Nano-Emulsions

4
Nicolas Anton and Thierry F. Vandamme

Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Nano-Emulsion Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Methods for Improving the Stability of Nano-Emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Nano-Emulsion Formulation Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Generalities on Emulsification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
High-Pressure Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Ultrasound-Based Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Low-Energy Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Characterization of Nano-Emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Dynamic Light Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Abstract
Nano-emulsion systems consist of a suspension of liquid nanodroplets stabilized
by surfactants. Nano-emulsions are very powerful and promising systems for
numerous applications, since there are very stable systems and their formulation
and characterization are not only very simple but also simply transposable for
industrial scale-up. Owing to a liquid core (mainly oily core), nano-emulsions
have appeared as an efficient solution to disperse and stabilize poorly water-
soluble compounds in aqueous media, through their nano-encapsulation. The
main emerging applications of nano-emulsions include the formulation of

N. Anton (*) • T.F. Vandamme

Faculty of Pharmacy, CNRS 7199 Laboratoire de Conception et Application de Molécules
Bioactives (CAMB), équipe de Pharmacie Biogalénique, University of Strasbourg,
Strasbourg, France
e-mail: [email protected]

# Springer International Publishing Switzerland 2016 93

M. Aliofkhazraei (ed.), Handbook of Nanoparticles,
DOI 10.1007/978-3-319-15338-4_2
94 N. Anton and T.F. Vandamme

innovative drug delivery systems and/or contrast agents. In this chapter, we will
present a state of the art of the different aspects of the nano-emulsion formulation:
processes of fabrication, optimization of the processes, impact of the chemical
nature of the compounds on the processes, stability, strategies for optimizing the
stability, and characterization. Likewise, we will discuss the emerging technolo-
gies aiming the surface treatment of nano-emulsions, including polymer coating
(e.g., for inducing stealth properties in vivo) or ligand grafting for active targeting.

Keywords
Nano-emulsions • Formulation • High-energy methods • Phase inversion tem-
perature method • Spontaneous emulsification

Introduction

Nano-emulsions constitute a very particular class of emulsions, typically due to

their size range, exhibiting hydrodynamic diameters from 10 to 20 nm up to
200–300 nm. Nano-emulsions are also frequently known as miniemulsions, fine-
dispersed emulsions, submicron emulsions, and so forth but are all characterized by
a great stability in suspension due to their very small size, essentially the conse-
quence of significant steric stabilization between droplets, which goes to explain
why the Ostwald ripening is the only adapted droplet destabilization process. In
general, emulsions and also nano-emulsions are not at the thermodynamic equilib-
rium. That means that at infinite time, all these emulsions will give a phase
separation. However, compared to classical emulsions, this destabilization time is
very slow and typically lasts several months. Actually, this kinetic stability is the
main typical characteristic of nano-emulsions, making them prime candidates for
numerous applications from nano-medicine, pharmaceutics, to agri-food industries.
In short, emulsions are thermodynamically unstable systems because the free
energy ΔGf is greater than zero. Considering the global expression ΔGf = γΔA –
TΔSf (with γ the water-oil interfacial tension, ΔA the water-oil interfacial area
gained with emulsification, TΔSf the entropy of droplet formation), the emulsion
instability only comes from ΔA, and ΔGf can be reduced with playing on the
interfacial tension γ. The physical destabilization of emulsions is related to the
spontaneous trend towards a minimal interfacial area between the two immiscible
phases. Therefore, a minimization of interfacial area is attained by two mecha-
nisms: (i) flocculation followed mostly by coalescence and (ii) Ostwald ripening.
In nano-emulsion systems, the very small size of droplets will prevent the
droplets from undergoing reversible processes like flocculation and creaming
(or sedimentation), thus preventing the coalescence. Eventually, Ostwald ripening
remains as the only destabilization process of nano-emulsions. On the other hand,
it is to keep in mind that the stability of nano-emulsions is actually a paradox
since, due to the very small droplet size, ΔA of nano-emulsions is definitively
higher than the one of macro-emulsions, thus resulting in even more increasing
ΔGf. However, stability of nano-emulsions is an experimental fact, almost a
4 Nano-Emulsions 95

Table 1 Main Type of article/application References

publications of our research
Fundamental on formulation and process [1–7]
group on nano-emulsions,
presented by type of Drug delivery systems [8–12]
publication and Contrast agent [13–18]
applications Reviews [19–22]

general rule, and very easily obtained with simple formulation procedures. Origins
of this stability were studied by many authors and found several plausible expla-
nations (summarized shortly below), but the most important ideas are that (1) sta-
bility is linked to the droplet submicron size, (2) the droplets always slowly grow
with time due to Ostwald ripening, and (3) some formulation strategies can be
followed to reduce the destabilization rate. Nano-emulsions are thermodynami-
cally unstable systems, but kinetically stable, and stable against fluctuations of
thermodynamic variables like temperature fluctuations or dilution, giving them
compatibility with a number of industrial applications. To sum up, nano-emulsions
are rather robust.
As a consequence, nano-emulsions naturally find numerous applications in
various fields, notably, since they can be fabricated with nontoxic compounds, in
nano-medicines as lipid nano-carriers. Nano-emulsions allow highly stable disper-
sion of lipid active ingredients, drugs, and contrast agents, in aqueous medium. The
main fundamental and applied works based on nano-emulsions, published by our
group, are reported in Table 1.

Nano-Emulsion Stability

Principles

Nano-emulsion flocculation is naturally prevented by steric stabilization due to the

sub-micrometric size of droplets. When interfacial layers of two different droplet
layers overlap, steric repulsion arises. It comes from two main origins [23, 24]:
the first one is the unfavorable mixing of the stabilizing chain of the adsorbed layer,
depending on the interfacial density, interfacial layer thickness δ, and Flory-Huggins
parameter χ 1,2, reflecting the interactions between the interfacial layer and solvent.
The second one is the reduction of the configurational entropy, due to the bending
stress of the chains, which occurs when inter-droplet distance h becomes lower than
δ. As well, the small droplet size reduces their deformability and the potential
formation of a thin film between two droplets (i.e., a permanent contact occurring
in the flocculation process). The sum of these contributions, i.e., energies of interac-
tions noted UT, can be represented in the function of the inter-droplet distance h as
shown in Fig. 1. This profile typically adopts a weak minimum around h = 2δ, for
which the depth of value |U0| depends on the particle radius r, the Hamaker constant
A, and the thickness of the adsorbed layer δ, with the result that the smaller the droplet
size, the lower the value of |U0| and thus the ability for the nano-emulsion droplets to
96 N. Anton and T.F. Vandamme

Fig. 1 Schematic illustration

of the influence of the nano-
emulsion droplet size on their
steric stabilization (Reprinted
adapted with permission from
Ref. [19])

flocculate. As well, it is worth noting that the small droplet sizes also induce
stabilization against sedimentation or creaming, insofar as the droplets are solely
under the influence of the Brownian motion. To summarize, nano-emulsion droplets
cannot have direct interaction or contact for physical reasons, and their behavior in
suspension trends towards a homogeneous and stable repartition in the whole
volume.
To sum up the destabilization of nano-emulsions is due only to a mass transfer
phenomenon between the droplets through the bulk phase, well described in the
literature the so-called Ostwald ripening in emulsions [25]. In order to understand
the different existing strategies for even improving the nano-emulsion stability, it is
important to know the physical phenomena related to Ostwald ripening. At the
origin of this destabilization process, the differences, however slight, of the droplet
radius induce differences in chemical potential of the material within the drops. The
reduction of free energy in the emulsion will result in the decrease in the interfacial
area and therefore in the growth of the bigger emulsion droplets at the expense of
the smaller ones. The dispersed phase migrates through the bulk from the smaller
droplets to the bigger ones, owing to the higher solubility in the bulk of the smaller
droplets. Ostwald ripening is initiated and will increase throughout the process. The
literature provides a number of theories dealing with calculations of the rate of
ripening, such as the most famous and complete given by Lifshitz, Slyozov, and
Wagner, the so-called LSW theory [26–28]. The diffusion of dispersed materials
through the continuous medium is assumed to be diffusion-controlled, i.e., crossing
4 Nano-Emulsions 97

Fig. 2 Illustration of Ostwald ripening as a function of time and storage temperature: (a) size
evolution; (b) Ostwald ripening rates ω (Reprinted adapted with permission from Ref. [30]. Copy-
right (2013) American Chemical Society)

the interface with ease. Details on LSW theories are fully developed and discussed
in the literature [24, 29] leading to the commonly used expression of the ageing
rate, ω, defined as

dr 3c
ω¼
dt
where rc is the critical radius of the system at any given time, at the frontier between
the growth and decrease in the droplets. Consequently, Ostwald ripening is reflected
by a linear relationship between the cube radius and time. The important idea to be
used in working out nano-emulsion stability studies is that if plotting (radius)3 = f
(time) is a linear function, the destabilization process is effectively Ostwald ripen-
ing, and the slope ω allows to quantify it. This is very useful in order to quantita-
tively evaluate the impact of the different strategies undertaken to reduce the
nano-emulsion destabilization rate. As an illustration, Fig. 2 (from Ref. [30]) pre-
sents the temporal evolution of oil-in-water nano-emulsion droplets’ hydrodynamic
98 N. Anton and T.F. Vandamme

diameter as a function of time for different temperatures (Fig. 2a), as well as

the evolution of the ripening rate (Fig. 2b). This simple system is an illustration
of a non-stabilized system, solely composed of medium-chain triglycerides
(oil core), nonionic surfactant (stabilizing agent), and water + NaCl
(as continuous phase). One can note that the ripening rates can be affected by
the storage temperature, but nevertheless remain suitable up to 40  C, and
relatively constant at room temperature. Moreover, this example is relatively
simple (only composed of oil + surfactant + water) and not further stabilized.
Eventually, this illustrates the huge potentials of nano-emulsions.

Methods for Improving the Stability of Nano-Emulsions

From the understanding of the processes driving the destabilization of nano-

emulsions (summed up in Ostwald ripening), various strategies were imagined
and studied [24, 30], in order to reduce or inhibit the destabilization kinetics.
Actually, Oswald ripening is due to the diffusion of the dispersed phase through
the continuous one, between two drops of different sizes. However, Ostwald
ripening can be significantly reduced both when a second compound is solubi-
lized in the dispersed phase and when this additive is significantly less soluble
in the bulk phase than the dispersed phase itself. The diffusion of such additive
limits the kinetics of the one of the dispersed phase, trending towards keeping
constant the chemical potential over the droplet population. However, as it is
done elsewhere [24], we will not describe in detail, here, the physical related
principles. The main idea to keep in mind is that the kinetics of the diffusion
between the droplets will be driven by the one of the additive, which should be
slower than the one of the continuous phase since it is chosen much less soluble
in the bulk one.
In industrial processes using nano-emulsions, the formulations commonly
include numerous additives like polymers, thickener, stabilizing agent, conserva-
tive agents, electrolyte, etc. They can also play the role of Ostwald inhibitors. In the
case of o/w nano-emulsions, various examples can be found in literature, such as
the influence of the nature of oil on the Ostwald ripening rate (reported in Fig. 3a) or
the impact of the concentration of additive (e.g., squalene) mixed with the dispersed
phase (Fig. 3b). On the other hand, it is important to note that Ostwald ripening can
be also affected by the composition of the continuous phase and notably by the
presence of surfactant micelles, which will promote the diffusion of the dispersed
phase (Fig. 3c). This is why, as we will see below, the amount of emulsifier has a
crucial role in the control of the size and size distribution, but too much sufactants
will also induce destabilization.
As a last remark, the structure and the density of the interface stabilizing layer
have also an influence on the Ostwald ripening rate. The diffusion of dispersed
phase can be decreased in function of the density and compactness of the surfactant
layer adsorbed at the water/oil interface of the droplets [31].
4 Nano-Emulsions 99

a 45
droplet r3 (nm3).10–5 40
35
30
25
20 Decane
15 Dodecane
10 Tetradecane
Hexadecane
5 Isohexadecane
0
0 50 100 150 200 250 300
Time (h)

b 25

20
r3•10–5 (nm3)

15
Isohex. / Sq. = 100/0
Isohex. / Sq. = 98/2
10
Isohex. / Sq. = 96/4
Isohex. / Sq. = 94/6
5 Isohex. / Sq. = 92/8
Isohex. / Sq. = 80/20
0
0 25 50 75 100
Time (h)
c 20
droplet r3(nm3).10–5

15

10
S: 4.0 wt%
S: 5.0 wt%
5 S: 6.0 wt%
S: 7.0 wt%
S: 8.0 wt%
0
0 50 100 150 200
Time (h)

Fig. 3 Influence of various experimental parameters on the Ostwald ripening rate of o/w nano-
emulsions: (a) nature of oil; (b) addition of insoluble oil in the dispersed phase; (c) concentration
of surfactant in the continuous phase (Adapted from Ref. [24])
100 N. Anton and T.F. Vandamme

Nano-Emulsion Formulation Processes

Generalities on Emulsification

Emulsification consists of dispersing one fluid into another nonmiscible one, as

seen above, creating interface (i.e., increasing ΔA). The physicochemical properties
of the resulting emulsion, like size distribution and stability, are not only closely
linked to the composition, surfactant properties, and ratio of viscosities of the
dispersed and bulk phases but also on the formulation protocol, temperature and
time of processing, shear rate, cooling time, and type of emulsification apparatus.
On the other hand, once the emulsion is fabricated (beside temperature and com-
position), the monodispersity of emulsions is an important criterion influencing
their stability. In the case of nano-emulsion, monodispersity will have a direct
impact on the inhibition of Ostwald ripening, since the difference of the pressure
between the bigger and smaller droplets will be reduced.
In view of the expression of ΔGf above (ΔGf = γΔA – TΔS), the formulation of
emulsions sizing below 100 nm is conditioned by the energy supplied by the
formulation device. Actually, most of the apparatuses currently used for the fabri-
cation of macro-emulsions do not allow decreasing the droplet size below 1 μm,
because of low emulsification yields due to the dissipation of the mechanical energy
in heat. It is the case of rotor/stator apparatuses like ULTRA-TURRAX ® and
colloidal mills. Only several methods allow the fabrication of nano-emulsions.
Table 2 (adapted from Ref. [32]) gathers the main methods for the formulation of
emulsions along with the range of the generated droplet sizes (with limiting ϕvd <
30% to avoid droplet recombination during processing).
In view of the possibilities offered by the emulsification devices (Table 2), we
have three possibilities for the formulation of nano-emulsions: (1) high-pressure
homogenization, (2) ultrasonication, and (3) low-energy emulsification. In the
following sections, these processes will be presented and the conditions to reach
nanometric-sized droplets will be discussed.
However, the industrial production of emulsions follows several well-defined steps.
After the preparation of the phase and all components like emulsifiers (e.g., weighting
and heating for decreasing the viscosities), (i) the first mechanical step consists in the
mixing or dispersing of the phases. This process is performed using stirring
(rotor/stator) devices and gives rise to a premix emulsion sizing around 100 μm.

Table 2 Summary of the Emulsification method Typical size

accessible sizes in function
Mechanical stirring 1 μm–15 μm
of the emulsification
apparatuses (in blue, the Colloidal mills 10 μm–50 μm
ones compatible with the High-pressure homogenization 50 nm–5 μm
formulation of nano- Membrane 0.2 μm–100 μm
emulsions) (Adapted from Microfluidizer ® 10 nm–1 μm
Ref. [32])
Ultrasonication 20 nm–1 μm
Low-energy methods 10 nm–200 nm
4 Nano-Emulsions 101

Fig. 4 Schematic of high-pressure homogenizer chamber

(ii) The second step is the homogenization step, and this is an essential step that will
give rise to fine and stable monodispersed emulsions. In industrial practice, the
formulation of stable emulsions generally passes by these two steps, giving rise to
nano-emulsions. One most famous and widespread example is the stabilization of milk
by high-pressure homogenization, allowing to decrease the size below the micrometer.
On the other hand, the low-energy methods can also be found in industrial processes
especially in cosmetic applications, but overall not as widespread as high-pressure
homogenizers.

High-Pressure Methods

High-pressure homogenization (also called microfluidization) is a very common

process in the field of agri-food liquids or semiliquid products. For instance, it
allows the stabilization of fatty emulsion (e.g., milk) by reducing the droplet sizes
and narrowing the size distribution. High-pressure homogenization is a physical
treatment during which a liquid or semiliquid system is projected under high
pressure (30–1’000 bar) through a homogenization head of particular geometry
(see Fig. 4). The premix emulsion undergoes a combination of elongation and shear
flows, impacts, and cavitations. Despite the complexity of the mechanisms
involved, the size distributions are usually reproducible with a mean size ranging
from 50 nm to 5 μm.
Emulsification by high-pressure homogenization results from a dynamic equi-
librium between droplet breakup (promoted by drop deformation resulting from the
high-speed flow) and recombination due to coalescence (promoted by collisions).
Numerous studies were performed in order to determine the effect on the droplet
size and emulsion stability, stabilizing agent concentration (protein or surfactant),
applied pressure, and number of passages in the homogenizer (cycles). Conceptu-
ally, two processing regimes can be distinguished, depending on the surfactant
concentration C.
102 N. Anton and T.F. Vandamme

(i) The first one corresponds to low surfactant concentration, for C < (critical
micelle concentration)/10. The applied pressure does almost not influence the
size of the homogenized droplets. Even if, during processing, drops are effi-
ciently fragmented, they undergo a strong coalescence owing to the inefficient
surface stabilization by surfactant due to their low concentration. As a result,
the droplet size cannot be decreased below 300 nm.
(ii) The second regime occurs at higher surfactant concentration, typically for C >
(critical micelle concentration)  10. This time, the newly fragmented droplets
are largely stabilized by surfactant before coalescence, allowing decreasing the
size up to 50 nm. This is due to the surfactant interfacial adsorption time being
shorter than the recombination and coalescence time. One can understand that
above a certain surfactant concentration, the interface of the newly formed
droplets is totally saturated, and thus, further increasing C will not have an
impact on the droplet size. Accordingly, in contrast with the case (i) above,
here, the value of the applied pressure P significantly impacts on the droplet
size, following a power law in the form (droplet size) / Pα, with the power
exponent α typically varying between 0.6 and 0.9 [33]. As surfactants are
adsorbed onto the droplet interface, the surface tension is globally gradually
decreased, thus favoring further rupturing.

To summarize, (i) for low surfactant concentration, the droplet size cannot be
decreased enough to generate nano-emulsions, and (ii) for higher surfactant con-
centration, the droplet size can be lowered in the nanometric range, with the size
depending on the pressure applied. It is to be noted that this behavior is transposable
to many surface-active compounds and macromolecules like protein or polymer.
Furthermore, several other parameters can significantly impact on the nano-
emulsion droplet size and distribution width, like (1) the number of passages in
the homogenizer reduces the distribution width; (2) the volume fraction ϕvd can
influence the mean droplet size, i.e., when ϕvd > 30 %, the number of droplet
collisions and recombinations during the emulsification step is increased; thus, the
mean droplet size is higher than the emulsion fabricated at low ϕvd values, and
above ϕvd < 30 %, this effect becomes negligible; (3) a second chamber (with the
pressure = 1/3 of the first one) is preferable, allowing a better control of the
cavitation process and of the quality and monodispersity of the emulsion; (4) finally,
it is to keep in mind that if the energy supplied is too high, it can induce the
degradation of the principles composing the emulsion, e.g., protein denaturation.
These different points are gathered in Fig. 5 (adapted from Ref. [34]): Fig. 5a, b
presents the effects on the number of passages on the droplet size, Fig. 5c the effects
of the value of ϕvd, and Fig. 5d the effects of the pressure applied.

Ultrasound-Based Methods

Power ultrasounds are acoustic waves of frequencies from 16 kHz (upper limit of
human acuteness) to 1 MHz. They are generally produced by a plane surface that
4 Nano-Emulsions 103

Fig. 5 High-pressure device for the formulation of nano-emulsion: influence various experimen-
tal parameters on the droplet size. (a, b) The effects on the number of passages on the droplet size,
(c) the effects of the value of φvd, and (d) the effects of the pressure applied (Adapted from Ref.
[34])

vibrates in a sinusoidal way. The amplitude is generally between 1 and 200 μm.
Cavitation bubbles alternatively undergo contraction and dilatations. However,
their diameters gradually increase up to a very fast implosion. It is to be noted
that such mechanical effects are favored at low frequencies (e.g., 20 kHz), at high
acoustic intensity, at high pressure, and at low temperature. The main mechanisms
that allow explaining the emulsification induced by ultrasounds consider that the
implosion of the cavitation bubble gives rise to the fragmentation of the droplets,
gradually decreasing their average diameter. It is to be noted that the ultrasonic
emulsification is only efficient in a small volume around the sonotrode (see Fig. 6,
left). That means that the fluid circulation in the sample is still necessary for
optimizing the process and involves a minimum time to be reached before stabili-
zation of the droplet sizes.
Furthermore, when ultrasound-based technologies are compared with high-
pressure homogenizers, the properties of the resulting emulsions are generally
similar. However, high-pressure homogenizers are widely used in industrial pro-
cesses, whereas ultrasonifiers are more adapted for the laboratory scale, research,
and development stages.
Once a premix emulsion is ultrasonified, the suspension undergoes a decrease
in the droplet size with the sonification time, up to a limit size. The visual aspect of
the flack along the nano-emulsification process is presented in Fig. 6 (right).
104 N. Anton and T.F. Vandamme

Sonication time

Macro- 200 nm 100 nm 70 nm 40 nm 40 nm

emulsion Nano-emulsions

Surfactant
layer
0.40
Sonotrode 180
Oil core 0.35

Polydispersity index (PDI)

160
0.30
140
0.25
120
dp (nm)

100 0.20

80 0.15
Emulsification
volume 60 0.10

40 0.05
Sample
20 0.00
0 5 10 15 20 25 30 35
Time (min)

Fig. 6 Left: schematic of a sonication-based nano-emulsification process. Right: evolution of

droplet size distribution along the sonication process: particle mean diameter (black disks) and
polydispersity index (open triangles). Schematic representation of a nano-emulsion droplet
(Reprinted adapted with permission from Ref. [30]. Copyright (2013) American Chemical
Society)

One can see the macro-emulsion or premix having a turbid aspect, and when the
droplet size decreases, a milky aspect appears and decreases with the droplet size.
The suspension becomes transparent for the smaller droplets. The nano-emulsion
mean size describes an exponential decay up to stabilization. This is as well
illustrated in Fig. 6 (right) above, for a simple system composed of medium-chain
triglycerides (oily core), nonionic surfactant (stabilizing agent), and water as
continuous phase. The figure shows the follow-up of the mean size and polydis-
persity indexes (PDI). PDI reflects the quality and monodispersity of the dispersion,
such that a suspension is considered of good monodispersity (good quality) if the
value of the PDI is below 0.2–0.15 and of very good monodispersity (very good
quality) if the PDI is below 0.1.
Actually, as a general rule for ultrasound-based nano-emulsification processes, this
behavior follows an exponential decay. Literature has highlighted several key points
impacting on the characteristic time of the exponential (i.e., time to reach the stabili-
zation (in the example of Fig. 6, it is ~10 min)) and impacting on the final droplet size:
to summarize the characteristic time is actually only due to the energy supplied.
4 Nano-Emulsions 105

Fig. 7 Left: size monitoring 200

for different sonication
powers. Right: size monitoring 180
as a function of the global 160 P20%
energy supplied (Reprinted P25%
adapted with permission from 140
P40%
Ref. [30]. Copyright (2013) 120

dp (nm)
American Chemical Society)
100
80
60
40
20
0
0 5 10 15 20 25 30
Sonication time (min)
150
140
130
120
110
dp (nm)

100
90
80 P = 25%
70 P = 30%
60 P = 40%
50
40
0 50000 100000 150000 200000 250000
Sonication Energy (J)

That is to say, the higher the power of the ultrasonifier (selected in the process by the
operator), the faster the nano-emulsification. This is illustrated in Fig. 7 below,
showing (left) the droplet size decrease in function of the sonication time, for the
same system in Fig. 6 (triglycerides/nonionic surfactant/water) at different powers.
Now, since the global energy supplied is given by the power of sonication  time of
sonication, the right part of this figure shows the droplet diameter in function (this
time) of the total energy supplied. The curves appear exactly similar. This result is
actually important for understanding the process, as well as its design and
optimization.
The second key parameter emphasized by the literature is the influence of the
surface-active agents used: both their chemical nature and concentration impact on
the final size of the nano-emulsion droplets (after the size stabilization time shown
above). The higher the surfactant amount, the lower the resulting nano-emulsion
size (as illustrated for instance in Fig. 8 (left) below). However, high and low
106 N. Anton and T.F. Vandamme

Fig. 8 Left: size as a function

of the emulsifier amount.
Right: impact of the

Diameter / nm
surfactant amount on the
100
coverage of the nano-
emulsion droplet interface
(Adapted from Ref. [35])

10
0.1 1 10
Emulsifier / %

Decreasing the surfactant concentration

Surface Area per molecules / nm2

6
region of miniemulsion
5

4 coexist.
with free
3 micelles
unstable
2

0
0 50 100 150 200
Particle Diameter / nm

surfactant concentrations have to be avoided in order to formulate stable nano-

emulsions. The reason is simple and illustrated in Fig. 8 (right): (i) for the higher
surfactant concentrations (i.e., smaller sizes), there is the concomitant presence of
droplets and micelles. This will decrease the suspension stability due to the
enhancement of Ostwald ripening (due to the inter-droplet transport of dispersed
phase induced by the micelles). (ii) On the other hand, along the nano-
emulsification process, the interfacial area increases in a dramatic manner, so that
for low surfactant concentrations, emulsion instability can come from the low
interface covering by surfactants. There are not enough molecules to cover the
entire available interface, and thus, the surface tension at the oil-water interface
increases. This remark is a general remark for the nano-emulsions formulated by
the high-energy method and may also be valuable for the nano-emulsions formu-
lated by high-pressure homogenization.
Likewise, the surface-active properties of the emulsifiers play a significant role
in the resulting size of nano-emulsion droplets.
To summarize, the ultrasonification process applied to a premix emulsion
induces a size decrease following an exponential decay (see Figs. 6 and 7). The
time necessary to reach the final stabilized size only relies on the energy supplied
(i.e., similar results will be obtained between low power and long time, or high
4 Nano-Emulsions 107

power and short time). Secondly, the size of the droplet size after stabilization
depends on the nature and concentration of the surfactant(s) used. Too high or too
low concentrations of surfactants have to be avoided, since these cases induce
instabilities in the nano-emulsion suspension. Finally, this sonication process
must be associated with a circulation of the liquid within the emulsification vessel.

Low-Energy Methods

Low-energy methods allow generating nano-emulsion droplets without energy

input, as it is the case for mechanical high-energy emulsification described above
(high pressure and sonication). In low-energy methods, the increase in ΔA is not
due to the supplied energy but rather is only obtained by taking benefit of the
intrinsic physicochemical properties of the different compounds. The slight energy
supplied is dedicated to mixing the sample and/or heating for optimizing the
spontaneous emulsification process. In the facts, important fundamental research
efforts are led to understand and control these low-energy formulation methods.
Such simple and cost-effective formulation processes are very attractive; however,
the low-energy methods are very much studied but almost only on the fundamental
point of view and much less used in industrial processes than high-energy ones.
This is likely due to several points, like the fact (1) that these methods only allow
nano-emulsification and not homogenization of premix (e.g., not suitable for
homogenizing milk), (2) that the physicochemical properties of the dispersion
cannot always be finely controlled, and finally (3) that they involve a
non-negligible amount of surfactants that is not always compatible with all the
specifications (impact on the taste in agri-food industries, induce a toxicity, etc.).

Spontaneous Emulsification
Spontaneous emulsification, or self-emulsification, is a process that occurs without
the need of an external energy supply when two immiscible fluids are brought in
contact and mixed. As a result, metastable nano-emulsion droplets are formed.
When, as seen above, increasing the interfacial area requires a significant energy
input, the spontaneous emulsification gives rise to similar gain in ΔA without
energy. Actually, the phenomena driving the spontaneous emulsification take
benefit of the physicochemical properties of the water, oil, and surfactant (plus
potentially other compounds like solvent, co-surfactant, additives), for creating the
emulsion droplets. However, in order to optimize the spontaneous process and
emulsification kinetics, the samples will be homogenized and/or heated during
emulsification. Of course, this still requires certain energy, but with no comparisons
with the one supplied for high-energy emulsifications.
Actually, although spontaneous emulsification was reported for more than
100 years, this phenomenon is still not fully understood. The literature has proposed
a plethora of explanation; however, it is only recently that a universal and simple
mechanism was proposed [3], explaining globally the spontaneous emulsification of
all the low-energy methods. The main mechanism is simple, as illustrated in Fig. 9,
108 N. Anton and T.F. Vandamme

Fig. 9 Schematic representation of the mechanism driving the spontaneous emulsification

(Adapted from Ref. [3])

and has been attributed to the penetration of the water phase in the oily one, inducing
the creation of nano-droplets by breaking up the oil phase at the molecular level.
The first step is the homogeneous mixing of surfactant in oil (i.e., solubilized or
dispersed) (Fig. 9a). Next, this surfactant/oil phase is mixed with the aqueous phase
(e.g., pure water) (Fig. 9b). Very rapidly, the water phase penetrates the former in
order to solubilize the surfactant molecules (Fig. 9c). Small nanometric oily drops
with a very narrow size distribution are immediately generated and stabilized with
surfactants (Fig. 9d). Actually, it appears that the mechanism is governed by the
physicochemical properties and solubility of the surfactant for the two oily and
aqueous phases. The first condition is a total solubilization of the surfactants in the
oily phase, and then, they must be also soluble in water. Actually, for an optimal
fractionation of the oil, during the water penetration step (Fig. 9c), surfactant
molecules must present significantly more affinities for water than for oil.
4 Nano-Emulsions 109

Fig. 10 Nano-emulsions formulated with oil and surfactant freely soluble at room temperature
(25  C). Surfactant = Cremophor ELP ®, oil = Labrafil M1944 CS ®. The hydrodynamic diameter
(filled circles) and PDI (open squares) are plotted against the surfactant-to-oil weight ratio. The
hatched parts indicate that the criteria of PDI quality are not met; the suspension cannot be
considered as a nano-emulsion. Inset presents the number of droplets as a function of SOR
(Adapted from Ref. [3])

Logically, the size of the nano-emulsion droplets depends on the concentration

of oil in the first surfactant/oil mixture (i.e., proportion of oil compared to surfac-
tant). The smaller is the oil amount, the higher is the size of the resulting droplets.
Besides, the amount of continuous phase in the process has been shown to not
influence the droplet size. In literature, as above described in emulsification mech-
anism, the compound inducing the diffusion of water is generally a surfactant, but it
can also be a hydrophilic solvent like ethanol (even if the presence of surfactants is
in all cases required for stabilizing the newly formed droplets). Therefore, the whole
process is based on the physicochemical properties of the compound inducing the
diffusion of water and thus on its affinities for the oil and water. An example is
presented in Fig. 10, showing the influence of the proportion of surfactant in the oil
plus surfactant mixture (noted SOR in the figure, surfactant-to-oil weight ratio), on
the properties of the w/o nano-emulsion generated, i.e., size (filled circles) and
polydispersity (open squares). Two main results emerge from this graph: (i) first, the
sizes of the emulsions are very finely controllable from all over the nano-emulsion
size range, i.e., from 200 nm up to 15 nm (for this example), and (ii) second,
the polydispersity indexes are very low, typically lower than 0.1, signifying that
nano-emulsions prepared by spontaneous emulsification are extremely
monodispersed. As a comparison with the example shown in Figs. 6 and 7
110 N. Anton and T.F. Vandamme

with a very similar system, where the values of the PDI stabilize between 0.10 and
0.15, here, the spontaneous method allows reducing the PDI below 0.05.
To summarize, the spontaneous emulsification process is due to the fractionation
of the phase to be dispersed by the continuous one. The success of the process
comes (i) from a good solubilization of the compound inducing the diffusion
(surfactant) into the phase to be dispersed (oil) and (ii) from an even higher affinity
of the surfactant with the continuous phase (water), therefore ensuring a very rapid
penetration of water in the surfactant/oil phase once mixed.
On the other hand, nonionic surfactants constitute the class of surfactants
predominantly used in spontaneous emulsification methods. This is actually due
to their particular physicochemical properties very compatible with the above-
described process, for the following reasons. The hydrophilic part of nonionic
surfactants is a hydrophilic polymer, generally a polyethylene glycol (PEG). This
polymer is sensitive to temperature and precisely will see its solubility changing
with the temperature. That is to say, the temperature modifies the surfactant
affinities for water and oil. Basically, at low temperature, the surfactants are
hydrosoluble. Upon a temperature increase, their solubility in water decreases,
and it becomes more soluble in oil (see general description of such affinities in [21]).
We saw above that the mechanism on which the spontaneous emulsification is
based is precisely linked to surfactant affinities for oil and water, needing first a
good solubilization in oil and then (when the surfactant/oil phase is mixed with
water) an even better affinity for water. This is actually often performed and/or
enhanced by the temperature. Indeed, if the surfactant/oil phase is heated, the
surfactant solubility in oil will be increased, resulting in a better homogeneous
dispersion of surfactant in oil. Then, when the hot surfactant/oil phase is mixed with
room-temperature water (i.e., colder water), the surfactant affinity will immediately
change towards the water, thus promoting the water penetration in the surfactant/oil
phase and promoting as well the nano-emulsification process.
This is a basic overview on the method, of course depending on the chemical
nature of each compound, predominantly on the transition temperature of the
nonionic surfactant (i.e., cloud point).

Phase Inversion Temperature Method

In literature, emulsification through “phase inversion” is also often considered as a
spontaneous emulsification process because it requires low energy input with
similar results than for spontaneous emulsification presented above. A second
advantage of this method is the possibility of producing concentrated emulsions.
Emulsification through phase inversion is often used industrially, especially in
cosmetics. The literature reports a large number of fundamental studies dealing
with the potential mechanisms governing the phase inversion emulsification
methods and actually providing as well a number of different theories explaining
these mechanisms. In order to understand how this emulsification process is worked
out, and what the influence of the formulation parameters on the process is, the two
following questions should be addressed: (i) what is emulsion phase inversion and
(ii) how can phase inversion induce nano-emulsification?
4 Nano-Emulsions 111

Fig. 11 Schematic emulsion

inversion map, in dynamic
conditions and at fixed
surfactant concentration
(simplified to the effect of
temperature) (Adapted from
Ref. [19])

(i) What is emulsion phase inversion? In contrast with the spontaneous emulsifi-
cation described above, oil, surfactant, and water are mixed right from the start,
forming a macroscopic emulsion. When we speak of emulsion phase inversion,
it is referred to the inversion of such macroscopic emulsions (and not to nano-
emulsions). However, the inversion process can be used for the generation of
nano-emulsions, and once nano-emulsions are generated, the emulsion phase
inversion process is over and cannot occur any more: the whole system is
converted to stable nano-emulsions. Emulsion inversion is produced when an
oil-in-water (o/w) emulsion becomes a water-in-oil (w/o) one, that is to say, the
continuous phase changes.

Beforehand, we have to notice that the nature of the continuous phase (if it is oily
of aqueous) is linked to the difference of the surfactant solubility for oil and water.
If the surfactants exhibit a higher solubility in one phase, the latter will be the
continuous phase of the emulsion (i.e., following the empirical Bancroft’s rule).
Thus, according to these principles, a general map of the emulsion inversion can be
drawn, as illustrated in Fig. 11.
The phase inversion temperature (PIT) is the temperature for which the surfac-
tant presents the same affinity for water and oil (horizontal gray line in the figure).
In this domain, the ternary system does not present anymore the structure of an
emulsion but becomes a bicontinuous (at the nanometric scale) system called
microemulsion. However, far from the PIT, the nonionic surfactant is hydrophilic
or lipophilic, respectively, forming and stabilizing o/w or w/o emulsions. It must be
noted that the inversion process can occur only for a roughly comparable amount of
water and oil, at the region corresponding to the horizontal gray line in the figure.
For higher proportions of oil, or water, the continuous phase will not invert but will
rather give instable double emulsions.

(ii) How can phase inversion induce nano-emulsification? The emulsion inversion
is a reversible process, that is to say, o/w emulsion can invert to give w/o upon
a temperature rise and vice versa upon a temperature decrease. In contrast,
the nano-emulsification based on the PIT method is an irreversible process,
112 N. Anton and T.F. Vandamme

i.e., once the nano-emulsions are generated, the ternary system has reached a
thermodynamic metastable state. The nano-emulsification process is actually
very simple and performed as follows: (1) the water/surfactant/oil system is
emulsified by a gentle stirring and heated above the PIT. The o/w emulsion
formed at T < PIT undergoes a phase inversion when T > PIT, giving rise to a
w/o emulsion. (2) The second step is an irreversible step consisting in a rapid
cooling of this emulsion. The system is broken up into nano-droplets, forming
the kinetically stable nano-emulsion.

In view of the literature that provides a plethora of explanations regarding the

potential emulsification mechanism, it seems not fully understood. However,
actually, the simple emulsification mechanism explaining the spontaneous emul-
sification presented above in section “Spontaneous Emulsification” can be
totally transposable to understand the PIT method. Indeed, at T > PIT, surfac-
tants are lipophilic, and according to the Bancroft’s rule, this induces oil is the
continuous phase. Moreover, in addition, at this temperature, the majority of free
surfactant (i.e., not adsorbed at the interface) is also lipophilic and will be
solubilized within the oily phase. This means that the so-called rapid cooling
step will induce a sudden change of the surfactant solubility, making them
hydrophilic. Finally, exactly like for spontaneous emulsification, this sudden
change in the surfactant affinities will result in the immediate penetration of
water within the oily phase (as illustrated in Fig. 9c), creating the nano-emulsion
droplets. This universality in the mechanisms has been proposed by comparing
the properties of different nano-emulsions, made with the same composition, but
formed with two different methods: spontaneous emulsification described in
section “Spontaneous Emulsification” and the PIT method. The results are
presented in Fig. 12 and show clearly comparable sizes whatever the formulation
method, suggesting that the mechanism of formation of the nano-emulsion
droplets is likely similar. As well, this confirms the mechanism proposed to
govern the spontaneous emulsification, eventually extended to low-energy
methods in general.

Characterization of Nano-Emulsions

When macroscopic emulsions can be characterized according to numerous

methods, from optical microscopy, optical counter, to static light scattering, all
these characterization methods are inapplicable with the nanoscaled emulsions.
Overall, the characterization techniques applied for characterizing emulsions sizing
> 1 μm are totally different than the ones sizing < 1 μm. In that way, the
characterization of nano-emulsions is mainly performed by dynamic light scatter-
ing (DLS) and sometimes by transmission electron microscopy (TEM). Represen-
tative examples we did with iodinated nano-emulsions are reported in Fig. 13,
showing the size distribution of nano-emulsions centered in 85 nm and performed
4 Nano-Emulsions 113

Spontaneous
emulsification PIT method

130

80
SOR = 20%
Diameter (nm)

SOR = 30%
SOR = 40%
60 SOR = 50%
SOR = 60%
SOR = 70%

40

20

50 60 70 80
WOR (%)

Fig. 12 A comparison between nano-emulsions generated by the PIT method and spontaneous
emulsification. Surfactant = Solutol HS 15 ®, oil = Labrafil M1944 CS ®, aqueous phase =
ultrapure Milli-Q water. The hydrodynamic diameter plotted against the WOR (water-to-oil
weight ratio) and the SOR (surfactant-to-oil weight ratio). One star = 0.1 < PDI < 0.2, no star
= PDI < 0.1 (Adapted from Ref. [3])

20 11 nm 71 nm
d = 85 nm
16 PDI = 0.16
Frequency (%)

12

0
10 100 1000
Diameter (nm)

Fig. 13 Characterization of nano-emulsions. (Left) Size distribution performed by dynamic light

scattering and (right) pictures done by transmission electron microscopy (Adapted from Ref. [16])

with a NanoSZ Malvern apparatus (left) and a picture of electron microscopy of the
same nano-emulsion droplets using a Philips Morgagni 268D electron microscope
(right).
114 N. Anton and T.F. Vandamme

Dynamic Light Scattering

The matter here is not to present the theory of autocorrelation giving size distribu-
tion of nano-dispersion, on which the method is based, but rather to describe the
experimental aspect of the characterization. Briefly, the sample is placed on a
thermostatic cell, crossed by a laser light. A photodetector (photomultiplier) at a
fixed angle, with respect to the light pathway, records the fluctuations of scattered
intensities over time (that are very slight). After a complex calculation, the appa-
ratus gives the distribution of the diffusion coefficients. This step involves knowing
the refractive indexes of the bulk and continuous phases. As the particles are very
small, we consider them only moving because of the Brownian motion, and this is
precisely why this method is valid in this case and not with bigger particles (for
which the gravitational component is not negligible). These diffusion coefficients
allow providing the size distribution according to the Stokes-Einstein relationship.
It is as well to be noticed that the Stokes-Einstein equation contains the temperature
and viscosity of the continuous phase.
To conclude, the method is not complicated for the operator since the appara-
tuses are generally built to automatically adapt the operating conditions to the
samples (e.g., by attenuating the incident light); the related software are very simple
to use; however, the measurements are valid, only if we know the values of the
refractive indexes of the bulk and continuous phases and the viscosity of the
continuous phase.

Electron Microscopy

Various electron microscopy technologies were developed, but only some of them
can be used for observing the structure of the nano-emulsion droplets. For the study
of liquid nano-droplets, only transmission electron microscopy, potentially cryo-
transmission microscopy, could be compatible. The liquid samples are placed on a
carbon grid and then dried. The dilution must be adapted in order to avoid droplet
aggregation. Measurements are performed under a strong vacuum. In contrast, the
cryo-microscopy will freeze the samples in a liquid state, allowing them to keep
their native structure (not affected by the evaporation stage). Actually, it is clear
that the more simple and appropriate routine characterization of nano-emulsions is
only dynamic light scattering.

Conclusion

Nano-emulsions constitute a very particular class of emulsions, with unique phys-

icochemical properties, high stability, and homogeneity. These features make nano-
emulsion a powerful and simple system for numerous domains and applications.
In this chapter, we proposed an overview of the main principles related to the nano-
emulsions and its main features, stability properties, and formulation methods.
4 Nano-Emulsions 115

For each experimental approach, high-energy methods (high-pressure and

ultrasound-based emulsification) and low-energy methods (spontaneous emulsifi-
cation and PIT methods) were detailed, and the impacts of the experimental
parameters on the resulting emulsification properties were discussed.

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