**5. Characterization of nanoemulsions**

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 and dielectric methods provide useful information at the macroscopic level. Viscosity meas‐ urements for example can indicate the presence of rod-like or worm-like reverse micelles and conductivity measurements provide the means of determining whether a nanoemulsion is oilcontinuous or water-continuous, as well as providing a means of monitoring phase inversion phenomena [99]. Dielectric measurements are a powerful means of probing both the structural and dynamic features of nanoemulsion system. Structural features of nanoemulsions have been studied using self-diffusion nuclear magnetic resonance (SD NMR) and small angle xray scattering (SAXS). Freeze fracture electron microscopy has also been used to study nanoemulsion structure, however extremely rapid cooling of the sample is required in order to maintain the structure and minimize the possibility of artifacts [101-103]. Nanoemulsion droplet polarity is also a very important factor in characterizing emulsification efficiency. The HLB, chain length and degree of unsaturation of fatty acids, molecular weight of the hydro‐ philic portion and concentration of the emulsifier have an impact on the polarity of the oil droplets. Polarity represents the affinity of the drug compound for oil and/ or water and the type of forces formed. Rapid release of the drug into the aqueous phase is promoted by the polarity. The charge of the oil droplets of nanoemulsions is another property that should be assessed. Usually it is negative due to the presence of free fatty acids; however, incorporation of a cationic lipid, such as oleylamine at a concentration range of 1-3%, will yield cationic nanoemulsions [104,105].

scattering. The polydispersity index indicates the quality or homogeneity of the dispersion [109]. PCS gives z-average particle diameter. Laser diffraction is another technique for measuring particle size. The fundamental particle size distribution derived by this technique is volume based and is expressed in terms of the volume of equivalent spheres (*DN*%) and weighted mean of the volume distribution (mass mean diameter). Since the laser diffraction system is used for this analysis, a rough equivalent of particle polydis‐ persity could be given by two factors/values namely, uniformity (how symmetrical the distribution is around the median point) and span (the width of the distribution). The

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Where *DN*% (*N=*10%, 50%, 90%), means that the volume percentage of particles with diameters up to *DN*% equals to *N*%. The smaller the span value the narrower the particle size distribution. **c. Viscosity:** This is carried out using a viscometer. The viscosity of nanoemulsions is a function of the surfactant, water and oil components and their concentrations. Increasing the water content lowers the viscosity, while decreasing the amount of surfactant and cosurfactant increases interfacial tension between water and oil resulting in increased viscosity. Viscosity is very important for stability and efficient drug release. Nanoemul‐ sion carrier formulations are basically oil-in-water and so in addition to being less greasy than water-in-oil formulations, often possess lower apparent viscosities. They are therefore expected to exhibit faster release of active ingredients and wash out easily after application on the skin surface. Various equipment and methods are available for assessment of rheological properties of nanoemulsion carriers. Monitoring of viscosity change is a method of assessing stability of liquid and semi-solid preparations including

**d.** *In vitro* **skin permeation:** Franz diffusion cell is used to obtain the drug release profile of the nanoemulsion formulation in the case of formulations for transdermal application. The extent or depth of skin penetration by the released content can be visualized by confocal scanning laser microscopy. *In vitro* drug release can be determined by dispersing an amount of the preparation in the donor compartment of a Franz cell having a membrane as barrier and monitoring the appearance of the encapsulated drug in the receptor compartment, usually containing phosphate buffer saline (PBS, pH 7.4) and stirring on a magnetic stirrer at 100 rpm at 37 ± 1 °C. Samples (1 ml) of the dispersion are withdrawn from the receptor medium and replaced with an equivalent amount of the medium at definite intervals. The withdrawn sample is then filtered using a 0.22-50 μm filter (e.g., Millipore, USA) and the drug released then analyzed using HPLC or UV-Vis spectroscopy at wavelength of peak absorption of the drug [110]. An alternative and popular method of *ex-vivo* release study is performed using diffusion cell. The skin is cut from the ear or abdomen and underlying cartilage and fats carefully removed. Appropriate size of skin is cut and placed on the diffusion cell which had earlier been filled with receptor solution.

Span 90% 10% / 50% = - (*DD D* ) (2)

span value is defined by the expression:

nanoemulsion formulations [99].

The following sub-headings could be used to discuss briefly the parameters commonly employed in the assessment of nanoemulsions:


scattering. The polydispersity index indicates the quality or homogeneity of the dispersion [109]. PCS gives z-average particle diameter. Laser diffraction is another technique for measuring particle size. The fundamental particle size distribution derived by this technique is volume based and is expressed in terms of the volume of equivalent spheres (*DN*%) and weighted mean of the volume distribution (mass mean diameter). Since the laser diffraction system is used for this analysis, a rough equivalent of particle polydis‐ persity could be given by two factors/values namely, uniformity (how symmetrical the distribution is around the median point) and span (the width of the distribution). The span value is defined by the expression:

and dielectric methods provide useful information at the macroscopic level. Viscosity meas‐ urements for example can indicate the presence of rod-like or worm-like reverse micelles and conductivity measurements provide the means of determining whether a nanoemulsion is oilcontinuous or water-continuous, as well as providing a means of monitoring phase inversion phenomena [99]. Dielectric measurements are a powerful means of probing both the structural and dynamic features of nanoemulsion system. Structural features of nanoemulsions have been studied using self-diffusion nuclear magnetic resonance (SD NMR) and small angle xray scattering (SAXS). Freeze fracture electron microscopy has also been used to study nanoemulsion structure, however extremely rapid cooling of the sample is required in order to maintain the structure and minimize the possibility of artifacts [101-103]. Nanoemulsion droplet polarity is also a very important factor in characterizing emulsification efficiency. The HLB, chain length and degree of unsaturation of fatty acids, molecular weight of the hydro‐ philic portion and concentration of the emulsifier have an impact on the polarity of the oil droplets. Polarity represents the affinity of the drug compound for oil and/ or water and the type of forces formed. Rapid release of the drug into the aqueous phase is promoted by the polarity. The charge of the oil droplets of nanoemulsions is another property that should be assessed. Usually it is negative due to the presence of free fatty acids; however, incorporation of a cationic lipid, such as oleylamine at a concentration range of 1-3%, will yield cationic

The following sub-headings could be used to discuss briefly the parameters commonly

**a. Morphology:** The morphology of nanoemulsions can be determined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). SEM gives a threedimensional image of the globules [105]. The samples are examined at suitable accelerat‐ ing voltage, usually 20 kV, at different magnifications. A good analysis of surface morphology of disperse phase in the formulation is obtained through SEM. Image analysis software may be employed to obtain an automatic analysis result of the shape and surface morphology [106]. In TEM, higher resolution images of the disperse phase are obtained. The sample is negatively stained with 1% aqueous solution of phosphotungstic acid or by dropping 2 % uranyl acetate solution onto a 200 μm mesh size Pioloform™-coated copper grid or a microscopic carbon-coated grid using a micropipette and the sample examined under a transmission electron microscope at appropriate voltage. Qualitative measure‐ ments of sizes and size distribution of TEM micrographs can be performed using a digital image processing programme [107]. More sophisticated techniques, such as x-ray or neutron scattering, atomic force microscopy, or cryo-electron microscopy are typically

required to explore the structure and behaviour of nanoemulsions [71].

**b. Droplet size, polydispersity and zeta potential:** Dynamic light scattering (DLS) otherwise called photon correlation spectroscopy (PCS) is used to analyze the fluctuations in the intensity of scattering by droplets/particles due to Brownian motion [108]. Nanoemulsion droplet size, polydispersity and zeta potential can be assessed by PCS using a particle size analyzer. This instrument also measures polydispersity index, which is a measure of the broadness of the size distribution derived from the cumulative analysis of dynamic light

nanoemulsions [104,105].

96 Application of Nanotechnology in Drug Delivery

employed in the assessment of nanoemulsions:

$$\text{Span} = \left(D90\% - D10\%\right) / D50\% \tag{2}$$

Where *DN*% (*N=*10%, 50%, 90%), means that the volume percentage of particles with diameters up to *DN*% equals to *N*%. The smaller the span value the narrower the particle size distribution.


Samples of the vesicular preparation are then applied on the dorsal surface of the skin and the instrument started. At intervals, up to 24 h, samples are withdrawn from the receptor medium and replaced with equal amounts of the medium and the withdrawn samples analyzed for the drug permeated using HPLC or UV spectroscopy [111,112]. Semipermeable membrane such as regenerated cellulose could be used in place of skin for *in vitro* release studies [113,114]. The flux *J*, of the drug across the skin or membrane is calculated from the formula:

$$\mathbf{J} \mathbf{=D} \mathbf{d} \mathbf{c} / \mathbf{d} \mathbf{x} \tag{3}$$

cosmetics because there is no chance of creaming, sedimentation, flocculation or coalescence, which is observed within microemulsions. The incorporation of potentially irritating surfac‐ tants can be avoided by using high-energy equipment during manufacturing process. PEGfree nanoemulsions for cosmetics has also been developed and formulations exhibited good

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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 organ‐ isms. 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

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

This medication delivery system uses nanotechnology to vaccinate against human immuno‐ deficiency 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 admin‐

stability [56-58,115,116].

antibiotics [56-58,115].

**6.3. Prophylactic in bio-terrorism attack**

**6.4. Nanoemulsions in vaccines delivery**

marketed as NANOSTAT™ (Nanobio Corp.) [56-58,115].

**6.2. Antimicrobial nanoemulsions**

Where D is the diffusion coefficient and is a function of the size, shape and flexibility of the diffusing molecule as well as the membrane resistance, c is the concentration of the diffusing species, x is the spatial coordinate [114].

**e.** *In vivo* **bioavailability/pharmacodynamic studies**: *In vivo* release study otherwise referred to as dermatopharmacokinetics, is carried out by applying or administering the preparation to whole live animal. Blood samples are then withdrawn at intervals, centrifuged and the plasma (deproteinated) analyzed for the drug content using HPLC. Results obtained from *in vitro* and *in vivo* studies are extrapolated to reflect bioavailability of the drug formulation. Moreover, the pharmacodynamic properties of nanoemulsion formulations are also assessed depending on the pharmacological properties of the incorporated drug [81-93,95,101].
