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Title Page

Nanoemulsions as optimized vehicles for essential oils

Thaís N. Barradas 1,2* and K. Gyselle de Holanda e Silva 3

1 Rio de Janeiro Federal Institute of Education, Science and Technology. R. Senador Furtado,
121-125 – Rio de Janeiro – Brazil, ZIP Code: 20270-021
2 Instituto de Macromoléculas Professora Eloisa Mano, Universidade Federal do Rio de Janeiro
(IMA/UFRJ), Centro de Tecnologia, Bl. J, Av. Horácio Macedo, 2030, Cidade Universitária, Ilha
do Fundão – Rio de Janeiro – Brazil, ZIP Code: 21941-598.
3 School of Pharmacy, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho, 373,
Cidade Universitária, Ilha do Fundão – Rio de Janeiro – Brazil, ZIP Code: 21941-902.

* Correspondent Author

Thaís Nogueira Barradas

Instituto de Macromoléculas Professora Eloisa Mano, Universidade Federal do Rio de Janeiro

(IMA/UFRJ), Centro de Tecnologia, Bl. J, Avenida Horácio Macedo, 2030, Cidade Universitária,
Ilha do Fundão – Rio de Janeiro – Brazil, CEP: 21941-598.
[email protected]. Tel: (+55) (21) 2562-7209
 2

Abstract

During the last few years, a great interested has been given in for nanotechnology applications

in pharmaceutical technology. At the same time, there is a growing research effort in the search

for healthier and safer products. In this context, the use of naturally originate raw materials,

essential oils for instance, rises as an interesting approach for replacing several synthetic active

pharmaceutical ingredients. However, these materials often feature low bioavailability,

uncontrolled volatility or low long-term stability, which require novel encapsulation techniques.

Therefore, research efforts have been focusing on nanoemulsion technology that is particularly

suited to produce novel products. Nanoemulsions constitute one interesting vehicle for enhancing

solubility, stability and delivering natural oils, by encapsulating them into nanosized micelles with

sizes ranging from 20 – 200 nm. They gather some unique characteristics as small size, increased

surface area and stability which can increase efficiency and biological effects of pharmaceutical

dosage forms.

The wide application of nanoemulsions to the encapsulation of bioactive molecules still require

much development in order to achieve the optimization of the obtention methods for large-scale

production. This chapter includes an overview about nanoemulsion stability characteristics and

the different approaches for obtaining nanoemulsions, including high energy methods like high-

pressure homogenization, microfluidizers and ultrasonic homogenization, which are the most

used approaches, and low energy methods such as phase inversion composition and phase

inversion temperature, simple but still less reproduceable methods. In addition, we present the

main aspects of nanoemulsion formulations, type of surfactants and oil phase, and the techniques

for characterizing nanoemulsions, for example, dynamic light scattering, zeta potential,

microscopy and X-ray diffraction. Moreover, applications of nanoemulsions to pharmaceutical

technological approaches for essential oils encapsulation, such as the improvement of bioactive

oils bioavailability and solubilization, masking unpleasant aspects of oils and enhancement of

essential oils pharmacological activity are also discussed.

Keywords,

Nanoemulsions, nanoencapsulation, surfactants, emulsification methods, essential oils, drug

delivery
 3

Contents (list of sections)

1. Introduction ............................................................................................ 4
2. Nanoemulsions: General aspects ............................................................... 6
3. Nanoemulsions: Composition ..................................................................... 9
3.1. Surfactants .............................................................................................................. 9
3.2 Types of surfactants ............................................................................................. 13
3.3 Oil phase ................................................................................................... 19
3.4 Essential oils .......................................................................................................... 20
4. Preparation of nanoemulsions .................................................................. 26
4.1 – High-energy methods ........................................................................................ 27
3.3 Comparison between obtention methods .............................................. 36
5. Stability of Nanoemulsions ....................................................................... 39
5.1 Instability phenomena........................................................................................... 40
6. Characterization of nanoemulsions .......................................................... 45
6.1 Dynamic Light Scattering ..................................................................................... 45
6.2 Zeta Potential ......................................................................................................... 47
6.4 Conductivity ............................................................................................................ 50
6.5 Phase behavior ...................................................................................................... 50
7. Application of nanoemulsions .................................................................. 51
8. Conclusion .................................................................................................. 55
9. Acknowledgements .................................................................................... 56
10. References ................................................................................................ 56
 4

1. Introduction

Recently, the use of naturally originate bioactive molecules, especially natural

antioxidants and anti-microbial agents, has been rising. The search for natural products has been

triggering more and more research in the development of suitable and stable formulations.

Essential oils are bioactive molecules, being considered safe and biocompatible, offering a great

range of benefits due to a complex composition of fatty acids, terpenes, triterpenes and many

other lipophilic components, that can offer protection against dehydration, solar radiation,

inflammation, insect attack, microorganisms, and viruses (Donsì and Ferrari 2016; Andreu et al.

2015; Bonferoni et al. 2017).

Many essential oils present proved antioxidant potential, preventing oxidation reaction

and reducing the formation of free radicals, which are risk factors for cellular damage and for

chronicle diseases as cancer. However, one of the main challenges to formulate essential oils

into a pharmaceutical product lies on low water solubility of most of oils, which impairs

bioavailability of lipophilic components and stability in hydrophilic formulations. Moreover, many

essential oils are highly volatile and labile, being unstable under many environmental conditions,

as light exposure and oxidation. Those restrictions must be overcome during the development of

pharmaceutical formulations (Badgujar, Patel, and Bandivdekar 2014; Herman and Herman 2015;

Moghimi et al. 2016).

Nanoemulsions are nanosized colloidal dispersions with droplets within the nanometric

scale, more often between 20 and 200 nm. Nanoemulsions are thermodynamically stable

isotropic systems in which two immiscible liquids are mixed to form a single phase by means of

an emulsifying agent, i.e., a surfactant (Helgeson 2016; C. Solans et al. 2005). Nanoemulsions

have been considered of a great potential of industrial application in pharmaceutical, food,

agrochemical, cosmetics and personal care products (David J. McClements and Jafari 2018).

Nanoemulsions are widely applied to encapsulate, protect and promote controlled release of

lipophilic bioactive molecules. Besides, nanoemulsions can also act as templates to produce

other types of nanoparticles, such as solid lipid nanoparticles (SLN), nanostructured lipid carriers

(NLC), multiple emulsions and hydrogel-thickened nanoemulsions (Sutradhar and Amin 2013;

David J. McClements and Jafari 2018; Barradas et al. 2017).

 5

Because of the reduced droplet size, nanoemulsions promote interesting characteristics

in comparison to conventional emulsions, i.e. macroemulsions. For instance, nanoemulsions

present much larger surface area of dispersed phase in relation to the total volume of the

dispersion than that observed in macroemulsions. Thus, droplet deformation-derived phenomena

are typically higher for nanoemulsions than for conventional emulsions (Helgeson 2016).

Besides, macroemulsions exhibit multiple light scattering, which provide them a white opaque

appearance. In contrast, in nanoemulsions, droplets are much smaller than the wavelength of

visible light. Hence, most of the nanoemulsions are optically transparent systems when droplet

size is small enough, usually below 100 nm (David Julian McClements and Rao 2011).

The development of nanoemulsions as nanostructured carriers for pharmaceutical

application requires an accurate selection of the components such as, surfactants, and oils as

well as the preparation method selected for nanoemulsion production. Special attention must be

given to oily phase used as inner phase in oil-in-water nanoemulsions. In order to obtain

nanoemulsions, a minimum energy input is necessary. Energy input can be achieved by applying

work to the system, usually called “high energy” methods, of by modulating composition or

temperature of the system, referred as “low energy” methods. Both methods require the same

amount of energy for providing nanosized droplets, regardless if the energy is provided

mechanically or thermodynamically (Helgeson 2016; David J. McClements and Jafari 2018).

Nanoemulsions and polymeric micelles can be considered promising systems for

encapsulation of essential oils since because of various advantages such as: sustained and

controlled release of bioactive molecules; solubilization of lipophilic substances as oils; they are

suitable for administration under different routes; protection from chemical, environmental and

enzymatic degradation of labile molecules; controlled volatilization of essential oils, reduction of

side effects and dose.

In this chapter, we provide an overview focused on formulation of oil-in-water

nanoemulsions, as they can provide encapsulation of lipophilic components, and diverse

approaches for nanoemulsions production. Additionally, we summarize the instability phenomena

related to nanoemulsions and the recent applications of nanoemulsions for the encapsulation of

natural oils.
 6

2. Nanoemulsions: General aspects

Emulsions are heterogeneous colloidal systems, consisting of an immiscible liquid

dispersed as droplets, i.e. disperse or inner phase in another immiscible liquid, i.e., continuous or

outer phase, stabilized by a surfactant. When the continuous phase is an aqueous solution and

the dispersed phase is an oil, the emulsion is called oil-in-water emulsion (Figure 1A). On the

other hand, when the dispersed phase is an aqueous solution and the continuous phase is

constituted by an oil, the emulsion is called water-in-oil emulsion (Figure 1B). In all cases, an

emulsifier agent or surfactant is needed to reduce interfacial tension between dispersed and

continuous phases and provide stable systems. The nature of the surfactant and the stabilizing

film provided by it, greatly influence general aspects of nanoemulsions, as long-term stability,

droplet size and charge, for example. In general, emulsified systems can be classified according

to some physicochemical properties such as droplet size and preparation methods into

macroemulsions, microemulsions and nanoemulsions.

Hydrophobic tail

Lipidic surfactant

Hydrophilic head

Water
Oil

Oil Water

A B

Figure 1: Oil-in-water nanoemulsion (A) and water-in-oil nanoemulsion (B), composed by oil and

aqueous phases, stabilized by surfactants. In oil-in-water nanoemulsion aqueous phase

constitutes the outer phase and oil phase is the inner phase, and the surfactant molecule self-

assemble into a micelle structure, with hydrophilic portion oriented towards the aqueous phase.

In water-in-oil nanoemulsions oil phase and water phase constitute the outer phase and inner

phase, respectively, and surfactants hydrophobic portion is in contact with the outer phase.
 7

The most important difference between nanoemulsions and macroemulsions lies in the

droplet size, since both are thermodynamically unstable systems. Nanoemulsions usually show

mean droplet size smaller than 200 nm, although there is no agreement regarding the size range

that comprise nanoemulsion and distinguish them from micro-sized and conventional-sized

macroemulsions. According to the literature, droplet size can vary from 20 nm to either 500, 300,

200, or yet 100 nm, being until now a controversial issue regarding nanoemulsions classification

(Conxita Solans and Solé 2012; David Julian McClements and Rao 2011).

In general, macroemulsions are optically opaque, due to the large droplet size, which

produces multiple light scattering, while nanoemulsions are often transparent or translucent, since

they present small droplet size scatter light weakly (David J. McClements and Jafari 2018; David

Julian McClements 2011). Nanoemulsions are kinetically stable systems and do not require the

addition of a cosurfactant, unlike microemulsions. Kinetic stability is attributed to the Brownian

motion of the droplets which overcomes the gravity force and prevents droplets sedimentation

and coalescence. In addition, the use of nonionic or polymeric surfactants may cause steric

repulsion between the droplets and contribute to the stability of the system (T. Tadros et al. 2004;

C. Solans et al. 2005).

Besides storage stability, there are other interesting physical properties that differentiate

nanoemulsions from macroemulsions. For example, nanoemulsions have dispersed phase

surface area relative to the total volume of the dispersion much larger than that observed in

macroemulsions. Due to the high relative surface area, all phenomena related to droplet

deformation are typically more relevant to nanoemulsions than to macroemulsions (Mason et al.

2006).

It is noteworthy the common misconception between nano and microemulsions. There

has been much effort to clarify the difference between these two colloidal systems in this scientific

field (Sonneville-Aubrun, Simonnet, and L’Alloret 2004; Anton and Vandamme 2011). Both

microemulsions and nanoemulsions contain droplets of diameters ranging from 20 to 200 nm,

differing in certain aspects, especially regarding the preparation method (Abismail et al. 1999;

David Julian McClements 2011). Although both systems can feature similar characteristics, as

nanosized droplets as inner phase dispersed in a continuous phase, they differ in therms of

stability and in physicochemical concepts (Anton and Vandamme 2011; David Julian McClements
 8

2012). Microemulsions is a term usually used to refer to thermodynamically stable isotropic

oil/surfactant/water systems, while nanoemulsions are kinetically stable conventional emulsions

constituted by nanosized micelles (David Julian McClements 2012). Microemulsions can feature

a wide range of structures with one, two, three ore more phases in equilibrium, depending on the

concentrations between all the components and upon certain temperatures. These different

structures can be water-continuous, oil-continuous or bicontinuous and nanometric swollen

micelles, which gives them a bluish and translucent nanoemulsion-like aspect. However, micelles

can form different geometries such as, worm-like, bicontinuous sponge-like, liquid crystalline,

hexagonal, lamellar, and spherical swollen micelles, which is the most often confused with

nanoemulsions (Anton and Vandamme 2011; David Julian McClements 2012).

Moreover, nanosized micelles from microemulsion are deeply afected in morphology and

size upon temperature and dilution, while nano-micelles from nanoemulsions remain unaltered

when submited to the same conditions, which is an important aspect to be considered when

microemulsions are intended to be applied as parenteral prooducts. Parenteral route can pose

some challenges as infinite dillution conditions and temperature, pH and osmolarity variations.

Under biological conditions, only nanoemulsions remain stable (Lefebvre et al. 2017; Hörmann

and Zimmer 2016; Anton and Vandamme 2011).

These misinterpretations are often found in literature, where one can find inadequate

methods for obtaining and characterizing micro and nanoemulsions. For example, ternary phase

diagrams to produce nanoemulsions constitutes a very frequent but inapropriate methodology

that can be more suitable to produce microemulsions. The simplicity of low-energy emulsifying

methods is the main aspect that can induce the confusion between nano and microemulsions.

Besides, in order produce nanoemulsions by low-energy methods, surfactant must be mixed in

oil phase before adding water, not being possible inverting the order of components incorporation.

Microemulsions can be formed no matter the order in which components are added (Anton and

Vandamme 2011). This difference between both systems is an easy and simple preliminary test

for classification of a system in whether nano or microemulsions.

The large surface area provided by the nanostructured droplets increases drug

absorption in biological membranes such as the intestinal epithelium, the cornea and skin for

example (Singh et al. 2017). Moreover, because they are transparent formulations,
 9

nanoemulsions present an interesting aesthetic characteristic, even containing significant

quantities of oil in the composition (Sonneville-Aubrun, Simonnet, and L’Alloret 2004; T. Tadros

et al. 2004).

3. Nanoemulsions: Composition

In general, a classical nanoemulsion formulation comprises an aqueous and an oil phase

stabilized by a surfactant. The physical-chemical properties of the components will greatly impact

the formation and stability of the nanoemulsions obtained from them. Typically, nanoemulsions

require relatively high amounts of surfactants, usually from 10-15 wt. %, in order to stabilize the

high surface area of nanosized micelles (Azeem et al. 2009; Mason et al. 2006). The composition

of nanoemulsions can be tuned by careful selection of the ingredients used to obtain them.

3.1. Surfactants

Surfactants are molecules whose structures comprise two parts of opposing affinities,

one having a hydrophilic character and the other with a hydrophobic character. Both hydrophilic

and lipophilic moieties should be in equilibrium in the surfactant molecule, so that it constitutes a

good emulsifier. This hydrophilic-lipophilic equilibrium of a both moieties in the surfactant

molecular structure is called hydrophilic-lipophilic balance (Abismail et al. 1999; Porras et al.

2004). Hydrophilic-lipophilic balance is a classifying system for all surfactants and provides a

good indication for the right the choice of emulsifiers suitable for obtaining stable emulsions.

During the production of nanoemulsions, surfactants molecules should be able to quickly adsorb

on newly formed droplet surface, while maintaining droplet integrity once two droplets collide,

avoiding droplet deformation or coalescence (Tadros 1994).

The formation of a stable nanoemulsion involves selecting an appropriate composition,

controlling the order of addition of the components and applying a minimum energy input capable

of promoting the deformation of droplets. This minimum energy input is related to the Young-

Laplace Theory, which shows that the difference between the external and internal pressures of

a drop is a direct function of the droplet radius (Equation 1). Therefore, to break a drop into

droplets of smaller size, the difference between the internal and external pressure to the drop

should be considerable (Tadros et al. 2004).

 10

𝜸
𝑷𝒊 − 𝑷𝒆 = (Equation 1)
𝟐𝐫

Where,

Pi = Internal pressure

Pe = External pressure

γ = Interfacial tension

r = Radius of droplet curvature

According to Equation 1, large amounts of surfactants are required to reduce surface

tension and stabilize the great interfacial surface produced. The production of nanoemulsions,

therefore, is a thermodynamic unfavorable and non-spontaneous process. Besides, to obtain

droplets of nanometric size it is necessary to supply energy to the system, usually provided by

mechanical devices or even by chemical potential of the constituents. Nanoemulsions often

present spherical droplets because both the high interfacial tension and small droplet size

combined provide high Laplace pressures, which triggers the minimization of the oil–water

interfacial area into a spherical shape (David Julian McClements 2011).

The success of developing a stable nanoemulsion depends on the right selection of the

surfactant used, which shall act on the interface between dispersed and continuous phases. The

choice of a surfactant for pharmaceutical purposes is one of the most critical steps and involves

consideration of the toxicity of substances that can be applied in large quantities. (Tadros 2009;

Wiedmann 2003). The selection of a surfactant can be considered the most important step to

produce nanoemulsions, since emulsifiers can influence both fabrication method and

performance of nanoemulsions.

Due to the amphiphilic molecular constitution, surfactants tend to migrate to the interfaces

between two immiscible fluids, in a way that polar groups are oriented to the aqueous phase and

the non-polar, to the organic phase. The polar group is of great importance because it defines

some properties, such as water solubility and the classification of the surfactants which can be

divided into ionic, which can be anionic and cationic, nonionic and amphoteric, or zwitterionic.
 11

At low concentrations, the surfactant molecules are solubilized within the solution as free

soluble surfactant molecules (Figure 2A). As the surfactant concentration increases, a decrease

in surface tension of the solution occurs, representing the surfactant adsorption at air-water

surface (Figure 2B). When reaching a certain concentration, it is observed that the variation of

the surface tension is minimal in relation to the increase of the concentration, which means that,

the saturation of the water-air interface is reached. At this stage, the adsorption of the surfactant

on the surface is no longer observed. The surfactant concentration that causes this phenomenon

is called the critical micelle concentration, where it can be observed the formation of molecular

aggregates, known as micelles (Figure 2C).

The moieties that are not soluble in the surrounding solvent attract strongly and produce

a stable compact vesicular morphology, as to expel the solvent from this environment. On the

other hand, groups that are very soluble, tend to be externally exposed producing a soluble

particle, i.e., a micelle (Myers 2005b). The size of the micelles and the number of surfactant

molecules per micelle depends on the type of surfactant and the physicochemical environment.

When the surfactant is adsorbed at the oil-water interface, interfacial tension decreases causing

a steric hindrance effect or electrostatic repulsion, both able to prevent coalescence or

aggregation phenomena. These barriers not only prevent emulsion droplets from coming into

direct contact, but also serve to stabilize the liquid film between two adjacent droplets (Myers

2005a)

Figure 2: Micellization steps: Surfactants as free soluble molecules, below critical micelle

concentration (A). As surfactant concentration raises, surfactants start to self-assemble as

monolayer in water-air interface (B). Above critical micelle concentration, water-air interface is
 12

saturated with surfactant molecules and interfacial tension no longer reduces. Then surfactants

start to self-assemble as micelles, in a thermodynamically favorable phenomenon (C).

The interfacial tension is related to the amount of surfactant adsorbed at the interface and

the nature of the interfacial layer. The interfacial tension decreases with increasing surface

charge, which is directly related to the concentration and size of the surfactant. However,

depending on the type of surfactant, many other effects are important (Tadros et al. 2004; Myers

2005a). It can be said, then, that surfactants play a very important role in the stabilization or

destabilization of nanoemulsions, increasing or reducing the electrostatic or steric repulsions of

the interface, which are dependent on structural aspects, such as double electric layer, branching,

aromaticity. Other factors like the presence and type of electrolytes, pH, temperature and

presence of additives (Myers 2005a). The most studied for the formation of nanoemulsions are

non-ionic surfactants.

Both liquids and solids feature surface tension due to the cohesive energy between their

molecules. Surface tension is resultant from unbalanced forces of attraction or non-equilibrated

in interfacial regions, in which there is a sudden variation of the density. Thus, the resultant force

on a molecule close to the surface liquid/vapor is different from that on a molecule that is in a

completely homogeneous region, in which the resulting force is null. The surface tension can also

be defined as the excess of surface energy (Tsujii 1998; Shaw 1992).

Liquids tend to self-assemble into forms that minimize surface area, that is, a greater

number of molecules are inside the liquid and, therefore, remain surrounded by other liquid

molecules. Liquid droplets therefore tend to be spherical because a sphere is the shape with the

smallest surface / volume ratio. Surface effects can be expressed according to the Helmholtz and

Gibbs energies (Equation 2), in which the bond between these amounts of energy and the surface

area is the work (force) needed to increase the surface area (σ) of a liquid in a given quantity

(Hunter 2001)

dW = γ.dθ (Equation 2)
 13

Where, W means work or applied force; γ (N.m-1) means superficial tension and θ

superficial tension.

The effect of surface tension is to minimize surface area, resulting in a curved surface,

as in a vesicle. The intensity of this tension depends on the liquid, solvent purity and temperature

in which the surfactant lies (Hunter 2001). The same considerations apply to the interface

between two immiscible liquids. Again, intermolecular forces in non-equilibrium are presented,

however with decreased intensity. The interfacial tensions usually lie between the individual

surface tension of the two liquids above mentioned (Shaw 1992).

3.2 Types of surfactants

There are several surfactants that have been used in the production of nanoemulsions,

since small-molecule synthetic surfactants, which may be non-ionic and ionic, and natural

surfactants such as phospholipids, proteins and polysaccharides. In general, the surfactants may

be: (i) non-ionic, (ii) amphoteric (iii) cationic, or (iv) anionic.

The class of the non-ionic surfactants comprise small molecules such as sorbitan esters,

like Span® and Tween® series and polymeric surfactants, such as poly(oxyethylene) ethers, for

example, Brij® or block copolymers as poly(ethylene oxide)-poly(propylene oxide). Small

molecules can form small-sized droplets because of the facility on easily adsorb on the surface

of droplets that they feature (Jafari et al. 2017).

Natural surfactants exhibit excellent surface activity, although they present molecular

structure being very voluminous compared to synthetic surfactants. Since natural surfactants are

naturally produced substances, they show advantages like biodegradability, are easily produced

from renewable resources, present high specificity and tend to less toxic (A. Salem and M. Ezzat

2018). Table 1 summarizes the most common types of natural and synthetic surfactants.

The use of natural surfactants, due to the innocuous character, provides some

advantages over synthetic surfactants, including biocompatibility and biodegradability,

multifunctional characteristics, stable activity under different environmental conditions, such as

high or low temperatures, pH, high pressure and osmolarity. Thus, natural surfactants may be

more effective in stabilizing nanoemulsions for the most diverse purposes (Dickinson 2003; Qian
 14

and McClements 2011). In general, most of natural surfactants are large molecules, which can

delay the ability to adsorb on the droplets surface and provide larger droplets. However, they can

provide good stability by both steric and electrostatic stabilization (Qian and McClements 2011).

Table 1: Main types of natural and synthetic surfactants and their general aspects

Characteristics/
Type Examples General aspects References
Charge
Mixture of natural (Klang and Valenta
Phospholipids polar phospholipids 2011; Hoeller,
Natural For example: Zwitterionic obtained from eggs or Sperger, and Valenta
Lecithin soybean. Amphoteric 2009; Khan and
behavior Krishnaraj 2014)
Provide good stability
either by electrostatic (Bai et al. 2016;
Polysaccharides Negatively or
or steric effects. Not Jafari et al. 2017;
Natural For example: Positively
suitable for low-energy Dickinson, Galazka,
Gum arabic charged
emulsification and Anderson 1991)
methods.
Provide good stability
(M. Sharma et al.
mainly by electrostatic
Amphiphilic 2017; Dickinson,
Charge repulsion and also
proteins, as Semenova, and
depends on pH steric effects under
Natural caseinate or Antipova 1998; Kuhn
relative to neutral pH. Not
whey protein and Cunha 2012;
isoelectric point suitable for low-energy
isolate David Julian
emulsification
McClements 2011)
methods
(Tian, Chen, and
Sodium Lauryl Used in both low and
Zhang 2016; Lémery
Sulfate (SLS) or Negatively high energy methods
Ionic surfactant et al. 2015; Rosety et
Sodium Dodecyl charged High irritation and
al. 2001; Bondi et al.
Sulfate (SDS) acute toxicity potential
2015)
Non-toxic,
biodegradable and
hydrophilic. Used to
Sucrose (Strickley 2004;
stabilize oil-in-water
Non-ionic monopalmitate Cerqueira-Coutinho
Sugar esters nanoemulsions by
surfactant Sorbitan et al. 2015; Rao and
high pressure
monooleate McClements 2011)
homogenization with
relatively low
surfactant-to-oil ratios.
Relatively low phase
Non-ionic Polyoxyethylene inversion temperature. (David Julian
Brij
surfactant alkyl esters Used in low-energy McClements 2011)
methods
Used in high-energy
methods. Differences (PATHANIA et al.
aliphatic chain length 2018; Dias et al.
Non-ionic Tweens and Ethoxylated
varies, providing 2014; Salvia-Trujillo
surfactant Spans Sorbian esters
several types of et al. 2015; Speranza
Tween and Span. Ex: et al. 2013)
Tween 20, 40, and 60.
 15

One of the most common natural surfactants used in the development of nanoemulsions

are phospholipids. Phospholipids can be classified as natural amphoteric surfactants and have

excellent biocompatibility because they are similar in composition to cell membranes. The term

lecithin is commonly used to designate a mixture of phospholipids obtained from both animal and

vegetable sources, as eggs and soybean, respectively (Shchipunov 2015). The composition of

lecithin may vary upon several environmental and processing conditions, however, the

phospholipids most commonly found in lecithin are: phosphatidylcholine,

phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, glycerol phospholipids of

complex fatty acid composition (Klang and Valenta 2011; Shchipunov 2015).

The polar portion of lecithin is comprised by positively charged phosphate groups, which

are bounded to lipophilic chains by ester groups. Moreover, nitrogen-containing moieties

bounded to phosphate groups can assume negative charges, providing phospholipids such as

lecithin an amphoteric character (Shchipunov 2015).

Biopolymers such as polysaccharides have been attracting attention as natural

surfactants for producing stable nanoemulsions (Dickinson 2003; Bai et al. 2016).

Polysaccharides feature a complex structure, water-soluble, with good emulsifying properties and

thickening properties, are widely used in food, pharmaceutical and paper industries.

Polysaccharides and gums such as gum arabic are often used as thickening or gelling agents in

pharmaceutical industry (Prajapati et al. 2013). The polysaccharides most commonly found are

hydrocolloids such as xanthan, modified starch, galactomannans and pectin (Dickinson 2003;

Prabaharan 2011; Sweedman et al. 2013; Prajapati et al. 2013). Recently, they have been

considered promising components to be used as natural surfactants as there is a growing demand

for natural and sustainable ingredients. The emulsification properties of polysaccharides are

related to the presence of non-polar groups and hydrophilic groups, providing the interaction with

both oil and water. The stabilization method related to polysaccharides can occur by both

electrostatic repulsion and steric effect, preventing droplet flocculation and coalescence (Jafari et

al. 2017).

Gum arabic is one of the most used polysaccharides for producing stable emulsions, due

to the formation of a stable layer around the droplets, providing steric repulsion among droplets

by forming steric layer around them (Yadav et al. 2007; McNamee, O’Riorda, and O’Sullivan
 16

1998). Other naturally occurring polysaccharides used in producing nanoemulsion comprise

pectin, a heterogenous anionic polysaccharide already used as gelling, thickening and stabilizing

properties (Bai et al. 2016) and maltodextrin (Sonu et al. 2018).

Some proteins, especially those obtained from bovine milk, as casein and whey proteins,

can be used as emulsifying and surfactants agents, since they feature both hydrophilic and

lipophilic residues and surface activity, providing cohesive and strong films around droplets

(Yerramilli and Ghosh 2017; Mayer, Weiss, and McClements 2013; Kuhn and Cunha 2012;

Adjonu et al. 2014). Proteins can stabilize droplets mainly by electrostatical repulsion because of

the presence of negatively charged groups, although steric effect can also be attributed to them

(Adjonu et al. 2014; Dickinson, Semenova, and Antipova 1998). As proteins are very large

molecules, the adsorption on droplet surface takes longer. On the other hand, proteins feature

the ability of forming a viscoelastic coating that prevent droplet deformation and, thus aggregation,

providing higher stability under several conditions (David Julian McClements 2011). One of the

most studied proteins used as natural surfactants is casein.

Casein comprises almost 80% of milk protein and it is a heterogeneous protein composed

by four main proteins fractions: αs1 (~44 wt.%), αs2 (~11 wt.%), β (~32 wt.%) and k (~11 wt.%)

with a strong aggregation behavior, which is fundamental to stabilize casein-based micelles (Lei

Wang and Zhang 2017; M. Sharma et al. 2017). Casein provides both electrostatic and steric

stabilization against coalescence under neutral pH values (Dickinson, Semenova, and Antipova

1998). At acidic pH values, the negative charge on caseinate structure is reduced and aggregation

between casein-based micelles occurs (M. Sharma et al. 2017).

Several works have reported the combination of proteins and polysaccharides-based

emulsifiers based on the ability to form polyelectrolyte complexes between these molecules and

the possibility of Maillard reaction, which contribute to increase stability of the layer around

nanosized micelles end prevent coalescence (Sonu et al. 2018; Farshi et al. 2019).

There is a growing demand on replacing synthetic surfactants for natural molecules.

However, the application of natural surfactants still is limited by several issues. For example,

Phospholipids and protein-based surfactants, are unstable under environmental conditions such

as pH, osmotic pressure and heating. Polysaccharides, on the other hand, provide stability under

environmental stressful conditions (J. Zhang, Peppard, and Reineccius 2015). Besides,
 17

polysaccharides can stabilize nanoemulsion by increasing the viscosity oh aqueous phase by

stablishing hydrogen bonds water molecules (Dickinson 2009). Moreover, according to Qian and

McClements 2011, small-molecule surfactants can provide smaller droplets than proteins, which

can be related to the ability to rapidly adsorb to the droplet surfaces during homogenization and

providing a more stable interface.

The solubility of synthetic or ionic surfactants is related to the interactions between the

ionic group, which is the polar part of the surfactant, and water. The ionic surfactants most used

in the production of nanoemulsion are summarized in Table 1. In general, ionic surfactants are

resistant to changes in temperature and quite soluble in water. The applications of ionic

surfactants are originated from these properties. Also, pH has great influence on the solubility of

the ionic surfactants due to the neutralization reactions that occur with this type of molecules.

In the case of anionic products, a reduction in pH causes a decrease in the degree of

dissociation and in the solubility of the surfactant due to the formation of the corresponding

undissociated acid, which has lower solubility. The electrical charge on droplet surface plays an

important role in stabilizing nanosized droplets as electrostatic repulsion, aggregation stability etc.

Moreover, surface charge can be explored to provide nanoemulsions novel application by

triggering the interaction of charged surfactants with other components in biological media (Tian,

Chen, and Zhang 2016). Quaternary ammonium salts belong to the class of cationic surfactants,

the best-known being hexadecyl trimethyl ammonium bromide and dodecyl ammonium bromide

(Bouchemal et al. 2004; Lawrence and Rees 2000).

In the case of ionic surfactants, the stability of nanoemulsions is mainly governed by

electrostatic interaction between the droplets. Surface charge and, consequently, zeta potential

can be modulated by mixing non-ionic and ionic surfactants and varying the proportion of both of

them, providing extremely long-term stable nanoemulsion (Tian, Chen, and Zhang 2016; Babchin

and Schramm 2012). Sodium lauryl sulfate (SLS) or sodium dodecyl sulfate is the most common

anionic surfactant used to produce emulsions. However, they can disorganize several cell

membrane organization and affect both protein and lipid structures, being highly irritant for skin

application and toxic for systemic administration (Elmahjoubi et al. 2009; Bondi et al. 2015).

Non-ionic surfactants are widely used for pharmaceutical purposes because they feature

low toxicity and greater stability against changes in ionic strength and pH of the common biological
 18

media. There are different types of non-ionic surfactants, the most used to provide pharmaceutical

nanoemulsions are polyglycerol alkyl ethers, glucosyl dialkyl ethers, crownethers, ester-linked

surfactants, polyoxyethylene alkyl ethers, ethoxylated hydrogenated castor oil, Brij, Spans, or

sorbitan esters and Tweens, or Polysorbates.

Synthetic non-ionic surfactants show no charges on the polar part of the molecular

structure, which is often composed of ethylene oxide groups. For this reason, solubility of

synthetic non-ionic surfactants in aqueous solution differs from most solutes: with increasing

temperature, these surfactants show phase separation, and this temperature is known as cloud

point temperature. This behavior occurs because of the increasing the size of the molecular

aggregates, known as micelles and the inter-micellar attraction. This can be explained by the

dehydration of the outer layer of the micelle of the nonionic surfactant with the increase in

temperature, which is promoted by the breaking of the hydrogen bonds between the ethylene

oxide group and the water (Prud’homme, Wu, and Schneider 1996; Alexandridis, Holzwarth, and

Hatton 1994).

Compared with low molecular weight surfactants, non-ionic polymeric surfactants

produce more stable micelles, have lower critical micelle concentration values, with a slower

dissociation rate, which allows controlling the release of the molecules contained in the nucleus

of nano-sized droplets and also achieve greater accumulation of the drug at a specific site. In

addition, these copolymers are capable of forming micelles in a wide variety of solvents (Xing and

Mattice 1997; Kataoka, Harada, and Nagasaki 2001).

Like Mao et al. studied the effect of different surfactants on interfacial tension, droplet

size, zeta potential and morphology of β-carotene-loaded nanoemulsions produced by high

pressure homogenization. High molecular weight surfactants, such as Tween 20®, decaglycerol

monolaurate, modified starch, whey protein isolate were evaluated. The results showed that

Tween 20® and decaglycerol monolaurate provided smaller droplet size, although with poorer

stability compared with modified starch and whey protein isolate, presenting droplets aggregates.

On the other hand, modified starch and whey protein isolate provided larger droplets with higher

stability against droplet aggregation due to the formation of stronger interfacial layers. Moreover,

the combination of Tween 20 and whey protein isolate provided nanoemulsions with higher
 19

stability regarding β-carotene content under storage conditions of 55oC for prolonged time (Like

Mao et al. 2009).

3.3 Oil phase

Oil phase composes the core of oil-in-water nanoemulsions and it can influence greatly

both formation and stability of nanoemulsions. Several aspects as polarity, water miscibility,

interfacial tension and viscosity can change nanoemulsions characteristics such as droplet size

and stability (David Julian McClements 2011). Different types of oil phases can be used for the

formation of nanoemulsions: synthetic, mineral, vegetable or essential oils. In this chapter the

origin and the chemical and physicochemical properties of essential oils will be the focus.

Triacylglycerols, free fatty acids, fixed/vegetable, essential and mineral oils, waxes alone or in

associations can be used as oil phase upon nanoemulsion development (Jafari 2017). The most

common the essential oils often found are clove, peppermint, sweet fennel and bergamot (Al-

Subaie et al. 2015; Saberi, Fang, and McClements 2014; Pengon et al. 2018; Strickley 2004; Wan

et al. 2019; Barradas et al. 2015). They can act as drug carriers or solvents or be used as the

only constitute of oil phase due to pharmacological properties commonly attributed to essential

oils.

Very often, oil phase of nanoemulsions are liquid, as natural oils. However, they can also

be solid as natural waxes and solid lipids (in the case of SLN and NLC) (Sutradhar and Amin

2013; David J. McClements and Jafari 2018). Physical state of oil droplets can influence creaming

stability, optical properties and release pattern of the encapsulates bioactive molecules. The right

selection of liquid and solid lipophilic components can provide the control over physical state of

oil droplets (David Julian McClements 2015).

In both oil-in-water and water-in-oil, oil phase is one of the most important issues, since

it can influence several physicochemical aspects, for example: viscosity, refractive index,

transparency, interfacial tension stability, sensorial aspects and droplet size. As a consequence,

a range of functional properties that are consequence of physicochemical parameters are

affected, as bioavailability, miscibility and digestibility (David J. McClements and Jafari 2018).

Moreover, molecular weight of the components, interfacial tension, density and viscosity of the oil
 20

phase also influence the properties of nanoemulsions and can lead the choice of a certain type

of emulsification method (David Julian McClements and Rao 2011).

Droplet stability is dependent on oil solubility, which is influenced by the amount of water

soluble and lipophilic oil components. Great amounts of water soluble components in oil phase

can lead to Ostwald ripening, the major source of instability in nanoemulsions (Chebil et al. 2013).

There are some strategies to retard droplet growth due to Ostwald ripening. Ripening inhibitors

can be added to oil phase of oil-in-water nanoemulsions prepared with highly soluble oils as

essential oils (Rao and McClements 2012; David J. McClements and Jafari 2018). Ripening

inhibitors are usually highly hydrophobic molecules almost water insoluble able to increase

hydrophobicity inside the droplets even upon diffusion of water-soluble molecules. As a

consequence, there is an increase in the concentration of the ripening inhibitor inside droplets,

promoting a concentration gradient in the system that triggers the diffusion of water-soluble

components from large back to small droplets, as the opposite of what occurs in Ostwald ripening

(David J. McClements and Jafari 2018).

Oils and lipophilic components can be encapsulated inside nanomicelles with the aim of

protecting labile and degradable bioactive molecules from environmental conditions, controlling

release, increasing bioavailability and providing an easier manipulation or incorporation of oils

into aqueous formulations. Moreover, encapsulation techniques can be applied to the modification

of physicochemical properties of oils, which makes it possible to convert solutions into fine

powders of to mask undesirable tastes when administrated by oral route.

3.4 Essential oils

Essential oils are aromatic substances extracted from aromatic plants or plants parts,

including leaves, fruits, barks and seeds. They considered raw materials of great importance for

the cosmetic, pharmaceutical, fragrance and food industries. These pure and extremely potent

organic substances are considered the main biochemical components of the therapeutic action

of medicinal and aromatic plants (PATHANIA et al. 2018; Pérez-Recalde, Ruiz Arias, and

Hermida 2018; Donsì and Ferrari 2016).

These are volatile substances extracted from aromatic plants, being extremely potent

organic substances and considered the main biochemical components of the therapeutic action
 21

of medicinal and aromatic plants. The methods of extraction vary according to the location of the

volatile oil in the plant and the proposed use of it. The most common methods are: enfleurage,

steam distillation, or azeotropic distillation; extraction with organic solvent in a continuous and

discontinuous way; pressing or supercritical CO2 extraction (Asbahani et al. 2015).

The designation of oil is given thanks to some physical-chemical characteristics, for

example, that they are often oily-appearing liquids at room temperature. The main characteristic

of essential oils is the volatility, which differs them from the fixed/vegetable oils, which are

mixtures of lipid substances normally obtained from seeds. Another major characteristic is given

thanks to the pleasant and intense aroma of most of the volatile oils, being therefore also called

essences. They are still soluble in apolar organic solvents, as ether, thus receiving the name of

ethereal oils or, in Latin, aetheroleum. Although they have a limited solubility in water, they have

been used to increase water solubility of poorly soluble drug by constituting the inner phase of oil-

in-water nanoemulsions (Barradas et al. 2017).

Essential oils constituents range from terpene hydrocarbons, simple and terpene

alcohols, aldehydes, ketones, phenols, esters, ethers, oxides, peroxides, furans, organic acids,

lactones, coumarins, to sulfur compounds. In the mixture, these compounds are present in

different concentrations, and usually one of them is the majority compound, others are in lower

levels and some in very low quantities (Donsì and Ferrari 2016)

Essential oils are well-known as flavors, antioxidant and antimicrobial products and have

been widely used as functional ingredients in food, pharmaceutical, and cosmetic formulations

(Jin, Xu, Liang, et al. 2016). In recent years, the application of essential oils in food products has

generated great interest because essential oils are generally recognized as safe regulatory

status, multiple functionalities, and wide acceptance by consumers. However, the poor solubility

in aqueous solutions and high volatility during processing are two major obstacles of utilizing EOs

in the industrial processes.

A superficial search in the main patent databases and indexed journal articles reveals the

growing interest in the use of nanoemulsions in the last decade as well as the use of these

systems in the pharmaceutical industry (Figures 3 and 4). It is possible to notice a considerable

increase in the number of publications describing essential oil-loaded nanoemulsions. The poor

solubility in aqueous solutions and high volatility during processing are two major obstacles of
 22

utilizing essential oils as sanitizing agents or as preservatives in food matrices. Oil-in-water

nanoemulsions carefully prepared using appropriate surfactants and emulsification processes are

common choices to deliver essential oils in aqueous systems, for several administration routes

and distinct applications. Nevertheless, there are still few studies that deal with the particularities

in the development, characterization and evaluation of the pharmaceutical properties of essential

oils-loaded nanoemulsions, especially regarding the evaluation of the industrial scale production.

Essential oils are generally obtained from the peeling of fruits, flowers or leaves. Several

essential oils are being used in the development of nanoemulsions, in Table 2 are listed some

nanosystems with proven activity. For example, clove oils are the essential oils obtained by

distillation of the flower buds, stems, and leaves of the clove tree (Syzygium aromaticum) (Goñi

et al. 2016). Among clove oils, clove bud oil (CBO) is widely used and well-known as a potent

antioxidant and for antibacterial, antifungal, and antiviral activities (Anwer et al. 2014; Chaieb et

al. 2007). Eugenol, 4-allyl-2-methoxyphenol, is the primary constituent, responsible for more than

80% of CBO and is the major contributor of the above biological functions of CBO (Jirovetz,

Buchbauer et al. 2006, Chaieb, Hajlaoui et al. 2007).

Figure 3: Patents related to nanoemulsions formulations from 2010 to 2019 using Boolean

operators AND (Espacenet search* 6/10/2019). Blue: nanoemulsions; Green: nanoemulsions

AND drugs; Purple: Nanoemulsions AND essential oil. * https://worldwide.espacenet.com

 23

Figure 4. Publications related to nanoemulsions formulations from 2010 to 2019 using

Boolean operators AND (Web of Science search** 6/10/2019). Blue: nanoemulsions; Green:

nanoemulsions AND drugs; Purple: nanoemulsions AND essential oil.

** https://webofknowlegde.com

Another example, Rosmarinus officinalis L. essential oil is usually isolated by

hydrodistillation, steam distillation, or extraction with organic solvents. The Rosemary oil is mainly

located in leaves and the flowers; it is known for antioxidant, antimicrobial, anti-inflammatory

properties and studies suggest that the one chemical compounds more frequently reported

molecules was 1,8-cineole, α-pinene, and camphor (Angioni et al. 2004; Hernández et al. 2016).

Basil and thyme are aromatic herbs that are also used extensively to add a distinctive

aroma and flavor. The essential oils are extracted from fresh leaves and flowers are very

recognized can be used as aroma additives in food, pharmaceuticals, and cosmetics (Q. X. Li

and Chang 2016; Mandal and DebManda 2016). Major compounds found in volatile extracts of

basil and thyme exhibited varying amounts of anti-oxidative activity in particular, eugenol, thymol,

carvacrol and 4-allylphenol (Marotti, Piccaglia, and Giovanelli 1996; Hudaib et al. 2002; Politeo,

Jukic, and Milos 2007; Hussain et al. 2008).

Recognized in traditional medicine, lemongrass essential oil is a potent antimicrobial and

antioxidant natural bioproduct. Lemongrass essential oil composition consists mainly of 70–85%
 24

geranial (citral), neral, geraniol, nerol, citronellol, 1,8-cineole (eucalyptol), α-terpineol, linalool,

geranyl acetate. Lemongrass oil is collected by steam distillation of the herbage. Lemongrass

essential oil is a viscous liquid, yellow to dark yellow or dark amber in color turning red on

prolonged storage (A. Sharma et al. 2018).

Also, among the essential oils, there are citrus fruits, which also have countless

therapeutic applications. Citrus fruits are well known and appreciated for centuries as they feature

pleasant aroma and appetizing flavor. The essential oils from these fruits are generally obtained

from the juice of the peels of fruits, but it can also be obtained from flowers or leaves (Asbahani

et al. 2015). Among the best-known essential oils are those of orange, lemon, mint, eucalyptus,

mint, citronella, clove, among others, as can be seen in Table 2.

The methods of extraction vary according to the location of the volatile oil in the plant and

the proposed use of it. The most common methods are: enfleurage, steam distillation; extraction

with organic solvent in a continuous and discontinuous way; pressing or supercritical CO2

extraction (Asbahani et al. 2015).

Table 2: Essential oils used in nanoemulsions with proven pharmaceutical or biological activities

Scientific Common Production Energy Reference

Surfactants Activity
plant name plant name Method supply

(Hasssanzadeh
Magnetic stirrer
Allium Low , Alizadeh, and
Garlic Polysorbate 80 and ultrasonic Antibacterial
sativum energy Rezazad Bari
water bath
2018)
Spontaneous (Osanloo,
Anethum Polysorbate 20 Low
Dill method without Larvicidal Sereshti, et al.
graveolens And ethanol energy
mechanical force 2018)
Polysorbate 80 (Botas et al.
Baccharis Magnetic stirring Low
- and sorbitan Larvicidal 2017)
reticularia DC energy
monooleate
Backhousia (Nirmal et al.
Lemon
citriodora / Span 80 and High 2018)
Myrtle / Ultra-sonication Antibacterial
Syzygium Tween 80 energy
Anise Myrtle
anisatum
(Kiaei,
Calendula Tween 80 and High Hajimohammad
Calendula Ultrasonication Antiinflametory
officinalis Span 80 energy i, and Hosseini
2018)
High-speed (Lou et al.
Citrus High
Pomelo Tween 20 emulsion Antibacterial 2017)
maxima energy
homogenizer
 25

Scientific Common Production Energy Reference

Surfactants Activity
plant name plant name Method supply
Different mixtures (Lou et al.
with Cremophor 2017; Z. Li et
Citrus medica EL, 1, 2- al. 2018)
Fingered Low Antioxidant /
L. var. propanediol, Self-emulsification
citron energy Antibacterial
sarcodactylis glycerol, Tween
80, ethanol, PEG-
400
Sorbitan (Seibert et al.
monooleate and Phase inversion 2019)
Cymbopogon Low
Lemongrass PEG 40 emulsification Antioxidant
densiflorus energy
Hydrogenated
Castor Oil
Polysorbate 80 (Alam et al.
and Labrasol Self- Low 2018)
Eucaliptus Eucaliptus Wound Healing
nanoemulsification energy

Sorbitan High-speed (Hussain et al.

Ocimum High
Basil monooleate and emulsion Antimicrobial 2008)
Basilicum energy
Polysorbate 80 homogenizer

Ocimum Polysorbate 80 Magnetic stirring Low (Sundararajan

Basil Larvicidal
Basilicum energy et al. 2018)

Origanum Pluronic F127 Self- Low (Taleb et al.

Oregano Antiacne
vulgate L nanoemulsification energy 2018)

Spontaneous (Hashem et al.

Pimpinella Low
Anise Polysorbate 80 method Insecticide 2018)
anisum L. energy
emulsification
Sorbitan (A. E. M. F. M.
Pterodon Sucupira monooleate/ Self- Low Oliveira et al.
Larvicidal
emarginatus branca polysorbate 80 nanoemulsification energy 2017)

Rosmarinus Low (Duarte et al.

Rosemary Polysorbate 20 Magnetic stirring Larvicidal
officinalis energy 2015)
Rosmarinus Self- Low Antiinflammatory (Borges et al.
Rosemary Polysorbate 20
officinalis nanoemulsification energy /Antialgic 2018)
Polysorbate 20 or (Mazarei and
80 and sorbitan Rafati 2019)
Satureja High
Satureja monolaurate or Ultra-sonication Antibacterial
khuzestanica energy
sorbitan
monooleate
Polysorbate 80 (Shahavi et al.
Syzygium and sorbitan High 2016)
Clove Ultra-sonication Antibacterial
aromaticum monooleate energy

Syzygium (Osanloo,
aromaticum / Clove / Polysorbate 80 Self- Low Sedaghat, et al.
Antimicrobial
Cinnamomum Cinnamon and ethanol nanoemulsification energy 2018)
verum
 26

Scientific Common Production Energy Reference

Surfactants Activity
plant name plant name Method supply
Syzygium Polysorbate-20 (S. Zhang et al.
aromaticum / Clove / and Castor Oil Self- Low 2017)
Antifungal
Cymbopogon Lemongrass Ethoxylate-40 nanoemulsification energy
densiflorus
Polysorbate 80 (A. Sharma et
Thymuns High
Thyme and lecithin Ultra-sonication Antibacterial al. 2018)
daenensis energy

Sodium dodecyl High- pressure (Moghimi et al.

Thymus Conehead High
sulfate and homogenizer Antibacterial 2016)
capitatus Thyme energy
Polysorbate 20

(Hashemi
Zataria High
Zataria Polysorbate 80 Ultra-sonication Antibacterial Gahruie et al.
Multiflora energy
2017)

4. Preparation of nanoemulsions

Typically, the emulsification process is based on the dispersion of an immiscible liquid

into another immiscible liquid as small droplets which are surrounded by a thin interfacial layer of

a surfactant that acts as an emulsifier, nanoemulsification methods are no different. In general,

the methods to produce nanoemulsions can be classified into high or low-energy methods. High-

energy methods have been widely used to produce nanoemulsions in large industrial scale (David

J. McClements and Jafari 2018). These methods use mechanical devices to produce nano-sized

droplets. Low-energy methods depend on internal energy of the components to spontaneous

produce nanoemulsions upon changes in the compositions or environmental conditions. In both

type of methods, surfactants play an important role in reducing the interfacial tension of the

system and thus they contribute to the reduction of the shear energy required to reduce the radius

of curvature of the formed droplets (T. Tadros et al. 2004). The choice of the method to obtain

nanoemulsion should be made based on the properties of the oil phase and the surfactant,

physicochemical properties and functional aspects requires for the final application (David J.

McClements and Jafari 2018).

A minimum energy input required is the same no matter the nanoemulsification method.

However, the preparation method may influence the properties of the produced nanoemulsions,

such as: droplet size and stability. Without affecting the final nature of the dispersion phases,

though (Gutiérrez et al. 2008). On the other hand, the choice of the technique adopted to produce
 27

nanoemulsions greatly affect droplet size and stability (A. Salem and M. Ezzat 2018). In this

section, we have a brief overview on the most commonly used high-energy and low-energy

approaches for nanoemulsion formation.

4.1 – High-energy methods

High-energy methods are also known as work-based methods, since they are dependent

of high amounts of intense energy, which are supplied by mechanical devices, like high pressure

homogenizers, microfluidizers and ultrasonicators, which generate shear forces able to disrupt oil

and water interface, providing nanosized droplets (C. Solans et al. 2005; Leong et al. 2009). The

size and stability of nanoemulsions produced can be influenced by the type and processing

conditions of the high-energy method, oil properties such as lipophilicity, viscosity and interfacial

tension, surfactant type and concentration.

High energy methods are carried out by the input of mechanical energy using mechanical

or ultrasonic equipment that generate high shear stress or pressure difference, disrupting and

breaking the droplets into smaller sizes (Abismail et al. 1999; Sonneville-Aubrun, Simonnet, and

L’Alloret 2004; Tadros et al. 2004). In general, the preparation of nanoemulsions through high-

energy methods can be divided into two distinct phases. In a first step the oil and aqueous phases

are emulsified with an homogenizer as Turrax® or Politron® and the coarse emulsion obtained

presents a submicron droplet size, between 500 and 1000 nm, depending on the equipment used

and the operating conditions (David Julian McClements and Rao 2011; David Julian McClements

2011). Then, the droplet diameter is progressively reduced to its minimal value, ranging from 20

to 200 nm by means of high pressure homogenizers or microfluidizers, depending on the energy

intensity of the homogenizer used, processing time and sample composition (T. Tadros et al.

2004; A. Salem and M. Ezzat 2018). It is worth noting that the minimum droplet size achievable

may not be stabilized if the surfactant is insufficient to cover the newly created interface, as

observed by Barradas et al. (2015). Final droplet size results from a balance between two

phenomena that happen at the same time during homogenization: Breaking of drops into fine

droplets and droplet coalescence after processing (Jafari et al. 2008). Higher surfactant

concentration, increasing shear intensity or duration can contribute to reducing droplet size

(Gupta et al. 2016b).

 28

The obtention of nanoemulsions through high pressure homogenizers, ultrasonic

cavitation and microfluidizers has been well described in the literature (Tadros et al. 2004;

Helgeson 2016; Dias et al. 2014), and is considered as a safe method for keeping nanoemulsions

from instability phenomena without the addition of stabilizers, thickeners, cosolvents or

cosurfactants (Anton, Benoit, and Saulnier 2008). Figure 5 summarizes the high-energy methods

reported in this chapter.

Ultrasonication techniques for the preparation of nanoemulsions has also been described

by several authors (Shahavi et al. 2015; Gupta et al. 2016a; Leong et al. 2009). The device

comprises a sonication probe constituted by piezoelectric crystals able to expand and contract as

a consequence to an altering electrical voltage (Figure 5A). In consequence to sonicator probe

vibration, ultrasonic high-frequency waves, higher than 20 KHz, are produced, which are able to

wield a cavitation effect and causing mechanical vibration and the formation of micro-sized

bubbles that collapse, causing the disruption of oil-water interface (David Julian McClements

2011). As a result, fine nanosized droplets are obtained after enough time of processing to ensure

homogeneous size distribution and polydispersity index (Schwarz et al. 2012; Shahavi et al. 2015;

Abismail et al. 1999; Sivakumar, Tang, and Tan 2014). The main parameters involved in this

emulsification procedure are the interfacial properties of the emulsion, which can be controlled by

the nature and concentration of surfactants and the oil properties, such as surface tension and

viscosity (Gupta et al. 2016a). Moreover, droplet size is dependent on processing time, sonication

intensity, type and concentration of surfactant (Dias et al. 2014).

High pressure homogenization features the advantage of being applied in the production

of industrial scale nanoemulsions and greater control in droplet size reduction (Abismail et al.

1999). However, it requires large amounts of energy input, being more expensive to perform. The

high-pressure valve homogenizer consists basically of a pump, which injects the liquid to be

homogenized under very high pressure in a restrictive homogenizing valve. Many aspects may

influence the physicochemical characteristics of the obtained nanoemulsions as temperature,

viscosity and oil phase concentration of the emulsions, which affects the choice of the operational

parameters that must be adjusted for each formulation (Lee and Norton 2013). In high pressure

homogenizer, the sample is forced through small channels under a pressure ranging from 500-

15,000 psi (Gupta et al. 2016a) (Figure 5B).

 29

The sample flows under high pressure through microchannels resulting in a very fine

emulsion, which causes the disruption of the dispersed droplets. Therefore, the radius of the

generated droplets decreases gradually according to the increase of the shear rate. However,

due to the lack of homogeneity of the flow, it is often necessary to process this fluid through the

device through various cycles, until adequate droplets of size and polydispersity index are

obtained. The pressure and number of processing cycles can be adjusted to produce

nanoemulsions with tunable droplet size (Ouzineb et al. 2006; Constantinides, Chaubal, and

Shorr 2008).

The use of high-pressure homogenizers and ultrasonicators can lead to nanoemulsions

of equivalent physical-chemical properties upon the optimization of the operational conditions and

the qualitative and quantitative composition of the formulations. However, some authors have

reported some disadvantages regarding the use of ultrasonication, such as excessive heating of

the sample, larger droplet distribution and low reproducibility in relation to the droplet diameter

and polydispersity index, in addition to problems related to the difficulties of scale up (Tadros et

al. 2004; Gutiérrez et al. 2008). Moreover, as they require high energy input, they are often

considered cost-inefficient (Conxita Solans and Solé 2012).

Microfluidizers are composed of an interaction chamber where the fluid is injected and

homogenized by cutting, impact and cavitation, in a design that resembles the high pressure

homogenizers (Figure 5C) (Lee and Norton 2013; Tadros et al. 2004; Salem and Ezzat 2018). In

this case, emulsification can occur inside the channels, since both dispersed and continuous

phases flow inside fine channels individually. Streams are conducted to an interaction chamber

under high pressures, where they are submitted to disruptive conditions, which provide the

formation of fine droplets (Lee and Norton 2013; David Julian McClements 2011).

In this method, several parameters can influence the obtention of small droplets: the

viscosity of both dispersed and continuous phases and the type of the surfactant used (David

Julian McClements 2011; A. Salem and M. Ezzat 2018). Droplet is shown to decrease as

homogenization pressure, surfactant concentration and number of processing passes increase

and viscosity decreases (David Julian McClements 2011).

 30

Coarse emulsion

A B
Seat valve

Impact ring

Fine droplets

High impact zone

High pressure valve

Pre-homogenization Microfluidization zone

zone

High shear zone

C High shear zones

Figure 5: High-energy methods based on mechanical devices, such as ultrasonicators (A),

composed by a probe able to produce a cavitation effect and nanosized droplets are obtained

after enough time of processing to ensure homogeneous size distribution and polydispersity

index: High pressure homogenizators (B) consist basically of a pump, which injects the liquid to

be homogenized under very high pressure in a restrictive homogenizing valve, which causes

the disruption of the dispersed droplets. Microfluidizers (C) are composed of an interaction

chamber where the fluid is injected and homogenized by cutting, impact and cavitation.

Emulsification can occur inside the channels, since streams are conducted to an interaction

chamber under high pressures, where they are submitted to disruptive conditions, which provide

the formation of fine droplets. Modified after Jafari 2017 and Rao and McClements 2011.

4.2 Low-energy methods

All low-energy emulsification methods are based on physicochemical properties and uses

the internal chemical energy of the components. In phase inversion methods nanoemulsification

achieve by spontaneous inversion of the surfactant's curvature providing small size globules (Jin
 31

et al. 2016; Solans and Solé 2012). Low-energy emulsifying methods are advantageous because

they are effective in providing small-sized droplets and allow nanoemulsification by simple stirring

(Conxita Solans and Solé 2012). Figure 6 summarizes the most common low-energy methods,

which are reviewed in this chapter.

Nanoemulsification occur by phase inversion in an coarse emulsion as a result of

dramatic changes in the environmental conditions, in which parameters affecting the hydrophile-

lipophilic balance (HLB) of the surfactant, as temperature and / or concentration are modified

(Tadros et al. 2004).

The spontaneous formation of nanoemulsions is achievable by various methods based

on diffusion of solutes between two phases, interfacial turbulence, surface tension gradient and

dispersion or condensation mechanisms (Salem and Ezzat 2018). The spontaneous

emulsification (Figure 6A), also referred as self-emulsification method is based on the diffusion of

a water-miscible component from the organic phase into aqueous phase when both phases are

put into contact, and one of the phases contains a component miscible in both phases. Surfactant,

cosurfactant or a polar organic solvent such as ethanol or acetone can be examples of dual-

soluble components (David Julian McClements 2011). As a consequence, some of the

components partially miscible in both phases diffuse from the original phase towards the other

one in a rapid diffusion movement, without no phase transition or change in the surfactant

spontaneous curvature (Conxita Solans and Solé 2012). The sudden diffusion of the components

provides an increased oil-water interfacial area, which trigger other phenomena such as interfacial

turbulence and thus, the spontaneous droplets assemble (Anton, Benoit, and Saulnier 2008; A.

Salem and M. Ezzat 2018).

Spontaneous nanoemulsification and droplet size can be highly influenced by the

composition of the formulation, some physical chemical properties and the mixing conditions

(Bouchemal et al. 2004; Rao and McClements 2012). The obtention of nanosized micelles can

be performed with the addition of high concentrations of water-miscible component into the oil

phase and very high solvent/oil ratio (David Julian McClements 2011; Conxita Solans and Solé

2012).

Spontaneous emulsification is often related to a special phenomenon called The Ouzo

effect with beverages based on sweet fennel oil, rich in trans-anethole, that allow surfactant-free
 32

self-emulsification (Carteau, Bassani, and Pianet 2008). When water is added to the alcoholic oil

solution, some of the ethanol molecules diffuse from the organic phase into aqueous phase, which

reduce sweet fennel oil solubility and small oil droplets spontaneously assemble. Spontaneous

emulsification has been recently explored to provide sweet fennel oil-based nanoemulsions with

very reduced amounts of surfactants (Barradas et al. 2017).

Moreover, nanoemulsions can also be obtained from dilution of surfactants aggregates,

as liquid crystalline particles and bicontinuous microemulsions. Solè et al. produced 20 nm oil-in-

water nanoemulsions upon dilution of oil-in-water and water-in-oil microemulsions by self-

emulsification method. The effect of dilution procedure, i.e., stepwise or at once, and cosurfactant

nature on nanoemulsion formation was studied. oil-in-water microemulsion provided small-sized

20 nm nanoemulsion regardless the dilution method and microemulsion composition. On the

other hand, water-in-oil microemulsion resulted in nanoemulsion when water dilution was

performed stepwise. Regarding cosurfactant nature, droplet size decreased as cosurfactant alkyl

chain size increased, which enhanced nanoemulsion stability (Solè et al. 2012).

Self-emulsification is being applied by pharmaceutical industry to obtain oil-in-water

nanoemulsions for being a low-cost technique. Moreover, spontaneous emulsification is being

highly explored to produce self-emulsifying drug delivery systems (SEDDS) and self-

nanoemulsifying drug delivery systems (SNEDDS). The main application of self-emulsifying drug

delivery systems comprises the very self-emulsification obtention method increase in drug

bioavailability and stability of the micelles (Wei et al. 2012; Shahba, Mohsin, and Alanazi 2012;

Anton and Vandamme 2011). However, one of the major limitation of this method is the very high

concentrations of surfactants needed to trigger self-emulsification, which can cause toxicity

(David Julian McClements 2011; Azeem et al. 2009).

Phase inversion-based methods are low-energy techniques that involve the inversion of

the surfactant curvature, passing through a transition phase in which surfactant curvature

achieves zero curvature, such as bicontinuous microemulsions or lamellar liquid crystalline

phases (Porras et al. 2008; T. Tadros et al. 2004; Fernandez et al. 2004; Sutradhar and Amin

2013; Mayer, Weiss, and McClements 2013; Conxita Solans and Solé 2012). They are based on

the chemical energy released from phase transitions phenomena during emulsification process.

Phase inversion methods occur when some dramatic change in the environmental conditions take
 33

place, i.e., temperature or composition. These methods require an extensive control in terms of

selecting the right surfactant, knowledge of surfactants phase behavior and the most adequate

thermodynamic method. Droplet size provided depends on the selected surfactant, the properties

of the intermediary phases formed and the interfacial properties between both fluids (Helgeson

2016; Maestro et al. 2008).

In phase inversion composition method (Figure 6B), phase inversion occurs by a major

change in composition of the system. There is an increase of the volumetric fraction of the

dispersed phase that is added to a microemulsion and then, the curvature of the surfactant is

altered. Maestro et al. showed that changes in salt concentration changed the spontaneous

curvature of ionic surfactants and prepared water-in-oil nanoemulsions from oil-in-water

emulsions by means of phase inversion composition. Phase inversion was achieved by the ability

of salt ions to screen the charges on surfactant groups (Maestro et al. 2008).

Changing pH is also a useful approach to produce nanoemulsions by phase inversion

composition. It can provide changes in electrical charge a stability of surfactants. Solè et al.

reported the production of nanoemulsions by phase inversion composition method using ionic

surfactant, potassium oleate, for example. At low pH values, the carboxyl groups from fatty acids

are non-ionized and, thus lipid soluble. Under this condition, these molecules can stabilize water-

in-oil emulsion. However, this situation is inverted if pH is raised. At higher pH values, carboxyl

groups become ionized which increases water-solubility and provides the stabilization of oil-in-

water emulsion (Solè et al. 2006).

Phase inversion composition method is considered an interesting approach for large-

scale production of nanoemulsions, since it relies only on the addition of one component to a

mixture of components without requiring high temperatures of high-energy input, being also

beneficial when temperature-sensitive components are used. Moreover, phase inversion

composition method is not limited a specific type of surfactant (Conxita Solans and Solé 2012).

For temperature-sensitive surfactants, a phase inversion can be achieved by changing

the temperature of the system, forcing the transition of an oil-in-water emulsion, prepared at low

temperatures, for a water-in-oil emulsion, formed at higher temperatures, due to changes in the

physicochemical properties of the surfactant. This is a typical phase inversion composition

example of a transient phase inversion, known as phase inversion temperature, in which

 34

nanoemulsions are formed under a fixed composition by changing temperature, by a drastic

change in surfactants water solubility, through an intermediate liquid crystalline or bicontinuous

microemulsion phase (Fernandez et al. 2004; David Julian McClements and Rao 2011).

This process is triggered by the changes in physicochemical properties of surfactant as

temperature changes. Polyethoxylated nonionic surfactants are highly temperature-dependent.

They tend to become lipophilic with increasing temperature due to dehydration of the chain of the

polar part of the surfactant, i.e., ethylene oxide groups. During heating, the ethylene oxide groups

responsible for the hydrophilic characteristic of the surfactant are "hidden" and, consequently,

there is modification of the affinity for the phases. As the system undergoes cooling, the surfactant

passes through a point of zero curvature and with minimum surface tension, which provides

conditions to the formation of nanoemulsions (Fernandez et al. 2004; T. Tadros et al. 2004).

The inversion point take place at a specific temperature, i.e., phase inversion

temperature, when the solubility of the surfactant in water and in oil reach equivalent values. As

temperature is continuously raised, the surfactant becomes more soluble in the oil phase than in

the aqueous phase (David Julian McClements 2011). Nanoemulsions are obtained by rapidly

breaking up microemulsions formed at phase inversion point by sudden cooling below phase

inversion temperature (David Julian McClements 2011). Through the phase inversion

temperature method very small droplet sizes and stable nanoemulsions are obtained. On the

other hand, the systems are highly prone to coalescence, being unstable, which makes cooling

step a critical aspect to obtain stable nanoemulsions (Tadros et al. 2004; Conxita Solans and Solé

2012).

In emulsion inversion point method (Figure 6C), one solvent, i.e. water or oil, is

continuously added under stirring to the dispersed phase until this solvent concentration becomes

predominant, at constant temperature. Once a critical amount of solvent is achieved, droplets are

so highly packed together that phase inversion occurs, and the emulsion reaches a phase

inversion point from water-in-oil to oil-in-water or vice-versa. This causes the spontaneous

surfactant curvature to change, i.e., micelles are assembled in reverse curvature, which disrupts

them into smaller structures thereby obtaining emulsions with nanometric droplets (Anton, Benoit,

and Saulnier 2008).

 35

Figure 6: Schematic diagram of low energy methods: The spontaneous emulsification (A),

is based on the diffusion of a water-miscible component from the organic phase into aqueous

phase when both phases are put into contact, and one of the phases contains a component

miscible in both phases. In phase inversion temperature (B), there is an increase of the volumetric

fraction of the dispersed phase that is added to a microemulsion and then, the curvature of the
 36

surfactant is altered. Emulsion inversion phase (C) is known to cause a catastrophic phase

inversion, which occurs through the inversion in oil-to-water ratio. Modified after Jin, Xu, Liang, et

al. 2016; Anton and Vandamme 2011 and Rao and McClements 2011.

Emulsion inversion point is considered to cause a catastrophic phase inversion, which

occurs through the inversion in oil-to-water ratio. The surfactants used to produce the

catastrophic phase inversion comprise small molecule synthetic emulsifiers able to stabilize both

oil-in-water and water-in-oil emulsions. In this method, droplet size depends on the stirring speed

and the solvent addition speed (David Julian McClements and Rao 2011).

Although the phase inversion temperature method facilitates the obtention of

nanoemulsions with nanometric droplet sizes by the fact that it is possible to achieve very low

values of interfacial tension, the dynamics of droplets coalescence can be extremely fast. In order

to obtain stable nanoemulsions, the water-oil dispersion formed from phase inversion temperature

method is readily cooled at a temperature just below it. Thus, the lamellar or the bicontinuous

system collapse forming small droplets kinetically stable, with very small droplet size and narrow

size droplet size. If the cooling process is not fast enough, the coalescence predominates and a

polydisperse mixture is obtained (Fernandez et al. 2004).

According to the literature, not only the minimum interfacial tension produced during

inversion of the curvature of the surfactant, but also the formation of the bicontinuous phase or

liquid-crystalline phase prior to the inversion locus is closely related to the formation of

nanoemulsions, in both emulsion inversion point and phase inversion temperature methods

(Fernandez et al. 2004; T. Tadros et al. 2004). Moreover, complete solubilization of the oil in the

bicontinuous phase or in the crystalline phase is an extremely important factor for the formation

of fine nanoemulsions.

3.3 Comparison between obtention methods

High energy emulsification processes are different regarding energy input and

emulsification time: the high-pressure homogenizer, for example, is a high energy and low

process time method; ultrasonication is a high energy and long process method; and rotor stator

is a mixer that uses less energy, requiring longer process times. On the other hand, low-energy
 37

processes can provide smaller droplet sizes compared to high-energy methods. However, there

are limited options for surfactants to be used in low-energy approaches, which exclude several

natural surfactants as proteins or polysaccharides (Rao and McClements 2012). Besides, low-

energy methods, as they cannot count on high shear energy devices, require large amounts of

surfactants, which can limit the pharmaceutical application of the obtained nanoemulsions.

Jafari, He, and Bhandari compared different methods of high energy emulsification, it was

observed that the average mechanical mixer produced emulsions with large droplet size, in a

range above 10000 nm, because, in this case, the main deformations forces are shear stresses

in laminar flow that are not able to efficiently break the droplets. The stator rotor mixer proved to

be more efficient than the average mechanical mixer in the formation of smaller droplets, of about

1000 nm, and with narrow size distribution, due to the incorporation of more forces on the shear

that can form smaller droplet sizes, for example, inertial forces (Jafari, He, and Bhandari 2007).

Considering the other two emulsification systems, the high-pressure homogenization, and

microfluidization, provided droplet sizes and size distribution smaller than the ultrasonication due

to the greater efficiency in the rupture of the drops, or with cavitation along with shear and inertial

forces, in addition to the higher energy input. From these results it can be shown that the

difference in droplets size obtained is directly related to the energy input (Jafari, He, and Bhandari

2007).

Comparing both high and low energy emulsification methods, it was verified the variation

of the droplet size as a function of time for nanoemulsions prepared using the phase inversion

temperature method and high-pressure homogenization. In both cases, an increase in droplet

hydrodynamic size after a certain time the preparation of nanoemulsions. However, when high

pressure homogenization is used, the droplet size can be maintained at lower values for longer

periods of time. On the other hand, phase inversion temperature method provides a faster

destabilization of the system (T. Tadros et al. 2004).

Phase inversion composition method and high-pressure homogenization were compared

in the production of oil-in-water efavirenz-loaded nanoemulsions. Phase inversion composition

method was performed by dilution in water. Nanoemulsions were evaluated regarding the

physical stability when submitted to heating-cooling cycles, centrifugation and freeze-thaw cycles,

water-dispersibility, droplet size, rheology and dilution impact on droplet size. Both
 38

nanoemulsions showed good physical stability and water dispersibility. Regarding viscosity, there

was no significant difference between nanoemulsions prepared by each method. Nanoemulsions

prepared through phase inversion composition method showed to be more transparent than those

prepared by high-pressure homogenization, this is due to the concentration of surfactant and

oil/surfactant ratio used to prepare nanoemulsions by each method. Droplet size showed no

significant difference between both methods. However, polydispersity Index was found to be

significantly different between the methods. Phase inversion temperature method provided

polydispersity index from 0.150 to 0.404, while high-pressure homogenization varied from 0.637

to 0.812. Both nanoemulsions proved to be stable under infinite dilution. Droplet size and

polydispersity index remained unaltered after sample dilution. Drug release also showed

differences between nanoemulsions made by high-energy and low-energy emulsification

methods. Drug release from nanoemulsion prepared by high-pressure homogenization was

smaller than that prepared by phase inversion composition method, which can be a consequence

of polydispersity index (Kotta et al. 2015).

Yang et al. compared microfluidization with spontaneous emulsification in the production

of food-grade nanoemulsions with 20% of oil phase and same type and concentration of

surfactants. The influence of the surfactant used end surfactant-oil ratio, stability and droplet size

were also evaluated. Microfluidization produced nanosized droplets with less than 100nm, using

lower surfactant-to-oil ratio than spontaneous emulsification. Besides, microfluidization showed a

linear decrease in droplet radius as surfactant concentrations increased until it reached a value

where no difference in droplet size is observed, suggesting that minimum droplet size was

achieved. Spontaneous emulsification also produces droplets smaller 100 nm, even though to do

so, it required considerably higher, i.e., 10-fold higher of surfactant concentration. Regarding the

kinetic stability, microfluidization provided stable droplets against aggregation and gravitational

separation phenomena for at least 30 days. Spontaneous emulsification, on the other hand,

provided droplets sizes that increased after one-month storage. Moreover, nanoemulsions

showed creaming instability signs (Yang et al. 2012).

 39

5. Stability of Nanoemulsions

As previously mentioned, nanoemulsions are characterized for being kinetically stable.

Nanoemulsions shelf life can be further improved with some stabilization strategies that aim at

maintaining bulk and droplet properties stable for longer periods of time. In order to achieve stable

nanoemulsions several stabilization methods can be used, such as electrostatic, steric and

mechanical stabilization (Cardoso-Ugarte, López-Malo, and Jiménez-Munguía 2016).

Repulsive electrostatic forces between droplets can ensure proper droplet separation and

prevent coalescence and/or flocculation. This is related to surfactant superficial charge and is

responsible for the high stability of ionically-charged nanoemulsions. Electrostatic stabilization is

more important with smaller droplets, since they have an increased superficial area (Helgeson

2016).

Superficial charge is often provided by the surfactants, especially natural surfactants, as

biopolymers with surface activity like proteins, polysaccharides, which can stablish electrostatical

intermolecular interactions depending on concentration, pH and isoelectric point, and ionic

strength of the solution (Cardoso-Ugarte, López-Malo, and Jiménez-Munguía 2016). When pH of

the surrounding solution is far from protein isoelectric point, proteins residues are charged and

there is an electrostatic repulsion that hinders droplets to aggregate (Dickinson, Galazka, and

Anderson 1991).

Non-ionic surfactants also can provide stabilization, even though they are not charged.

Steric stabilization is preponderant in the case of non-ionic surfactants, particularly with

amphiphilic non-ionic polymers, which can form voluminous interfacial films around droplets able

to prevent coalescence. Amphiphilic block polymers are advantageous since less amounts of

surfactants are required to stabilize droplets. On the other hand, in some cases, macromolecular

surfactants can have difficult diffusion towards droplets interface due to their size and molecular

mobility (Bouyer et al. 2012; T. Tadros 2009; Qian and McClements 2011).

Steric stabilization comprises three main mechanisms: (i) non-adsorbent macromolecules

which provide an elastic film around droplets that avoid droplet collision and deformation, (ii)

branched macromolecules that form a voluminous surface that prevent droplets from

approaching, (iii) stabilization due to hydrophobic interactions between adsorbed macromolecular

surfactants (Cardoso-Ugarte, López-Malo, and Jiménez-Munguía 2016).

 40

The addition of viscosity agents and gelling polymers in the outer phase of nanoemulsions

is often called as mechanical stabilization, since they can reduce droplets mobility by providing a

mechanical network that serves as a barrier to aggregation. Viscosity agents are often called as

stabilizers and produce semi-solid or gel-like systems. However, stabilizers should be used with

caution, since important properties as optical appearance, droplet size and encapsulation

efficiency can be modified (Behrend, Ax, and Schubert 2000; Dickinson 2009).

5.1 Instability phenomena

As nanoemulsions are formed from non-spontaneous process they tend to undergo

instability phenomena such as flocculation, coalescence and Ostwald ripening and gravitational

phase separation as conventional emulsions. However, the small size of the droplets in a

nanoemulsion confers enhanced kinetic stability, compared to ordinary emulsions (David Julian

McClements 2011).

Thus, nanoemulsions can be destabilized by several different phenomena: (i) irreversible

phenomena, related to the permanent modification of the droplet size and may lead to complete

phase separation; (ii) reversible flocculation of droplets, which may be followed by creaming or

sedimentation, according to the respective densities of the dispersed and continuous phases

(Tadros et al. 2004; Sing et al. 1999; Abismail et al. 1999). In general, nanoemulsions tend to be

more stable to gravitational separation, flocculation and coalescence and more susceptible to

Ostwald ripening (David Julian McClements 2011).

Reversible phenomena involve aggregation and migration of droplets, as flocculation and

creaming. Irreversible phenomena are related to the modification of droplet size, for examples,

coalescence. Droplets can co-exist in nanoemulsions as individually separated entities or as flocs,

i.e., droplets aggregates formed as consequence of attractive interactions among them,

characterizing a flocculated system (David Julian McClements 2015; David J. McClements and

Jafari 2018).

Flocculation is a reversible phenomenon in which the droplets dispersed in an emulsion

aggregate and migrate, aiming to reach the thermodynamic equilibrium by decreasing the

chemical potential differences that exist throughout the system. During this process, the droplets

collide randomly and can remain in contact after these shocks, producing aggregates or flocs
 41

(Katsumoto et al. 2001; Starov and Zhdanov 2003). In flocculation, droplets aggregate without

the rupture of the interfacial surfactant film, being a reversible phenomenon. The flocculation rate

depends on attractive forces between droplets, the frequency of collisions between droplets and

how long they remain in contact (Lijuan Wang et al. 2009). In a dilute system, flocculation may

accelerate gravitational phase separation as a consequence of the increase in droplet size. On

the contrary, in concentrated systems, flocculation can prevent gravitational phase separation,

since the aggregates may produce a tree-dimensional network which may be a barrier to phase

segregation (David J. McClements and Jafari 2018).

According to the difference of density between both inner and outer phases, the

aggregates formed in the flocculation phenomenon may show gravitational separation such as

sedimentation or creaming. When less dense aggregates are formed, they can rise to the surface,

characterizing the phenomenon of creaming. Contrarywise, when inner phase shows higher

density, denser aggregates are formed and deposited at the bottom of the system, constituting

the sedimentation process (Figure 7). Both sedimentation and creaming phenomena are related

to gravitational forces that can influence whether the aggregates will move upwards or

downwards, depending on the difference of density between inner or outer phases. Hence, both

phenomena are most prone to occur with droplets with increased droplet size, as larger objects

are more susceptible to gravitational forces. Thus, gravitational separation can be reduced with

smaller droplet sizes, increasing the viscosity of continuous phase or by reducing the density

difference between dispersed and continuous phases (David Julian McClements 2011; David J.

McClements and Jafari 2018).

 42

Figure 7: Instability phenomena in emulsions and nanoemulsions. Reversible phenomena

comprise aggregation and density-driven migration of droplets, as flocculation and creaming.

Irreversible phenomena are related to a definitive increase of droplet size, for example:

coalescence, Ostwald ripening and, the most drastic, phase inversion.

Modified after David J. McClements and Jafari 2018 and Taylor 1998.

Some surfactants as proteins and polysaccharides can change droplet density by forming

a shell layer on droplet surface, which can prevent gravitational separation phenomena such

creaming, since it reduces density differences between inner and outer phases. Moreover, it can

be possible to produce tunable-density droplets by controlling droplet size and thickness of

surfactant layer (David Julian McClements 2011).

Irreversible phenomena include the Ostwald ripening, phase separation and

coalescence, which may lead to the eventual complete phase separation (Sing et al. 1999).

Coalescence can occur when droplets collide and merge, producing one larger droplet.

Coalescence can lead to complete phase separation but can be avoided when repulsive
 43

interactions are provided by either electrostatic or steric effects, which is achievable by the right

choice of surfactant (David J. McClements and Jafari 2018).

Ostwald ripening is often considered as the main cause of instability of a nanoemulsion

(Liu et al. 2006). This process is dependent on the polydispersity of the system, the solubility of

dispersed phase in the continuous phase and the difference in solubility between droplets of

different sizes. Ostwald ripening is the process by which the larger particles grow from the smaller

droplets due to the greater solubility of the smaller droplets and by the molecular diffusion of the

continuous phase.

In polydisperse nanoemulsions, there is a difference between the capillary pressure from

different-sized droplets which makes the small droplets of the dispersed phase to diffuse into the

large droplets (Chebil et al. 2013; Taylor 1998). As a consequence, the larger droplets grow from

the smaller ones that have greater chemical potential. Thus, the droplets content diffuses through

the dispersing phase due to the greater solubility of the smaller droplets, which does not require

contact between the droplets. This phenomenon aims to decrease the total energy of the system

by reducing the total interfacial area, which can be avoided with the use of insoluble oils and with

the choice of suitable polymeric surfactants (T. Tadros et al. 2004; C. Solans et al. 2005;

Constantinides, Chaubal, and Shorr 2008).

The rate of droplet growth depends on the product of the solubility of the dispersed oil in

the aqueous continuous phase and oil diffusion coefficient and is explained by the Lifshitz-Slezov-

Wagner theory (Taylor 1998). It predicts that the Ostwald ripening rate presents a linear

relationship between the dispersed phase droplet radius and time. Lifshitz-Slezov-Wagner theory

assumes that the droplets of the dispersed phase are spherical and furthermore the distance

between them is greater than the diameters of these droplets and the kinetics is controlled by

molecular diffusion of the dispersed phase in the continuous phase. Also according to this theory,

the Ostwald ripening rate in oil-in-water nanoemulsions, although is predicted to be lower than in

conventional emulsions, is directly proportional to the solubility of the oil in the aqueous phase

(Helgeson 2016).

However, the decrease in droplet size causes the increase of oil solubility in water, which

is the driving force of Ostwald ripening. As the main factor for Ostwald ripening is the solubility of

oil phase in water, it does not configure a real issue when it comes to poorly water-soluble oils.
 44

On the other hand, for nanoemulsions formulated with oils with some water-solubility, Ostwald

ripening might represent the main instability event (David Julian McClements 2011).

Tadros et al. presented two methods that tended to delay the increase in droplet size. In

the first, a further dispersed phase that is insoluble in the continuous phase is added to the

nanoemulsion (T. Tadros et al. 2004). The fact that it is insoluble causes the droplets to remain

almost unchanged in size, generating a balance in the chemical potential due to the partitioning

of the dispersed phases. This method has limited application because of the difficulty of finding a

phase that fully meets this process. From these results, reduction of the Ostwald ripening process

can be observed by the addition to the system of a small amount of a second oil with low solubility

in the aqueous phase

The second method consists in the modification of the interfacial film between the oil

phase and water, i.e. outer phase, by adding another surfactant, preferably a block copolymer A-

B-A type. The effect would be the adsorption of this copolymer at the oil-in-water interface

reducing interfacial tension and balancing curvature effects, which would reduce the diffusion rate

of the dispersed phase, minimizing droplet ripening (T. Tadros et al. 2004). In addition, for a

system containing a nonionic surfactant based on poly (ethylene oxide), the Ostwald ripening rate

can also be decreased by adding a second surfactant having the same hydrophobic group and

with a higher hydrophilic content, i.e., ethylene oxide chains, than the primary surfactant (C.

Solans et al. 2005).

Several studies were performed to describe and to modulate the Ostwald ripening

mechanism. They suggested that the Ostwald ripening can also be delayed or disrupted by size,

interfacial viscosity or elasticity of the droplets. This occurs when the interfacial tension between

the dispersed phase and the continuous phase equals zero. For a number of drops of the

emulsion it was also shown that the Ostwald ripening could be disrupted by interfacial elasticity,

even at finite interfacial tensions (Meinders and van Vliet 2004; Liu et al. 2006).

Repeated collisions might trigger coalescence, which is the mechanical fusion of two

droplets (Helgeson 2016). Coalescence occurs after prolonged contact between the particles,

when the adhesion energy between two droplets is greater than the turbulent energy that causes

the dispersion. The mechanism is based on the rupture of the thin film between adjacent droplets,

which leads to the joining of two droplets, forming one of larger size. The origin of the rupture of
 45

this film can occur due to a mechanical instability in the emulsion. When a large number of

particles coalesce, the result is complete separation of the phases. The greater the extent of this

phenomenon, the greater the tendency to complete phase separation (Sing et al. 1999).

Irreversible changes caused by Ostwald ripening lead to the formation of larger droplets,

i.e., the formation of less stable emulsions, which may be responsible for phase separation.

Phase inversion may occur due to temperature variation and/or change in composition of the

emulsion.

The composition of nanoemulsions can be tuned towards to produce kinetically stable

systems. The adequate selection of oil and aqueous phase and, most importantly, surfactants are

essential to an ideal nanoemulsion design.

6. Characterization of nanoemulsions

A wide range of analytical methodologies are required in order to fully characterize

nanoemulsions droplet size, aggregation, charge and physical state and bulk properties as

rheology and stability. The most used are described in more details here.

6.1 Dynamic Light Scattering

Droplet size distribution are some of the most important physical characteristics of

nanoemulsions and can influence a great number of characteristics, since droplet size can

influence bulk properties, such as optical properties, rheology and stability (David Julian

McClements 2011) and also other functional properties as solubility, release profile and

bioavailability (Bourbon et al. 2018). In general, when nanoemulsions are produced, a range of

different droplet sizes are distributed all over the system. Hence, droplet size is often reported as

mean droplet size and polydispersity index (PdI). PdI values below 0.3 is indicative of a narrow

size distribution whereas values close to 1.0 are indicative of very high size heterogeneity (Klang

and Valenta 2011).

Dynamic light scattering (DLS) (λ = 500 nm) is based on measurements of photon

correlation spectroscopy caused by the Brownian motion of droplets. The diffusion of small

isometric particles is rapid, causing faster fluctuations in the intensity of scattering light compared
 46

to large particles that diffuse more slowly. The particle/droplet size analyzer is based on the typical

principle of DLS. This principle comprises four main components. The first of all is the laser beam,

which is used to provide the light source to illuminate the particles inside the vial. Most of the laser

beam passes rectilinearly through the sample, but another part of it is scattered by particles or

droplets dispersed in the medium (Dorfmüller 1987).

A detector is used to measure the intensity of scattered light. As one single particle

scatters light in all directions, it is possible to place a detector at any position and still detect the

scattering. In DLS, the position of 90°, from the detector to the incident light beam, is a classical

detection arrangement. However, it provides a narrower detectable size range and thus, can only

be used for low sample concentrations (Dorfmüller 1987).

Another common device used for particle size analysis feature the position of the detector

in 173° from the transmitted light beam. The scattered light intensity must be within a specific

range for the detector to measure it successfully. When the amount of light detectable is out of

this range attenuators should be applied to reduce the light intensity of the laser and thus reduce

the scattered intensity (Dorfmüller 1987).

The intensity of the scattering signal is transmitted to the detector by a digital signal

processor called correlator. The correlator compares the scattering intensity at successive time

intervals to derive the rate at which the intensity varies. The detection optics of this equipment

measure the information of the scattering near 180º, being known as backscatter detection,

patented technology known as non-invasive backscatter (Porras et al. 2004).

Analysis of apparent hydrodynamic radius (rh) are often performed under the dilute

regimen, which makes it possible to obtain the diffusion coefficient D as the Equation 3, where η

is the viscosity of the medium (Lijuan Wang et al. 2008).

𝑟ℎ = 𝐾𝑇/6𝜋𝜂𝐷 (Equation 3)

With very concentrated samples the apparent droplet size can be overestimated because

of multiple scattering due to long-length colloidal interactions among close droplets. In this

context DLS measurements can be often limited by the necessity of diluting the samples.
 47

The mean droplet size obtained from DLS can be determined by different manner, for

instance, number, intensity (Z-average) and volume values. Hence, it is fundamental to be clear

about the type of determination applied to droplet size characterization, since these values can

be very discrepant, depending on sample concentration, polydispersity and refraction index.

In order to characterize droplet size, techniques such as electron microscopy, X-Ray and

Neutron Scattering of liquid emulsions, spectro-turbidity and dynamic light scattering can be used,

the latter being the most used in the characterization of nanoemulsions (David Julian McClements

2015).

Droplet size can also be characterized by transmission electron microscopy, which can

visually provide droplet size and distribution in nanoemulsions of nanometric droplet sizes. From

this technique it is also possible to observe morphological spherical shape of droplets and

evaluate the destabilization of the nanoemulsions, in which droplet coalescence can observed.

However, transmission electron microscopy has also limitations, which are often related to sample

preparation that can induce to coalescence upon solvent evaporation during drying step. Besides,

contrast with heavy metals as drying can also alter droplets environment and induce to some

instability phenomena. High-energy electron beams can ruin nanostructure of droplets, which can

be overcome by Cryo- transmission electron microscopy techniques.

6.2 Zeta Potential

Nanoemulsions formulated with ionic surfactants feature superficial charge. The type and

magnitude of the superficial charge of droplets are responsible for many characteristics of

nanoemulsions, such as stability against aggregation phenomena and functional properties like

mucoadhesivity and absorption (David Julian McClements 2015).

The electrical characteristics of nanoemulsion can range from strongly positive, to neutral

and strongly negative depending on the surfactant charge and the surrounding conditions. Droplet

superficial charge can be characterized by zeta potential. Zeta potential describes the electro-

kinetic potential in a system. Charged nanodroplets dispersed in a liquid system attract opposite-

charged ions close to surface, providing a double layer with a certain thickness that can vary

depending on the type and concentration of counter-ions (Jin, Xu, Liang, et al. 2016). With the

distribution of counter-ions in the surrounding droplet layer, any movement between rigid and
 48

mobile phases provides an electrokinetic potential (Bourbon et al. 2018). The stern layer plays

an important role in the stabilization of nanoemulsions, since stern layer is responsible for the

repulsive forces that prevent droplets from aggregate.

Zeta potential measurements results are presented in millivolts (mV) and is carried in an

electrophoretic cell, where two electrodes create an electrical field and migration of colloidal

particles or droplets is measured. Zeta potential can be strongly affected by pH and ionic strength

of the system (Bhattacharjee 2016). Other factors can greatly influence zeta potential as the state

oh hydration and droplet morphology. According to literature, zeta potential values above 60 mV

suggest excellent electrostatic stability, while results from 60 to 30 mV indicate good stability and

from 5 to 15 mV suggest a region of limited flocculation and from 3 to 5 mV correspond to

maximum flocculation (Bourbon et al. 2018). When zeta potential values are high in module,

droplets would repulse each other and the system is less prone to coalescence (E. F. de Oliveira,

Paula, and Paula 2014; Junyaprasert et al. 2009; Svetlichny et al. 2017).

Nanoemulsion stabilized by anionic surfactants show negative superficial charge and

those stabilized by cationic surfactants present positive superficial charge. By controlling the

amount and the type of surfactant used when formulating nanoemulsions, it is possible to tune

the superficial charge according to the characteristics needed.

6.3 Rheological behavior

Bulk physicochemical properties, as rheological behavior can deeply impact

nanoemulsions applicability, particularly for some applications routes as topical or dermal

application (Barradas et al. 2018). Rheology is an important tool to characterize nanoemulsion

stability. In general, flocculated systems are more viscous than a non-flocculated nanoemulsion

with the same droplet size and concentration (David Julian McClements and Rao 2011).

Moreover, rheological viscoelastic properties reflect flow behavior and the deformation during

nanoemulsions industrial production in processing steps as flow through pipes, mixing and

packaging (Chung and McClements 2018).

Flow behavior of nanoemulsions are measured apparent viscosity values as a function of

shear rate or shear stress. Viscoelastic properties are determined under dynamic oscillation

modes and are presented as viscous (G`) and elastic (G´´) modulus as a function of deformation
 49

of frequency. Typically, dilute small-sized nanoemulsions can show low viscosity and a newtonian

behavior as previously published by Barradas et al (Barradas et al. 2017). The elastic modulus

provides information about the elastic properties and the stored energy, being considered a solid-

like property. On the other hand, the viscous modulus indicates the viscous properties and the

energy dissipated as heat being a liquid-like property.

Nanoemulsions viscosity increases as droplet increases and when dispersed phase is

more concentrated. As droplet concentration increases, there is a tendency to aggregation

among them, which can increase both viscosity and viscoelastic behavior (T. Tadros 2004).

Droplet aggregation can change flow behavior from newtonian to shear thinning and increase

elastic modulus of concentrated nanoemulsions. Shift on rheological behavior occur once critical

droplet concentration is achieved. Besides, rheological behavior of nanoemulsions can be altered

by strong electrostatic attractive or repulsive interactions among the droplets, which can provide

a three-dimensional network and increase viscoelastic behavior.

The viscosity of oil phase can also greatly influence rheology and viscosity of

nanoemulsions. Longer fatty acids chains lengths are able to increase viscosity of vegetable oils.

As oil degradation occur, the size of fatty acids chains can be reduced by hydrolysis, which often

causes the reduction in nanoemulsions viscosity as reported by Barradas et al. 2017. Thus,

rheology can be an important tool to access nanoemulsion stability.

Rheology can also be a useful approach to characterize nanoemulsions microstructure

and classify them as viscous, viscoelastic or semi-solid materials. Transient non-destructive tests

are often applied to study structural recovery time of nanoemulsions after a constant stress is

applied. This measurement can provide important information regarding the strength of

intermolecular bonds and microstructure of the sample. For example, for injectable

nanoemulsions it is fundamental to predict formulation viscosity, rheological behavior and

structural integrity after being submitted to high deformation upon passing through a syringe.

Dynamic oscillatory tests can determine viscoelastic properties of the samples, allowing

to classify them into fluid-like, solid-like, gel-like and semi-solid materials (Helgeson 2016; Chung

and McClements 2018). Both frequency and strain stress can be tuned to obtain information about

the macrostructure of materials. Non-destructive experiments are often performed under small

strains and low frequencies in order to maintain internal structure unaltered, within linear
 50

viscoelastic region. Largely destructive measurements are conducted to reproduce stresses to

which nanoemulsions can be submitted during production or practical application. High stresses

are applied to cause flow or large deformations. In these cases, for liquid-like fluid nanoemulsions,

viscosity is reported as apparent viscosity at a particular shear stress (Chung and McClements

2018).

6.4 Conductivity

Calderò et al. produced nanoemulsions oil-in-water nanoemulsions from water-in-oil

emulsions by phase inversion composition method as a template for polymeric nanoparticles by

solvent evaporation. Conductivity determinations were used to confirm phase inversion from

water-in-oil to oil-in-water (Calderó, García-Celma, and Solans 2011). As water concentration

was increased, conductivity was significantly raised until a maximum, when it dropped. It was

justified by the fact that at lower water concentrations, conductivity increases as the electrolytes

in the system become gain mobility. However, with higher water concentrations, a dilution effect

in the charged components takes place, which causes the conductivity (Calderó, García-Celma,

and Solans 2011).

6.5 Phase behavior

Small-angle neutron scattering (SANS) has been quite explored for structural

characterization for phase behavior and surfactant curvature during low-energy emulsification

methods (Conxita Solans and Solé 2012). Moreover, it is considered a valuable tool for

characterizing bulk properties of nanoemulsions, since neutron wavelengths can probe nanosized

materials (Graves et al. 2005). SANS provide the identification of ordered regions within the

sample, which can be observed as spots in the scattering pattern. In contrast, amorphous and

disordered regions show a diffuse pattern distributed all over the scattering profile. Moreover, a

color-detector provide different intensity levels, which can emphasize scattering from ordered

liquid crystalline regions in the presence of disordered phases (Sonneville-Aubrun et al. 2009).

Compared with DLS, SANS provides information of droplet size in a wide range of sizes

and regarding the internal structure of nanosized droplets, by performing selective deuteration of
 51

the components. Besides droplet size information, SANS provide information regarding droplet

structure, such as core-shell structure, globular drops or bicontinuous microemulsion (Lijuan

Wang et al. 2008).

Sonneville-Aubrun et al. produced 100 nm oil-in-water nanoemulsions by performing the

addition water to a water-in-oil reverse microemulsions by phase transition methods and used

SANS as a tool to study phase inversion stages. Measurements were performed immediately

after the addition of water to the microemulsion. The SANS profiles showed that in the early

stages of water addition, the initial formation of mesophase occurred, suggesting that a liquid

crystalline region was formed as a transition phase. Moreover, SANS showed a strong change

in surfactant curvature progressively or abruptly as the hydration of the surfactant polar group

occurs, depending on the speed of water addition (Sonneville-Aubrun et al. 2009).

SANS has also been used to study silicone anionic nanoemulsions produced with

different oil volume fractions, from 0.008 to 0.6. At dilute oil volume fractions, droplets are

spherical. As silicon volume fractions increased above a critical jamming point, the primary peak

increased, suggesting more frequent interactions and deformations among droplets due to

electrostatic repulsion of neighboring droplets (Graves et al. 2005).

7. Application of nanoemulsions

Nanoemulsions feature the most diverse applications in the cosmetics and

pharmaceutical industry (Singh et al. 2017; Sutradhar and Amin 2013; Jaiswal, Dudhe, and

Sharma 2015). The small droplet size, increased surface area, high kinetic stability and optical

transparency of nanoemulsions, compared to conventional emulsions, offer them advantages in

many technological applications, some of them are explored below. The ability to encapsulate

lipophilic bioactive molecules and improve solubility and stability of lipophilic components, makes

oil-in-water nanoemulsions a useful tool to enhance the delivery of natural oils. Qian et al.

produced β-lactoglobulin-stabilized nanoemulsion for encapsulation of carotenoids, natural

antioxidant easily chemically degradable. The influence of ionic strength, temperature and pH on

chemical stability of β-carotene and physical stability of nanoemulsions was studied. Color fading

due to chemical degradation of β-carotene was significantly smaller to β-lactoglobulin-stabilized

 52

nanoemulsions, which showed to be an effective approach to increase chemical stability of

bioactive molecules, such as β-carotene (Qian et al. 2012).

The encapsulation of lipophilic components, as vegetable oils provides easier handling

or administration and incorporation into a pharmaceutical secondary formulation, for example,

gels, lotions or creams. Besides, nanoencapsulation of vegetable oils can also increase

bioavailability by increasing water solubility and due to small droplet size, promote rate and site-

controlled delivery and protect from environmental degradation and prevent early evaporation

(Dias et al. 2014; Sutradhar and Amin 2013).

The encapsulation of bioactive molecules and vegetable oils can be useful in developing

novel pharmaceutical formulations for masking unpleasant taste or smell of some drugs, which is

especially useful for pediatrics formulations (Amin et al. 2018). Moreover, nanoemulsions are

useful tools to improve bioavailability of both synthetic drugs and biologically active lipids or

probiotics, as polyphenols and oil-soluble vitamins, for example, which can improve the

pharmacological effect (Chen et al. 2011; A. Salem and M. Ezzat 2018). Yen et al developed

nanoemulsions for improving bioavailability of andrographolide, a poorly water-soluble anti-

inflammatory. The results indicated a significantly enhanced bioavailability of 6-fold from

nanoemulsions in comparison with conventional drug suspension. The ability of preventing

gastrointestinal inflammatory disorders was much higher for nanoemulsions then for drug

suspension (Yen et al. 2018). Besides, there is a general understanding that both solubility and

bioavailability of poorly soluble drugs increase with reduced droplet size. The most plausible

explanation is the enhancement of surface contact area, which can increase contact with solvents

or cells.

Lane et al. studied the bioavailability of omega-3-rich algal oil encapsulated by

nanoemulsions as a prophylactic strategy to prevent coronary heart disease and cerebrovascular

disease in 11 subjects, which were fed with the formulations. This study investigated whether the

ingestion of omega-3-loaded nanoemulsions increased bioavailability in comparison with free oil.

The results showed that bioavailability of omega-3 and polyunsaturated oils was drastically

enhanced in patients fed with nanoemulsions (Lane et al. 2014).

The solubility of oil encapsulated in nanoemulsions increases with the reduction of droplet

size. According to McClements studies (2011), the solubility of a typical oil was increased by 2.24,
 53

1.08, 1.01 and 1.00 when droplet sizes of 10, 100, 1000 and 10 000 nm, respectively, were

obtained. It is due to modification in oil-water partition coefficient of the encapsulated substance

(David Julian McClements 2011).

A great number of studies have reported that oil-in-water nanoemulsions can increase

antimicrobial activity of essential oils against several microorganisms as bacteria and fungus

(Salvia-Trujillo et al. 2015; Sonu et al. 2018; Chuesiang et al. 2019). The encapsulation of

essential oils into nanosized droplets can lead to a great disruptive activity of essential oils on cell

membranes of microorganisms (David J. McClements and Jafari 2018). Chuesiang et al.

produced cinnamon oil-loaded oil-in-water nanoemulsions by phase inversion temperature

method and investigated water-miscibility and antimicrobial activity against Escherichia coli,

Salmonella enterica serova Triphimurim, Staphylococcus aureus and Vibrio parahaemolyticus.

Cinnamon oil features biological activities, which is related to cinnamaldehyde, cinnamon oil main

constituent, that is able to interact with bacterial cell membrane (Chuesiang et al. 2019).

Nanoemulsions increased antimicrobial activity probably due to the encapsulation of

cinnamaldehyde, which is prone to chemical degradation in its free form. Besides, water

dispersibility of cinnamon oil was enhanced by nanoencapsulation, which can allow the use as a

natural preservative to be incorporated in food or beverages. Moreover, smaller droplets seemed

to be more efficiently transported through bacterial membranes, and thus provided high

antimicrobial activity in comparison with larger droplets, even though the latter contained higher

amounts of cinnamon oil (Chuesiang et al. 2019).

A wide range of bioactive natural oils feature important antioxidant properties, however,

as they are highly lipophilic molecules, the incorporation of these components into many aqueous

pharmaceutical formulations can be limited. In this context nanoemulsions rise as an

encapsulation approach to provide protection to the droplet content, while preserving bioactive

molecules functional properties. Rinaldi et al. produced neem oil-loaded nanoemulsions by

ultrasound sonication Tween 20® as surfactant. The antioxidant activity of neem oil alone and

encapsulated into nanoemulsions were quite similar, suggesting that nanoemulsions are efficient

in encapsulating bioactive molecules, while maintaining functional activity of neem oil. Moreover,

cytotoxicity was significantly reduced when Neem oil was incorporated in droplets in comparison

with free Neem oil (Rinaldi et al. 2017).

 54

Nanoemulsions can be applied in the controlled release of bioactive molecules in the

pharmaceutical and cosmetic area. This is due to the large surface area and low interfacial tension

of droplets, which allows the effective penetration of active pharmaceutical ingredient. Because

of the small size of nanosized oil-loaded droplets, they can penetrate the stratum corneum and

can they also be applied in alcohol-free perfume formulations (Rai et al. 2018). The encapsulation

of vegetable oils can be particularly beneficial for volatile components, such as essential oils and

aromas. The encapsulation approach can control the release and evaporation rate of bioactive

molecules and volatile components, which can bring important for aroma perception and duration

(David Julian McClements 2015). Time-controlled oil release can be tuned by modulating the

lipophilicity of the inner phase. High lipophilicity can lead to a more sustained oil release. Droplet

size in the case of nanoemulsions is not a limiting parameter for release profile (David Julian

McClements 2011).

Because the nanoemulsions are often transparent, they are related to freshness, purity

and simplicity even when carrying great amounts of oil. This characteristic has been very much

valued in both pharmaceutical and cosmetic industries. It is only achieved when droplet size is

too small (< 70 nm) to avoid strong light scattering and ensure optical transparency, which is also

dependent on polydispersity. In that context, optically transparent products can be produced when

small droplet size and narrow size distribution are obtained and maintained for a considerable

period of time (Wooster, Golding, and Sanguansri 2008). Transparent nanoemulsions can be

prepared by both high-energy and low-energy methods by adjusting and optimizing oil and

aqueous phase composition, surfactants and processing parameters to achieve small droplet

sizes and prevent Ostwald ripening and aggregation phenomena (David Julian McClements

2011).

Nanoemulsions with small droplet size can be sterilized through filtration and lead to a

wide variety of water-based pharmaceutical products. A wide variety of products are obtained

with the use of nanoemulsions, for example: lotions, moisturizers and transparent gels, with

different rheological behavior (Helgeson 2016). Parenteral, or injectable, administration of

nanoemulsions is employed for a variety of purposes, i.e., nutrition, for example in the

administration of vegetable oils, vitamins, among others, and topical or systemic drug release.

Nanoemulsions are advantageous for intravenous administration because of the rigid

 55

requirements of this route of administration, particularly the need for a droplet size in the

formulation below 1 μm (Hörmann and Zimmer 2016). The benefit of nanoemulsions in oral drug

administration has also been reported in the absorption of the emulsion in the gastrointestinal

tract which has been correlated with droplet size (Bali, Ali, and Ali 2011).

8. Conclusion

Nanoemulsions are unique nanocarriers for the delivery lipophilic components as they

provide a more stable, bioavailable, readily manufacturable, and acceptable formulation. They

also impart good protection to the entrapped bioactive molecules against the effects of external

conditions, as they encapsulate bioactive molecules in the core of nanosized micelles. In addition,

nanoemulsions exhibit high surface area, stability and tunable rheology, which can improve drug

bioavailability, making many treatments less toxic and invasive. Recently, a growing interest in

the use of natural oils has been taking place, since they are proving to feature antimicrobial,

antioxidants and anti-inflammatory properties, among others. In this chapter the main aspects on

nanoemulsion formulation, obtention methods, characterization techniques and applications were

presented.

Both high-energy and low-energy emulsification methods provide nanoemulsions with

small droplet size and stability. However, much research is still needed to the achieving scaling

up of these processes and to understand the impact on size and stability aspects for

nanoemulsions. The difficult to scale up nanoemulsions production is responsible for so few

nanoemulsion-based products are commercially available in contrast with so much research

being published in this field.

Before nanoemulsions become widespread in pharmaceutical field, other challenges

must be overcome. First, pharmaceutical-grade excipients should be ideally chosen, such as

synthetic polymers and surfactants. Next, there are some safety concerns involving

nanotechnological products as nanoemulsion and nano-based products toxicological profile is

different from conventional emulsion. In this context further research is needed to promote wide

nanoemulsions production and utilization.

 56

9. Acknowledgements

This material is based upon the work supported by the Coordination for the Improvement of Higher

Education Personnel (CAPES) and National Council for Scientific and Technological

Development (CNPq).

10. References

A. Salem, Mohamed, and Shahira M. Ezzat. 2018. “Nanoemulsions in Food Industry.” In Some

New Aspects of Colloidal Systems in Foods. IntechOpen.

https://doi.org/10.5772/intechopen.79447.

Abismail, B, J P Canselier, a M Wilhelm, H Delmas, and C Gourdon. 1999. “Emulsification by

Ultrasound- Drop Size Distribution and Stability.Pdf.” Ultrasonics Sonochemistry 6: 75–83.

Adjonu, Randy, Gregory Doran, Peter Torley, and Samson Agboola. 2014. “Formation of Whey

Protein Isolate Hydrolysate Stabilised Nanoemulsion.” Food Hydrocolloids 41 (December):

169–77. https://doi.org/10.1016/j.foodhyd.2014.04.007.

Al-Subaie, Mutlaq M., Khaled M. Hosny, Khalid Mohamed El-Say, Tarek A. Ahmed, and Bader

M. Aljaeid. 2015. “Utilization of Nanotechnology to Enhance Percutaneous Absorption of

Acyclovir in the Treatment of Herpes Simplex Viral Infections.” International Journal of

Nanomedicine. https://doi.org/10.2147/IJN.S83962.

Alam, Prawez, Faiyaz Shakeel, Md Khalid Anwer, Ahmed I. Foudah, and Mohammed H.

Alqarni. 2018. “Wound Healing Study of Eucalyptus Essential Oil Containing

Nanoemulsion in Rat Model.” Journal of Oleo Science 67 (8): 957–68.

https://doi.org/10.5650/jos.ess18005.

Alexandridis, Paschalis, Josef F. Holzwarth, and T. Alan Hatton. 1994. “Micellization of

Poly(Ethylene Oxide)-Poly(Propylene Oxide)-Poly(Ethylene Oxide) Triblock Copolymers in

Aqueous Solutions: Thermodynamics of Copolymer Association.” Macromolecules 27 (9):

2414–25. https://doi.org/10.1021/ma00087a009.

Amin, Fazli, Shahzeb Khan, Syed Muhammad Hassan Shah, Haroon Rahim, Zahid Hussain,

Muhammad Sohail, Riaz Ullah, Mansour S. Alsaid, and Abdelaaty A. Shahat. 2018. “A

New Strategy for Taste Masking of Azithromycin Antibiotic: Development,

Characterization, and Evaluation of Azithromycin Titanium Nanohybrid for Masking of

 57

Bitter Taste Using Physisorption and Panel Testing Studies.” Drug Design, Development

and Therapy Volume 12 (November): 3855–66. https://doi.org/10.2147/DDDT.S183534.

Andreu, Vanesa, Gracia Mendoza, Manuel Arruebo, and Silvia Irusta. 2015. “Smart Dressings

Based on Nanostructured Fibers Containing Natural Origin Antimicrobial, Anti-

Inflammatory, and Regenerative Compounds.” Materials 8 (8): 5154–93.

https://doi.org/10.3390/ma8085154.

Angioni, Alberto, Andrea Barra, Elisabetta Cereti, Daniela Barile, Jean Daniel Coïsson, Marco

Arlorio, Sandro Dessi, Valentina Coroneo, and Paolo Cabras. 2004. “Chemical

Composition, Plant Genetic Differences, Antimicrobial and Antifungal Activity Investigation

of the Essential Oil of Rosmarinus Officinalis L.” Journal of Agricultural and Food

Chemistry 52 (11): 3530–35. https://doi.org/10.1021/jf049913t.

Anton, Nicolas, Jean-Pierre Benoit, and Patrick Saulnier. 2008. “Design and Production of

Nanoparticles Formulated from Nano-Emulsion Templates—A Review.” Journal of

Controlled Release 128 (3): 185–99. https://doi.org/10.1016/j.jconrel.2008.02.007.

Anton, Nicolas, and Thierry F. Vandamme. 2011. “Nano-Emulsions and Micro-Emulsions:

Clarifications of the Critical Differences.” Pharmaceutical Research 28 (5): 978–85.

https://doi.org/10.1007/s11095-010-0309-1.

Anwer, Md Khalid, Shahid Jamil, Elmutasim Osman Ibnouf, and Faiyaz Shakeel. 2014.

“Enhanced Antibacterial Effects of Clove Essential Oil by Nanoemulsion.” Journal of Oleo

Science. https://doi.org/10.5650/jos.ess13213.

Asbahani, A. El, K. Miladi, W. Badri, M. Sala, E.H. Aït Addi, H. Casabianca, A. El Mousadik, et

al. 2015. “Essential Oils: From Extraction to Encapsulation.” International Journal of

Pharmaceutics 483 (1–2): 220–43. https://doi.org/10.1016/j.ijpharm.2014.12.069.

Azeem, Adnan, Mohammad Rizwan, Farhan J. Ahmad, Zeenat Iqbal, Roop K. Khar, M. Aqil,

and Sushama Talegaonkar. 2009. “Nanoemulsion Components Screening and Selection:

A Technical Note.” AAPS PharmSciTech 10. https://doi.org/10.1208/s12249-008-9178-x.

Babchin, Alexander J., and Laurier L. Schramm. 2012. “Osmotic Repulsion Force Due to

Adsorbed Surfactants.” Colloids and Surfaces B: Biointerfaces.

https://doi.org/10.1016/j.colsurfb.2011.10.050.

Badgujar, Shamkant B., Vainav V. Patel, and Atmaram H. Bandivdekar. 2014. “Foeniculum
 58

Vulgare Mill: A Review of Its Botany, Phytochemistry, Pharmacology, Contemporary

Application, and Toxicology.” BioMed Research International.

https://doi.org/10.1155/2014/842674.

Bai, Long, Siqi Huan, Jiyou Gu, and David Julian McClements. 2016. “Fabrication of Oil-in-

Water Nanoemulsions by Dual-Channel Microfluidization Using Natural Emulsifiers:

Saponins, Phospholipids, Proteins, and Polysaccharides.” Food Hydrocolloids 61

(December): 703–11. https://doi.org/10.1016/j.foodhyd.2016.06.035.

Bali, Vikas, Mushir Ali, and Javed Ali. 2011. “Nanocarrier for the Enhanced Bioavailability of a

Cardiovascular Agent: In Vitro, Pharmacodynamic, Pharmacokinetic and Stability

Assessment.” International Journal of Pharmaceutics.

https://doi.org/10.1016/j.ijpharm.2010.10.018.

Barradas, T.N., V.E.B. de Campos, J.P. Senna, C.S.C. Coutinho, B.S. Tebaldi, K.G.H. Silva,

and C.R.E. Mansur. 2015. “Development and Characterization of Promising o/w

Nanoemulsions Containing Sweet Fennel Essential Oil and Non-Ionic Sufactants.”

Colloids and Surfaces A: Physicochemical and Engineering Aspects 480.

https://doi.org/10.1016/j.colsurfa.2014.12.001.

Barradas, T.N., J.P. Senna, S.A. Cardoso, K.G. de Holanda e Silva, and C.R. Elias Mansur.

2018. “Formulation Characterization and in Vitro Drug Release of Hydrogel-Thickened

Nanoemulsions for Topical Delivery of 8-Methoxypsoralen.” Materials Science and

Engineering C 92. https://doi.org/10.1016/j.msec.2018.06.049.

Barradas, Thaís Nogueira T.N., J.P. Juliana Perdiz Senna, S.A. Stephani Araujo Cardoso, Sara

Nicoli, Cristina Padula, Patrizia Santi, Francesca Rossi, K.G. Gyselle de Holanda e Silva,

and Claudia R. Elias C.R.E. Mansur. 2017. “Hydrogel-Thickened Nanoemulsions Based

on Essential Oils for Topical Delivery of Psoralen: Permeation and Stability Studies.”

European Journal of Pharmaceutics and Biopharmaceutics 116 (November): 38–50.

https://doi.org/10.1016/j.ejpb.2016.11.018.

Behrend, O., K. Ax, and H. Schubert. 2000. “Influence of Continuous Phase Viscosity on

Emulsification by Ultrasound.” Ultrasonics Sonochemistry. https://doi.org/10.1016/S1350-

4177(99)00029-2.

Bhattacharjee, Sourav. 2016. “DLS and Zeta Potential - What They Are and What They Are
 59

Not?” Journal of Controlled Release. https://doi.org/10.1016/j.jconrel.2016.06.017.

Bondi, Cara Am, Julia L Marks, Lauren B Wroblewski, Heidi S Raatikainen, Shannon R Lenox,

and Kay E Gebhardt. 2015. “Human and Environmental Toxicity of Sodium Lauryl Sulfate

(SLS): Evidence for Safe Use in Household Cleaning Products.” Environmental Health

Insights 9: 27–32. https://doi.org/10.4137/EHI.S31765.

Bonferoni, Maria Cristina, Silvia Rossi, Antonia Icaro Cornaglia, Barbara Mannucci, Pietro

Grisoli, Barbara Vigani, Francesca Saporito, et al. 2017. “Essential Oil-Loaded Lipid

Nanoparticles for Wound Healing.” International Journal of Nanomedicine Volume 13

(December): 175–86. https://doi.org/10.2147/IJN.S152529.

Borges, Raphaelle Sousa, Emerson Silva Lima, Hady Keita, Irlon Maciel Ferreira, Caio Pinho

Fernandes, Rodrigo Alves Soares Cruz, Jonatas Lobato Duarte, et al. 2018. “Anti-

Inflammatory and Antialgic Actions of a Nanoemulsion of Rosmarinus Officinalis L.

Essential Oil and a Molecular Docking Study of Its Major Chemical Constituents.”

Inflammopharmacology 26 (1): 183–95. https://doi.org/10.1007/s10787-017-0374-8.

Botas, Gisele, Rodrigo Cruz, Fernanda de Almeida, Jonatas Duarte, Raquel Araújo, Raimundo

Souto, Ricardo Ferreira, et al. 2017. “Baccharis Reticularia DC. and Limonene

Nanoemulsions: Promising Larvicidal Agents for Aedes Aegypti (Diptera: Culicidae)

Control.” Molecules 22 (11): 1990. https://doi.org/10.3390/molecules22111990.

Bouchemal, K., S. Briançon, E. Perrier, and H. Fessi. 2004. “Nano-Emulsion Formulation Using

Spontaneous Emulsification: Solvent, Oil and Surfactant Optimisation.” International

Journal of Pharmaceutics 280 (1–2): 241–51.

https://doi.org/10.1016/j.ijpharm.2004.05.016.

Bourbon, Ana I., Raquel F.S. Gonçalves, António A. Vicente, and Ana C. Pinheiro. 2018.

“Characterization of Particle Properties in Nanoemulsions.” In Nanoemulsions, 519–46.

Elsevier. https://doi.org/10.1016/B978-0-12-811838-2.00016-3.

Bouyer, Eléonore, Ghozlene Mekhloufi, Véronique Rosilio, Jean Louis Grossiord, and Florence

Agnely. 2012. “Proteins, Polysaccharides, and Their Complexes Used as Stabilizers for

Emulsions: Alternatives to Synthetic Surfactants in the Pharmaceutical Field?”

International Journal of Pharmaceutics. https://doi.org/10.1016/j.ijpharm.2012.06.052.

Calderó, Gabriela, María José García-Celma, and Conxita Solans. 2011. “Formation of
 60

Polymeric Nano-Emulsions by a Low-Energy Method and Their Use for Nanoparticle

Preparation.” Journal of Colloid and Interface Science 353 (2): 406–11.

https://doi.org/10.1016/j.jcis.2010.09.073.

Cardoso-Ugarte, Gabriel Abraham, Aurelio López-Malo, and María Teresa Jiménez-Munguía.

2016. “Application of Nanoemulsion Technology for Encapsulation and Release of

Lipophilic Bioactive Compounds in Food.” In Emulsions, 227–55. Elsevier.

https://doi.org/10.1016/B978-0-12-804306-6.00007-6.

Carteau, David, Dario Bassani, and Isabelle Pianet. 2008. “The ‘Ouzo Effect’: Following the

Spontaneous Emulsification of Trans-Anethole in Water by NMR.” Comptes Rendus

Chimie. https://doi.org/10.1016/j.crci.2007.11.003.

Cerqueira-Coutinho, Cristal, Ralph Santos-Oliveira, Elisabete dos Santos, and Claudia Regina

Mansur. 2015. “Development of a Photoprotective and Antioxidant Nanoemulsion

Containing Chitosan as an Agent for Improving Skin Retention.” Engineering in Life

Sciences. https://doi.org/10.1002/elsc.201400154.

Chaieb, Kamel, Hafedh Hajlaoui, Tarek Zmantar, Amel Ben Kahla-Nakbi, Mahmoud Rouabhia,

Kacem Mahdouani, and Amina Bakhrouf. 2007. “The Chemical Composition and

Biological Activity of Clove Essential Oil, Eugenia Caryophyllata (Syzigium Aromaticum L.

Myrtaceae): A Short Review.” Phytotherapy Research. https://doi.org/10.1002/ptr.2124.

Chebil, Asma, Jacques Desbrières, Cécile Nouvel, Jean-Luc Six, and Alain Durand. 2013.

“Ostwald Ripening of Nanoemulsions Stopped by Combined Interfacial Adsorptions of

Molecular and Macromolecular Nonionic Stabilizers.” Colloids and Surfaces A:

Physicochemical and Engineering Aspects 425 (May): 24–30.

https://doi.org/10.1016/j.colsurfa.2013.02.028.

Chen, Huabing, Chalermchai Khemtong, Xiangliang Yang, Xueling Chang, and Jinming Gao.

2011. “Nanonization Strategies for Poorly Water-Soluble Drugs.” Drug Discovery Today.

https://doi.org/10.1016/j.drudis.2010.02.009.

Chuesiang, Piyanan, Ubonrat Siripatrawan, Romanee Sanguandeekul, Jason Szuhao Yang,

David Julian McClements, and Lynne McLandsborough. 2019. “Antimicrobial Activity and

Chemical Stability of Cinnamon Oil in Oil-in-Water Nanoemulsions Fabricated Using the

Phase Inversion Temperature Method.” LWT 110 (August): 190–96.

 61

https://doi.org/10.1016/j.lwt.2019.03.012.

Chung, Cheryl, and David J. McClements. 2018. “Characterization of Physicochemical

Properties of Nanoemulsions: Appearance, Stability, and Rheology.” In Nanoemulsions,

547–76. Elsevier. https://doi.org/10.1016/B978-0-12-811838-2.00017-5.

Constantinides, Panayiotis P., Mahesh V. Chaubal, and Robert Shorr. 2008. “Advances in Lipid

Nanodispersions for Parenteral Drug Delivery and Targeting.” Advanced Drug Delivery

Reviews 60 (6): 757–67. https://doi.org/10.1016/j.addr.2007.10.013.

Dias, Daiane de O, Mariana Colombo, Regina G. Kelmann, Samuel Kaiser, Letícia G. Lucca,

Helder F. Teixeira, Renata P. Limberger, Valdir F. Veiga, and Letícia S. Koester. 2014.

“Optimization of Copaiba Oil-Based Nanoemulsions Obtained by Different Preparation

Methods.” Industrial Crops and Products 59: 154–62.

https://doi.org/10.1016/j.indcrop.2014.05.007.

Dickinson, Eric. 2003. “Hydrocolloids at Interfaces and the Influence on the Properties of

Dispersed Systems.” Food Hydrocolloids. https://doi.org/10.1016/S0268-005X(01)00120-

5.

———. 2009. “Hydrocolloids as Emulsifiers and Emulsion Stabilizers.” Food Hydrocolloids 23

(6): 1473–82. https://doi.org/10.1016/j.foodhyd.2008.08.005.

Dickinson, Eric, Vanda B. Galazka, and Douglas M.W. Anderson. 1991. “Emulsifying Behaviour

of Gum Arabic. Part 1: Effect of the Nature of the Oil Phase on the Emulsion Droplet-Size

Distribution.” Carbohydrate Polymers 14 (4): 373–83. https://doi.org/10.1016/0144-

8617(91)90003-U.

Dickinson, Eric, Maria G. Semenova, and Anna S. Antipova. 1998. “Salt Stability of Casein

Emulsions.” Food Hydrocolloids 12 (2): 227–35. https://doi.org/10.1016/S0268-

005X(98)00035-6.

Donsì, Francesco, and Giovanna Ferrari. 2016. “Essential Oil Nanoemulsions as Antimicrobial

Agents in Food.” Journal of Biotechnology. Elsevier B.V.

https://doi.org/10.1016/j.jbiotec.2016.07.005.

Dorfmüller, Th. 1987. “R. Pecora (Ed.): Dynamic Light Scattering - Applications of Photon

Correlation Spectroscopy , Plenum Press, New York and London 1985. 420 Seiten, Preis:

$ 59.90.” Berichte Der Bunsengesellschaft Für Physikalische Chemie 91 (4): 498–99.

 62

https://doi.org/10.1002/bbpc.19870910455.

Duarte, Jonatas L., Jesús R.R. Amado, Anna E.M.F.M. Oliveira, Rodrigo A.S. Cruz, Adriana M.

Ferreira, Raimundo N.P. Souto, Deborah Q. Falcão, José C.T. Carvalho, and Caio P.

Fernandes. 2015. “Evaluation of Larvicidal Activity of a Nanoemulsion of Rosmarinus

Officinalis Essential Oil.” Revista Brasileira de Farmacognosia 25 (2): 189–92.

https://doi.org/10.1016/j.bjp.2015.02.010.

Elmahjoubi, Eman, Yakov Frum, Gillian M. Eccleston, Simon C. Wilkinson, and Victor M.

Meidan. 2009. “Transepidermal Water Loss for Probing Full-Thickness Skin Barrier

Function: Correlation with Tritiated Water Flux, Sensitivity to Punctures and Diverse

Surfactant Exposures.” Toxicology in Vitro. https://doi.org/10.1016/j.tiv.2009.06.030.

Farshi, Parastou, Mahnaz Tabibiazar, Marjan Ghorbani, Mohammadamin Mohammadifar,

Maryam Bannazadeh Amirkhiz, and Hamed Hamishehkar. 2019. “Whey Protein Isolate-

Guar Gum Stabilized Cumin Seed Oil Nanoemulsion.” Food Bioscience 28 (April): 49–56.

https://doi.org/10.1016/j.fbio.2019.01.011.

Fernandez, Patrick, Valérie André, Jens Rieger, and Angelika Kühnle. 2004. “Nano-Emulsion

Formation by Emulsion Phase Inversion.” Colloids and Surfaces A: Physicochemical and

Engineering Aspects. https://doi.org/10.1016/j.colsurfa.2004.09.029.

Goñi, María G., Sara I. Roura, Alejandra G. Ponce, and María R. Moreira. 2016. “Clove

(Syzygium Aromaticum) Oils.” In Essential Oils in Food Preservation, Flavor and Safety,

edited by Victor R. Preedy. Academic Press.

Graves, S., K. Meleson, J. Wilking, M. Y. Lin, and T. G. Mason. 2005. “Structure of

Concentrated Nanoemulsions.” The Journal of Chemical Physics 122 (13): 134703.

https://doi.org/10.1063/1.1874952.

Gupta, Ankur, H. Burak Eral, T. Alan Hatton, and Patrick S. Doyle. 2016a. “Controlling and

Predicting Droplet Size of Nanoemulsions: Scaling Relations with Experimental

Validation.” Soft Matter. https://doi.org/10.1039/C5SM02051D.

———. 2016b. “Nanoemulsions: Formation, Properties and Applications.” Soft Matter 12 (11).

https://doi.org/10.1039/C5SM02958A.

Gutiérrez, J. M., C. González, A. Maestro, I. Solè, C. M. Pey, and J. Nolla. 2008. “Nano-

Emulsions: New Applications and Optimization of Their Preparation.” Current Opinion in

 63

Colloid and Interface Science. https://doi.org/10.1016/j.cocis.2008.01.005.

Hashem, Ahmed S., Samir S. Awadalla, Gamal M. Zayed, Filippo Maggi, and Giovanni Benelli.

2018. “Pimpinella Anisum Essential Oil Nanoemulsions against Tribolium Castaneum—

Insecticidal Activity and Mode of Action.” Environmental Science and Pollution Research

25 (19): 18802–12. https://doi.org/10.1007/s11356-018-2068-1.

Hashemi Gahruie, Hadi, Esmaeil Ziaee, Mohammad Hadi Eskandari, and Seyed Mohammad

Hashem Hosseini. 2017. “Characterization of Basil Seed Gum-Based Edible Films

Incorporated with Zataria Multiflora Essential Oil Nanoemulsion.” Carbohydrate Polymers

166 (June): 93–103. https://doi.org/10.1016/j.carbpol.2017.02.103.

Hasssanzadeh, Hamed, Mohammad Alizadeh, and Mahmoud Rezazad Bari. 2018.

“Formulation of Garlic Oil-in-Water Nanoemulsion: Antimicrobial and Physicochemical

Aspects.” IET Nanobiotechnology 12 (5): 647–52. https://doi.org/10.1049/iet-

nbt.2017.0104.

Helgeson, Matthew E. 2016. “Colloidal Behavior of Nanoemulsions: Interactions, Structure, and

Rheology.” Current Opinion in Colloid and Interface Science.

https://doi.org/10.1016/j.cocis.2016.06.006.

Herman, Anna Andrzej P., and Anna Andrzej P. Herman. 2015. “Essential Oils and Their

Constituents as Skin Penetration Enhancer for Transdermal Drug Delivery: A Review.”

Journal of Pharmacy and Pharmacology. https://doi.org/10.1111/jphp.12334.

Hernández, M. D., J. A. Sotomayor, Á. Hernández, and M. J. Jordán. 2016. “Rosemary

(Rosmarinus Officinalis L.) Oils.” In Essential Oils in Food Preservation, Flavor and Safety,

edited by V. R. Preedy, 677–88. San Diego.

Hoeller, Sonja, Andrea Sperger, and Claudia Valenta. 2009. “Lecithin Based Nanoemulsions: A

Comparative Study of the Influence of Non-Ionic Surfactants and the Cationic

Phytosphingosine on Physicochemical Behaviour and Skin Permeation.” International

Journal of Pharmaceutics 370 (1–2): 181–86.

https://doi.org/10.1016/j.ijpharm.2008.11.014.

Hörmann, Karl, and Andreas Zimmer. 2016. “Drug Delivery and Drug Targeting with Parenteral

Lipid Nanoemulsions — A Review.” Journal of Controlled Release 223 (February): 85–98.

https://doi.org/10.1016/j.jconrel.2015.12.016.
 64

Hudaib, Mohammad, Ester Speroni, Anna Maria Di Pietra, and Vanni Cavrini. 2002. “GC/MS

Evaluation of Thyme (Thymus Vulgaris L.) Oil Composition and Variations during the

Vegetative Cycle.” Journal of Pharmaceutical and Biomedical Analysis 29 (4): 691–700.

http://www.ncbi.nlm.nih.gov/pubmed/12093498.

Hunter, Robert J. 2001. Foundations of Colloid Science. 2nd ed. New York: Oxford University

Press.

Hussain, Abdullah Ijaz, Farooq Anwar, Syed Tufail Hussain Sherazi, and Roman Przybylski.

2008. “Chemical Composition, Antioxidant and Antimicrobial Activities of Basil (Ocimum

Basilicum) Essential Oils Depends on Seasonal Variations.” Food Chemistry 108 (3): 986–

95. https://doi.org/10.1016/j.foodchem.2007.12.010.

Jafari, Seid Mahdi. 2017. Nanoencapsulation Technologies for the Food and Nutraceutical

Industries. Academic Print. https://doi.org/10.1016/B978-0-12-809436-5/00014-8.

Jafari, Seid Mahdi, Elham Assadpoor, Yinghe He, and Bhesh Bhandari. 2008. “Re-Coalescence

of Emulsion Droplets during High-Energy Emulsification.” Food Hydrocolloids.

https://doi.org/10.1016/j.foodhyd.2007.09.006.

Jafari, Seid Mahdi, Yinghe He, and Bhesh Bhandari. 2007. “Production of Sub-Micron

Emulsions by Ultrasound and Microfluidization Techniques.” Journal of Food Engineering

82 (4): 478–88. https://doi.org/10.1016/j.jfoodeng.2007.03.007.

Jafari, Seid Mahdi, Paraskevi Paximada, Ionna Mandala, Eltham Assadpour, and Mohammad

A. Mehrnia. 2017. “Encapsulation by Nanoemulsions.” In Nanoencapsulation Technologies

for the Food and Nutraceutical Industries.

Jaiswal, Manjit, Rupesh Dudhe, and P. K. Sharma. 2015. “Nanoemulsion: An Advanced Mode

of Drug Delivery System.” 3 Biotech. Springer Verlag. https://doi.org/10.1007/s13205-014-

0214-0.

Jin, Weiping, Wei Xu, Hongshan Lian, Yan Li, Shilin Liu, and Bin Li. 2016. “Nanoemulsions for

Food: Properties, Production, Characterization and Applications.” In Emulsions, edited by

Alexandru Grumezescu, 1–29. London: Elsevier Inc.

Jin, Weiping, Wei Xu, Hongshan Liang, Yan Li, Shilin Liu, and Bin Li. 2016. “Nanoemulsions for

Food: Properties, Production, Characterization, and Applications.” In Emulsions, 1–36.

Elsevier. https://doi.org/10.1016/B978-0-12-804306-6.00001-5.
 65

Junyaprasert, Varaporn B., Veerawat Teeranachaideekul, Eliana B. Souto, Prapaporn Boonme,

and Rainer H. Müller. 2009. “Q10-Loaded NLC versus Nanoemulsions: Stability, Rheology

and in Vitro Skin Permeation.” International Journal of Pharmaceutics.

https://doi.org/10.1016/j.ijpharm.2009.05.020.

Kataoka, K, a Harada, and Y Nagasaki. 2001. “Block Copolymer Micelles for Drug Delivery:

Design, Characterization and Biological Significance.” Advanced Drug Delivery Reviews

47 (1): 113–31.

Katsumoto, Y., H. Ushiki, J. Lachaise, and A. Graciaa. 2001. “Time Evolution of the Size

Distribution of Nano-Sphere Droplets in the Hexadecane-in-Water Miniemulsion Stabilized

by Nonionic Surfactants.” Colloid & Polymer Science 279 (2): 122–30.

https://doi.org/10.1007/s003960000395.

Khan, Mohammed Shuaib, and K. Krishnaraj. 2014. “Phospholipids: A Novel Adjuvant in Herbal

Drug Delivery Systems.” Critical Reviews in Therapeutic Drug Carrier Systems 31 (5):

407–28. https://doi.org/10.1615/CritRevTherDrugCarrierSyst.2014010634.

Kiaei, Narges, Reza Hajimohammadi, and Morteza Hosseini. 2018. “Investigation of the Anti-

Inflammatory Properties of Calendula Nanoemulsion on Skin Cells.” Bioinspired,

Biomimetic and Nanobiomaterials 7 (4): 228–37. https://doi.org/10.1680/jbibn.17.00033.

Klang, V., and C. Valenta. 2011. “Lecithin-Based Nanoemulsions.” Journal of Drug Delivery

Science and Technology 21 (1): 55–76. https://doi.org/10.1016/S1773-2247(11)50006-1.

Kotta, Sabna, Abdul Wadood Khan, S. H. Ansari, R. K. Sharma, and Javed Ali. 2015.

“Formulation of Nanoemulsion: A Comparison between Phase Inversion Composition

Method and High-Pressure Homogenization Method.” Drug Delivery 22 (4): 455–66.

https://doi.org/10.3109/10717544.2013.866992.

Kuhn, K.R., and R.L. Cunha. 2012. “Flaxseed Oil – Whey Protein Isolate Emulsions: Effect of

High Pressure Homogenization.” Journal of Food Engineering 111 (2): 449–57.

https://doi.org/10.1016/j.jfoodeng.2012.01.016.

Lane, Katie E., Weili Li, Chris Smith, and Emma Derbyshire. 2014. “The Bioavailability of an

Omega-3-Rich Algal Oil Is Improved by Nanoemulsion Technology Using Yogurt as a

Food Vehicle.” International Journal of Food Science & Technology 49 (5): 1264–71.

https://doi.org/10.1111/ijfs.12455.
 66

Lawrence, M.Jayne, and Gareth D. Rees. 2000. “Microemulsion-Based Media as Novel Drug

Delivery Systems.” Advanced Drug Delivery Reviews 45 (1): 89–121.

https://doi.org/10.1016/S0169-409X(00)00103-4.

Lee, Laura, and Ian T. Norton. 2013. “Comparing Droplet Breakup for a High-Pressure Valve

Homogeniser and a Microfluidizer for the Potential Production of Food-Grade

Nanoemulsions.” Journal of Food Engineering 114 (2): 158–63.

https://doi.org/10.1016/j.jfoodeng.2012.08.009.

Lefebvre, G., J. Riou, G. Bastiat, E. Roger, K. Frombach, J-C. Gimel, P. Saulnier, and B.

Calvignac. 2017. “Spontaneous Nano-Emulsification: Process Optimization and Modeling

for the Prediction of the Nanoemulsion’s Size and Polydispersity.” International Journal of

Pharmaceutics 534 (1–2): 220–28. https://doi.org/10.1016/j.ijpharm.2017.10.017.

Lémery, Emmanuelle, Stéphanie Briançon, Yves Chevalier, Claire Bordes, Thierry Oddos,

Annie Gohier, and Marie Alexandrine Bolzinger. 2015. “Skin Toxicity of Surfactants:

Structure/Toxicity Relationships.” Colloids and Surfaces A: Physicochemical and

Engineering Aspects. https://doi.org/10.1016/j.colsurfa.2015.01.019.

Leong, T. S.H., T. J. Wooster, S. E. Kentish, and M. Ashokkumar. 2009. “Minimising Oil Droplet

Size Using Ultrasonic Emulsification.” Ultrasonics Sonochemistry.

https://doi.org/10.1016/j.ultsonch.2009.02.008.

Li, Q. X., and C. L. Chang. 2016. “Basil (Ocimum Basilicum L.) Oils.” In Essential Oils in Food

Preservation, Flavor and Safety, edited by V. R. Preedy, 231–38. San Diego: Academic

Press.

Li, Ze-hua, Ming Cai, Yuan-shuai Liu, and Pei-long Sun. 2018. “Development of Finger Citron

(Citrus Medica L. Var. Sarcodactylis) Essential Oil Loaded Nanoemulsion and Its

Antimicrobial Activity.” Food Control 94 (December): 317–23.

https://doi.org/10.1016/j.foodcont.2018.07.009.

Like Mao, Duoxia Xu, Jia Yang, Fang Yuan, Yanxiang Gao, and Jian Zhao. 2009. “Effects of

Small and Large Molecule Emulsifiers on the Characteristics of B-Carotene

Nanoemulsions Prepared by High Pressure Homogenization.” Food Technology

Biotechnology 47 (3): 336–42.

Liu, Weirong, Dejun Sun, Caifu Li, Qian Liu, and Jian Xu. 2006. “Formation and Stability of
 67

Paraffin Oil-in-Water Nano-Emulsions Prepared by the Emulsion Inversion Point Method.”

Journal of Colloid and Interface Science 303 (2): 557–63.

https://doi.org/10.1016/j.jcis.2006.07.055.

Lou, Zaixiang, Jie Chen, Fuhao Yu, Hongxin Wang, Xingran Kou, Chaoyang Ma, and Song Zhu.

2017. “The Antioxidant, Antibacterial, Antibiofilm Activity of Essential Oil from Citrus

Medica L. Var. Sarcodactylis and Its Nanoemulsion.” LWT 80 (July): 371–77.

https://doi.org/10.1016/j.lwt.2017.02.037.

Maestro, Alicia, Isabel Solè, Carmen González, Conxita Solans, and José M. Gutiérrez. 2008.

“Influence of the Phase Behavior on the Properties of Ionic Nanoemulsions Prepared by

the Phase Inversion Composition Method.” Journal of Colloid and Interface Science 327

(2): 433–39. https://doi.org/10.1016/j.jcis.2008.07.059.

Mandal, S., and M. DebManda. 2016. “Thyme (Thymus Vulgaris L.) Oils.” In Essential Oils in

Food Preservation, Flavor and Safety., edited by V. R. Preedy, 25–834. San Diego:

Academic Press.

Marotti, Mauro, Roberta Piccaglia, and Enrico Giovanelli. 1996. “Differences in Essential Oil

Composition of Basil ( Ocimum Basilicum L.) Italian Cultivars Related to Morphological

Characteristics.” Journal of Agricultural and Food Chemistry 44 (12): 3926–29.

https://doi.org/10.1021/jf9601067.

Mason, T G, J N Wilking, K Meleson, C B Chang, and S M Graves. 2006. “Nanoemulsions:

Formation, Structure, and Physical Properties” 18 (18): 635–66.

https://doi.org/10.1088/0953-8984/18/41/R01.

Mayer, Sinja, Jochen Weiss, and David Julian McClements. 2013. “Vitamin E-Enriched

Nanoemulsions Formed by Emulsion Phase Inversion: Factors Influencing Droplet Size

and Stability.” Journal of Colloid and Interface Science.

https://doi.org/10.1016/j.jcis.2013.04.016.

Mazarei, Zeinab, and Hasan Rafati. 2019. “Nanoemulsification of Satureja Khuzestanica

Essential Oil and Pure Carvacrol; Comparison of Physicochemical Properties and

Antimicrobial Activity against Food Pathogens.” LWT 100 (February): 328–34.

https://doi.org/10.1016/j.lwt.2018.10.094.

McClements, David J., and Seid Mahdi Jafari. 2018. “General Aspects of Nanoemulsions and
 68

Their Formulation.” In Nanoemulsions, 3–20. Elsevier. https://doi.org/10.1016/B978-0-12-

811838-2.00001-1.

McClements, David Julian. 2011. “Edible Nanoemulsions: Fabrication, Properties, and

Functional Performance.” Soft Matter 7 (6): 2297–2316.

https://doi.org/10.1039/C0SM00549E.

———. 2012. “Nanoemulsions versus Microemulsions: Terminology, Differences, and

Similarities.” Soft Matter 8 (6): 1719–29. https://doi.org/10.1039/C2SM06903B.

———. 2015. Food Emulsions: Principles, Practices, and Techniques, Third Edition. 3rd ed.

CRC Press.

McClements, David Julian, and Jiajia Rao. 2011. “Food-Grade Nanoemulsions: Formulation,

Fabrication, Properties, Performance, Biological Fate, and Potential Toxicity.” Critical

Reviews in Food Science and Nutrition 51 (4): 285–330.

https://doi.org/10.1080/10408398.2011.559558.

McNamee, Brian F., E. Dolores O’Riorda, and Michael O’Sullivan. 1998. “Emulsification and

Microencapsulation Properties of Gum Arabic.” Journal of Agricultural and Food

Chemistry. https://doi.org/10.1021/jf9803740.

Meinders, Marcel B.J, and Ton van Vliet. 2004. “The Role of Interfacial Rheological Properties

on Ostwald Ripening in Emulsions.” Advances in Colloid and Interface Science 108–109

(May): 119–26. https://doi.org/10.1016/j.cis.2003.10.005.

Moghimi, Roya, Lida Ghaderi, Hasan Rafati, Atousa Aliahmadi, and David Julian McClements.

2016. “Superior Antibacterial Activity of Nanoemulsion of Thymus Daenensis Essential Oil

against E. Coli.” Food Chemistry 194 (March): 410–15.

https://doi.org/10.1016/j.foodchem.2015.07.139.

Myers, Drew. 2005a. “Surfactants in Solution.” In Surfactant Science and Technology, 3rd ed.,

107–42. New Jersei: John Wiley & Sons.

———. 2005b. “The Organic Chemistry of Surfactants.” In Surfactant Science and Technology,

1–448. New Jersei.

Nirmal, Nilesh Prakash, Ram Mereddy, Li Li, and Yasmina Sultanbawa. 2018. “Formulation,

Characterisation and Antibacterial Activity of Lemon Myrtle and Anise Myrtle Essential Oil

in Water Nanoemulsion.” Food Chemistry 254 (July): 1–7.

 69

https://doi.org/10.1016/j.foodchem.2018.01.173.

Oliveira, Anna E.M.F.M., Desirane C. Bezerra, Jonatas L. Duarte, Rodrigo A.S. Cruz, Raimundo

N.P. Souto, Ricardo M.A. Ferreira, Jeane Nogueira, et al. 2017. “Essential Oil from

Pterodon Emarginatus as a Promising Natural Raw Material for Larvicidal Nanoemulsions

against a Tropical Disease Vector.” Sustainable Chemistry and Pharmacy 6 (December):

1–9. https://doi.org/10.1016/j.scp.2017.06.001.

Oliveira, Erick F. de, Haroldo C.B. Paula, and Regina C.M.de Paula. 2014. “Alginate/Cashew

Gum Nanoparticles for Essential Oil Encapsulation.” Colloids and Surfaces B:

Biointerfaces. https://doi.org/10.1016/j.colsurfb.2013.08.038.

Osanloo, Mahmoud, Mohammad Mehdi Sedaghat, Fariba Esmaeili, and Amir Amani. 2018.

“Larvicidal Activity of Essential Oil of Syzygium Aromaticum (Clove) in Comparison with Its

Major Constituent, Eugenol, against Anopheles Stephensi.” Journal of Arthropod-Borne

Diseases 12 (4): 361–69. http://www.ncbi.nlm.nih.gov/pubmed/30918905.

Osanloo, Mahmoud, Hassan Sereshti, Mohammad Mehdi Sedaghat, and Amir Amani. 2018.

“Nanoemulsion of Dill Essential Oil as a Green and Potent Larvicide against Anopheles

Stephensi.” Environmental Science and Pollution Research 25 (7): 6466–73.

https://doi.org/10.1007/s11356-017-0822-4.

Ouzineb, Keltoum, Catherine Lord, Nathalie Lesauze, Christian Graillat, Philippe A. Tanguy,

and Timothy McKenna. 2006. “Homogenisation Devices for the Production of

Miniemulsions.” Chemical Engineering Science 61 (9): 2994–3000.

https://doi.org/10.1016/j.ces.2005.10.065.

PATHANIA, RUHI, HUMA KHAN, RAVINDER KAUSHIK, and MOHAMMED AZHAR KHAN.

2018. “Essential Oil Nanoemulsions and Their Antimicrobial and Food Applications.”

Current Research in Nutrition and Food Science Journal.

https://doi.org/10.12944/crnfsj.6.3.05.

Pengon, Sirikarn, Nawinda Chinatangkul, Chutima Limmatvapirat, and Sontaya Limmatvapirat.

2018. “The Effect of Surfactant on the Physical Properties of Coconut Oil Nanoemulsions.”

Asian Journal of Pharmaceutical Sciences 13 (5): 409–14.

https://doi.org/10.1016/j.ajps.2018.02.005.

Pérez-Recalde, Mercedes, Ignacio E. Ruiz Arias, and Élida B. Hermida. 2018. “Could Essential
 70

Oils Enhance Biopolymers Performance for Wound Healing? A Systematic Review.”

Phytomedicine 38 (January): 57–65. https://doi.org/10.1016/j.phymed.2017.09.024.

Politeo, O., M. Jukic, and M. Milos. 2007. “Chemical Composition and Antioxidant Capacity of

Free Volatile Aglycones from Basil (Ocimum Basilicum L.) Compared with Its Essential

Oil.” Food Chemistry 101 (1): 379–85. https://doi.org/10.1016/j.foodchem.2006.01.045.

Porras, M., C. Solans, C. González, and J. M. Gutiérrez. 2008. “Properties of Water-in-Oil (W/O)

Nano-Emulsions Prepared by a Low-Energy Emulsification Method.” Colloids and

Surfaces A: Physicochemical and Engineering Aspects.

https://doi.org/10.1016/j.colsurfa.2008.04.012.

Porras, M., C. Solans, C. González, A. Martínez, A. Guinart, and J. M. Gutiérrez. 2004. “Studies

of Formation of W/O Nano-Emulsions.” In Colloids and Surfaces A: Physicochemical and

Engineering Aspects. https://doi.org/10.1016/j.colsurfa.2004.08.060.

Prabaharan, M. 2011. “Prospective of Guar Gum and Its Derivatives as Controlled Drug

Delivery Systems.” International Journal of Biological Macromolecules.

https://doi.org/10.1016/j.ijbiomac.2011.04.022.

Prajapati, Vipul D., Girish K. Jani, Naresh G. Moradiya, and Narayan P. Randeria. 2013.

“Pharmaceutical Applications of Various Natural Gums, Mucilages and Their Modified

Forms.” Carbohydrate Polymers. https://doi.org/10.1016/j.carbpol.2012.11.021.

Prud’homme, Robert K., Guangwei Wu, and Dieter K. Schneider. 1996. “Structure and

Rheology Studies of Poly(Oxyethylene−oxypropylene−oxyethylene) Aqueous Solution.”

Langmuir 12 (20): 4651–59. https://doi.org/10.1021/la951506b.

Qian, Cheng, Eric Andrew Decker, Hang Xiao, and David Julian McClements. 2012. “Physical

and Chemical Stability of β-Carotene-Enriched Nanoemulsions: Influence of PH, Ionic

Strength, Temperature, and Emulsifier Type.” Food Chemistry 132 (3): 1221–29.

https://doi.org/10.1016/j.foodchem.2011.11.091.

Qian, Cheng, and David Julian McClements. 2011. “Formation of Nanoemulsions Stabilized by

Model Food-Grade Emulsifiers Using High-Pressure Homogenization: Factors Affecting

Particle Size.” Food Hydrocolloids 25 (5): 1000–1008.

https://www.sciencedirect.com/science/article/pii/S0268005X10002328.

Rai, Vineet Kumar, Nidhi Mishra, Kuldeep Singh Yadav, and Narayan Prasad Yadav. 2018.
 71

“Nanoemulsion as Pharmaceutical Carrier for Dermal and Transdermal Drug Delivery:

Formulation Development, Stability Issues, Basic Considerations and Applications.”

Journal of Controlled Release. https://doi.org/10.1016/j.jconrel.2017.11.049.

Rao, Jiajia, and David Julian McClements. 2011. “Food-Grade Microemulsions, Nanoemulsions

and Emulsions: Fabrication from Sucrose Monopalmitate &amp; Lemon Oil.” Food

Hydrocolloids 25 (6): 1413–23. https://doi.org/10.1016/j.foodhyd.2011.02.004.

———. 2012. “Food-Grade Microemulsions and Nanoemulsions: Role of Oil Phase

Composition on Formation and Stability.” Food Hydrocolloids 29 (2): 326–34.

https://doi.org/10.1016/j.foodhyd.2012.04.008.

Rinaldi, Federica, Patrizia Nadia Hanieh, Catia Longhi, Simone Carradori, Daniela Secci,

Gokhan Zengin, Maria Grazia Ammendolia, et al. 2017. “Neem Oil Nanoemulsions:

Characterisation and Antioxidant Activity.” Journal of Enzyme Inhibition and Medicinal

Chemistry 32 (1): 1265–73. https://doi.org/10.1080/14756366.2017.1378190.

Rosety, M, F J Ordóñez, M Rosety-Rodríguez, J M Rosety, I Rosety, C Carrasco, and A

Ribelles. 2001. “Acute Toxicity of Anionic Surfactants Sodium Dodecyl Sulphate (SDS)

and Linear Alkylbenzene Sulphonate (LAS) on the Fertilizing Capability of Gilthead

(Sparus Aurata L.) Sperm.” Histology and Histopathology 16 (3): 839–43.

https://doi.org/10.14670/HH-16.839.

Saberi, Amir Hossein, Yuan Fang, and David Julian McClements. 2014. “Stabilization of Vitamin

E-Enriched Mini-Emulsions: Influence of Organic and Aqueous Phase Compositions.”

Colloids and Surfaces A: Physicochemical and Engineering Aspects.

https://doi.org/10.1016/j.colsurfa.2014.02.042.

Salvia-Trujillo, Laura, Alejandra Rojas-Graü, Robert Soliva-Fortuny, and Olga Martín-Belloso.

2015. “Physicochemical Characterization and Antimicrobial Activity of Food-Grade

Emulsions and Nanoemulsions Incorporating Essential Oils.” Food Hydrocolloids.

https://doi.org/10.1016/j.foodhyd.2014.07.012.

Schwarz, Julia C., Angelika Weixelbaum, Elisabeth Pagitsch, Monika Löw, Guenter P. Resch,

and Claudia Valenta. 2012. “Nanocarriers for Dermal Drug Delivery: Influence of

Preparation Method, Carrier Type and Rheological Properties.” International Journal of

Pharmaceutics. https://doi.org/10.1016/j.ijpharm.2012.08.003.
 72

Seibert, Janaína Brandão, Ivanildes Vasconcelos Rodrigues, Simone Pinto Carneiro, Tatiane

Roquete Amparo, Juliane Sousa Lanza, Frédéric Jean G. Frézard, Gustavo Henrique

Bianco de Souza, and Orlando David Henrique dos Santos. 2019. “Seasonality Study of

Essential Oil from Leaves of Cymbopogon Densiflorus and Nanoemulsion Development

with Antioxidant Activity.” Flavour and Fragrance Journal 34 (1): 5–14.

https://doi.org/10.1002/ffj.3472.

Shahavi, Mohammad Hassan, Morteza Hosseini, Mohsen Jahanshahi, Rikke Louise Meyer, and

Ghasem Najafpour Darzi. 2015. “Evaluation of Critical Parameters for Preparation of

Stable Clove Oil Nanoemulsion.” Arabian Journal of Chemistry.

https://doi.org/10.1016/j.arabjc.2015.08.024.

———. 2016. “Clove Oil Nanoemulsion as an Effective Antibacterial Agent: Taguchi

Optimization Method.” Desalination and Water Treatment 57 (39): 18379–90.

https://doi.org/10.1080/19443994.2015.1092893.

Shahba, Ahmad Abdul-Wahhab, Kazi Mohsin, and Fars Kaed Alanazi. 2012. “Novel Self-

Nanoemulsifying Drug Delivery Systems (SNEDDS) for Oral Delivery of Cinnarizine:

Design, Optimization, and In-Vitro Assessment.” AAPS PharmSciTech 13 (3): 967–77.

https://doi.org/10.1208/s12249-012-9821-4.

Sharma, Abhishek, Naveen Kumar Sharma, Ankit Srivastava, Arti Kataria, Saurabh Dubey,

Satyawati Sharma, and Bishwajit Kundu. 2018. “Clove and Lemongrass Oil Based Non-

Ionic Nanoemulsion for Suppressing the Growth of Plant Pathogenic Fusarium Oxysporum

f.Sp. Lycopersici.” Industrial Crops and Products 123 (November): 353–62.

https://doi.org/10.1016/j.indcrop.2018.06.077.

Sharma, Minaxi, Bimlesh Mann, Rajan Sharma, Rajesh Bajaj, S. Athira, Prabin Sarkar, and

Ramesh Pothuraju. 2017. “Sodium Caseinate Stabilized Clove Oil Nanoemulsion:

Physicochemical Properties.” Journal of Food Engineering 212 (November): 38–46.

https://doi.org/10.1016/j.jfoodeng.2017.05.006.

Shaw, Duncan J. 1992. “The Colloidal State.” In Introduction to Colloid and Surface Chemistry,

4th ed., 320. Elsevier. https://doi.org/10.1016/C2009-0-24070-0.

Shchipunov, Yurii A. 2015. “Lecithin.” In Encyclopedia of Surface and Colloid Science, edited by

P. Somasundaran, 3674–93. Boca Raton: CRC Press. https://doi.org/10.1081/E-ESCS3.

 73

Sing, A.J.F, A Graciaa, J Lachaise, P Brochette, and J.L Salager. 1999. “Interactions and

Coalescence of Nanodroplets in Translucent O/W Emulsions.” Colloids and Surfaces A:

Physicochemical and Engineering Aspects 152 (1–2): 31–39.

https://doi.org/10.1016/S0927-7757(98)00622-0.

Singh, Yuvraj, Jaya Gopal Meher, Kavit Raval, Farooq Ali Khan, Mohini Chaurasia, Nitin K.

Jain, and Manish K. Chourasia. 2017. “Nanoemulsion: Concepts, Development and

Applications in Drug Delivery.” Journal of Controlled Release.

https://doi.org/10.1016/j.jconrel.2017.03.008.

Sivakumar, Manickam, Siah Ying Tang, and Khang Wei Tan. 2014. “Cavitation Technology - A

Greener Processing Technique for the Generation of Pharmaceutical Nanoemulsions.”

Ultrasonics Sonochemistry. https://doi.org/10.1016/j.ultsonch.2014.03.025.

Solans, C., P. Izquierdo, J. Nolla, N. Azemar, and M. J. Garcia-Celma. 2005. “Nano-Emulsions.”

Current Opinion in Colloid and Interface Science.

https://doi.org/10.1016/j.cocis.2005.06.004.

Solans, Conxita, and Isabel Solé. 2012. “Nano-Emulsions: Formation by Low-Energy Methods.”

Current Opinion in Colloid & Interface Science 17 (5): 246–54.

https://doi.org/10.1016/j.cocis.2012.07.003.

Solè, I., A. Maestro, C.M. Pey, C. González, C. Solans, and J.M. Gutiérrez. 2006. “Nano-

Emulsions Preparation by Low Energy Methods in an Ionic Surfactant System.” Colloids

and Surfaces A: Physicochemical and Engineering Aspects 288 (1–3): 138–43.

https://doi.org/10.1016/j.colsurfa.2006.02.013.

Solè, I., C. Solans, A. Maestro, C. González, and J.M. Gutiérrez. 2012. “Study of Nano-

Emulsion Formation by Dilution of Microemulsions.” Journal of Colloid and Interface

Science 376 (1): 133–39. https://doi.org/10.1016/j.jcis.2012.02.063.

Sonneville-Aubrun, O., D. Babayan, D. Bordeaux, P. Lindner, Gabriel Rata, and B. Cabane.

2009. “Phase Transition Pathways for the Production of 100 Nm Oil-in-Water Emulsions.”

Phys. Chem. Chem. Phys. 11 (1): 101–10. https://doi.org/10.1039/B813502A.

Sonneville-Aubrun, O., J. T. Simonnet, and F. L’Alloret. 2004. “Nanoemulsions: A New Vehicle

for Skincare Products.” Advances in Colloid and Interface Science.

https://doi.org/10.1016/j.cis.2003.10.026.
 74

Sonu, K. S., Bimlesh Mann, Rajan Sharma, Rajesh Kumar, and Richa Singh. 2018. “Physico-

Chemical and Antimicrobial Properties of d-Limonene Oil Nanoemulsion Stabilized by

Whey Protein–Maltodextrin Conjugates.” Journal of Food Science and Technology 55 (7):

2749–57. https://doi.org/10.1007/s13197-018-3198-7.

Speranza, A., M. G. Corradini, T. G. Hartman, D. Ribnicky, A. Oren, and M. A. Rogers. 2013.

“Influence of Emulsifier Structure on Lipid Bioaccessibility in Oil–Water Nanoemulsions.”

Journal of Agricultural and Food Chemistry 61 (26): 6505–15.

https://doi.org/10.1021/jf401548r.

Starov, Victor M., and Vjacheslav G. Zhdanov. 2003. “Viscosity of Emulsions: Influence of

Flocculation.” Journal of Colloid and Interface Science 258 (2): 404–14.

https://doi.org/10.1016/S0021-9797(02)00149-2.

Strickley, Robert G. 2004. “Solubilizing Excipients in Oral and Injectable Formulations.”

Pharmaceutical Research. https://doi.org/10.1023/B:PHAM.0000016235.32639.23.

Sundararajan, Balasubramani, Anil Kumar Moola, K. Vivek, and B.D.Ranjitha Kumari. 2018.

“Formulation of Nanoemulsion from Leaves Essential Oil of Ocimum Basilicum L. and Its

Antibacterial, Antioxidant and Larvicidal Activities (Culex Quinquefasciatus).” Microbial

Pathogenesis 125 (December): 475–85. https://doi.org/10.1016/j.micpath.2018.10.017.

Sutradhar, Kumar Bishwajit, and Lutful Amin. 2013. “Nanoemulsions: Increasing Possibilities in

Drug Delivery.” European Journal of Nanomedicine. https://doi.org/10.1515/ejnm-2013-

0001.

Svetlichny, G., I. C. Külkamp-Guerreiro, D. F. Dalla Lana, M. D. Bianchin, A. R. Pohlmann, A.

M. Fuentefria, and S. S. Guterres. 2017. “Assessing the Performance of Copaiba Oil and

Allantoin Nanoparticles on Multidrug-Resistant Candida Parapsilosis.” Journal of Drug

Delivery Science and Technology 40: 59–65. https://doi.org/10.1016/j.jddst.2017.05.020.

Sweedman, Michael C., Morgan J. Tizzotti, Christian Schäfer, and Robert G. Gilbert. 2013.

“Structure and Physicochemical Properties of Octenyl Succinic Anhydride Modified

Starches: A Review.” Carbohydrate Polymers.

https://doi.org/10.1016/j.carbpol.2012.09.040.

Tadros, Th F. 1994. “Fundamental Principles of Emulsion Applications.” Colloids and Surfaces

Physicochemical and Engineering Aspects 91: 39–55.

 75

Tadros, Tharwat. 2004. “Application of Rheology for Assessment and Prediction of the Long-

Term Physical Stability of Emulsions.” Advances in Colloid and Interface Science.

https://doi.org/10.1016/j.cis.2003.10.025.

———. 2009. “Polymeric Surfactants in Disperse Systems.” Advances in Colloid and Interface

Science 147–148: 281–99. https://doi.org/10.1016/j.cis.2008.10.005.

Tadros, Tharwat, P. Izquierdo, J. Esquena, and C. Solans. 2004. “Formation and Stability of

Nano-Emulsions.” Advances in Colloid and Interface Science.

https://doi.org/10.1016/j.cis.2003.10.023.

Taleb, Mohammed, Nourtan Abdeltawab, Rehab Shamma, Sherein Abdelgayed, Sarah

Mohamed, Mohamed Farag, and Mohammed Ramadan. 2018. “Origanum Vulgare L.

Essential Oil as a Potential Anti-Acne Topical Nanoemulsion—In Vitro and In Vivo Study.”

Molecules 23 (9): 2164. https://doi.org/10.3390/molecules23092164.

Taylor, P. 1998. “Ostwald Ripening in Emulsions.” Advances in Colloid and Interface Science 75

(2): 107–63. https://doi.org/10.1016/S0001-8686(98)00035-9.

Tian, Yonghong, Lianghong Chen, and Wanping Zhang. 2016. “Influence of Ionic Surfactants on

the Properties of Nanoemulsions Emulsified by Nonionic Surfactants Span 80/Tween 80.”

Journal of Dispersion Science and Technology 37 (10): 1511–17.

https://doi.org/10.1080/01932691.2015.1048806.

Tsujii, Kaoru. 1998. Surface Activity - Principles, Phenomena, and Applications. Edited by

Toyoichi Tanaka. 1st ed. San Diego: Academic Press.

Wan, Jing, Shaobin Zhong, Paul Schwarz, Bingcan Chen, and Jiajia Rao. 2019. “Physical

Properties, Antifungal and Mycotoxin Inhibitory Activities of Five Essential Oil

Nanoemulsions: Impact of Oil Compositions and Processing Parameters.” Food Chemistry

291: 199–206. https://doi.org/10.1016/j.foodchem.2019.04.032.

Wang, Lei, and Yue Zhang. 2017. “Eugenol Nanoemulsion Stabilized with Zein and Sodium

Caseinate by Self-Assembly.” https://doi.org/10.1021/acs.jafc.7b00194.

Wang, Lijuan, Kevin J. Mutch, Julian Eastoe, Richard K. Heenan, and Jinfeng Dong. 2008.

“Nanoemulsions Prepared by a Two-Step Low-Energy Process.” Langmuir 24 (12): 6092–

99. https://doi.org/10.1021/la800624z.

Wang, Lijuan, Rico Tabor, Julian Eastoe, Xuefeng Li, Richard K. Heenan, and Jinfeng Dong.
 76

2009. “Formation and Stability of Nanoemulsions with Mixed Ionic–Nonionic Surfactants.”

Physical Chemistry Chemical Physics 11 (42): 9772. https://doi.org/10.1039/b912460h.

Wei, Yinghui, Xiaoli Ye, Xiaoguang Shang, Xuan Peng, Qiang Bao, Minchen Liu, Manman Guo,

and Fanzhu Li. 2012. “Enhanced Oral Bioavailability of Silybin by a Supersaturatable Self-

Emulsifying Drug Delivery System (S-SEDDS).” Colloids and Surfaces A: Physicochemical

and Engineering Aspects. https://doi.org/10.1016/j.colsurfa.2011.12.025.

Wiedmann, Timothy. 2003. “Surfactants and Polymers in Drug Delivery.” In Journal of

Controlled Release, 93:415. https://doi.org/10.1016/j.jconrel.2002.11.001.

Wooster, Tim J., Matt Golding, and Peerasak Sanguansri. 2008. “Impact of Oil Type on

Nanoemulsion Formation and Ostwald Ripening Stability.” Langmuir 24 (22): 12758–65.

https://doi.org/10.1021/la801685v.

Xing, Li, and Wayne L. Mattice. 1997. “Strong Solubilization of Small Molecules by Triblock-

Copolymer Micelles in Selective Solvents.” Macromolecules 30 (6): 1711–17.

https://doi.org/10.1021/ma961175p.

Yadav, Madhav P., J. Manuel Igartuburu, Youchun Yan, and Eugene A. Nothnagel. 2007.

“Chemical Investigation of the Structural Basis of the Emulsifying Activity of Gum Arabic.”

Food Hydrocolloids. https://doi.org/10.1016/j.foodhyd.2006.05.001.

Yang, Ying, Christopher Marshall-Breton, Martin E. Leser, Alexander A. Sher, and David Julian

McClements. 2012. “Fabrication of Ultrafine Edible Emulsions: Comparison of High-

Energy and Low-Energy Homogenization Methods.” Food Hydrocolloids 29 (2): 398–406.

https://doi.org/10.1016/j.foodhyd.2012.04.009.

Yen, Ching-Chi, Yi-Chen Chen, Ming-Tsang Wu, Chia-Chi Wang, and Yu-Tse Wu. 2018.

“Nanoemulsion as a Strategy for Improving the Oral Bioavailability and Anti-Inflammatory

Activity of Andrographolide.” International Journal of Nanomedicine Volume 13 (January):

669–80. https://doi.org/10.2147/IJN.S154824.

Yerramilli, Manispuritha, and Supratim Ghosh. 2017. “Long-Term Stability of Sodium Caseinate-

Stabilized Nanoemulsions.” Journal of Food Science and Technology 54 (1): 82–92.

https://doi.org/10.1007/s13197-016-2438-y.

Zhang, Jian, Terry L. Peppard, and Gary A. Reineccius. 2015. “Preparation and

Characterization of Nanoemulsions Stabilized by Food Biopolymers Using

 77

Microfluidization.” Flavour and Fragrance Journal 30 (4): 288–94.

https://doi.org/10.1002/ffj.3244.

Zhang, Shengjiang, Min Zhang, Zhongxiang Fang, and Yaping Liu. 2017. “Preparation and

Characterization of Blended Cloves/Cinnamon Essential Oil Nanoemulsions.” LWT 75

(January): 316–22. https://doi.org/10.1016/j.lwt.2016.08.046.

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