Advanced Nanoemulsions: Further

PC63CH22-Mason ARI 5 March 2012 10:11
ANNUAL
REVIEWS Further
Advanced Nanoemulsions
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3
• Top cited articles California NanoSystems Institute, University of California, Los Angeles, California 90095;
email: mason@chem.ucla.edu
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Annu. Rev. Phys. Chem. 2012. 63:493–518 Keywords

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rupturing
This article’s doi:
10.1146/annurev-physchem-032210-103436 Abstract
Copyright c 2012 by Annual Reviews. Recent advances in the growing field of nanoemulsions are opening up
All rights reserved
new applications in many areas such as pharmaceuticals, foods, and cosmet-
0066-426X/12/0505-0493$20.00 ics. Moreover, highly controlled nanoemulsions can also serve as excellent
model systems for investigating basic scientific questions about soft matter.
Here, we highlight some of the most recent developments in nanoemulsions,
focusing on methods of formation, surface modification, material properties,
and characterization. These developments provide insight into the substan-
tial advantages that nanoemulsions can offer over their microscale emulsion
counterparts.
493
INTRODUCTION
Because nanoemulsions can offer enhanced material properties and versatility, they are replac-
Nanoemulsion ing microscale emulsions in a growing number of applications. Nanoemulsions are metastable
[nanoscale emulsion dispersions of sub-100-nm droplets of one liquid in a different immiscible liquid, and they have
(NEM)]: one liquid been produced for decades using a variety of methods, primarily high-flow emulsification. Recent
dispersed by a
improvements in formulation, emulsification, and characterization are leading to advanced na-
nonequilibrium
emulsification process noemulsions that have desirable functionality and well-controlled physical properties. Although
as sub-100-nm some properties of nanoemulsions resemble those of their larger micron and submicron emul-
droplets in a different sion counterparts, there are a number of important differences that emerge as the droplet size is
immiscible continuous reduced into the nanoscale regime. Mechanical, optical, and transport properties of long-lived,
liquid phase; droplets
metastable nanoemulsions can be tuned and controlled, and other desirable materials, such as
are typically stabilized
against subsequent coatings, biomolecules, and chemical additives, can simultaneously be incorporated. Overall, these
fusion by a surfactant advances provide guiding principles that enable better design of nanoemulsions having desirable
Emulsion: one liquid compositions and properties.

Many basic concepts about emulsions (1–7) apply to nanoemulsions as well. Beyond microscale
dispersed as droplets
in a continuous phase and larger emulsions, several reviews specifically dealing with nanoemulsion systems have been
of a different written within the past decade (e.g., 8–11). Some aspects of the formation and stability of na-
immiscible liquid and
noemulsions have been discussed, focusing on the roles that surfactants can play and on coarsening
typically stabilized by a
surfactant to inhibit of the droplet size distribution. Additionally, low-energy emulsification (12), optimal conditions
droplet coalescence, for emulsification (13), and skincare applications (14) have been discussed. Beyond a review on
thereby forming a simple nanoemulsions (11), the focus of this review is on highlighting some of the most recent and
metastable dispersion exciting developments in nanoemulsions, emphasizing physical methods of formation to further
that can often be
reduce droplet size toward the micellar scale, nanodroplets serving as templates for self-assembly,
long-lived
controlled access to double nanoemulsions via amphiphile design, advanced real-space character-
Surfactant: an
ization, and design of optical and mechanical properties.
amphiphilic molecular
species that Long-lived metastable nanoemulsions are different than thermodynamically stable microemul-
preferentially resides sions, also known as lyotropic microemulsion phases (9, 15, 16). Swollen micellar microemulsions
at droplet interfaces, can resemble nanoemulsions, yet microemulsions have a fundamentally different route of for-
lowers interfacial mation, namely thermodynamic self-assembly, and thus represent a different physical system.
tension, and typically
Typically, nanoemulsions require a route of fabrication that involves energy input, usually in the
provides an interfacial
repulsion between form of fluid flow. Applied fluid stresses overcome interfacial tension σ between the two immisci-
droplets that inhibits ble liquids and rupture larger droplets down to a smaller size, thereby creating a high total droplet
coalescence and surface area per volume (16). Interfacially active components, such as surfactants and cosurfac-
stabilizes an emulsion tants, can be added to reduce the interfacial tension (17) and consequently help to decrease the
Droplet size size of droplets resulting from flow-induced rupturing. In most stable nanoemulsions, at least
distribution: one component within the dispersed droplet phase is insoluble in the continuous liquid phase out-
probability
side the droplets. Typically, a short-range repulsive interaction between surfactant-coated droplet
distribution p(a) of
finding a droplet interfaces inhibits droplet coalescence (i.e., fusion), even over very long time scales (7), making
having a given radius nanoemulsions long-lived metastable states. In contrast, for most microemulsion phases (18, 19),
a, typically measured there is usually a high level of relative solubility of the molecular components, so these systems
as either volume- can spontaneously self-assemble into a variety of nanostructures having sizes and morphologies
weighted or number-
dictated by composition and temperature (20–23). Thus, microemulsion phases are appropriate
weighted, depending
on the type of in equilibrium phase diagrams; however, by the most commonly accepted definition, the term
experiment nanoemulsion should never appear in any equilibrium phase diagram.
A wide range of emulsification methods can be used to produce either oil-in-water (O/W; i.e.,
direct) nanoemulsions or water-in-oil (W/O; i.e., inverse) nanoemulsions. High-flow (i.e., high-
energy) emulsification provides a simple route to forming nanoscale droplets wherein externally
494 Fryd · Mason

applied shear and/or elongational flow overcome interfacial and internal viscous stresses to rupture
bigger droplets into smaller droplets; examples include high-pressure microfluidic homogeniza-
tion (16, 24–26) and ultrasonic emulsification (27–29).
Emulsification: the
Alternatively, a different approach to nanoemulsion formation is through low-flow emulsifica- process by which an
tion. One low-flow method that has been known for decades is the phase inversion temperature emulsion is formed,
(PIT) method (30). In this method, a composition is chosen for the interfacially active compo- typically involving
nent or components such that the interfacial properties of the emulsion change dramatically with flow-induced
rupturing of the
only modest changes in temperature near the PIT. Moving through this temperature, a transition
dispersed phase of
from a direct to an indirect emulsion (or vice versa) may occur. Most often, nonionic surfac- larger droplets into
tants are chosen to achieve this purpose. Within this surfactant class, ethoxylated surfactants have smaller droplets within
typically been chosen because of their inherent ability to become more hydrophobic with in- the continuous phase
creasing temperature (11, 31). A microscale emulsion is then formed using the chosen surfactant in the presence of a
surfactant that inhibits
and brought near the PIT to drastically lower the interfacial tension. At this temperature, the
subsequent
application of low flow, combined with a temperature quench, can be used to produce nanoemul- coalescence
sions (12). A potential drawback of the low-flow PIT and microemulsion temperature quenching
Microemulsion:
methods is that often the resulting nanoemulsion is not very stable over long periods of time, a thermodynamically
because of the relatively high solubility of the components even at the lower final temperature. stable lyotropic phase
For this reason, surfactant displacement methods are being developed to stabilize nanoemulsions that can be composed
(32). of materials similar
to those used for
Many other methods of nanoemulsion formation also exist. These include phase inversion
emulsions; greater
composition (33–36), flow focusing (37, 38), satellite droplets (39, 40), membrane emulsification mutual solubility of
(41, 42), and liquid-liquid nucleation (43–45). Figures 1 and 2 summarize and categorize methods components enables
of nanodroplet creation according to higher throughput or lower throughput, respectively. Some spontaneous
higher-throughput methods often offer the advantage of producing very concentrated nanoemul- self-assembly (not
a nanoemulsion)
sions that have higher droplet volume fractions φ, well beyond the dilute regime.
Nanoemulsions are a subset of what have been traditionally called colloidal emulsions and Coalescence: fusion
of two or more
mini-emulsions (46, 47). Only relatively recently has the nano nomenclature been adopted by
droplets when the
many in place of the traditional colloidal nomenclature. A generally accepted range of lengths, droplets’ surfaces
when using the term nanoscale, is between roughly 1 nm and 100 nm (48). Although one can come into physical
debate whether the radius or the diameter of an undeformed spherical droplet should be chosen contact; coalescence
as the appropriate measure of length, emulsions having radii between approximately 100 nm and causes coarsening of
the droplet size
1,000 nm are at best submicron emulsions, not nanoemulsions. Regardless of the nomenclature
distribution toward
used, nanoscale colloidal emulsions typically exhibit physical properties that are interesting and larger sizes
useful, beyond the range accessible by microscale emulsion systems.
Solubility:
equilibrium
thermodynamic
CONTROLLING DROPLET SIZE AND POLYDISPERSITY concentration of one
molecular species
When designing nanoemulsions, controlling the droplet size distribution, especially the average (i.e., solute) in a liquid
droplet radius <a> and the polydispersity δa/<a> (associated with the effective width of the phase of a different
distribution), is of vital importance. Here, we focus on recent methods for obtaining the smallest molecular species
(i.e., solvent); for
nanodroplets possible.
emulsions, molecules
Ostwald ripening, a form of coarsening, is a process by which the higher Laplace pressure of the dispersed phase
of smaller droplets causes net migration of dispersed-phase molecules from the smaller droplets typically have low
through the continuous phase into larger droplets that have lower Laplace pressures (49, 50). solubility in the
When dispersed-phase molecules have extremely low solubility in the continuous phase, Ostwald continuous phase
ripening is suppressed, and the emulsion does not coarsen. Ostwald ripening of nanoemulsions can
be very rapid compared with microscale emulsions because the differences in Laplace pressures
www.annualreviews.org • Advanced Nanoemulsions 495

High-throughput Process concept Typical Typical

nanoemulsion advantages disadvantages References
and schematic
formation method
Premix Microscale Nanoemulsion 1. Appropriate for a 1. More energy input 16, 24–29
emulsion emulsion wide range of required compared to
,... compositions beyond low-flow methods
the dilute ϕ limit
2. Apparatus can require
, 2. Droplet size significant up-front
Extreme distribution tailored investment
through both flow
emulsification rate and 3. Viscous heating can
compositional control become appreciable at
high-flow rates
Low flow High flow 3. Scalable to very large
volumes
Extreme flow exerts stresses that overcome

interfacial tension, thereby rupturing microscale
droplets into nanoscale droplet
1. Versatility of routes 1. More limited range of 33–36

W/O Lyotropic O/W compositions compared
emulsion microemulsion nanoemulsion a. Vary liquid ratio to high-flow methods
b. Change
[electrolyte] a. Limited ϕ range
c. Mix surfactants b. Limited [electrolyte]
range
Phase inversion 2. Low-energy process c. Limited surfactant
composition 3. Not necessary to types
supply heat 2. More prone to Ostwald
ripening
Low flow is applied as interfacial tension is
reduced, causing, e.g., a transition from W/O to 3. Good mutual solubility
O/W, thereby producing nanodroplets of oil and water is
needed
Lyotropic 1. Low-flow process 1. Limited to mostly 11, 12, 30, 31

microemulsion nonionic surfactants
2. Requires heat energy
input
3. Limited stable T range
Phase inversion 4. More prone to Ostwald
temperature ripening
5. Good mutual solubility
of oil and water is
Reduction in interfacial tension near the PIT needed
enables nanodroplet formation by low flow and
quenching temperature T.
Water droplet Oil Beaker Low-flow mixer Oil surfactant

solution
Aqueous surfactant Oil droplet Ultrasonic High-flow

solution agitation homogenizer
Figure 1
Concept, schematic, typical advantages, and typical disadvantages of several important high-throughput nanoemulsification methods:
extreme emulsification, phase inversion composition, and phase inversion temperature. Abbreviations: O/W, oil-in-water; W/O,
water-in-oil. Copyright 2011 Michael M. Fryd and Thomas G. Mason.
496 Fryd · Mason

driving the molecular transport through the continuous phase are greater. In most cases, Ostwald
ripening is undesirable because it undermines long-term shelf stability.
By increasing the complexity of the formulation, Ostwald ripening can be slowed or arrested
Phase inversion
(51). The most common approach is the so-called two-component, dispersed phase method, also temperature (PIT):
known as the trapped species method (52, 53). A first solvent-like dispersed-phase liquid, which temperature at which
would exhibit Ostwald ripening after emulsification, is mixed with a second dispersed-phase liquid, an emulsion system
which has negligible solubility in the continuous phase. After emulsification, the second dispersed- can invert from
oil-continuous to
phase liquid is essentially a trapped species inside a droplet and gives rise to an internal osmotic
water-continuous or
pressure that depends on its concentration. Thus, as the solvent-like dispersed-phase liquid leaves vice-versa under
a droplet, the increasing osmotic pressure ultimately surpasses the increasing Laplace pressure moderate flow; near
of the droplet, thereby stabilizing the droplet’s size and inhibiting any further net transport. the PIT, the interfacial
Experimental studies have characterized how the mixing ratio of first and second dispersed-phase tension and film
stability are typically
liquid influences the ripening rate (26, 54–56). For example, negligible ripening occurs for oil-in-
strongly reduced
water nanoemulsions of peanut oil mixed with solvent-like tricaprylin when the internal volume
Droplet volume
fraction of the peanut oil is greater than 40% (26).
fraction: volume of
To overcome potential disadvantages of the trapped-species method, namely the limited re- dispersed phase
duction in size of the smallest droplets and also the presence of much larger droplets that continue divided by the total
to coarsen, a different method called evaporative ripening has been developed to further reduce the volume of the
size of nanoemulsions (24) and has been applied to food nanoemulsions (25). Analogous to solvent dispersed and
continuous phases;
evaporation methods, which have been thus far used to create solid nanoparticles (57), evaporative
typically denoted φ
ripening of nanoemulsions can be used to make droplets containing a single dispersed-phase liquid
Polydispersity:
down to micellar dimensions. Here, we illustrate this method for oil-in-water nanoemulsions. A
nonuniformity of the
mixture of a first volatile solvent-like oil (L1) and a second non-volatile oil (L2) is emulsified into an size distribution of
aqueous surfactant solution. In addition to having a higher vapor pressure, L1 also has significant droplets or particles,
solubility in the continuous phase; L2 has a much lower vapor pressure and negligible solubility in typically defined as
the continuous phase. After high-flow emulsification, L1 from the resulting nanoemulsion is evap- δa/<a>, where δa is
the standard deviation
orated, typically by heating and stirring, while replenishing the continuous phase; evaporation of
of the distribution of
L1 effectively provides an infinite sink for L1 that overcomes the internal osmotic pressure caused droplet radii and <a>
by L2. Thus, as has been demonstrated, all L1 can eventually be removed by evaporative ripening. is the average droplet
A schematic of this process is shown in Figure 3a. The temporal dependence of droplet size is radius
shown in Figure 3b for an O/W nanoemulsion system consisting of L1 = 0.65 cSt polydimethyl- Ostwald ripening:
siloxane (PDMS), L2 = 100 cSt PDMS, in an aqueous sodium dodecyl sulfate (SDS) solution (24). coarsening of an
Plotting the absolute value of the initial rate of droplet size reduction against the initial ψ 2 reveals emulsion through
migration of molecules
a quadratic relation, which has yet to be explained theoretically. As experimentally demonstrated,
of the dispersed phase
droplet sizes have been reduced down to micellar dimensions, and extremely viscous liquids can be through the
nanoemulsified. Furthermore, the evaporated volatile oil L1 may be recovered through distillation continuous phase;
and separation and subsequently reused, creating an environmentally friendly and cost-effective molecules are driven
process (24). This approach, in its simplest form without recovery of L1, has been used in a food from smaller droplets
to larger droplets
application to produce small final droplet sizes for a system containing corn oil (L2), ethyl acetate
through differences in
(L1), and stabilizing whey protein isolate (25). Laplace pressures
Another route to nanoemulsion size reduction is through the addition of viscosity modifiers
to the continuous phase in combination with high-flow emulsification. The viscosity ratio ηd /ηc
(dispersed to continuous phases) can be altered to maximize droplet rupturing. Several studies have
shown that smaller nanoemulsions can be produced as ηc is increased while ηd is fixed (16, 26).
Maintaining high surfactant concentrations in the continuous phase, while reducing surface ten-
sion and increasing droplet stability, also provides a substantial degree of modification to ηc that can
lead to smaller droplets (16). For instance, a peanut O/W nanoemulsion exhibits smaller droplet
sizes as polyethylene glycol (PEG) is added to the continuous phase, as shown in Figure 3c (26).

Droplet polydispersity of nanoemulsions can be reduced by ensuring that all droplets

experience the peak shear rate generated by a flow-producing device during emulsification. This
can be achieved, for instance, in a homogenizer by recirculating the emulsion multiple times (i.e.,
Laplace pressure:
the difference in increasing the number of passes) (16). A second way of reducing a nanoemulsion’s polydispersity
pressure between the is through post-emulsification fractionation using a physical process that separates larger from
dispersed liquid phase smaller nanodroplets. For instance, by applying a high effective acceleration through centrifuga-
inside a droplet and tion, a solid plug having gradients in φ and a may be formed (58). By repeating the process of cutting
the continuous phase
these plugs cross-sectionally, combining sections of plugs having similar φ and a, redispersing to
outside a droplet
arising from surface lower the droplet volume fraction, and centrifuging multiple times, nanoemulsions with a poly-
tension and curvature dispersity approaching δa/<a> ≈ 0.1 have been achieved. This polydispersity is low enough that
Self-assembly: the resulting nanoemulsions are suitable for scientific studies that require near-monodispersity.
a process by which
thermally driven
building blocks, such VIRUS-LIKE DROPLETS: CURVED NANODROPLET TEMPLATES

as atoms, molecules, or
FOR MOLECULAR SELF-ASSEMBLY
colloids, interact to
spontaneously form a Because some types of proteins, notably amphiphilic proteins, contain both hydrophobic and
larger-scale hydrophilic segments, they adsorb on droplet interfaces and therefore have commonly been used
superstructure
as emulsifiers and stabilizers (59–61). Many naturally occurring proteins, such as casein, have been
Dispersed phase: studied in emulsion formation (62). However, some proteins are not strongly amphiphilic; they can
for emulsions, the
coexist in the continuous aqueous phase along with oil droplets yet do not adsorb significantly onto
disconnected liquid
material inside oil-water interfaces. Among these types of hydrophilic proteins are the coat proteins of certain
droplets that have plant viruses. For instance, a cowpea chlorotic mottle virus consists simply of a shell, or capsid,
been dispersed into the composed of coat proteins that protects the internal genetic material (e.g., RNA) from enzymatic
immiscible liquid of degradation. This protein shell is typically composed of ordered multiprotein ring-like subunits,
the continuous phase
called capsomers, which self-assemble to form a water-permeable, often icosahedral, capsid cage
Continuous phase: (63, 64). By altering the pH and ionic strength, viruses can be disassembled, and RNA separated
for emulsions, the
from coat proteins, yielding purified protein (65).
interconnected liquid
material into which Purified capsid proteins have been self-assembled around anionically stabilized silicone oil
droplets of the nanodroplets to create virus-like droplets (VLDs), as shown in Figure 4a (66). The proteins as-
immiscible dispersed semble to encage (i.e., encapsidate) the nanodroplets through droplet-templated self-assembly,
phase are emulsified not through standard amphiphilic adsorption. Negatively charged droplets effectively act as sur-
rogates for RNA, and the capsid proteins assemble in the aqueous phase around the droplets’
anionic surfaces when pH and ionic strength are set using dialysis. This process creates protein-
caged VLDs that have liquid cores; these cores can be loaded with drug molecules, and even the
carrier oil can be chosen for biocompatibility or bioavailability. This self-assembly of viral pro-
tein cages around nanodroplets is distinctly different from a simple polymer adsorption process
of amphiphilic proteins at the oil/water interface, and it represents a new route to a decorated
nanodroplet structure. VLDs are the dispersed liquid analogue of virus-like particles (VLPs),
which contain surface-modified solid nanoparticles around which a protein cage has been formed
(67). Charge-stabilized quantum dots (68), polymers (69), and gold nanoparticles (70) have been
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 2
Schematic, typical advantages, and typical disadvantages of low-throughput nanoemulsification methods for (a) nanoemulsions (NEMs)
and (b) microemulsions. (a) Micro/nanofluidic flow focusing, satellite droplets, membrane emulsification, and liquid-liquid nucleation.
(b) Microemulsions are thermodynamically stable lyotropic phases that can have swollen spherical micelles similar in size to
nanoemulsion droplets; however, microemulsions are not metastable nanoemulsions and cannot be classified as a long-lived metastable
system. Abbreviation: W/O, water-in-oil. Copyright 2011 Michael M. Fryd and Thomas G. Mason.
498 Fryd · Mason

a
Low-throughput Typical Typical
nanoemulsion Process schematic References
advantages disadvantages
formation method
Oil flow 1. Forms monodisperse 1. Requires microfluidic 37, 38

Jet droplets apparatus
Micro/nanofluidic Water flow 2. Moderate flow 2. Limited range of flow rate
Flow ratios that produce
flow focusing NEM-sized droplets
Droplets
Oil flow 3. NEM-sized droplets
demonstrated for W/O
only
1. Moderate flow 1. Satellite droplet size and 39, 40

Stretching production are limited by
a. Viscosity ratio ηd /ηc

Satellite Pinch off b. Radius before
breakup
droplets
c. Interfacial tension
2. Producing a NEM from
satellite droplets requires
Moderate flow separation from the larger
Satellite droplet droplets
Pores 1. Forms monodisperse 1. Requires Shirasu or other 41, 42

Porous membrane droplets if a controlled porous glass
membrane having a membrane
Membrane narrow pore size
distribution is used 2. Coalescence of droplets
emulsification Oil near pores can occur—
2. Low-pressure process wetting must be
N2 pressure overcome and flow of
outer liquid is needed to
Stir bar aid droplet detachment
1. Low-energy process 1. Limited compositions: 43–45

Supersaturation Continuous phase
requires high mutual
Liquid-liquid solubility.
nucleation 2. Need small solubility of oil
in one continuous phase
(Ouzo effect) component
3. Generates only very
dilute ϕ
b
Swollen micellar Lamellar phase, 1. Self-assembled 1. Subject to phase changes 18, 20, 21
with changes in, e.g.,
Lyotropic phase, L1 Lα 2. Thermodynamically temperature, composition
microemulsion stable phase
2. Typically requires high
phase 3. Variety of different surfactant concentrations
phases may be
formed depending 3. Nondroplet morphologies
Not a metastable … on, e.g., temperature, are common, e.g.,
T1 composition lamellae
nanoemulsion T2
Water Oil molecule Water-miscible solvent
Surfactant Oil surfactant Water-miscible solvent

solution + water

a b Size reduction by
i evaporative ripening
100
Heat Heat
+ + 90
stir stir
80
<a> (nm)
70
ii 60
50
40
30 nm 30
0 2 4 6 8 10
t (h)
iii ψ2,i = 0.3 ψ2,i = 0.7
ψ2,i = 0.5 ψ2,i = 1

1.3 cm
c Size reduction by
ηd /ηc viscosity ratio
120
i) SDS
100 ii) Polysorbate 80
Diameter, D1 (nm)
80
60
40
20
0
0 5 10 15 20
Aqueous phase PEG 6600 content (wt%)
Figure 3
(a) Evaporative ripening process of a nanoemulsion that shrinks nanodroplet sizes over time: (row i ) microscale schematic, (row ii )
nanoscale schematic, and (row iii ) sequential macroscale photographs of a nanoemulsion at different stages during an evaporative
ripening process with 0.65 cSt polydimethylsiloxane (PDMS) as volatile dispersed liquid L1 and 100 cSt PDMS as nonvolatile dispersed
liquid L2 held at ≈60◦ C at times t = 30 s, 50 min, and 2.5 h. Conditions are as follows: initial overall droplet volume fraction φ i = 0.2,
initial L2 volume fraction within the dispersed phase ψ 2,i = 0.1, homogenizer inlet air pressure pa = 75 psi, sodium dodecyl sulfate
concentration CSDS = 100 mM, and number of passes N = 6. Magenta spheres represent micelles; magenta spheres with wavy lines
represent surfactant molecules; orange wavy lines represent L1 molecules; and blue wavy lines represent L2 molecules. (b) Average
droplet radius evolution over time for the evaporative ripening process under the same emulsification conditions as panel a, row iii.
(c) Effect of raising the continuous phase viscosity through addition of polyethylene glycol on mean droplet diameter for φ = 0.15 with
peanut oil as the dispersed phase stabilized with 5.6 wt% polysorbate 80 and sodium dodecyl sulfate (SDS). Panels a and b adapted with
permission from 24, copyright 2010 by the American Chemical Society, and panel c reprinted with permission from 26, copyright 2008
by the American Chemical Society.
encapsidated to form VLPs that contain nonliquid cores. Both VLDs and VLPs are potentially
useful for vaccine development (71).
Figure 4b shows transmission electron microscopy (TEM) micrographs of purified cowpea
chlorotic mottle virus (CCMV) protein that has been self-assembled around SDS-stabilized
silicone oil nanodroplets for various ionic strength and pH levels (66). Native CCMV is an
icosahedral-shaped virus composed of five-fold and six-fold protein rings called capsomers. Perfect
500 Fryd · Mason

icosahedral symmetry occurs when the number of proteins in the shell coincides with integer mul-
tiples of 60 (i.e., allowed Caspar-Klug T-numbers) (72). In the observed VLDs, varying degrees
of order and disorder in the protein cages are observed for nanodroplets having different radii.
Double emulsion: an
Certain buffer conditions even lead to the self-assembly of multi-shell protein structures around emulsion in which the
nanodroplets. However, the dominant structural motifs (i.e., ordered capsomers or disordered droplets of the
non-wild-type configurations) in the CCMV cages around droplets appear to be dependent on dispersed phase also
droplet size and therefore curvature, as well as buffer conditions. For larger nanodroplets, well contain one or more
inner droplets of an
beyond the native size of the virus, however, these ordered structures give way to other structures
immiscible liquid that
such as incomplete capsomers, scars, and hexagonal webs, as shown in Figure 4c. The origin of may be the same as or
the scar-like defects of proteins may be analogous to scar defects in Pickering emulsions (73, 74). different than the
The degree of order and disorder in the presentation of multiple assembled proteins that encage continuous phase
the nanodroplets is likely to have an important impact on the effectiveness of VLDs in vaccine,
targeting, and therapy applications.
DOUBLE NANOEMULSIONS:
MOLECULARLY DESIGNED AMPHIPHILES
By contrast to standard single emulsions, a double emulsion consists of dispersed liquid phase
droplets, each of which contains one or more internal droplets of a different immiscible liquid
phase. For example, a water-in-oil-in-water (W/O/W) emulsion consists of an aqueous dispersion
of oil droplets, each of which contains at least one internal water droplet. Beyond double emulsions,
multiple emulsions that have even greater hierarchies of internal droplets exist (75).
Double emulsions can be prepared in a variety of ways. Traditionally, a two-step fabrication
method has been used (76). First, an inverse W/O emulsion is made under high flow using a
surfactant having a low hydrophilic-lipophilic balance (HLB) dissolved in the oil to stabilize the
emulsion in accordance with the Bancroft rule (77). Next, the W/O emulsion is progressively
added to water containing a high HLB surfactant and emulsified under low flow rates to avoid
coalescence and destruction of the internal water droplets, yielding a W/O/W double emulsion
that typically has substantial polydispersity yet can be made in large quantities. Monodisperse
double emulsions have been created through microfluidic flow focusing (78) and T-junctions (79).
By varying flow rates and/or orifice sizes, the number of internal droplets in addition to the internal
droplet size may be controlled, yet throughput is rather limited, as each double emulsion droplet
must be formed individually. The lower bound achieved for outer droplet size is several microns
because the channel dimensions are large and the flow rates are much lower than those present in
high-pressure microfluidic homogenizers.
Nanoscale W/O/W double emulsions have been fabricated using high-flow emulsification in
combination with molecularly designed diblock copolypeptide (DBC) amphiphiles (80). The outer
droplets in these double nanoemulsions (DNEMs) are roughly an order of magnitude smaller than
previously achieved by other methods relying on different stabilizers. The DBC functions as the
emulsifier and can be, for instance, composed of poly(L-lysine · HBr)x -b-poly(racemic-leucine)y
[i.e., Kx (rac-L)y ], as shown in Figure 5a, where x is the number of lysine chain segments typically
ranging from 20–100 and y is the number of leucine segments typically ranging from 5–30. A
three-dimensional rendering of a double nanoemulsion system and a two-dimensional cross-
section of an individual double nanoemulsion droplet are shown in Figure 5b. The hydrophobic
leucine and charged hydrophilic lysine segments lead to preferential adsorption onto droplet
interfaces. Surprisingly, DNEMs are formed in a single-step process using a single type of DBC
amphiphile with common emulsification devices without relying on multistage emulsification
involving two or more surfactants. Additional studies are necessary to identify the molecular origin

a Capsid protein + droplet Encapsidated droplet
Na+ – – Na+ Dialysis

– – – pH, I
+ Oil
– Oil
–
– SDS
–
Na+ – – Na+
b i ii
E D
1.0
I (M)
M H R 20 nm
0.1
4.8 6.2 7.2

20 nm pH
iii
E D
1.0
20 μm
I (M)
M H R
0.1
4.8 6.2 7.2

20 nm pH
c 56 64
44 49
24 37
20 nm
3 4 7 9 12 13 16 T#
28 32 43 48 56 58 65 d (nm)
502 Fryd · Mason

of internal droplet stability and how internal water droplets are incorporated into the oil droplets
during emulsification. At least regarding the formation and stabilization of DNEMs, racemic
leucine in DBCs appears to be important (Figure 5a) because nonracemic leucine yields only
single emulsions under the same conditions. Furthermore, DBCs can be made using a variety of
peptides having different functionality groups; anionic as well as cationic blocks have been shown
to provide stable DNEMs, thereby increasing the range of potential biological applications. A
cryo-transmission electron microscopy (C-TEM) micrograph of a DNEM is shown in Figure 5c.
In Figure 5d, an optical micrograph of a microscale double emulsion, also made using DBCs in a
single-step process, is shown. Various fluorophore labels identify cargo loading in different parts
of the emulsion. Further advances in molecular design and emulsification offer the potential to
access other topologies while also tailoring the biointeractivity of nanoemulsions.
ATTRACTIVE NANOEMULSIONS
For dilute nanoemulsions composed of nanodroplets that interact only through short-range re-
pulsions, the nanodroplets diffuse through Brownian motion in the continuous phase. However,
not all nanoemulsion compositions yield repulsive nanodroplets. Deep secondary attractive wells
in the interaction potential between droplets can be created by modifying the composition of a
repulsive nanoemulsion. For instance, adding an appropriate quantity of salt to an O/W emulsion,
whether microscale or nanoscale, can transform a repulsive system of charged droplets into an
attractive system in which droplets aggregate yet do not coalesce (81). This fundamental change in
the droplet-droplet interaction potential can produce large macroscopic and microscopic changes
in a number of important physical properties, as shown in Figure 6a. In the attractive regime, the
pair interaction potential between two droplets develops a secondary minimum that is deep enough
relative to thermal energy, kB T, that the droplets do not unbind once they have encountered each
other, but not so deep that the secondary minimum merges with the primary minimum, thereby
leading to droplet coalescence. Salt-induced aggregation can be highly temperature-dependent;
above a critical temperature, T ∗ , thermal energy becomes greater than the secondary potential
well depth and the droplets redisperse (82). The origin of this salt-induced attraction lies beyond
simple Derjaguin-Landau-Verwey-Overbeek theory (83, 84), and a full quantitative understand-
ing remains to be developed. Attractive interactions between water nanodroplets in a continuous
oil phase can also exist (see the sidebar Inverse Nanoemulsions).
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 4
(a) An anionically stabilized nanoemulsion droplet can be encapsidated (i.e., caged) by purified cowpea
chlorotic mottle virus (CCMV) protein that self-assembles around the droplet interface as a result of changes
in ionic strength, I, and pH. (b) Negatively stained transmission electron micrographs of isolated CCMV
protein (i ), a bare nanoemulsion droplet (ii ), and encapsidated nanoemulsion droplets (iii ). Subpanels i and
iii show isolated protein self-assembly and self-assembly of the CCMV protein around a droplet, respectively,
under different I and pH buffer conditions: E = empty shell, D = dimer, M = multishell, H = hexagonal
sheet, R = reassembly. The subpanel iii inset shows green fluorescing fluorescein isothiocyanate–labeled
CCMV protein coating microscale emulsion droplets. (c) Structural surface features of the CCMV protein
self-assembled on nanoemulsion droplets as a function of droplet diameter. Allowed Caspar-Klug T-numbers
for perfect icosahedral virus geometries and corresponding estimated diameters are shown in the bottom
scale. As droplet sizes approach that of the native CCMV (T# = 3, diameter d = 28 nm), more completely
ordered protein capsomers (white rings) are found. These capsomers may order into sixfold arrangements, as
shown within the blue circle. For larger droplet sizes, scars (red circle) and hexagonal web-like structures
( purple circle) are also more frequently observed. Figure reprinted with permission from Reference 66,
copyright 2008 by the American Chemical Society. Abbreviation: SDS, sodium dodecyl sulfate.

a +
O R'
+ H
+ N H
+ R N x
N y
+ H H
K x(rac-L)y R O
+ O R'
+ H
N H
+ R N N y
+ H x H
+ KxLy R O
R= (CH2)4NH+3 Br –
R' = CH2CH(CH3)2
b
c d
5 μm
Figure 5
(a) Molecular structure and schematic three-dimensional structure of lysine-leucine diblock co-polypeptides
Kx (rac-L)y and Kx Ly . (b) Schematic of double nanoemulsions on a microscopic scale and an individual
double emulsion droplet with Kx (rac-L)y coating both the inner water droplet and the outer oil droplet
surfaces. (c) Cryo-transmission electron micrograph of diluted double nanoemulsion droplets prepared using
a microfluidic homogenizer: φ = 0.2, C = 1 mM K40 (rac-L)20 , pa = 130 psi, and N = 6. (d ) Micrograph
of microscale double emulsion droplets created at φ = 0.2 and C = 0.1 mM K40 (rac-L)10 and subsequently
diluted. The oil contains 0.01 M pyrene that fluoresces blue; the internal water droplets contain 2 μM
InGaP quantum dots that fluoresce red; and the K40 (rac-L)20 molecules have been labeled with fluorescein
isothiocyanate that fluoresces green. This demonstrates loading of the oil droplet and internal water droplet
with different oilborne and waterborne cargos, respectively. Panels a, c, and d first published in Reference 80,
Nature Publishing Group.
Strong secondary attractive interactions between droplets can also be generated using a deple-
tion attraction (85). The entropically induced attractive potential, which depends on the ratio of
the droplet radius a to the depletion agent am , may be fine-tuned to size-fractionate microscale
emulsions. Although classic micelle-induced depletion-attraction fractionation works well for mi-
croscale and some smaller submicron emulsions (7), the attractive potential is often too weak to
effectively size-fractionate nanoemulsions. Indeed, nanoemulsions are so small that they have also
been used as the depletion agent in the self-assembly of microscale particles (86).
Attractions between dispersed objects can lead to a variety of aggregation scenarios. In classic
diffusion-limited aggregation (DLA), a cluster grows from spheres that diffuse one at a time and
504 Fryd · Mason

a Attractive NEM Repulsive NEM c i ii iii

ϕ = 0.2 ϕ = 0.2
10 2 s –1 10 3 s –1
103
0.8 cm
102
Attractive 10 4 s–1
NEM gel
η (P) 101
10 μm 10 μm h
0.010 Å–1
0
b
10 –1
10 0 101 10 2 10 3 10 4
·
γ (s –1)
0 25 50
n/1,000
Figure 6
Attractive droplet interactions that create a secondary minimum in the pair potential can significantly alter the structure and properties
of nanoemulsions (NEMs). (a) Macroscale (upper) and microscale (lower) images of an attractive (Csalt = 400 mM NaCl) and a repulsive
nanoemulsion (Csalt = 0) having φ = 0.2 and C = 270 mM sodium dodecyl sulfate. The attractive nanoemulsion appears white owing
to multiple scattering of light from microscale inhomogeneities in the gel of aggregated nanodroplets, whereas the repulsive
nanoemulsion appears bluish-white owing to scattering from droplets having length scales that are smaller than the wavelength of
visible light. (b) Computer simulation results of slippery diffusion-limited aggregation (S-DLA). Shown is an example of an S-DLA
aggregate that has been formed as a result of slippery aggregation of 50,000 spheres, each added one at a time through a random
diffusion process to the growing cluster. The color scale indicates the order in which the n-th sphere was added. (c) Nanoemulsion gels
can be disrupted by high shear flow. Shown are the structure and rheology of an attractive nanoemulsion gel made under steady shear:
during (open blue circles) and independent of ( filled blue circles) simultaneous SANS measurements. Gel is formed with <a> = 40 nm,
φ = 0.44, CSDS = 10 mM, and Csalt = 700 mM NaCl. The lower left inset shows a schematic of an attractive nanoemulsion gel
network under shear in a Couette geometry with a gap, h. The shear direction of the gel is normal to the incident neutron beam (red
bar). The upper right insets show two-dimensional scattering intensity profiles for γ̇ = 102 , 103 , and 104 s−1 . Panel b reprinted with
permission from Reference 90, copyright 2007 by the American Physical Society. Panel c adapted with permission from Reference 91,
copyright 2011 by the American Chemical Society.
stick rigidly to the cluster (87). In a scenario more closely related to experimental quenching of
an attractive interaction in a uniform dispersion of spheres, aggregating clusters can themselves
diffuse and join together to form a shear-rigid–bonded network through diffusion-limited cluster
aggregation (DLCA) (88).
A related type of aggregation process, called slippery diffusion-limited cluster aggregation
(S-DLCA), has been found while studying dynamics of gel formation in attractive nanoemulsions
(89). In S-DLCA, clusters of four droplets, each of which retains the ability to rearrange or slip
around another, will form a tetramer before jamming together with other tetramer clusters to form
a gel. Time-resolved small angle neutron scattering (SANS) studies of the structure factor, S(q), on
S-DLCA of nanoemulsions reveal the mechanism of formation of the salt-induced gel network. As
Rheology: the study
time increases, the low-q value for S(q) increases as larger-length-scale features form; meanwhile, in
of deformation and
the mid-q range, the S(q) minimum decreases as smaller individual droplets aggregate to form larger flow of a material
clusters. At high q, cluster peaks are seen in S(q); these peaks have a higher intensity corresponding

INVERSE NANOEMULSIONS
Although inverse water-in-oil (W/O) nanoemulsions are not the emphasis of this review, important advances have
been made in W/O nanoemulsions. For instance, W/O nanoemulsions are being studied as agents for intravesicle
delivery of drugs (92), for use as a reaction polymerization medium to fabricate solid nanoparticles (93), and
for potential use to enhance thermal conductivity (94). For delivery of bioactive materials to the lungs, water-
in-fluorocarbon nanoemulsions are being studied (95). Fluorocarbons, from a biomedical standpoint, have the
advantage of being biologically inert, and they can dissolve an appreciable concentration of oxygen.
Low-energy emulsification is a method often employed for creating W/O nanoemulsions, typically using a
mixture of nonionic surfactants (96–98). Much effort has been put into characterizing the type of emulsion formed
under a variety of emulsification conditions and compositions. Depending on the relative proportions of oil, water,
and surfactants, different flow and compositional pathways may lead to microemulsions, microscale emulsions, or
nanoemulsions. Typically, in these W/O systems, an increase in water droplet size is seen over time, and Ostwald
ripening has been identified as the main cause. Opportunities exist for developing new types of inverse nanoemulsions
in which coarsening is strongly inhibited.
to more nearest neighbors than those of classic DLCA. This behavior of S(q) has motivated the
creation of the S-DLCA model for describing this system. Subsequent computer simulations of
S-DLA in the limit of extremely dilute φ confirm that interlocking tetramers within a single
isolated cluster confer rigidity, as shown in Figure 6b (90).
Rheological small angle neutron scattering (rheo-SANS) has also been used to probe a similar
attractive nanoemulsion system (91). Three different regimes are found according to shear rate at
φ = 0.44 (Figure 6c). At low shear rates, the originally jammed gel network is broken up into large
clusters, leading to shear-thinning (regime i). At intermediate shear rates, the clusters break and
reform while tumbling under shear and increasingly become smaller as γ̇ is increased (regime ii).
Finally, at high shear rates, strong shear-thinning is again seen, corresponding to droplet clusters
breaking into individual droplets (regime iii). An effective volume fraction argument related to a
model of tumbling clusters is presented to explain the cluster breakup.
OPTICAL PROPERTIES: TAILORING VISUAL APPEARANCE

One of the appealing aspects of nanoemulsions is that their optical properties can be tuned
to yield an appearance that ranges from nearly transparent to white primarily by controlling
<a>, φ, and the refractive index difference between the continuous and dispersed phases n
(Figure 7a). This tunable appearance of nanoemulsions contrasts with common emulsions hav-
ing a > 0.2 μm, which typically appear milky white, owing to multiple scattering of polychromatic
light. The visual appearance of a repulsive nanoemulsion changes based on the viewing angle with
respect to the light source (Figure 7b) in a manner reminiscent of Rayleigh scattering. More-
over, by changing a repulsive nanoemulsion into an attractive one (Figure 7c), its appearance can
be varied from translucent to white, because light is multiply scattered from larger microscale
aggregates and gel droplet network structures in attractive nanoemulsions.
Optical techniques have long been used to characterize colloidal dispersions, and nanoemul-
sions of undeformed droplets have optical properties that mimic dispersions of polymer latex
spheres. For instance, dynamic light scattering from a highly dilute, nonattractive nanoemulsion
is commonly used to characterize its effective average hydrodynamic radius and polydispersity
(99). Moreover, the optical transmission as a function of wavelength of a nanoemulsion loaded in
506 Fryd · Mason

a Repulsive NEM b Side illumination d

i ii iii 0.030
0.025
0.020
Transmission χ 0.015
iv v vi illumination
0.010
0.005
0
0 0.1 0.2 0.3
0.55 cm 1.25 cm ϕ
CSDS = 25 mM CSDS = 500 mM
CSDS = 400 mM Serial dilution curve
c Repulsive NEM Attractive NEM

i ii iii iv v vi
0.8 cm
Increasing [NaCl]
Figure 7
(a) A series of nanoemulsions (NEMs, i–vi ) at φ = 0.3 having decreasing droplet sizes <a> ≈ 101, 75, 59, 51, 42, and 35 nm and
increasing CSDS = 10, 25, 50, 100, 200, and 400 mM, respectively. Even at high φ, nanoemulsions become increasingly transparent
and preferentially scatter blue light owing to scattering by droplets having very small <a> relative to the shortest visible wavelengths.
(b) A nanoemulsion’s visual appearance can change dramatically when viewed at different angles relative to the direction of incident
light. A nanoemulsion when illuminated from the side appears bluish-white, whereas when illuminated in transmission, it appears
reddish-white. (c) A series of nanoemulsions at φ = 0.2 and CSDS = 270 mM having increasing Csalt from left to right: 0, 50, 100, 200,
300, and 400 mM NaCl, respectively. Between 200 and 300 mM NaCl, the nanoemulsion becomes dominantly attractive (i.e., forms a
gel network of aggregated nanodroplets), and the nanoemulsion’s appearance turns from blue to white because multiple scattering from
larger-microscale aggregate and gel structures occurs. (d ) The normalized refractive index difference, χ = (n − n0 )/n0 , where n is the
measured refractive index of a polydimethylsiloxane (PDMS) oil-in-water nanoemulsion and n0 is the measured refractive index of pure
water, plotted as a function of φ for a PDMS nanoemulsion. Linear fits are to the equation χ (φ,C) = χ C ∗ (C/C∗ )(1 − φ) + χ φ φ, where
C∗ is the critical micelle concentration and χ C ∗ and χ φ are coefficients inherent to the type of surfactant and oil used, respectively. A
serial dilution curve ( filled black circles and dashed line) of χ (φ,C) can be used to determine both φ and C of a previously uncharacterized
nanoemulsion. Panels a–c copyright 2011 Michael M. Fryd and Thomas G. Mason. Panel d reproduced by permission of the PCCP
Owner Societies from Reference 104.

a thin optical cuvette has been used to measure the effective scattering mean free path, l ∗ , which
depends primarily on <a>, φ, and n (100). These measurements compare reasonably well with
predictions given by Mie theory. Either very dilute or very concentrated nanoemulsions having
the smallest droplet sizes provide the greatest degree of transparency and largest l∗ for visible
light. Significant scattering can still be present in the UV, since UV wavelengths are closer to the
droplet size.
An optical refractometer can be used to obtain the refractive index of a homogeneous nanoemul-
sion to a very high degree of accuracy and precision, provided the droplets are small enough that
effective medium theory holds. Similar refractive index measurements have been made by the
solid nanoparticulate dispersion (101, 102) and microemulsion (103) communities. The normal-
ized refractive index difference, χ = (n − n0 )/n0 , where n is the measured refractive index of a
nanoemulsion and n0 is the measured refractive index of pure water, of an SDS-stabilized silicone
O/W nanoemulsion is plotted as a function of φ for a series of different SDS concentrations in Fig-
ure 7d (104). Volume-weighted effective medium predictions of χ explain the measured trends,
independent of <a>, provided <a> < 100 nm. Moreover, an unknown nanoemulsion’s surfac-
tant concentration C and also its φ may be deduced simultaneously from a series of nondestructive
measurements of χ after diluting repeatedly with the pure continuous phase.
The visual appearance of nanoemulsions can also be altered using additives. For instance,
incorporating a molecular additive that aids in refractive index matching will reduce scattering from
the droplet interfaces and make a nanoemulsion more transparent. Alternatively, dyes, whether
absorbing or fluorescing, can be included in the dispersed or continuous phases to color the
emulsion. Thus, a wide range of desirable and attractive appearances of nanoemulsions can be
achieved by varying their composition and size distribution.
MECHANICAL PROPERTIES: CONTROLLING RHEOLOGY

Nanoemulsions can exhibit stronger elasticity than microscale emulsions because the elastic stor-
age modulus G of a concentrated repulsive emulsion is proportional to the Laplace pressure of an
undeformed droplet,
L = 2σ /a and therefore is inversely proportional to droplet size. More-
over, a microscale emulsion at φ well below random close packing, which flows like a viscous
liquid, can be transformed into a strongly elastic nanoemulsion without altering the composition
by applying high flow rates that cause extreme droplet rupturing that leads to jamming (105). This
dramatic and irreversible flow-induced elastification (i.e., termed vitrification because the result-
ing positional structure of nanodroplets is disordered) is an interesting development in the field
of soft matter, both from scientific and applied viewpoints. Figure 8a illustrates this process, and
macrophotographs, shown in Figure 8b, demonstrate the increase in elasticity as <a> is reduced
further into the nanoscale regime. Measurements of G (a,φ) of direct silicone oil nanoemulsions
stabilized by anionic SDS confirm that a Debye-screened repulsion between droplet interfaces
plays an essential role in the nanoemulsion’s elasticity and the elastic vitrification effect (105).
Later experiments using a PDMS, glycerol, ethanol, fatty acid, anionic surfactant system have also
shown elastification (106). Consequently, a stable, repulsive O/W nanoemulsion’s composition
can be mostly water and yet exhibit elasticity characteristic of microscale emulsions having much
higher oil content, as shown by the plateau storage modulus G p (φ) in Figure 8c. Emulsions that
are made elastic by attractive interactions often suffer from gravitational instabilities, so repulsive
nanoemulsions offer a route to higher elasticity at lower φ while maintaining good stability against
gravitational separation over long periods of time.
The elasticity of charge-stabilized repulsive nanoemulsions can be strongly reduced by adding
modest concentrations of ionic species, thereby further screening the charge interactions between
508 Fryd · Mason

a c
105
,… , ,…
G'p (dyn cm–2 )

,
104
103
t t ϕ:
Constant
C ϕ High
Hi h flow
fl Hi
High
h fl
flow Microscale
Nanoemulsions emulsion
102
0.2 0.4 0.6 0.8
ϕ
<a> = 28 nm <a> = 740 nm
<a> = 47 nm
<a> = 73 nm
b Viscous Slowly relaxing Elastic d

<a> ≈ 82 nm <a> ≈ 70 nm <a> ≈ 62 nm

105
G'p (dyn cm–2 )

ϕ = 0.40 N = 2 ϕ = 0.40 N = 4 ϕ = 0.40 N = 8 104
G' ≈ 370 dyn cm–2 G' ≈ 1.8 × 103 dyn cm–2 G' ≈ 5.4 × 103 dyn cm–2
103 Increasing [NaCl]
0.3 0.4 0.5

ϕ
Csalt = 0 mM Csalt = 40 mM
Csalt = 10 mM Csalt = 90 mM
Figure 8
(a) Schematic of flow-induced vitrification (i.e., solidification into a disordered elastic solid) of an ionically stabilized nanoemulsion.
At constant φ, high flow and droplet rupturing cause the distances between droplet interfaces to approach the Debye screening length
(represented by the dark blue spherical shell around the orange oil droplets), thereby causing droplets to repulsively jam in a disordered
configuration. (b) Photographs of nanoemulsions made at constant φ = 0.40 as a function of an increasing number of passes N through
a microfluidic homogenizer. The images, which show the influence of gravity on a vial filled with nanoemulsion that has been tipped
sideways, illustrate the increasing elasticity as the droplet size decreases toward the Debye screening length. Measurements of the
elastic shear modulus G are reported at frequency ω = 10 rad s−1 . (c) The plateau storage modulus G p as a function of φ for
polydimethylsiloxane (PDMS) nanoemulsions at CSDS = 10 mM and, for comparison, a microscale emulsion having <a> = 740 nm
(open black circles). A nanoemulsion’s elastic onset occurs at much lower φ than that of a similar microscale emulsion and well below the
maximal random jamming point (i.e., random close packing) of monodisperse spheres at φ MRJ ≈ 0.64. (d ) G p as a function of φ for
PDMS nanoemulsions at CSDS = 10 mM and <a> = 47 nm, with added sodium chloride salt concentrations. As [Na+ ] and [Cl− ]
increase, the Debye screening length decreases, and the once-solid nanoemulsion is effectively melted into a liquid-like material. Panels
b and d reprinted with permission from Reference 107, and panel c reprinted with permission from Reference 105, copyright 2007 by
the American Physical Society.
droplet interfaces. For instance, a solid direct nanoemulsion can be effectively melted by adding
aqueous solutions of NaCl, as shown from the G p (φ,[NaCl]) in Figure 8d (107). Electrolyte
control thus provides another mechanism for tailoring a nanoemulsion’s mechanical properties
when ionic concentrations are varied in the continuous aqueous phase. As [NaCl] is increased
beyond the range associated with melting, the nanoemulsion can form a gel due to attractive
droplet interactions. If the attractive nanoemulsion remains stable and is not destroyed by droplet
coalescence, one would observe re-entrant elasticity in G ([NaCl]).

ADVANCED CHARACTERIZATION: SCATTERING AND IMAGING

Optical, X-ray, and neutron scattering are powerful techniques that aid in the determination of
Dynamic light droplet size, shape, volume fraction, and interaction in nanoemulsions. These techniques are also
scattering: an optical widely used for many dispersed materials other than nanoemulsions, yet the interpretation of the
technique that can scattering typically requires a model and may not be unique. Optical scattering techniques may be
provide a
divided into many subcategories, including dynamic light scattering, diffusing-wave spectroscopy,
measurement of the
distribution of effective static light scattering, and refractive index measurements. For example, dynamic light scattering
hydrodynamic radii of (99) typically provides a mean hydrodynamic radius of colloids, such as nanodroplets, diffusing at
thermally diffusing extremely dilute φ (Figure 9a) (24). Diffusing-wave spectroscopy (108) extends the interpretation
droplets in a dilute of dynamic light scattering from the single scattering limit into the multiple scattering regime, as
emulsion by detecting
has been shown with bubbles in foam (109). Static light scattering (110) can be used to determine
and interpreting
fluctuations in the droplet sizes in microscale emulsions and can potentially be extended to nanoemulsions (111).
scattered laser light In addition, small-angle X-ray scattering (SAXS) (112) can provide the wavenumber-dependent
intensity intensity I(q) of silicone O/W nanoemulsions (113); X-ray contrast is adequate without relying
on any contrast enhancement agents. Droplet dynamics may also be studied using X-ray photon
correlation spectroscopy (113) (Figure 9b). Similarly, differences in neutron scattering length
density of some nanoemulsion components can provide adequate contrast for rapid data collection
using SANS; selective molecular deuteration can be used to control contrast in neutron scattering
(114). The structure factor and the form factor may also be obtained from I(q) measurements
(115) (Figure 9c). In fact, neutron scattering studies of concentrated nanoemulsions provided the
first observations of subunity peaks in the effective structure factor (9). These peaks have been
explained using a polydisperse Percus-Yevick theory (116).
Although yielding average structure of a large ensemble of droplets, scattering techniques
do not provide real-space images of nanoemulsion droplets. By contrast, microscopy techniques
do. Typically, optical microscopies are very useful for characterizing microscale emulsions, but
nanoemulsions are effectively invisible because of their small droplet size. Other microscopy
methods do, however, provide spatial resolution that enables direct imaging of nanoscopic objects
(117–120). For example, various forms of electron microscopy have been used or can be extended to
obtain real-space images of nanoemulsions: negative-staining TEM (Figure 9d ), freeze-fracture–
scanning electron microscopy (121), freeze-fracture TEM (122) (Figure 9e), and C-TEM (80)
(Figure 9f ). As these images demonstrate, electron microscopies are powerful techniques that
can reveal surface structure and internal morphologies of nanoemulsions.
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 9
(a) Volume-weighted mean hydrodynamic radii as determined by dynamic light scattering for the
evaporative ripening method as a function of time for the nonvolatile polydimethylsiloxane (PDMS) liquid
L2 having various viscosities. (b) X-ray scattering intensity as a function of the scattering wavenumber q for a
PDMS oil, sodium dodecyl sulfate (SDS) stabilized, oil-in-water (O/W) nanoemulsion having φ eff = 0.60 as
measured for many waiting times after loading the cell. (c) Small-angle neutron scattering intensities as a
function of q for a PDMS oil, SDS stabilized, O/W nanoemulsion for a series of different φ (successive
curves have been shifted down by a factor of 5 as φ increases for clarity). (d ) Transmission electron
micrograph of a PDMS oil, SDS stabilized, O/W nanoemulsion negatively stained with uranyl acetate. (e)
Freeze fracture–transmission electron micrograph of a snake oil, lecithin stabilized nanoemulsion. ( f )
Cryo-transmission electron micrograph of a PDMS water-in-oil-in-water nanoemulsion coated with a
diblock copolypeptide, K40 (rac-L)10 . Panel a reprinted with permission from Reference 24, copyright 2010
by the American Chemical Society. Panel b reprinted with permission from Reference 113, copyright 2007
by the American Physical Society. Panel c reprinted with permission from Reference 115, copyright 2006 by
the American Chemical Society. Panel e reprinted with permission from Reference 122, Springer. Panel f
first published in Reference 80, Nature Publishing Group.
510 Fryd · Mason

When using traditional non-cryo-TEM to probe nanoemulsions containing nonvolatile

droplets in a volatile continuous phase, it is often necessary to negatively stain the nanoemul-
sion using a high-Z material, such as uranyl acetate (UA), to provide enough contrast. The UA is
also used to stain biomolecules, so for virally caged nanodroplets (VLDs), the UA also provides
contrast of the protein cage around individual droplets (66). Despite the potential drawbacks of
traditional TEM (i.e., imaging under vacuum, the potential for radiation damage, and the neces-
sity of staining), it offers a powerful platform for assessing droplet size distributions and droplet
surface morphologies.
a b
η2 = 100 cSt <a> = 36 nm
70
η2 = 1,000 cP 1 ϕ = 0.60
η2 = 60,000 cP
60
<a> (nm)
28 min
I (q)
71 min
50 0.1 133 min
184 min
245 min
40 427 min
0.01 588 min
30 0.05 0.1 0.2 0.3 0.4
0 2 4 6 8 10
t (h) q [nm –1]
c d
1010
10 8
I (q) (cm –1)
10 6
10 4
10 2
10 0 –3 100 nm
10 10 –2 10 –1
q (Å–1)
ϕ = 0.17 ϕ = 0.39 ϕ = 0.60
ϕ = 0.24 ϕ = 0.45 ϕ = 0.67
ϕ = 0.32 ϕ = 0.52 ϕ = 0.72
e f
200 nm
200 nm

FRONTIERS AND CONCLUSION

Although recent achievements in controlling a combination of composition, structure, and phys-
ical properties of nanoemulsions via a number of synthetic routes are impressive, such advances
provide access to yet other new, exciting frontiers in nanoemulsion research and applications.
Composition-structure-property-function relationships for nanoemulsions pose an array of very
broad problems in many different application areas, ranging from medical science to advanced
building materials. It would be desirable to systematically design nanoemulsions that optimize cer-
tain desired end-uses, based on rules that govern, for example, their transport, stability, rheological,
and optical properties, while maintaining wanted chemical compositions and functionalities.
Decorated nanoemulsions, such as the examples of virus-like droplets and synthetic
polypeptide-coated droplets, are likely to provide new avenues for achieving desired functionality
of nanoemulsions in the pharmaceutical area. Indeed, we anticipate that future research on
decorated, functionalized, and internally structured nanoemulsions as vaccines or drug delivery
vehicles is likely to provide enhanced targeting and response in biological systems. In vitro and
in vivo studies using advanced nanoemulsions are also likely to yield advances in the medical area
for certain therapeutic and diagnostic applications, whether topical or internal.

Although the recent advances in fabricating nanodroplets down to the micellar scale are already
useful, controlling and reducing the polydispersity of the droplet size distribution through high-
throughput, direct emulsification remains a frontier. For some emulsification methods, the poly-
dispersity can be reduced significantly, but not all of these methods are amenable to a wide range
of potential compositions. Ultimately, it would be desirable to reduce the polydispersity of a wide
variety of emulsification methods to the low single-digit percentage level that is routinely achieved
for monodisperse polymer particles. It is conceivable that colloidal crystals of charge-stabilized
or sterically stabilized monodisperse nanoemulsions could be achieved. For some applications, a
glassy structure of disordered nanodroplets can be desirable, so being able to control the droplet
polydispersity for a wide variety of compositions and emulsification conditions would be useful.
From a physical point of view, well-controlled nanoemulsions serve as excellent model systems
for studying the scientific properties of soft materials via SANS and SAXS experiments. The
examples of X-ray photon correlation spectroscopy (113) and rheo-SANS (91) of nanoemulsions
show that they are well suited for studying composition-structure-rheology relationships. Sim-
ilarly, because nanoscale droplets do not scatter visible light very strongly, a nanoemulsion laced
with a small number of much larger polystyrene probe spheres could be used for microrheology
experiments of glassy materials involving particle tracking or light scattering.
From an applications perspective, understanding the compatibility of nanoemulsions in
complex formulations, which may involve solid particulates, micelles, polymeric structures, and
dissolved species, is crucial to designing useful products. Much work is still needed to explore
how nanoemulsions interact with other colloidal dispersions and with surfaces under a wide range
of environmental conditions, and one might anticipate widely different results depending on the
particular formulation used.
Overall, nanoemulsions are very promising and flexible soft-matter systems, and they offer
outstanding potential for new advances in basic science, customized colloidal design, and
high-value applications.
SUMMARY POINTS
1. Nanoemulsions offer a range of desirable physical properties and chemical compositions
that can provide significant advantages over microscale emulsions.
512 Fryd · Mason

2. A variety of advanced techniques, including postflow evaporative ripening, have been

developed for making extremely fine nanoemulsions with droplets approaching micellar
dimensions for a very wide range of compositions, including very viscous liquids.
3. Nanodroplets can serve as highly curved templates that influence self-assembly and the
resulting structures of biomolecules, such as viral capsid proteins, thereby demonstrating
surface functionalization by bioactive molecular materials through a process other than
simple adsorption of amphiphilic molecules.
4. Molecular design of amphiphiles, such as di-block copolypeptides, can lead to new stable
nanoemulsion topologies, such as double nanoemulsions, that offer attractive chemical
surface functionality as well as loading and targeted delivery of hydrophilic and hy-
drophobic bioactive materials.
5. Aggregates and gels of attractive nanoemulsion droplets can be formed by introducing

strong slippery interactions, such as through the addition of electrolytes to a charge-
stabilized direct nanoemulsion. Thus, composition can strongly influence the mechanical
and optical properties of nanoemulsions through the droplet pair interaction potential.
6. The visual appearance of nanoemulsions can be tailored for specific applications by
controlling droplet size, droplet volume fraction, droplet interaction potential, and sur-
factant concentration, all of which influence light scattering, and by adding dyes to the
continuous and/or dispersed phases.
7. Elastic nanoemulsions may be made through high-flow emulsification that causes extreme
droplet rupturing, leading to elastic vitrification in systems of repulsive droplets. The
onset of appreciable elasticity in a repulsive nanoemulsion system occurs at much lower
droplet volume fractions than in microscale emulsions. Certain ionically stabilized elastic
nanoemulsions can be effectively melted by adding electrolytes, thereby yielding liquid-
like behavior.
8. Advanced nanoemulsions offer control over droplet size, polydispersity, topology, chem-
ical functionality, and composition that can be tailored for a very wide range of applica-
tions, especially in foods, personal care products, and pharmaceuticals.
DISCLOSURE STATEMENT
M.M.F. is an author of a UCLA patent application related to the evaporative ripening of na-
noemulsions, and T.G.M. is an author of several UCLA patent applications related to nanoemul-
sion formation, VLDs, evaporative ripening, and D-NEMs. T.G.M. has a financial interest in
NanoPacific Holdings, Inc., that has licensed the D-NEM technology from UCLA.
ACKNOWLEDGMENTS
The authors thank J.N. Wilking for discussions about nanoemulsion rheology and X. Zhu for
discussions about refractive index measurements of nanoemulsions.
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PC63-FrontMatter ARI 27 February 2012 8:1
Annual Review of
Physical Chemistry
Contents Volume 63, 2012
Membrane Protein Structure and Dynamics from NMR Spectroscopy

Mei Hong, Yuan Zhang, and Fanghao Hu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
The Polymer/Colloid Duality of Microgel Suspensions

L. Andrew Lyon and Alberto Fernandez-Nieves p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p25
Relativistic Effects in Chemistry: More Common Than You Thought

Pekka Pyykkö p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p45
Single-Molecule Surface-Enhanced Raman Spectroscopy
Eric C. Le Ru and Pablo G. Etchegoin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p65
Singlet Nuclear Magnetic Resonance
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Environmental Chemistry at Vapor/Water Interfaces: Insights from
Vibrational Sum Frequency Generation Spectroscopy
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Extensivity of Energy and Electronic and Vibrational Structure
Methods for Crystals
So Hirata, Murat Keçeli, Yu-ya Ohnishi, Olaseni Sode, and Kiyoshi Yagi p p p p p p p p p p p p p p 131
The Physical Chemistry of Mass-Independent Isotope Effects and
Their Observation in Nature
Mark H. Thiemens, Subrata Chakraborty, and Gerardo Dominguez p p p p p p p p p p p p p p p p p p 155
Computational Studies of Pressure, Temperature, and Surface Effects
on the Structure and Thermodynamics of Confined Water
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Orthogonal Intermolecular Interactions of CO Molecules on a
One-Dimensional Substrate
Min Feng, Chungwei Lin, Jin Zhao, and Hrvoje Petek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 201
Visualizing Cell Architecture and Molecular Location Using Soft
X-Ray Tomography and Correlated Cryo-Light Microscopy
Gerry McDermott, Mark A. Le Gros, and Carolyn A. Larabell p p p p p p p p p p p p p p p p p p p p p p p p p 225
vii
Deterministic Assembly of Functional Nanostructures Using

Nonuniform Electric Fields
Benjamin D. Smith, Theresa S. Mayer, and Christine D. Keating p p p p p p p p p p p p p p p p p p p p p 241
Model Catalysts: Simulating the Complexities
of Heterogeneous Catalysts
Feng Gao and D. Wayne Goodman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 265
Progress in Time-Dependent Density-Functional Theory
M.E. Casida and M. Huix-Rotllant p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 287
Role of Conical Intersections in Molecular Spectroscopy
and Photoinduced Chemical Dynamics
Wolfgang Domcke and David R. Yarkony p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 325
Nonlinear Light Scattering and Spectroscopy of Particles

and Droplets in Liquids

Sylvie Roke and Grazia Gonella p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 353
Tip-Enhanced Raman Spectroscopy: Near-Fields Acting
on a Few Molecules
Bruno Pettinger, Philip Schambach, Carlos J. Villagómez, and Nicola Scott p p p p p p p p p p p 379
Progress in Modeling of Ion Effects at the Vapor/Water Interface
Roland R. Netz and Dominik Horinek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 401
DEER Distance Measurements on Proteins
Gunnar Jeschke p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 419
Attosecond Science: Recent Highlights and Future Trends
Lukas Gallmann, Claudio Cirelli, and Ursula Keller p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 447
Chemistry and Composition of Atmospheric Aerosol Particles
Charles E. Kolb and Douglas R. Worsnop p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 471
Advanced Nanoemulsions
Michael M. Fryd and Thomas G. Mason p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 493
Live-Cell Super-Resolution Imaging with Synthetic Fluorophores
Sebastian van de Linde, Mike Heilemann, and Markus Sauer p p p p p p p p p p p p p p p p p p p p p p p p p p 519
Photochemical and Photoelectrochemical Reduction of CO2
Bhupendra Kumar, Mark Llorente, Jesse Froehlich, Tram Dang,
Aaron Sathrum, and Clifford P. Kubiak p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 541
Neurotrophin Signaling via Long-Distance Axonal Transport
Praveen D. Chowdary, Dung L. Che, and Bianxiao Cui p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 571
Photophysics of Fluorescent Probes for Single-Molecule Biophysics
and Super-Resolution Imaging
Taekjip Ha and Philip Tinnefeld p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 595
viii Contents
Ultrathin Oxide Films on Metal Supports:

Structure-Reactivity Relations
S. Shaikhutdinov and H.-J. Freund p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 619
Free-Electron Lasers: New Avenues in Molecular Physics and
Photochemistry
Joachim Ullrich, Artem Rudenko, and Robert Moshammer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 635
Dipolar Recoupling in Magic Angle Spinning Solid-State Nuclear
Magnetic Resonance
Gaël De Paëpe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 661
Indexes
Cumulative Index of Contributing Authors, Volumes 59–63 p p p p p p p p p p p p p p p p p p p p p p p p p p p 685

Cumulative Index of Chapter Titles, Volumes 59–63 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 688
Errata
An online log of corrections to Annual Review of Physical Chemistry chapters (if any,
1997 to the present) may be found at http://physchem.AnnualReviews.org/errata.shtml
Contents ix

Advanced Nanoemulsions: Further

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PC63CH22-Mason ARI 5 March 2012 10:11

• Top downloaded articles

Annu. Rev. Phys. Chem. 2012. 63:493–518 Keywords

Emulsion: one liquid compositions and properties.

494 Fryd · Mason

www.annualreviews.org • Advanced Nanoemulsions 495

High-throughput Process concept Typical Typical

Extreme flow exerts stresses that overcome

1. Versatility of routes 1. More limited range of 33–36

Lyotropic 1. Low-flow process 1. Limited to mostly 11, 12, 30, 31

Water droplet Oil Beaker Low-flow mixer Oil surfactant

Aqueous surfactant Oil droplet Ultrasonic High-flow

496 Fryd · Mason

www.annualreviews.org • Advanced Nanoemulsions 497

Droplet polydispersity of nanoemulsions can be reduced by ensuring that all droplets

building blocks, such VIRUS-LIKE DROPLETS: CURVED NANODROPLET TEMPLATES

498 Fryd · Mason

Oil flow 1. Forms monodisperse 1. Requires microfluidic 37, 38

1. Moderate flow 1. Satellite droplet size and 39, 40

a. Viscosity ratio ηd /ηc

Pores 1. Forms monodisperse 1. Requires Shirasu or other 41, 42

1. Low-energy process 1. Limited compositions: 43–45

Water Oil molecule Water-miscible solvent

Surfactant Oil surfactant Water-miscible solvent

www.annualreviews.org • Advanced Nanoemulsions 499

ψ2,i = 0.5 ψ2,i = 1

500 Fryd · Mason

www.annualreviews.org • Advanced Nanoemulsions 501

a Capsid protein + droplet Encapsidated droplet

Na+ – – Na+ Dialysis

4.8 6.2 7.2

4.8 6.2 7.2

502 Fryd · Mason

www.annualreviews.org • Advanced Nanoemulsions 503

504 Fryd · Mason

a Attractive NEM Repulsive NEM c i ii iii

www.annualreviews.org • Advanced Nanoemulsions 505

in which coarsening is strongly inhibited.

OPTICAL PROPERTIES: TAILORING VISUAL APPEARANCE

506 Fryd · Mason

a Repulsive NEM b Side illumination d

c Repulsive NEM Attractive NEM

www.annualreviews.org • Advanced Nanoemulsions 507

MECHANICAL PROPERTIES: CONTROLLING RHEOLOGY

508 Fryd · Mason

G'p (dyn cm–2 )

b Viscous Slowly relaxing Elastic d

<a> ≈ 82 nm <a> ≈ 70 nm <a> ≈ 62 nm

G'p (dyn cm–2 )

103 Increasing [NaCl]

0.3 0.4 0.5

www.annualreviews.org • Advanced Nanoemulsions 509

ADVANCED CHARACTERIZATION: SCATTERING AND IMAGING

510 Fryd · Mason

When using traditional non-cryo-TEM to probe nanoemulsions containing nonvolatile

www.annualreviews.org • Advanced Nanoemulsions 511

FRONTIERS AND CONCLUSION

for certain therapeutic and diagnostic applications, whether topical or internal.

512 Fryd · Mason

2. A variety of advanced techniques, including postﬂow evaporative ripening, have been

5. Aggregates and gels of attractive nanoemulsion droplets can be formed by introducing

www.annualreviews.org • Advanced Nanoemulsions 513

13. Jafari SM, He Y, Bhandari B. 2007. Optimization of nano-emulsions production by microﬂuidization.