Design and Production of Nanoparticles Formulated From Nano-Emulsion

Journal of Controlled Release 128 (2008) 185199
Contents lists available at ScienceDirect
Journal of Controlled Release

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Review
Design and production of nanoparticles formulated from nano-emulsion

templatesA review
Nicolas Anton a,b, Jean-Pierre Benoit a,c,, Patrick Saulnier a
a
Inserm U646, Ingnierie de la vectorisation particulaire, 10 rue A. Boquel, F-49100 Angers, France; University of Angers, F-49100 Angers, France
ESPCI, Laboratoire Collodes et Matriaux Diviss, ParisTech, 10 rue Vauquelin, Paris, F-75005 France; CNRS, UMR 7612, Paris, F-75005 France; Universit Pierre et Marie Curie-Paris 6,
Paris, F-75005 France
c
cole pratique des hautes tudes (EPHE), 12 rue Cuvier, F-75005 Paris, France
b
A R T I C L E
I N F O
Article history:
Received 3 December 2007
Accepted 11 February 2008
Available online 23 February 2008
Keywords:
Nano-emulsion
Nanoparticle
Nanocapsule
Colloidal carrier
High-energy method
Low-energy method
Drug delivery
Solvent displacement
Phase inversion temperature
PIT method
A B S T R A C T
A considerable number of nanoparticle formulation methods are based on nano-emulsion templates, which
in turn are generated in various ways. It must therefore be taken into account that active principles and drugs
encapsulated in nanoparticles can potentially be affected by these nano-emulsion formulation processes.
Such potential differences may include drug sensitivity to temperature, high-shear devices, or even contact
with organic solvents. Likewise, nano-emulsion formulation processes must be chosen in function of the
selected therapeutic goals of the nano-carrier suspension and its administration route. This requires the
nanoparticle formulation processes (and thus the nano-emulsion formation methods) to be more adapted to
the nature of the encapsulated drugs, as well as to the chosen route of administration. Offering a
comprehensive review, this paper proposes a link between nano-emulsion formulation methods and
nanoparticle generation, while at the same time bearing in mind the above-mentioned parameters for active
molecule encapsulation. The rst part will deal with the nano-emulsion template through the different
formulation methods, i.e. high energy methods on the one hand, and low-energy ones (essentially
spontaneous emulsication and the phase inversion temperature (PIT) method) on the other. This will be
followed by a review of the different families of nanoparticles (i.e. polymeric or lipid nanospheres and
nanocapsules) highlighting the links (or potential links) between these nanoparticles and the different nanoemulsion formulation methods upon which they are based.
2008 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The great stability of nano-emulsions . . . . . . . . . . . . . . . . . . . . . .
High-energy emulsication methods . . . . . . . . . . . . . . . . . . . . . .
3.1.
Devices and processes . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
The choice of surfactants, monomers, aqueous and oily phases . . . . . . .
3.3.
On the potentialities, advantages and disadvantages of high-energy methods
Low-energy emulsication methods . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Spontaneous nano-emulsication . . . . . . . . . . . . . . . . . . . . .
4.1.1.
The diffusion mechanism and diffusion path theory . . . . . . . .
4.1.2.
The emulsion inversion point (EIP) method . . . . . . . . . . . .
4.2.
Phase inversion temperature (PIT) method . . . . . . . . . . . . . . . .
The generation of nanoparticles from the nano-emulsion template . . . . . . . .
5.1.
On nanoparticle denition . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Polymeric nanospheres . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.1.
in situ polymerization . . . . . . . . . . . . . . . . . . . . .
5.2.2.
Formulations with preformed polymers . . . . . . . . . . . . .
5.3.
Solid lipid nanoparticles (SLNs) . . . . . . . . . . . . . . . . . . . . .
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Corresponding author. Inserm U646, Ingnierie de la vectorisation particulaire, 10 rue A. Boquel, F-49100 Angers, France; University of Angers, F-49100 Angers, France.
E-mail addresses: nicolas.anton@espci.fr (N. Anton), jean-pierre.benoit@univ-angers.fr (J.-P. Benoit), patrick.saulnier@univ-angers.fr (P. Saulnier).
0168-3659/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2008.02.007
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N. Anton et al. / Journal of Controlled Release 128 (2008) 185199
5.4.
Nanocapsules (NC) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.1.
Polymeric nanocapsules: in situ interfacial polymer synthesis . . .
5.4.2.
Polymeric nanocapsules: nanoprecipitation of preformed polymers .
5.4.3.
Lipid nanocapsules (LNC) . . . . . . . . . . . . . . . . . . . .
6.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Over the past few decades, extensive research has been done on the
study of nanoparticle generating processes. Owing to the variety of the
application elds of such colloidal objects (from nanomedicine, drug
delivery and cosmetics, to printing ink or petroleum sciences...), and as
existing nanoparticles are now innumerable, a thorough knowledge of
the formulating processes (and their potentialities) is essential in
order to achieve the given purposes and needs for research. Likewise,
in that most nanoparticle formulations are effectively based on
nanometric-scaled emulsions, so-called nano-emulsions, the study
of nanoparticle formulation has to include knowledge of nanoemulsion formation governing phenomena. Nano-emulsions are
nanometric-sized emulsions, typically exhibiting diameters of up to
500 nm. Nano-emulsions are also frequently known as miniemulsions, ne-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 consequence of signicant steric
stabilization between droplets, which goes to explain why the
Ostwald ripening is the only adapted droplet destabilization process
(detailed below). It follows therefore that nano-emulsion systems
can be regarded as a template for nanoparticle generation, even if
these two steps can often be combined into one. Therefore, the
innumerable variants of nanoparticle formulation are mainly based
on three different groups of methods for the generation of nanoemulsions, i.e. high-energy methods, the low-energy spontaneous
emulsication method, and the low-energy phase inversion temperature (PIT) method.
The different kinds, or morphologies, of the nanoparticles
generated, can be broken down into polymeric nanospheres, solid
lipid nanoparticles (SLNs), or polymeric or lipid nanocapsules. The
links between nano-emulsion formulation processes and nanoparticle
morphology are neither obvious nor systematic and should be tackled
with particular detachment: Such is the purpose of the current review.
Indeed, by establishing a link between the formulation of nanoemulsions and nanoparticle generation, our intention has been to
highlight the extent to which experimental processes can be adapted
to given specications.
In the rst part, the nano-emulsion template is presented by a
thorough description of the mechanisms and phenomena governing
its formation, including a comprehensive review of the different
existing methods. Special attention has been given to the low-energy
processes, since they constitute a privileged way to prevent the
potential degradation of encapsulated molecules during processing
and are also important for, (among others) energy yields in the case of
industrial scale-up.
In the second part, nanoparticle formulation processes are
reviewed with regard to the place of the nano-emulsion generation
methods (amongst others) that they are based on. Furthermore, their
potential adaptation to other nano-emulsion forming methods is
discussed and their potential signicance highlighted. Accordingly, the
choice of the energy-type method, the use of organic solvent in the
formulation, the choice of the polymer according to its biocompatibility or biodegradability, even the choice of an in situ synthesis during
nanoparticle generation, as well as the use of preformed polymers and
nally the choice of nanoparticle morphology, all of these parameters
must be thoroughly considered and closely adapted to the therapeutic
objectives.
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194
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196
196
In the case of polymers in situ synthesized with polymeric

nanospheres of nanocapsule creation, the potential interactions
between drugs (or active molecules to be encapsulated) and the
polymers being formed must be systematically considered. Indeed,
covalent bonds may be established between drugs and polymers, and
thus their potential mutual reactivity must be taken into account in
the choice of monomers and nanoparticle-generating systems.
The last remark concerns nano-emulsion and nanoparticle characterization. Given the numerous papers and reviews (e.g. [13])
where this aspect has been covered in great detail, we have not dealt
with it in the present review. The droplet size distribution is essentially
disclosed by dynamic light scattering (DLS), but also by transmission
electronic microscopy (TEM) coupled with negative staining, or cryoTEM, freeze-fracturing followed by replication plus TEM, or capillary
hydrodynamic fractionation (CHDF), etc. More detailed information on
surface particle characterization may be obtained by surface potential
characterization (such as [13] potential) and an indication of the
nanoparticle surface morphology may be highlighted by specic
approximations of electrophoretic models (e.g. soft particle model [4]).
Finally, small-angle neutron scattering (SANS) or small-angle X-ray
scattering (SAXS) may be useful for investigating the internal
morphology of such colloidal objects.
2. The great stability of nano-emulsions
The main particularity of nano-emulsions, making them prime
candidates for nanoparticle engineering, is their great stability of droplet
suspension. A kinetic stability that lasts for months, stability against
dilution or even against temperature changes, totally unlike the
(thermodynamically stable) microemulsions. Emulsions are thermodynamically unstable systems, due to the free energy of emulsion
formation (Gf) greater than zero. The large positive interfacial energy
term (A) outweighs the entropy of droplet formation (TSf), also
positive. The terms and A respectively represent the surface tension
and the surface area gained with emulsication. Emulsion instability is
therefore induced by the positive sign of Gf (Eq. (1)).
DGf gDA TDSf
Accordingly, 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 mechanisms: (i) Flocculation followed mostly by
coalescence, and (ii) Ostwald ripening.
In nano-emulsion systems, occulation is naturally prevented by
steric stabilization, essentially due to the sub-micrometric droplet
size. In short [57], when interfacial droplet layers overlap, steric
repulsion occurs,from two main origins. The rst one is the unfavorable mixing of the stabilizing chain of the adsorbed layer, depending on
the interfacial density, interfacial layer thickness , and FloryHuggins
parameter 1,2 (which reects the interactions between the interfacial
layer and solvent). The second one is the reduction of the congurational entropy, due to the bending stress of the chains, which occurs
when inter-droplet distance h becomes lower than .
Generally, the sum of the energies of interaction UT adopts a typical
shape of systems wherein molecules repel and particles attract each
other, showing a weak minimum, around h= 2, and a very rapid
increase below this value (see Fig. 1 for illustration). The depth of the
Fig. 1. Diagram of the inuence of emulsion droplet radius on steric stabilization.
minimum |U0| will induce predispositions for coagulate in the colloidal

suspension, that is to say, it is intimately linked to the stability of the
suspension. |U0| is shown to be dependent on the particle radius r, the
Hamaker constant A, and the adsorbed layer thickness , with the result
that the higher the /r ratio, the lower the value of |U0|. Now, in the case
of nano-emulsion droplets, /r becomes very high in comparison with
macro-emulsions, which in the end totally inhibits its ability to coagulate. On the other hand, it is worth noting that the small droplet sizes
also induce stabilization against sedimentation or creaming, in so far as
the droplets are solely under the inuence of the Brownian motion.
Taking all this into account, 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 [8] as Ostwald
ripening in emulsions. At theorigin 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 of 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. As an
illustration and under the assumption that only one component
composes the dispersed phase, the solubility, C(r), of the dispersed
material throughout the dispersion medium is expressed as a function
of the droplet radius r, from the Kelvin equation [9], Eq. (2),

2gM
C r Cl exp
qRTr
where C is the bulk solubility of the dispersed phase, M its molar

mass, and its density. In most studies, the follow-up of Ostwald
ripening as the temporal evolution of the droplet diameter still
remains well tted, even under the approximations involved in Eq. (2).
In addition, 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 and Slezov [10,11] and Wagner [12], the socalled LSW theory. Besides the consideration of Eq. (2), the diffusion of
dispersed materials through the continuous medium is assumed to be
diffusion-controlled, i.e. crossing the interface with ease. Details on
LSW theories are fully developed and discussed in the literature
[13,14,8] leading to the commonly used expression of the ageing rate,
, in Eq. (3),
x
drc3 8DCl gM
dt
9q2 RT
where rc is the critical radius of the system at any given time, at the
frontier between the growth and decrease of the droplets. Conse-
187
quently, Ostwald ripening is reected by a linear relationship between

the cube radius and time.
In processes involved in nanoparticle engineering, i.e. for multicomponent emulsion droplets, by adding monomer, polymer, or simply
surfactant or co-surfactant, the above approximation is surpassed. The
rate of ripening can be reduced by several orders of magnitude when the
additive has a substantially lower solubility in the bulk phase than the
main component of the droplet. This phenomenon has been widely
studied [1522], since it appears to be an efcient method to reduce the
Ostwald ripening rate, even when using small amounts of additives. In
short, it is explained by the difference of solubility in the continuous
phase between the dispersed phase noted (1) and the additive (2), less
soluble in this example. The rst step remains similar to the ripening
without additives, since only the component (1) diffuses from the
smaller to the larger droplets, due to the higher chemical potential of the
materials within the smaller drops. Gradually, the chemical potential in
the larger droplets increases due to the presence of the component (2),
until the diffusion process of (1) is stopped. Equilibrium is reached
between the two opposing effects and the limiting process becomes the
diffusion of the less soluble additive (2), signicantly reducing the
ripening rate and the nano-emulsion destabilization.
A nal remark, which may be of importance here, concerns the
inuence on the nano-emulsion destabilization of layer density and
structure in the interfacial zone. Indeed, up to now it has been
considered that Ostwald ripening is a diffusion-controlled process, but
this assumption does not take into account the fact that surfactants,
polymeric emulsiers or stabilizers can create a thick steric barrier at
the droplet interface [23,24]. As a consequence, the diffusion of the
inner material of the droplets may be slowed down, reducing the
ripening rate.. The substantial difference in stability between nanoemulsions and nanocapsules for instance, appears essentially from
such details.
3. High-energy emulsication methods
In this section, we will consider emulsication methods involving
high (mechanical) energy used in the formation of nano-emulsion,
that is to say, the use of devices to force the creation of huge interfacial
areas. Nano-emulsion generation is very commonly performed with
such high-energy emulsication methods, particularly exploited in
nano-emulsion polymerization [1,2]. The formation of such nanometric-scaled droplets is governed by directly controllable formulation parameters such as the quantity of energy, amount of surfactant
and nature of the components, unlike the low-energy methods
(presented in the following sections), governed by the intrinsic
physicochemical properties and behavior of the systems. It follows
therefore that high-energy methods present natural predispositions
to preserve the formation processes of nano-emulsions droplets,
against even the slightest potential modications of the formulation
like the addition of monomer, initiator, surfactant, etc.
3.1. Devices and processes
The mechanical processes generating nanometric emulsions
include, as a rst step of the drop creation, the deformation and
disruption of macrometric initial droplets, followed by the surfactant
adsorption at their interface to insure the steric stabilization discussed
above. The challenge of these mechanical nano-emulsication
methods is to combine these two steps, in order to allow and optimize
nano-emulsion generation. Three main groups of devices are used in
the literature: The rotor/stator devices, which appear in the rst
articles on nano-emulsions, and then high-efciency devices, including ultrasound generators and high-pressure homogenizers.
Rotor/stator type apparatuses, such as Omni-mixerpsy or Ultraturraxpsy, do not provide a good dispersion in terms of droplet size
and monodispersity [25] in comparison with the nano-emulsions
188
generated by thetwo others kinds of devices (and also with the lowenergy methods). Indeed, the energy provided is mostly dissipated,
generating heat and being wasted in viscous friction [26,27]. Therefore, the additional free energy Gf necessary to create the huge
interfacial area of nano-emulsions is not obtained.
Nano-emulsions generated by soniers are generally attributed to
a mechanism of cavitation [28,29], but are not as yet understood well
enough. The ultrasound waves in liquid macroscopic dispersion, result
in a succession of mechanical depressions and compressions,
generating cavitation bubbles, which tend irremediably to implode.
Subsequently, this shock provides sufcient energy locally to increase
A corresponding to nanometric-scaled droplets. Efciency of nanoemulsication by sonication (considered as the nal size of the nanoemulsion droplets as well as the time needed to attain this asymptotic
size), depends both on the composition of the emulsion and the power
device. Indeed, the addition of surfactants and/or monomers has been
shown an important parameter to efciently reduce droplet sizes [30].
Sonication is thus the most popular way to produce nano-emulsions
and nanoparticles for research purposes. It does not, however, appear
practical for use on an industrial scale, for which high-pressure [31]
devices (and low-energy methods) are often preferred.
High-pressure homogenizers, generally Microuidizer or Manton
Gaulin devices, are designed in order to force macro-emulsions to pass
through narrow gaps, by imposing high pressures. The uid
accelerates dramatically,reaching, in the microchannels of Microuidizers [2] for instance, a velocity of around 300 ms 1. As a result,
shear, impact and cavitation forces are applied on very small volumes
and generate nano-scaled nano-emulsion droplets (closely related to
the phenomena involved in the use of soniers).
3.2. The choice of surfactants, monomers, aqueous and oily phases
The nature and amount of the surfactant, monomer or hydrophobe
used in the formulation completely determine the size distribution,
structure and stability of the resulting nano-emulsions and nanoparticles. Thus, the different components are chosen in function of the
formulation strategies undertaken. For instance, Landfester [3236]
presented from nano-emulsions (by sonication), (i) the formulation of
inorganic particles by playing on thephysicochemical properties of
molten salt droplets, (ii) the formulation of polymeric nanospheres by
in situ polymer synthesis within nano-emulsion droplets, (iii) the
combination of both these types of technology to provide hybrid
nanoparticles, and nally (iv) the use of oil as a hydrophobe to
generate core-shell nanocapsules, by polymer-specic synthesis and
segregation to the oil/water region [35], or by interfacial nanoprecipitation [36]. As regards high-energy methods for generating
nanoparticles, the literature extensively reports comparisons between
the different devices, hydrophobes, surfactants and monomers, for
instance in Ref. [1]. In the current paper, by describing the various
reported strategies for generating nanoparticles and nanocapsules, we
draw a parallel between high-energy technologies and those involving
only low-energy methods. Our purpose here is thus to propose further
insights into the possible transpositions from high-energy to lowenergy nanoparticle-generating methods.
3.3. On the potentialities, advantages and disadvantages of high-energy
methods
In general, high-energy nano-emulsication methods present a
good potential for polymeric nanoparticle generation, since the
formulation parameters are directly controllable. Thus the addition
of monomers, initiators, or encapsulating molecules appears not to
inuence the emulsication process, governed by the high shear
processes. If anything, it may be and additional molecules to be
encapsulated, monomers, initiators, or stabilizing agents that interfere
with the emulsication process, unlike for the low-energy methods in
which nano-emulsication is totally governed by the physicochemical

behaviors of the surfactants. However, when the purpose of the experiment is the encapsulation of fragile molecules such as peptides,
proteins, ornucleic acid, often encountered in pharmaceutical or medical
research, high-energy methods may give rise to drug degradation,
denaturation or activity loss during processing. Moreover, in the case of
an industrial scale-up, it is of importance to consider the energetic yield,
which is incomparable between high and low-energy methods [37,7].
This is especially true for sonication, since during the emulsication
process, only the near-volume of the sonier nip is affected by ultrasonic
waves, and for high volumes, a weak additional mechanical stirring is
needed to homogenize the sizes and generate nano-emulsions. In
concrete terms, the emulsication time (i.e. energy) to provide
homogeneous nano-emulsions increases in function of the volume to
nano-emulsify, which is fundamentally not the case for all low-energy
methods.
4. Low-energy emulsication methods
Let us now move on to nano-emulsication methods, involving only
a low quantity of applied energy to generate nano-emulsions. Nanometric-scaled emulsion droplets may be obtained by diverting the
intrinsic physicochemical properties of the surfactants, co-surfactants
and excipients composing the formulation Two groups of methods are
proposed in the literature and developed below: (i) The rst one
describes emulsication as a spontaneous phenomenon [3847], which
uses the rapid diffusion of water-soluble solvent, solubilized rst in the
organic phase, moving towards the aqueous one when the two phases
are mixed. Among the works on spontaneous emulsication, the literature emphasizes the solvent displacement method [451], also called
the Ouzo effect [52], which consists in nano-emulsion formulation due to
the specic and very rapid diffusion of an organic solvent (e.g. acetone,
ethanol...) from the oily phase to the aqueous one. In theory,
the spontaneous nano-emulsication process can provide as much oilin-water as water-in-oil nano-emulsions, but the majority of the
reported studies concern o/w generation. (ii) Secondly, the so-called
phase inversion temperature (PIT) method [5364], which uses the
specic properties of polyethoxylated surfactants to modify their
partitioning coefcient as a function of the temperature, and leads to
the creation of bicontinuous systems when the temperature is close to
the PIT, broken-up to generate nano-emulsions. Practically, it leads to o/
w nano-emulsions.
4.1. Spontaneous nano-emulsication
4.1.1. The diffusion mechanism and diffusion path theory
This section describes the main principles of spontaneous emulsication, underlining the governing phenomena and mechanisms and
considering the suitability of this method for nanoparticle generation.
It is interesting to note rst that the spontaneous features of such
phenomena are simply the results of the initial non-equilibrium states
of the two bulk liquids when they are brought into contact without
stirring. It is only under specic conditions that spontaneous emulsication occurs and in some cases, nanometric-scaled droplets are
generated. The spontaneous emulsication process itself increases
entropy and thus decreases the Gibbs free energy of the system [65].
The establishment of phase diagrams, as well as video-microscopy
experiments is essential for evidencing the conditions related to
spontaneous emulsication [45]. Evolution of the system is basically
promoted by diffusion of a solute into the phase in which it has greater
solubility. Thus, spontaneous emulsication behaviors can potentially
be predicted by following the diffusion pathway within the phase
diagram.. The different cases are described below, and of course, this
theory has been widely supported by experiments reported in the
literature. The video-microscopy technique was introduced to observe
the behavior of the liquid at the interface of the two immiscible phases
brought into contact without stirring. Thus, the regions of spontaneous emulsication may be disclosed, as well as their respective
location towards the interface.
The source of energy of spontaneous emulsication reportedly
stemmed mainly from interfacial turbulences, closely related to the
surface tension gradient induced by the diffusion of solutes between
two phases. Likewise, the interfaces are subject to capillary waves
from thermal origins, gradually amplied as the surface tension
decreases [45]. The drops are created as a result of sufciently large
interfacial corrugations, similar to the dynamic behavior of the
frontier between microemulsion and the bulk phase in multi-phase
equilibrium systems, i.e. a continuous coalescence and break-off of
emulsion droplets [66]. Such a phenomenon has been called dispersion, spontaneously increasing the entropy and decreasing the Gibbs
free energy of the system. The other (complementary) spontaneous
emulsication mechanism, known as condensation, is also assumed to
be intimately linked to the uctuation of the interfacial amphiphile
concentration. Owing to theregion of local supersaturation (overconcentration of surfactant at interface) induced by the diffusion
process, spontaneous interfacial expansion takes place, resulting in
the nucleation and growth of drops. These conditions appear
analogous to the system behavior in the two-phase microemulsion
regions, for instance, where drops are continuously nucleated, grow,
by similar spontaneous phenomena and disappear by coalescing
(maintaining the two-phase equilibrium).
The theoretical mechanism was proposed by Ruschak and Miller
[67], and has since been broadly corroborated by experiments and by
the literature. These authors have put forward their theory from the
solution of the diffusion equations for thesemi-innite phase, and
under some assumptions detailed below. The variation of composition
in each phase (aqueous and oily) is directly represented on the ternary
phase diagram by straight lines, from the semi-innite reservoirs, to
the interfacial concentration. Such a schematic representation of the
evolution of the concentration within each phase is called a diffusion
path. This model, however, is based on the following assumptions, (i)
that non-equilibrium phases are brought into contact, and eventually
some species should diffuse into the opposite phase, (ii) that for semi
innite phases, the theory is limited to time, for which some
proportions of both contacted phases retain their initial composition,
(iii) that the diffusion coefcients of all components are equal in a
phase, which is precisely the condition for representing the diffusion
paths as straight lines, and nally (iv) that the interface presents a
local equilibrium. Thus, spontaneous emulsication only depends on
the diffusion path with regards to the equilibrium phase diagram. On
the other hand, stirring the two phases brought into contact has no
inuence on the own mechanism of spontaneous emulsication, even
though it increases the rate of emulsication by increasing the
interfacial area A.
The rst illustration is provided by the study of the water/alcohol/
oil ternary system, presented in Fig. 2 inspired from Ref. [45], where a
pure water {w} phase (point 1) is brought into contact with an alcohol
plusoil {a + o} phase (point 4). The local equilibrium at the interface is
shown via the dotted segment (23) at the frontier of the two-phase
equilibrium region (in the phase diagram). Depending on the initial
composition and on the nature of the alcohol, the location of the
interfacial equilibrium appears to condition spontaneous emulsication, as illustrated by the difference between the Fig. 2a and b. Actually,
it allows the diffusion path to cross the two-phase microemulsion
equilibrium region (i.e. spontaneous emulsication (SE) region). This
may indicate that the maximum intensity of spontaneous emulsication is not necessarily near the interface, but at the maximum depth
within the SE region. Hence, the deeper the diffusion path within the
SE region, the higher the interfacial turbulences and dispersion,
diffusion or condensation phenomena. It is therefore easy to imagine a
bridge with the droplet size of the forming emulsion intimately linked
to the intensity of the spontaneous phenomenon. Nano-emulsion
189
Fig. 2. Diffusion path in a typical water/alcohol/oil system. Segment (12), diffusion

path of the aqueous phase. Segment (34), diffusion path of the oily phase. Segment (2
3), interfacial local equilibrium. (a) Case where no spontaneous emulsication occurs.
(b) Spontaneous emulsication occurs when the diffusion path crosses the two-phase
equilibrium region, segment (1'2).
droplets appear to be formed in this way, with the use of a high

quantity of diffusing solvent in the oily phase [4851]. Bouchemal et al.
[51], for instance, proposed a study on the optimization of the solvent
displacement method formulating nano-emulsions for cosmetic and
pharmaceutical applications, in which the overall solvent/oil ratio was
around 0.01. Likewise, these authors disclosed the important inuence
on the nano-emulsication process, of oil viscosity, surfactant HLB, and
the nature of the solvent (also as a function of its toxic potential) and
miscibility with water.
Of course, in all these optimized systems for nano-emulsion (and
nanoparticle) formulation by such spontaneous emulsication methods, the systems are more complex than the three-component model
described above. In fact, this model only accounts for the droplet
formation, nonetheless unstable and highly subject to destabilization
after formation (even the nanometric-scaled droplets). Therefore,
after creation, the newly formed interfaces have to be stabilized by
surfactant adsorption. Hence, the initial phase diagrams are modied
accordingly, and a more complex diffusion path has to be considered
between the different phases potentially formed in the interfacial
region. It is to be noted that the presence of liquid crystalline (LC)
phases are acknowledged as playing a decisive role in these
spontaneous-forming formulations. Two examples presenting the
spontaneous emulsication of such quaternary systems are proposed
in Fig. 3, for both surfactants having a negative and positive Winsor R
ratio. R is dened as the ratio between the inter-molecular interactions per unit interfacial area, surfactantoil/surfactantwater [68].
In the case shown in Fig. 3a, of the rather hydrophilic surfactant
(R b 1), the formation of the LC phase in the semi-innite aqueous one
(point 1) appears in equilibrium with a pure aqueous sub-phase,
190
established microemulsion structures, the surfactant hydration is

potentially changed, as well as their afnity for the aqueous phase, and
thus instabilities are created in the microemulsion network, resulting
in its break-up into nano-emulsions. The addition of water {w}, for
instance, in oil plus surfactant {o + s} continuous medium [7880] gives
rise to weak oil/water interfacial curvature uctuations, thereby
inducing the system to fall into the thermodynamically favorable
state of nano-emulsion at this time. Of course after formation, other
destabilizing mechanisms affect the nano-emulsion, such as Ostwald
ripening, mentioned in the previous sections.
Once again, in order to determine suitable conditions for generating
nano-emulsion, the equilibrium phase diagram needs to be carefully
studied and the phases analyzed and characterized. This time, it is the
dilution pathway which indicates the best conditions to form nanoemulsions, i.e. the conditions for which the emulsion droplets formed
are the smallest. The main results appear to show that nanometric-sized
emulsion droplets are formed when the whole phase to be dispersed
appears solubilizedin the bicontinuous system in the phase diagram
[59,60,80]. These phases are reported to be either bicontinuous
microemulsion, or lamellar liquid crystals L. To nish, regarding the
formulation of nanoparticles from nano-emulsions, this method is very
similar to that of solvent diffusion, owing to the fact that the process may
be very dependent and sensitive to phase behavior. Thus, adapting the
process to some formulation specications (addition of monomers,
initiators, drugs to be encapsulated...) is likely to modify the phase
diagram, and thus potentially disrupt the nano-emulsion formation
process.
4.2. Phase inversion temperature (PIT) method
Fig. 3. Diffusion path in a typical water/alcohol+surfactant/oil system (see details in the

text).
segment (23), before establishing the local interface equilibrium with

the oil-rich phase, segment (45). The diffusion path is simpler and
presents the spontaneous emulsication of hydrophilic droplets in the
oil segment (55'). Symmetric phenomena are also conceivable in the
case where the rather lipophilic surfactant (R N 1) is used, presented in
Fig. 3b, where an isotropic phase is generally formed in the surfactant/
oil-rich region. Subsequently, spontaneous emulsication of oily
droplets in water arises within the segment (1'2).
The study of nano-emulsion formation using this method implies a
thorough establishment of the phase diagram to disclose the potential
feasibility diagram and optimization. In this context, it follows that the
potential inuence of additional components (like monomers and
polymers, whether or not they are neutral in the formulation), need to
be investigated, both on the phase diagrams and on the diffusion
pathway. This may, to some extent imply restrictions in terms of ease
of handling, modifying and adapting the nanoparticle formulation to
the given needs. However, the generation of nanocapsules and
nanospheres by nanoprecipitation or in situ polymerization, from
nano-emulsions using the solvent displacement method, has provided
a great number of examples developed below, e.g. the works of Fessi
et al. [6973].
4.1.2. The emulsion inversion point (EIP) method
Another spontaneous emulsication method, known as the emulsion inversion point method, has been reported in numerous works. At
a constant temperature, it consists in diverting the intrinsic features of
thermodynamically stable microemulsions D or Liquid crystals LC to
be nano-structured by a progressive dilution with water or oil, in order
to create thermodynamically unstable but kinetically stable, respectively direct or inverse nano-emulsions [7482]. In fact, these authors
explain that by slightly changing the water or oil proportion within
The phase inversion temperature (PIT) method is particularly

interesting since it is an organic, solvent-free and low-energy method.
The latter two experimental conditions are potentially the most
suitable for application in the elds of nano-medicine, pharmaceutical
sciences and cosmetics, to prevent the drug to be encapsulated from
degradation during processing. Likewise, since the process is relatively
simple and low-energy consuming, it allows easy industrial scale-up.
The PIT concept was introduced in the last decade by Shinoda and Saito
[53,54], using the specic ability of surfactants, usually nonionic, (NS)
such as polyethoxylated surfactants, to modify their afnities for water
and oil in function of the temperature, andtherefore to undergo a phase
inversion. Indeed, the so-called transitional emulsion phase inversion
occurs when, at xed composition, the relative afnity of the
surfactant for the different phases is changed, resulting in a gradual
modication of the temperature. For example, an oil-in-water (o/w)
emulsion is subjected to a phase inversion, giving rise to a water-in-oil
(w/o) one, when the temperature rises. Within the transitional region
between macro-emulsions, i.e. for the temperatures at which the
nonionic surfactants exhibit a similar afnity for the two immiscible
phases, the ternary system shows an ultralow interfacial tension and
curvature, typically creating microemulsions, bicontinuous and nanoscaled systems [56,8389]. Therefore, the PIT method consists in
suddenly breaking-up the chosen bicontinuous microemulsion maintained at the PIT, by a rapid cooling [59,62,64] or by a sudden dilution in
water or oil [57,61,90,91]. Nano-emulsions are immediately generated.
These bi-continuous systems have been thoroughly and widely
characterized by establishing phase diagrams at equilibrium and formulation maps under dynamic conditions. The inuence of the formulation (electrolyte concentration, temperature...) and composition
parameters (surfactant amount or water/oil weight ratio, WOR = 100
water / (water + oil)), has been largely reported on the potentialities to
formulate nanometric-scaled emulsion droplets [57,59,60,6264,83,92].
During the emulsion inversion phenomena, the respective afnity
of the NS for both immiscible phases is given by the difference
between the chemical potentials of the surfactants in each phase.
According to the physicochemical denition of De Donder, Eq. (4), the
surfactant afnity difference (SAD) is dened with Eq. (5), at the

physicochemical equilibrium, considering the activity coefcients
close to the unity. It follows that the SAD is closely linked to the NS
partitioning coefcient.
Ai ABi RTlnai Ci
i is the chemical potential of the NS in phase i, i are the standards, a

the activity coefcients, and C the surfactant concentration.

Coil
SAD ABwater ABoil RTln
Cwater
In the case of ionic surfactants, the emulsion inversion corresponds to

the SAD= 0, but this is not the case for nonionic surfactants and corresponds to a given reference noted SADref. Hence, this deviation with
regards to the optimum formulation was dened with an adimensional
variable [9397], known as the hydrophilic lipophilic deviation (HLD),
given for ionic and nonionic surfactants, and for a hydrocarbon n-alcane
oily phase by the following Eqs. (6) and (7), respectively.
HLD SAD=RT r lnS kACN tDT aA

HLD SAD SADref =RT
a EON bS kACN tDT aA
where EON is the number of ethylene oxide groups for NS, S is the weight
percentage of electrolytes in the aqueous phase, ACN the amount of
carbon numbers of the n-alcane composing the oily phase, T the
temperature difference from the reference temperature (25 C), A the
weight percentage of alcohol potentially added (not necessary for the
PIT method), , , k, t parameters in function of the used surfactant, a a
constant given from the types of alcohol and surfactant, and nally b a
constant function of the nature of the added electrolytes.
Thus, the correlation between the HLD empirical expressions (6)
and (7), and the SAD denition (5), gives the link between the
temperature variation and the amphiphile partitioning coefcient
[98], and thereby the surfactant behavior regarding the water/oil
interface when using the PIT method. Hence, when NS is mainly used
for generating nano-emulsions by the PIT method, formulationcomposition maps are typically built, as reported in Fig. 4a. Under
constant stirring and for a xed surfactant amount in the formulation,
the emulsion gradually undergoes a phase inversion, as the HLD is
changed by temperature variation. According to the HDL variation, at a
constant WOR, the process is called transitional phase inversion.
Moreover, for the lowest and highest WOR, emulsion inversion
does not occur, due to the excessively rich water and oil regions. The
emulsion morphology changes from the normal to the abnormal
emulsion types, to form simple or multiple emulsions, respectively in
191
accordance with Bancroft's rule and not. The illustration is provided in

Fig. 4a with the transitions between (i) o/w and w/o/w, and (ii) w/o and
o/w/o. In this case, even if the emulsion does not clearly exhibit a phase
inversion, conditions are still suitable to perform the PIT method,
where particular microemulsion structures can form at HLD = 0, thus
also leading to the generation of nano-emulsions, see for instance Ref.
[99].
The study of emulsion inversion involving only the variation of
WOR at a constant HLD is known as catastrophic phase inversion, and
has been extensively studied [100105]. It regards the transitions
(horizontal pathways in Fig. 4a) between (i) w/o and w/o/w for HLD N 0
and (ii) o/w and o/w/o for HLD b 0, therefore this phenomenon is
basically not included in the PIT method.
Fig. 4b and c show the corresponding equilibrium phase diagrams,
exhibiting the different thermodynamic equilibriums Winsor I to IV in
function of the temperature. Kahlweit-sh diagram [57,60,63,64,83]
nally traces as well, such an evolution of the system morphology. For
instance, in the case of WOR = 50, a rise in temperature crosses the sh
body in Fig. 4b, and crosses the caudal n in Fig. 4c. According to the
comprehensive study proposed by Morales et al. [60], optimum conditions for nano-emulsion generation are closely linked to the ability of
the microemulsion, precisely at the PIT, to solubilize all thephases to be
dispersed. Indeed in most cases, this corresponds to the Winsor II and
IV microemulsion formation, when the system is maintained at the PIT.
In the basic cases of Fig. 4b and c, the nano-emulsions will be generated
from the systems exhibiting W IV equilibrium microemulsions, or
potentially W IV + LC, at the PIT, essentially for systems with higher
surfactant amounts, Fig. 4c. Finally, the process implied in the PIT
method of generating nano-emulsions, which suddenly breaks-up the
microemulsions, can essentially be considered as irreversible since the
nano-suspension formed is kinetically stable. It appears to go beyond
the connes of the studied ternary system from the phase diagrams
and formulation-composition maps, to create a kinetically stable nanoemulsion state. Of course, it should only be interpreted as a quasistable state, even if it is stable for months, and when achieved, the
destabilization will provide the phase equilibrium considered above.
Establishing the phase diagram is a requisite preliminary study in
order to grasp and analyze the conditions suitable for nano-emulsion
formulation.
In this context, the link between EIP and PIT methods is claried,
highlighting the latter (PIT) as the one providing suitable experimental conditions to attain the nanometric structuring of the ternary
system (in function of the formulation variables), similar to structures
already established with mixing the components using the EIP
method at room temperature (see above).
Thus, the PIT method appears exclusively governed by the PEOsurfactant phase behavior with regards to the formulation variables, and
particularly the temperature. In this context, it is totally conceivable to
Fig. 4. (a) Typical formulation-composition map for water/nonionic surfactant/oil system, showing the emulsion inversion zones. Typical equilibrium phase diagrams for the same
system, HLD as a function of the composition, (b) for low surfactant amounts, and (c) for high surfactant amounts. The frontier between both behaviors is roughly dened at 10 wt.%.
192
add neutral components to the formulation, which inuence neither the

system phase behavior, nor the forming domain of nano-emulsions. For
instance, some formulations based on the PIT method [99,106109],
include the addition of neutral amphiphiles for the formulation (such as
phospholipids), in order to play on the nal nanoparticle properties and
structure: In these examples, the presence of phospholipids increases
the lipid nanocapsule stability and acts as a framework on the nal shell
structure.
To summarize, the phase inversion mechanisms of emulsions stabilized by PEO-surfactants, appear to establish close links between the NS
partitioning coefcients and the temperature variation. Furthermore,
many works elucidate this solubilizing behavior of NS moving towards
the aqueous phase as the consequence of the hydration state of the
surfactant ethylene oxide (EO) chains [110117]. In concrete terms, the
water solubility and self association of NS are totally governed by the
structuring state of water molecules, associated by hydrogen bonds into
ickering clusters, wrapping the surfactant polar head. This well documented, volumic surfactant behavior was transposed at the water/oil
interface, showing the analogy between cloud point and PIT [118,119],
and more recently [120] a comprehensive study presented this molecular behavior of structured water, within the inversion of emulsions.
In these works, NS behavior appears related to a salting-out effect, itself
induced by the formulation variables and by the temperature pathway
imposed on the system.
To conclude, the low-energy and solvent-free PIT method generally
appears relatively adaptable, easy to handle. The incorporation of
additional molecules (to a certain extent of course) in most cases has
been shown to be neutral for the formulation, or only slightly inuence
the global trends (weak PIT shift, weak modication of the nanoemulsion droplet size...), but not the global phenomena governing the
process. It follows then that the potentiality of the PIT method to generate
nanoparticles at a low energetic cost, free from the toxicity of organic
solvent, with a potentially low amount of surfactant (e.g. at 5 wt.% in [90]),
makes such a process essentially one of the most appealing methods.
However, as presented below, the literature mainly reports formulation
of nanoparticles based on nano-emulsion templates formed by highenergy methods and by the solvent displacement method. Transposition
and adaptation to the low-energy PIT method thus remains anecdotal.
5. The generation of nanoparticles from the nano-emulsion
template
5.1. On nanoparticle denition
Nanoparticles are frequently dened [121] as solid colloidal
particles ranging in size from 10 nm to 1 m. Nanoparticles are built
from macromolecular and/or molecular assemblies, in which the active
principle is dissolved, entrapped, encapsulated, or even adsorbed or
attached to the external interface. Thus, one fundamental advantage of
nanoparticles with regard to other colloidal drug delivery systems
(liposomes, niosomes, microemulsions etc.) and a fortiori to nanoemulsions, is their great kinetic stability and rigid morphology.
Therefore, nanoparticles can be divided into two main families:
nanospheres, which have a homogeneous structure in the whole
particle, and nanocapsules, which exhibit a typical core-shell structure.
A main challenge of the formulation of nanoparticles isadapting the
choice of their own structure to the nal aims of drug delivery:
Biocompatibility of the polymer, physicochemical properties of the
drug, and therapeutic goals. Hence, the following sections will focus on
polymeric or lipid nanospheres and nanocapsules, thus presenting (i) a
general overview on the different formulation methods, the strategies
undertaken and the process-governing phenomena, (ii) a few illustrations by way of a non-exhaustive list of examples, and (iii) the extent to
which the high- and low-energy methods are used or could be used for
similar results. Finally, the partitioning coefcient will govern the
choice of the formulation, and even the choice of particle morphology.
5.2. Polymeric nanospheres

5.2.1. in situ polymerization
The generation of polymeric nanospheres formed by in situ
polymerization is exclusively provided by the literature from nanoemulsions formed using high-energy methods. The chemical reactions
are principally described as the radical polymerization of droplets,
monomers are generally used as hydrophobe and specic surfactants are
chosen for generating nano-emulsions.Thus, the nano-emulsions are
composed of pure monomer droplets surrounded by the adsorbed,
stabilizing surfactants. Subsequently, polymerization starts in the
droplets themselves by the addition, in most cases, of the initiator in
the continuous phase, chosen for the hydrophobic phase in function of
its partial solubility. The initiation of droplet polymerization can also be
UV-induced [122], or ultrasonically induced [123], or even enzymeinduced [124]. As regards the prevalent way for initiating the polymerization process by adding initiator molecules, the widely accepted
mechanism [1,2] for successful nano-emulsion polymerization is
described as the droplet nucleation mechanism. It suggests that the
radicals enter each one of the monomer droplets taken as individual
reaction sites, and in that way, the particle number and size do not
change during polymerization. This is consistent with the trend, which
correlates the use of an oil-soluble initiator to the improvement in the
number of nucleated droplets. Initiator molecules can also be included
from the start within the nano-emulsion droplets. Polymerization is
then started up by raising the temperature, e.g. in Ref. [125].
Azobisisobutyronitrile (AIBN) is added in the oil prior to ultrasonication,
whereas potassium persulfate (KPS) is subsequently added in the
external phase for the same system. Inverse nano-emulsion polymerization is also conceivable [126128], and hydrophilic components may
thus be entrapped in the droplets.
This type of radical polymerization of nano-emulsions is frequently
chosen in the literature, and it is a non-negligible detail to consider,
since it could also be aggressive for the potentially encapsulated
fragile molecules. Antonietti and Landlester [1] briey reviewed their
works dealing with non-radical polymerization in nano-emulsions,
which include polyaddition [34,129], anionic polymerization [130], or
metal catalysed polymerization reactions [131]. For instance in
[34,129], they made polyurethane latex nanospheres by direct nanoemulsication (sonication) of a mixture of the two lipophilic species:
diisocyanate and activated diol, which react to eachother within the
nano-emulsion droplets. The successful reaction is due to the fact that
(i) the reactants have to exhibit low water solubility, (ii) polymerization kinetics is lower than emulsication time, (iii) the diisocyanate
has to show a higher reactivity with the other hydrophobe reactant
(diol) than with the water that forms the continuous phase. Finally, a
number of examples of nano-emulsion polymerizations using such
polymerization technology (radical or not) and high-energy methods,
are reported, for instance in the literature reviewed in great detail by
Antonietti and Landlester [1], or by Asua [2]. Such a listing is not our
purpose in the current paper.
Does this nanosphere technology seem adaptable to low-energy
methods? The literature provides few examples of nanosphere
formulation by in situ polymerization with low-energy methods. The
reason is simple: The hydrophobic phase in high-energy processes is
only composed of the monomer itself, whereas in low-energy
methods, it includes the oil. Theoretically speaking, adapting the
former to the latter requires similar behavior and interaction of
surfactants/oil and surfactants/hydrophobic monomer, which appears
relatively unlikely. Nevertheless, as proposed by Magdassi and
Spernath [132], it is only when such conditions are met that it may
be conceivable to perform the PIT nano-emulsication method easily,
even then only after thorough characterization of the systems. As a
result, monomer nano-emulsion droplets are formed and the
initiation process can be carried out until the formation of polymeric
nanospheres.
Finally, as reported in the works of Gallardo et al. [133], during the

formulation process of nanocapsules using a reactive alkylcyanoacrylate monomer and a solvent diffusion method, nanospheres can be
generated and favored by the use of a protic solvent [134]. These
objects should in fact have a mixed oil/polymer morphology, a porous
matrix and should potentially be surrounded by a polymer capsule.
5.2.2. Formulations with preformed polymers
This section presents formulations in which the macromolecules
are dissolved in the phase to be dispersed (mainly organic solvent). The
process irremediably involves the removal of the organic (and volatile)
solvent from the formulation and therefore polymer precipitation
within the organic phase template. Removing the solvent can be
performed by evaporation or diffusion shock. The main difference from
the previous process appears to be the fact that not only are synthetic
polymers used, but also natural macromolecules, such as chitosan,
polysaccharides, alginate, gelatin etc, hence increasing their biocompatibility with the potential therapeutic objectives. The second point
differing from the previous section is the number of examples in the
literature proposing the formulation of nanoparticles by low-energy
method, especially the solvent displacement method.
Let us rst consider a few examples of nanosphere formulation using
classical sonication methods. Polymeric nano-dispersion is created
simply by dissolving the polymer in the organic phase, and by nanoemulsication using an appropriate method of sonication. Then, the
polymer-containing nano-droplets formed are gradually evaporated to
generate nanoparticles, as described in Ref. [135]. Kietzke et al. [136]
proposed novel approaches working with semiconducting polymer
blends. Likewise, Yang et al. [137] used chloroform to dissolve a mainchain liquid crystalline polymer (MCLCP), and after a 5 min-sonication
phase to establish the nano-emulsion, the solvent was removed by
evaporation and a very stable suspension of nanoparticles was generated.Today, numerous examples are provided in the eld of drug
delivery and in the controlled release of lipophilic and hydrophilic
components [138] such as protein in PLGA nanospheres [139]. Perez et
al. [140] proposed an original method for encapsulating plasmid DNA
under PLA-PEG nanoparticles with a double emulsion-like structure (w/
o/w), by a two-step sonication, followed by solvent (ethyl acetate/
methylene chloride) evaporation in order to induce polymer precipitation. An alternative of their second sonication consists in a solvent
diffusion which creates the nal nanoparticles (i.e. spontaneous
emulsication by solvent displacement), inducing polymer precipitation: This could constitute a bridge to the low-energy, nanoparticlegenerating methods. We can also cite some works studying the
phenomena of crystallization and undercooling of poly(ethylene
oxide) [141,142] within the nano-emulsion droplets. Finally, working
on the intrinsic solubility and melting point (Tm) of a particular polymer
without the use of an organic solvent, Quaglia et al. [143] reported a
method known as melting/sonication (MS) which consists in the nanoemulsifation by sonication in water, of a uid, non water-miscible
copolymer at T N Tm. Cooling to room temperature then hardens the
copolymer. As a result, spherical and non-aggregated particles are
formed, and depending on the macromolecule properties, particular
structures (i.e. core-shell...) can be adopted by the nanospheres.
Most nanosphere engineering using preformed polymers
described in the literature, is shown to be performed by low-energy
spontaneous nano-emulsication, the so-called solvent displacement
method described above. The general idea is to consider the
macromolecules dissolved in organic solvent (plus possibly hydrophobes, like oil), as neutral for the spontaneous nano-emulsication
process. Thus, the solvent diffusion towards the aqueous phase,
generating nano-emulsions causes the polymer to precipitate uniformly within the nano-emulsion template. Classical examples are the
works of Fessi et al. [144147] or Leroux et al. [49,71,148]. Many
different polymers and organic solvents are commonly used. We can
nd for instance the couples {poly(D,L-lactic acid)/ethyl acetate} in
193
[145], {Eudragitpsy L/acetone, dimethyl sulfoxide, isopropyl alcohol,

ethanol or ethyl lactate} in [146], {poly(D,L-lactic acid), Eudragitpsy E,
cellulose acetate phthalate, cellulose acetate trimelitate/ethyl acetate,
2-butanone} in [49], or {PLGA, PLGA:poloxamer, PLGA:poloxamine/
dichloromethane, methylene chloride} in [149152].
As regards such formulation through the phase inversion temperature methods, it is the process itself that seems ill adapted to the concept
of desolubilizing the polymer from the nano-emulsion droplets to create
nanoparticles. In fact, a non-volatile dispersed phase is generally used,
and avoiding it does not appear physically possible. However, by substituting oil with a volatile solvent, into which the polymer has been
introduced beforehand, cf Ref. [132], the authors established nanoemulsion followed by solvent evaporation below the PIT to generate
nanoparticles. To date, the literature does not provide other formulations of polymeric nanospheres using the PIT method. Likewise, in that
using dissolved polymers involves the use of harmfulorganic solvents,
the main advantage of PIT methods, that of being organic solvent-free,
is lost: There appears to be no further interest to change the phase
inversion temperature method in such a way.
5.3. Solid lipid nanoparticles (SLNs)
Solid lipid nanoparticles [153155] are commonly dened as nanoscaled lipid matrices, solid at physiological temperatures and stabilized
by surfactants. Owing to the choice of lipids (generally biocompatible
and biodegradable) and given that the particles are stable for years, this
type of nanoparticle appears to be a privileged, promising drug delivery
system, especially for the parenteral method. However, the limits lie in
the fact that the drug molecules to be encapsulated generally have a poor
solubility in lipids, and are thus rapidly expelled after polymorphic
transition [156]. Ongoing research is looking to increase encapsulation
rates and lower these problems, using nanostructured lipid matrices
[157] or lipid-drug conjugates [158]. Here, the formulation of SLNs is
provided by both high- and low-energy methods.
High-energy production methods of SLNs [159,160] for nanoemulsion generation may be similar to those described above for
copolymers, such as the melting/sonication method: It consists in (i)
maintaining the lipid phase (plus potentially solubilized drug) 510 C
above its melting point, (ii) premixing it in aqueous surfactant
solution at the same temperature, (iii) nano-emulsifying the preemulsion using a high-energy method (high pressure homogenizer
[153155,159161] or sonication [162166]), and nally (iv) cooling it
down to room temperature to crystallize the lipids. Special care has to
be taken to avoid the lipid memory effect, making new crystallization
possible [167]. These processes are reviewed in detail by Mller et al.
[154]. These authors illustrate the advantages of such a protocol even
for encapsulating thermo-sensitive drugs, since exposure to an
increased temperature is relatively short. For highly thermo-sensitive
or hydrophilic molecules, they propose an alternative method known
as the cold homogenization technique [154].
Low-energy methods are also used for generating solid-matrix
lipid nanospheres. These methods are mainly based on the formulation of microemulsions (still above the melting point), followed by
water dilution which induces a cooling of the system and lipidnanoparticle precipitation [168171]. This process can be compared to the
EIP method (presented above) for nano-emulsion generation, by
substituting oil for melt lipid. Additional cooling gives rise to lipid
crystallization and the generation of SLNs.
Other processes found in the literature are based on the solvent
diffusion spontaneous emulsication method [172178]. Like nanoemulsions, SLNSs are generated by the solvent displacement method,
but again substituting oil for melt lipids, for instance, glyceryl
monostearate,used in Ref. [172]. Of course, it means controling the
temperature of the organic phase solubilizing the lipid, typically 5
10 C above the lipid melting point. The suspension of SLNs is then
quickly formed and the lipids crystallized after pouring this hot
194
organic solution into an aqueous one at room temperature. In Ref.

[172], the authors solubilized glyceryl monostearate in a mixture of
ethanol and acetone at 50 C, before generating SLNs by dilution into
an acidic aqueous solution. The nanoparticles formed exhibited
diameters from 50 to 500 nm, depending on the experimental
conditions (pH, drug encapsulated, etc.).
Thus, it is conceivable that when oil is added with the lipids in the
formulation process at temperatures above the lipid melting point, a
certain structuring may be induced in the particle, as it is the case in
the formulation of nanocapsules by solventdiffusion (see below).
Therefore, the addition of oil will create a porous lipid matrix as with
the nanoparticle structure, in which the porosity will be controlled by
the amount of oil [178]. Such a particle appears to be a relatively good
candidate for drug delivery, since it offers a ne arrangement of the
release proles from the specications. As a last remark, high-energy
methods may be used to incorporate this oil, in view of controlling the
particle morphology.
The PIT method also appears easily adaptable to the process of
generating SLNs and to our knowledge, no work of this nature has yet
been reported. Excipients should be chosen in function of their
physicochemical properties: (i) to allow the system to undergo a
transitional phase inversion, and (ii) to exhibit a sufciently high PIT,
compared to the lipid melting point. Hence, strong system characterization and optimization has to be done for the nely dispersed SLN
generation. Finally, it may also be possible to produce the abovementioned porous lipid matrix using the solvent diffusion method by
the further incorporation of oil in the oily phase.
5.4. Nanocapsules (NC)
Let us now move on to the last category of nanoparticles formulated
from the nano-emulsion template covered in the present review:
Nanocapsules (NC). It consists of colloidal objects exhibiting a core-shell
structure. The core acts as a liquid reservoir for drugs, mainly lipophilic
solvent (and usually oil) but also aqueous core NC, Both are described
below. The shell is generally made of polymers, preferentially biodegradable, i.e. dense and rigid, even if on the nanometric scale, such a
concept is still being discussed. For the past 20 years, as reviewed in Ref.
[3], Couvreur's team have been doing extensive research on nanocapsules as drug carriers and on their therapeutic applications.
The advantages of such a structure are: Firstly, the high drug
encapsulation efciency due to the optimized drug solubility in the
nanoparticle core and low polymer content compared to polymeric
nanospheres. Secondly, since the drug is protected within the NC core,
tissue irritation at the administration site as well as the burst effect are
lowered, and the drug itself remains protected against degradation.
Experimentally speaking, several methods are usually used to
establish such a core-shell structure on a nanometric scale. As for
nanospheres, these include: In situ polymerization at the nano-emulsion
droplet interface, nanocapsule generation using preformed polymer and
the generation of lipid nanocapsules. The NC morphology and the
formulation strategy totally depend on the therapeutic objectives and on
the drug to encapsulate. Mainly, lipophilic and hydrophilic cores of the
NC are distinguished, requiring the NC to be dispersed respectively in
water and in oil.Thus, for the parenteral administration route, it is
evidently the aqueous dispersion medium which will be adopted, the
aqueous-core NC dispersed in oil being an unsuitable system. Therefore
over the past few years, much effort has been made to formulate
aqueous-core NC as a hydrophilic drug carrier, eventually with a
polymerosome or vesicule-like structure, as presented below.
5.4.1. Polymeric nanocapsules: in situ interfacial polymer synthesis
The rst remark about this strategy of nanocapsule formulation is
the universality of the method with regard to the formulation of the
nano-emulsion template. Since polymer synthesis is performed at a
specic location (the droplet interface), and after the formation of
nano-emulsion, this process may be considered independent from the

method chosen to generate nano-emulsions. Hence, regarding the
choice of monomers and the chemical reactions during polymerization, the nano-emulsion template can constitute the starting point of
the process. Thus, all the methods used to formulate nano-emulsions,
both low and high-energy, can be considered.
It is possible to introduce the monomers (i) in the continuous phase,
reacting with the material constituting the droplets, (ii) vice versa in the
droplets, reacting with the continuous phase, or polymerization
initiated by adding initiator in the continuous phase, and nally the
combination (iii) in both phases, reacting together to induce an interfacial polycondensation. These three points are developed below. In
cases (ii) and (iii), the monomers have to be included in the formulation
process. However, the weak amounts generally added do not inuence
nano-emulsion formulation.
(i) In the rst case, monomers can be added in the external phase
after completing the nano-emulsion formulation. Thus, the monomer
is freely soluble in the continuous medium and a reagent towards the
dispersed phase itself. The interfactial polymerization reaction is
initiated at contact with the droplet and the morphology of the
polymer shell is in function of the initial quantity of the monomer. For
instance the polymerization of oil-soluble alkylcyanoacrylate on
aqueous droplets of inverted nano-emulsion, are part of a process
initiated by Lambert et al. [179182] for the formulation of aqueouscore nanocapsules. Nanometric-scaled emulsion droplets are generated using a mechanical method and monomer isobutyl-cyanoacrylate
is then added, a very reactive species towards the aqueous droplets.
The reaction is catalyzed (and initiated) by the presence of nucleophiles such as hydroxyl ions. Interfacial polymerization is completed
within seconds and w/o nanocapsules are generated. The authors then
extract colloidal objects from the organic phase and perform a resuspension in water. Various hydrophilic drugs have been encapsulated in this way, such as oligonucleotides [179181,183] including
antisenses [182], or siRNA [184]. Overall a relatively high encapsulation
efcacy has been observed. More recently, this process has even been
optimized by Hillaireau et al. [185,186] by polymerizing onto a w/o
microemulsion template (surely similarly to water-loaded inverted
micelle suspension), instead of mechanically established nanometric
aqueous dispersion.
Prime candidate monomers for performing such nanocapsule
formulation strategies appear to have both a good solubility in the
external phase and a sufcient reactivity with the phase constituting the
dispersed nano-emulsion droplets. The morphology of theresulting
nanocapsules will be in function of the amount of monomer added as
well as the polymerization time. Thus, molecules other than alkylcyanoacrylates could be considered as candidates for nano-emulsion in situ
interfacial polymer synthesis. Take for example diisocyanate lipophilic
molecules, in which the isocyanate functions are possibly hydrolizable by
an electrophile site-rich solvent such as water, leading to amine formation.
The amine, then reacts rapidly with another monomer molecule, since it is
more reactive than water: The polycondensation reaction occurs exactly
at the interface. Our recent works [187] report the formation of aqueouscore nanocapsules, which work on the same principle, but through
different formulation strategies, i.e. by low-energy (PIT method), and
interfacial nano-emulsion diisocyanate polycondensation.
However, such interfacial polycondensation is nothing other than
the adaptation to nanometric dispersion of the interfacial polycondensation reported for microcapsule generation, for instance by Pens
et al. [188,189].
To summarize: When the monomer is added in the continuous
phase (case (i)), interfacial polymerization does not appear dependent
on the method chosen to generate the nano-emulsion templates,
high- and low-energy, PIT method, spontaneous emulsication, etc.
(ii) In the second case, a great number of publications present the
formulation of nanocapsules by interfacial polymerization, where the
monomer is included in the dispersed nano-emulsion droplets. Let us
begin by the high-energy formulations of Tiarks et al. [35,32] (see also

[190]), with the formulation of polymeric nanospheres: The hydrophobe is now composed of a monomer, an initiator and oil, dispersed
in water by sonication, generating nano-emulsions. Polymerization is
initiated via the temperature, the monomer precipitates in oil and
there is gradually segregation towards the water/oil interface, thus
generating nanocapsules. In fact, this phenomenon has given rise to
the theory of droplets composed of binary mixture [191], and the
polymer/oil system can adopt several congurations (i.e. the polymer
can engulf the oil totally, partially or not at all). Nanocapsule generation requires the polymer to totally engulf the oil core after its
segregation. Such segregating behavior is governed by the respective
interfacial tensions between the three immiscible species.
As regards the solvent displacement method, (still) concerning
alkylcyanoacrylate, a large number of papers have been reported,
initiated by Al Khouri Fallouh et al. [192,193] and thereafter widely
studied and optimized [134,194199]. These authors include alkylcyanoacrylate monomers within the (water miscible) organic solvent
plus oil phase, and polymerization is initiated along with the
instantaneous diffusion of the solvent to the aqueous bulk phase.
Since both the generation of nano-emulsions and the interfacial
polymerization reaction are extremely fast, it is assumed that they are
produced simultaneously.
(iii) Finally, in the third case, the nano-emulsion is rst prepared
including a monomer within the oil droplets (for instance diisocyanate) and the polycondensation reaction proceeds at the water/oil
interface. Thus again, it is possible to generatenano-emulsions using
both high- and low-energy methods, as long as the inner monomer is
included and without further reaction of the latter with the aqueous
phase. The main challenge of these studies is to adapt the interfacial
polycondensation processes, well known for micrometric droplets
[200202], to a nanometric dispersion exhibiting a huge water/oil
interface, as well as to the characterization methods used, in order to
ensure that the polymer coating is successfully achieved.
For example, Takasu and Kawaguchi [203] propose the synthesis of
a polyurea shell, by coating nano-emulsion made of styrene in the
hydrophobe phase (before generating latex core-shell nanoparticles
via a second styrene polymerization). Nano-emulsions containing
diisocyanates (such as isophorone diisocyanate or tolylene-2,4diisocyanate) are established by sonication and the polycondensation
reaction begins subsequently, when the aqueous solution of diamine
(isophorone diamine or hexamethylene diamine) is added to the o/w
nano-emulsion. The reaction mixture is then slowly stirred for no
more than 2 h to complete the water-insoluble polyurea coating.
Montasser et al. [204206] and Bouchemal et al. [72] have provided some examples regarding such an in situ interfacial polycondensation, for which the nano-emulsion template is generated by
a low-energy method: Solvent displacement. The reaction is performed by a stepwise reaction between for instance, diisocyanate
molecules within oil droplets and activated diols, poly(ethylene
oxide), or even diamine, solubilized in the aqueous phase, in order
to generate, respectively, polyurethane or poly(ether urethane) shells,
or polyurea. It is to be noted that the diols have to be activated with
the help, for instance, of diazobicyclo, 2-2-2,octane molecules.
Finally about the PIT method, it appears conceivable to incorporate
diisocyanate into the droplets in order to induce similar interfacial
chemical reactions (as in the case (i)), but only when the selected
diisocyanate exhibits low reactivity with the aqueous phase during
processing until completion of nano-emulsion generation.
5.4.2. Polymeric nanocapsules: nanoprecipitation of preformed polymers
The general protocol to specically induce the precipitation of
preformed polymers at the droplet interface appears principally based
on the solvent displacement nano-emulsifying method. It is the only
experimental procedure which combines (i) the use of solvents
efcient enough to solubilize the macromolecules, and (ii) its specic
195
displacement towards the bulk phase, during which the precipitating

polymers will be deposed according to the prole of the nanometric
oil droplets being formed. Earlier works on such a process were
proposed by Fessi et al. [69,70] using poly(D,L-lactic acid) (PLA) or poly
(alkylcyanoacrylate) polymers and acetone as the displacing solvent.
Of course, these pioneer works were followed by a large number of
variants, applications and patents (e.g. [73]), using different polymers
such as for instance poly(isobutyl-cyanoacrylate) [207], PLA, Eudragitpsy E, poly(-caprolactone) (PCL) [208210], poly(lactic-coglycolic acid) (PLGA) [211] and so forth.
The rst advantage thus appears to be the apparent lack of
potential and aggressive drug-monomer interaction owing to the
absence of chemical reaction, in comparison with other methods in
which the polymerization (interfacial or not) is in direct contact with
the drug. Secondly, as various types of polymers are used (see also [3]),
this method appears relatively adaptable to given specic and
therapeutic purposes, etc. However, this low-energy process inexorably implies the use of organic solvents and their specic drawbacks
for pharmaceutical applications.
Another method (somewhat anecdotal in comparison with the one
described above), presents the generation of polymeric nanocapsules
with preformed polymers in two steps: (i) Firstly, by preparing the
nano-emulsion template, whatever the (high- or low-energy) method
used and (ii) secondly, by coating it with the polymer deposition on the
water/oil nano-emulsion surface. The polymers are added in the
continuous phase (even after nano-emulsion is complete) and their
precipitation onto the nano-emulsion droplets is induced by solvent
evaporation. For instance, Paiphansiri et al. [36] propose the encapsulation of an antiseptic agent, a chorhexidine digluconate solution,
within a w/o nano-emulsion generated by sonication. The polymers
they use, poly(methyl methacrylate) (PMMA), poly(methacrylate)
(PMA) or PCL, are solubilzed in dichloromethane (DCM) gradually
added in the continuous organic phase of the nano-emulsion. Next, the
temperature is maintained, with slow stirring, above boiling point
until the DCM has completely evaporated. The polymers precipitate
onto the nano-emulsion water droplets, thus forming nanocapsules.
The authors present such a mechanism of specic segregation on the
droplet interface, similar to their other works presented above (Section
5.4.1) dealing with polymer synthesis and specic segregation and
therefore totally engulng the nanocapsule core, depending solely on
the respective interfacial tension between the three species [35].
Finally, another strategy was developed by Prego et al. [212214],
reporting specic chitosan or PEG-chitosan nano-emulsion coating.
Nano-emulsions are obtained by the low-energy solvent displacement
method, and stabilized by lecithin. After eliminating the solvent, the
nanocapsules are created by simple incubation with the polymer.
These nanocapsules have been used as carriers for hydrophilic
molecules such as peptides (e.g. salmon calcitonin in Refs. [213,214]),
simply by including the peptide within the organic solvent before it is
poured into the aqueous phase to diffuse and generate nanoemulsions. The peptides are entrapped within the o/w nano-emulsion,
and the surrounding chitosan wall acts as a barrier, preventing extensive release.
To summarize, the formulation of nanocapsules using preformed
polymers are mainly performed (in the rst case) by combining the
solvent displacement method and the specic nanoprecipitation of
polymers onto the water/oil interface of the forming oil droplets. These
objects are exclusively o/w nanocapsules. A second strategy consists in
establishing rst the nano-emulsion template, and then forcing the
polymer (solubilized in the continuous phase) to specically precipitate
to the nano-emulsion interface. Oily-core as well as aqueous-core
nanocapsules can be formulated. Furthermore, since the two formulation steps can be dissociated, nano-emulsion formulation can be
performed whatever the method chosen. Thus, if the specications are
respected, it is conceivable to include the solvent-free and low-energy
PIT method for nano-emulsion generation.
196
5.4.3. Lipid nanocapsules (LNC)

Lipid nanocapsules were introduced by Heurtault et al. [106
108,215], and are commonly dened as exhibiting a core-shell structure
composed of a liquid oily core and an amorphous surfactant shell. The
formulation process is in fact based on the PIT method plus the
temperature cycling treatment. Biocompatible excipients are chosen for
the formulation and for nanocapsule administration via a parenteral
route, mainly medium-chain triglycerides (caprilic triglycerides) as
the oil phase, a polyoxyethylene-660-12-hydroxy stearate as the PEO
nonionic surfactant and MilliQpsy water plus NaCl as the aqueous
phase. An additional, somewhat neutral component, lecithin, was
introduced in the formulation, and has been shown [216218] to increase the nanocapsule stability signicantly, creating a framework in
the shell. Therefore, the own formulation process was actually established as a function of the physicochemical properties of such
adequate components. Since the temperature cycling process has been
shown [90] to increase the quality of the nano-emulsions (in terms of
size and PDI) by increasing the surfactant amount at the water/oil
interface, the generation of nano-emulsions by the PIT method plus
temperature cycling leads, in fact, to droplets exhibiting an important
quantity of surfactants in the interfacial region. In addition to the present
case, the fact that the nal formulation is reached by a sudden dilution
with water at temperature below the nonionic surfactant melting point
(~30 C), indicate that shell crystallization could occur. Finally, as
discussed above concerning droplets stabilized with macromolecular
assemblies or thick polymeric species [23,24], Ostwald ripening is
signicantly reduced even more than for the simple nano-emulsions,
and it is precisely the case for LNC. In view of all these results, these
particular nano-emulsions, formulated by temperature cycling, are
denominated as lipid nanocapsules. Nevertheless, the structure of these
LNC being specic and typical of the formulating method and of the
properties of the used nonionic surfactants, it is hard to imagine the
formulation of similar objects using methods other than the PIT method.
6. Conclusions
The formulation of nanoparticulate drug carriers, based on nanoemulsion formulation, appears at rst to require adaptation to the
therapeutic aims and specicity of the drug to encapsulate. That is to
say, both high- and low-energy nano-emulsion formulation methods,
whether or not they include the use of organic solvents, have to be
adapted according to the active molecule properties, i.e. sensitivity to
temperature, high-shear devices, contact with organic solvents, etc.
This review has presented the extent to which these nanoparticulate
drug carriers are generated using various nano-emulsion formulation
processes. High- and low-energy methods used to establish nanoemulsions are presented in the rst part. The second part proposes a
link towards the generation of nanoparticles. The formulation of the
main groups of nanoparticles, i.e. polymeric nanospheres, solid lipid
nanoparticles and nanocapsules, are reviewed in the light of the nanoemulsion formation method as well as conceivable alternatives (not
necessarily reported in the literature). The global aim of this review is
to provide the formulator with a broad basis on the adaptation, or
even the carrying out of a process, according to specic needs.
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Design and Production of Nanoparticles Formulated From Nano-Emulsion

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Journal of Controlled Release 128 (2008) 185199

Contents lists available at ScienceDirect

Journal of Controlled Release

Design and production of nanoparticles formulated from nano-emulsion

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

In the case of polymers in situ synthesized with polymeric

Accordingly, the physical destabilization of emulsions is related to the

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

Fig. 1. Diagram of the inuence of emulsion droplet radius on steric stabilization.

minimum |U0| will induce predispositions for coagulate in the colloidal

where C is the bulk solubility of the dispersed phase, M its molar

quently, Ostwald ripening is reected by a linear relationship between

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

which nano-emulsication is totally governed by the physicochemical

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

Fig. 2. Diffusion path in a typical water/alcohol/oil system. Segment (12), diffusion

droplets appear to be formed in this way, with the use of a high

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

established microemulsion structures, the surfactant hydration is

Fig. 3. Diffusion path in a typical water/alcohol+surfactant/oil system (see details in the

segment (23), before establishing the local interface equilibrium with

The phase inversion temperature (PIT) method is particularly

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

surfactant afnity difference (SAD) is dened with Eq. (5), at the

i is the chemical potential of the NS in phase i, i are the standards, a

In the case of ionic surfactants, the emulsion inversion corresponds to

accordance with Bancroft's rule and not. The illustration is provided in

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

add neutral components to the formulation, which inuence neither the

5.2. Polymeric nanospheres

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

Finally, as reported in the works of Gallardo et al. [133], during the

[145], {Eudragitpsy L/acetone, dimethyl sulfoxide, isopropyl alcohol,

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

organic solution into an aqueous one at room temperature. In Ref.

nano-emulsion, this process may be considered independent from the

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

begin by the high-energy formulations of Tiarks et al. [35,32] (see also

displacement towards the bulk phase, during which the precipitating

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

5.4.3. Lipid nanocapsules (LNC)

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

[77] A. Forgiarini, J. Esquena, C. Gonzlez, C. Solans, Formation of nano-emulsions by

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

[139] M.D. Blanco, M.J. Alonso, Development and characterization of protein-loaded

N. Anton et al. / Journal of Controlled Release 128 (2008) 185199

[196] M. Gallardo, G. Couarraze, B. Denizot, L. Treupel, P. Couvreur, F. Puisieux, Study of the

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