), 50 % (w/w) of surfactant mixture (Tween-80 and Transcutol-P) and 40 % (w/w) water. The anti-inflammatory effects of the formulation showed a significant increase (p < 0.05) in inhibition after 24 hours compared to celecoxib gel and nanoemulsion gel on carrageenan-induced paw edema in rats. These results suggested that nanoemulsions are potential vehicles for improved transdermal delivery of celecoxib.

Harwansh et al evaluated an isotropic and thermodynamically stable nanoemulsion formulation for transdermal delivery of glycyrrhizin, with minimum surfactant and cosurfactant concentrations that could improve its solubility, permeation enhancement, and stability [147]. A significant increase in permeability parameters such as steady-state flux and permeability coefficient was observed in the optimized nanoemulsion formulation, which consisted of 1 % w/w of mono ammonium glycyrrhizinate, 32.4 % Span 80, 3.7 % Brij 35, 10 % isopropyl alcohol, 46.5 % soyabean oil and 6.4 % distilled water. No obvious skin irritation was observed for the studied nanoemulsion formulation or the gel. The results indicated that nanoemulsions are promising vehicles for transdermal delivery of glycyrrhizin through human cadaver skin, without the use of additional permeation enhancers, because excipients of nanoemulsions act as permeation enhancers themselves.

Inayat et al. developed a potential of nanoemulsion formulation for transdermal delivery of tamoxifene citrate for breast cancer [148]. Transdermal permeation of tamoxifene citrate through rat skin was determined by Keshary-Chien diffusion cell. A significant increase in permeability parameter such as steady–state flux was observed in optimized nanoemulsion formulation, which consist of 5 % w/w of drug, 4.12 % w/w of oil phase, 37.15 % w/w of surfactant (mix) and 58.73 % w/w of distilled water. It possessed a mean globule size of 68 nm. Transmission electron microscopy demonstrated spherical particle morphology and DSC and FTIR study revealed the compatibility among the ingredient. These results proposed that the prepared system could be promising to improve transdermal efficacy of the tamoxifen citrate.

Shakeel et al investigated the potential of a nanoemulsion formulation for transdermal delivery of aceclofenac [149]. Transdermal permeation of aceclofenac through rat abdominal skin was determined by Franz diffusion cell. The in vitro skin permeation profile of optimized formulations was compared with that of aceclofenac conventional gel and nanoemulsion gel. A significant increase in permeability parameters such as steady-state flux, permeability coefficient, and enhancement ratio was observed in optimized nanoemulsion formulation, which consisted of 2 % w/w of aceclofenac, 10% w/w of Labrafil R, 5 % w/w of Triacetin R, 35.33 % w/w of Tween 80 R, 17.66 % w/w of Transcutol PR, and 32 % w/w of distilled water. The anti-inflammatory effects of optimized formulation showed a significant increase in percent inhibition value after 24 hours when compared with aceclofenac conventional gel and nanoemulsion gel on carrageenan-induced paw edema in rats. These results suggested that nanoemulsions are potential vehicles for improved transdermal delivery of aceclofenac.

Shakeel et al presented an overview of the efforts that have been made in the last decade by various researchers in exploring new types of nanoemulsion-based drug delivery system for dermal and transdermal delivery of many hydrophobic compounds [150]. This area of research would be very advantageous for formulation scientists in order to develop some nanoemulsion-based formulations for their commercial exploitation and clinical applications. Moreover, Harwansh et al reviewed efforts made by various researchers in the delivery of phytopharmaceuticals using nanoemulsions [151].

6.10. Nanoemulsion in cancer therapy and in targeted drug delivery

Another interesting application, which is experiencing an active development, is the use of nanoemulsion formulations, for controlled drug delivery and targeting [82]. Because of their submicron size, they can easily be targeted to the tumor area. Although nanoemulsions are chiefly seen as vehicles for administering aqueous insoluble drugs, they have more recently received increasing attention as colloidal carriers for targeted delivery of various anticancer drugs, photosensitizers, neutron capture therapy agents, or diagnostic agents. The development of magnetic nanoemulsions is an innovative approach for cancer therapy. These can deliver photosensitizers like Foscan^® to deep tissue layers across the skin thereby inducing hyperthermia for subsequent free radical generation. This methodology can be used for the treatment of cancer in the form of photodynamic therapy [152].

6.11. Nanoemulsions and intranasal drug delivery

Intranasal drug delivery system has now been recognized as a reliable route for the administration of drugs next to parenteral and oral routes. Nasal mucosa has emerged as a therapeutically viable channel for the administration of systemic drugs and also appears to be a favourable way to overcome the obstacles for the direct entry of drugs to the target site [153]. This route is also painless, non-invasive and well tolerated. The nasal cavity is one of the most efficient sites because of its reduced enzymatic activity, high availability of immunoactive sites and its moderately permeable epithelium [154]. There are several problems associated with targeting drugs to brain, especially the hydrophilic ones and those of high molecular weight. This is because of the impervious nature of the endothelium, which divides the systemic circulation and barrier between the blood and brain [155]. The olfactory region of the nasal mucosa provides a direct connection between the nose and brain, and by the use of nanoemulsions loaded with drugs, conditions such as Alzheimer’s disease, migraine, depression, schizophrenia, Parkinson’s diseases, meningitis, etc. can be treated [156,157]. Preparation of nanoemulsions containing risperidone for its delivery to the brain via nose has been reported [158]. It is inferred that this emulsion is more effective through the nasal rather than intravenous route. Another application of intranasal drug delivery system in therapeutics is their use in development of vaccines. Immunity is achieved by the administration of mucosal antigen. Currently, the first intranasal vaccine has been marketed [158]. Among the possible delivery systems, the use of nano based carriers hold a great promise to protect the biomolecules, promote nanocarrier interaction with mucosae and to direct antigen to the lymphoid tissues. Therefore the use of nanoemulsions in intranasal drug delivery system is set to bring about significant results in targeting drugs to the brain in treatment of diseases related to the central nervous system [159]. Bhanushali et al developed intranasal nanoemulsion and gel formulations for rizatriptan benzoate for prolonged action [160]. Various mucoadhesive agents were tried out to form thermo-triggered mucoadhesive nanoemulsions. Mucoadhesive gel formulations of rizatriptan were prepared using different ratios of HPMC and Carbopol 980. Comparative evaluation of intranasal nanoemulsions and intranasal mucoadhesive gels indicated that greater brain-targeting could be achieved with nanoemulsions. Other drugs which have been formulated for nasal delivery are insulin and testosterone [161].

6.12. Nanoemulsions and parenteral drug delivery

This is one of the most common and effective routes of drug administration usually adopted for actives with low bioavailability and narrow therapeutic index. Their capacity to dissolve large quantities of hydrophobics, together with their mutual compatibility and ability to protect the drugs from hydrolysis and enzymatic degradation make nanoemulsions ideal vehicles for the purpose of parenteral transport. Further, the frequency and dosage of injections can be reduced throughout the drug therapy period as these emulsions guarantee the release of drugs in a sustained and controlled mode over long periods of time. Additionally, the lack of flocculation, sedimentation and creaming, combined with a large surface area and free energy, offer obvious advantages over emulsions of larger particle size, for this route of administration [69]. Their very large interfacial area positively influences the drug transport and their delivery, along with targeting them to specific sites. Major clinical and pre-clinical trials have hence been carried out with parenteral nanoemulsion based carriers. The advances in these novel drug delivery systems have been reviewed by Patel and Patel [162]. Nanoemulsions loaded with thalidomide have been synthesized where a dose as low as 25 mg leads to plasma concentrations which can be therapeutic [163]. However, a significant decrease in the drug content of the nanoemulsion was observed at 0.01% drug formulation after two months storage which could be overcome by the addition of polysorbate 80. Chlorambucil, a lipophilic anticancer agent has been used against breast and ovarian cancer. Its pharmacokinetics and anticancer activity has been studied by loading it in parenteral emulsions prepared by high energy ultrasonication method. Treatment of colon adenocarcinoma in the mouse with this nanoemulsion leads to higher tumor suppression rate compared to plain drug solution treatment, concluding that the drug loaded emulsion could be an effective carrier for its delivery in cancer treatment [164]. Carbamazepine, a widely used anticonvulsant drug had no parenteral treatment available for patients due to its poor water solubility. Kelmann et al. have developed a nanoemulsion for its intravenous delivery, which showed favorable in vitro release kinetics [165]. Parenteral nanoemulsion formulations of the following drugs have been documented as well: diazepam, propofol, dexamethasone, etomidate, flurbiprofen and prostaglandin E1 [166].

The high lipophilicity of diazepam (an anxiolytic and sedative) makes the use of solvents (such as propylene glycol phenyl carbinol and ethanol) for the dissolution of the drug in conventional aqueous preparations (Valium^® and Stesolid^®) necessary, leading to pain and thrombophlebitis on the patient during the injection. The development of a nanoemulsion, commercially available under the name of Diazemuls^® (Kabi-Pharmacia) allows for the reduction of these adverse effects, keeping stages of distribution and elimination similar to Valium^®. However, higher doses of Diazemuls^® are necessary to obtain the same effect as Valium^® since this leads to higher free fraction of plasma diazepam [167,168].

The solution for intravenous administration of etomidate (hypnotic short) due to stability problems, its composition contains 35 % propylene glycol (Hypnomidate^®) [169,170]. Due to the presence of high osmolarity of the solvent, the administration is associated with various adverse effects such as hemolysis, thrombosis, thrombophlebitis and pain at the site of application [171,172]. A nanoemulsion containing 2 mg/ml Lipofundin^® etomidate in medium chain triglyceride named Lipuro-etomidate^® (B. Braun) was developed [173]. The emulsion allowed the reduction of the hemolytic and venous sequelae, besides the pain at the time of application [169-171].

The pharmacokinetics and pharmacodynamics of propofol (anesthetic) are complex. It has an initial rapid distribution of about 2-3 minutes, with high variability between patients and reduced concentrations to subtherapeutic levels within minutes. However, due to its high lipophilicity, it has a high volume of distribution and its complete elimination from the body can take days [174]. Due to the occurrence of anaphylactic effects associated with Cremophor EL, present in the original formulation of propofol nanoemulsion as vehicle for this drug-containing composition in soybean oil, glycerol, egg yolk lecithin and disodium edentate, this vehicle helped to reduce the volume of distribution of the drug, accelerating their processes of clearance by the responsible agencies. This formulation also allowed the use of minimal effective dose need to produce the needed therapeutic effect, allowing a rapid onset and recovery from anesthesia, when compared to a non-lipid (ethanol) solution, thereby generating greater security administration, due to the lower continuous accumulation of the drug, and eliminating the need for constant adjustment of the dose. This product was approved in 1989 in the United States, under the name of Diprivan^® 1 or 2 % (AstraZeneca / APP Pharmaceuticals) [175]. In Brazil, the product is available as Lipuro 1% (B. Braun) and Diprivan^® 1 and 2% (AstraZeneca), besides the generic 1% (Eurofarma Labs.) [176]. The various generic formulations currently available are constituted by an additional factor of variability in response between individuals in the induction of anesthesia, apart from the pharmacokinetic characteristics of the drug itself [174] and the differences in lipoprotein profile of each patient, due to the high binding of propofol to low density lipoprotein and albumin [177]. Due to related pain at the injection site and increased triglyceride levels after administration for long periods, some changes in the formulation of Diprivan^® adverse effects have been proposed, including some already being marketed as Propofol^® Lipuro (B. Braun) as oil core which contains a mixture of oils [178]. The addition of more oil to the formulation allowed the reduction of pain on injection due to increased incorporation of the drug in the oily core and the lower amount of free propofol phase the external aqueous emulsion [171,177-180]. Alternative formulations have been developed, for example, the incorporation of higher concentrations of propofol (6 %) in the nanoemulsion [180-182], or the development of a propofol prodrug in solution (Aquavan^®) [183].

Furthermore, despite the excellent anti-inflammatory activity of dexamethasone, the clinical use of corticosteroids is limited by numerous side effects [184,185]. ​​To circumvent these drawbacks, lipophilic prodrugs in the body that are gradually hydrolyzed to the active metabolite can be used (thus presenting prolonged anti-inflammatory effect). The advantage is the use of lower doses than those used in conventional water soluble form (dexamethasone phosphate), reducing the risks of adverse effects. Considering that nanoemulsions are picked up by inflammatory cells of the mononuclear phagocytic system, nanoemulsions were used as a vehicle for lipophilic prodrug of dexamethasone (palmitate), which is commercially available as Limethason^® (Green Cross Co./Mitsubishi Tanabe Pharma Co.). Limethason^® showed excellent results in the treatment of rheumatoid arthritis, West syndrome, inflammatory diseases and other autoimmune diseases. While the solution of dexamethasone phosphate is rapidly distributed in water-rich tissues, such as muscles, the nanoemulsion is accumulated mainly in tissues inflamed organs such as liver and spleen. The biodistribution profile is different even if the elimination pattern is similar between the two. Limethason^® removes over 80 % of the phagocytic activity of macrophages at a concentration of 0.03 mg/mL [185].

Flurbiprofen (non-steroidal anti-inflammatory oral use), a lipophilic drug, is used to treat rheumatoid arthritis and other inflammatory diseases associated or not with cancer [186]. The non-availability of oral and/or various gastrointestinal effects caused by this drug often require the use of parenteral route. Considering the severe local irritation caused by the sodium salt of flurbiprofen, it was developed as a prodrug of flurbiprofen (cefuroxime) and because of the lipophilicity of the latter especially in soybean oil, it was incorporated in nanoemulsions for parenteral use (Ropion^®, Kaken Pharmaceuticals Co., Lipfen^®, GreenCross Co.), and is commercially available in the Japanese market since 1992. Administration of Ropion^® resulted in an increase in area under the concentration-time curve and reduced clearance when compared to the solution. The incorporation of the drug into nanoemulsions containing unesterified ethyl oleate, lecithin and modified egg yolk led to a lower drug accumulation in organs such as the liver and spleen due to the lower uptake by the mononuclear phagocyte system [166].

Prostaglandin E1, which is synthesized in several places of the body, is responsible for various physiological effects such as vasodilatation, lowering of blood pressure, angiogenesis and inhibition of platelet aggregation [187,188]. When administered for the treatment of various diseases, it has a short half-life; high doses are needed, leading to numerous adverse effects such as hypotension, diarrhea, local irritation and pain [187]. In this context, nanoemulsions were made commercially available in 1975, PGE1 complexed to cyclodextrins and, in 1985, prostaglandin E1 incorporated in lipid nanoemulsions (Liple^®, Mitsubishi Tanabe Pharma Corporation, Palux^®, Taisho Pharmaceutical) [189]. Lipid formulations are used to treat cardiovascular diseases because they accumulate in the walls of injured vessels, transporting the drug to the site of vascular injury and to protect it from rapid inactivation by the lungs [187-190].

6.13. Nanoemulsions and pulmonary drug delivery

The lung is an attractive target for drug delivery due to noninvasive administration via inhalation aerosols, avoidance of first-pass metabolism, direct delivery to the site of action for the treatment of respiratory diseases, and the availability of a huge surface area for local drug action and systemic absorption of drug. Colloidal carriers (ie, nanocarrier systems) in pulmonary drug delivery offer many advantages such as the potential to achieve relatively uniform distribution of drug dose among the alveoli, achievement of improved solubility of the drug from its own aqueous solubility, a sustained drug release which consequently reduces dosing frequency, improves patient compliance, decreases incidence of side effects, and the potential of drug internalization by cells [191]. Until now, the submicron emulsion system has not yet been fully exploited for pulmonary drug delivery and very little has been published in this area [191]. Bivas-Benita et al. reported that cationic submicron emulsions are promising carriers for deoxyribonucleic acid vaccines to the lung since they are able to transfect pulmonary epithelial cells, which possibly induce cross priming of antigen-presenting cells and directly activate dendritic cells, resulting in stimulation of antigen-specific T-cells [192]. Therefore the nebulization of submicron emulsions will be a new and upcoming research area. However, extensive studies are required for the successful formulation of inhalable submicron emulsions due to possible adverse effects of surfactants and oils on lung alveoli function (adverse interactions with lung surfactant). A novel pressurized aerosol system has been devised for the pulmonary delivery of salbutamol using lecithin-stabilized microemulsions formulated in trichlorotrifluoroethane [193].

6.14. Nanoemulsions as gene delivery vector

Emulsion systems have been introduced as alternative gene transfer vectors to liposomes [194]. Other emulsion studies for gene delivery (non-pulmonary route) have shown that binding of the emulsion/DNA complex was stronger than liposomal carriers [195]. This stable emulsion system delivered genes more efficiently than liposomes [196]. Silva et al evaluated factors that influence DNA compaction in cationic lipid nanoemulsions [cationic nanoemulsions containing stearylamine (a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions, and in dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface conferring a positive charge to them)] [197]. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by dynamic light scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery.