Journal of Controlled Release 295 (2019) 31–49

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Review article

Double emulsions prepared by two–step emulsification: History, state-of- T

the-art and perspective
Shukai Dinga,b, Christophe A. Serrab, , Thierry F. Vandammec, Wei Yub, Nicolas Antonc,
⁎ ⁎

a
Materials Institute of Atomic and Molecular Science, Shaanxi University of Science & Technology, Xi'an, Shaanxi CN-710021, China
b
Université de Strasbourg, CNRS, ICS UPR 22, Strasbourg F-67000, France
c
Université de Strasbourg, CNRS, CAMB UMR 7199, Strasbourg F-67000, France

ARTICLE INFO ABSTRACT

Keywords: Attractive interest on double emulsions comes from their unique morphology, making them general multi-
Double emulsions functional carriers able to encapsulate different hydrophilic and lipophilic molecules in the same particle. Over
Two-step emulsification the past century, two different types of methods were followed to prepare double emulsions for pharmaceutics
Polymeric particles applications, so-called “one-step” and “two-step” processes. The two-step approach, consisting in two different
Drug release
emulsifications successively performed, allows the optimal and more efficient formulations due to simplicity of
Microfluidic system
principle and controllability of the process. In this review, focused on the formulation of double emulsions by
Encapsulation of hydrophilic materials
two-step process, we recount the historical development of this approach, along with the state-of-the-art, in-
cluding a discussion on the role of the formulation parameters, surfactants, amphiphilic polymers, interface
stabilization, volume fraction, and so forth, on the final formulation stability, morphology and properties as drug
delivery system. Discussion was also extended to polymeric microparticles and nanoparticles made by solvent
diffusion, on the basis of double emulsions made by two-step process, along with literature review on the impact
of different formulation and processing parameters. In addition, the properties of the polymers used in the
microparticles matrix (molecular weight, chemical nature) potentially impacting on the ones of the micro-
particles formed (drug release kinetics, stability, morphology), were also discussed. Finally, the future trends in
double emulsions application were addressed, emphasizing some new advances made in the emulsifications
method as potentially able to open the range of applications, for example to nanoscale with spontaneous
emulsification or low energy microfluidic emulsification.

1. Introduction generation of micelles and liposomes [11]. However, the common

Double emulsions, so-called Water-in-Oil-in-Water emulsions (W/
O/W emulsions), consist of water droplets dispersed in oil or oil/
polymer globules, themselves ultimately dispersed in an aqueous phase
(Fig. 1). Such a structure appeared highly interesting to encapsulate
very different and even incompatible molecules (e.g. hydrophobic and
hydrophilic) in a single carrier. Over the past thirty years, double
emulsions has known an important interest in the formulation of
pharmaceutics, including drug carriers in cocktail therapy [1,2], sub-
stitute blood [3], vaccines [4], vitamins [5], and enzymes [6]. There-
fore, the design of double emulsions was extensively modified in
function of specifications and the diversity of the aimed applications.
Indeed, different types of double emulsions have been developed such
as oil-based double emulsion [7], polymeric microparticles [8], nano-
particles [9], nanocapsules [10], double emulsions template for the
particularity of all these systems lies in the fact that they were prepared
by a two-step emulsification process. It follows therefrom that a deep
understanding of this “two-step” emulsification process appears crucial
for preparation, adaptation and optimization of double emulsions.
Literature reviewed several specific aspects related to double
emulsions, such as their structural stability [12], the transport phe-
nomena between the different phases [13], double emulsion based
polymeric carrier [14], microfluidic formulation of double emulsions
[15] and so forth. To our best knowledge, even if it is a fundamental
aspect of the design of double emulsion based particulate carriers, the
two-step emulsification method has never been specifically reviewed.
This aspect will precisely be the main interest of the current review,
which namely will focus on two aspects: (1) the effect of formulation
parameters, nature of the phases, volume fractions, additives' con-
centration, and (2) the effect of processing parameters of

⁎
Corresponding authors.
E-mail addresses: ca.serra@unistra.fr (C.A. Serra), nanton@unistra.fr (N. Anton).

https://doi.org/10.1016/j.jconrel.2018.12.037
Received 6 September 2018; Received in revised form 18 December 2018; Accepted 19 December 2018
Available online 21 December 2018
0168-3659/ © 2019 Elsevier B.V. All rights reserved.
S. Ding et al.
Fig. 1. Schematic drawing of a double emulsion structure.
emulsification. These two aspects of the process will be discussed in
relation with their impact on their physico-chemical properties, size,
size distribution and stability. In addition, since they are quite different
from classical W/O/W double emulsions, the other complex specific
systems like oil-in-oil-in-water (O/O/W) or water-in-water-in-oil (W/
W/O) emulsions, will not be included in the scope of the current re-
view.
In this context, the discussion will be organized around the evolu-
tion of two-step emulsification process, in relation with the types, sizes
and nature of the emulsions, and type of encapsulated molecules.
Specific aspects herein reviewed will include: the given advantages of
two-step over one-step processes, the efforts undertaken regarding the
stabilization of double emulsions, aspects related to the controlled drug
release, surface modification, the precise control of the numbers of
inner water droplets, and finally to microfluidic tools used to prepare
polymeric particles or double emulsions.
Fig. 2. Schematic drawings of the two-step processe to produce double emulsions.
32
Journal of Controlled Release 295 (2019) 31–49
1.1. History of double emulsions by two-step emulsification
1.1.1. Advantages of double emulsions and two-step emulsification
The double emulsions were firstly presented in 1925 by William
Seifriz [16], included in research works investigating the impact of oil
density on the type of emulsions formed. For instance, an emulsion
prepared with straw-oil (having a density of 0.882 kg.L−1) exhibits an
atypical behavior since it can form a complex system consisting in a
coarse oil-in-water emulsion made of oil globules (about 1 mm) that
encapsulate a fine water-in-oil emulsion. This example was actually the
first model of double emulsions reported. However, these double dro-
plets rapidly destabilized and turned to single emulsions, thus were
considered as a transitional and instable state between Water-in-Oil
(W/O) and Oil-in-Water (O/W) emulsions [17]. The reasons of this
intrinsic instability have been explained by physical reasons, i.e. ori-
ginated from osmotic pressure and Laplace pressure [18]. The differ-
ence of solute concentration (related to osmotic pressure) between
inner water and continuous aqueous phases induces a water migration
between these two phases in order to re-equilibrate osmotic pressures.
Higher osmotic pressure in external phase induces the swelling of inner
droplets, while, lower osmotic pressure results in the shrinkage of inner
droplets. Accordingly, concentration or osmotic pressure is a crucial
parameter as regards to the double emulsion stability. As such the La-
place pressure (that depends on the droplets size and surface tension)
induces a very high pressure inside inner droplets compared to the one
of the oil globule. This difference leads to the pressure re-equilibration
and ultimately to the collapse and rupture of double emulsions struc-
ture. Interestingly, these particularities of double emulsions, that make
them fragile in some extent, were also taken beneficial as a way to
control the release of encapsulated materials stimulated by osmotic
pressure. Thanks to their versatile and instable structure, the first ap-
plication was designed for encapsulating insulin as drug carrier in the
late 1960s, in fact thirty-five years after discovery of double emulsions
[19]. However, the main advantage of double emulsions lies in the
possibility they offer to encapsulate several types of molecules si-
multaneously.
In 1976, Matsumoto et al. [20] firstly reported the two-step emul-
sification standard procedure for preparing double emulsions (Fig. 2).
First, a W/O emulsion is prepared in accordance with the Bancroft rule,
and then the double W/O/W emulsion is obtained by using the former
W/O emulsion as the oil phase for the second emulsification step. Since
then, the two-step emulsification method became the most prevalent
method to get double emulsions [21]. On the other hand, the one-step
emulsification process, in which the double emulsion spontaneously
form, has always got attraction owing to the simplicity of the process
S. Ding et al.
[22]. This approach however suffers of limitations such as the en-
capsulation efficiency that only remains quite low (ratio between en-
capsulated and total amount of species).
1.1.2. Choosing surfactants: 1. Impact on the double emulsions stability
Especially for the two-step emulsification process, the choice of the
surfactants is a crucial parameter to insure the stability of each in-
dependent emulsion. In principle, a lipophilic/hydrophobic surfactant
characterized by a HLB (Hydrophilic Lipophilic Balance) value lower
than 7 (commonly around 3–4) should be chosen for the preparation of
the primary W/O emulsions. Reverse droplets are in general adhesive
and subject to flocculation and coalescence, the use of very-low HLB or
lipophilic macromolecules are preferred to prevent this destabilization
of internal droplets. On the other hand, hydrophilic surfactant (HLB
value higher than 10) is necessary to stabilize the external surface of
double globules after second emulsification. Eventually, choice of sur-
factants is essential to formulate stable double emulsions, but also to
play on their size, encapsulation efficiency.
Historically, the stability of double emulsions was the main key
parameter taken into account in the quality of double emulsions. As a
result, an important research effort was dedicated to this aspect and
resulted in several efficient formulation strategies to increase double
emulsions stability. To this end, the so-called optimal HLB was defined
and corresponded to the HLB of surfactant mixture giving the most
stable emulsion. Garti et al. [23] reported the preparation of stable W/
O emulsions obtained by tailoring the HLB value of surfactant mixture,
the so-called required HLB related to the oil chosen in the formulation.
In their work, W/O emulsion with the best stability was simultaneously
obtained with value HLB close to 4–5, either using a single amphiphile
brij 93 at 8 wt% (polyethylene glycol oleyl ether), with HLB value equal
to 4.9, or a mixture of Span 80 (sorbitan oleate) and Span 85 (sorbitane
trioleate) at 10 wt% with a HLB value equal to 4. Any separation of
inner water droplets from the oil phase was observed after 30 days, and
regarding the water droplets size, only a slight increase from 0.5 μm to
3 μm was reported [23].
On the other hand, the stability of W/O emulsions can be also af-
fected and strengthened with the addition of a large amount of hy-
drophobic surfactants. Thus, a stable W/O emulsion was obtained with
Span 80 at a concentration as high as 30 wt% [20] but it was believed,
as a result of the migration of the Span 80 towards the external inter-
face, that its global effective concentration was reduced. Consequently,
the corresponding high concentration of hydrophilic surfactant ap-
peared necessary to balance and stabilize the double structure, while
the opposite effect was observed with high concentration of hydrophilic
surfactant, i.e. low encapsulation efficiency, uncontrollable droplets
size and stability. It thus seemed imperative to develop alternative
methods to stabilize such a fragile structure. Garti et al. [24] proposed a
pioneer study using polymeric surfactant to increase the stability of W/
O emulsions, resulting in a more stable interface and lower desorption
and migration of stabilizing molecules. A polymeric surfactant was
synthesized through a reaction between polymerized soybean oil and
polyglycerol, giving rise to molecular weights ranging from 881 to
3758 g/mol. Importantly, these authors showed that the stability of W/
O emulsions increased with the molecular weight. In addition, an ex-
cellent stability of W/O emulsions can be obtained at weight fractions
of internal phase as high as 50 wt%, even at low concentration of
polymeric surfactant, around 3–5 wt%. In comparison, to reach a si-
milar level of stability with non-polymeric (i.e. classical) surfactants,
concentrations must be increased up to 20–25 wt% [24].
Additionally, the concentration of hydrophilic surfactant in the ex-
ternal phase can also play a significant role on the double emulsions
stability. As a result of an increased concentration of hydrophilic sur-
factants, it has been observed that the double droplets were smaller
with an increased stability, and in the same time the encapsulation
efficiency was decreased [25]. This phenomenon was explained by the
effects of these surfactant excess, even in the internal droplets interface
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Journal of Controlled Release 295 (2019) 31–49
(stabilized by hydrophilic surfactant) affecting the established balance
among three phases. Solving this trade-off problem was found in using a
mixture of ionic-surfactant and nonionic hydrophilic surfactants, and
showed significant improvements in terms of encapsulation efficiency
and stability [23,26]. This result is likely due to the much more de-
creased solubility of the ionic surfactants in the lipophilic phases, along
with, on the other hand, some intrinsic incompatibilities in term of
potential interactions with encapsulated materials or toxicity.
Playing a crucial role in the encapsulation efficiency and stability,
the choice of hydrophobic surfactant is fundamental. Generally con-
sidered through its low HLB value (HLB ≤ 4), some other factors need
to be considered in order to optimize the results [27,28]: firstly the
“rigid” molecular structure (for example with a high degree of un-
saturation), and secondly, better compatibility or solubility of the sur-
factant in the oil phase (for example oil having a structure close to the
aliphatic part of the surfactant), are important factors that will increase
the stability of primary W/O emulsion. It follows that a lipophilic
surfactant with an important degree of unsaturation, formulated with
unsaturation oil made with a similar structure, could be optimized
conditions to increase the double emulsion stability.
1.1.3. Choosing surfactants: 2. Impact on the encapsulation efficiency
The second major challenge regarding the formulation of double
emulsion is the encapsulation efficiency, actually intimately related to
the stability. High encapsulation efficiency, according to literature
[29], corresponds to an optimum HLB value between hydrophilic and
lipophilic surfactants in external phase and in oil, respectively. In the
cited example, the authors shows the similar encapsulation efficiency
for different concentrations of surfactants (mixture of Span 20 and
Tween 80) but formulated at the same global mixture HLB. The concept
is actually similar to the one followed in the stabilization of emulsions
so-called HLB method, that consists in equalizing the mixture HLB with
the required HLB of the oil/water couple, and eventually revealed that
a high encapsulation efficiency is a direct consequence of a good sta-
bility of the double emulsion.
A second notable factor impacting on the double emulsion stability
[30] is the molecular weight of the hydrophobic surfactant. Three dif-
ferent surfactants were employed and compared to stabilize W/O
emulsions: 1) low molecular weight classical emulsifiers such as Span
80; 2) medium molecular weight macromolecules such as polyglycerol
polyricinoleate (ETD or PGPR) and 3) high molecular weight grafted
silicone lipophilic surfactant (Abil EM-90). As a result the higher the
molecular weight, the higher the encapsulation efficiency: highest with
Abil EM-90 then PGPR, and finally Span 80. The explanation proposed
was that higher molecular weight will induce a higher effective con-
centration in oil at same concentration owing to the lower migration
rates towards external interface and phase.
1.1.4. Choosing Surfactants 3. Impact of the types of double emulsions
The different types of double emulsions have been classified based
on the number of water droplets in global droplets: 1) Microcapsules:
the oil droplet includes only one inner aqueous droplet (Fig. 3A); 2)
Multivesicular, droplets for which the oil droplets encapsulates nu-
merous inner aqueous droplets (Fig. 3B); 3) Finally, microsphere,
double droplets with a complex inner structure inside oil droplet
(Fig. 3C). A. T. Florence's group [31] demonstrated that the different
types of double emulsions can be obtained by a proper selection of the
hydrophilic surfactant. Three types of double emulsions were also
prepared with three types of hydrophilic surfactant:1) Polyethylene
glycol dodecyl ether (Briji 30, 2 wt%) 2) Octyl phenol ethoxylate
(Triton X-165, 2 wt%) 3) Combined surfactant by span 80 and tween
80, in which span 80 (5 wt%) was used as hydrophobic surfactant in
middle phase. The three types of double emulsions are illustrated in
Fig. 3.
On the other hand, different concentrations of span 80 in middle
phase were also used to obtain different types of double emulsions,
S. Ding et al.
Fig. 3. Three typical types of double emulsions Left: Schematic drawings of
double emulsions (A) Microcapsule, (B) Multivesicular, (C) Microsphere; Right:
Three types of double emulsions as seen by an optical microscope [31]; Scale
bar is 10 μm.
tween 80 (1 wt%) and additional stabilizing agent (bovine serum al-
bumin, BSA) was used in external phase [32]. It was demonstrated that
the type of double emulsions can also be impacted by the concentration
of span 80 ranging from 1 to 10 wt%, resulting in morphology mod-
ification, e.g. from microcapsules to microspheres. By now, three main
types of double emulsions have been shown and potentially controlled
by the nature of hydrophilic surfactants or concentration of hydro-
phobic surfactants.
Regarding the selection and concentration of surfactants, it can be
summarized as follows: 1) for the stabilization of internal W/O emul-
sions, low HLB, high surfactant rigidity, and similarity between the
nature of oil and aliphatic part of the surfactant, are required. In ad-
dition, increasing the hydrophobic surfactant concentration gives rise
to more stable double emulsion, higher encapsulation efficiency, and
complex structure of double emulsion (microsphere); 2) Then for the
formulation of the double structure of W/O/W emulsions, the main
parameters that impact on the emulsion stability are high HLB and/or
surfactant concentration of hydrophilic surfactants. However, in-
creasing concentration makes the size and the encapsulation efficiency
decrease at a trade-off problem. Emulsion stability and encapsulation
efficiency are also related to the mixing HLB related to the optimum
formulation determination and required HLB of the oil 3) Finally, the
types of hydrophilic surfactant and the concentration of hydrophobic
surfactants will significantly influence the type of double emulsions.
1.1.5. Additional methods for stabilizing double emulsions
Even though the stability of double emulsions was increased by
adjustment of surfactant nature and concentration, in practice this ap-
proach could rapidly reach limitations. Florence et al. [33] proposed
the transformation of aqueous phase (inner phase or external phase) to
a polymeric gel for improving the stability of double emulsions. For
instance, a polyoxyethylene-polyoxypropylene-polyoxyethylene ABA
block copolymer (poloxamer) and acrylamide were added into inner
phase or external phase, respectively. After emulsification, the gel is
formed by crosslinking ABA block copolymer or polymerizing acryla-
mide with UV irradiations [34]. The formation of such hydrogel gel
gave a more stable double emulsion, with however, a potential pitfall
lying in the impact of UV irradiations on sensitive molecules en-
capsulated.
Some other strategies were developed based on the formation of
complexes at interface between macromolecules and nonionic
34
Journal of Controlled Release 295 (2019) 31–49
surfactants [35]. A type of crosslinked polyacrylic acid or BSA was
added in the inner phase to create a complex with hydrophobic sur-
factant (poloxamer), forming a strong interfacial film stabilizing the
inner water droplets.
Such a poloxamer/protein complex resulted in an important slowing
down of the release kinetics, from 40% to 10% total released of en-
capsulated sulphane blue after 6 h, without and with this interfacial
stabilization, respectively [35]. A similar example was proposed with
an interfacial stabilization with BSA and a small molecular weight li-
pophilic surfactant (Span 80), following the encapsulation efficiency of
sodium chloride. The results evidenced an increase of the encapsulation
efficiency around 20% to finally reach a value of 80% [32], and con-
served over 60% after 30 days. Garti et al. [36] have shown that this
BSA/Span 80 system significantly improved the stability of the double
emulsions, giving release rate around 8% and 20% for 5 h and 20 h,
much smaller compared to the ones without stabilization about 35% to
52%% for 5 h and 20 h. Another effect of the addition of BSA, was a
smaller size of double droplet, along with a size stability not changing
after 6 weeks. This effect was explained by the interfacial stability
conferred by the closed-packed BSA layer at the external oil/water in-
terface, likely improving the interfacial elasticity and resistance, thus
preventing the double droplets coalescence and rupture of inner water
droplets. In this context, double emulsion stabilization by such mac-
romolecules have attracted an important interest. In this line, the use of
colloidal microcrystal based of cellulose was associated to “mechanical
stabilizers” of double emulsions [37]. Some examples have also de-
scribed the formulation of double emulsions using different biopoly-
mers to stabilize internal droplets, following different mechanisms such
as gelation, caseins, whey protein, chitosan and cyclodextrins
[24,38–50]. Apart from molecules or macromolecules, double emul-
sions were also stabilized by nanoparticles, as Pickering double emul-
sions, comprehensively reviewed in literature by Clegg et al. [22].
A complementary approach have been shown, by Kawashima et al.
[51], that the fact making hyperosmotic internal droplets improves the
stability and encapsulation efficiency. Indeed, increasing sodium
chloride or glucose in internal water, in a certain extent, induces the
migration from external towards internal water phases, likely along
with phenomena that induce the interfacial concentration and stabili-
zation.
1.2. Development of double emulsions by two-step emulsification
1.2.1. Polymeric microparticles synthesized by the two-step emulsification-
evaporation method
Due to the real advantages of biocompatible polymers (such as Poly
(lactide-co-glycolide) (PLGA), Poly(lactic acid) (PLA) and Poly-ε-ca-
prolactone (PCL)) in the encapsulation and controlled release of hy-
drophilic molecules (like protein or peptides), such microparticles were
initially prepared with double emulsion as template according to a two-
step emulsification method [52]. Ogawa et al. [53] have firstly de-
scribed the standard experimental procedure as follows (schematically
described in Fig. 4): 1) Polymer is dissolved in the organic solvent,
immiscible with water in which are solubilized hydrophilic drugs. The
W/O emulsion is fabricated with these two phases; 2) The double W/O/
W emulsion is then formulated with (another) external water phase; 3)
Last stage consists of the evaporation of the organic solvent, making
precipitating the polymer in the form of solid microcapsules. Initially,
Ogawa et al. [54] synthesized PLA or PLGA to be dissolved in di-
chloromethane used as oil phase, and a hydrophilic analogue of lutei-
nizing hormone was encapsulated in the matrix of polymeric micro-
particles according to the above-described method. Since no
hydrophobic surfactant was added into the oil phase, a broad size dis-
tribution of microparticles was obtained (from 30 to 125 μm), size was
only controlled by the processing parameter (using sieves of different
apertures).
To optimize the properties of polymeric particles, all strategies
S. Ding et al.
Fig. 4. Schematic drawing of the two-step process to produce polymeric particles by the modified two-step emulsification method.
described in the previous sections for oil-based double emulsions can be
followed. For example, gelation of internal droplets using gelatin have
shown the clear increase in the encapsulation efficiency, from 6.7 to
70.7%, without and with gelatin, respectively. Eventually, irrespective
to the oil-based double emulsions, when polymers are used, the stability
is much more increased, and the stability and drug release profiles will
be more related to droplet morphology, degradation and destruction of
the polymer [54].
1.2.2. Impact of the size of inner and middle phases in the case of polymeric
microparticles
Rosca et al. [55] proposed a study on the respective impact on the
carrier properties, of the size of inner water droplets, global double
droplets, and the morphology of the precipitated PLGA. These authors
showed that in function of the protocol followed during evaporation,
the size of the droplet may vary, and thus the resulting properties of the
double globule, i.e. mainly depending on the inner droplets dimeter and
(Di) and double globule diameter (Dg). When Di ≈ Dg, the polymeric
layer was so thin that it could not resist to the inward forces originating
from the Laplace pressure and osmotic pressure between the inner and
external phases. Thus, this polymeric layer broke down during the
solvent evaporation, which results in the impossibility to create the
global particle as seen in Fig. 5 (I). When Di < < Dg, a thicker poly-
meric barrier protects and stabilizes the double droplets (Fig. 5 (II)).
These droplets properties were shown to be controllable by the pro-
cessing and formulation parameters like emulsification energy input,
polymer concentration, surface active agent concentration, phase vo-
lumes, phase viscosities and so forth [55].
1.2.3. Progress degradation and destruction of polymeric microparticles
Langer et al. [52,56] reported encapsulated proteins (BSA and te-
tanus toxoid) in PLGA microparticles (Fig. 6 (I)). In the case of PLGA,
drug release typically comes from the PLGA degradation, as shown in
Fig. 6 (II). Initially, the PLGA microparticles had a smooth surface. After
one day, small micropores (diameter < 0.1 μm) appeared all over the
surface. After 4 days in maintained in aqueous release medium, the size
of the pores increased. After 14 days, the microspheres were highly
porous, however, keeping their spherical shape until 76 days. The in-
fluence of hydrophobic surfactant, type and molecular weight of
polymer, on the size and morphology, degradability of microparticles,
has been investigated. For example, small and porous particles were
formed when using hydrophobic surfactant (e.g. L-α-phosphatidylcho-
line) in the organic solvent. On the other hand, bigger microparticles
were obtained when using higher molecular weights polymer [52].
Degradability of microparticles was investigated in function of the
PLGA/PLA molecular weight (Fig. 6 (III)), showing a higher stability for
PLA, compared to PLGA. Regarding the release of model hydrophilic
protein, PLGA microparticles exhibited a slower release rate, for which
35
Journal of Controlled Release 295 (2019) 31–49
only 8% has been released, while it is 30% with PLA, after 3 days. It
indicated that the release of protein strongly depended on the diffusion
through small channels right from the initial stage, in which the re-
duction in molecular weight has a lower impact on the structure of
microparticles. Along the degradation, a critical increase of porosity
likely induced a higher release rate.
1.2.4. Effect of parameters on polymeric microparticles 1. Interface
stabilization by chelation
As discussed above for W/O/W double emulsions, the formation of
chelates at the W/O interface has a strong stabilization effect on the
resulting double emulsion. Similarly it has also been observed in the
fabrication of polymeric double droplets, in addition to an important
impact on the drug release kinetics.
Nihant et al. [57] studied the influence of different parameters on
the properties of PLA microparticles. BSA was chosen as stabilizer for
the inner aqueous phase. The BSA concentration showed an important
influence on the microparticle morphology, differentiating three types
as depicted above in Fig. 3. Yang et al. [8] prepared PCL microparticles
using PVA this time (instead of BSA) in order to strengthen the inter-
face, owing to the higher hydrophobicity of PCL (compare to PLGA).
The effect of PVA concentration in inner phase was investigated on the
inner structure of microparticles. It was shown that low PVA con-
centration in inner phase (0.025%) induced a higher rate of internal
droplet coalescence giving rise to a relatively low encapsulation effi-
ciency around 42.8%. On the other hand, a higher PVA concentration
(0.5%) prompted a significantly increase of the encapsulation efficiency
up to 70 wt% (Fig. 7 (I)), likely related to the internal water interfacial
polymer coverage.
1.2.5. Effect of parameters on polymeric microparticles 2. different volume
ratios, concentration of polymer
Different volume ratios of inner phases, middle phase and con-
centrations of polymer were studied. When a lower volume ratio and a
higher concentrated polymer solution was applied, it appeared more
difficult to break bigger global droplets into small ones during the
second emulsification step. These experimental conditions resulted in
an increase of the size of the microparticles. When BSA was used as a
marker to investigate the encapsulation efficiency and loading rate of
microparticles, higher concentrations of BSA were found to increase the
loading rate (the ratio of mass of encapsulated molecule and mass of
particles) while the encapsulation efficiency was decreased, which re-
sulted from a higher loss of BSA during the emulsification. Finally, the
size of microparticles was controlled between 60 and 120 μm, the en-
capsulation efficiency was modified from 40 to70 wt% respectively [8].
Moreover, different volume ratios of inner and middle phase were
investigated related to the morphology and inner structure of micro-
particles (Fig. 7 (III)). The higher ratio of inner phase and middle phase,
S. Ding et al. Journal of Controlled Release 295 (2019) 31–49

Fig. 5. Evolution from double emulsions to solid

PLGA microparticles and relation between the dia-
meters of two phases and type of microparticles (I) a.
Double emulsions synthesized for PLGA at 10% w/v,
1% w/v, 500 rpm by homogenizer for second step
(II) a. Double emulsions synthesized for PLGA at 5%
w/v, 1% w/v, 8000 rpm by homogenizer for second
step. All SEM micrographs b. correspond to the same
sample in a after solvent evaporation [55].

Fig. 6. (I) Picture of typical PLGA microparticles

prepared with the modified two-step emulsification
method by phase contrast light microscopy [56]. (II)
Typical degradation processes of PLGA micro-
particles prepared with the two-step emulsification-
evaporation methods without hydrophobic surfac-
tant by SEM, (A) immediately after preparation, (B)
after 1 day, (C) after 4 days, (D) after 7 days, (E),
after 14 days (F) and after 76 days [56]. (III) Typical
polymer degradation rate of mciroparticles prepared
with the two-step emulsification-evaporation
methods between PLA and PLGA [52,56]. (IV) Cu-
mulative release of tetanus toxoid from microspheres
prepared with different polymers, ○, PLA (Mw
50,000); ▲, PLGA (Mw 100,000); ●, PLA (Mw
3000) [52,56].

36
S. Ding et al.
Fig. 7. (I) Effect of different concentrations of hydrophilic surfactant (PVA) in inner phase on the distribution of drug (BSA) inside PCL (Mn 10,000) microparticles
(A) 0.025 wt% (B) 0.1 wt% [8]; (II) cross-sectional of PLGA microparticles by SEM with different temperatures [58]; (III) Surface morphology and inner structure of
PLGA microparticles with different ratio of inner phase and solvent phase [59];(IV) Size distribution of PLGA microspheres with respect to the temperature during
solvent evaporation [58]
the higher porosity of the surface and inner network. Besides, in-
creasing the volume ratio results in the increasing the globule size from
61.4 to 81.9 μm, slightly affecting the encapsulation efficiency, but
highly impacting on the burst release that increased from 9.75 to 76.6%
[58,59].
1.2.6. Effect of parameters on polymeric microparticles: 3. Evaporation
temperature
Yang et al. [58,59] reported on the influence of solvent evaporation
temperature on the property of PLGA microspheres during the solidi-
fication process. The whole process was recorded by microscope. The
size and size distribution of microparticles increased with temperature.
When the range of temperature was fixed from 5 °C to 42 °C, the size of
the microparticles changed from 88 to 130 μm respectively (Fig. 7 (IV)).
It was explained that the global droplets kept the low viscosity solution
during evaporation at low temperature until the critical concentration
of polymer in organic solvent was reached. At this critical temperature,
the low viscous solution transformed into solid state. Thus, low visc-
osity solution of droplets had ability to keep homogeneous size under
shearing force. In contrast, as the microparticles rapidly transformed
37
Journal of Controlled Release 295 (2019) 31–49
from a low viscosity solution to a high viscosity state at higher tem-
perature and the rapidly evaporation of solvent, the size and size dis-
tribution of microparticles have been significantly influenced by
shearing forces induced by stirring. In addition, the morphology of
microparticles was similarly investigated with different temperature. It
was observed that the prepared microparticles had a porous structure. It
resulted from the low concentration of PLGA (3% w/v) and PVA (0.05%
w/v) in inner phase. More porous and thicker polymeric layer were
obtained at lower temperature, whereas the microparticles prepared at
higher temperature presented a more homogeneous polymeric layer
(Fig. 7 (II)).
1.2.7. Polymeric microcapsules
Though the polymeric microparticles have been produced by two-
step emulsification method, the preparation of polymeric micro-
particles with single inner core remained quite complicated. Gao et al.
[60] firstly adopted the coalescence of inner droplets after preparation
of double emulsions to obtain the polymeric microcapsules by batch
method, in which the method was denoted as “emulsion ripening”
(Fig. 8). To prevent the breaking of the middle phase during the
S. Ding et al.
Fig. 8. (a) Flowchart of the preparation of templated double emulsion microcapsules, (b) evolution of transformation of double emulsion with different ripening
times [60].
ripening process of the emulsions, the middle phases were polymerized
and crosslinked with methyl methacrylate as monomer, trimethylol-
propane trimethacrylate as crosslinker and Azobis(2,4-dimethylvaler-
onitrile) as radical photoinitiator.
1.2.8. Double emulsions by microfluidic systems
Microfluidic technologies have been widely developed over the past
decades. They have been the cornerstone of intensive research for the
production of particles owing to finely controlling segments of fluids.
Due to a vigorous mixing in batch processes, the turbulent shear force
resulted in a broad size distribution of the inner water droplets and
double structure droplets. In addition, it is difficult to precisely control
the number of inner droplets in the oil droplets by conventional
methods [61]. However, the control of the number of inner droplets is
very important to control the release rate of encapsulated molecular.
Thanks to the precise manipulation of droplets and producing droplets
of uniform size, there is an increasing interest to produce double
emulsions by microfluidic methods [62,63].
Nisisako et al. [64] firstly reported the formation of monodisperse
double emulsions prepared by microfluidic systems with two T-shaped
microchannels and Pyrex glass as chip materials, in which W/O emul-
sions and W/O/W emulsions were formed at a first T-shaped micro-
channels and second T-shaped microchannels, respectively (Fig. 9 (I)).
In this work, two types of microfluidic chips were fabricated to produce
double emulsions. 1) A microfluidic chip composed of two T-shaped
microchannels with different surface properties (Hydrophilic or hy-
drophobic surface); 2) Two T-shaped chips with different surface
properties joined with Polytetrafluoroethylene (PTFE) tubes. To obtain
38
Journal of Controlled Release 295 (2019) 31–49
a hydrophobic surface of T-junction, the surface was treated by a saline-
coupling agent to prevent a phase inversion during the preparation of
W/O emulsions (water phase flowing along the wall of the device). As
results, the number of water droplets was controlled in one oil globule
by modifying the flow rate of middle phase from 12 to 1 mL/h with
such microfluidic system. The size of water droplets also was modified
from 10 to 45 μm, and the oil globule size was controlled from 95 to
220 μm (Fig. 9 (II)).
Weitz et al. [7] designed a microfluidic system, which was based on
flow focusing [65] and selective withdrawal technique [66], to syn-
thesize double emulsions by assembling cylindrical glass capillary
nested within a square glass tube (Fig. 9 (III)). The inner and middle
phases were pumped through the capillary tube and around the capil-
lary respectively while the external phase was pumped through the
square glass tube with opposite direction. The double emulsions were
delicately formed at the entrance of the collection tube. Through the
assembly of different sizes capillaries and collection tubes, different
morphologies of double droplets can be prepared (Fig. 9 (IV)). The
thickness of middle phase in the double emulsion can be also modified.
In addition, the design actually prevented the device from partial sur-
face modification.
To increase the production rate, the parallelization of several single
microfluidic system was commonly adopted [68]. Different types of
microfluidic systems based on the aforementioned two microfluidic
systems have been developed to address different drawbacks in mi-
crofluidic systems such as partial surface modification [69,70] or low
production rate [71–73]. Those microfluidic systems for the production
of double emulsions have been reviewed by Chong et al. [15].
S. Ding et al. Journal of Controlled Release 295 (2019) 31–49

Fig. 9. (I) Basic concept for preparing double emulsions (W/O/W) using T-shaped microchannels [64]; (II) different types of double emulsion synthesized by T-
junction microfluidic system [64]; (III) Schematic diagram of the coaxial microcapillary fluidic device [7]; (IV) different types of double emulsions synthesized by
coaxial microcapillary fluidic device [7]; (V) Microcapsules prepared by microfluidic coaxial flow method. Arrows represent the dewetting phenomenon [67].

Moreover, due to the easy control of the fluid elements, the preparation which have been applied to prepare anisotropic particles by double
of double emulsions showed exceptional flexibility in obtaining other emulsions [84].
types of particles [74], such as lipid vesicles [75], mesoporous hydro-
xyapatite [76], polymersomes [77], microgels [78,79], gas-filled mi-
1.3. State-of-the-art and perspective of double emulsions
croparticles, non-spherical colloidosomes [80], core-shell particles
[81], hollow particles [82] and high-order multiple emulsions [61].
1.3.1. Double emulsions in state-of-the-art for nanocarriers
To show the versatility of microfluidic systems, Weitz et al. [67]
As delving into many diseases such as Cancer, Alzheimer's disease
firstly prepared homogeneous polymeric microcapsules with single
and so on, drugs have been asked to be delivery into specific tissue even
inner core using a microfluidic coaxial flow method (Fig. 9(III)).
single cell. It brought the new challenge for pharmaceutists and biol-
Polymeric microcapsules have been produced with two types of diblock
ogists. Due to the potentiality of nanocarriers in these aspects, the en-
polymers: polystyrene-block-poly(ethylene oxide)(PS-PEO) and PBA-
capsulation of hydrophilic molecules (such as insulin, protein and DNA)
PAA poly(normal-butyl acrylate)-poly(acrylic acid) [83]. The research
in nanocarriers has stimulated an extensive research activity. Blanco
revealed the solidification process of microparticles by microscope,
et al. firstly modified the two-step emulsification method to get nano-
which help deeply understanding on the origin of transformation. The
carriers [85,86]. The typical picture of PLA nanoparticles prepared is
forming of microcapsules resulted from the polymeric shell, which
presented in Fig. 10(I). For preparation of nanoparticles, high-energy
slowly separates from the organic solvent, which was coined by de-
emulsification methods such as ultrasonication, microfluidic emulsifi-
wetting phenomena (Fig. 9(V)). The different concentrations of poly-
cation, were used instead of the homogenization or mechanical stirring.
mers in organic solvent resulted in different results, which have been
Thus, BSA-loaded PLGA nanoparticles have been successfully prepared
classified into three categories: low concentration (0.01 wt%), middle
from 320 nm to 530 nm depending on the molecular weight of PLGA. It
concentration (0.1 wt/%) and high concentration (1–1.5 wt%). Firstly,
showed us that the two-step emulsification has the ability to produce
it was not possible to form stable polymeric microcapsules at low
polymeric particles at the nanoscale.
concentrations. When the concentration of polymer was increased up to
Compared to microparticles, the molecular weight and the structure
the middle concentration, stable microcapsules were obtained due to
of polymer have become the most important key on the properties of
the dewetting phenomena along with the resolubilization of polymeric
nanocarriers, which strongly relate to interaction between molecules.
barrier. At high concentrations of polymer, the microcapsules will be
Thus, the researches have been focused on the influence of the polymer
breakup into smaller microcapsules due to the dewetting instability,
molecular weight. Tobío et al. [86] used poly(lactic acid)-b-poly

39
S. Ding et al.
Fig. 10. (I) TEM microphotograph of typical BSA-loaded PLGA nanospheres [85]; (II) TEM microphotograph of the nanocapsules prepared via the double emulsion
approach [10].
Fig. 11. Evolution of the relationship between double emulsions and process.
(‫ )׀‬Double emulsions prepared by one-step emulsification with hand-shaking
[16]; (‫ )׀׀‬Double emulsions prepared by two-step emulsifications with me-
chanical stirring [93]; (‫ )׀׀׀‬Double emulsions prepared by two-step emulsifica-
tions with small vibrating mixer [31] (‫׀‬v) Double emulsions prepared by Ul-
traturrax (X1020, Ystral) [95] (v) Double emulsions prepared by a fractionated
crystallization technique [97] and Ultraturrax [25]; (v‫ )׀‬Double emulsions
prepared by extrusion of a primary emulsions prepared by method (‫ )׀׀‬through a
porous membrane [93]. (v‫ )׀׀‬Double emulsions prepared with pin-mixer and
Ultraturrax (Tokushu Kikakogyo Co., Japan) for W/O and W/O/W emulsions,
respectively [20]. (v‫ )׀׀׀‬Double emulsions prepared bya couette mixer [99]. (‫׀‬x)
Double emulsions synthesized by Microfludizer [102].
(ethylene glycol) (PLA-PEG), a block copolymer, to encapsulate tetanus
toxoid (TT, a model protein antigen) by the modified two-step emul-
sification method. PLA-PEG polymer nanoparticle size was controlled at
40
Journal of Controlled Release 295 (2019) 31–49
142.8 nm compared to PLA nanoparticle at 153 nm. Bonneaux et al.
[87] used a two-step ultrasonication method to prepare double emul-
sions by using different molecular weights of PLA (from 1938 to
90,000 Da) and varying different process parameters. In results, using
rotating evaporator did not influence the size of the nanoparticles at
room temperature. Finally, the size of PLA nanoparticles reached
around 200 nm. Human Serum Albumin (HSA) was used as an en-
capsulated molecule to calculate encapsulation efficiency, which varied
from 20 to 30% by increasing volume ratio of inner phase. As a result,
different phenomena are observed for PLGA and PLA nanoparticles
with respect to different molecular weights. The nanoparticles size in-
creased with a decrease in the molecular weight of PLA and an increase
in the molecular weight of PLGA. It was explained that the nanoparticle
sizes depend on the property and the molecular weight of polymer at
same time. PLA has more hydrophobic property than PLGA. Lowering
the molecular weight of PLA or heightening the molecular weight of
PLGA resulted in more hydrophilic property of polymer, which fa-
cilitated the faster exchange of polymer between global droplets. Ma
et al. [88] conjugated bis(β-cyclodextrin) (7-membered sugar mole-
cular ring, commonly used as solubilizing agents to increase water-so-
lubility of lipophilic compounds and enhance bioavailability of drug)
into PLGA to encapsulate BSA by two-step emulsification method with
sonication. It was found that the encapsulation efficiency, ranging from
80 to 90% wt., has apparently increased compared to the encapsulated
HSA PLA nanoparticles (encapsulation efficiency of 20 to 30% wt.) and
encapsulated HSA PLGA nanoparticle (encapsulation efficiency at
around 60% wt.). However, the smallest size of nanoparticles was
around 300 nm. It is to be noted that the nanoparticles were synthesized
with sonication (30 W), bigger sizes might be resulting from the applied
lower energy during the process.
In addition, Doelker et al. [89] studied the influence of sonication
parameters on the preparation of nanoparticles. The exposure time and
energy of sonication were systematically investigated. Simultaneously,
the nanoparticles were prepared by vortex mixing instead of sonication
at each emulsification step. The results were compared with the na-
noparticles prepared only with sonication. It was observed that the
exposure time at the second emulsification step has a greater influence
on the nanoparticle size than the one at the first step emulsification.
The energy of sonication has two thresholds for the preparation of the
nanoparticles. On one hand, if the applied energy is less than the low-
energy threshold, this one cannot produce nanoparticles. On the other
hand, if the applied energy is higher than the high-energy threshold, it
will result in a high polydispersity index of nanoparticles due to the
non-uniform energy dissipation. In this paper, the threshold was found
between 20 and 65 W. Concerning the use of a vortex mixing during the
first step emulsification, similar nanoparticle sizes to those produced
 Table 1
Formulations, processes and properties for double emulsions in literatures.
S. Ding et al.

Author Year Oil Encapsulated Inner phase Inter phase External phase Process Size of W/O Inner phase: Size of W/ W/O Encapsulation
molecule emulsion Inter phase (v/ O/W emulsions: efficiency (wt
v) emulsion External phase %)
(v/v)

R.H. Engel [19] 1968 Trioctanoin Insulin ZnCl2 (0.003 M) Palmitic acid (0.03 M) in Sodium lauryl Sonifier N/A 7.2:10.8 N/A 4.5:13.5 l N/A
Insulin (100 u/ml) trioctanion sulphate (1 w/v %)
solution solution
Sachio 1976 Liquid Glucose Glucose 0.5 wt% Span-80 (30 wt%) in liquid Tween-20 0.5% Pin-mixer for W/O 2 μm 72:28 about 2 μm 51:49 90
Matsumoto paraffine aqueous phase paraffin aqueous solution emulsions;
[20] (same as light Ultraturrax for W/
mineral in O/W emulsions.
wiki)
A. T. Florence 1981 Isopropyl N/A Water Oil containing Span 80 Water containing Small vibrating 2–3 μm 1:1 8–25 μm 1:1 N/A
[31] myristate (5 wt%) 2% wt/wt mixer
surfactant as
follows: Brij 30,
Triton X-165, or a
3:1 Span 80:Tween
80 mixture.
Nissim Garti 1983 Paraffinic oil Chloropromazin Aqueous drug Paraffinic oil containing Water(3–5 wt%, Magnetic stirring 0.5–3 μm 3:7 5–15 μm 3:7 10
[23] hydrochloride solution (55 mg/ 8–10 wt%, of oil soluble emulsifier) Span
ml of the drug) emulsifiers 20 + Tween 80,
Triethanolamine
+ Oleic acid (1:1
wt)
Nissim Garti 1984 Light mineral NaCl NaCl solution (1 wt Brij 92 in light mineral oil Span 20 + Tween Homogenization N/A 30: 70-x (Here, N/A 20:80-X (wt%) 30

41
[27] oil %) 80 (HLB = 11) (Silverson x represent the Here, x
glucose solution homogenizer) for concentration represent the
(5.5 wt%) W/O emulsions, of surfactant) concentration
(equalizing the Magnetic strring for of surfactant
osmotic pressure W/O/W emulsions
caused by the
NaCl.)
A.T. Florence 1986 Isopropyl NaCl BSA (0.05–2.0 wt Span 80 (1–10 wt%) in Water containing Whirlimixer 2–3 μm 1:1 8–25 μm 1:1 50–80
[32] myristate %) NaCl 1.25 wt% isopropyl myristate 1% w/v Tween 80
solution adding sorbitol to
the external
aqueous phase
Kamlesh P. Oza 1989 Heavy NaCl Avicel RC591 Span 80 (2 wt%) or Span 85 CMCC (2 wt% or Polytron N/A 1:9 or 4:6 24–30 μm 1:4 or 1:1 N/A
[37] mineral oil colloidal in heavy oil phase 1 wt%) dispersion Homogenization
microcrystalline
cellulose (CMCC
2 wt% or 1 wt%)
solution
Yoshiaki 1991 liquid paraffin NaCl NaCl 0.02 M 70 wt% liquid paraffin and Tween 20 (0.5% Mechanical stirring 2.25 μm or 5:3 3.64 μm or 1:1 67–98
Kawashima aqueous solutions 30 wt% w/v) aqueous (390 rpm) for W/O 2.5 μm 4.41 μm
[93] Span 80 solution emulsions, 630 rpm
for W/O/W
emulsions.
Prepared W/O
emulsions or W/O/
W emulsions is
extruded by porous
membranes
(continued on next page)
Journal of Controlled Release 295 (2019) 31–49
 Table 1 (continued)

Author Year Oil Encapsulated Inner phase Inter phase External phase Process Size of W/O Inner phase: Size of W/ W/O Encapsulation
S. Ding et al.

molecule emulsion Inter phase (v/ O/W emulsions: efficiency (wt

v) emulsion External phase %)
(v/v)

Takayuki 1993 liquid paraffin Secretin Sodium Span 80 (10% w/v) in PSML 20 (1.5% w/ Ultraturrax with U- 100–200 nm 8:5 (v/v) 14–20 μm 1:1 N/A
Ohwaki alkylsulfonate in liquid paraffin v) buffer solution shaped blade
[95] buffer(PH 6.33),
sodium chloride
(0–1.8% w/v)
coccine (0.2% w/
v) or Secretin
(0.064% w/v)
solution
Nissim Garti 1994 Paraffinic oil NaCl NaCl solution Span 80, ETD, PGPR Or Hydrophilic poly Ultraturrax Below 3:7 4.5 μm 1:4 N/A
[30] Grafted silicone lipophilic (siloxane)-graft- homogenizer 0.5 μm
surfactant (Silicone-based poly(oxyethylene) (8000–24,500) rpm
poly(ethylene-glycol) solution, for W/O emulsions,
copolymer in paraffinic oil, concentration of Microfluidizer for
concentration of surfactant surfactant set 2 wt W/O/W emulsions
set at 10 wt% %
Chong-Kook Kim 1995 Liquid Cytarabine Cytarabine Span (30 wt%, Span 20, 80) Tween (0.5 wt% Ultrasonicator N/A 2:5 1.3–1.6 μm 1:4 60–80
[94] paraffin solution in liquid paraffin Tween 20, 80)
solution
F. Leal-Calderon 1998 Dodecane NaCl NaCl (0.1 M) in Span 80 in the dodecane SDS, TTAB Mechanical stirring 400 nm 1:1 After 10 μm 1:9 N/A
[25] water (1:1 Weight mixture). (0.0008 mol/l) and then Fractionated diluted in
Tween 80 in water crystallization dodecane to
technique for W/O 10% by volume

42
emulsions,
Ultraturrax for W/
O/W emulsions
Hideaki Okochi 2000 The mixture Vancomycin VCM (5% w/v) HCO-40 (5%) in the Pluronic F-88 Complex of 250–500 nm 1:4 20–100 μm 1:1 55–90
[96] of several oils (VCM) saline solution mixture of soybean oil (0.5 wt% ~5 wt%) homogenization
(70 wt%) and Lipiodol solution and Ultrasonicator
(30 wt%) for W/O emulsions,
Homogenization for
W/O/W emulsions
F. Leal-Calderon 2001 Dodecane or NaCl NaCl (0.2 M) in Admul Wol 1403 (10 wt%, NaCl solution or Couette-type mixer 0.3 μm 2:3–3:1 2–7 μm 7:3 or 3:2 95
[99] Commercial water Polyrycinoleate of glucose (0.4 M).
sunflower oil polyglycerol), Arlacel p135
(Stora) (polyethylene-30
dipolyhydroxystearate);
Arlacel 186 (glyceryl
monooleate) in oil phase
Nissim Garti 2002 Mixture of Vitamin B1 Glucose (16.7 wt PGPR (11.4 wt%) in Whey protein Complex of N/A 3:47 w/w 3–4 μm 1:5 w/w 60–90
[39] mono %) aqueous phase Mixture of mono glyceride isolate (WPI homogenization
glyceride oleate (GMO 2.8 wt%) and 2–6 wt%) and and High pressure
oleate (GMO) medium chain fatty acid Xanthan gum (0.1 homogenizer for W/
and medium trglycerides (MCT, or 0.5 wt%) water O emulsions, High
chain fatty C8~C10) solution pressure
acid Homogenizer for
trglycerides W/O/W emulsions
(MCT,
C8~C10)
(continued on next page)
Journal of Controlled Release 295 (2019) 31–49
S. Ding et al. Journal of Controlled Release 295 (2019) 31–49

Encapsulation with the sonicator were obtained. It suggested it is not necessary to use
efficiency (wt sonication for both emulsification steps. In contrast, replacing sonica-
tion by vortex mixing at second step cannot produce individual nano-
particles as polymer aggregation. To summarize the nanoparticle pre-
N/A
%)

paration mainly depends on the emulsification method and processing

parameters [56,90]. Fessi et al. [9] adopted PCL to synthesize nano-
External phase

particles by two-step of sonication (500 W, 20 kHz). They also studied

emulsions:

the influence of the exposure time of sonication during the first step
emulsification and the second step emulsification on the size of the
(v/v)
W/O

1:10

obtained polymeric nanoparticles. These experimental studies allowed

to conclude that the exposure time of sonication at the first step has not
Size of W/

0.7–2 μm
emulsion

significant influence on the size of the obtained nanoparticles. Also, it

O/W

was observed that, the size of the PCL nanoparticles decreased with the
exposure time of sonication during the second step emulsification. So-
nication frequency, PVA concentration in the external phase, PCL
Inter phase (v/
Inner phase:

concentration in the middle phase and the volume of external phase

1:1 or 1:5

have been investigated. All of these parameters in the two-step emul-

sification have a maximum limitation of influence on the size of par-
v)

ticles. Over the threshold, the nanoparticle sizes cannot be further de-
Size of W/O

creased. Finally, from this study, the smallest PCL nanoparticles were
emulsion

synthesized at 219 nm.

0.1 μm

Aforementioned polymeric nanoparticles were considered as nano-

spheres, which included different size of hydrophilic vesicles; not only
one, dispersed into the matrix of the polymeric network. In contrast,
Homogenizer or

Gao et al. [10] showed nanocapsules which have one hydrophilic core
polycarbonate

emulsification

in the polymeric network. These nanocapsules were prepared by two-

sonication,

membrane

step emulsification methods (Fig. 10(II)). The more complex structure

Process

of nanocapsules has been demonstrated by carefully choosing lipids as

surfactants in double emulsions. Clearly, the double structure has been
proven by negative staining TEM [91].
External phase

NaCl (0.1 M)

1.3.2. Emulsification methods for the future of double emulsions

In summary, different materials have been used for the middle
solution

phases such as mineral oil, vegetable oil or polymer. Different para-

meters in process and materials presented huge influence on the phy-
sicochemical and encapsulation properties, release profile of the en-
Arlacel P 135 dodecane

capsulated product. The two step emulsification process, thanks to the

easy modulation of the formulation parameters (interfacial properties,
volume ratio), will allow obtaining very controllable double emulsions.
Among considerable properties of double emulsions, the suitable size
Inter phase

and size distribution are prerequisite parameters for potential applica-

solution

tions in pharmaceutics. These properties can influence many functions

including encapsulation efficiency, degradation, flow properties,
clearance and uptake mechanisms of carriers [92]. From our point of
(9*10−5 M) water

view, the emulsification method during two-step emulsification sig-

NaCl (0.1 M)
Inner phase

nificantly affect the size and size distribution of the double emulsion.
solution
Dextran

Based on this consideration, the development of emulsification methods

should attract much attention from researchers, because it will defini-
tively affect the characteristics of the final product. To clarify the de-
velopment of emulsification, the relation between emulsification
Encapsulated

methods and size of carriers have been clearly shown in Fig. 11. Be-
sides, for more details information of formula, all of results are clearly
molecule

shown in Table 1. The double emulsions, firstly, have been observed by

N/A

William Seifriz [16]. Hand shaking was used to prepare double emul-
sions in (Fig. 11(I)). The results presented a coarse emulsion. There-
after, in the early stage, double emulsions were prepared by the simple
Dodecane

mechanical stirring (Fig. 11 (III)), in which the detail information on

equipment are presented in Table 2. The size of such double emulsions
Oil

ranged from 5 to 30 μm. The double emulsions size strongly depended

2006
Year

on the stirring rate. Kawashima et al. used an extremely low stirring

Table 1 (continued)

rate such as 390 rpm and 630 rpm to prepare W/O emulsion and W/O/
W emulsions, respectively [93].In this last example, the size of double
Hu Gang [101]

emulsions was large and polydisperse (8 to90 μm, Fig. 11 (II)). On the
contrary, Matsumoto et al. [20] declared that the size of synthesized
Author

double emulsions can achieve 2 μm by using a special pin-mixer and a

high speed homogenization (Fig. 11(VII)). However, these authors

43
 Table 2
Formulations, processes and properties for polymeric microparticles in literatures.
S. Ding et al.

Author Year Organic solvent Polymer Process of emulsification Encapsulated molecule Inner phase Inter phase

Yasuaki Ogawa [53,54] 1988 Dichloromethane PLA (Mw 22,500), PLGA Homogenizer for W/O leuprolide acetate Leuprolide acetate(8% PLA or PLGA
(Mw 14,000) emulsions, turbine- w/v) water or gelatin dichloromethane
shaped mixer for W/O/ solution solution
W emulsions
Robert Langer [56] 1991 Methylene PLGA (75:25 Mw 5000, Vortex mixer or probe Fluorescein Protein (20% w/v) PLGA (100% w/v)
chloride 10,000, 14,000) sonicated for W/O isothiocyanate-lablled solution methylene chloride
emulsions. Magnetic bovine serum albumin solution
stirring for W/O/W and FITC-labelled
emulsions horseradish peroxidase
Robert Langer [52] 1993 Methylene PLA (Mw 3000, 50,000) and Sonication (50 W 10 s) Tetanus toxoid Tetanus toxoid solution Polymer (PLA
chloride PLGA (50:50 Mw 100,000 for W/O emulsion, 20–50% w/v PLGA
Resomer RG 506) Homogenization at 10–20% w/v)
15000 rpm for 10 s ethylene chloride
solution or Polymer
L-α-
phosphatidylcho-
line (0.3% w/v) in
chloroform
Nicole Nihant [57] 1994 Methylene PLA (Mn 51,000) Ultraturrax for W/O BSA BSA solution PLA (9 wt%)
chloride emulsions, A four- methylene chloride
pitched blade impeller with or without
(800 rpm) for W/O/W Pluronic F68
emulsions
M.J. Alonso [84] 1997 Ethyl acetate PLGA (50:50 Mw 15,000 Sonication (15 W 15 s) FITC-BSA FITC-BSA (4% w/v) PLGA (20% w/v)
Resomer RG 502, Mw solution ethyl acetate

44
43,000 RG 503 and Mw solution
50,000 503H)
M.J. Alonso [85] 1998 Ethyl acetate PLA (Mn 50,000) PLA-PEG Sonication (15 W 15 s) Tetanus toxoid Tetanus toxoid (10% w/ PLA or PLA-PEG
(PLA Mn 45,000 PEG Mn v) solution or gelatine (5% w/v) ethyl
5000) (2%) added as stabilizer acetate solution
F. Bonneaux [87] 1998 Methylene PLA (Mw 90,000, 50,000, Sonication HSA Water or Human serum PLA (2.5 wt%)
chloride 17,400, 1938) albumin (HSA 2%) methylene chloride
solution used as a 20% solution
injectable solution)
Yi-Yan Yang [58,59] 2000 Dichloromethane PLGA(65:35 Mw Sonication for W/O BSA BSA (4% w/v) PLGA (3% w/v)
40,000–75,000) emulsions, Mechanic phosphate-buffered methylene chloride
stirring for W/O/W saline (PBS) (pH 7.4) solution
emulsions solution containing PVA
(0.05% w/v)
Eric Doelker [89] 2003 Ethyl acetate PLGA (50:50 Mw Sonicator (20–65 W Methylene blue Water PLGA (20% w/v)
34,000 Da, Resomer RG 2–20 s) for W/O ethyl acetate
503) emulsions, Sonicator/ solution
Vortex for W/O/W
emulsions
Iosif Daniel Rosca [55] 2004 Dichloromethane PLGA(50:50 Mw Homogenizer for W/O N/A PVA (1% w/v) water PLGA
45,000–75,000) emulsion, or solution dichloromethane
Homogenizer or solution
Mechanic stirring W/O/
W
(continued on next page)
Journal of Controlled Release 295 (2019) 31–49
 Table 2 (continued)

Author Year Organic solvent Polymer Process of emulsification Encapsulated molecule Inner phase Inter phase
S. Ding et al.

Omid C. Fraokhzad [91] 2011 Dichloromethane PLGA (Viscosity of Sonication for W/O Small interference RNA Water PLGA, 1,2-
0.26–0.54 dL/g) emulsions, W/O/W (siRNA) Dimyristoleoyl-sn-
emulsions glycero-3-
ethylphospho-
choline(EPC 14:1)
dichloromethane
solution
XiaoHu Gao [10] 2012 Chloroform Poly(styrene-allyl alcohol), N/A Dye-labelled plasmid PVA, MW 9000 PS16-PAA10, Oleic
PS16-PAA10, Mw 2200) DNA, Water-soluble acid Solution
quantum dots,
Doxorubicin

Author External phase Diluted phase Inner phase: Inter W/O emulsions: Double emulsion/ Size of polymeric Encapsulation
phase (v/v) External phase (v/ Diluted emulsion particles efficiency (wt%)
v) (v/v)

Yasuaki Ogawa [53,54] Polyvinyl alcohol N/A 1:4 1:8 N/A 125,88, 74, 44, and 2–70
(0.5%) solution 37 μm

Robert Langer [56] PVA (1%) solution PVA (0.1%) 1:20 1:2 1:100 55–99 μm 60–90
solution
Robert Langer [52] PVA(1%) solution PVA (0.1%) 1:20 1:1 1:100 10–50 μm N/A
solution
Nicole Nihant [57] PVA (2.5 wt%) PVA (0.1%) PBS 1:5(v/g) 1:25 (v/g/v) N/A > 100 μm N/A
solution solution

45
M.J. Alonso [84] PVA (1% w/v) PVA (0.3% w/v) 1:20 1:2 1:50 320–530 nm 20–70 (with PVA
obtained 20 wt%)
M.J. Alonso [85] Sodium cholate (1% Sodium cholate 1:20 1:2 1:50 130–150 nm 30–35
w/v) solution (0.3% w/v)
F. Bonneaux [87] PVA (5% w/v) PVA (0.1% w/v) 1:20 1:2 1:20 200 ± 5 nm 20–30
solution solution
Yi-Yan Yang [58,59] PVA (0.05 wt%) 640 ml PBS with 1:24 1:20 25:64 88–130 μm 58
PBS solution 0.05% PVA
Eric Doelker [89] PVA (2%) solution PVA (0.3%) 1:20 1:2 1:5 250–350 nm 16–20
solution
Iosif Daniel Rosca [55] PVA solution 500 ml of 0.1% w/v 1:10 1:6 1:16 10 μm N/A
PVA solution
Omid C. Fraokhzad [91] 1,2-Distearoyl-sn- DSPE-PEGm and N/A N/A N/A 225 nm 78–82
glycero-3- Lecithin solution
phosphoethanola-
mine-N-methoxy
(polyethylene
glycol) (DSPE-PEG)
and Lecithin
solution
XiaoHu Gao [10] PVA solution N/A N/A N/A N/A 260–290 nm 60 for DNA,
20–53 for
Doxorubicin
Journal of Controlled Release 295 (2019) 31–49
S. Ding et al. Journal of Controlled Release 295 (2019) 31–49

thought that the obtained results were originated from the incorpora-

Size of W/O/W
tion of high concentrations of Span 80 (30% wt.). Further, Kawashima

50–500 μm
et al. [93] introduced a method to decrease the polydispersity and the

emulsion

83 μm
size of double emulsions, in which the double emulsions were firstly
synthesized by a conventional method and then were extruded through
a porous membrane. Finally, the obtained double emulsion was redis-

in one oil globule)

4.7–11.9 droplets
persed in an additional external phase (Fig. 11(VI)). The size of double

52 μm (including
emulsion was decreased down to 3.64 μm. Kim et al. [94] reported

Size of W/O
double emulsions synthesized by two-step emulsification with sonica-

10–50 μm
emulsion
tion. The double emulsion size varied from 1.3 to 1.6 μm. Ohwaki et al.
[95] claimed a size of W/O emulsions ranging from 100 to 200 nm
when they were formulated by using a high pressure homogenizer.

decaglycerol monostearate
Sodium dodecyl sulfate or

(0.5 wt%) water solution

However, the prepared double emulsions after a second emulsification
were still in the micron range (Fig. 11(IV)). Okochi et al. [96] proposed
the concept of the mixed emulsification for the formation of a single

External phase
emulsion. The size of W/O emulsion was around 256 nm by using a
combination of sonication and homogenizer. It was concluded that the
production of fine and homogeneous emulsions can be reached by the

water
combination of multi emulsification process, in which size and size
distribution of emulsions can be reduced step by step. Besides, double

Norland Optical Ahesiive) 30% acetone or

condensed ricinoleic acid ester (1.0 wt%,

Polymer (70% can be photopolymerized

Poly(butyl acrylate)-b-poly(acrylic acid)

emulsions were synthesized by using the membrane emulsification

toluene and tetrahydrofuran (7:3 v/v)

method. The results presented a size distribution profile of membrane

Lecithin (0.5 wt%) or tetraglycerin-

method, which was sharper than the one obtained with the stirring
method (Table 3).
Leal-Calderon et al. [25] used a new technology to get mono-
disperse 400 nm W/O emulsions after a coarse emulsification by me-

(PBA-PAA) (2 wt%)
CR-310) in corn oil
chanical mixing, which was called fractionated crystallization tech-
nique (Fig. 11(V)). However, the method involved multi-purification Inter phase

[97]. Following the pioneering work of Talyor [98], an efficient method

for producing quasi-monodisperse double emulsions was proposed by
using a couette mixer (Fig. 11 (VIII)) [99]. When polyols and PGPR
Formulations, processes and properties for polymeric microparticles prepared by microfluidic system in literatures.

were incorporated in the middle phase, extremely small W/O emulsion

sizes were produced by the combination of a rotor-stator emulsifier and
Water
phase

water
Inner

a high-pressure homogenizer. The size of W/O emulsions (0.2 μm) ob-

tained with this method was much lower compared to the W/O emul-
sions size of 1.8 μm produced without any polyols and that of 0.8 μm
Encapsulated

with high-pressure homogenizer without any polyols [100].

molecule

By high-pressure microfluidizer, a new type of emulsification called

N/A

N/A

a dual-feed process was applied to a second step emulsification to ob-

tain double emulsions. The size of the double emulsion was decreased
the injection tube range from 10 to 50 μm, and the orifice
in the collection tube of the injection tube range from 50
focusing, (Typical inner diameters of the tapered end of
85*35225*100 Two-chip module:80*40 (130−220)*90

down to 3–4 μm with an incorporation of polysaccharides and proteins

Microcapillary device, Based on coaxial flow and flow
Micro-chips which based on T-junction: One-chip and

in the external phase [39]. Yafei et al. [101] reported an extremely

width*depth) Type1:first junction:60*25 μm Second

complicated process to obtain submicron size double emulsion (0.7 to

juntion:130*65 μm;type2: 40*10180*75,type3:
two chip One-chip module (Number represent

2 μm). In this process, the production rate seemed to be excessively low

since a multi-fractionation centrifugation stage was applied after each
step of sequential emulsifications.
In contrast, for polymeric particles, Ogawa et al. [54] firstly in-
troduced the two-step emulsification to encapsulate a hydrophilic drug
in the matrix of polymeric microparticle. Rotor-stator emulsifier and a
Process of emulsification

turbine-shaped mixer were employed for the two-steps. The prepared

microparticles had extremely broad size distribution so that the sieves
had to be used to separate the different sizes of microparticles. As the
development of emulsification methods, more advanced technologies
to 500 μm)

were considered for the preparation of polymeric double particles

which allowed to reduce and narrow the size and size distribution of
microparticles respectively [52]. Encapsulated hydrophilic molecules in
Corn oil

nanocarriers have been also considered. Alonso et al. synthesized na-

N/A

noscale polymeric particles by two-step emulsification with ultra-

Oil

sonication [85]. It was found that the emulsification methods employed

2004

2005
Year

had a great influence on the properties of double emulsions during

process.
D. A. Weitz [83]

Recently, the advantages of microfluidic systems assembling T-type

Takasi Nisisako

microchannel or co-axial capillary have completely solved the poor

[64]

control drawback of the two-step emulsification method for the pre-

Author
Table 3

paration of double emulsions at micron sizes [64]. The microfluidic

systems stimulated the new prospective for a precise control of double

46
S. Ding et al.
emulsions, in which the number of water droplets included in one
global droplet can be accurately controlled by the flow rate of the
middle phase and the inner phase. Nevertheless, the biggest challenge
still lies in the development of efficient methods that can produce na-
noscale double emulsions. To date, for effectively preparation of na-
noemulsions, two methods mainly were involved, namely emulsifica-
tion of premixed emulsions by ultrasonic agitation and high pressure
microfluidic system [103]. Yet, for the preparation of nano double
emulsion, the situation became more complex due to the extra re-
quirement for balancing Laplace-pressure and osmotic pressure be-
tween the inner phase and the middle phase. Thus, it is almost im-
possible to complete this mission, though they have been shown by
means of special surfactants [102] (Fig. 11 (IX)) or gelation of inner
phase [104]. Besides, for controlling drug release or targeting, the in-
volved high-energy processes will damage fragile molecules such as
peptides, proteins, nucleic acids. So, the preparation of nano double
emulsions by the moderate method is the other challenge we face in
future. Flow-focusing [105] and spontaneous emulsification [106] are
known as potential low energy methods, which should be paid attention
to develop moderate two-step emulsification for nano double emul-
sions.
2. Conclusion
Preparation of stable double emulsions with highly controllable
properties has become the development direction of two-step emulsi-
fication methods. However, microfluidic systems have well solved the
broadness of the size distribution and non-controllable structures' pro-
blems at microscale. Furthermore, the complex of protein, poly-
saccharides and polymeric surfactant or pickering stabilizing agents
allowed obtaining stable microscale double emulsions. In contrast,
polymeric materials tend to be the privileged strategy to obtain na-
noscale carriers although some researchers still tried to find methods to
get double nanoemulsions without polymer. To avoid the destabiliza-
tion of the W/O emulsion during the second step emulsification, low
energy methods could be considered in conjunction with an efficient
emulsification method for the first step (like the high pressure micro-
fluidizer). Among low energy methods, the spontaneous emulsification
presents some advantages. The emulsification is promoted by the ad-
dition of an external compound dissolved in the external phase (water)
that will trigger the nanoemulsification and thus can be carried out
under gentle stirring.
Thus, it is believed that the next breakthrough in the production of
double nanoemulsions may come from the breakthrough of emulsifi-
cation methods and complex for stabilization.
Acknowledgments
SD would like to acknowledge the China Scholarship Council for his
Ph.D fellowship. Natural Science Foundation of ShaanXi Province in
China (Grant No. 2018JQ5164 supported this work). Natural Science
Foundation of Educational Department in ShaanXi Province, China
(Grant No. 18JK0114 supported this work).
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